Mecanismo y diseño de vacunas para el sars-cov-2, revisión narrativa / Mechanism and design of vaccines against sars-cov-2, narrative review / Mecanismo e desenho de vacinas para sars-cov-2, revisão narrativa

La infección por el virus SARS-CoV-2, conocida como COVID-19, ha causado alta morbilidad y mortalidad en el mundo. Después de haber descifrado el código genético del virus y haber desarrollado un gran trabajo investigativo en la creación de vacunas, con diversas estrategias de acción, se ha logrado disminuir la morbi mortalidad. Fue necesario acelerar el proceso de producción de vacunas, lo cual estuvo facilitado por el avanzado conocimiento científico en el campo de la genética y la virología, para brindar a la especie humana una protección eficaz y segura contra la agresiva y progresiva infección. Las vacunas se clasifican de acuerdo con su mecanismo de acción, existen vacunas basadas en vectores virales que no se replican, vacunas recombinantes, otras basadas en virus atenuados y virus inactivos, y (la gran novedad de la ciencia actual) las vacunas basadas en ARN mensajero y ADN. Estas últimas han demostrado una gran eficacia y seguridad en la prevención de la infección por el SARS-CoV-2, también han impactado de manera fuerte, por lo que han reducido la infección y la mortalidad en la población. En consecuencia, cada día que pasa desde que se inició el periodo de vacunación mundial, se evidencia una reducción en la curva de contagio y mortalidad por COVID-19.

The infection produced by the SARS-CoV-2 virus, known as COVID-19, has caused high morbidity and mortality across the world. After having deciphered the virus's genoma and carried out investigative endeavors that led to the creation of a variety of vaccines with different mechanisms of action, it has been possible to decrease the morbidity and mortality associated with the virus. It was necessary to accelerate the vaccine production process, which was facilitated by advanced scientific knowledge within the disciplines of genetics and virology, in order to provide the human species with a safe and effective form of protection against the aggressive and progressive infection. Vaccines are classified differently depending on their action mechanisms: there are some based on non-replicating viral vectors, recombinant vaccines, ones that are based on attenuated or inactivated viruses, and (the greatest novelty of current scientific developments) vaccines based on DNA and messenger RNA. The latter has demonstrated significant efficacy and safety in the prevention of the SARS-CoV-2 infection as observed in preliminary studies, and they have meaningfully impacted the population by reducing the rates of infection and mortality. As a result, decreased levels of spread of and mortality from COVID-19 have been evidenced across the globe following the beginning of the vaccine distribution period.

A infecção pelo vírus SARS-CoV-2, conhecido como COVID-19, tem causado elevada morbidade e mortalidade no mundo. Depois de ter decifrado o código genético do virus e de ter realizado um grande trabalho de investigação na criação de vacinas, com diversas estratégias de ação, a morbilidade e a mortalidade foram reduzidas. Foi necessário acelerar o processo de produção de vacinas, facilitado por conhecimentos científicos avançados no domínio da genética e da virologia, para proporcionar à espécie humana uma proteção eficaz e segura contra a infecção agressiva e progressiva. As vacinas são classificadas de acordo com seu mecanismo de ação, existem vacinas baseadas em vetores virais que não se replicam, vacinas recombinantes, outras baseadas em virus atenuados e vírus inativos, e (a grande novidade da ciência atual) vacinas baseadas em RNA mensageiro e ADN. Estas últimas demonstraram grande eficácia e segurança na prevenção da infecção por SARS-CoV-2, mas também tiveram um forte impacto, razão pela qual reduziram a infecção e a mortalidade na população. Consequentemente, a cada dia que passa desde o início do período global de vacinação, fica evidente uma redução na curva de contágio e mortalidade por COVID-19.

Recent advances in drug screening methods of SARS-CoV-2 spike protein / 药学学报

The pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a serious impact on global public health and the economy. SARS-CoV-2 infiltrates host cells via its surface spike protein, which binds to angiotensin-converting enzyme 2 on the host cell membrane. As a result, small molecules targeting spike protein have emerged as a hotspot in anti-SARS-CoV-2 drug research. Activity screening is an important step in seeking small molecule drugs. Therefore, this article aims to review the biological activity evaluation methods of small molecule inhibitors targeting SARS-CoV-2 spike protein, with the goal of laying the foundation for the discovery of new anti-SARS-CoV-2 drugs.

Immunogenicity of mucosal COVID-19 vaccine candidates based on the highly attenuated vesicular stomatitis virus vector (VSVMT) in golden syrian hamster

COVID-19 is caused by coronavirus SARS-CoV-2. Current systemic vaccines generally provide limited protection against viral replication and shedding within the airway. Recombinant VSV (rVSV) is an effective vector which inducing potent and comprehensive immunities. Currently, there are two clinical trials investigating COVID-19 vaccines based on VSV vectors. These vaccines were developed with spike protein of WA1 which administrated intramuscularly. Although intranasal route is ideal for activating mucosal immunity with VSV vector, safety is of concern. Thus, a highly attenuated rVSV with three amino acids mutations in matrix protein (VSVMT) was developed to construct safe mucosal vaccines against multiple SARS-CoV-2 variants of concern. It demonstrated that spike protein mutant lacking 21 amino acids in its cytoplasmic domain could rescue rVSV efficiently. VSVMT indicated improved safeness compared with wild-type VSV as the vector encoding SARS-CoV-2 spike protein. With a single-dosed intranasal inoculation of rVSVΔGMT-SΔ21, potent SARS-CoV-2 specific neutralization antibodies could be stimulated in animals, particularly in term of mucosal and cellular immunity. Strikingly, the chimeric VSV encoding SΔ21 of Delta-variant can induce more potent immune responses compared with those encoding SΔ21 of Omicron- or WA1-strain. VSVMT is a promising platform to develop a mucosal vaccine for countering COVID-19.

Screening and identification of single-chain fragment variable against the receptor-binding domain of spike protein of SARS-CoV-2 / 中国生物制品学杂志

@#Objective To construct a single-chain fragment variable(scFv)phage display library against receptor-binding domain(RBD)of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)spike protein(S)to screen specific scFv and identify the function.Methods m RNA was extracted from spleen cells of mice immunized with RBD protein and reversely transcribed into c DNA,with which as template,genes of the hight chain fragment of variable(VH)and light chain fragment of variable(VL)of scFv were amplified and then assembled into scFv gene fragment through splicing overlap extension PCR(SOE-PCR).The scFv gene fragment was inserted to phage vector to construct scFv phage display library.After four rounds of biopanning,the scFv gene with strong binding ability to RBD was screened and expressed recombinantly,purified and identified for biological activity.Results The constructed scFv phage library showed a titer of 6.0×10(11)pfu/m L.After four rounds of biopanning,four scFv strains with strong binding to RBD were selected,namely scFv11,scFv12,scFv25and scFv28.scFv was mainly expressed in the form of inclusion body with a relative molecular mass of about 27 000,a concentration of 2.4 mg/m L and a purity of about 90%,which bound specifically to mouse monoclonal antibody against His labeled by HRP after purification.All four scFv strains bound specifically to RBD recombinant protein,among which the other 3 scFv strains bound to the S protein of wild type and multiple mutant strains except scFv28.All four strains showed dose-dependent interaction with RBD,with affinity dynamic fitting dissociation constants(K_Ds)8.9,5.92,10.67and 2.36 nmol/L,and steady-state fitting dissociation constants(K_Ds)of 5.3,6.5,8.7 and 5.8 nmol/L,respectively.scFv11,scFv12 and scFv25 simultaneously identified three independent RBD polypeptides,including RBD2(S(11)pfu/m L.After four rounds of biopanning,four scFv strains with strong binding to RBD were selected,namely scFv11,scFv12,scFv25and scFv28.scFv was mainly expressed in the form of inclusion body with a relative molecular mass of about 27 000,a concentration of 2.4 mg/m L and a purity of about 90%,which bound specifically to mouse monoclonal antibody against His labeled by HRP after purification.All four scFv strains bound specifically to RBD recombinant protein,among which the other 3 scFv strains bound to the S protein of wild type and multiple mutant strains except scFv28.All four strains showed dose-dependent interaction with RBD,with affinity dynamic fitting dissociation constants(K_Ds)8.9,5.92,10.67and 2.36 nmol/L,and steady-state fitting dissociation constants(K_Ds)of 5.3,6.5,8.7 and 5.8 nmol/L,respectively.scFv11,scFv12 and scFv25 simultaneously identified three independent RBD polypeptides,including RBD2(S(334～353)),RBD9(S(334～353)),RBD9(S(439～458))and RBD13(S(439～458))and RBD13(S(499～518)).Homologous model of scFv constructed by online server SWISS-MODEL showed a good quality and was used for molecular docking.The interface at which scFv11 interacted with RBD only partially coincided with the interaction interface of human angiotensin converting enzyme 2(ACE2)and RBD,and the interaction interfaces of scFv12 and scFv25 with RBD were quite different from that of ACE2.Conclusion In this study,scFv specifically bound to SARS-Co V-2 RBD was screened and prepared through constructing scFv phage library against SARS-CoV-2 RBD,which provided experimental basis for further development of anti-SARS-CoV-2 drugs and detection reagents.

Research progress of angiotensin converting enzyme 2 co-expression in non-small cell lung cancer and SARS-CoV-2 / 中国胸心血管外科临床杂志

@#Since the first case of corona virus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at the end of 2019, the virus has spread rapidly around the world and has become a global public health problem. In the process of this virus epidemic, compared with the general population, cancer patients are considered to be highly susceptible people, especially the lung cancer patients. Some studies have shown that angiotensin converting enzyme 2 (ACE2) may be the pathway for SARS-CoV-2 to infect the host. At the same time, ACE2 is often abnormally expressed in non-small cell lung cancer. Therefore, understanding the respective mechanisms of ACE2 in COVID-19 and non-small cell lung cancer has extremely important reference value for the study of vaccines and therapeutic drugs, and also provides meaningful guidance for the protection of patients with lung cancer during the epidemic. This article reviews the possible invasive mechanism of ACE2 in SARS-CoV-2 and its abnormal expression in non-small cell lung cancer.

Overexpression of SARS-CoV-2 spike protein mediates growth inhibition in human retinal pigment epithelial cells / 中华眼底病杂志

Objective:To observe the inhibition of SARS-CoV-2 spike protein (S-protein) on the proliferation of human retinal pigment epithelium (RPE) cells.Methods:SARS-CoV-2 S-protein gene fragment expression plasmid (p3xflag-S) was constructed and transfected into human RPE, HEK293 cells. DNA sequencing was used for identification, and the expression of Flag-S was detected by Western blot. HEK293 cells were divided into the cells 1, 2, 3 and 4 and transfected with GFP11 plasmid and vector, GFP1-10 plasmid and vector, transfected with GFP11 and pCMV-HA-ACE2 plasmid, GFP1-10 and p3xflag-S plasmid. Cell 1 was co-cultured with cell 2 (control group 1), cell 2 with cell 3 (control group 2), cell 3 with cell 4 (observation group), and cell 1 mixed with cells 2, 3 and 4 (control group 3). Bright-field microscopy and fluorescence microscopy were used to observe cell fusion. RPE cells were divided into control group and overexpression S-protein group. The cell cycle was detected by flow cytometry; the cell proliferation level was detected by Counting Kit 8 (CCK-8); and the S-protein expression level in RPE cells was detected by Western blot. The Student’s t-test was performed for comparison between groups. Results:DNA sequence assay showed that S-protein cDNA was fused with flag-tagged protein. Western blot assay showed that S-protein-related expression was elevated in transfected HEK293 cells compared with untransfected p3xflag-S cells. Large, multinucleated fused cell clusters were visible under bright-field microscopy; multiple nuclear with distinct green fluorescence were visible in the fused cells under fluorescence microscopy. Western blot assay showed elevated S-protein-related expression in transfected p3xflag-S plasmid RPE cells compared to untransfected p3xflag-S plasmid RPE cells. CCK-8 results showed that the proliferative capacity of RPE cells in the S-protein overexpression group was significantly reduced compared with the control group, with statistically significant differences ( t=22.70, 16.75, 23.38; P<0.000 1). The results of flow cytometry showed that the G1 phase cells in the control and overexpression S-protein groups were 41.1 % and 67.0%, respectively; compared with the control group, the G1 phase cells in the overexpression S-protein group were significantly higher, and the difference was statistically significant ( t=4.76, P=0.018). The apoptosis rate was significantly increased in the S-protein overexpression group compared with the control group, and the difference was statistically significant ( t=4.91, P=0.008). Conclusion:Overexpression of the SARS-CoV-2 spike protein reduced the proliferation of human RPE cells.

Characteristics of whole genome analysis of severe acute respiratory syndrome coronavirus 2 during different epidemic periods in Wuxi City / 中华传染病杂志

Objective:To understand the genome sequence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and spike protein variations during different epidemic periods in Wuxi City.Methods:Nucleic acid was extracted from the nasopharyngeal swab samples of six local cases of coronavirus disease 2019 (COVID-19) (from January to February, 2020) and 13 imported cases of COVID-19 (from March to September, 2021) in Wuxi City, and the whole genome was amplified to construct the sequencing library. The second-generation sequencer was used for sequencing. The CLC Genomics Workbench (21 version) software was used to analyze the offline data with NC_045512.2 as the reference strain, and MEGA 7.0 software was used to construct the phylogenetic tree.Identification of type was conducted by Nextstrain typing method and phylogenetic assignment of named global outbreak lineages (Pangolin) typing method.Results:There were five subtypes in Nextstrain and seven subtypes in Pangolin of the nineteen patients with COVID-19. Compared with NC_045512.2, the median nucleotide mutation sites were 29 (range 0 to 42) and amino acid mutation sites were 20 (range 0 to 34). The six local and 13 imported cases had no common nucleotide mutation sites and were in different evolutionary branches. The sequences of the six local cases were highly homologous with the reference strain sequences (NC_045512.2) at the early stage of the pandemic, and the evolutionary distance was close to that of the reference strain. The 13 imported cases were obviously divided into three evolutionary branches (Alpha, Beta, Delta variant).The four Beta variants shared eight amino acid mutation sites in spike protein, and the two Alpha variants shared eight amino acid mutation sites in spike protein, and the seven Delta variants shared five amino acid mutation sites in spike protein.Conclusions:New mutations of SARS-CoV-2 are constantly emerging during the epidemic. The increase of the nucleotide sites number may result in the change of spike protein amino acid. Therefore, the whole-genome sequencing analysis plays an important role in the accurate tracing of epidemic origin and adjustment of prevention and control measures.

Highly Sensitive Poly-N-isopropylacrylamide Microgel-based Electrochemical Biosensor for the Detection of SARS-COV-2 Spike Protein / 生物医学与环境科学（英文）

OBJECTIVE@#Late 2019 witnessed the outbreak and widespread transmission of coronavirus disease 2019 (COVID-19), a new, highly contagious disease caused by novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Consequently, considerable attention has been paid to the development of new diagnostic tools for the early detection of SARS-CoV-2.@*METHODS@#In this study, a new poly-N-isopropylacrylamide microgel-based electrochemical sensor was explored to detect the SARS-CoV-2 spike protein (S protein) in human saliva. The microgel was composed of a copolymer of N-isopropylacrylamide and acrylic acid, and gold nanoparticles were encapsulated within the microgel through facile and economical fabrication. The electrochemical performance of the sensor was evaluated through differential pulse voltammetry.@*RESULTS@#Under optimal experimental conditions, the linear range of the sensor was 10 -13-10 -9 mg/mL, whereas the detection limit was 9.55 fg/mL. Furthermore, the S protein was instilled in artificial saliva as the infected human saliva model, and the sensing platform showed satisfactory detection capability.@*CONCLUSION@#The sensing platform exhibited excellent specificity and sensitivity in detecting spike protein, indicating its potential application for the time-saving and inexpensive detection of SARS-CoV-2.

Multi-level scoring approach to discover multi-targeting potency of medicinal plant phytochemicals against protein targets in SARS-CoV-2 and human ACE-2 receptor

SARS-CoV-2 pandemic has become a major threat to human healthcare and world economy. Due to the rapid spreading and deadly nature of infection, we are in a situation to develop quick therapeutics to combat SARS-CoV-2. In this study, we have adopted a multi-level scoring approach to identify multi-targeting potency of bioactive compounds in selected medicinal plants and compared its efficacy with two reference drugs, Nafamostat and Acalabrutinib which are under clinical trials to treat SARS-CoV-2. In particular, we employ molecular docking and implicit solvent free energy calculations (as implemented in the Molecular Mechanics -Generalized Born Surface Area approach) and QM fragmentation approach for validating the potency of bioactive compounds from the selected medicinal plants against four di?erent viral targets and one human receptor (Angiotensin-converting enzyme 2 -ACE-2) which facilitates the SARS-CoV-2entry into the cell. The protein targets considered for the study are viral 3CL main protease (3CLpro), papain-like protease (PLpro), RNA dependent RNA polymerase (RdRp), and viral spike protein-human hACE-2 complex (Spike:hACE2)including human protein target (hACE-2). Herein, thereliable multi-level scoring approach was used to validate the mechanism behind the multi-targeting potency of selected phytochemicals from medicinal plants. The present study evidenced that the phytochemicals Chebulagic acid, Stigmosterol, Repandusinic acid and Geranin exhibited efficient inhibitory activity against PLpro while Chebulagic acid was highly active against 3CLpro. Chebulagic acid andGeranin also showed excellent target specific activity against RdRp.Luteolin, Quercetin, Chrysoeriol and Repandusinic acid inhibited the interaction of viral spike protein with human ACE-2 receptor. Moreover Piperlonguminine and Piperine displayed significant inhibitory activity against human ACE-2 receptor. Therefore, the identified compounds namely Chebulagic acid, Geranin and Repandusinic acid can serve as potent multi-targeting phytomedicine for treating COVID-19

Correlación entre dos metodologías para la medición de anticuerpos contra la proteína spike del coronavirus del Síndrome Respiratorio Agudo Grave 2 / Correlation between two methodologies for the measurement of antibodies against the spike protein of coronavirus of the Severe Acute Respiratory Syndrome 2

Resumen El coronavirus del Síndrome Respiratorio Agudo Grave 2 (SARS-CoV-2) posee diversas proteínas estructurales que incluyen la proteína spike (S), principal blanco de las vacunas actuales. Existen diversas metodologías para la medición de anticuerpos contra ésta que brindan información acerca de la respuesta inmune frente a la vacunación. El objetivo de este trabajo fue determinar la correlación entre quimioluminiscencia (CLIA) y enzimoinmunoanálisis de adsorción (ELISA) para la medición de anticuerpos IgG anti-proteína S (IgG anti-S). Se recolectaron resultados serológicos de 169 individuos y se determinaron los niveles de anticuerpos por ambas metodologías. Del total de muestras, 106 arrojaron un resultado positivo por ambas metodologías y 15 resultaron discordantes (CLIA+, ELISA-), con índice Kappa de 0,80. La correlación entre ambas metodologías fue buena. Este estudio podría aportar al manejo y seguimiento de la población vacunada, con la finalidad de obtener un valor de corte para evaluar la aplicación de una dosis adicional.

Abstract Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) has several structural proteins including the spike (S) protein, which is the main target of current vaccines. There are various methodologies for the measurement of antibodies against it that provide information about the immune response to vaccination. The objective of this study was to determine the correlation between chemiluminescence (CLIA) and enzyme-linked immunoassay (ELISA) for the measurement of IgG anti-S protein (IgG anti-S) antibodies. Serological results were collected from 169 individuals and antibody levels were determined by both methodologies. Out of the total samples, 106 were positive by both methodologies and 15 were discordant (CLIA+, ELISA-), with a Kappa index of 0.80. The correlation between both methodologies was good. This study could contribute to the management and follow-up of the vaccinated population, in order to obtain a cut-off value to evaluate the application of an additional dose.

Resumo O coronavírus da Síndrome Respiratória Aguda Grave 2 (SARS-CoV-2) possui várias proteínas estruturais, incluindo a proteína spike (S), principal alvo das vacinas atuais. Existem várias metodologias para medir anticorpos contra ela que fornecem informações sobre a resposta imune diante da vacinação. O objetivo deste trabalho foi determinar a correlação entre quimioluminescência (CLIA) e enzimoimunoanálise de absorção (ELISA) para a medição de anticorpos IgG anti-proteína S (IgG anti-S). Foram coletados resultados sorológicos de 169 indivíduos e os níveis de anticorpos foram determinados por ambas as metodologias. Do total de amostras, 106 deram resultados positivos nas duas metodologias e 15 foram discordantes (CLIA+, ELISA-), com índice Kappa de 0,80. A correlação entre as duas metodologias foi boa. Este estudo poderia contribuir para a gestão e seguimento da população vacinada, visando a obter um valor de corte para avaliar a aplicação de uma dose adicional.

Variation of SARS-CoV-2 spike protein and its effects on neutralization sensitivity / 中华微生物学和免疫学杂志

The COVID-19 epidemic that occurred at the end of 2019 spreads rapidly to all parts of the world, putting the global public health system to a severe test. With the continuation of the epidemic, SARS-CoV-2 variants are constantly emerging. In particular, the mutation of the spike protein can cause changes in the infectivity and antigenicity of the virus, resulting in an increase in the infectivity and a decline in the protective efficacy of existing vaccines, and even the replacement of epidemic strains. This is also one of the reasons why the epidemic has not been effectively controlled so far. Nowadays, the main circulating variants have changed their characteristics to a certain extent, and the neutralization sensitivity of some variants to neutralizing monoclonal antibodies, immune sera and convalescent sera has decreased to a certain extent compared with the original strains. The emergence of variants is not only related to the characteristics of the virus itself, but also to the changes of transmission host and the chronic infection in people with deficient immunity. The emerging variants should be closely monitored, and their functional characteristics should be systematically studied so as to provide data for vaccine research and development and the designation of immunization strategies.

Inhaled heparin polysaccharide nanodecoy against SARS-CoV-2 and variants

The heparin polysaccharide nanoparticles block the interaction between heparan sulfate/S protein and inhibit the infection of both wild-type SARS-CoV-2 pseudovirus and the mutated strains through pulmonary delivery.Image 1.

Optimization of SARS-CoV-2 spike protein receptor binding domain expression in Pichia pastoris and evaluation of its immunogenicity / 中华微生物学和免疫学杂志

Objective:To effectively express the receptor binding domain (RBD) of SARS-CoV-2 spike protein in Pichia pastoris and to evaluate its immunogenicity. Methods:The gene encoding the RBD protein was synthesized and cloned into the pPICZαA plasmid. After linearization, the plasmid was transferred and integrated into the genome of Pichia pastoris. The expressed RBD protein in culture supernatant was analyzed by Western blot and Biolayer interferometry. After screening, a single clone expressing the RBD protein was selected. The high-level expression of RBD protein was achieved by optimizing the fermentation process, including the salt concentration adjusting of the medium and induction condition optimization (pH, temperature and duration). The immunogenicity of the expressed RBD protein was evaluated in a mouse model. Results:A single clone with a high expression level of RBD protein was obtained and named RBD-X33. The expression level of RBD protein in the fermentation supernatant reached up to 240 mg/L after optimization of the induction condition (HBSM medium, pH=6.5±0.3, 22℃ and 120 h). In the mouse experiment, the recombinant RBD protein was formulated with Alum+ CpG dual adjuvant and injected into mice. The binding IgG antibody levels were up to 2.7×10 6 tested by ELISA and the neutralizing antibody levels were up to 726.8 tested by live virus neutralizing antibody assay (prototype). Conclusions:The RBD protein could be efficiently expressed in Pichia pastoris and induce stronger immune response in animals. This study suggested that the recombinant SARS-CoV-2 RBD protein expressed in Pichia pastoris could serve as a candidate antigen in the development of SARS-CoV-2 vaccine.

Comparison of IgG Antibody Levels in Mice Immunized with SARS-CoV-2 RBD Proteins Produced from Two Expression Systems / 中国生物化学与分子生物学报

The COVID-19 pandemic is still spreading around the world,posing a severe challenge to public health security and economic development in China and beyond. The pivotal process of SARS-COV-2 invasion is that the receptor binding domain (RBD) of Coronavirus spike protein binds with the angioten-sin converting enzyme 2 (ACE2) receptor on host cells. Therefore, RBD is closely related to our major anti-epidemic strategies such as rapid pathogen detection and development of vaccines and antiviral drugs. In this study, we aim to compare the anti-RBD IgG by immunizing mice with the recombinant RBDs of novel Coronavirus spike protein, and evaluate the antibody titers and duration elicited by RBD produced from different host cells and used in different doses, which can be used for vaccine evaluation, drug development, and pathogen detection. SDS-PAGE and mass spectrometry analysis showed that the molecular weight of bRBD (produced by insect cells) and hRBD (produced by mammalian cells) were slightly different, which are mainly caused by different glycosylation modification. Flow cytometry revealed that the binding rates of bRBD and hRBD to HEK293-ACE2-overexpressing cells were 88. 5% and 92. 7%, respectively, indicating that both antigens have favorable bioactivity. Balb/ c mice are immunized intramuscularly with 10 μg and 20 μg per mouse of RBD proteins, which were mixed with Quick Antibody adjuvant previously, and blood samples collected from the tail at 2, 4, 6, 8, 12 and 24 weeks after the primary immunization. Enzyme-linked immunosorbent assay (ELISA) showed that both RBD immunization with 10 μg or 20 μg could induce a rapid immune response to produce IgG antibodies. The antibody titers reached the peak at 4-6 weeks post first immunization, and persisted over a long time up to 6 months. No significant difference was observed in the induced antibody titers by bRBD and hRBD as well as their different doses. Virus Neutralization Assays showed that specific antibodies induced by RBD-immunized mice could inhibit the infection of ACE2-positive cells by SARS-CoV-2 pseudovirus. These results suggest that both RBD proteins through commendable adjuvant inoculation can rapidly induce an effective humoral immune response and generate specific neutralizing antibodies. It is expected to be helpful for rapid preparation of antibodies against the future various RBD mutants during the pandemic.

Desenvolvimento de teste diagnóstico não invasivo capaz de detectar anticorpos anti-SARS-CoV-2 em urina / Development of a non-invasive diagnostic test capable of detecting anti-SARS-CoV-2 antibodies in urine

A plataforma de ELISA (ensaio de imunoabsorção por ligação enzimática) tem sido amplamente utilizada para detectar anticorpos anti-SARS-CoV-2 gerados após a exposição ao vírus ou à vacinação. A amostra comumente utilizada para a realização do teste é o soro. Até o momento, nenhum estudo havia investigado a urina do paciente como amostra para detectar anticorpos específicos para o vírus SARS-CoV-2. A urina é um espécime biológico que traz vantagens significativas inerentes ao tipo de amostra, que compreende coleta não invasiva, de fácil manuseio e armazenamento. Neste trabalho, propomos um ELISA indireto in house baseado no uso de urina e proteínas recombinantes do Nucleocapsídeo (N) ou da Spike (S) do vírus SARS-CoV-2. As proteínas recombinantes (r) de SARS-CoV-2, N e as subunidades da proteína S (S-Glic, S1-NGlic e RBD-NGlic), foram avaliadas usando um painel composto por aproximadamente 200 amostras de urina e de soro. A presença de anticorpos anti-SARS-CoV-2 na urina foi detectada com sensibilidade e especificidade similares ou superiores ao soro, nas quais foram obtidos valores de sensibilidade de 94,0%, 75,0%, 81,38% e 89,66%, e especificidade de 100%, 96,0%, 96,77% e 96,77%, frente às proteínas rSARS-CoV-2 N, S-Glic, S1-NGlic e RBDNGlic, respectivamente. Dessa forma, os dados apresentados sugerem que a urina poderia ser considerada como uma potencial amostra biológica para aplicação em plataformas de imunodiagnóstico para a infecção por SARS-CoV-2, trazendo benefícios tanto no contexto individual quanto populacional.

The Enzyme-linked immunosorbent assay (ELISA) method has been widely used to detect anti-SARS-CoV-2 antibodies generated after exposure to the virus or vaccination. The sample usually used to perform the test is the serum. Thus far, no study has investigated the urine of patients as biological sample to detect specific SARS-CoV-2 antibodies. Urine is a biological specimen with significant advantages inherent to the type of sample, which comprises non-invasive collection, easy handling and storage. In this work, we propose an in house urine-based indirect ELISA using recombinant proteins from Nucleocapsid (N) and Spike (S) of the SARSCoV-2 virus. SARS-CoV-2 recombinant N and S protein subunits (Gly-S, NonGly-S1 and NonGly-RBD) were evaluated in an ELISA platform with a panel composed about 200 urine and serum samples. The presence of anti-SARS-CoV-2 antibodies in urine was detected with similar or superior sensitivity and specificity to serum, in which sensitivity values of 94.0%, 75.0%, 81.38% and 89.66% were obtained, while specificity values were of 100.0%, 96.0%, 96.77% and 96.77%, respectively, against rSARS-CoV-2 N, S-Glic, S1-NGlic and RBD-NGlic proteins. In conclusion, the data presented suggest that urine could be considered as a potential biological sample for application in immunodiagnostic platforms for SARS-CoV-2 infection, with benefits to the individual and population context.

The impact of COVID-19 in medical practice. A review focused on Urology

ABSTRACT COVID-19 pandemic is a rapidly spreading virus that is changing the World and the way doctors are practicing medicine. The huge number of patients searching for medical care and needing intensive care beds led the health care system to a burnout status especially in places where the care system was already overloaded. In this setting, and also due to the absence of a specific treatment for the disease, health authorities had to opt for recommending or imposing social distancing to relieve the health system and reduce deaths. All other medical specialties nondirectly related to the treatment of COVID-19 had to interrupt or strongly reduce their activities in order to give room to seriously ill patients, since no one knows so far the real extent of the virus damage on human body and the consequences of doing non deferrable procedures in this pandemic era. Despite not been a urological disease, the urologist needs to be updated on how to deal with these patients and how to take care of himself and of the medical team he works with. The aim of this article is to review briefly some practical aspects of COVID-19 and its implications in the urological practice in our country.

Recommendations for providing nutritional care to COVID-19 inpatients. A literature review / Recomendaciones para la atención nutricional hospitalaria en pacientes con COVID-19. Revisión de la literatura

Resumen Introducción. La enfermedad por coronavirus 2019 (COVID-19) puede llevar a la hospitalización de los pacientes en los casos más graves; por tanto, es necesario establecer recomendaciones teórico-prácticas de intervención nutricional hospitalaria. Objetivo. Identificar las recomendaciones dirigidas a los equipos de salud hospitalarios para brindar atención nutricional a pacientes hospitalizados mayores de 18 años e infectados con COVID-19. Materiales y métodos. Se realizó una revisión de la literatura en PubMed mediante la siguiente estrategia de búsqueda: periodo de publicación: diciembre 1 de 2019 a marzo 21 de 2020; idiomas de publicación: inglés y chino; términos de búsqueda: "Coronavirus Infection", "Severe Acute Respiratory Syndrome" y "Nutrition for Vulnerable Groups". Resultados. Se identificaron 283 artículos en la búsqueda inicial, de los cuales se seleccionaron 6 para analizar las prácticas de atención nutricional hospitalaria. Las áreas de investigación de estas publicaciones fueron soporte nutricional; evaluación de intervenciones para abordar y disminuir la severidad de las complicaciones pulmonares y hepáticas, y efectos de antioxidantes y ácidos grasos omega-3 en adultos con síndrome de dificultad respiratoria aguda. Conclusión. Las recomendaciones para la atención nutricional hospitalaria en pacientes con COVID-19 se basan en procesos de cuidado nutricional y en la gestión de los servicios de alimentación; según estas recomendaciones, se deben establecer estrategias de evaluación de riesgo nutricional, realizar intervenciones de soporte nutricional que reduzcan el riesgo de desnutrición y vigilar los riesgos asociados a la administración del soporte nutricional y las alteraciones metabólicas asociadas a esta enfermedad.

Abstract Introduction: Coronavirus disease (COVID-19) may require the hospitalization of patients in the most severe cases. Therefore, it is necessary to establish theoretical and practical recommendations for nutritional interventions at hospitals. Objective: To identify recommendations given to hospital health care teams for providing nutritional care to inpatients over 18 years of age infected with COVID-19. Materials and methods: A literature review was conducted in PubMed using the following search strategy: publication period: December 2019 to March 2020; publication language: English and Chinese; search terms: "Coronavirus Infection", "Severe Acute Respiratory Syndrome" and "Nutrition for Vulnerable Groups". Results: A total of 283 articles were retrieved after completing the initial search, of which 6 were selected to analyze nutrition care practices in the hospital setting. The areas of research of these publications were nutritional support, the evaluation of interventions aimed to manage and reduce the severity of pulmonary and hepatic complications, and the effects of antioxidants and omega-3 fatty acids in adults with acute respiratory distress syndrome. Conclusion: Recommendations for providing nutritional care to COVID-19 inpatients are based on nutritional care processes and food service management. According to these recommendations, nutritional risk assessment strategies should be established, nutritional support interventions aimed at reducing the risk of malnutrition should be carried out, and the risks associated with the administration of nutritional support, as well as COVID-19-re-lated metabolic disorders, should be monitored.

Proteína de la espícula del virus SARS-CoV-2 y su relación con la enzima convertidora de angiotensina-2 / Proteína de pico do vírus SARS-CoV-2 e sua relação com a enzima conversora de angiotensina-2 / Spike protein of the SARS-CoV-2 virus and its relationship with the angiotensin-converting enzyme-2

Introducción: La COVID-19 causada por el virus del SARS-CoV-2 es una pandemia que ha cobrado la vida de millones de personas y sobrecargado los servicios sanitarios de todo el mundo. Objetivo: Describir la relación entre la proteína de la espícula (proteína S, proteína espicular o spike) del SARS-CoV-2 y enzima convertidora de angiotensina 2 como desencadenante primario de la infección por la COVID-19. Método: Se realizó una búsqueda bibliográfica en Google Académico, SciELO y PubMed, con los descriptores iniciales COVID-19 y SARS-CoV-2. El periodo de publicación seleccionado fue entre los años 2019-2021, sin restricciones en cuanto al tipo de artículo. Los trabajos debieron estar disponibles en español e inglés a texto completo. Resultados: La proteína de la espícula del SARS-CoV-2, que desempeña un papel clave en el reconocimiento del receptor y en el proceso de fusión de la membrana celular, está compuesta por dos subunidades, S1 y S2. La subunidad S1 contiene un dominio de unión al receptor RBD (por sus siglas en inglés, receptor-binding domain) que se une al receptor del huésped, la enzima convertidora de angiotensina 2, mientras que la subunidad S2 interviene en la fusión de la membrana viral y celular. La ubicuidad tisular de la enzima convertidora de angiotensina 2 explica las múltiples manifestaciones clínicas de la enfermedad. Conclusiones: El conocimiento de la relación entre el SARS-CoV-2 y su receptor enzima convertidora de angiotensina 2 permite no solo conocer la fisiopatología de la COVID-19, sino el diseño de fármacos antivirales y vacunas que contribuyen a la prevención y tratamiento de esta enfermedad viral.

Introduction: COVID-19 caused by the SARS-CoV-2 virus is a pandemic that has claimed the lives of millions of people and overloaded health services around the world. Objective: To describe the relationship between the spike protein (S) of SARS-CoV-2 and the angiotensin-converting enzyme 2 as the primary trigger of COVID-19 infection. Method: A bibliographic search was carried out in Google Scholar, SciELO and PubMed, with the initial descriptors COVID-19 and SARS-CoV-2. The publication period selected was between the years 2019 to 2021, without restrictions regarding the type of article. The papers had to be available in full text in Spanish and English. Results: The spike protein of SARS-CoV-2, which plays a key role in receptor recognition and in the cell membrane fusion process, is composed of two subunits, S1 and S2. The S1 subunit contains a receptor-binding domain (RBD) that binds to the host's receptor, angiotensin-converting enzyme 2, while the S2 subunit is involved in the viral and cellular membrane fusion. The tissue ubiquity of angiotensin converting enzyme 2 explains the multiple clinical manifestations of the disease. Conclusions: The knowledge of the relationship between SARS-CoV-2 and its receptor the angiotensin-converting enzyme 2, allows not only to know the pathophysiology of COVID-19, but also the design of antiviral drugs and vaccines that contribute to the prevention and treatment of this viral disease.

Introdução: COVID-19 causada pelo vírus SARS-CoV-2 é uma pandemia que ceifou a vida de milhões de pessoas e sobrecarregou os serviços de saúde em todo o mundo. Objetivo: Descrever a relação entre a proteína spike (S) do SARS-CoV-2 e a enzima conversora de angiotensina 2 como o principal fator desencadeante da infecção por COVID-19. Método: Foi realizada uma busca bibliográfica no Google Scholar, SciELO e PubMed, com os descritores iniciais COVID-19 e SARS-CoV-2. O período de publicação selecionado foi entre os anos de 2019 a 2021, sem restrições quanto ao tipo de artigo. Os artigos deveriam estar disponíveis na íntegra em espanhol e inglês. Resultados: A proteína spike do SARS-CoV-2, que desempenha um papel fundamental no reconhecimento do receptor e no processo de fusão da membrana celular, é composta por duas subunidades, S1 e S2. A subunidade S1 contém um domínio de ligação ao receptor (RBD) que se liga ao receptor do hospedeiro, a enzima conversora de angiotensina 2, enquanto a subunidade S2 está envolvida na fusão da membrana viral e celular. A onipresença tecidual da enzima conversora da angiotensina 2 explica as múltiplas manifestações clínicas da doença. Conclusões: O conhecimento da relação entre o SARS-CoV-2 e seu receptor, a enzima conversora de angiotensina 2, permite não só conhecer a fisiopatologia da COVID-19, mas também o desenho de antivirais e vacinas que contribuam para a prevenção e tratamento desta doença viral.

The impact of spike mutated variants of SARS-CoV2 [Alpha, Beta, Gamma, Delta, and Lambda] on the efficacy of subunit recombinant vaccines

ABSTRACT Since the first described human infection with SARS-CoV-2 in December of 2019 many subunit protein vaccines have been proposed for use in humans. Subunit vaccines use one or more antigens suitable for eliciting a robust immune response. However, the major concern is the efficacy of subunit vaccines and elicited antibodies to neutralize the variants of SARS-CoV-2 like B.1.1.7 (Alpha), B.1.351 (Beta) and P1 (Gamma), B.1.617 (Delta) and C.37 (Lambda). The Spike protein (S) is a potential fragment for use as an antigen in vaccine development. This protein plays a crucial role in the first step of the infection process, as it binds to Angiotensin-Converting Enzyme 2 (ACE2) receptor and enters the host cell after binding. Immunization-induced specific antibodies against the receptor binding domain (RBD) may block and effectively prevent virus invasion. The focus of this review is the impact of spike mutated variants of SARS-CoV2 (Alpha, Beta, Gamma, Delta, and Lambda) on the efficacy of subunit recombinant vaccines. To date, a low or no significant impact on vaccine efficacy against Alpha and Delta variants has been reported. Such an impact on vaccine efficacy for Beta, Delta, Gamma, and Lambda variants may be even greater compared to the Alpha variant. Nonetheless, more comprehensive analyses are needed to assess the real impact on vaccine efficacy brought about by SARS-CoV-2 variants.

SARS-CoV-2 cell entry and targeted antiviral development

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the pandemic coronavirus disease 2019 (COVID-19), which threatens human health and public safety. In the urgent campaign to develop anti-SARS-CoV-2 therapies, the initial entry step is one of the most appealing targets. In this review, we summarize the current understanding of SARS-CoV-2 cell entry, and the development of targeted antiviral strategies. Moreover, we speculate upon future directions toward next-generation of SARS-CoV-2 entry inhibitors during the upcoming post-pandemic era.