Structural basis for the participation of the SARS-CoV-2 nucleocapsid protein in the template switch mechanism and genomic RNA reorganization.

The COVID-19 pandemic has resulted in a significant toll of deaths worldwide, exceeding seven million individuals, prompting intensive research efforts aimed at elucidating the molecular mechanisms underlying the pathogenesis of SARS-CoV-2 infection. Despite the rapid development of effective vaccines and therapeutic interventions, COVID-19 remains a threat to humans due to the emergence of novel variants and largely unknown long-term consequences. Among the viral proteins, the nucleocapsid protein (N) stands out as the most conserved and abundant, playing the primary role in nucleocapsid assembly and genome packaging. The N protein is promiscuous for the recognition of RNA, yet it can perform specific functions. Here, we discuss the structural basis of specificity, which is directly linked to its regulatory role. Notably, the RNA chaperone activity of N is central to its multiple roles throughout the viral life cycle. This activity encompasses double-stranded RNA (dsRNA) annealing and melting and facilitates template switching, enabling discontinuous transcription. N also promotes the formation of membrane-less compartments through liquid-liquid phase separation, thereby facilitating the congregation of the replication and transcription complex. Considering the information available regarding the catalytic activities and binding signatures of the N protein-RNA interaction, this review focuses on the regulatory role of the SARS-CoV-2 N protein. We emphasize the participation of the N protein in discontinuous transcription, template switching, and RNA chaperone activity, including double-stranded RNA melting and annealing activities.

The assembly-defective phenotype of an FIV Gag polyprotein containing the SIV nucleocapsid zinc finger motifs is reversed by including the SIV SP2 domain in the chimeric protein.

To gain insight into the functional relationship between the nucleocapsid (NC) domains of the Gag polyproteins of feline and simian immunodeficiency viruses, FIV and SIV, respectively, we generated two FIV Gag chimeric proteins containing different SIV NC and gag sequences. A chimeric FIV Gag protein (NC1) containing the SIV two zinc fingers motifs was incapable of assembling into virus-like particles. By contrast, another Gag chimera (NC2) differing from NC1 by the replacement of the C-terminal region of the FIV NC with SIV SP2 produced particles as efficiently as wild-type FIV Gag. Of note, when the chimeric NC2 Gag polyprotein was expressed in the context of the proviral DNA in feline CrFK cells, wild-type levels of virions were produced which encapsidated 50% of genomic RNA when compared to the wild-type virus.

Utilizing COVID-19 as a Model for Diagnostics Using an Electrochemical Sensor.

This paper reports a rapid and sensitive sensor for the detection and quantification of the COVID-19 N-protein (N-PROT) via an electrochemical mechanism. Single-frequency electrochemical impedance spectroscopy was used as a transduction method for real-time measurement of the N-PROT in an immunosensor system based on gold-conjugate-modified carbon screen-printed electrodes (Cov-Ag-SPE). The system presents high selectivity attained through an optimal stimulation signal composed of a 0.0 V DC potential and 10 mV RMS-1 AC signal at 100 Hz over 300 s. The Cov-Ag-SPE showed a log response toward N-PROT detection at concentrations from 1.0 ng mL-1 to 10.0 µg mL-1, with a 0.977 correlation coefficient for the phase (θ) variation. An ML-based approach could be created using some aspects observed from the positive and negative samples; hence, it was possible to classify 252 samples, reaching 83.0, 96.2 and 91.3% sensitivity, specificity, and accuracy, respectively, with confidence intervals (CI) ranging from 73.0 to 100.0%. Because impedance spectroscopy measurements can be performed with low-cost portable instruments, the immunosensor proposed here can be applied in point-of-care diagnostics for mass testing, even in places with limited resources, as an alternative to the common diagnostics methods.

Evidence of SARS-CoV-2 infection in companion animals from owners who tested positive for COVID-19 in the Valley of Mexico.

BACKGROUND: Little is known about the companion animals which tested positive in Mexico for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection. Due to this, it is that we have documented the infection of companion animals, via an exploratory approach in two localities of the Valley of Mexico, in which the companion animal owners tested positive for COVID-19. METHODS: Oropharyngeal and nasopharyngeal swabs were collected from 21 companion animals. Also, a Reverse-Transcription Quantitative Polymerase Chain Reaction was used to test five probes in three SARS-CoV-2 genes. More than one-third (5/14) of these samples were positive for SARS CoV-2 corresponding to dogs. RESULTS: This research translates into the first available report on companion animals with SARS-CoV-2 infection in the most populated area of Mexico. Samples were added chronologically to previous reports prepared in other areas of the country, from February through November 2022. CONCLUSION: Although SARS-CoV-2 infection in dogs is not as common as in other animals, our results suggest that it can be transmitted to dogs by their owners to a greater extent than previously reported.

Case Report of a Young Adult with Fatal COVID-19 and Abundant SARS-CoV-2 Nucleocapsid Protein and Lipofuscin Accumulation in Tissues.

This is a case report of a young adult who died of COVID-19 twelve days after admission, with coronavirus nucleocapsid protein and lipofuscin found in the heart and kidney tissues, providing further evidence of the role of SARS-CoV-2 in cellular senescence.

Distinct anti-NP, anti-RBD and anti-Spike antibody profiles discriminate death from survival in COVID-19.

Introduction: Infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces rapid production of IgM, IgA, and IgG antibodies directed to multiple viral antigens that may have impact diverse clinical outcomes. Methods: We evaluated IgM, IgA, and IgG antibodies directed to the nucleocapsid (NP), IgA and IgG to the Spike protein and to the receptor-binding domain (RBD), and the presence of neutralizing antibodies (nAb), in a cohort of unvaccinated SARS-CoV-2 infected individuals, in the first 30 days of post-symptom onset (PSO) (T1). Results: This study included 193 coronavirus disease 2019 (COVID-19) participants classified as mild, moderate, severe, critical, and fatal and 27 uninfected controls. In T1, we identified differential antibody profiles associated with distinct clinical presentation. The mild group presented lower levels of anti-NP IgG, and IgA (vs moderate and severe), anti-NP IgM (vs severe, critical and fatal), anti-Spike IgA (vs severe and fatal), and anti-RBD IgG (vs severe). The moderate group presented higher levels of anti-RBD IgA, comparing with severe group. The severe group presented higher levels of anti-NP IgA (vs mild and fatal) and anti-RBD IgG (vs mild and moderate). The fatal group presented higher levels of anti-NP IgM and anti-Spike IgA (vs mild), but lower levels of anti-NP IgA (vs severe). The levels of nAb was lower just in mild group compared to severe, critical, and fatal groups, moreover, no difference was observed among the more severe groups. In addition, we studied 82 convalescent individuals, between 31 days to 6 months (T2) or more than 6 months (T3), PSO, those: 12 mild, 26 moderate, and 46 severe plus critical. The longitudinal analyzes, for the severe plus critical group showed lower levels of anti-NP IgG, IgA and IgM, anti-Spike IgA in relation T3. The follow-up in the fatal group, reveals that the levels of anti-spike IgG increased, while anti-NP IgM levels was decreased along the time in severe/critical and fatal as well as anti-NP IgG and IgA in several/critical groups. Discussion: In summary, the anti-NP IgA and IgG lower levels and the higher levels of anti-RBD and anti-Spike IgA in fatal compared to survival group of individuals admitted to the intensive care unit (ICU). Collectively, our data discriminate death from survival, suggesting that anti-RBD IgA and anti-Spike IgA may play some deleterious effect, in contrast with the potentially protective effect of anti-NP IgA and IgG in the survival group.

SARS-CoV-2 Rapid Antigen Test Based on a New Anti-Nucleocapsid Protein Monoclonal Antibody: Development and Real-Time Validation.

SARS-CoV-2 diagnostic tests have become an important tool for pandemic control. Among the alternatives for COVID-19 diagnosis, antigen rapid diagnostic tests (Ag-RDT) are very convenient and widely used. However, as SARS-CoV-2 variants may continuously emerge, the replacement of tests and reagents may be required to maintain the sensitivity of Ag-RDTs. Here, we describe the development and validation of an Ag-RDT during an outbreak of the Omicron variant, including the characterization of a new monoclonal antibody (anti-DTC-N 1B3 mAb) that recognizes the Nucleocapsid protein (N). The anti-DTC-N 1B3 mAb recognized the sequence TFPPTEPKKDKKK located at the C-terminus of the N protein of main SARS-CoV-2 variants of concern. Accordingly, the Ag-RDT prototypes using the anti-DTC-N 1B3 mAB detected all the SARS-CoV-2 variants-Wuhan, Alpha, Gamma, Delta, P2 and Omicron. The performance of the best prototype (sensitivity of 95.2% for samples with Ct ≤ 25; specificity of 98.3% and overall accuracy of 85.0%) met the WHO recommendations. Moreover, results from a patients' follow-up study indicated that, if performed within the first three days after onset of symptoms, the Ag-RDT displayed 100% sensitivity. Thus, the new mAb and the Ag-RDT developed herein may constitute alternative tools for COVID-19 point-of-care diagnosis and epidemiological surveillance.

Obtaining a high titer of polyclonal antibodies from rats to the SARS-CoV-2 nucleocapsid protein and its N- and C-terminal domains for diagnostic test development.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is an enveloped, plus-stranded RNA virus responsible for the Coronavirus Disease 2019 (COVID-19). Patients infected with COVID-19 may be asymptomatic or have symptoms ranging from mild manifestations to severe cases of the disease that could lead to death. The SARS-CoV-2 genome encodes 4 structural proteins, including the Spike protein (S), the Nucleocapsid protein (N), Membrane protein (M) and, the Envelope protein (E). The N protein forms a major component of the ribonucleoprotein complex within the virus particle and play a vital role in its transcription and replication. Nevertheless, the S protein was the most important protein in the development of vaccines against COVID-19. However, the decrease in number of registered immunizations against the disease and the rapid drop in neutralizing antibody titers together with looser preventive measures for virus transmission, favored the rapid appearance of new variants of concerns (VOCs) that primarily show mutations in the S protein. This fact makes the N protein a good candidate for the development of diagnostic tests, due to its stability, amino acid conservation, high immunogenicity, and the smaller likelihood of mutation. With the aim of developing a new diagnostic kit based on the N protein, we evaluated the humoral response in female Wistar rats against this target. Three constructions of the N protein were used to inoculate the animals: the full-length protein (Cfull), the N- (NTD), and the C-terminal (CTD) portion of the protein. The immunizations induced the animal's immune response, with specific polyclonal IgG antibodies against the Cfull protein and its fragments. There were not non-specific bind to the protein used as negative control. Anti-Cfull antibodies demonstrated high efficiency in binding to the NTD protein and the antibodies present in the anti-CTD and anti-NTD sera have recognized the Cfull protein, but they were not able to recognize the NTD and CTD proteins, respectively. Our results indicate an efficient protocol for obtaining high antibody titers against the N recombinant protein of SARS-CoV-2 and its fragments highlighting the Cfull protein, which can be used in the development of new diagnostic kits.

Antibody response following the fourth SARS-CoV-2 vaccine dose during the Omicron wave in Brazil.

We investigated the impact of the fourth dose with ChAdOx1 nCoV-19 (AstraZeneca) in the humoral immune response to SARS-CoV-2 during a 9-month follow-up period in which Omicron was the predominant variant in Brazil. IgG for the SARS-CoV-2 spike protein (S) and nucleocapsid (N) proteins were analyzed in samples collected before and after the fourth dose. All participants were tested monthly for SARS-CoV-2 infection by RT-qPCR. The antibody response induced by the fourth dose of the coronavirus disease 2019 vaccine was evaluated and compared with the response induced by the second and third doses. The additional antibody response to the viral S protein after the fourth dose was smaller than those after the third vaccine dose. In contrast, an increase in the N IgG levels could be observed after the fourth dose compared to other vaccine doses. In the comparison of the antibody response before and after the fourth dose, an increase in both S-and-N IgG was noted, mainly in the positive qPCR group. We did not observe a significant decline in IgG levels after the fourth dose, as observed after the second and third doses, therefore, a sustained humoral response to both S and N proteins seems to be achieved.

A highly sensitive nanobody-based immunoassay detecting SARS-CoV-2 nucleocapsid protein using all-recombinant reagents.

Antigen tests have been crucial for managing the COVID-19 pandemic by identifying individuals infected with SARS-CoV-2. This remains true even after immunity has been widely attained through natural infection and vaccination, since it only provides moderate protection against transmission and is highly permeable to the emergence of new virus variants. For this reason, the widespread availability of diagnostic methods is essential for health systems to manage outbreaks effectively. In this work, we generated nanobodies to the virus nucleocapsid protein (NP) and after an affinity-guided selection identified a nanobody pair that allowed the detection of NP at sub-ng/mL levels in a colorimetric two-site ELISA, demonstrating high diagnostic value with clinical samples. We further modified the assay by using a nanobody-NanoLuc luciferase chimeric tracer, resulting in increased sensitivity (detection limit = 61 pg/mL) and remarkable improvement in diagnostic performance. The luminescent assay was finally evaluated using 115 nasopharyngeal swab samples. Receiver Operating Characteristic (ROC) curve analysis revealed a sensitivity of 78.7% (95% confidence interval: 64.3%-89.3%) and specificity of 100.0% (95% confidence interval: 94.7%-100.0%). The test allows the parallel analysis of a large number of untreated samples, and fulfills our goal of producing a recombinant reagent-based test that can be reproduced at low cost by other laboratories with recombinant expression capabilities, aiding to build diagnostic capacity.

Assembly of the Tripartite and RNA Condensates of the Respiratory Syncytial Virus Factory Proteins In Vitro: Role of the Transcription Antiterminator M2-1.

A wide variety of viruses replicate in liquid-like viral factories. Non-segmented negative stranded RNA viruses share a nucleoprotein (N) and a phosphoprotein (P) that together emerge as the main drivers of liquid-liquid phase separation. The respiratory syncytial virus includes the transcription antiterminator M2-1, which binds RNA and maximizes RNA transcriptase processivity. We recapitulate the assembly mechanism of condensates of the three proteins and the role played by RNA. M2-1 displays a strong propensity for condensation by itself and with RNA through the formation of electrostatically driven protein-RNA coacervates based on the amphiphilic behavior of M2-1 and finely tuned by stoichiometry. M2-1 incorporates into tripartite condensates with N and P, modulating their size through an interplay with P, where M2-1 is both client and modulator. RNA is incorporated into the tripartite condensates adopting a heterogeneous distribution, reminiscent of the M2-1-RNA IBAG granules within the viral factories. Ionic strength dependence indicates that M2-1 behaves differently in the protein phase as opposed to the protein-RNA phase, in line with the subcompartmentalization observed in viral factories. This work dissects the biochemical grounds for the formation and fate of the RSV condensates in vitro and provides clues to interrogate the mechanism under the highly complex infection context.

Combination of Recombinant Proteins S1/N and RBD/N as Potential Vaccine Candidates.

Despite all successful efforts to develop a COVID-19 vaccine, the need to evaluate alternative antigens to produce next-generation vaccines is imperative to target emerging variants. Thus, the second generation of COVID-19 vaccines employ more than one antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to induce an effective and lasting immune response. Here, we analyzed the combination of two SARS-CoV-2 viral antigens that could elicit a more durable immune response in both T- and B-cells. The nucleocapsid (N) protein, Spike protein S1 domain, and receptor binding domain (RBD) of the SARS-CoV-2 spike surface glycoproteins were expressed and purified in a mammalian expression system, taking into consideration the posttranscriptional modifications and structural characteristics. The immunogenicity of these combined proteins was evaluated in a murine model. Immunization combining S1 or RBD with the N protein induced higher levels of IgG antibodies, increased the percentage of neutralization, and elevated the production of cytokines TNF-α, IFN-γ, and IL-2 compared to the administration of a single antigen. Furthermore, sera from immunized mice recognized alpha and beta variants of SARS-CoV-2, which supports ongoing clinical results on partial protection in vaccinated populations, despite mutations. This study identifies potential antigens for second-generation COVID-19 vaccines.

Antigen-Specific Antibody Signature Is Associated with COVID-19 Outcome.

Numerous studies have focused on inflammation-related markers to understand COVID-19. In this study, we performed a comparative analysis of spike (S) and nucleocapsid (N) protein-specific IgA, total IgG and IgG subclass response in COVID-19 patients and compared this to their disease outcome. We observed that the SARS-CoV-2 infection elicits a robust IgA and IgG response against the N-terminal (N1) and C-terminal (N3) region of the N protein, whereas we failed to detect IgA antibodies and observed a weak IgG response against the disordered linker region (N2) in COVID-19 patients. N and S protein-specific IgG1, IgG2 and IgG3 response was significantly elevated in hospitalized patients with severe disease compared to outpatients with non-severe disease. IgA and total IgG antibody reactivity gradually increased after the first week of symptoms. Magnitude of RBD-ACE2 blocking antibodies identified in a competitive assay and neutralizing antibodies detected by PRNT assay correlated with disease severity. Generally, the IgA and total IgG response between the discharged and deceased COVID-19 patients was similar. However, significant differences in the ratio of IgG subclass antibodies were observed between discharged and deceased patients, especially towards the disordered linker region of the N protein. Overall, SARS-CoV-2 infection is linked to an elevated blood antibody response in severe patients compared to non-severe patients. Monitoring of antigen-specific serological response could be an important tool to accompany disease progression and improve outcomes.

Longitudinal study comparing IgG antibodies induced by heterologous prime-boost COVID-19 vaccine.

Vaccines are critical cost-effective tools to control the COVID-19 pandemic. The heterologous prime-boost vaccination has been used by many countries to overcome supply issues, so the effectiveness and safety of this strategy need to be better clarified. This study aims to verify the effect of heterologous prime-boost COVID-19 vaccination on healthcare professionals from Dante Pazzanese Hospital in Brazil. It was performed serological assays of vaccinated individuals after 2-dose of CoronaVac (Sinovac; n = 89) or ChAdOx1 nCoV-19 (Oxford-AstraZeneca; n = 166) followed by a BNT162b2 booster (Pfizer-BioNTech; n = 255). The serum antibodies anti-S (spike), anti-N (nucleocapsid), and anti-RBD (receptor binding domain) were assessed by enzyme-linked immunosorbent assay. The heterologous booster dose induced a 10-fold higher anti-Spike antibody regardless of the 2-dose of a prime vaccine. It was strikingly observed that BNT162b2 enhanced levels of anti-spike antibodies, even in those individuals who did not previously respond to the 2-dose of CoronaVac. In conclusion, the heterologous scheme of vaccination using mRNA as a booster vaccine efficiently enhanced the antibody response against SARS-CoV-2, especially benefiting those elderly who were seronegative with a virus-inactivated vaccine.

SARS-CoV-2 rapid antigen test based on a new anti-nucleocapsid protein monoclonal antibody: development and real-time validation

SARS-CoV-2 diagnostic tests have become an important tool for pandemic control. Among the alternatives for COVID-19 diagnosis, antigen rapid diagnostic tests (Ag-RDT) are very convenient and widely used. However, as SARS-CoV-2 variants may continuously emerge, the replacement of tests and reagents may be required to maintain the sensitivity of Ag-RDTs. Here, we describe the development and validation of an Ag-RDT during an outbreak of the Omicron variant, including the characterization of a new monoclonal antibody (anti-DTC-N 1B3 mAb) that recognizes the Nucleocapsid protein (N). The anti-DTC-N 1B3 mAb recognized the sequence TFPPTEPKKDKKK located at the C-terminus of the N protein of main SARS-CoV-2 variants of concern. Accordingly, the Ag-RDT prototypes using the anti-DTC-N 1B3 mAB detected all the SARS-CoV-2 variants—Wuhan, Alpha, Gamma, Delta, P2 and Omicron. The performance of the best prototype (sensitivity of 95.2% for samples with Ct ≤ 25; specificity of 98.3% and overall accuracy of 85.0%) met the WHO recommendations. Moreover, results from a patients’ follow-up study indicated that, if performed within the first three days after onset of symptoms, the Ag-RDT displayed 100% sensitivity. Thus, the new mAb and the Ag-RDT developed herein may constitute alternative tools for COVID-19 point-of-care diagnosis and epidemiological surveillance.

Distinct anti-NP, anti-RBD and anti-spike antibody profiles discriminate death from survival in COVID-19

Introduction: Infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces rapid production of IgM, IgA, and IgG antibodies directed to multiple viral antigens that may have impact diverse clinical outcomes. Methods: We evaluated IgM, IgA, and IgG antibodies directed to the nucleocapsid (NP), IgA and IgG to the Spike protein and to the receptor-binding domain (RBD), and the presence of neutralizing antibodies (nAb), in a cohort of unvaccinated SARS-CoV-2 infected individuals, in the first 30 days of post-symptom onset (PSO) (T1). Results: This study included 193 coronavirus disease 2019 (COVID-19) participants classified as mild, moderate, severe, critical, and fatal and 27 uninfected controls. In T1, we identified differential antibody profiles associated with distinct clinical presentation. The mild group presented lower levels of anti-NP IgG, and IgA (vs moderate and severe), anti-NP IgM (vs severe, critical and fatal), anti-Spike IgA (vs severe and fatal), and anti-RBD IgG (vs severe). The moderate group presented higher levels of anti-RBD IgA, comparing with severe group. The severe group presented higher levels of anti-NP IgA (vs mild and fatal) and anti-RBD IgG (vs mild and moderate). The fatal group presented higher levels of anti-NP IgM and anti-Spike IgA (vs mild), but lower levels of anti-NP IgA (vs severe). The levels of nAb was lower just in mild group compared to severe, critical, and fatal groups, moreover, no difference was observed among the more severe groups. In addition, we studied 82 convalescent individuals, between 31 days to 6 months (T2) or more than 6 months (T3), PSO, those: 12 mild, 26 moderate, and 46 severe plus critical. The longitudinal analyzes, for the severe plus critical group showed lower levels of anti-NP IgG, IgA and IgM, anti-Spike IgA in relation T3. The follow-up in the fatal group, reveals that the levels of anti-spike IgG increased, while anti-NP IgM levels was decreased along the time in severe/critical and fatal as well as anti-NP IgG and IgA in several/critical groups. Discussion: In summary, the anti-NP IgA and IgG lower levels and the higher levels of anti-RBD and anti-Spike IgA in fatal compared to survival group of individuals admitted to the intensive care unit (ICU). Collectively, our data discriminate death from survival, suggesting that anti-RBD IgA and anti-Spike IgA may play some deleterious effect, in contrast with the potentially protective effect of anti-NP IgA and IgG in the survival group.

Expressão e purificação da proteína nucleocapsídica recombinante de SARS-CoV-2

Known as the Covid-19 Pandemic, caused by the SARS-Cov 2 virus, it became known as one of the great challenges of the 21st century and brought to industry and research the need to create defensive parameters and of rapid effectiveness for the biological study of possible threats to the health of the general population. The Sars-COV-2 Nucleocapsid protein, responsible for stabilizing the nucleocapsid, in addition to being involved in RNA synthesis and translation, and acting as an interferon antagonist and RNA interference suppressor, favoring viral replication, became the target of this study with the objective of creating parameters for its production in the laboratory in the form of recombinant protein. Aiming at high yield and good quality, through its expression in competent bacteria Escherichia coli BL21(DE3) and the improvement in the steps of its creation, counting on sonication of the expressed bacteria, its purification and protein concentration. The present study reached the objectives for the production of this protein for the Laboratório de Imunoquímica of the Instituto Butantan, also being able to guide the production of this and other recombinant proteins that end up becoming of interest for study and research in search of the health of the population.

Denominada como Pandemia do Covid-19, causada pelo vírus SARS-Cov 2, ficou conhecida como um dos grandes desafios do século XXI e trouxe à indústria e à pesquisa a necessidade de criar parâmetros defensivos e de rápida efetividade para o estudo biológico de possíveis ameaças à saúde da população em geral. A proteína Nucleocapsídica do Sars-COV-2, responsável por estabilizar o nucleocapsídeo, além de estar envolvida na síntese e tradução do RNA, e atuar como antagonista do interferon e supressor de interferência do RNA, favorecendo a replicação viral, se tornou alvo deste estudo com o objetivo de criar parâmetros para a sua produção em laboratório em forma de proteína recombinante. Visando o alto rendimento e boa qualidade, através de sua expressão em bactéria competente Escherichia coli BL21(DE3) e a melhora nos passos de sua criação, contando com sonicação das bactérias expressas, sua purificação e concentração proteica. O presente estudo alcançou os objetivos para produção dessa proteína para o Laboratório de Imunoquímica do Instituto Butantan, também sendo capaz de guiar a produção dessa e de outras proteínas recombinantes que acabam se tornando de interesse para estudo e pesquisa em busca da saúde da população.

Equine Anti-SARS-CoV-2 Serum (ECIG) Binds to Mutated RBDs and N Proteins of Variants of Concern and Inhibits the Binding of RBDs to ACE-2 Receptor.

The COVID-19 pandemic caused by the severe acute syndrome virus 2 (SARS-CoV-2) has been around since November 2019. As of early June 2022, more than 527 million cases were diagnosed, with more than 6.0 million deaths due to this disease. Coronaviruses accumulate mutations and generate greater diversity through recombination when variants with different mutations infect the same host. Consequently, this virus is predisposed to constant and diverse mutations. The SARS-CoV-2 variants of concern/interest (VOCs/VOIs) such as Alpha (B.1.1.7), Beta (B.1.351), Gamma (B.1.1.28/P.1), Delta (B.1.617.2), and Omicron (B.1.1.529) have quickly spread across the world. These VOCs and VOIs have accumulated mutations within the spike protein receptor-binding domain (RBD) which interacts with the angiotensin-2 converting enzyme (ACE-2) receptor, increasing cell entry and infection. The RBD region is the main target for neutralizing antibodies; however, other notable mutations have been reported to enhance COVID-19 infectivity and lethality. Considering the urgent need for alternative therapies against this virus, an anti-SARS-CoV-2 equine immunoglobulin F(ab')2, called ECIG, was developed by the Butantan Institute using the whole gamma-irradiated SARS-CoV-2 virus. Surface plasmon resonance experiments revealed that ECIG binds to wild-type and mutated RBD, S1+S2 domains, and nucleocapsid proteins of known VOCs, including Alpha, Gamma, Beta, Delta, Delta Plus, and Omicron. Additionally, it was observed that ECIG attenuates the binding of RBD (wild-type, Beta, and Omicron) to human ACE-2, suggesting that it could prevent viral entry into the host cell. Furthermore, the ability to concomitantly bind to the wild-type and mutated nucleocapsid protein likely enhances its neutralizing activity of SARS-CoV-2. We postulate that ECIG benefits COVID-19 patients by reducing the infectivity of the original virus and existing variants and may be effective against future ones. Impacting the course of the disease, mainly in the more vulnerable, reduces infection time and limits the appearance of new variants by new recombination.

Development of monoclonal antibody based IgG and IgM ELISA for diagnosis of severe fever with thrombocytopenia syndrome virus infection.

INTRODUCTION: Severe fever with thrombocytopenia syndrome virus (SFTSV) is a newly emerged virus that poses a great threat to human health because of high fatality rate. METHODS: To develop sensitive and specific sero-diagnostic systems for SFTSV infections, monoclonal antibodies (MAbs) against recombinant SFTSV nucleocapsid (rSFTSV-N) protein were developed by immunizing BALB/C mice with rSFTSV-N protein and fusing the spleen cells with SP2/0 myeloma cells. Three hybridoma cell lines secreting MAbs against rSFTSV-N were obtained. MAb based IgG sandwich enzyme linked immunosorbent assay (ELISA) and IgM capture ELISA systems were established by using the newly developed MAbs. One hundred fifteen clinical suspected SFTS patients serum samples were used to evaluate the newly established systems by comparing with the total antibody detecting sandwich ELISA system and indirect ELISA systems. RESULTS: The MAbs based sandwich IgG ELISA was perfectly matched with that of the total antibody sandwich ELISA and the indirect IgG ELISA. IgM capture ELISA results perfectly matched with that of the total antibody sandwich ELISA while detecting eight additional positive samples missed by the indirect IgM ELISA. CONCLUSIONS: The MAbs against rSFTSV-N protein offer new tools for SFTSV studies and our newly developed MAb-based IgG and IgM capture ELISA systems would offer safe and useful tools for diagnosis of SFTS virus infections and epidemiological investigations.

SARS-CoV-2 Seroconversion in Response to Infection and Vaccination: a Time Series Local Study in Brazil.

The investigation of antibodies raised against different severe acute respiratory syndrome coronavirus (SARS-CoV-2) antigens can help to determine the extent of previous SARS-CoV-2 infections in the population and track the humoral response to vaccination. Therefore, serological surveys can provide key information to better manage the pandemic and/or to implement the most effective vaccination program. Here we describe a time series anti-nucleocapsid, anti-spike IgG serological survey analysis in the city of Matinhos, PR, Brazil during the year of 2021. Seroconversion rates to the nucleocapsid antigen were not influenced by gender or age. The serological data support that the coronavirus disease 2019 (COVID-19) infection rate is ~50% higher than official numbers. Furthermore, by applying serological data, the corrected infection fatality rate was estimated to be lower than 2.4% in contrast with the official estimative of 3.6%. The rates of IgG reactive to spike antigen resembled the curve of the fraction the population that had taken the second vaccine dose. Up to 82% of spike seroconversion was detected in the end of 2021, confirming the effectiveness of the COVID-19 vaccination program in the city. This SARS-CoV-2 serological study unraveled the SARS-CoV-2 infection rates and the response to vaccination in the city of Matinhos. IMPORTANCE The investigation of antibodies raised against SARS-CoV-2 can help to determine the extent of previous SARS-CoV-2 infections and track the humoral response to vaccination. Here we describe a time series anti-nucleocapsid, anti-spike IgG serological survey in the city of Matinhos, PR, Brazil during the year of 2021. The data depicted the progression of SARS-CoV-2 infections in the city allowing the correction of the number of citizens who experienced COVID-19 and the disease fatality rate. The seroconversion rates to the spike antigen resembled the curve of the fraction of the population that had taken the second vaccine dose, thereby confirming the effectiveness of the COVID-19 vaccination program in the city.