Recombinant Nucleocapsid Protein Research Articles

SARS-CoV-2 cross-sectional serosurvey across three HIV-1 therapeutic clinical trials in Africa.

Data on the impact of COVID-19 in people living with HIV (PWH) are lacking in resource-constrained settings. We utilised existingrandomised clinical trials (RCTs) on antiretroviral therapies (ART) in HIV-1 infection to conduct a SARS-CoV-2 serosurvey, between January and March 2021, while characterising participants' features. Cross-sectional serosurvey. Demographic characteristics, medical history and a serum sample were collected from consenting PWH. Samples were analysed centrally for immunoglobulin G antibodies to recombinant nucleocapsid and spike proteins derived from SARS-CoV-2 using a Luminex based assay. The 549participants recruited in 9 sites across Africa had a median age of 40 years (IQR [34-45]); 63.0% (346) were female. All were on ART; 81.8% (449) had an HIV-1 viral load <50 copies/mL, with CD4 count median at 478/mm3 (IQR [320-677]). None had received vaccination against SARS-CoV-2. Forty participants (7.3%) had a prior SARS-CoV-2 PCR testing, of whom 10 were positive (1.8%). Crude SARS-CoV-2 seroprevalence was 36.2% (; 95%CI [32.2-40.4]). In the explorative multivariable analysis, comparison of the characteristics of PWH with a positive SARS-CoV-2 serology with those with a negative or indeterminate serology: PWH with a body mass index (BMI)≥30 kg/m2 were more likely to have a positive serology than those with a BMI≤25 (aOR = 2.39 [1.48-3.86], p < 0.001); and PWH living in Cameroon were less likely to have a positive serology. This study demonstrates a substantial seroprevalence level of SARS-CoV-2 in PWH in the first quarter of 2021, with a marked disparity with the number of COVID-19 PCR tests reported positive.

Ultra-rapid detection of nuclear protein of severe fever with thrombocytopenia syndrome virus by colloidal gold immunochromatography assay.

In 2009, severe fever with thrombocytopenia syndrome virus (SFTSV), also known as the Dabie bandavirus (DBV), was first discovered in Henan, China. It is a tick-borne zoonotic virus with a fatality rate ranging from 6% to 30%. Currently, we lack safe and effective vaccines or antiviral drugs to treat SFTSV infection. Therefore, the development of a specific, sensitive, and cost-effective detection method is crucial. Using inactivated SFTSV and recombinant SFTSV nucleocapsid protein (SFTSV-NP), we repeatedly immunized mice with different adjuvants and obtained two monoclonal antibodies against SFTSV-NP, which were used to develop a colloidal gold immunochromatographic assay (ICA) rapid test kit for SFTSV. Compared with nucleic acid testing (gold standard), the ICA test strips are 97.67% accurate in testing clinical serum samples (36 cases of clinical serum samples and seven cases of whole blood samples). The test kit was 100% accurate in detecting different SFTSV strains. No false-positive results were generated when detecting other arboviruses. Therefore, our developed SFTSV test kit conveniently, rapidly, and effectively detects SFTSV.

N-protein vaccine is effective against COVID-19: Phase 3, randomized, double-blind, placebo-controlled clinical trial

Despite the success of first-generation COVID-19 vaccines targeting the spike (S) protein, emerging SARS-CoV-2 variants have led to immune escape, reducing the efficacy of these vaccines. Additionally, some individuals are unable to mount an effective immune response to S protein-based vaccines. This has created a need for alternative vaccine strategies that are less susceptible to mutations and capable of providing broad and durable protection. This study aimed to evaluate the efficacy and safety of a novel COVID-19 vaccine based on the full-length recombinant nucleocapsid (N) protein of SARS-CoV-2. We conducted a prospective, multicenter, randomized, double-blind, placebo-controlled phase 3 clinical trial (NCT05726084) in Russia. Participants (n=5229) were adults aged 18 years and older, with a BMI of 18.5-30kg/m², and without significant clinical abnormalities. They were randomized in a 2:1 ratio to receive a single intramuscular dose of either the N protein-based vaccine (50µg) or placebo. Randomization was done through block randomization, and masking was ensured by providing visually identical formulations of vaccine and placebo. The primary outcome was the incidence of symptomatic COVID-19 confirmed by PCR more than 15 days after vaccination within a 180-day observation period, analyzed on an intention-to-treat basis. Between May 18, 2023, and August 9, 2023, 5229 participants were randomized, with 3486 receiving the vaccine and 1743 receiving the placebo. Eight cases of PCR-confirmed symptomatic COVID-19 occurred in the vaccine group (0.23%) compared to 27 cases in the placebo group (1.55%), yielding a vaccine efficacy of 85.2% (95% CI: 67.4-93.3; p<0.0001). Adverse events were mostly mild and included local injection site reactions. There were no vaccine-related serious adverse events. The N protein-based COVID-19 vaccine demonstrated significant efficacy and a favorable safety profile, suggesting it could be a valuable addition to the global vaccination effort, particularly in addressing immune escape variants and offering an alternative for those unable to respond to S protein-based vaccines. These results support the continued development and potential deployment of N protein-based vaccines in the ongoing fight against COVID-19.

Monoclonal Antibodies for Rift Valley Fever Virus Nucleocapsid: Application in IgG/IgM ELISA for Sero-Diagnosis.

Rift Valley fever virus (RVFV) belonging to the Phenuiviridae family is responsible for a zoonotic disease called Rift Valley fever (RVF). Currently, RVFV has spread from Africa to Asia, and due to its ability to cause high mortality rates, it has significantly impacted human health and economic development in many societies. Highly specific and sensitive systems for sero-diagnosis of RVFV infection are needed for clinical use. BALB/c mice were immunized with recombinant RVFV nucleocapsid (rRVFV-N) protein and the spleen cells fused with SP2/0 myeloma cells to create hybridoma cell lines. The secreted monoclonal antibodies (MAbs) were purified and characterized. Enzyme-linked immunosorbent assay (ELISA) systems for the detection of IgG and IgM using the new MAbs were established and evaluated. Serum samples from 96 volunteers and 93 patients of suspected RVF from Kenya were tested compared with the ELISA systems based on inactivated viruses and the rabbit polyclonal antibody. Three monoclonal antibodies against rRVFV-N protein were established. The performance of the MAb-based sandwich IgG ELISA and the IgM capture ELISA perfectly matched the ELISA systems using the inactivated virus or the polyclonal antibody. Recombinant RVFV-N protein-specific MAbs were developed and they offer useful tools for RVFV studies. The MAb-based ELISA systems for detecting IgG and IgM offer safe and useful options for diagnosing RVFV infections in humans.

Single-Domain Antibody-Gold Nanoparticle Bioconjugates as Immunosensors for the Detection of Hantaviruses.

Hantavirus, a zoonotic pathogen, causes severe syndromes like hemorrhagic fever with renal syndrome (HFRS), sometimes fatal in humans. Considering the importance of detecting the hantavirus antigen, the construction of an immunosensor is essential. The structural and functional characteristics of camelid nanobodies (VHHs) encourage their application in the areas of nanobiotechnology, therapeutics, diagnostics, and basic research. Therefore, this study aimed to standardize stable bioconjugates using gold nanoparticles (AuNPs) and VHHs, in order to develop immunobiosensors for the diagnosis of hantavirus infection. Immobilized metal affinity chromatography (IMAC) was performed to obtain purified recombinant anti-hantavirus nucleocapsid nanobodies (anti-prNΔ85 VHH), while AuNPs were synthesized for bioconjugation. UV-visible spectrophotometry and transmission electron microscopy (TEM) analysis were employed to characterize AuNPs. The bioconjugation stability parameters (VHH-AuNPs), analyzed by spectrophotometry, showed that the ideal pH value and VHH concentration were obtained at 7.4 and 50 μg/mL, respectively, after addition of 1 M NaCl, which induces AuNP aggregation. TEM performed before and after bioconjugation showed uniform, homogeneous, well-dispersed, and spherical AuNPs with an average diameter of ~ 14 ± 0.57 nm. Furthermore, high-resolution images revealed a thin white halo on the surface of the AuNPs, indicating the coating of the AuNPs with protein. A biosensor simulation test (dot blot-like [DB-like]) was performed in stationary phase to verify the binding and detection limits of the recombinant nucleocapsid protein from the Araucária hantavirus strain (prN∆85). Using AuNPs/VHH bioconjugates, a specific interaction was detected between 5 and 10 min of reaction in a dose-dependent manner. It was observed that this test was sensitive enough to detect prNΔ85 at concentrations up to 25 ng/μL. Considering that nanostructured biological systems such as antibodies conjugated with AuNPs are useful tools for the development of chemical and biological sensors, the stability of the bioconjugate indicates proficiency in detecting antigens. The experimental results obtained will be used in a future immunospot assay or lateral flow immunochromatography analysis for hantavirus detection.

Natural Antibodies Produced in Vaccinated Patients and COVID-19 Convalescents Hydrolyze Recombinant RBD and Nucleocapsid (N) Proteins.

Antibodies are protein molecules whose primary function is to recognize antigens. However, recent studies have demonstrated their ability to hydrolyze specific substrates, such as proteins, oligopeptides, and nucleic acids. In 2023, two separate teams of researchers demonstrated the proteolytic activity of natural plasma antibodies from COVID-19 convalescents. These antibodies were found to hydrolyze the S-protein and corresponding oligopeptides. Our study shows that for antibodies with affinity to recombinant structural proteins of the SARS-CoV-2: S-protein, its fragment RBD and N-protein can only hydrolyze the corresponding protein substrates and are not cross-reactive. By using strict criteria, we have confirmed that this proteolytic activity is an intrinsic property of antibodies and is not caused by impurities co-eluting with them. This discovery suggests that natural proteolytic antibodies that hydrolyze proteins of the SARS-CoV-2 virus may have a positive impact on disease pathogenesis. It is also possible for these antibodies to work in combination with other antibodies that bind specific epitopes to enhance the process of virus neutralization.

The Nucleocapsid Protein of SARS-CoV-2, Combined with ODN-39M, Is a Potential Component for an Intranasal Bivalent Vaccine with Broader Functionality.

Despite the rapid development of vaccines against COVID-19, they have important limitations, such as safety issues, the scope of their efficacy, and the induction of mucosal immunity. The present study proposes a potential component for a new generation of vaccines. The recombinant nucleocapsid (N) protein from the SARS-CoV-2 Delta variant was combined with the ODN-39M, a synthetic 39 mer unmethylated cytosine-phosphate-guanine oligodeoxynucleotide (CpG ODN), used as an adjuvant. The evaluation of its immunogenicity in Balb/C mice revealed that only administration by intranasal route induced a systemic cross-reactive, cell-mediated immunity (CMI). In turn, this combination was able to induce anti-N IgA in the lungs, which, along with the specific IgG in sera and CMI in the spleen, was cross-reactive against the nucleocapsid protein of SARS-CoV-1. Furthermore, the nasal administration of the N + ODN-39M preparation, combined with RBD Delta protein, enhanced the local and systemic immune response against RBD, with a neutralizing capacity. Results make the N + ODN-39M preparation a suitable component for a future intranasal vaccine with broader functionality against Sarbecoviruses.

Repurposing naproxen as a potential nucleocapsid antagonist of beta-coronaviruses: targeting a conserved protein in the search for a broad-spectrum treatment option

Ongoing mutations in the coronavirus family, especially beta-coronaviruses, raise new concerns about the possibility of new unexpected outbreaks. Therefore, it is crucial to explore new alternative treatments to reduce the impact of potential future strains until new vaccines can be developed. A promising approach to combat the virus is to target its conserved parts such as the nucleocapsid, especially via repurposing of existing drugs. The possibility of this approach is explored here to find a potential anti-nucleocapsid compound to target these viruses. 3D models of the N- and C-terminal domains (CTDs) of the nucleocapsid consensus sequence were constructed. Each domain was then screened against an FDA-approved drug database, and the most promising candidate was selected for further analysis. A 100 ns molecular dynamics (MD) simulation was conducted to analyze the final candidate in more detail. Naproxen was selected and found to interact with the N-terminal domain via conserved salt bridges and hydrogen bonds which are completely conserved among all Coronaviridae members. MD analysis also revealed that all relevant coordinates of naproxen with N terminal domain were kept during 100 ns of simulation time. This study also provides insights into the specific interaction of naproxen with conserved RNA binding pocket of the nucleocapsid that could interfere with the packaging of the viral genome into capsid and virus assembly. Additionally, the in-vitro binding assay demonstrated direct interaction between naproxen and recombinant nucleocapsid protein, further supporting the computational predictions. Communicated by Ramaswamy H. Sarma

Safety and Immunogenicity of the Convacell® Recombinant N Protein COVID-19 Vaccine.

We have developed Convacell®-a COVID-19 vaccine based on the recombinant nucleocapsid (N) protein of SARS-CoV-2. This paper details Convacell's® combined phase I/II and IIb randomized, double-blind, interventional clinical trials. The primary endpoints were the frequency of adverse effects (AEs) and the titers of specific anti-N IgGs induced by the vaccination; secondary endpoints included the nature of the immune response. Convacell® demonstrated high safety in phase I with no severe AEs detected, 100% seroconversion by day 42 and high and sustained for 350 days anti-N IgG levels in phase II. Convacell® also demonstrated a fused cellular and humoral immune response. Phase IIb results showed significant post-vaccination increases in circulating anti-N IgG and N protein-specific IFNγ+-producing PBMC quantities among 438 volunteers. Convacell® showed same level of immunological efficacy for single and double dose vaccination regimens, including for elderly patients. The clinical studies indicate that Convacell® is safe and highly immunogenic.

Exploring immune response and lab markers across COVID-19 severity levels

&lt;p&gt;This retrospective cohort study investigated antigen-specific antibody responses to SARS-CoV-2 and laboratory markers in coronavirus disease 2019 (COVID-19) patients of varying severities. Serum or plasma samples collected early in the pandemic were analyzed for the presence of IgG antibodies and IgG subclasses which target the recombinant spike (S) and nucleocapsid (N) proteins of SARS-CoV-2. Correlation analyses were conducted to assess the possible relationship between the IgG subclasses to SARS-CoV-2 proteins, inflammatory markers, and the severity of the disease. It was shown that the severity of the disease positively correlated with the C-reactive protein (CRP), but most of all with the neutrophil/lymphocyte ratio (NLR) levels. IgG titers against the S- and N-proteins decreased in the most severe COVID-19 cases, which potentially attributed to a delayed IgG formation compared to milder infections. The presence of anti-spike IgG displayed a medium negative correlation trend (not statistically significant, which might be due to the small sample size in our study) with laboratory markers (such as CRP, Fibrinogen, Degree) which are suggestive of infection severity. anti-S IgG correlated positively but weakly with the serum histamine levels. The presence of anti-N IgG at the time of hospitalization correlated with the subsequent outcome. At the same time, anti-N IgG1 correlated with the detection of the viral RNA in the blood. Thus, seroconversion may occur later in patients with a more severe pneumonia. The summary data suggests correlations between the expression of inflammatory markers and the antibody responses, which can also serve as early clinical markers.&lt;/p&gt;

Development and Clinical Performance of InteliSwab® COVID-19 Rapid Test: Evaluation of Antigen Test for the Diagnosis of SARS-CoV-2 and Analytical Sensitivity to Detect Variants of Concern Including Omicron and Subvariants.

Timely detection of SARS-CoV-2 infection with subsequent contact tracing and rapid isolation are considered critical to containing the pandemic, which continues with the emergence of new variants. Hence, there is an ongoing need for reliable point-of-care antigen rapid diagnostic tests (Ag-RDT). This report describes the development, evaluation, and analytical sensitivity of the diagnostic performance of the InteliSwab® COVID-19 Rapid Test. Methods: Samples from 165 symptomatic subjects were tested with InteliSwab® and the results were compared to RT-PCR to determine the antigen test performance. The analytical sensitivity of InteliSwab® for the detection of different variants was assessed by limit of detection (LOD) determination using recombinant nucleocapsid proteins (NPs) and testing with virus isolates. Western immunoblot independently confirmed that each monoclonal Ab is capable of binding to all variants tested thus far. The overall positivity rate by RT-PCR was 37% for the 165 symptomatic subjects. Based on RT-PCR results as the reference standard, InteliSwab® showed clinical sensitivity and specificity of 85.2% (95% CI, 74.3-92.0%) and 98.1% (95% CI, 93.3-99.7%), respectively. The overall agreement was 93.3% (Kappa index value 0.85; 95% CI, 0.77-0.74) between RT-PCR and InteliSwab® test results. Furthermore, the evaluation of analytical sensitivity for different SARS-CoV-2 variants by InteliSwab® was comparable in the detection of all the variants tested, including Omicron subvariants, BA.4, BA.5, and BQ.1. Due to the surge of infections caused by different variants from time to time, there is a critical need to evaluate the sensitivity of rapid antigen-detecting tests for new variants. The study findings showed the robust diagnostic performance of InteliSwab® and analytical sensitivity in detecting different SARS-CoV-2 variants, including the Omicron subvariants. With the integrated swab and excellent sensitivity and variant detection, this test has high potential as a point-of-care Ag-RDT in various settings when molecular assays are in limited supply and rapid diagnosis of SARS-CoV-2 is necessary.

Study of the Immunogenicity of Combination of Recombinant RBD (Omicron) and Nucleocapsid Proteins of SARS-CoV-2 Expressed in Pichia Pastoris

Background: SARS-CoV-2 is a virus responsible for the COVID-19 pandemic that began in late 2019. This pandemic has had a devastating impact worldwide, resulting in over 6.95 million deaths. The development of effective vaccines against the virus is crucial for preventing infection and reducing the severity of the disease. Objective: This study aimed to obtain the recombinant receptor-binding domain (RBD) and the nucleocapsid (N) proteins of SARS-CoV-2 as well as assess the immunogenicity of the combination of these recombinant proteins. Methods: The recombinant plasmids encoding the receptor-binding domain (RBD) of the spike protein of the Omicron variant and the nucleocapsid protein of SARS-CoV-2 were cloned into the yeast Pichia pastoris. The optimal fermentation conditions were established for recombinant P. pastoris strains. The methods for the isolation and purification of the target recombinant RBD and nucleocapsid proteins were developed. The immunogenicity of the purified recombinant proteins was evaluated by injecting them into mice and analyzing the specific IgG antibody responses using ELISA. Results: The study found that RBD and N proteins, as well as their combination, showed antigenic specificity and were highly immunogenic in mice. The immunogenicity was measured by determining the antibody titer, which represents the concentration of antibodies produced in response to the antigen. The antibody titers were 1:60000 for both RBD and N proteins, and 1:80000 for their combination. Conclusion: These findings suggest that the expressed proteins could be potential candidates for the development of vaccines or immunological diagnostic test kits for combatting or detecting the Omicron variant of SARS-CoV-2.

Abstract 14877: Spike Protein of SARS-CoV-2 Virus Induces Mitochondrial Dysfunction in Swine Heart via Redox Impairment of Heme Proteins and Increasing Superoxide Generation

The cellular entry of the SARS-CoV-2 virus depends on the binding of spike (S) protein to its biological ligand, ACE2. Although the virus commonly causes respiratory distress, cardiac injury can occur in COVID-19 patients, which is consistent with the expression of ACE2 in myocytes. Previous reports indicate SARS-CoV-2 proteins can target cardiac mitochondria and suppress mitochondrial function via enhancing MPTP pore opening and perturbing cardiac bioenergetics. To explore the underlying mechanism of the effect of SARS-CoV-2 on cardiac mitochondria, we measured the interactions of SARS-CoV-2 proteins with swine heart mitochondria in vitro. Incubation of recombinant S protein with isolated mitochondria significantly decreased state-3 oxygen consumption rate (OCR, 95.10 vs 65.68 nmol/min/mg) and FCCP uncoupling OCR (82.94 vs 66.95 nmol/min/mg). We further detected that S protein impaired the enzymatic activities of electron transport chain (ETC) (by 15.62% to 34.44%). However, S protein had no effect on TCA cycle enzymes, indicating the involvement of mitochondrial membrane components in decreased OCR by S proteins. Recombinant nucleocapsid (N) protein of SAR-CoV-2 had no effect on the OCR and ETC activities of swine mitochondria. S proteins decreased the intensity of mitochondrial heme spectrum determined by dithionite reduction with UV/VIS spectroscopy (by 17.52% heme a , 15.82% heme c 1 ). The results were further assessed using isolated complex III (Cx3) and complex IV (Cx4). Treatment of isolated Cx3 and Cx4 with S protein decreased the spectral intensities of heme a and heme c 1 . The spectra of both Cx3 and Cx4 were not affected by N protein. The results suggested S protein downregulates redox potentials of ETC in swine mitochondria. Treatment of swine mitochondria with S proteins enhanced superoxide ( . O 2 – ) generation by Cx1 (by 43.7%) and by Cx3 (by 10.9-fold) assessed by EPR and cytochrome c reduction assays. However, we detected S proteins modestly decreased . O 2 – generation by swine mitochondria under state-2 condition (by 9.52%), indicating impairing pH gradient by S protein. In conclusion, the spike protein of SARS-CoV-2 virus mediates mitochondrial dysfunction of swine heart via impairing the redox function and increasing . O 2 – generation.

Electrochemical biosensing based comparative study of monoclonal antibodies against SARS-CoV-2 nucleocapsid protein

In this study, we are reporting an electrochemical biosensor for the determination of three different clones of monoclonal antibodies (mAbs) against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) recombinant nucleocapsid protein (rN). The nucleocapsid protein was chosen as a system component identifying and discriminating antibodies that occur after virus infection instead of S protein used in serological tests to measure antibodies raised after vaccination and infection. The sensing platform was based on a screen-printed carbon electrode (SPCE) covered with gold nanoparticles (AuNP) and subsequently modified with a self-assembled monolayer (SAM) to ensure the covalent immobilization of the rN. The interaction between the protein and three clones of mAbs against SARS-CoV-2 rN with clone numbers 4G6, 7F10, and 1A6, were electrochemically registered in the range of concentrations. Three techniques, cyclic voltammetry (CV), differential pulse voltammetry (DPV), and pulse amperometric detection (PAD) were used for the detection. A gradual change in the responses with an increase in mAbs concentration for all techniques was observed. To assess the performance of the developed electrochemical biosensor, ‘complexation constant’ (KC), limit of detection (LOD), and limit of quantification (LOQ) were calculated for all assessed clones of mAbs and all used techniques. Our results indicated that DPV possessing higher fitting accuracy illustrated more significant differences in KC constants and LOD/LOQ values. According to the DPV results, 7F10 clone was characterized with the highest KC value of 1.47 ± 0.07 μg/mL while the lowest LOD and LOQ values belonged to the 4G6 clone and equaled 0.08 ± 0.01 and 0.25 ± 0.01 μg/mL, respectively. Overall, these results demonstrate the potential of electrochemical techniques for the detection and distinguishing of different clones of mAbs against SARS-CoV-2 nucleocapsid protein.

Cloning, expression, purification and characterization of the recombinant nucleocapsid protein from SARS-CoV-2 and its combination with a CpG ODN-39M

Several studies have reported the ability of the nucleocapsid (N) protein from the SARS-CoV-2 virus to interact with RNA in vitro. Nevertheless, its capacity to interact with deoxyribonucleic acid has yet to be widely described. In the present work, the nucleocapsid protein from the SARS-CoV-2 Delta variant was obtained in E. coli. The recombinant protein was purified, immune identified and incubated with different quantities of the ODN-39M, a CpG ODN with adjuvant properties. As a result, a curve of precipitation was obtained. The analysis by agarose gel electrophoresis of the mixtures revealed that ODN-39M retarded its mobility, whereas the protein increased its migration. When a sample of N+ODN-39M was crosslinked and analyzed by SDS-PAGE, high molecular weight aggregates were detected. Accordingly, upon analysis by Transmission Electron Microscopy, particles of approximately 20 nm were visualized in both samples of N protein, with and without ODN-39M. However, aggregates of particles were abundant in the mixture. Notably, such structures kept the ability of N protein to be recognized by human sera of COVID-19 convalescent donors. We can assert that nucleocapsid protein can bind to deoxyribonucleic acid. Such interaction changed the protein conformation, contributing to the previously reported immunogenicity in mice of N+ODN-39M complexes. Keywords: Nucleocapsid, SARS-CoV-2, ODN-39M, Particles

Comparative assessment of homemade ELISA and lateral flow assay (LFA)in the rapid, specific and sensitive detection of SARS-CoV-2 anti-nucleocapsid protein in sera of Egyptian patients

ABSTRACT Several diagnostic measures have been employed to precisely detect the SARS-CoV-2 viral infection using viral antigens, nucleic acids, and other serological approaches. The sensitivity and specificity of the serological tests remain a challenging need. Here, we describe the detection of human anti-SARS-CoV-2 IgG and IgM antibodies qualitatively through two optimized in-house ELISA and lateral flow immunoassay. Both approaches are based on the prokaryotic expression of 50 kDa SARS-CoV-2 recombinant nucleocapsid protein. This SARS-CoV-2rN-6×His was used either to coat ELISA plates or to be conjugated to gold nanoparticles followed by colorimetric detection of bound human IgG or IgM. In the LFA, we show the optimization of nanoparticle size, protein-binding capacity, membrane treatment, and finally testing the potential capacity of using either the optimized ELISA or LFA in detecting antibodies raised against viral infection. Assessment of both methods was carried out using human sera-positive and negative SARS-CoV-2 antibodies. The ELISA and LFA tests showed 86%, 96.5% sensitivity, 92%, 93.75% specificity, 97%, 98.2% PPV, and 64%, 88.2% NPV, respectively. In conclusion, both approaches were able to successfully detect human antibodies against SARS-CoV-2 nucleocapsid protein. The importance of both protocols cannot be overstated in the detection and diagnosis of viral infections, especially in developing countries.

Large Diffusion of Severe Acute Respiratory Syndrome Coronavirus 2 After the Successive Epidemiological Waves, Including Omicron, in Guinea and Cameroon: Implications for Vaccine Strategies.

We aimed to estimate the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) seroprevalence among the general population in Conakry, Guinea and Yaounde, Cameroon after the coronavirus disease 2019 Omicron wave. We conducted population-based, age-stratified seroprevalence surveys in Conakry and Yaounde (May and June 2022). We collected demographic and epidemiologic information and dried blood spot samples that were tested for SARS-CoV-2 immunoglobulin G (IgG) antibodies using recombinant nucleocapsid and spike proteins with Luminex technology. Samples were obtained from 1386 and 1425 participants in Guinea and Cameroon, respectively. The overall age-standardized SARS-CoV-2 IgG seroprevalence against spike and nucleocapsid proteins was 71.57% (95% confidence interval [CI], 67.48%-75.33%) in Guinea and 74.71% (95% CI, 71.99%-77.25%) in Cameroon. Seroprevalence increased significantly with age categories. Female participants were more likely than male participants to be seropositive. The seroprevalence in unvaccinated participants was 69.6% (95% CI, 65.5%-73.41%) in Guinea and 74.8% (95% CI, 72.04%-77.38%) in Cameroon. In multivariate analysis, only age, sex, and education were independently associated with seropositivity. These findings show a high community transmission after the different epidemiological waves including Omicron, especially among people aged >40 years. In addition, our results suggest that the spread of SARS-CoV-2 has been underestimated as a significant proportion of the population has already contracted the virus and that vaccine strategies should focus on vulnerable populations.

Process development and characterization of recombinant nucleocapsid protein for its application on COVID-19 diagnosis

COVID-19 pandemic was caused by the severe acute respiratory syndrome coronavirus 2 (Sars-CoV-2). The nucleocapsid (N) protein from Sars-CoV-2 is a highly immunogenic antigen and responsible for genome packing. Serological assays are important tools to detect previous exposure to SARS-CoV-2, complement epidemiological studies, vaccine evaluation and also in COVID-19 surveillance. SARS-CoV-2 N (r2N) protein was produced in Escherichia coli, characterized, and the immunological performance was evaluated by enzyme-linked immunosorbent assay (ELISA) and beads-based array immunoassay. r2N protein oligomers were evidenced when it is associated to nucleic acid. Benzonase treatment reduced host nucleic acid associated to r2N protein, but crosslinking assay still demonstrates the presence of higher-order oligomers. Nevertheless, after RNase treatment the higher-order oligomers reduced, and dimer form increased, suggesting RNA contributes to the oligomer formation. Structural analysis revealed nucleic acid did not interfere with the thermal stability of the recombinant protein. Interestingly, nucleic acid was able to prevent r2N protein aggregation even with increasing temperature while the protein benzonase treated begin aggregation process above 55 °C. In immunological characterization, ELISA performed with 233 serum samples presented a sensitivity of 97.44% (95% Confidence Interval, CI, 91.04%, 99.69%) and a specificity of 98.71% (95% CI, 95.42%, 99.84%) while beads-based array immunoassay carried out with 217 samples showed 100% sensitivity and 98.6% specificity. The results exhibited an excellent immunological performance of r2N protein in serologic assays showing that, even in presence of nucleic acid, it can be used as a component of an immunoassay for the sensitive and specific detection of SARS-CoV-2 antibodies.

Analytical sensitivity of COVID-19 rapid antigen tests: A case for a robust reference standard

Aggressive diagnostic testing remains an indispensable strategy for health and aged care facilities to prevent the transmission of SARS-CoV-2 in vulnerable populations. The preferred diagnostic platform has shifted towards COVID-19 rapid antigen tests (RATs) to identify the most infectious individuals. As such, RATs are being manufactured faster than at any other time in our history yet lack the relevant quantitative analytics required to inform on absolute analytical sensitivity enabling manufacturers to maintain high batch-to-batch reproducibility, and end-users to accurately compare brands for decision making. Here, we describe a novel reference standard to measure and compare the analytical sensitivity of RATs using a recombinant GFP-tagged nucleocapsid protein (NP-GFP). Importantly, we show that the GFP tag does not interfere with NP detection and provides several advantages affording streamlined protein expression and purification in high yields as well as faster, cheaper and more sensitive quality control measures for post-production assessment of protein solubility and stability. Ten commercial COVID-19 RATs were evaluated and ranked using NP-GFP as a reference standard. Analytical sensitivity data of the selected devices as determined with NP-GFP did not correlate with those reported by the manufacturers using the median tissue culture infectious dose (TCID50) assay. Of note, TCID50 discordance has been previously reported. Taken together, our results highlight an urgent need for a reliable reference standard for evaluation and benchmarking of the analytical sensitivity of RAT devices. NP-GFP is a promising candidate as a reference standard that will ensure that RAT performance is accurately communicated to healthcare providers and the public.

Evaluation of a high-sensitivity SARS-CoV-2 antigen test on the fully automated light-initiated chemiluminescent immunoassay platform.

To describe a high-sensitivity SARS-CoV-2 antigen test that is based on the fully automated light-initiated chemiluminescent immunoassay (LiCA®), and to validate its analytical characteristics and clinical agreement on detecting SARS-CoV-2 infection against the reference molecular test. Analytical performance was validated and detection limits were determined using different types of nucleocapsid protein samples. 798-pair anterior nasal swabspecimens were collected from hospitalized patients and asymptomatic screening individuals. Agreement between LiCA® antigen and real-time reverse transcription polymerase chain reaction (rRT-PCR) was evaluated. Repeatability and within-lab precision were 1.6-2.3%. The C5∼C95 interval was-5.1-4.6% away from C50. Detection limits in average (SD) were 325 (±141) U/mL onthenational reference panel, 0.07 (±0.04) TCID50/mL on active viral cultures, 0.27 (±0.09) pg/mL on recombinant nucleocapsid proteins and 1.07 (±1.01) TCID50/mL on inactivated viral suspensions, respectively. LiCA detected a median of 374-fold (IQR 137-643) lower levels of the viral antigen than comparative rapid tests. As reference to the rRT-PCR method, overall sensitivity and specificity were determined to be 97.5% (91.4-99.7%) and 99.9% (99.2-100%), respectively. Total agreement between both methods was 99.6% (98.7-99.9%) with Cohen's kappa 0.98 (0.96-1). A positive detection rate of 100% (95.4-100%) was obtained as Ct≤37.8. The LiCA® system provides an exceptionally high-sensitivity and fully automated platform for the detection of the SARS-CoV-2 antigen in nasal swabs. Theassay may have high potential use for large-scale population screening and surveillance of COVID-19 as an alternative to the rRT-PCR test.