The frequency and function of nucleoprotein-specific CD8+ T cells are critical for heterosubtypic immunity against influenza virus infection.

Cytotoxic T lymphocytes (CTLs) mediate host defense against viral and intracellular bacterial infections and tumors. However, the magnitude of CTL response and their function needed to confer heterosubtypic immunity against influenza virus infection are unknown. We addressed the role of CD8+ T cells in the absence of any cross-reactive antibody responses to influenza viral proteins using an adenoviral vector expressing a 9mer amino acid sequence recognized by CD8+ T cells. Our results indicate that both CD8+ T cell frequency and function are crucial for heterosubtypic immunity. Low morbidity, lower viral lung titers, low to minimal lung pathology, and better survival upon heterosubtypic virus challenge correlated with the increased frequency of NP-specific CTLs. NP-CD8+ T cells induced by differential infection doses displayed distinct RNA transcriptome profiles and functional properties. CD8+ T cells induced by a high dose of influenza virus secreted significantly higher levels of IFN-γ and exhibited higher levels of cytotoxic function. The mice that received NP-CD8+ T cells from the high-dose virus recipients through adoptive transfer had lower viral titers following viral challenge than those induced by the low dose of virus, suggesting differential cellular programming by antigen dose. Enhanced NP-CD8+ T-cell functions induced by a higher dose of influenza virus strongly correlated with the increased expression of cellular and metabolic genes, indicating a shift to a more glycolytic metabolic phenotype. These findings have implications for developing effective T cell vaccines against infectious diseases and cancer. IMPORTANCE: Cytotoxic T lymphocytes (CTLs) are an important component of the adaptive immune system that clears virus-infected cells or tumor cells. Hence, developing next-generation vaccines that induce or recall CTL responses against cancer and infectious diseases is crucial. However, it is not clear if the frequency, function, or both are essential in conferring protection, as in the case of influenza. In this study, we demonstrate that both CTL frequency and function are crucial for providing heterosubtypic immunity to influenza by utilizing an Ad-viral vector expressing a CD8 epitope only to rule out the role of antibodies, single-cell RNA-seq analysis, as well as adoptive transfer experiments. Our findings have implications for developing T cell vaccines against infectious diseases and cancer.

Unraveling the assembly mechanism of SADS-CoV virus nucleocapsid protein: insights from RNA binding, dimerization, and epitope diversity profiling.

The swine acute diarrhea syndrome coronavirus (SADS-CoV) has caused significant disruptions in porcine breeding and raised concerns about potential human infection. The nucleocapsid (N) protein of SADS-CoV plays a vital role in viral assembly and replication, but its structure and functions remain poorly understood. This study utilized biochemistry, X-ray crystallography, and immunization techniques to investigate the N protein's structure and function in SADS-CoV. Our findings revealed distinct domains within the N protein, including an RNA-binding domain, two disordered domains, and a dimerization domain. Through biochemical assays, we confirmed that the N-terminal domain functions as an RNA-binding domain, and the C-terminal domain is involved in dimerization, with the crystal structure analysis providing visual evidence of dimer formation. Immunization experiments demonstrated that the disordered domain 2 elicited a significant antibody response. These identified domains and their interactions are crucial for viral assembly. This comprehensive understanding of the N protein in SADS-CoV enhances our knowledge of its assembly and replication mechanisms, enabling the development of targeted interventions and therapeutic strategies. IMPORTANCE: SADS-CoV is a porcine coronavirus that originated from a bat HKU2-related coronavirus. It causes devastating swine diseases and poses a high risk of spillover to humans. The coronavirus N protein, as the most abundant viral protein in infected cells, likely plays a key role in viral assembly and replication. However, the structure and function of this protein remain unclear. Therefore, this study employed a combination of biochemistry and X-ray crystallography to uncover distinct structural domains in the N protein, including RNA-binding domains, two disordered domains, and dimerization domains. Additionally, we made the novel discovery that the disordered domain elicited a significant antibody response. These findings provide new insights into the structure and functions of the SADS-CoV N protein, which have important implications for future studies on SADS-CoV diagnosis, as well as the development of vaccines and anti-viral drugs.

Designing a Conserved Immunogenic Peptide Construct from the Nucleocapsid Protein of Puumala orthohantavirus.

Puumala orthohantavirus (PUUV) is an emerging zoonotic virus endemic to Europe and Russia that causes nephropathia epidemica, a mild form of hemorrhagic fever with renal syndrome (HFRS). There are limited options for treatment and diagnosis of orthohantavirus infection, making the search for potential immunogenic candidates crucial. In the present work, various bioinformatics tools were employed to design conserved immunogenic peptides containing multiple epitopes of PUUV nucleocapsid protein. Eleven conserved peptides (90% conservancy) of the PUUV nucleocapsid protein were identified. Three conserved peptides containing multiple T and B cell epitopes were selected using a consensus epitope prediction algorithm. Molecular docking using the HPEP dock server demonstrated strong binding interactions between the epitopes and HLA molecules (ten alleles for each class I and II HLA). Moreover, an analysis of population coverage using the IEDB database revealed that the identified peptides have over 90% average population coverage across six continents. Molecular docking and simulation analysis reveal a stable interaction with peptide constructs of chosen immunogenic peptides and Toll-like receptor-4. These computational analyses demonstrate selected peptides' immunogenic potential, which needs to be validated in different experimental systems.

Development and standardization of an enzyme-linked inmunosorbent for the detection of orthohantavirus infection in Argentina based on its bacterial-expressed nucleocapside protein.

We conducted a development and standardization of an IgG ELISA assay for serological detection of human orthohantavirus infections using the recombinant antigen rLECH13 produced in bacterial and derived from the LECHV. The evaluation and standardization were carried out by analyzing serum samples from a total of 50 patients with confirmed Hantavirus Pulmonary Syndrome (HPS) diagnosis through the reference technique, 50 negative sera, and 53 patients with other medical conditions. The data from the assay analysis showed a diagnostic sensitivity value of 95% and a diagnostic specificity of 80%. The high sensitivity of this novel assay leads us to conclude that rLECH13 is a feasible option for use in the immunodiagnostic of orthohantavirus infection. Additionally, it is crucial to have an antigen that can be produced under conditions that do not require highly complex laboratories. Furthermore, the new assay is cost-effective, reproducible, and demonstrates excellent performance.

The Key to Increase Immunogenicity of Next-Generation COVID-19 Vaccines Lies in the Inclusion of the SARS-CoV-2 Nucleocapsid Protein.

Vaccination is one of the most effective prophylactic public health interventions for the prevention of infectious diseases such as coronavirus disease (COVID-19). Considering the ongoing need for new COVID-19 vaccines, it is crucial to modify our approach and incorporate more conserved regions of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to effectively address emerging viral variants. The nucleocapsid protein is a structural protein of SARS-CoV-2 that is involved in replication and immune responses. Furthermore, this protein offers significant advantages owing to the minimal accumulation of mutations over time and the inclusion of key T-cell epitopes critical for SARS-CoV-2 immunity. A novel strategy that may be suitable for the new generation of vaccines against COVID-19 is to use a combination of antigens, including the spike and nucleocapsid proteins, to elicit robust humoral and potent cellular immune responses, along with long-lasting immunity. The strategic use of multiple antigens aims to enhance vaccine efficacy and broaden protection against viruses, including their variants. The immune response against the nucleocapsid protein from other coronavirus is long-lasting, and it can persist up to 11 years post-infection. Thus, the incorporation of nucleocapsids (N) into vaccine design adds an important dimension to vaccination efforts and holds promise for bolstering the ability to combat COVID-19 effectively. In this review, we summarize the preclinical studies that evaluated the use of the nucleocapsid protein as antigen. This study discusses the use of nucleocapsid alone and its combination with spike protein or other proteins of SARS-CoV-2.

Assessment of CD8+ T-cell mediated immunity in an influenza A(H3N2) human challenge model in Belgium: a single centre, randomised, double-blind phase 2 study.

BACKGROUND: Protection afforded by inactivated influenza vaccines can theoretically be improved by inducing T-cell responses to conserved internal influenza A antigens. We assessed whether, in an influenza controlled human infection challenge, susceptible individuals receiving a vaccine boosting T-cell responses would exhibit lower viral load and decreased symptoms compared with placebo recipients. METHODS: In this single centre, randomised, double-blind phase 2 study, healthy adult (aged 18-55 years) volunteers with microneutralisation titres of less than 20 to the influenza A(H3N2) challenge strain were enrolled at an SGS quarantine facility in Antwerp, Belgium. Participants were randomly assigned double-blind using a permuted-block list with a 3:2 allocation ratio to receive 0·5 mL intramuscular injections of modified vaccinia Ankara (MVA) expressing H3N2 nucleoprotein (NP) and matrix protein 1 (M1) at 1·5 × 108 plaque forming units (4·3 × 108 50% tissue culture infectious dose [TCID50]; MVA-NP+M1 group) or saline placebo (placebo group). At least 6 weeks later, participants were challenged intranasally with 0·5 mL of a 1 × 106 TCID50/mL dose of influenza A/Belgium/4217/2015 (H3N2). Nasal swabs were collected twice daily from day 2 until day 11 for viral PCR, and symptoms of influenza were recorded from day 2 until day 11. The primary outcome was to determine the efficacy of MVA-NP+M1 vaccine to reduce the degree of nasopharyngeal viral shedding as measured by the cumulative viral area under the curve using a log-transformed quantitative PCR. This study is registered with ClinicalTrials.gov, NCT03883113. FINDINGS: Between May 2 and Oct 24, 2019, 145 volunteers were enrolled and randomly assigned to the MVA-NP+M1 group (n=87) or the placebo group (n=58). Of these, 118 volunteers entered the challenge period (71 in the MVA-NP+M1 group and 47 in the placebo group) and 117 participants completed the study (71 in the MVA-NP+M1 group and 46 in the placebo group). 78 (54%) of the 145 volunteers were female and 67 (46%) were male. The primary outcome, overall viral load as determined by quantitative PCR, did not show a statistically significant difference between the MVA-NP+M1 (mean 649·7 [95% CI 552·7-746·7) and placebo groups (mean 726·1 [604·0-848·2]; p=0·17). All reported treatment emergent adverse events (TEAEs; 11 in the vaccination phase and 51 in the challenge phase) were grade 1 and 2, except for two grade 3 TEAEs in the placebo group in the challenge phase. A grade 4 second trimester fetal death, considered possibly related to the MVA-NP+M1 vaccination, and an acute psychosis reported in a placebo participant during the challenge phase were reported. INTERPRETATION: The use of an MVA vaccine to expand CD4+ or CD8+ T cells to conserved influenza A antigens in peripheral blood did not affect nasopharyngeal viral load in an influenza H3N2 challenge model in seronegative, healthy adults. FUNDING: Department of Health and Human Services; Administration for Strategic Preparedness and Response; Biomedical Advanced Research and Development Authority; and Barinthus Biotherapeutics.

Ultra-Sensitive Detection of the SARS-CoV-2 Nucleocapsid Protein via a Clustered Regularly Interspaced Short Palindromic Repeat/Cas12a-Mediated Immunoassay.

Tracking trace protein analytes in precision diagnostics is an ongoing challenge. Here, we developed an ultrasensitive detection method for the detection of SARS-CoV-2 nucleocapsid (N) protein by combining enzyme-linked immunosorbent assay (ELISA) with the clustered regularly interspaced short palindromic repeat/CRISPR-associated protein (CRISPR/Cas) system. First, the SARS-CoV-2 N protein bound by the capture antibody adsorbed on the well plate was sequentially coupled with the primary antibody, biotinylated secondary antibody, and streptavidin (SA), followed by biotin primer binding to SA. Subsequently, rolling circle amplification was initiated to generate ssDNA strands, which were targeted by CRISPR/Cas12a to cleave the FAM-ssDNA-BHQ1 probe in trans to generate fluorescence signals. We observed a linear relationship between fluorescence intensity and the logarithm of N protein concentration ranging from 3 fg/mL to 3 × 107 fg/mL. The limit of detection (LOD) was 1 fg/mL, with approximately nine molecules in 1 µL of the sample. This detection sensitivity was 4 orders magnitude higher than that of commercially available ELISA kits (LOD: 5.7 × 104 fg/mL). This method was highly specific and sensitive and could accurately detect SARS-CoV-2 pseudovirus and clinical samples, providing a new approach for ultrasensitive immunoassay of protein biomarkers.

Identification of B-cell epitope on the N protein of type 1 Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) using monoclonal antibody and construction of epitope-mutated virus.

The escalating epidemic of PRRSV-1 in China has prompted widespread concern regarding the evolution of strains, disparities in pathogenicity to herds, and immunological detection of emerging strains. The nucleocapsid (N) protein, as a highly conserved protein with immunogenic properties in PRRSV, is a subject of intensive study. In this research, the recombinant His-N protein was expressed based on the N gene of PRRSV-1 using a prokaryotic expression system and then administered to BALB/c mice. A cell fusion protocol was implemented between SP2/0 cells and splenocytes, resulting in the successful screening of a monoclonal antibody against the N protein, designated as mAb 2D7, by indirect ELISA. Western Blot analysis and Indirect Immunofluorescence Assay (IFA) confirmed that mAb 2D7 positively responded to PRRSV-1. By constructing and expressing a series of truncated His-fused N proteins, a B-cell epitope of N protein, 59-AAEDDIR-65, was identified. A sequence alignment of two genotypes of PRRSV revealed that this epitope is relatively conserved in PRRSV, yet more so in genotype 1. Cross-reactivity analysis by Western blot analysis demonstrated that the B-cell epitope containing D62Y mutation could not be recognized by mAb 2D7. The inability of mAb 2D7 to recognize the epitope carrying the D62Y mutation was further determined using an infectious clone of PRRSV. This research may shed light on the biological significance of the N protein of PRRSV, paving the way for the advancement of immunological detection and development of future recombinant marker vaccine.

[Prokaryotic expression of N protein of akabane disease virus and preparation of monoclonal antibody].

In order to generate monoclonal antibodies against the akabane virus (AKAV) N protein, this study employed a prokaryotic expression system to express the AKAV N protein. Following purification, BALB/c mice were immunized, and their splenocytes were fused with mouse myeloma cells (SP2/0) to produce hybridoma cells. The indirect ELISA method was used to screen for positive hybridoma cells. Two specific hybridoma cell lines targeting AKAV N protein, designated as 2C9 and 5E9, were isolated after three rounds of subcloning. Further characterization was conducted through ELISA, Western blotting, and indirect immunofluorescence assay (IFA). The results confirmed that the monoclonal antibodies specifically target AKAV N protein, exhibiting strong reactivity in IFA. Subtype analysis identified the heavy chain of the 2C9 mAb's as IgG2b and its light chain as κ-type; the 5E9 mAb's heavy chain was determined to be IgG1, with a κ-type light chain. Their ELISA titers reached 1:4 096 000. This study successfully developed two monoclonal antibodies targeting AKAV N protein, which lays a crucial foundation for advancing diagnostic methods for akabane disease prevention and control, as well as for studying the function of the AKAV N protein.

An indirect ELISA for detecting anti-SARS-CoV-2 antibodies in human sera using a baculovirus-expressed recombinant nucleocapsid antigen.

This study focuses on the development and initial assessment of an indirect IgG enzyme-linked immunosorbent assay (ELISA) specifically designed to detect of anti-SARS-CoV-2 antibodies. The unique aspect of this ELISA method lies in its utilization of a recombinant nucleocapsid (N) antigen, produced through baculovirus expression in insect cells. Our analysis involved 292 RT-qPCR confirmed positive serum samples and 54 pre-pandemic healthy controls. The process encompassed cloning, expression, and purification of the SARS-CoV-2 N gene in insect cells, with the resulted purified protein employed in our ELISA tests. Statistical analysis yielded an Area Under the Curve of 0.979, and the optimized cut-off exhibited 92 % sensitivity and 94 % specificity. These results highlight the ELISA's potential for robust and reliable serological detection of SARS-CoV-2 antibodies. Further assessments, including a larger panel size, reproducibility tests, and application in diverse populations, could enhance its utility as a valuable biotechnological solution for diseases surveillance.

Development and application of a blocking ELISA based on a N protein monoclonal antibody for the antibody detection against porcine reproductive and respiratory syndrome virus 2.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most widespread illnesses in the world's swine business. To detect the antibodies against PRRSV-2, a blocking enzyme-linked immunosorbent assay (B-ELISA) was developed, utilizing a PRRSV-2 N protein monoclonal antibody as the detection antibody. A checkerboard titration test was used to determine the optimal detection antibody dilution, tested pig serum dilution and purified PRRSV coated antigen concentration. After analyzing 174 negative pig sera and 451 positive pig sera, a cutoff value of 40 % was selected to distinguish between positive and negative sera using receiver operating characteristic curve analysis. The specificity and sensitivity of the assay were evaluated to equal 99.8 % and 96 %, respectively. The method had no cross-reaction with PCV2, PRV, PPV, CSFV, PEDV, TGEV, and PRRSV-1 serum antibodies, and the coefficients of variation of intra-batch and inter-batch repeatability experiments were both <10 %. A total of 215 clinical serum samples were tested, and the relative coincidence rate with commercial ELISA kit was 99.06 %, and the kappa value was 0.989, indicating that these two detection results exhibited high consistency. Overall, the B-ELISA should serve as an ideal method for large-scale serological investigation of PRRSV-2 antibodies in domestic pigs.

A novel linear B cell epitope of the canine coronavirus nucleocapsid protein identified by a monoclonal antibody.

The infection of canine coronavirus (CCoV) causes a highly contagious disease in dogs with acute gastroenteritis. The efficient serological diagnostics is critical for controlling the disease caused by CCoV. Nucleocapsid (N) protein of CCoV is an important target for developing serological approaches. However, little is known about the antigenic sites in the N protein of CCoV. In this study, we generated a monoclonal antibody (mAb) against the N protein of CCoV, designated as 13E8, through the fusion of the sp2/0 cells with the spleen cells from a mouse immunized with the purified recombinant GST-N protein. Epitope mapping revealed that mAb 13E8 recognized a novel linear B cell epitope in N protein at 294-314aa (named as EP-13E8) by using a serial of truncated N protein through Western blot and ELISA. Sequence analysis showed that the sequence of EP-13E8 was highly conserved (100 %) among different CCoV strains analyzed, but exhibited a low similarity (31.8-63.6 %) with the responding sequence in other coronaviruses of the same genus such as FCoV, PEDV and HCoV except for TGEV (95.5 % identity). Structural assay suggested that the epitope of EP-13E8 were located in the close proximity on the surface of the N protein. Overall, the mAb 13E8 against N protein generated and its epitope EP-13E8 identified here paid the way for further developing epitope-based serological diagnostics for CCoV.

Characterization of non-neutralizing human monoclonal antibodies that target the M1 and NP of influenza A viruses.

IMPORTANCE: Currently, many groups are focusing on isolating both neutralizing and non-neutralizing antibodies to the mutation-prone hemagglutinin as a tool to treat or prevent influenza virus infection. Less is known about the level of protection induced by non-neutralizing antibodies that target conserved internal influenza virus proteins. Such non-neutralizing antibodies could provide an alternative pathway to induce broad cross-reactive protection against multiple influenza virus serotypes and subtypes by partially overcoming influenza virus escape mediated by antigenic drift and shift. Accordingly, more information about the level of protection and potential mechanism(s) of action of non-neutralizing antibodies targeting internal influenza virus proteins could be useful for the design of broadly protective and universal influenza virus vaccines.

SARS-CoV-2 spike and nucleocapsid proteins fail to activate human dendritic cells or γδ T cells.

γδ T cells are thought to contribute to immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but the mechanisms by which they are activated by the virus are unknown. Using flow cytometry, we investigated if the two most abundant viral structural proteins, spike and nucleocapsid, can activate human γδ T cell subsets, directly or in the presence of dendritic cells (DC). Both proteins failed to induce interferon-γ production by Vδ1 or Vδ2 T cells within fresh mononuclear cells or lines of expanded γδ T cells generated from healthy donors, but the same proteins stimulated CD3+ cells from COVID-19 patients. The nucleocapsid protein stimulated interleukin-12 production by DC and downstream interferon-γ production by co-cultured Vδ1 and Vδ2 T cells, but protease digestion and use of an alternative nucleocapsid preparation indicated that this activity was due to contaminating non-protein material. Thus, SARS-CoV-2 spike and nucleocapsid proteins do not have stimulatory activity for DC or γδ T cells. We propose that γδ T cell activation in COVID-19 patients is mediated by immune recognition of viral RNA or other structural proteins by γδ T cells, or by other immune cells, such as DC, that produce γδ T cell-stimulatory ligands or cytokines.

Transient and durable T cell reactivity after COVID-19.

This study analyzed whole blood samples (n = 56) retrieved from 30 patients at 1 to 21 (median 9) mo after verified COVID-19 to determine the polarity and duration of antigen-specific T cell reactivity against severe acute respiratory syndrome coronavirus 2-derived antigens. Multimeric peptides spanning the entire nucleocapsid protein triggered strikingly synchronous formation of interleukin (IL)-4, IL-12, IL-13, and IL-17 ex vivo until ∼70 d after confirmed infection, whereafter this reactivity was no longer inducible. In contrast, levels of nucleocapsid-induced IL-2 and interferon-γ remained stable and highly correlated at 3 to 21 mo after infection. Similar cytokine dynamics were observed in unvaccinated, convalescent patients using whole-blood samples stimulated with peptides spanning the N-terminal portion of the spike 1 protein. These results unravel two phases of T cell reactivity following natural COVID-19: an early, synchronous response indicating transient presence of multipolar, antigen-specific T helper (TH) cells followed by an equally synchronous and durable TH1-like reactivity reflecting long-lasting T cell memory.

Reduction of Influenza A Virus Transmission in Mice by a Universal Intranasal Vaccine Candidate is Long-Lasting and Does Not Require Antibodies.

Vaccination against influenza virus infection can protect the vaccinee and also reduce transmission to contacts. Not all types of vaccines induce sterilizing immunity via neutralizing antibodies; some instead permit low-level, transient infection. There has been concern that infection-permissive influenza vaccines may allow continued spread in the community despite minimizing symptoms in the vaccinee. We have explored that issue for a universal influenza vaccine candidate that protects recipients by inducing T cell responses and nonneutralizing antibodies. Using a mouse model, we have shown previously that an adenoviral vectored vaccine expressing nucleoprotein (NP) and matrix 2 (M2) provides broad protection against diverse strains and subtypes of influenza A viruses and reduces transmission to contacts in an antigen-specific manner. Here, we use this mouse model to further explore the mechanism and features of that reduction in transmission. Passive immunization did not reduce transmission from infected donors to naive contact animals to whom passive serum had been transferred. Vaccination of antibody-deficient mIgTg-JHD-/- mice, which have intact T cell responses and antigen presentation, reduced transmission in an antigen-specific manner, despite the presence of some virus in the lungs and nasal wash, pointing to a role for cellular immunity. Vaccination at ages ranging from 8 to 60 weeks was able to achieve reduction in transmission. Finally, the immune-mediated reduction in transmission persisted for at least a year after a single-dose intranasal vaccination. Thus, this infection-permissive vaccine reduces virus transmission in a long-lasting manner that does not require antibodies. IMPORTANCE Universal influenza virus vaccines targeting antigens conserved among influenza A virus strains can protect from severe disease but do not necessarily prevent infection. Despite allowing low-level infection, intranasal immunization with adenovirus vectors expressing the conserved antigens influenza nucleoprotein (A/NP) and M2 reduces influenza virus transmission from vaccinated to unvaccinated contact mice. Here, we show that antibodies are not required for this transmission reduction, suggesting a role for T cells. We also show that transmission blocking could be achieved in recipients of different ages and remained effective for at least a year following a single-dose vaccination. Such vaccines could have major public health impacts by limiting viral transmission in the community.

Single Immunization with Recombinant ACAM2000 Vaccinia Viruses Expressing the Spike and the Nucleocapsid Proteins Protects Hamsters against SARS-CoV-2-Caused Clinical Disease.

Increasing cases of SARS-CoV-2 breakthrough infections from immunization with current spike protein-based COVID-19 vaccines highlight the need to develop alternative vaccines using different platforms and/or antigens. In this study, we expressed SARS-CoV-2 spike and nucleocapsid proteins based on a novel vaccinia virus (VACV) ACAM2000 platform (rACAM2000). In this platform, the vaccinia virus host range and immunoregulatory gene E3L was deleted to make the virus attenuated and to enhance innate immune responses, and another host range gene, K3L, was replaced with a poxvirus ortholog gene, taterapox virus 037 (TATV037), to make virus replication competent in both hamster and human cells. Following a single intramuscular immunization, the rACAM2000 coexpressing the spike and nucleocapsid proteins induced significantly improved protection against SARS-CoV-2 challenge in comparison to rACAM2000 expressing the individual proteins in a hamster model, as shown by reduced weight loss and shorter recovery time. The protection was associated with reduced viral loads, increased neutralizing antibody titer, and reduced neutrophil-to-lymphocyte ratio. Thus, our study demonstrates that rACAM2000 expressing a combination of the spike and nucleocapsid antigens is a promising COVID-19 vaccine candidate, and further studies will investigate if the rACAM2000 vaccine candidate can induce a long-lasting immunity against infection by SARS-CoV-2 variants of concern. IMPORTANCE Continuous emergence of SARS-CoV-2 variants which cause breakthrough infection from the immunity induced by current spike protein-based COVID-19 vaccines highlights the need for new generations of vaccines that will induce long-lasting immunity against a wide range of the variants. To this end, we investigated the protective efficacy of the recombinant COVID-19 vaccine candidates based on a novel VACV ACAM2000 platform, in which an immunoregulatory gene, E3L, was deleted and both the SARS-CoV-2 spike (S) and nucleocapsid (N) antigens were expressed. Thus, it is expected that the vaccine candidate we constructed should be more immunogenic and safer. In the initial study described in this work, we demonstrated that the vaccine candidate expressing both the S and N proteins is superior to the constructs expressing an individual protein (S or N) in protecting hamsters against SARS-CoV-2 challenge after a single-dose immunization, and further investigation against different SARS-CoV-2 variants will warrant future clinical evaluations.

Rapid Biosensor of SARS-CoV-2 Using Specific Monoclonal Antibodies Recognizing Conserved Nucleocapsid Protein Epitopes.

Coronavirus disease 2019 (COVID-19), the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is characterized by symptoms such as fever, fatigue, a sore throat, diarrhea, and coughing. Although various new vaccines against COVID-19 have been developed, early diagnostics, isolation, and prevention remain important due to virus mutations resulting in rapid and high disease transmission. Amino acid substitutions in the major diagnostic target antigens of SARS-CoV-2 may lower the sensitivity for the detection of SARS-CoV-2. For this reason, we developed specific monoclonal antibodies that bind to epitope peptides as antigens for the rapid detection of SARS-CoV-2 NP. The binding affinity between antigenic peptides and monoclonal antibodies was investigated, and a sandwich pair for capture and detection was employed to develop a rapid biosensor for SARS-CoV-2 NP. The rapid biosensor, based on a monoclonal antibody pair binding to conserved epitopes of SARS-CoV-2 NP, detected cultured virus samples of SARS-CoV-2 (1.4 × 103 TCID50/reaction) and recombinant NP (1 ng/mL). Laboratory confirmation of the rapid biosensor was performed with clinical specimens (n = 16) from COVID-19 patients and other pathogens. The rapid biosensor consisting of a monoclonal antibody pair (75E12 for capture and the 54G6/54G10 combination for detection) binding to conserved epitopes of SARS-CoV-2 NP could assist in the detection of SARS-CoV-2 NP under the circumstance of spreading SARS-CoV-2 variants.

Kinetics of Glycoprotein-Specific Antibody Response in Patients with Severe Fever with Thrombocytopenia Syndrome.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tickborne disease in East Asia that is causing high mortality. The Gn glycoprotein of the SFTS virus (SFTSV) has been considered to be an essential target for virus neutralization. However, data on anti-Gn glycoprotein antibody kinetics are limited. Therefore, we investigated the kinetics of Gn-specific antibodies compared to those of nucleocapsid protein (NP)-specific antibodies. A multicenter prospective study was performed in South Korea from January 2018 to September 2021. Adult patients with SFTS were enrolled. Anti-Gn-specific IgM and IgG were measured using an enzyme-linked immunosorbent assay. A total of 111 samples from 34 patients with confirmed SFTS were analyzed. Anti-Gn-specific IgM was detected at days 5-9 and peaked at day 15-19 from symptom onset, whereas the anti-NP-specific IgM titers peaked at days 5-9. Median seroconversion times of both anti-Gn- and NP-specific IgG were 7.0 days. High anti-Gn-specific IgG titers were maintained until 35-39 months after symptom onset. Only one patient lost their anti-Gn-specific antibodies at 41 days after symptom onset. Our data suggested that the anti-Gn-specific IgM titer peaked later than anti-NP-specific IgM, and that anti-Gn-specific IgG remain for at least 3 years from symptom onset.

Monoclonal antibodies to Cache Valley virus for serological diagnosis.

Cache Valley virus (CVV) is a mosquito-borne virus in the genus Orthobunyavirus, family Peribunyaviridae. It was first isolated from a Culiseta inorata mosquito in Cache Valley, Utah in 1956 and is known to circulate widely in the Americas. While only a handful of human cases have been reported since its discovery, it is the causative agent of fetal death and severe malformations in livestock. CVV has recently emerged as a potential viral pathogen causing severe disease in humans. Currently, the only serological assay available for diagnostic testing is plaque reduction neutralization test which takes several days to perform and requires biocontainment. To expand diagnostic capacity to detect CVV infections by immunoassays, 12 hybridoma clones secreting anti-CVV murine monoclonal antibodies (MAbs) were developed. All MAbs developed were found to be non-neutralizing and specific to the nucleoprotein of CVV. Cross-reactivity experiments with related orthobunyaviruses revealed several of the MAbs reacted with Tensaw, Fort Sherman, Tlacotalpan, Maguari, Playas, and Potosi viruses. Our data shows that MAbs CVV14, CVV15, CVV17, and CVV18 have high specific reactivity as a detector in an IgM antibody capture test with human sera.