Search Results (505)

Background/Objectives: An initial COVID-19 candidate vaccine containing a purified ancestral SARS-CoV-2 spike antigen was characterized with an ELISA using recombinant monoclonal antibodies (mAbs) generated against this variant. Upon the emergence of a new Beta (B.1.351) spike variant early in the pandemic, the assessment of a bivalent vaccine containing ancestral and Beta spike antigens began. Due to accelerated project timelines, mAbs generated specifically against the Beta spike antigen were not available at the time to address assay development and vaccine testing requirements. Methods: Using only the initial mAb panel raised against the ancestral spike antigen, an epitope-blocking ELISA strategy was developed to independently measure Beta spike antigen in bivalent vaccine formulations. To facilitate this, epitope profiling of spike antigens from both ancestral and Beta variants was performed with biolayer interferometry and hydrogen–deuterium exchange mass spectrometry using the original panel of mAbs. Results: The resulting blocking ELISA was precise and specific for the Beta spike antigen and detected the expected amount of this antigen in bivalent vaccine formulations. The specific amount of ancestral spike protein in the bivalent vaccine was also confirmed using the original ELISA developed at the onset of the pandemic. Conclusions: This epitope-blocking strategy helped to overcome key reagent availability issues and could be applied to other projects involving related proteins. Full article

Background/Objectives: SARS-CoV-2 vaccine candidates comprising the receptor binding domain (RBD) of the spike protein have been shown to confer protection against infection. Previous research evaluating vaccine candidates with SARS-CoV-2 RBD fused to ferritin (RBD-ferritin) and other scaffolds suggested that multimeric assemblies of RBD can enhance antigen presentation to improve the potency and breadth of immune responses. Though RBDs directly fused to a self-assembling scaffold can be delivered as messenger RNA (mRNA) formulated with lipid nanoparticles (LNPs), reports of SARS-CoV-2 vaccine candidates that combine these approaches remain scarce. Methods: Here, we designed RBD fused to AP205 or TIP60 self-assembling nanoparticles following a search of available structures focused on several scaffold properties. RBD-AP205 and RBD-TIP60 were tested for antigenicity following transfection and for immunogenicity and neutralization potency when delivered as mRNA in mice, with RBD-ferritin as a direct comparator. Results: All scaffolded RBD constructs were readily secreted to transfection supernatant and showed antigenicity in ELISA, though clear heterogeneity in assembly was observed. RBD-AP205 and RBD-TIP60 also exhibited robust antibody binding and neutralization titers in mice that were comparable to those elicited by RBD-ferritin or a full-length membrane-bound spike. Conclusions: These data suggest that AP205 and TIP60 can present RBD as effectively as ferritin and induce similar immune responses. By describing additional scaffolds for multimeric display that accommodate mRNA delivery platforms, this work can provide new tools for future vaccine design efforts. Full article

Background/Objectives: Rapid development of vaccines against SARS-CoV-2 was pivotal to controlling the COVID-19 pandemic. The emergency also provided a rare opportunity to test novel vaccine platforms such as mRNA in large clinical trials. Most of the early vaccines used SARS-CoV-2 Spike protein as the target antigen. Nevertheless, subsequent studies have shown that Receptor Binding Domain (RBD) of Spike also can yield efficacious vaccines, and we previously demonstrated that chemical conjugation of RBD to a carrier protein, EcoCRM^®, enhanced antibody responses and induced strong virus neutralization activity in mice. Methods: Here, we compared the immunogenicity of this conjugate to that of an approved mRNA vaccine from Pfizer/BioNTech in rhesus macaques over a period of nine months. Results: AS01-adjuvanted RBD conjugate induced a similar or better antibody response, receptor binding inhibition, and virus neutralization activity against different variants of SARS-CoV-2, compared to mRNA. IgG subclass profiles induced by conjugate and mRNA vaccines were initially dominated by IgG1 and IgG3 then switched to IgG2 and IgG4 dominant profiles during the subsequent six-month period. Polyclonal immune sera from the conjugate and mRNA had similar antibody avidity at multiple time points. Conclusions: In summary, antibody responses in rhesus macaques induced by the RBD-EcoCRM conjugate and the Spike mRNA vaccine are very similar. These results demonstrate the potential for the RBD-EcoCRM conjugate as a vaccine against SARS-CoV-2. Full article

Community-based serosurveillance for emerging zoonotic viruses can provide a powerful and cost-effective measurement of cryptic spillovers. Betacoronaviruses, including SARS-CoV, SARS-CoV-2 and MERS-CoV, are known to infect bats and can cause severe respiratory illness in humans, yet remain under-surveyed in high-risk populations. This study aimed to determine the seroprevalence of betacoronaviruses in an occupational cohort in contact with bats before and after the emergence of SARS-CoV-2. Serum samples from pre- and post-COVID-19 pandemic were screened using antigen-based multiplex microsphere immunoassays (MMIAs) and a multiplex surrogate virus neutralization test (sVNT). Pre-pandemic samples showed no SARS-CoV-2 antibodies, while post-pandemic samples from vaccinated participants displayed binding and neutralizing antibodies against SARS-CoV-2 and a related bat CoV. Furthermore, one participant (1/237, 0.43%) had persistent antibodies against MERS-CoV in 2017, 2018 and 2021 but was seronegative in 2023, despite reporting no history of traveling abroad or severe pneumonia. The observed sustained antibody levels indicate a possible exposure to MERS-CoV or a MERS-CoV-like virus, although the etiology and clinical relevance of this finding remains unclear. Ongoing surveillance in high-risk populations remains crucial for understanding virus epidemiology and mitigating zoonotic transmission risk. Full article

Background/Objectives: The COVID-19 pandemic, caused by SARS-CoV-2, required the rapid development of diagnostic tests. SARS-CoV-2, part of the betacoronavirus genus, shares characteristics with SARS-CoV-1, including its ability to survive on surfaces, facilitating the spread of the infection. This study analyzes the technique of nasopharyngeal secretion collection for SARS-CoV-2 diagnosis and compares the accuracy of rapid antigen and molecular tests. Methods: This study had two components: study A assessed the healthcare personnel training in collecting nasopharyngeal secretions and the discomfort associated with applying a questionnaire. Study B compared rapid antigen test accuracy with RT-PCR among children, through a retrospective analysis. The data were statistically analyzed to assess compliance with the testing protocols. Results: In study A, 88 healthcare workers achieved an average compliance score of 7.60 out of 10 regarding the collection procedure. Over 70% of participants correctly followed the fundamental steps of the procedure. Many patients who underwent sample collection reported pain and symptoms such as coughing or sneezing. In study B, 198 pediatric patients were tested using rapid antigen tests, collected simultaneously with RT-PCR. The rapid tests showed a 50% sensitivity and 97.5% specificity. Conclusions: This study indicates that nasopharyngeal specimen collection techniques are based on international recommendations, but improvements could be made to reduce discomfort. Rapid antigen tests are helpful for screening due to their high specificity and negative predictive value. Continuous healthcare personnel training and the monitoring of diagnostic techniques remain essential in managing SARS-CoV-2 and other viral infections. Full article

Background/Objectives: Identification and characterization of broadly neutralizing monoclonal antibodies from individuals exposed to SARS-CoV-2, either by infection or vaccination, can inform the development of next-generation vaccines and antibody therapeutics with pan-SARS-CoV-2 protection. Methods: Through single B cell sorting and RT-PCR, monoclonal antibodies (mAbs) were isolated from a donor who experienced a BA.5 or BF.7 breakthrough infection after three doses of inactivated vaccines. Their binding and neutralizing capacities were measured with ELISA and a pseudovirus-based neutralization assay, respectively. Their epitopes were mapped by competition ELISA and site-directed mutation. Results: Among a total of 67 spike-specific mAbs cloned from the donor, four mAbs (KXD643, KXD652, KXD681, and KXD686) can neutralize all tested SARS-CoV-2 variants from wild-type to KP.3. Moreover, KXD643, KXD652, and KXD681 belong to a clonotype encoded by IGHV5-51 and IGKV1-13 and recognize the cryptic and conserved RBD-8 epitope on the receptor-binding domain (RBD). In contrast, KXD686 is encoded by IGHV1-69 and IGKV3-20 and targets a conserved epitope (NTD Site iv) outside the antigenic supersite (NTD Site i) of the N-terminal domain (NTD). Notably, antibody cocktails containing these two groups of mAbs can neutralize SARS-CoV-2 more potently due to synergistic effects. In addition, bispecific antibodies derived from KXD643 and KXD686 demonstrate further improved neutralizing potency compared to antibody cocktails. Conclusions: These four mAbs can be developed as candidates of pan-SARS-CoV-2 antibody therapeutics through further antibody engineering. On the other hand, vaccines designed to simultaneously elicit neutralizing antibodies towards RBD-8 and NTD Site iv have the potential to provide pan-SARS-CoV-2 protection. Full article

Cases of new COVID-19 infection, which manifested in 2019 and caused a global socioeconomic crisis, still continue to be registered worldwide. The high mutational activity of SARS-CoV-2 leads to the emergence of new antigenic variants of the virus, which significantly reduces the effectiveness of COVID-19 vaccines, as well as the sensitivity of diagnostic test systems based on variable viral antigens. These problems may be solved by focusing on highly conserved coronavirus antigens, for example nucleocapsid (N) protein, which is actively expressed by coronavirus-infected cells and serves as a target for the production of virus-specific antibodies and T cell responses. It is known that anti-N antibodies are non-neutralizing, but their protective potential and functional activity are not sufficiently studied. Here, the protective effect of anti-N antibodies was studied in Syrian hamsters passively immunized with polyclonal sera raised to N(B.1) recombinant protein. The animals were infected with 10⁵ or 10⁴ TCID₅₀ of SARS-CoV-2 (B.1, Wuhan or BA.2.86.1.1.18, Omicron) 6 h after serum passive transfer, and protection was assessed by weight loss, clinical manifestation of disease, viral titers in the respiratory tract, as well as by the histopathological evaluation of lung tissues. The functional activity of anti-N(B.1) antibodies was evaluated by complement-dependent cytotoxicity (CDC) and antibody-dependent cytotoxicity (ADCC) assays. The protection of anti-N antibodies was evident only against a lower dose of SARS-CoV-2 (B.1) challenge, whereas almost no protection was revealed against BA.2.86.1.1.18 variant. Anti-N(B.1) monoclonal antibodies were able to stimulate both CDC and ADCC. Thus, anti-N(B.1) antibodies possess protective activity against homologous challenge infection, which is possibly mediated by innate Fc-mediated immune reactions. These data may be informative for the development of N-based broadly protective COVID-19 vaccines. Full article

Background and Objectives: Group A Streptococcus (GAS) is a leading cause of acute pharyngitis with seasonal outbreaks. The coronavirus disease 2019 (COVID-19) pandemic significantly altered respiratory infection trends; however, its impact on GAS pharyngitis (GAS-P) incidence remains unclear. Additionally, data on co-infections with GAS and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are limited. In this study, temporal trends in GAS-P incidence and characteristics of GAS–SARS-CoV-2 co-infections in Japan were examined. Materials and Methods: In this observational study, data from patients who visited the Tokyo Shinagawa Hospital between January 2019 and December 2024 were retrospectively analyzed. Data on GAS and SARS-CoV-2 test results and patient demographics were extracted from medical records. The study period was categorized based on COVID-19-related public health measures as follows: pre-COVID-19 social period (January 2019–April 2020), restricted social period (May 2020–April 2023), and post-restriction period (May 2023–December 2024). GAS incidence stratified by sex, age, and period was calculated. Clinical characteristics of patients co-infected with GAS and SARS-CoV-2 were analyzed. Results: Among 4837 GAS tests, 463 (9.6%) were positive. GAS positivity rates varied significantly: 11.4% (pre-COVID-19), 7.1% (restricted social period), and 12.6% (post-restriction period; p < 0.001). The proportion of pediatric cases decreased significantly during the restricted social period (24.8–5.3%) before rising sharply in the post-restriction period (47.1%, p < 0.001). Among 151 patients tested for GAS and SARS-CoV-2, 14 (9.3%) had co-infections, which were identified exclusively after July 2022. Most patients exhibited mild symptoms, primarily fever and sore throat, with decreased lymphocyte counts despite normal white blood cell counts. Conclusions: In our cohort, the incidence of GAS pharyngitis temporarily declined during COVID-19-related public health measures and subsequently increased, particularly among children, after restrictions were lifted. Limited testing may contribute to the underdiagnosis of GAS–SARS-CoV-2 co-infections. Further large-scale studies are warranted to assess microbial interactions, disease severity, and long-term outcomes. Full article

The ongoing demand for reliable, cost-effective, and scalable diagnostic solutions during the COVID-19 pandemic emphasized the need for innovative production platforms. In this study, we present a plant-based molecular farming (PMF) strategy for the production of the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein fused with an Fc region (RBDw-Fc). The RBDw-Fc antigen was transiently expressed in the Nicotiana benthamiana plant, achieving high yields and purity. Its functionality was assessed through antigen–antibody binding assays. The purified antigen was subsequently employed in the development of a rapid diagnostic blot assay capable of screening plasma EDTA samples from pre- and post-vaccinated as well as pre- and post-infected individuals, demonstrating high sensitivity and specificity. Our results show that the RBDw-Fc-based assay is effective for SARS-CoV-2 detection and offers considerable advantages in terms of production speed, scalability, and cost efficiency compared to traditional systems, such as cell-culture-based production. The assay delivers accurate results in just a few minutes, making it particularly suitable for clinical and resource-limited settings. This study highlights the versatility of PMF as a platform for producing high-quality reagents, with promising applications beyond SARS-CoV-2 diagnostics. The RBDw-Fc antigen-based method provides a model for the rapid, economical, and flexible development of screening tools for emerging infectious diseases and future pandemics. Full article

The COVID-19 pandemic has necessitated urgent measures to curb its spread, with vaccination emerging as a pivotal strategy. This prospective observational study evaluated breakthrough COVID-19 infections among healthcare workers (HCWs) vaccinated with Comirnaty (Pfizer-BioNTech) at a tertiary care hospital in Greece. Over an 8-month period, from February to September 2021, 1958 fully vaccinated HCWs were monitored. Rapid antigen tests and RT-PCR tests were conducted weekly for asymptomatic HCWs. Contact tracing and whole-genome sequencing were performed. Results showed that 2.75% (54 cases) of HCWs experienced breakthrough infections. Among these, 25 (45%) were asymptomatic, mild symptoms occurred in 21 (37%), while 7 (13%) had a fever (≥38 °C) alone and 3 (5%) developed high fever (≥39 °C) with respiratory symptoms. Physicians and nursing staff were the most affected groups. Dominant SARS-CoV-2 variants detected included Delta, British, and Wild type variants. Comparison with existing literature underscored the effectiveness of Comirnaty in reducing breakthrough infections. The findings emphasize the importance of continued booster vaccinations and ongoing surveillance to mitigate breakthrough infections among HCWs. Full article

Background: The effective management of vaccination schedules requires thorough knowledge of an individual’s immunoprotection level, including the interaction and persistence of immune responses at both the humoral and cellular levels following SARS-CoV-2 vaccination. This study aimed to investigate the potential relationship between the levels and duration of the SARS-CoV-2 T cell response and IgG measurements in a cohort of COVID-19-naive individuals who had received the SARS-CoV-2 vaccine. Methods: We performed a retrospective descriptive analysis utilizing data retrieved from the electronic medical records of consecutive COVID-19-naive and vaccinated adult individuals who underwent COVID-19 immunity screening at a private healthcare center from September 2021 to September 2022. T cell response was evaluated using the IGRA methodology T-SPOT^®.COVID (Oxford Immunotec, Abingdon, Oxfordshire, UK). Results: A retrospective analysis was conducted on a cohort of 262 individuals, comprising 148 females (56.5%) and 114 males (43.5%), with ages ranging from 17 to 92 years (mean age: 59.47 ± 15.5 years). Among the participants, 216/262 (82.4%) tested negative for SARS-CoV-2 IgG antibodies (group A), while 46/262 (17.6%) tested positive (group B). No significant difference was observed between the two groups in the time period post vaccination, with the mean times after vaccination being 136.38 ± 78.68 days in group A and 140.6 ± 79.5 days in group B (T-test, p = 0.74). Among the two groups, a positive T cell reaction against the S antigen was reported in 132/216 (61.1%) participants in group A and 39/46 (84.8%) in group B (X² test, p = 0.002). Additionally, individuals with a positive antibody response demonstrated statistically significant higher T SPOT results compared to those with undetectable antibody levels, with a mean SFC count of 125.70 for group A and 158.73 for group B (Mann–Whitney test, p = 0.006). Conclusions: Our findings suggest that T cell immunity may persist even when antibodies are undetectable, highlighting the potential role of cellular immunity in providing protection against COVID-19 over time. Additionally, this study demonstrates a significant correlation between humoral and cellular immune response levels to SARS-CoV-2, suggesting that the activation of humoral immunity following SARS-CoV-2 vaccination is associated with higher levels of cellular immunity compared to individuals with undetectable antibody levels. Full article

The COVID-19 pandemic has highlighted the need for rapid and accurate tests to detect SARS-CoV-2. Objectives: This study evaluates the effectiveness of the Panbio™ COVID-19 Antigen Self-Test rapid test compared to reverse transcriptase polymerase chain reaction (RT-PCR). Methods: A prospective diagnostic testing study was performed. Patients with respiratory symptoms who provided informed consent were included. Results: We included 205 patients with COVID-19 symptoms who underwent both tests. The mean age was 35.55 ± 12.62 years, and 64% of the participants were female. Sensitivity and specificity were 71.2% (95% CI: 62.5–79.9%) and 100% (95% CI: 96.4–100%), respectively. Conclusions: If a test is positive within the first 72 h after the onset of symptoms, we can be sure that it is a case of COVID-19; however, when the antigen test is negative, confirmation with RT-PCR is necessary. Its ease of use and results with moderate precision make it a valuable tool for early detection. Full article

Widespread and rapidly evolving SARS-CoV-2 posed an unprecedented challenge to vaccine developers. GeoVax has designed a multiantigen SARS-CoV-2 vaccine, designated GEO-CM02 based on a Modified Vaccinia Virus (MVA) vector that expresses spike (S), membrane (M), and envelope (E) antigens. This experimental vaccine was tested in the hACE2 transgenic mouse model to assess immunogenicity and efficacy. Administration of the vaccine in a two-dose regimen elicited high levels of neutralizing antibodies and provided complete protection, effectively reducing lung, olfactory bulb, and brain viral load and reducing lung inflammation following infection with original B.1 virus and the B.1.1.529 variant. In addition, GEO-CM02 conferred 80% protection against a lethal infection with the B.1.351 variant. GEO-CM02 vaccine efficacy studies also demonstrated a complete level of vaccine-induced protection with a single dose against the original B.1 virus and B.1.1.529 variant. GEO-CM02 effectively elicited functional T-cell responses in both prime and prime–boost groups. These data indicate that vaccination with the GEO-CM02 vaccine can induce immune responses that protect against severe disease induced by SARS-CoV-2 and its variants in a highly relevant pre-clinical model. Full article

(1) Background: Human Leukocyte Antigen (HLA) genetics substantially affect viral infection outcomes. SARS-CoV-2 continues to evolve, potentially escaping HLA presentation and hindering immune control. However, studies on HLA alleles in diverse non-Western populations remain limited. Therefore, we aimed to investigate whether mutations in successive SARS-CoV-2 variants have led to viral escape from common HLA class I alleles in the Saudi Arabian population. (2) Methods: The binding affinities of spike protein epitopes for common Saudi HLA alleles (HLA-A02:01, HLA-C06:02, and HLA-B51:01) were predicted across major SARS-CoV-2 strains using NetMHCpan. One-way ANOVA, one-sample t-tests, and pairwise chi-square analyses were performed to assess the differences in binding affinities and epitope binding categories among strains. (3) Results: One-way ANOVA revealed significant differences in binding affinities among SARS-CoV-2 strains for HLA-A02:01 and HLA-C06:02, but not for HLA-B51:01. One-sample t-tests revealed significant differences in mean binding affinity scores compared to a theoretical mean of 0 for all strain–HLA allele combinations, except for HLA-B51:01. Pairwise chi-square analyses identified significant differences in the epitope binding category distribution between Alpha and Epsilon strains, as well as between Epsilon and Gamma strains for HLA-B51:01. (4) Conclusions: The evolution of SARS-CoV-2 has enabled its escape from common HLA alleles in Saudis. Tracking population-specific HLA binding profiles is crucial for the elucidation of associated evasion mechanisms and guiding the design of future vaccines against COVID-19. Full article

Lateral flow tests (LFTs) had a pivotal role in combating the spread of the SARS-CoV-2 virus throughout the COVID-19 pandemic thanks to their affordability and ease of use. Most of LFT devices were based on nitrocellulose membrane strips whose industrial upscaling to billions of devices has already been extensively demonstrated. Nevertheless, the assay option in an LFT format is largely restricted to qualitative detection of the target antigens. In this research, we surveyed the potential of UV nanoimprint lithography (UV-NIL) and extrusion coating (EC) for the high-throughput production of disposable capillary-driven, foil-based tests that allow multistep assays to be implemented for quantitative readout to address the inherent lack of on-demand fluid control and sensitivity of paper-based devices. Both manufacturing technologies operate on the principle of imprinting that enables high-volume, continuous structuring of microfluidic patterns in a roll-to-roll (R2R) production scheme. To demonstrate the feasibility of R2R-fabricated foil chips in a point-of-care biosensing application, we adapted a commercial chemiluminescence multiplex test for COVID-19 antibody detection originally developed for a capillary-driven microfluidic chip manufactured with injection molding (IM). In an effort to build a complete ecosystem for the R2R manufacturing of foil chips, we also recruited additional processes to streamline chip production: R2R biofunctionalization and R2R lamination. Compared to conventional fabrication techniques for microfluidic devices, the R2R techniques highlighted in this work offer unparalleled advantages concerning improved scalability, dexterity of seamless handling, and significant cost reduction. Our preliminary evaluation indicated that the foil chips exhibited comparable performance characteristics to the original IM-fabricated devices. This early success in assay translation highlights the promise of implementing biochemical assays on R2R-manufactured foil chips. Most importantly, it underscores the potential utilization of UV-NIL and EC as an alternative to conventional technologies for the future development in vitro diagnostics (IVD) in response to emerging point-of-care testing demands. Full article