Search Results (80)

Objectives: Membrane-enveloped virus-like particles (VLPs) constitute a versatile vaccine platform allowing for the display of heterologous viral surface antigens. The density of displayed antigens is paramount for the efficient elicitation of a strong cellular and humoral immune response. SARS-CoV-2 spike protein variants with engineered cytoplasmic tails (CTs) were generated to enhance decoration efficiency on the surface of VLPs formed by the HIV core protein Gag. These HIV (SARS-CoV-2) chimeric particles serve as a vaccine component prototype. Methods: Spike variants were first analyzed for cellular and surface expression as well as incorporation into extracellular vesicles (EVs) and VLPs using flow cytometric analysis and Western blot analysis. Receptor binding, fusogenicity, i.e., mediating the fusion of spike-positive with receptor-containing membranes, and the proteins’ potential to mediate lentiviral vector gene transduction into susceptible target cells was examined by employing syncytia-formation assays and vector titration experiments. The display of a neutralization-sensitive epitope was examined utilizing immuno-precipitation using a neutralizing antibody. Results: All four variants were shown to be cell-surface expressed, to recruit the cognate receptor, to mediate membrane fusion and cell entry of lentiviral pseudotype vector particles and to decorate VLPs and EVs. However, the spike variant encompassing a truncated CT derived from the gibbon ape leukemia virus (GaLV) transmembrane (TM) envelope protein was most efficiently incorporated into HIV Gag-formed VLPs. All variants exposed a neutralization-sensitive epitope in the receptor binding domain. Conclusions: Engineering of the CTs of viral surface antigens can enhance VLP decoration, while required functionality of the ecto-domain such as receptor recognition, fusogenicity and neutralization-sensitive epitope presentation are not abrogated. This indicates the preservation of the structural integrity of the antigen required to elicit a neutralizing humoral immunity upon vaccination. The identified truncated CT of GaLV TM may be of utility to improve the incorporation of other viral surface antigens into a variety of membrane-enveloped VLPs derived from a range of different parental viruses. Full article

Background/Objectives: Marine-derived bioactive compounds have attracted increasing interest due to their potential antiviral properties. This study investigated in vitro antiviral activity of oil extracted from the green-lipped mussel (Perna canaliculus, GLM) and low-molecular-weight (LMW) fucoidan from Undaria pinnatifida against three human viruses in mammalian cell systems. herpes simplex virus-1 (HSV-1), respiratory syncytial virus (RSV), and SARS-CoV-2. These marine compounds were selected with the longer-term aim of evaluating their combination as a potential synergistic antiviral strategy. Methods: Antiviral efficacy was assessed using complementary assay platforms, including plaque reduction assays in mammalian cell systems and a lentiviral pseudovirus system delivering a bioluminescent reporter gene in HEK293/ACE2 cells pseudotyped with the SARS-CoV-2 spike glycoprotein. Cytotoxicity was assessed in parallel, and the selectivity index (SI) was calculated as the ratio of CC₅₀ to IC₅₀ for each compound and virus tested. Results: GLM oil showed potential antiviral activity against SARS-CoV-2 pseudovirus (SI > 6.20), with limited activity against RSV (SI > 3.48) and HSV-1 (SI > 2.28). In contrast, LMW fucoidan did not demonstrate antiviral activity against any of the tested viruses. Conclusions: These findings support further investigation of GLM-derived bioactive compounds as potential antiviral agents, including studies to elucidate their mechanisms of action and in vivo studies to confirm their antiviral efficacy. Combination studies were not pursued in the present work as both compounds require further optimisation individually; however, future studies should evaluate their combined antiviral potential, as synergistic or additive effects remain plausible. Full article

In this study, the phenotypic consequences of naturally occurring single nucleotide polymorphisms (SNPs) in the Middle East respiratory syndrome coronavirus (MERS-CoV) Spike protein were investigated. The impact of Spike mutations on the syncytia formation and neutralisation of contemporary MERS-CoV strains is not currently well understood. Mutations were identified by aligning 584 MERS-CoV Spike sequences from either human clinical isolates collected between 2012 and 2024 or from a clinical isolate that had been passaged in human cells. Fifteen SNPs of interest occurring in the N-terminal domain (NTD), receptor binding domain (RBD) and adjacent to the S1/S2 cleavage site were selected for further characterisation based on their location in the Spike protein, frequency and identification in previous studies. A contemporary clade B, lineage 5 wildtype Spike sequence, obtained from a human MERS-CoV clinical isolate, was used as the backbone in this study. The mutations of interest were introduced to the wildtype backbone to generate Spike variants. Spike variants were characterised via cell–cell fusion assays, and a lentiviral pseudotyping system was used to investigate the impact of these Spike mutations on neutralisation. The I529T, E536K and L745F mutations were shown to increase fusion and syncytia formation. The L411F, T424I, L506F, L745F and T746K mutations were found to increase resistance to neutralisation by pooled patient sera. This study has identified novel naturally occurring Spike mutations that resulted in phenotypic differences in the syncytia formation and neutralisation of contemporary MERS-CoV strains. Continued investigation of the phenotypic consequences of MERS-CoV Spike mutations is essential for assessing the risk to public health, especially given the pandemic potential of this virus. Full article

Background/Objectives: SARS-CoV-2, the causative agent of COVID-19, has been responsible for more than seven million deaths worldwide since its emergence. The Bacillus Calmette–Guérin (BCG) vaccine, used for over 100 years to prevent tuberculosis, induces a Th1-prominent immune response that is important for protection against Mycobacterium tuberculosis, other mycobacteria, and intracellular pathogens. BCG has also been shown to induce innate immune memory and heterologous protection against non-related infections. Additionally, BCG has been used as a vector to express heterologous proteins, showing protective effects against various diseases, particularly respiratory viral infections, including SARS-CoV-2. In this report, we constructed two recombinant BCG strains as potential vaccine candidates based on the receptor-binding domain (RBD) of the Spike antigen: one expressing only the RBD protein (rBCG-RBD) and another expressing the RBD protein in fusion with the LTB (Escherichia coli Labile Toxin subunit B) adjuvant (rBCG-LTB-RBD). Methods: We evaluated the induction of SARS-CoV-2-specific humoral and cellular immune responses using these vaccine candidates in a prime–boost strategy with a booster dose using the rRBD protein (produced in cell culture) and the Alum adjuvant. Antisera were evaluated for neutralization of the Wuhan and Omicron SARS-CoV-2 pseudotyped virus. Results: Either immunization scheme (rBCG-RBD/rRBD or rBCG-LTB-RBD/rRBD) induced high IgG antibody titers, with antibody neutralization against a Wuhan SARS-CoV-2 pseudotyped virus after 10 weeks. The antibody levels induced by rBCG-RBD/rRBD were maintained for up to 9 months. Interestingly, only the sera from mice receiving the prime–boost with rBCG-LTB-RBD/rRBD showed cross-reactive neutralization against the Omicron SARS-CoV-2 pseudotyped virus. Immunization with rBCG-RBD or rBCG-LTB-RBD and a rRBD booster dose promoted the induction of specific CD4+ and CD8+ T cells producing Th1/Th2 cytokines (IL-4, TNF-α and IFN-γ). Conclusions: Our study highlights the potential of the prime–boost immunization strategy using rBCG-RBD/rRBD to induce long-term immunity and rBCG-LTB-RBD/rRBD to induce cross-protection against different variants, both of which could serve as promising vaccine candidates. Full article

SARS-CoV-2 virus contains two highly conserved domains, the papain-like protease (PLpro) and main protease (Mpro), which play important roles in virus replication, immune suppression, and the induction of inflammation in host tissue. In this study, we applied small-molecule chip screening, enzymatic assays, SARS-CoV-2 spike pseudotyped virus detection and molecular docking to find potential Mpro or PLpro inhibitors. Two small molecules, oxytocin and risedronate sodium, stood out in drug repurposing. Oxytocin and risedronate sodium were shown to influence the activities of Mpro and PLpro, thereby preventing the virus from replication, which may alleviate SARS-CoV-2 infection. Thus, oxytocin, risedronate sodium, and cephalosporins may expand the drug library for treating coronavirus infection. Full article

Since its discovery in 2019, SARS-CoV-2 has continued to be detected in both humans and animals worldwide. Currently there is limited research focusing on serological surveillance of wildlife under human care. Here we tested 230 serum samples of 134 animals from two zoological institutions collected between 2015 and 2024. To assess prior exposure and antibody responses from natural infection or vaccination, we used three serological assays: a nucleocapsid protein-based ELISA (N-ELISA), a surrogate virus neutralization test (sVNT) for spike (S) protein and a neutralization assay with S-pseudotyped viral particles. Among the 114 samples collected from 58 animals at Fort Wayne Zoo in Indiana, 37 samples from 20 vaccinated animals were sVNT-positive, and 2 of the positive animals had 2 samples prior to vaccination that tested positive by N-ELISA. Of the 116 samples from 76 animals at Brookfield Zoo in Illinois, 20 samples of 20 animals were sVNT-positive, and 19 of the positive animals had been vaccinated. Among these 20 sVNT-positive samples, only one sample from a South American Tapir was positive from prior to vaccination and 1 sample from a sloth bear was also positive by N-ELISA, marking the first documented cases of SARS-CoV-2 exposure in both species. Neutralization assays with S-pseudotyped virus revealed that some of the sVNT-positive samples have strong activity against the WH1-S pseudovirus but showed significantly reduced neutralization against the Omicron LP.8.1-S pseudovirus. These results underscore the need for updated vaccines tailored to emerging variants. Overall, our findings highlight the importance of continued serological surveillance across multiple species to detect new SARS-CoV-2 exposures and monitor vaccine-induced immunity in captive animal populations. Full article

SARS-CoV-2, the agent responsible for coronavirus disease in 2019 (COVID-19), has caused extensive global health and socioeconomic impact due to its transmissibility and pathology. As a result, it was classified as a Risk Group 3 human pathogen, and handling samples containing live virus requires enhanced biological containment facilities (i.e., CL3) to reduce the potential of laboratory infection to personnel and the spread of the virus into the community. While the use of an authentic live virus remains the gold standard for biological assays, alternative methods have been developed to effectively evaluate neutralization activity in the absence of a replicating viral agent. Here, we describe a cell-based spike–ACE2 binding assay as a surrogate for neutralization of SARS-CoV-2 spike to identify potential neutralizing antibodies. A main advantage of this approach is the exclusion of infectious viral particles, increasing biosafety for laboratory personnel. The interaction of recombinant SARS-CoV-2 trimeric spike protein with ACE2 is monitored and quantified by flow cytometry. Notably, our previous studies have demonstrated the utility of this assay for other viruses, beyond SARS-CoV-2. The methodology presented here has exhibited a strong correlation to other widely accepted methods, such as pseudotyped lentiviral and live virus neutralization assays, in identifying neutralizing antibodies. Full article

Glycyrrhizic acid (GA) and its derivatives have been reported to have potent pharmacological effects against viral infections, including SARS-CoV and SARS-CoV-2. However, their antiviral mechanisms against coronaviruses are not fully understood. In this study, we found that diammonium glycyrrhizinate (DG) can effectively reduce infections of several human coronaviruses, including HCoV-OC43, HCoV-229E, and SARS-CoV-2, as well as newly emerged variants, with EC₅₀ values ranging from 115 to 391 μg/mL being recorded. Time-of-addition and pseudotype virus infection studies indicated that DG treatment dramatically inhibits the process of virus entry into cells. Furthermore, we demonstrated that DG broadly binds to the RBD of human coronaviruses, thereby blocking spike-mediated cellular entry, by using TR-FRET-based receptor-binding domain (RBD)-ACE2 interaction assay, capillary electrophoresis (CE), and surface plasmon resonance (SPR) assay. In support of this notion, studies of molecular docking and amino acid mutation showed that DG may directly bind to a conserved hydrophobic pocket of the RBD of coronaviruses. Importantly, intranasal administration of DG had a significant protective effect against viral infection in a HCoV-OC43 mouse model. Finally, we found that combinations of DG and other coronavirus inhibitors exhibited antiviral synergy. In summary, our studies strongly reveal that DG exerts broad-spectrum antiviral activity against human coronaviruses by interrupting spike-mediated cellular entry, demonstrating the pharmacological feasibility of using DG as a candidate for alternative treatment and prevention of coronavirus infection. Full article

Background/Objectives: Patients suffering from inflammatory bowel diseases (IBDs) undergoing treatment with anti-TNF antibodies mount a diminished humoral immune response to vaccination against SARS-CoV-2 compared to healthy controls. The characterization of variant-specific immune responses is particularly warranted among immunosuppressed patients, where reduced responses may necessitate further medical interventions. Methods: This pilot study investigated the humoral immune response of vaccinated IBD patients on anti-TNF medication and a comparable group of healthy individuals against the viral variants Alpha, Beta, Gamma, Delta, and Omicron BA.1 and BA.5. While total IgG antibodies targeting the receptor binding site of the spike protein of SARS-CoV-2 were quantified using a chemiluminescence microparticle immunoassay (CMIA), their potential neutralizing capacity was determined using commercial and variant-specific in-house surrogate virus neutralization tests (sVNTs) against a variant-specific in-house VSV-pseudotyped virus neutralization test (pVNT) as the gold standard. Results: Employing variant-specific assays recapitulated the immune escape functions of virus variants. Conspicuously, antibody reactivity against Alpha and Omicron BA.1 and BA.5 was strikingly poor in IBD patient sera post-initial vaccination compared to healthy individuals. A comparison of the diagnostic performance of assays with the pVNT revealed that identification of patients with inadequate humoral responses by CMIA and sVNT may require adjustments to cut-off values and end-point titration of sera. Following adaptation of cut-off values, patient sera exhibited reduced reactivity against all tested variants. The assay panel used substantiated the impact of anti-TNF therapy in IBD patients as to reduced strength, function, and breadth of the immune response to several SARS-CoV-2 variants. The immune response measured following the second vaccination was comparable to the antibody response observed in healthy individuals following the first vaccination. Conclusion: Variant-specific sVNTs and pVNTs have the potential to serve as valuable tools for evaluating the efficacy of adapted vaccines and to inform clinical interventions in the care of immunosuppressed patients. Anti-TNF-treated individuals with antibody levels below the optimized CMIA threshold should be considered for early booster vaccination and/or close immunological monitoring. Full article

Nanobodies, or single-domain antibodies (V_HHs) from camelid heavy-chain-only antibodies, offer significant advantages in therapeutic and diagnostic applications due to their small size and ability to bind cryptic protein epitopes inaccessible to conventional antibodies. In this study, we examined nanobodies specific to regions of the SARS-CoV-2 spike glycoprotein, including the receptor-binding domain (RBD), N-terminal domain (NTD), and subunit 2 (S2). Using flow virometry, a high-throughput technique for viral quantification, we achieved the efficient detection of pseudotyped viruses expressing the spike glycoprotein. RBD-targeting nanobodies showed the most effective staining, followed by NTD-targeting ones, while S2-specific nanobodies exhibited limited resolution. The simple genetic structure of nanobodies enables the creation of multimeric formats, improving binding specificity and avidity. Bivalent V_HH-Fc constructs (V_HHs fused to the Fc region of human IgG) outperformed monovalent formats in resolving viral particles from background noise. However, S2-specific monovalent V_HHs demonstrated improved staining efficiency, suggesting their smaller size better accesses restricted antigenic sites. Furthermore, direct staining of cell supernatants was possible without virus purification. This versatile nanobody platform, initially developed for antiviral therapy against SARS-CoV-2, can be readily adapted for flow virometry applications and other diagnostic assays. Full article

Background: Porcine epidemic diarrhea virus (PEDV) is a significant pathogen in swine, causing substantial economic losses worldwide. Despite the availability of existing vaccines, there is a critical need for novel vaccine platforms that ensure robust protection while maintaining safety. Methods: A recombinant replication-deficient vesicular stomatitis virus (VSV) vaccine, rVSV∆G-PEDV-S, was developed by pseudotyping the virus with the spike (S) protein from PEDV. To achieve high-titer pseudotyped rVSV particles, a stable Huh7 cell line expressing the PEDV S protein (Huh7-PEDV-S) was generated. The infectivity and replication capacity of rVSV∆G-PEDV-S were evaluated in PEDV-susceptible cell lines and Huh7-PEDV-S cells. The vaccine’s immunogenicity and safety were assessed in BALB/c mice vaccinated intramuscularly with rVSV∆G-PEDV-S. Results: The pseudotyped rVSV∆G-PEDV-S demonstrated infectivity in PEDV-susceptible cell lines and robust replication in Huh7-PEDV-S cells, while remaining replication-deficient in non-complementary cells. In vaccinated BALB/c mice, the vaccine elicited a strong humoral immune response, characterized by high levels of PEDV S1-specific IgG and neutralizing antibodies. No adverse effects, including weight loss or behavioral changes, were observed in the vaccinated mice, confirming the vaccine’s safety. Conclusions: The rVSV∆G-PEDV-S vaccine represents a promising platform for controlling PEDV outbreaks. Its replication-deficient design and pseudotyping methodology ensure safety and adaptability to emerging PEDV variants. These findings highlight the potential of rVSV∆G-PEDV-S as a safe and effective solution to the ongoing challenges posed by PEDV. Full article

Background/Objectives: Virus-like particles (VLPs) represent an attractive platform for delivering vaccine formulations, combining a high biosafety profile with a potent immune-stimulatory ability. VLPs are non-infectious, non-replicating, self-assembling nanostructures that can be exploited to efficiently expose membrane-tethered glycoproteins such as the SARS-CoV-2 Spike (S) protein, the main target of approved preventive vaccines. Here, we describe the development and preclinical validation of Simian Immunodeficiency Virus (SIV)-based GFP-labeled VLPs displaying S from the B.1.617.2 (Delta) variant (VLP/S-Delta) for inducing persistent anti-SARS-CoV-2 neutralizing antibodies (nAbs) and S-specific T cell responses in mice. Methods: SIV-derived VLP/S-Delta were produced by co-transfecting a plasmid expressing SIVGag-GFP, required for VLP assembly and quantification by flow virometry, a plasmid encoding the Delta S protein deleted in the cytoplasmic tail (CT), to improve membrane binding, and a VSV.G-expressing plasmid, to enhance VLP uptake. Recovered VLPs were titrated by flow virometry and characterized in vitro by transmission electron microscopy (TEM) and confocal microscopy (CLSM). BALB/c mice were immunized intramuscularly with VLP/S-Delta following a prime–boost regimen, and humoral and cellular immune responses were assessed. Results: VLP/S-Delta were efficiently pseudotyped with CT-truncated S-Delta. After BALB/c priming, VLP/S-Delta elicited both specific anti-RBD IgGs and anti-Delta nAbs that significantly increased after the boost and were maintained over time. The prime–boost vaccination induced similar levels of cross-nAbs against the ancestral Wuhan-Hu-1 strain as well as cross-nAbs against Omicron BA.1, BA.2 and BA.4/5 VoCs, albeit at lower levels. Moreover, immunization with VLP/S-Delta induced S-specific IFNγ-producing T cells. Conclusions: These data suggest that SIV-based VLPs are an appropriate delivery system for the elicitation of efficient and sustained humoral and cellular immunity in mice, paving the way for further improvements in the immunogen design to enhance the quality and breadth of immune responses against different viral glycoproteins. Full article

Since the onset of the COVID-19 (COronaVIrus Disease 19) pandemic, SARS-CoV-2 has exhibited a high transmission rate, further enhanced by new variants able to better adapt to humans. Addressing this issue has been challenging due to viral resistance and side effects associated with antiviral drugs and vaccines. As a result, there has been a growing interest in plant-derived compounds with antiviral properties. Our study revealed that pistachio extracts significantly inhibited SARS-CoV-2 viral entry. Employing pseudotyped particles bearing the S protein of SARS-CoV-2, we demonstrated that treatment with pistachio extracts inhibited binding of alpha (α) and omicron (ο) SARS-CoV-2 variants. Furthermore, our study revealed that the pistachio carotenoid zeaxanthin exhibited a different inhibitory activity against two SARS-CoV-2 variants. In silico analyses demonstrated a strong interaction between zeaxanthin and the receptor-binding domain (RBD) domain of the omicron spike (S) protein, thus reducing pseudovirus entry. However, zeaxanthin’s weaker interaction with the alpha variant’s RBD was insufficient to inhibit entry. Additionally, zeaxanthin suppressed the expression of the host protease TMPRSS2 at the protein level, thereby limiting the internalization of the alpha variant, which relies on TMPRSS2 for cellular entry. Full article

Background: This study assessed the long-term dynamics of neutralizing antibodies in a Ugandan cohort primarily exposed to the A.23.1 SARS-CoV-2 variant, examining how this shaped immune breadth and potency against diverse strains following infection and prototype-based vaccination. Methods: We conducted a 427-day retrospective analysis of 41 participants across multiple SARS-CoV-2 waves, assessing binding and neutralizing antibody responses using in-house ELISA and pseudotyped virus neutralization assays. We quantified immune responses to key SARS-CoV-2 variants, A.23.1, D614G, Delta, and BA.4, capturing evolving immunity across the pandemic. Results: Neutralizing antibody titers against A.23.1 remained significantly higher than those against D614G, Delta, and BA.4, highlighting the solid immune memory following A.23.1 infection. Consistently lower titers were observed for BA.4 across all time points, aligning with its strong immune-evasion capability. Correlations between neutralizing titers and spike-directed IgG (S-IgG) concentrations were significantly stronger for A.23.1 than for D614G, with no correlation for BA.4. ChAdOx1-S vaccination substantially elevated the neutralizing titers across all variants, most notably BA.4, highlighting the essential role of vaccination in boosting immunity, even in individuals with initially low titers. Conclusions: Initial exposure to the A.23.1 variant triggered potent immune responses, shaping neutralizing antibody dynamics during subsequent exposures. These findings highlight the importance of accounting for early viral exposures in vaccine development and public health planning. The distinctly lower immune response to BA.4 highlights the need for continuous antigenic monitoring and timely vaccine updates for protection against emerging variants. Vaccination remains essential for reinforcing and sustaining immunity against evolving variants. Full article

Background: The continuous evolution of SARS-CoV-2 and the emergence of novel variants with numerous mutations have heightened concerns surrounding the possibility of cross-species transmission and the establishment of natural animal reservoirs for the virus, but the host range of emerging SARS-CoV-2 variants has not been fully explored yet. Methods: We employed an in vitro model comprising VSV∆G* pseudotyped viruses bearing SARS-CoV-2 spike proteins to explore the plausible host range of SARS-CoV-2 emerging variants. Results: The overall host tropism of emerging SARS-CoV-2 variants are consistent with that of the SARS-CoV-2 wuhan-hu-1 strain with minor difference. Pseudotyped viruses bearing spike protein from RaTG13 and RmYN02 can enter cell cultures from a broad range of mammalian species, revealing that mink and hamsters may act as potential intermediate hosts. We further investigated 95 potential site-specific mutations in the SARS-CoV-2 spike protein that could impact viral infectivity across different species. The results showed that 13 of these mutations notably increased the transduction rates by more than two-fold when compared to the wild-type spike protein. Further examination of these 13 mutations within cell cultures from 31 different species revealed heightened sensitivity in cells derived from palm civets, minks, and Chinese horseshoe bats to the VSV∆G*-SARS2-S mutants. Specific mutations, such as L24F, R158G, and L212I, were seen to significantly enhance the capacity for SARS-CoV-2 of cross-species transmission. Conclusions: This study offers critical insights for the ongoing surveillance and monitoring efforts of SARS-CoV-2 evolution, emphasizing the need for the vigilant monitoring of specific mutations in both human and animal populations. Full article