Recombinant SARS-CoV-2 S protein expression was examined in Vero cells by imaging using the human monoclonal antibody panel (PD4, PD5, sc23, and sc29). The PD4 and sc29 antibodies recognised conformational specific epitopes in the S2 protein subunit at the Endoplasmic reticulum and Golgi complex. While PD5 and sc23 detected conformationally specific epitopes in the S1 protein subunit at the Golgi complex, only PD5 recognised the receptor binding domain (RBD). A comparison of the staining patterns of PD5 with non-conformationally specific antibodies that recognises the S1 subunit and RBD suggested the PD5 recognised a conformational structure within the S1 protein subunit. Our data suggests the antibody binding epitopes recognised by the human monoclonal antibodies formed at different locations in the secretory pathway during S protein transport, but a conformational change in the S1 protein subunit at the Golgi complex formed antibody binding epitopes that are recognised by virus neutralising antibodies.

A recombinant lineage of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant, named XBB, appeared in late 2022 and evolved descendants that successively swept local and global populations. XBB lineage members were noted for their improved immune evasion and transmissibility. Here, we determine cryoelectron microscopy (cryo-EM) structures of XBB.1.5, XBB.1.16, EG.5, and EG.5.1 spike (S) ectodomains to reveal reinforced 3-receptor binding domain (RBD)-down receptor-inaccessible closed states mediated by interprotomer RBD interactions previously observed in BA.1 and BA.2. Improved XBB.1.5 and XBB.1.16 RBD stability compensated for stability loss caused by early Omicron mutations, while the F456L substitution reduced EG.5 RBD stability. S1 subunit mutations had long-range impacts on conformation and epitope presentation in the S2 subunit. Our results reveal continued S protein evolution via simultaneous optimization of multiple parameters, including stability, receptor binding, and immune evasion, and the dramatic effects of relatively few residue substitutions in altering the S protein conformational landscape.

This study aims to develop an effective bivalent subunit vaccine that is promising to prevent both porcine deltacoronavirus (PDCoV) and porcine epidemic diarrhea virus (PEDV). The receptor-binding domains (RBDs) of PDCoV and PEDV were fused and cloned into the eukaryotic expression vector pCDNA3.1(+). The fusion protein PDCoV-RBD-PEDV-RBD (pdRBD-peRBD) was expressed by the ExpiCHOTM expression system and purified. Mice were immunized with the fusion protein at three different doses (10, 20, and 30 μg). The humoral immune response and cellular immune response induced by the fusion protein were evaluated by ELISA and flow cytometry. The neutralization titers of the serum of immunized mice against PDCoV and PEDV were determined by the microneutralization test. The results showed that high levels of IgG antibodies were induced in the three different dose groups after booster immunization, and there was no significant difference in the antibody level between different dose groups, indicating that the immunization dose of 10 μg could achieve the fine immune effect. The results of flow cytometry showed that the immunization groups demonstrated increased proportion of CD3+CD4+ T cells and decreased proportion of CD3+CD8+ T cells, which was consistent with the expectation about the humoral immune response induced by the subunit vaccine. At the same time, the levels of interleukin (IL)-2, IL-4, and interferon (IFN)-γ in the serum were determined. The results showed that the fusion protein induced both humoral immune effect and cellular immune response. The results of the neutralization test showed that the antibody induced by 10 μg fusion protein neutralized both PDCoV and PEDV in vitro, with the titers of 1:179.25 and 1:141.21, respectively. The above results suggested that the pdRBD-peRBD could induce a high level of humoral immune response at a dose of 10 μg, and the induced antibody could neutralize both PDCoV and PEDV. Therefore, the fusion protein pdRBD-peRBD is expected to be an effective subunit vaccine that can simultaneously prevent PDCoV and PEDV.

The spike protein of the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is responsible for infecting host cells. It has two segments, S1 and S2. The S1 segment has a receptor-binding domain (RBD) that attaches to the host receptor angiotensin-converting enzyme 2 (ACE2). The S2 segment helps in the fusion of the viral cell membrane by creating a six-helical bundle through the two-heptad repeat domain. To develop effective vaccines and therapeutics against COVID-19, it is critical to express and purify the SARS-CoV-2 Spike protein. Extensive studies have been conducted on expression of a complete recombinant spike protein or its fragments. This review provides an in-depth analysis of the different expression systems employed for spike protein expression, along with their advantages and disadvantages.

The Coronavirus Disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has incurred devastating human and economic losses. Vaccination remains the most effective approach for controlling the COVID-19 pandemic. Nonetheless, the sustained evolution of SARS-CoV-2 variants has provoked concerns among the scientific community regarding the development of next-generation COVID-19 vaccines. Among these, given their safety, immunogenicity, and flexibility to display varied and native epitopes, virus-like particle (VLP)-based vaccines represent one of the most promising next-generation vaccines. In this review, we summarize the advantages and characteristics of VLP platforms, strategies for antigen display, and current clinical trial progress of SARS-CoV-2 vaccines based on VLP platforms. Importantly, the experience and lessons learned from the development of SARS-CoV-2 VLP vaccines provide insights into the development of strategies based on VLP vaccines to prevent future coronavirus pandemics and other epidemics.

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) is a prevalent swine pathogen, which has caused adverse impact on the global swine industry for almost 30 years. However, due to the immune suppression caused by the virus and the genetic diversity in PRRSV, no virus-targeting broad neutralizing strategy has been successfully developed yet. Antiviral peptide and nanobody have attracted extensive attention with the ease in production and the efficacy in practice. In this study, four new fusion proteins named nanobody peptide conjugates (NPCs) were developed by combining PRRSV specific non-neutralizing nanobodies with CD163-derived peptides targeting the receptor binding domain (RBD) of PRRSV proteins.
RESULTS: Four NPCs were successfully constructed using two nanobodies against PRRSV N and nsp9 individually, recombining with two antiviral peptides 4H7 or 8H2 from porcine CD163 respectively. All four NPCs demonstrated specific capability of binding to PRRSV and broad inhibitory effect against various lineages of PRRSV in a dose-dependent manner. NPCs interfere with the binding of the RBD of PRRSV proteins to CD163 in the PRRSV pre-attachment stage by CD163 epitope peptides in the assistance of Nb components. NPCs also suppress viral replication during the stage of post-attachment, and the inhibitory effects depend on the antiviral functions of Nb parts in NPCs, including the interference in long viral RNA synthesis, NF-κB and IFN-β activation. Moreover, an interaction was predicted between aa K31 and T32 sites of neutralizing domain 4H7 of NPC-N/nsp9-4H7 and the motif 171NLRLTG176 of PRRSV GP2a. The motif 28SSS30 of neutralizing domain 8H2 of NPC-N/nsp9-8H2 could also form hydrogens to bind with the motif 152NAFLP156 of PRRSV GP3. The study provides valuable insights into the structural characteristics and potential functional implications of the RBD of PRRSV proteins. Finally, as indicated in a mouse model, NPC intranasally inoculated in vivo for 12-24 h sustains the significant neutralizing activity against PRRSV. These findings inspire the potential of NPC as a preventive measure to reduce the transmission risk in the host population against respiratory infectious agents like PRRSV.
CONCLUSIONS: The aim of the current study was to develop a peptide based bioactive compound to neutralize various PRRSV strains. The new antiviral NPC (nanobody peptide conjugate) consists of a specific nanobody targeting the viral protein and a neutralizing CD163 epitope peptide for virus blocking and provides significant antiviral activity. The study will greatly promote the antiviral drug R&D against PRRSV and enlighten a new strategy against other viral diseases.

The coronavirus disease 2019 pandemic due to severe acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2) seriously affected global public health security. Studies on vaccines, neutralizing antibodies (NAbs) and small molecule antiviral drugs are currently ongoing. In particular, NAbs have emerged as promising therapeutic agents due to their well‑defined mechanism, high specificity, superior safety profile, ease of large‑scale production and simultaneous application for both prevention and treatment of viral infection. Numerous NAb therapeutics have entered the clinical research stages, demonstrating promising therapeutic and preventive effects. These agents have been used for outbreak prevention and control under urgent authorization processes. The present review summarizes the molecular targets of SARS‑CoV‑2‑associated NAbs and screening and identification techniques for NAb development. Moreover, the current shortcomings and challenges that persist with the use of NAbs are discussed. The aim of the present review is to offer a reference for the development of NAbs for any future emergent infectious diseases, including SARS‑CoV‑2.

The Omicron variant and its sub-lineages are the only current circulating SARS-CoV-2 viruses worldwide. In this study, the conformational stability of the isolated Receptor Binding Domain (RBD) of Omicron\'s spike protein is examined in detail. The parent Omicron lineage has over ten mutations in the ACE2 binding region of the RBD that are specifically associated with its β hairpin loop domain. It is demonstrated through biophysical molecular computations that the mutations in the β hairpin loop domain significantly increase the intra-protein interaction energies of intra-loop and loop-RBD interactions. The interaction energy increases include the formation of new hydrogen bonds in the β hairpin loop domain that help stabilize this critical ACE2 binding region. Our results also agree with recent experiments on the stability of Omicron\'s core β barrel domain, outside of its loop domain, and help demonstrate the overall conformational stability of the Omicron RBD. It is further shown here through dynamic simulations that the unbound state of the Omicron RBD remains closely aligned with the bound state configuration, which was not observed for the wild-type RBD. Overall, these studies demonstrate the significantly increased conformational stability of Omicron over its wild-type configuration and raise a number of questions on whether conformational stability could be a positive selection feature of SARS-CoV-2 viral mutational changes.

Parvovirus B19 (B19V) is a human pathogen that is the causative agent of several diseases in infants and adults. Due to a lack of antivirals against this virus, treatment options are limited. The minor capsid protein of B19V has a unique N terminus, named VP1u, which is essential for infection. The VP1u encodes a receptor binding domain (RBD), necessary for host cell entry, and a phospholipase A2 (PLA2) domain, crucial for endosomal escape during cellular trafficking. Both domains are indispensable for infection, making the RBD a plausible drug target for inhibitors against B19V, as it is located on the exterior surface of the virus. To date, no experimental structural information has been available for the VP1u component for any Parvovirus. Here we report the backbone NMR resonance assignments for the RBD of B19V and demonstrate it forms a stable structure. The backbone chemical shifts are in good agreement with a structure predicted by AlphaFold, validating that the RBD contains three helices connected by tight turns. This RBD construct can now be used for further NMR studies, including assignment of full-length VP1u, determination of protein-protein interaction interfaces, and development of B19 antivirals specific to the RBD domain.

Parvovirus B19 (B19V) is a human pathogen that is the causative agent of several diseases in infants and adults. Due to a lack of antivirals against this virus, treatment options are limited. The minor capsid protein of B19V has a unique N terminus, named VP1u, which is essential for infection. The VP1u encodes a receptor binding domain (RBD), necessary for host cell entry, and a phospholipase A2 (PLA2) domain, crucial for endosomal escape during cellular trafficking. Both domains are indispensable for infection, making the RBD a plausible drug target for inhibitors against B19V, as it is located on the exterior surface of the virus. To date, no experimental structural information has been available for the VP1u component for any Parvovirus. Here we report the backbone NMR resonance assignments for the RBD of B19V and demonstrate it forms a stable structure. The backbone chemical shifts are in good agreement with a structure predicted by AlphaFold, validating that the RBD contains three helices connected by tight turns. This RBD construct can now be used for further NMR studies, including assignment of full-length VP1u, determination of protein-protein interaction interfaces, and development of B19 antivirals specific to the RBD domain. Database: BMRB submission code: 52440.