Search Results (19)

Background: Rabies is a highly fatal zoonotic disease that causes approximately 59,000 human deaths worldwide each year. Current inactivated rabies vaccines require multiple doses and are associated with high costs. The full-length rabies virus glycoprotein (RVG), a membrane protein, exhibits substantial instability in its trimeric structure during recombinant expression. This instability makes it difficult to obtain high-purity, correctly folded antigens. Objectives: This study focuses on the preparation of a full-length recombinant RVG subunit vaccine candidate expressed in a glycoengineered Pichia pastoris system with mammalian-like glycosylation. Methods: The full-length RVG gene (including the transmembrane domain and cytoplasmic tail) from the Challenge Virus Standard-11 (CVS-11) strain was codon-optimized and inserted into the pPICZαA vector to construct the recombinant expression plasmid pPICZαA-RVG. The plasmid was transformed into glycoengineered Pichia pastoris X33-7 (low-mannose type) by electroporation for inducible expression. The target protein was purified by nickel affinity chromatography, anion-exchange chromatography, and Superdex-200 size-exclusion chromatography. The structural characteristics of the purified protein were analyzed by dynamic light scattering (DLS) and transmission electron microscopy (TEM). The purified antigen was formulated with the adjuvants AS03 or MF59. BALB/c mice (n = 5 per group) were immunized intramuscularly following a four-dose schedule (days 0, 7, 14, and 28). Antigen-specific IgG antibody titers were measured by ELISA, and neutralizing antibody titers were determined using the rapid fluorescent focus inhibition test (RFFIT). Results: Glycoengineered Pichia pastoris yeast strains expressing wild-type RVG (RVG-WT) or a mutant variant (RVG-M6: R84S, R199S, H270P, R279S, K300S, and R463S) were successfully constructed. The purified RVG antigen formed nanoparticles with an average particle size of approximately 75 nm. Immunized mice generated robust RVG-specific IgG responses, with titers reaching approximately 6.31 × 10⁵ for RVG-WT after the fourth immunization, compared to 3.16 × 10³ for RVG-M6 and 5.62 × 10³ for the RVG-WT-PEG control. Two weeks after the fourth immunization, RVG-WT formulated with AS03 or MF59 induced significant neutralizing antibody responses compared with the control group (p < 0.0001 and p < 0.01, respectively). The neutralizing antibody titers reached 1:79.43 in the AS03 group and 1:33.11 in the MF59 group, whereas the WT-PEG + AS03 control group showed a low titer of 1:3.72. In contrast, RVG-M6 formulated with MF59 failed to induce detectable neutralizing antibodies (1:3.02). Furthermore, RVG-WT + AS03 induced significantly higher neutralizing antibody responses than the WT-PEG + AS03 control group (p < 0.0001), and a significant difference was also observed between the RVG-WT + MF59 and RVG-M6 + MF59 groups (p < 0.01). Conclusions: The glycoengineered Pichia pastoris expression system successfully produced uniform full-length rabies virus glycoprotein nanoparticles with high purity. When formulated with the AS03 adjuvant, RVG-WT induced high-titer neutralizing antibodies in mice, suggesting a promising strategy for the development of recombinant subunit vaccines against rabies. However, this study is limited by the absence of challenge studies and validation in target animal species, which will be further investigated in future work. Full article

The continuous evolution of SARS-CoV-2 affects its infectivity and ability to evade the immune system. The XBB.1.5 subvariant carries numerous mutations compared to previous Omicron variants and exhibits significant evasion of polyclonal neutralizing antibodies. In this study, the mechanistic effects of mutations in the XBB.1.5 spike protein on structural stability, antigenic markers, and antibody epitopes were analyzed using homology modeling, epitope prediction, protein stability analysis, coarse-grained dynamic simulations, and chain-specific interface mapping. Thirty-eight amino acid substitutions were identified relative to Wuhan-Hu-1, including 22 in the receptor-binding region. The prefusion trimeric fold was conserved, with localized rearrangements in the N-terminal domain, receptor-binding domain, and S1/S2 region. Linear B-cell epitope prediction yielded similar epitope counts and length distributions in wild-type and XBB.1.5, but only moderate residue-level overlap (Jaccard ≈ 0.40–0.62), indicating epitope turnover and alteration of four conserved antigenic determinants. Functional screening suggested that ~45% of substitutions could affect protein function. Chain-specific interface analysis of the A–B protomer interface indicated preserved inter-protomer coupling with modest repacking of the polar/directional contacts. Overall, XBB.1.5 appears to maintain ACE2 engagement while redistributing antibody targets, underscoring the need for updated vaccine formulations and therapeutic antibodies. Full article

This study focuses on biochemical pathways of complex biochemical formation, taking into account various thermodynamic parameters that change as the complexity and molecular weight of complex molecules increase. We conducted a study of the co-direction of changes in thermodynamic quantities such as $lg\left[K_d\right]$, $T\Delta S$, $\Delta (\Delta W)$, and $lg\left(\mathrm{cond}\right(W\left)\right)$ during the transition from a monomer to a dimer and then to a trimer and tetramer. In this work, we assume that the co-direction of changes in thermodynamic quantities as the final molecular formation being achieved signals a higher affinity of molecules among themselves than there is for a biochemical formation, which is characterized by the lack of coordination of the biochemical pathway directions of the final molecular compound. As the studied molecular complexes, we took [LGP2-8dsRNA-LGP2], [VP35]₂-dsRNA-[VP35]₂, and MARV NPcore proteins with peptides and the complex of MJ20 with antigens from the Bundibugyo strain of Ebola virus. Calculations of biochemical reaction paths were conducted. Full article

Nipah virus (NiV), of the Paramyxoviridae family, causes highly fatal infections in humans and is associated with severe neurological and respiratory diseases. Currently, no commercial vaccine is available for human use. Here, eight structure-based mammalian-expressed recombinant proteins harboring the NiV surface proteins, fusion glycoprotein (F), and the major attachment glycoprotein (G) were produced. Specifically, prefusion NiV-F and/or NiV-G glycoproteins expressed in monomeric, multimeric (trimeric F and tetra G), or chimeric forms were evaluated for their properties as sub-unit vaccine candidates. The antigenicity of the recombinant NiV glycoproteins was evaluated in intramuscularly immunized mice, and the antibodies in serum were assessed. Predictably, all homologous immunizations exhibited immunogenicity, and neutralizing antibodies to VSV-luciferase-based pseudovirus expressing NiV-GF glycoproteins were found in all groups. Comparatively, neutralizing antibodies were highest in vaccines designed in their multimeric structures and administered as bivalent (GMYtet + GBDtet) and trivalent (Ftri + GMYtet + GBDtet). Additionally, while all adjuvants were able to elicit an immunogenic response in vaccinated groups, bivalent (GMYtet + GBDtet) and trivalent (Ftri + GMYtet + GBDtet) induced more potent neutralizing antibodies when administered with oil-in-water nano-emulsion adjuvant, AddaS03. For all experiments, the bivalent GMYtet + GBDtet was the most immunogenic vaccine candidate. Results from this study highlight the potential use of these mammalian-expressed recombinant NiV as vaccine candidates, deserving further exploration. Full article

Efforts to develop vaccine and immunotherapeutic countermeasures against the COVID-19 pandemic focus on targeting the trimeric spike (S) proteins of SARS-CoV-2. Vaccines and therapeutic design strategies must impart the characteristics of virion S from historical and emerging variants onto practical constructs such as soluble, stabilized trimers. The virus spike is a heterotrimer of two subunits: S1, which includes the receptor binding domain (RBD) that binds the cell surface receptor ACE2, and S2, which mediates membrane fusion. Previous studies suggest that the antigenic, structural, and functional characteristics of virion S may differ from current soluble surrogates. For example, it was reported that certain anti-glycan, HIV-1 neutralizing monoclonal antibodies bind soluble SARS-CoV-2 S but do not neutralize SARS-CoV-2 virions. In this study, we used single-molecule fluorescence correlation spectroscopy (FCS) under physiologically relevant conditions to examine the reactivity of broadly neutralizing and non-neutralizing anti-S human monoclonal antibodies (mAbs) isolated in 2020. Binding efficiency was assessed by FCS with soluble S trimers, pseudoviruses and inactivated wild-type virions representing variants emerging from 2020 to date. Anti-glycan mAbs were tested and compared. We find that both anti-S specific and anti-glycan mAbs exhibit variable but efficient binding to a range of stabilized, soluble trimers. Across mAbs, the efficiencies of soluble S binding were positively correlated with reactivity against inactivated virions but not pseudoviruses. Binding efficiencies with pseudoviruses were generally lower than with soluble S or inactivated virions. Among neutralizing mAbs, potency did not correlate with binding efficiencies on any target. No neutralizing activity was detected with anti-glycan antibodies. Notably, the virion S released from membranes by detergent treatment gained more efficient reactivity with anti-glycan, HIV-neutralizing antibodies but lost reactivity with all anti-S mAbs. Collectively, the FCS binding data suggest that virion surfaces present appreciable amounts of both functional and nonfunctional trimers, with neutralizing anti-S favoring the former structures and non-neutralizing anti-glycan mAbs binding the latter. S released from solubilized virions represents a nonfunctional structure bound by anti-glycan mAbs, while engineered soluble trimers present a composite structure that is broadly reactive with both mAb types. The detection of disparate antigenicity and immunoreactivity profiles in engineered and virion-associated S highlight the value of single-virus analyses in designing future antiviral strategies against SARS-CoV-2. Full article

Immunotoxins (ITXs) are chimeric molecules that combine the specificity of a targeting domain, usually derived from an antibody, and the cytotoxic potency of a toxin, leading to the selective death of tumor cells. However, several issues must be addressed and optimized in order to use ITXs as therapeutic tools, such as the selection of a suitable tumor-associated antigen (TAA), high tumor penetration and retention, low kidney elimination, or low immunogenicity of foreign proteins. To this end, we produced and characterized several ITX designs, using a nanobody against EGFR (V_HH 7D12) as the targeting domain. First, we generated a nanoITX, combining V_HH 7D12 and the fungal ribotoxin α-sarcin (αS) as the toxic moiety (V_HHEGFRαS). Then, we incorporated a trimerization domain (TIE^XVIII) into the construct, obtaining a trimeric nanoITX (TriV_HHEGFRαS). Finally, we designed and characterized a bispecific ITX, combining the V_HH 7D12 and the scFv against GPA33 as targeting domains, and a deimmunized (DI) variant of α-sarcin (BsITXαSDI). The results confirm the therapeutic potential of α-sarcin-based nanoITXs. The incorporation of nanobodies as target domains improves their therapeutic use due to their lower molecular size and binding features. The enhanced avidity and toxic load in the trimeric nanoITX and the combination of two different target domains in the bispecific nanoITX allow for increased antitumor effectiveness. Full article

The SARS-CoV-2 pandemic commenced in 2019 and is still ongoing. Neither infection nor vaccination give long-lasting immunity and, here, in an attempt to understand why this might be, we have compared the neutralizing antibody responses to SARS-CoV-2 with those specific for human immunodeficiency virus type 1 (HIV-1) and respiratory syncytial virus (RSV). Currently, most of the antibodies specific for the SARS-CoV-2 S protein map to three broad antigenic sites, all at the distal end of the S trimer (receptor-binding site (RBD), sub-RBD and N-terminal domain), whereas the structurally similar HIV-1 and the RSV F envelope proteins have six antigenic sites. Thus, there may be several antigenic sites on the S trimer that have not yet been identified. The epitope mapping, quantitation and longevity of the SARS-CoV-2 S-protein-specific antibodies produced in response to infection and those elicited by vaccination are now being reported for specific groups of individuals, but much remains to be determined about these aspects of the host–virus interaction. Finally, there is a concern that the SARS-CoV-2 field may be reprising the HIV-1 experience, which, for many years, used a virus for neutralization studies that did not reflect the neutralizability of wild-type HIV-1. For example, the widely used VSV-SARS-CoV-2-S protein pseudotype has 10-fold more S trimers per virion and a different configuration of the trimers compared with the SARS-CoV-2 wild-type virus. Clarity in these areas would help in advancing understanding and aid countermeasures of the SARS-CoV-2 pandemic. Full article

The spread of SARS-CoV-2 and its variants leads to a heavy burden on healthcare and the global economy, highlighting the need for developing vaccines that induce broad immunity against coronavirus. Here, we explored the immunogenicity of monovalent or bivalent spike (S) trimer subunit vaccines derived from SARS-CoV-2 B.1.351 (S1-2P) or/and B.1. 618 (S2-2P) in Balb/c mice. Both S1-2P and S2-2P elicited anti-spike antibody responses, and alum adjuvant induced higher levels of antibodies than Addavax adjuvant. The dose responses of the vaccines on immunogenicity were evaluated in vivo. A low dose of 5 μg monovalent recombinant protein or 2.5 μg bivalent vaccine triggered high-titer antibodies that showed cross-activity to Beta, Delta, and Gamma RBD in mice. The third immunization dose could boost (1.1 to 40.6 times) high levels of cross-binding antibodies and elicit high titers of neutralizing antibodies (64 to 1024) prototype, Beta, Delta, and Omicron variants. Furthermore, the vaccines were able to provoke a Th1-biased cellular immune response. Significantly, at the same antigen dose, S1-2P immune sera induced stronger broadly neutralizing antibodies against prototype, Beta, Delta, and Omicron variants compared to that induced by S2-2P. At the same time, the low dose of bivalent vaccine containing S2-2P and S1-2P (2.5 μg for each antigen) significantly improved the cross-neutralizing antibody responses. In conclusion, our results showed that monovalent S1-2P subunit vaccine or bivalent vaccine (S1-2P and S2-2P) induced potent humoral and cellular responses against multiple SARS-CoV-2 variants and provided valuable information for the development of recombinant protein-based SARS-CoV-2 vaccines that protect against emerging SARS-CoV-2 variants. Full article

We investigated the spike IgG levels of HIV+ patients on antiretroviral therapy six months after they received their second dose (T2) and six months after the third dose (T3) of the BNT162b2 mRNA vaccine, as well as the influence of different levels of plasma HIV viremia of overall CD4+ cell count and nadir value on the humoral time course. One hundred eighty-four patients were enrolled. The median age was 55 years, the median CD4+ cell count was 639 cells/mm³ and the median nadir value was 258 cells/mm³. On the basis of all tests performed during the study period, persistently undetectable plasma HIV RNA (PUD) was found in 66 patients, low-level viremia (LLV) in 57 and ongoing viremia (OV) in 61. Serum levels of IgG antibodies against a trimeric S-protein antigen were tested with DiaSorin Liaison SARS-CoV-2 TrimericS IgG and the response was classified as optimal (>75th percentile), intermediate (50th–25th percentile) and low (<25th percentile). The frequencies of the three different patterns of plasma HIV viremia (PUD, LLV and OV) were comparable in patients with low, intermediate and optimal IgG response evaluated at T2, with no difference in overall CD4+ cell count or nadir count. At T3, 92.9% of patients achieved an optimal response: T2 response proved to be the most important factor in predicting T3 optimal response in patients with LLV and OV.A nadir value ≤ 330 cells/mm³ had 100% sensitivity in predicting a non-optimal response. In conclusion, we demonstrated the persistence of anti-spike IgG, with high serum levels occurring in most patients six months after the third dose of the BNT162b2 mRNA vaccine and a predictive role of humoral response at T2 in subjects with detectable plasma HIV viremia. Immunological alterations related to past immunodeficiency may persist despite immune reconstitution, and the nadir value could be a useful tool for elaborating personalized vaccine schedules. Full article

The pandemic of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has posed great threat to the world in many aspects. There is an urgent requirement for an effective preventive vaccine. The receptor binding domain (RBD), located on the spike (S) gene, is responsible for binding to the angiotensin-converting enzyme 2 (ACE2) receptor of host cells. The RBD protein is an effective and safe antigen candidate. The six-helix bundle (6HB) “molecular clamp” is a novel thermally-stable trimerization domain derived from a human immunodeficiency virus (HIV) gp41 protein segment. We selected the baculovirus system to fuse and express the RBD protein and 6HB for imitating the natural trimeric structure of RBD, named RBD-6HB. Recombinant RBD-6HB was successfully obtained from the cell culture supernatant and purified to high homogeneity. The purity of the final protein preparation was more than 97%. The results showed that the protein was identified as a homogeneous polymer. Further studies showed that the RBD-6HB protein combined with AL/CpG adjuvant could stimulate animals to produce sustained high-level antibodies and establish an effective protective barrier to protect mice from challenges. Our findings highlight the importance of trimerized SARS-CoV-2 S protein RBD in designing SARS-CoV-2 vaccines and provide a rationale for developing a protective vaccine through the induction of antibodies against the RBD domain. Full article

During the sustained COVID-19 pandemic, global mass vaccination to achieve herd immunity can prevent further viral spread and mutation. A protein subunit vaccine that is safe, effective, stable, has few storage restrictions, and involves a liable manufacturing process would be advantageous to distribute around the world. Here, we designed and produced a recombinant spike (S)-Trimer that is maintained in a prefusion state and exhibits a high ACE2 binding affinity. Rodents received different doses of S-Trimer (0.5, 5, or 20 μg) antigen formulated with aluminum hydroxide (Alum) or an emulsion-type adjuvant (SWE), or no adjuvant. After two vaccinations, the antibody response, T-cell responses, and number of follicular helper T-cells (Tfh) or germinal center (GC) B cells were assessed in mice; the protective efficacy was evaluated on a Syrian hamster infection model. The mouse studies demonstrated that adjuvating the S-Trimer with SWE induced a potent humoral immune response and Th1-biased cellular immune responses (in low dose) that were superior to those induced by Alum. In the Syrian hamster studies, when S-Trimer was adjuvanted with SWE, higher levels of neutralizing antibodies were induced against live SARS-CoV-2 from the original lineage and against the emergence of variants (Beta or Delta) with a slightly decreased potency. In addition, the SWE adjuvant demonstrated a dose-sparing effect; thus, a lower dose of S-Trimer as an antigen (0.5 μg) can induce comparable antisera and provide complete protection from viral infection. These data support the utility of SWE as an adjuvant to enhance the immunogenicity of the S-Trimer vaccine, which is feasible for further clinical testing. Full article

The COVID-19 pandemic, which began at the end of 2019 in Wuhan, has affected 220 countries and territories to date. In the present study, we studied humoral immunity in samples of the blood sera of COVID-19 convalescents of varying severity and patients who died due to this infection, using native SARS-CoV-2 and its individual recombinant proteins. The cross-reactivity with SARS-CoV (2002) was also assessed. We used infectious and inactivated SARS-CoV-2/human/RUS/Nsk-FRCFTM-1/2020 strain, inactivated SARS-CoV strain (strain Frankfurt 1, 2002), recombinant proteins, and blood sera of patients diagnosed with COVID-19. The blood sera from patients were analyzed by the Virus Neutralization test, Immunoblotting, and ELISA. The median values and mean ± SD of titers of specific and cross-reactive antibodies in blood sera tested in ELISA were mainly distributed in the following descending order: N > trimer S > RBD. ELISA and immunoblotting revealed a high cross-activity of antibodies specific to SARS-CoV-2 with the SARS-CoV antigen (2002), mainly with the N protein. The presence of antibodies specific to RBD corresponds with the data on the neutralizing activity of blood sera. According to the neutralization test in a number of cases, higher levels of antibodies that neutralize SARS-CoV-2 were detected in blood serum taken from patients several days before their death than in convalescents with a ranging disease severity. This high level of neutralizing antibodies specific to SARS-CoV-2 in the blood sera of patients who subsequently died in hospital from COVID-19 requires a thorough study of the role of humoral immunity as well as comorbidity and other factors affecting the humoral response in this disease. Full article

The ongoing coronavirus disease 2019 (COVID-19) pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Most of the currently approved SARS-CoV-2 vaccines use the prototype strain-derived spike (S) protein or its receptor-binding domain (RBD) as the vaccine antigen. The emergence of several novel SARS-CoV-2 variants has raised concerns about potential immune escape. In this study, we performed an immunogenicity comparison of prototype strain-derived RBD, S1, and S ectodomain trimer (S-trimer) antigens and evaluated their induction of neutralizing antibodies against three circulating SARS-CoV-2 variants, including B.1.1.7, B.1.351, and B.1.617.1. We found that, at the same antigen dose, the RBD and S-trimer vaccines were more potent than the S1 vaccine in eliciting long-lasting, high-titer broadly neutralizing antibodies in mice. The RBD immune sera remained highly effective against the B.1.1.7, B.1.351, and B.1.617.1 variants despite the corresponding neutralizing titers decreasing by 1.2-, 2.8-, and 3.5-fold relative to that against the wild-type strain. Significantly, the S-trimer immune sera exhibited comparable neutralization potency (less than twofold variation in neutralizing GMTs) towards the prototype strain and all three variants tested. These findings provide valuable information for further development of recombinant protein-based SARS-CoV-2 vaccines and support the continued use of currently approved SARS-CoV-2 vaccines in the regions/countries where variant viruses circulate. Full article

Stabilization of the HIV-1 Envelope glycoprotein trimer (Env) in its native pre-fusion closed conformation is regarded as one of several requirements for the induction of neutralizing antibody (nAb) responses, which, in turn, will most likely be a prerequisite for the development of an efficacious preventive vaccine. Here, we systematically analyzed how the stepwise stabilization of a clade C consensus (ConC) Env immunogen impacts biochemical and biophysical protein traits such as antigenicity, thermal stability, structural integrity, and particle size distribution. The increasing degree of conformational rigidification positively correlates with favorable protein characteristics, leading to optimized homogeneity of the protein preparations, increased thermal stability, and an overall favorable binding profile of structure-dependent broadly neutralizing antibodies (bnAbs) and non-neutralizing antibodies (non-nAbs). We confirmed that increasing the structural integrity and stability of the Env trimers positively correlates with the quality of induced antibody responses by the immunogens. These and other data contribute to the selection of ConCv5 KIKO as novel Env immunogens for use within the European Union’s H2020 Research Consortium EHVA (European HIV Alliance) for further preclinical analysis and phase 1 clinical development. Full article

In eukaryotic cells, DNA replication licensing is precisely regulated to ensure that the initiation of genomic DNA replication in S phase occurs once and only once for each mitotic cell division. A key regulatory mechanism by which DNA re-replication is suppressed is the S phase-dependent proteolysis of Cdt1, an essential replication protein for licensing DNA replication origins by loading the Mcm2-7 replication helicase for DNA duplication in S phase. Cdt1 degradation is mediated by CRL4^Cdt2 ubiquitin E3 ligase, which further requires Cdt1 binding to proliferating cell nuclear antigen (PCNA) through a PIP box domain in Cdt1 during DNA synthesis. Recent studies found that Cdt2, the specific subunit of CRL4^Cdt2 ubiquitin E3 ligase that targets Cdt1 for degradation, also contains an evolutionarily conserved PIP box-like domain that mediates the interaction with PCNA. These findings suggest that the initiation and elongation of DNA replication or DNA damage-induced repair synthesis provide a novel mechanism by which Cdt1 and CRL4^Cdt2 are both recruited onto the trimeric PCNA clamp encircling the replicating DNA strands to promote the interaction between Cdt1 and CRL4^Cdt2. The proximity of PCNA-bound Cdt1 to CRL4^Cdt2 facilitates the destruction of Cdt1 in response to DNA damage or after DNA replication initiation to prevent DNA re-replication in the cell cycle. CRL4^Cdt2 ubiquitin E3 ligase may also regulate the degradation of other PIP box-containing proteins, such as CDK inhibitor p21 and histone methylase Set8, to regulate DNA replication licensing, cell cycle progression, DNA repair, and genome stability by directly interacting with PCNA during DNA replication and repair synthesis. Full article