Search Results (77)

Background: Trimeric envelope-proximal domains in viral class I fusion proteins are conserved targets of broadly neutralizing antibodies (bNAbs), but it has proven difficult to develop vaccines against those targets. The HIV-1 gp41 membrane-proximal external region (MPER) is one such target. Induction of a neutralizing response likely depends on the immunogen having a close-to-native structure. Methods: Native sequence MPER was displayed on genome-reduced bacteria as a coiled-coil homotrimer using a Haemophilus influenzae Hia trimeric autotransporter. Vaccine designs incorporated additional features, including trimerization domains to stabilize MPER, tandem MPER repeats to increase antigen valency, and immunomodulatory elements. Antigen exposure was assessed by flow cytometry, antibody responses were evaluated by ELISA, and functional activity was measured using HIV-1 pseudovirus neutralization assays. Results: Trimer stabilization improved MPER exposure, but immunogen visibility alone did not predict neutralization. After three immunizations, neutralizing activity was detected only in the most extensively engineered vaccine, which neutralized tier 2 virus CNE55. After five immunizations, the same vaccine also neutralized the tier 2 virus 25710-2.43. A further design modification that included an extended Hia-derived spacer increased MPER exposure and antibody binding, with neutralization detected against MN.3, X1632_S2_B10, and 25710-2.43 viruses in subsets of animals. Conclusions: As a proof-of-concept, native-sequence MPER can induce detectable, though modest and virus-dependent, HIV-1 neutralizing activity when displayed in a carefully controlled trimeric bacterial surface-display platform. The results show that MPER vaccine performance depends not only on antigen exposure, but also on multimeric organization, immunomodulatory context, and antigen-scaffold geometry. Analogous coiled-coil trimeric bacterial surface display immunogens may inform vaccine development for stem/stalk regions of other Class I fusion protein viruses. Full article

Nanoplastic particles are increasingly detected in aquatic environments, yet whether cellular stress responses persist after exposure cessation remains unclear. Here, Lateolabrax maculatus rostral (LMR) cells from spotted sea bass were continuously exposed for up to 30 passages (105 days) to nominal 20 nm carboxylated fluorescent polystyrene particles (20 μg/mL), followed by 10 particle-free passages (recovery). Long-term exposure was associated with reduced proliferation and pronounced changes in cell surface morphology and ultrastructure. Although NP-associated fluorescence became undetectable during recovery, several nuclear-associated alterations persisted throughout the 10-passage recovery period, including TEM-observed nuclear-envelope alterations, filamentous actin (F-actin) reorganization, and sustained elevation of phosphorylated H2AX (γ-H2AX) foci. Several nuclear pore complex (NPC) genes were downregulated during exposure and rebounded after particle removal, whereas the nuclear-to-cytoplasmic distribution of proliferating cell nuclear antigen (PCNA) remained shifted. Together, these findings indicate that prolonged exposure to this nanoscale polystyrene particle formulation was associated with nuclear stress responses that did not fully resolve within the 10-passage recovery window in fish cells. Because bulk-polymer and chemical-extract (leachate) controls were not included, nanoscale-specific effects cannot be distinguished from contributions of polymer chemistry, surface functionalization, fluorescent dye, or other formulation-related effects. Full article

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: Vibrio harveyi is a major bacterial pathogen threatening turbot aquaculture, necessitating the development of more effective vaccines. Bacterial ghosts (BGs), which are empty bacterial envelopes with preserved surface antigens, offer a promising alternative to traditional formaldehyde-killed vaccines that often suffer from reduced immunogenicity. Methods: We developed an optimized BGs vaccine for V. harveyi by combining the nonionic surfactant NP-40 with sodium hydroxide (NaOH). This NP-40/NaOH combination demonstrated a synergistic lytic effect, halving the minimum inhibitory concentration of NaOH required for complete inactivation. Results: The resulting BGs exhibited intact cellular morphology with transmembrane pores, efficient removal of cytoplasmic contents, and significantly better preservation of lipopolysaccharide structure compared to NaOH-alone treatment. Vaccination trials in turbot demonstrated that the NP-40/NaOH BGs provided the highest relative percent survival (RPS = 58.8%) upon challenge, outperforming both NaOH-alone BGs (RPS = 55.0%) and a traditional formaldehyde-killed vaccine (RPS = 34.8%). The superior protection was correlated with the induction of a more robust and sustained immune response, characterized by significantly higher levels of specific IgM antibodies, elevated lysozyme activity, and increased total serum protein. Conclusions: This study establishes the NP-40/NaOH protocol as an effective strategy for producing high-quality BGs with enhanced immunogenicity, presenting a potent vaccine candidate for controlling vibriosis in aquaculture. Full article

Background/Objectives: Ocular graft-versus-host disease (oGVHD) is a chronic, immune-mediated complication of allogeneic hematopoietic stem cell transplantation that can progress to corneal ulceration or perforation. These cases are often refractory to standard therapy and present a high risk of graft failure after keratoplasty. We report a case of oGVHD-related corneal perforation successfully managed with a novel amniotic membrane-assisted “envelope” technique during corneal transplantation. Case Report: A 42-year-old man with chronic oGVHD and a full-thickness corneal perforation underwent urgent repair with a lamellar patch graft completely wrapped in cryopreserved amniotic membrane, followed by penetrating keratoplasty (PKP) using an amniotic membrane envelope surrounding the donor lenticule. Results: The amniotic membrane provided a 360° biological barrier that isolated graft antigens from the inflammatory environment while supporting epithelial healing and stromal remodeling. Despite recurrent inflammatory episodes and multiple procedures—including cataract extraction, pars plana vitrectomy, and multilayer amniotic membrane transplantation—the graft remained clear and stable at 12-month follow-up, achieving a best-corrected visual acuity of 20/40. Conclusions: The amniotic membrane envelope technique may represent a valuable adjunct in managing high-risk corneal perforations secondary to oGVHD. By combining immune modulation and regenerative support, this approach can enhance tectonic stability, reduce rejection risk, and promote durable surface recovery, potentially delaying or avoiding keratoprosthesis in refractory cases. Full article

The HIV-1 envelope glycoprotein gp120 is prominently exposed on the surface of both HIV-1 virions and infected host cells, serving as a key marker of infection. gp120 plays a pivotal role in viral entry by interacting with the primary receptor, CD4, on host cells. Therapeutic strategies targeting the HIV-1 reservoir, such as anti-gp120 antibodies that trigger antibody-dependent cellular cytotoxicity (ADCC) and chimeric antigen receptor T (CAR-T) cells, rely on the presence of gp120 on the surface of infected cells to exert their effects. Consequently, accurate monitoring of gp120 expression on infected cells is essential for evaluating the pharmacological efficacy of these interventions. In this study, a sensitive, specific, and inexpensive enzyme-linked immunosorbent assay (ELISA) for quantifying HIV-1 gp120 glycoprotein was developed using a selected pair of anti-gp120 antibodies. The assay achieved a lower limit of quantitation (LLOQ) of 0.16 pM, demonstrating sensitivity comparable to that of the digital single molecule array (Simoa) platform, which exhibited a LLOQ of 0.23 pM and requires specialized instrumentation. The binding specificity of the antibodies used in the novel assay was confirmed using liquid chromatography–mass spectrometry (LC-MS), and the assay was pharmacologically validated with lysates obtained from 2D10 and MOLT IIIB cell lines. Furthermore, treatment of HIV-infected human primary CD4⁺ T cells with a targeted activator of cell kill (TACK) compound significantly reduced gp120 concentration in CD4⁺ T cell lysate compared to controls. The gp120 marker from infected cell lysates correlated with the number of gp120-positive cells detected by immunocytochemistry, as well as with HIV-1 p24 levels and cell-associated viral RNA measurements. In summary, a novel, simple, and sensitive HIV-1 gp120 ELISA has been developed and validated. This assay holds potential for investigating HIV-1 persistence and evaluating the efficacy of therapeutic agents targeting infected cells. Full article

Background: Human Metapneumovirus (HMPV) is a respiratory virus in the Pneumoviridae family. HMPV is an enveloped, negative-sense RNA virus encoding three surface proteins: SH, G, and F. The highly immunogenic fusion (F) protein is essential for viral entry and a key target for vaccine development. The F protein exists in two conformations: prefusion and postfusion. The prefusion form is highly immunogenic and considered a potent vaccine antigen. However, this conformation needs to be stabilized to improve its immunogenicity for effective vaccine development. Specific mutations are necessary to maintain the prefusion state and prevent it from changing to the postfusion form. Methods: In silico mutagenesis was performed on the C-terminal domain of the pre-F protein, focusing on five amino acids at positions 469 to 473 (LVDQS), using the established pre-F structure (PDB: 8W3Q) as the reference. The amino acid sequence was sequentially mutated based on hydrophobicity, resulting in mutants M1 (IIFLL), M2 (LLIVL), M3 (WWVLL), and M4 (YMWLL). Increasing hydrophobicity was found to enhance protein stability and structural rigidity. Results: Epitope mapping revealed that all mutants displayed significant B and T cell epitopes similar to the reference protein. The structure and stability of all mutants were analyzed using molecular dynamics simulations, free energy calculations, and secondary structure analysis. Based on the lowest RMSD, clash score, MolProbity value, stable radius of gyration, and low RMSF, the M1 mutant demonstrated superior structural stability. Conclusions: Our findings indicate that the M1 mutant of the pre-F protein could be the most stable and structurally accurate candidate for vaccine development against HMPV. Full article

Historically, DENV-4 has been rarely associated with epidemics and has been less well-studied than DENV-1 to -3. Epidemic dengue struck several South and Central American countries in 2022, with Nicaragua reporting the highest incidence. In an acute febrile illness (AFI) cohort enrolled from June to September 2022, 58 (34%) of 172 patients had PCR-confirmed dengue, of which 46 (79%) were serotyped as DENV-4. In this cohort, acute dengue, as a proportion of AFI, increased from 8% in June to a peak of 58% in August. Genome sequencing and phylogenetic analysis identified a lineage of DENV-4 Genotype IIb (GIIb) with six amino acid substitutions on the surface-exposed regions of the envelope (E) protein as compared to a reference sequence from 2005. Indeed, two of these mutations appear to be novel and located at G172E or near N174K, an antigenic epitope on domain I. Most (90%, 43/48) DENV-4 patients had pre-existing DENV IgG (secondary dengue), at the acute phase. Secondary dengue was associated with the male sex (prevalence ratio (PR)), 6.88) and being younger than 11 years of age (PR, 8.38). Further analysis showed no association between past Zika exposure and DENV-4 acute illness in older subjects (≥12 years of age). In conclusion, our study describes an epidemic of DENV-4 in León, Nicaragua, associated with a novel lineage of genotype GIIb, which contains two amino acid changes not observed in DENV-4 before 2022. Full article

The attractiveness of OMVs derived from Gram-negative bacteria lies in the fact that they have two biomembranes sandwiching a peptidoglycan layer. It is well known that the envelope of OMVs consists of the outer bacterial membrane [OM] and not of the inner one [IM] of the source bacterium. This implies that all outer membranous molecules found in the OM act as antigens. However, under specific conditions, some of the inner membrane proteins can be exported into the outer membrane layer and perform as antigens. A key information was that the used purification procedures for OMVs, the induction methods to increase the production of OMVs as well as the specific mutant strains obtained via genetic engineering affect the composition of potential antigens on the surface and in the lumen of the OMVs. The available literature allowed us to list the major antigens that could be defined on OMVs. The functions of the antigens within the source bacterium are discussed for a better understanding of the various available hypotheses on the biogenesis of vesicle formation. Also, the impacts of OMV antigens on the immune system using animal models are assessed. Furthermore, information on the pathways of OMVs entering the host cell is presented. An example of a bacterial infection that causes epidemic diseases, namely via Neisseria meningitidis, is used to demonstrate that OMVs derived from this pathogen elicit protective immune responses when administered as a vaccine. Furthermore, information on OMV vaccines under development is presented. The assembled knowledge allowed us to formulate a number of reasons why OMVs are attractive as vaccine platforms, as their undesirable side effects remain small, and to provide an outlook on the potential use of OMVs as a vaccine platform. Full article

Background and Objectives: The measurement of hepatitis B surface antigen (HBsAg) is essential for managing chronic hepatitis B virus infection (CHB). HBsAg consists of three different surface envelope proteins: large, middle, and small HB surface proteins. However, in clinical practice, it is not common to evaluate each of these HB surface proteins separately. Materials and Methods: In this study, we investigated preS1 expression using seven monoclonal antibodies (mAbs) in 68 CHB patients, as well as examining their antigenicity. Results: Although the seven mAbs had been derived from genotype (Gt) C, they could recognize preS1 with Gts A to D. The epitopes were concentrated within the aa33-47 region of preS1, and their antigenicity was significantly reduced by an aa45F substitution. We found that preS1 expression remained consistent regardless of HBsAg levels and different Gts in CHB patients, in contrast to what was observed in SHBs. Conclusions: These results suggest that the antigenic epitope is preserved among different Gts and that the expression pattern of preS1 is altered during CHB, highlighting its vital role in the HBV infection cycle. Our present results suggest preS1 is a promising therapeutic target in CHB. Full article

T help (Th), stimulation of toll-like receptors (pathogen-associated molecular patterns, PAMPs), and antigen organization and repetitiveness (pathogen-associated structural patterns, PASPs) were shown numerous times to be important in driving B-cell and antibody responses. In this study, we dissected the individual contributions of these parameters using newly developed “Immune-tag” technology. As model antigens, we used eGFP and the third domain of the dengue virus 1 envelope protein (DV1 EDIII), the major target of virus-neutralizing antibodies. The respective proteins were expressed alone or genetically fused to the N-terminal fragment of the cucumber mosaic virus (CMV) capsid protein—nCMV, rendering the antigens oligomeric. In a step-by-step manner, RNA was attached as a PAMP, and/or a universal Th-cell epitope was genetically added for additional Th. Finally, a PASP was added to the constructs by displaying the antigens highly organized and repetitively on the surface of CMV-derived virus-like particles (CuMV VLPs). Sera from immunized mice demonstrated that each component contributed stepwise to the immunogenicity of both proteins. All components combined in the CuMV VLP platform induced by far the highest antibody responses. In addition, the DV1 EDIII induced high levels of DENV-1-neutralizing antibodies only if displayed on VLPs. Thus, combining multiple cues typically associated with viruses results in optimal antibody responses. Full article

The human hepatitis delta virus (HDV) is a satellite RNA virus that depends on hepatitis B virus (HBV) surface proteins (HBsAg) to assemble into infectious virions targeting the same organ (liver) as HBV. Until recently, the evolutionary origin of HDV remained largely unknown. The application of bioinformatics on whole sequence databases lead to discoveries of HDV-like agents (DLA) and shed light on HDV’s evolution, expanding our understanding of HDV biology. DLA were identified in heterogeneous groups of vertebrates and invertebrates, highlighting that the evolution of HDV, represented by eight distinct genotypes, is broader and more complex than previously foreseen. In this study, we focused on the characterization of three mammalian DLA discovered in woodchuck (Marmota monax), white-tailed deer (Odocoileus virginianus), and lesser dog-like bat (Peropteryx macrotis) in terms of replication, cell-type permissiveness, and spreading pathways. We generated replication-competent constructs expressing 1.1-fold over-length antigenomic RNA of each DLA. Replication was initiated by transfecting the cDNAs into human (HuH7, HeLa, HEK293T, A549) and non-human (Vero E6, CHO, PaKi, LMH) cell lines. Upon transfection and replication establishment, none of the DLA expressed a large delta antigen. A cell division-mediated viral amplification assay demonstrated the capability of non-human DLA to replicate and propagate in hepatic and non-hepatic tissues, without the requirement of envelope proteins from a helper virus. Remarkably L-HDAg but not S-HDAg from HDV can artificially mediate envelopment of WoDV and DeDV ribonucleoproteins (RNPs) by HBsAg to form infectious particles, as demonstrated by co-transfection of HuH7 cells with the respective DLA expression constructs and a plasmid encoding HBV envelope proteins. These chimeric viruses are sensitive to HDV entry inhibitors and allow synchronized infections for comparative replication studies. Our results provide a more detailed understanding of the molecular biology, evolution, and virus–host interaction of this unique group of animal viroid-like agents in relation to HDV. Full article

Currently, no effective vaccine to prevent human immunodeficiency virus (HIV) infection is available, and various platforms are being examined. The vesicular stomatitis virus (VSV) vaccine vehicle can induce robust humoral and cell-mediated immune responses, making it a suitable candidate for the development of an HIV vaccine. Here, we analyze the protective immunological impacts of recombinant VSV vaccine vectors that express chimeric HIV Envelope proteins (Env) in rhesus macaques. To improve the immunogenicity of these VSV-HIV Env vaccine candidates, we generated chimeric Envs containing the transmembrane and cytoplasmic tail of the simian immunodeficiency virus (SIV), which increases surface Env on the particle. Additionally, the Ebola virus glycoprotein was added to the VSV-HIV vaccine particles to divert tropism from CD4 T cells and enhance their replications both in vitro and in vivo. Animals were boosted with DNA constructs that encoded matching antigens. Vaccinated animals developed non-neutralizing antibody responses against both the HIV Env and the Ebola virus glycoprotein (EBOV GP) as well as systemic memory T-cell activation. However, these responses were not associated with observable protection against simian-HIV (SHIV) infection following repeated high-dose intra-rectal SHIV SF162p3 challenges. Full article

We sought to determine the long-term outcomes of chronic hepatitis B (CHB) cases switching to tenofovir alafenamide (TAF, n = 104, median age = 63.5 years). Data at switching to TAF (baseline) and those at 1, 2, 3, 4, and 5 years from switching to TAF were compared. At baseline, HB envelop antigen (HBeAg) seropositivity was found in 20 patients (19.2%), and undetectable HBV-DNA in 77 patients (74.0%). Percentage of detectable HBV-DNA significantly reduced at any time point. HB surface antigen (HBsAg) levels significantly reduced at 3, 4, and 5 years. The percentage of HBeAg seropositivity significantly reduced at 5 years. HB core related antigen levels did not significantly change. In patients with baseline HbeAg seropositivity, HbsAg levels significantly reduced at any time point, and a similar trend was found in patients without HBeAg seropositivity. In patients with baseline FIB4 index >1.85, HBsAg levels significantly reduced at 3, 4, and 5 years, and in patients with baseline FIB4 index <1.85, HBsAg levels significantly reduced at any time point. The estimated glomerular filtration rate significantly reduced only at 5 years. The discontinuation rate owing to the side effects of TAF was 0%. In conclusion, switching to TAF therapy in patients with CHB may be effective and safe at least up to 5 years. Full article

Extracellular vesicles (EVs) are small membrane-enclosed structures that have gained much attention from researchers across varying scientific fields in the past few decades. Cells secrete diverse types of EVs into the extracellular milieu which include exosomes, microvesicles, and apoptotic bodies. These EVs play a crucial role in facilitating intracellular communication via the transport of proteins, lipids, DNA, rRNA, and miRNAs. It is well known that a number of viruses hijack several cellular pathways involved in EV biogenesis to aid in their replication, assembly, and egress. On the other hand, EVs can also trigger host antiviral immune responses by carrying immunomodulatory molecules and viral antigens on their surface. Owing to this intricate relationship between EVs and viruses, intriguing studies have identified various EV-mediated viral infections and interrogated how EVs can alter overall viral spread and longevity. This review provides a comprehensive overview on the EV-virus relationship, and details various modes of EV-mediated viral spread in the context of clinically relevant enveloped and non-enveloped viruses. Full article