Search Results (82)

Glycans on the surface of all immune cells are the product of diverse post-translational modifications (glycosylation) that affect almost all proteins and possess enormous structural heterogeneity. Their bioinformational content is decoded by glycan-binding proteins (lectins, GBPs), such as C-type lectins, including selectins, galectins, and Siglecs. Glycans located on the surface of immune cells are involved in many immunological processes through interactions with GBPs. Lectins recognize changes in the glycan epitopes; distinguish among host (self), microbial (non-self), and tumor (modified self) antigens; and consequently regulate immune responses. Understanding GBP–glycan interactions accelerates the development of glycan-targeted therapeutics in severe diseases, including inflammatory and autoimmune diseases and cancer. This review will discuss N- and O-glycosylations and glycosyltransferases involved in the biosynthesis of carbohydrate epitopes and address how interactions between glycan epitopes and GBPs are crucial in immune responses. The pivotal role of the glycan antigen tetrasaccharide sialyl Lewis x in mediating immune and tumor cell trafficking into the extravascular site will be discussed. Next, the role of glycans in modulating bacterial, fungal, viral, and parasitic infections and cancer will be surveyed. Finally, the role of glycosylation in antibodies and carbohydrate vaccines will be analyzed. Full article

Background/Objectives: In the past, FhSAP-2, an 11.5 kDa recombinant protein belonging to the Fasciola hepatica saposin-like/NK-lysin family, has been shown to induce over 60% partial protection in immunized rabbits and mice when challenged with F. hepatica metacercariae. However, despite FhSAP-2 being a promising vaccine candidate, its hydrophobic nature has made its purification a challenging process. The present study aimed to determine whether a modified 9.8 kDa variant of protein (mFhSAP-2), lacking a string of 16 hydrophobic amino acids at the amino terminus and a dominant Th1 epitope, could retain its immunogenic and Th1-inducing properties. Methods: RAW264.7 cells were stimulated with mFhSAP-2, and TNFα levels were determined. C57BL/6 mice were immunized with mFhSAP-2 alone or emulsified with Montanide ISA50. Total anti-mFhSAP-2 IgG subtypes, along with their avidity and titers, were measured using ELISA. The T cell proliferation index and levels of CD4+/CD8+ and IFNγ/IL-4 ratios were determined. Results: In vitro, mFhSAP-2 induced dose-dependent TNFα production in RAW264.7 cells. In vivo, mice immunized with mFhSAP-2 or mFhSAP-2+ISA50 developed high-avidity IgG2a and IgG2c antibodies at levels that were significantly higher than IgG1 antibody levels. However, the mFhSAP-2+ISA50 formulation induced higher and more homogenous antibody titers than mFhSAP-2, suggesting that an adjuvant may be required to enhance mFhSAP-2 immunogenicity. Immunization with mFhSAP-2+ISA50 also induced significantly higher activated CD4+/CD8+ T cell ratios and IFNγ/IL-4 ratios compared to naïve mice. Conclusions: Our results demonstrate that mFhSAP-2 retained its immunogenicity and Th1-polarizing properties, which were enhanced by the Montanide ISA50 adjuvant. The present study highlights the feasibility of inducing Th1-associated immune responses in mice using mFhSAP-2 as an antigen. Further studies are required to assess the potential application of the mFhSAP-2+ISA50 formulation as a vaccine against F. hepatica in natural hosts such as cattle and sheep, which could contribute to improved control and aid in the prevention and eradication of F. hepatica infection. Full article

Background/Objectives: The revolution for the treatment of melanoma came with the approval of checkpoint blockade antibodies. However, a substantial proportion of patients show primary or secondary resistance to this type of immunotherapy, indicating the need for alternative therapeutic strategies. Dendritic cells (DCs) of the skin are prime targets for vaccination approaches due to their potential to prime naïve T cells and their accessibility. This study aimed to develop and evaluate novel vaccines targeting the C-type lectin receptor DEC-205 to deliver melanoma-associated antigenic peptides to skin DCs. Methods: We cloned MHC-I-restricted peptides from the glycoprotein (gp)100_25–33 and Tyrosinase-related protein (trp)2_180–188 into the DEC-205 antibody sequence with modified peptide cutting sites from the OVA_257–264 SIINFEKL peptide. We tested their potential to induce CD8⁺ T cell responses in both in vitro and in vivo settings. Tumor growth inhibition was evaluated in the transplantable B16.OVA melanoma murine model using a multi-epitope DC-based vaccine combining both peptides. Results: The cross-presentation of both gp100 and trp2 peptides was confirmed in vivo when peptide sequences were flanked by the OVA_257–264 peptide cutting sites. Moreover, the combination of both antigenic peptides into a multi-epitope DC vaccine was required to inhibit B16.OVA melanoma growth. Conclusions: Our findings suggest that a DC-targeted vaccination approach using multiple epitopes deriving from melanoma antigens could represent a promising strategy for melanoma therapy. Full article

Background/Objectives: Porcine reproductive and respiratory syndrome virus (PRRSV) is classified into various lineages based on the phylogenetic variation of orf5, which encodes a major surface glycoprotein GP5 containing both neutralizing and non-neutralizing linear epitopes. Several positively selected sites have been identified on the GP5 ectodomain, indicating host immune pressure on these sites. This present study aimed to investigate the kinetics of antibody responses to GP5 and to map the epitope-specific response to the GP5 ectodomain from different PRRSV lineages after vaccination with commercially available modified live virus (MLV) vaccines. Methods: Post-weaning pigs were vaccinated with MLV vaccines derived from either lineage 1D (Prevacent PRRS^®) or lineage 5 (Ingelvac PRRS^®). Animals were challenged with a heterologous (lineage 1A) strain at 64 days post-vaccination (dpv). Blood samples were collected at various times post-vaccination and challenge. Kinetics of antibody response to different PRRSV antigens were monitored and virus neutralization against archetypal and contemporary strains belonging to lineage 5 and 1A were evaluated. In addition, antibody responses to peptides derived from the GP5 ectodomain of different viral lineages were assessed. Results: Our results showed that the GP5-specific antibody response observed between 18 and 35 dpv was delayed compared to responses to the viral nucleocapsid protein. The polyclonal antibody response in both vaccinated groups showed similar levels of binding to variant GP5 peptides from different sub-lineages. Notably, in both vaccinated groups, the antibody directed to a peptide representing the GP5 ectodomain of a lineage 1C strain (variant 1C.5) displayed a rise in titer at 64 dpv, which was further increased by the challenge with the lineage 1A strain. Less than 50% of animals developed heterologous neutralizing antibodies post-vaccination with both MLV vaccines. However, higher neutralization titers were observed in all vaccinated animal post-challenge. Conclusions: Together, these data provide insights into the antibody responses to the GP5 ectodomain in MLV-vaccinated swine herds. Full article

Fluorination of carbohydrates is a promising strategy to produce glycomimetics with improved pharmacological properties, such as increased metabolic stability, bioavailability and protein-binding affinity. Fluoroglycans are not only of interest as inhibitors and chemical probes but are increasingly being used to develop potential synthetic vaccine candidates for cancer, HIV and bacterial infections. Despite their attractiveness, the synthesis of fluorinated oligosaccharides is still challenging, emphasizing the need for efficient protocols that allow for the site-specific incorporation of fluorine atoms (especially at late stages of the synthesis). This is particularly true for the development of fully synthetic vaccine candidates, whose (modified) carbohydrate antigen structures (glycotopes) per se comprise multistep synthesis routes. Based on a known minimal protective epitope from the capsular polysaccharide of S. pneumoniae serotype 8, a panel of six novel F-glycotope mimetics was synthesized, equipped with amine linkers for subsequent conjugation to immunogens. Next to the stepwise assembly via fluorinated building blocks, the corresponding 6F-substituted derivatives could be obtained by microwave-assisted, nucleophilic late-stage fluorination of tri- and tetrasaccharidic precursors in high yields. The described synthetic strategy allowed for preparation of the targeted fluorinated oligosaccharides in sufficient quantities for future immunological studies. Full article

Background/Objectives: Incidence of vector-borne diseases, including rickettsioses and anaplasmosis, has been increasing in many parts of the world. The obligate intracellular nature of rickettsial pathogens has hindered the development of robust genetic tools for the study of gene function and the identification of therapeutic targets. Transposon mutagenesis has contributed to recent progress in the identification of virulence factors in this important group of pathogens. Methods: Combining the efficiency of the himar1 transposon method with a recombinase-mediated system, we aimed to develop a genetic tool enabling the exchange of the transposon with a cassette encoding non-native sequences. Results: This approach was used in Rickettsia parkeri to insert a himar1 transposon encoding fluorescent protein and antibiotic resistance genes for visualization and selection, flanked by mismatched loxP sites to enable subsequent recombinase-mediated cassette exchange (RMCE). RMCE mediated by a plasmid-encoded Cre recombinase was then employed to replace the transposon with a different cassette containing alternate fluorescent and selection markers and epitopes of Anaplasma phagocytophilum antigens. The resulting genetically modified R. parkeri was trialed as a live-attenuated vaccine against spotted fever rickettsiosis and anaplasmosis in mice. Conclusions: The use of this system provides a well-established and relatively efficient way of inserting non-native sequences into the rickettsial genome, with applications for the study of gene function and vaccine development. Full article

This study aimed to create a new recombinant virus by modifying the EV-A71 capsid protein, serving as a useful tool and model for studying human Enteroviruses. We developed a new screening method using EV-A71 pseudovirus particles to systematically identify suitable insertion sites and tag types in the VP1 capsid protein. The pseudovirus’s infectivity and replication can be assessed by measuring postinfection luciferase signals. We reported that the site after the 100th amino acid within the VP1 BC loop of EV-A71 is particularly permissive for the insertion of various tags. Notably, the introduction of S and V5 tags at this position had minimal effect on the fitness of the tagged pseudovirus. Furthermore, recombinant infectious EV-A71 strains tagged with S and V5 epitopes were successfully rescued, and the stability of these tags was verified. Computational analysis suggested that viable insertions should be compatible with capsid assembly and receptor binding, whereas non-viable insertions could potentially disrupt the capsid’s binding with heparan sulfate. We expect the tagged recombinant EV-A71 to be a useful tool for studying the various stages of the enterovirus life cycle and for virus purification, immunoprecipitation, and research in immunology and vaccine development. Furthermore, this study serves as a proof of principle and may help develop similar tags in enteroviruses, for which there are fewer available tools. Full article

Intranasal immunization is one of the most effective methods for eliciting lung mucosal immunity. Multiple intranasal immunization with bacterial polypeptide, termed as a modified PnxIIIA (MP3) protein, is known to elicit production of a specific antibody in mice. In this study, a nasal immuno-inducible sequence (NAIS) was designed to remove the antigenicity of the MP3 protein that can induce mucosal immunity by intranasal immunization, and was examined to induce antigen-specific antibodies against the fused bacterial thioredoxin (Trx) as a model antigen. A NAIS was modified and generated to remove a large number of predicted MHC (Major Histocompatibility Complex)-I and MHC-II binding sites in parent protein PnxIIIA and MP3 in order to reduce the number of antigen epitope sites. For comparative analysis, full-length NAIS291, NAIS230, and NAIS61 fused with Trx and 6× His tag and Trx-fused 6× His tag were used as antigen variants for the intranasal immunization of BALB/c mice every two weeks for three immunizations. Anti-Trx antibody titers in serum and bronchoalveolar lavage fluid (BALF) IgA obtained from NAIS291-fused Trx-immunized mice were significantly higher than those from Trx-immunized mice. The antibody titers against NAIS alone were significantly lower than those against Trx alone in the serum IgG, serum IgA, and BALF IgA. These results indicate that the NAIS contributes to antibody elicitation of the fused antigen as an immunostimulant in intranasal vaccination vaccines. The results indicate that the NAIS and target inactivated antigen fusions can be applied to intranasal vaccine systems. Full article

Background/Objectives: Influenza viruses and SARS-CoV-2 are currently cocirculating with similar seasonality, and both pathogens are characterized by a high mutational rate which results in reduced vaccine effectiveness and thus requires regular updating of vaccine compositions. Vaccine formulations combining seasonal influenza and SARS-CoV-2 strains can be considered promising and cost-effective tools for protection against both infections. Methods: We used a licensed seasonal trivalent live attenuated influenza vaccine (3×LAIV) as a basis for the development of a modified 3×LAIV/CoV-2 vaccine, where H1N1 and H3N2 LAIV strains encoded an immunogenic cassette enriched with conserved T-cell epitopes of SARS-CoV-2, whereas a B/Victoria lineage LAIV strain was unmodified. The trivalent LAIV/CoV-2 composition was compared to the classical 3×LAIV in the golden Syrian hamster model. Animals were intranasally immunized with the mixtures of the vaccine viruses, twice, with a 3-week interval. Immunogenicity was assessed on day 42 of the study, and the protective effect was established by infecting vaccinated hamsters with either influenza H1N1, H3N2 or B viruses or with SARS-CoV-2 strains of the Wuhan, Delta and Omicron lineages. Results: Both the classical 3×LAIV and 3×LAIV/CoV-2 vaccine compositions induced similar levels of serum antibodies specific to all three influenza strains, which resulted in comparable levels of protection against challenge from either influenza strain. Protection against SARS-CoV-2 challenge was more pronounced in the 3×LAIV/CoV-2-immunized hamsters compared to the classical 3×LAIV group. These data were accompanied by the higher magnitude of virus-specific cellular responses detected by ELISPOT in the modified trivalent LAIV group. Conclusions: The modified trivalent live attenuated influenza vaccine encoding the T-cell epitopes of SARS-CoV-2 can be considered a promising tool for combined protection against seasonal influenza and COVID-19. Full article

The virions of plant viruses and their structurally modified particles (SP) represent valuable platforms for recombinant vaccine epitopes and antitumor agents. The possibility of modifying their surface with biological compounds makes them a tool for developing medical biotechnology applications. Here, we applied a new type of SP derived from virions and virus-like particles (VLP) of Alternanthera mosaic virus (AltMV) and well-studied SP from Tobacco mosaic virus (TMV). We have tested the ability of SP from AltMV (AltMV SP_V) and TMV virions also as AltMV VLP to bind to and penetrate Ewing sarcoma cells. The adsorption properties of AltMV SP_V and TMV SP are greater than those of the SP from AltMV VLP. Compared to normal cells, AltMV SP_V adsorbed more effectively on patient-derived sarcoma cells, whereas TMV SP were more effective on the established sarcoma cells. The AltMV SP_V and TMV SP were captured by all sarcoma cell lines. In the established Ewing sarcoma cell line, the effectiveness of AltMV SP_V penetration was greater than that of TMV SP. The usage of structurally modified plant virus particles as a platform for drugs and delivery systems has significant potential in the development of anticancer agents. Full article

Background. Influenza and SARS-CoV-2 viruses are two highly variable pathogens. We have developed a candidate bivalent live vaccine based on the strain of licensed A/Leningrad/17-based cold-adapted live attenuated influenza vaccine (LAIV) of H3N2 subtype, which expressed SARS-CoV-2 immunogenic T-cell epitopes. A cassette encoding fragments of S and N proteins of SARS-CoV-2 was inserted into the influenza NA gene using the P2A autocleavage site. In this study, we present the results of preclinical evaluation of the developed bivalent vaccine in a non-human primate model. Methods. Rhesus macaques (Macaca mulatta) (n = 3 per group) were immunized intranasally with 7.5 lg EID₅₀ of the LAIV/CoV-2 bivalent vaccine, a control non-modified H3N2 LAIV or a placebo (chorioallantoic fluid) using a sprayer device, twice, with a 28-day interval. The blood samples were collected at days 0, 3, 28 and 35 for hematological and biochemical assessment. Safety was also assessed by monitoring body weight, body temperature and clinical signs of the disease. Immune responses to influenza virus were assessed both by determining serum antibody titers in hemagglutination inhibition assay, microneutralization assay and IgG ELISA. T-cell responses were measured both to influenza and SARS-CoV-2 antigens using ELISPOT and flow cytometry. Three weeks after the second immunization, animals were challenged with 10⁵ PFU of Delta SARS-CoV-2. The body temperature, weight and challenge virus shedding were monitored for 5 days post-challenge. In addition, virus titers in various organs and histopathology were evaluated on day 6 after SARS-CoV-2 infection. Results. There was no toxic effect of the immunizations on the hematological and coagulation hemostasis of animals. No difference in the dynamics of the average weight and thermometry results were found between the groups of animals. Both LAIV and LAIV/CoV-2 variants poorly replicated in the upper respiratory tract of rhesus macaques. Nevertheless, despite this low level of virus shedding, influenza-specific serum IgG responses were detected in the group of monkeys immunized with the LAIV/CoV-2 bivalent but not in the LAIV group. Furthermore, T-cell responses to both influenza and SARS-CoV-2 viruses were detected in the LAIV/CoV-2 vaccine group only. The animals were generally resistant to SARS-CoV-2 challenge, with minimal virus shedding in the placebo and LAIV groups. Histopathological changes in vaccinated animals were decreased compared to the PBS group, suggesting a protective effect of the chimeric vaccine candidate. Conclusions. The candidate bivalent vaccine was safe and immunogenic for non-human primates and warrants its further evaluation in clinical trials. Full article

Classical Swine Fever (CSF), a highly contagious viral disease affecting pigs and wild boar, results in significant economic losses in the swine industry. In endemic regions, prophylactic vaccination and stamping-out strategies are used to control CSF outbreaks. However, sporadic outbreaks and persistent infections continue to be reported. Although the conventional attenuated CSF vaccines protect pigs against the disease, they do not allow for the differentiation of infected from vaccinated animals (DIVA), limiting their use as an eradication tool. In this study, three targeted attenuation strategies were employed to generate vaccine candidates based on the current prevalent CSFV group 2 strains GD18 and QZ07: a single deletion of H79 in E^rns (QZ07-sdErnsH-KARD), double deletion of H79 and C171 in E^rns (GD18-ddErnsHC-KARD and QZ07-ddErnsHC-KARD), and deletion of H79 in E^rns combined with a 5–168 amino acids deletion of N^pro (GD18-ddNpro-ErnsH-KARD). Additionally, a negative serological marker with four substitutions in a highly conserved epitope in E2 recognized by the monoclonal antibody 6B8 was introduced in each candidate for DIVA purposes. The safety of these four resulting vaccine candidates was evaluated in pregnant sows. Two candidates, GD18-ddErnsHC-KARD and QZ07-sdErnsH-KARD were found to be safe for pregnant sows and unlikely to cause vertical transmission. Both candidates also demonstrated potential to be used as DIVA vaccines, as was shown using a proprietary blocking ELISA based on the 6B8 monoclonal antibody. These results, together with our previous work, constitute a proof-of-concept for the rational design of CSF antigenically marked modified live virus vaccine candidates. Full article

The continuously evolving PRRSV has been plaguing pig farms worldwide for over 30 years, with conventional vaccines suffering from insufficient protection and biosecurity risks. To address these challenges, we identified 10 PRRSV-specific CTL epitopes through enzyme-linked immunospot assay (ELISPOT) and constructed a multi-epitope peptide (PTE) by linking them in tandem. This PTE was then fused with a modified porcine Fc molecule to create the recombinant protein pFc-PTE. Our findings indicate that pFc-PTE effectively stimulates PRRSV-infected specific splenic lymphocytes to secrete high levels of interferon-gamma (IFN-γ) and is predicted to be non-toxic and non-allergenic. Compared to PTE alone, pFc-PTE not only induced a comparable cellular immune response in mice but also extended the duration of the immune response to at least 10 weeks post-immunization. Additionally, pFc-PTE predominantly induced a Th1 immune response, suggesting its potential advantage in enhancing cellular immunity. Consequently, pFc-PTE holds promise as a novel, safe, and potent candidate vaccine for PRRSV and may also provide new perspectives for vaccine design against other viral diseases. Full article

Nanoparticles (NPs) have been surfacing as a pivotal platform for vaccine development. In our previous work, we developed a cholera toxin B subunit (CTB)-based self-assembled nanoparticle (CNP) and produced highly promising bioconjugate nanovaccines by loading bacterial polysaccharide (OPS) in vivo. In particular, the Klebsiella pneumoniae O2 serotype vaccine showcased a potent immune response and protection against infection. However, extremely low yields limited its further application. In this study, we prepared an efficient Klebsiella pneumoniae bioconjugate nanovaccine in Escherichia coli with a very high yield. By modifying the 33rd glycine (G) in the CNP to aspartate (D), we were able to observe a dramatically increased expression of glycoprotein. Subsequently, through a series of mutations, we determined that G33D was essential to increasing production. In addition, this increase only occurred in engineered E. coli but not in the natural host K. pneumoniae strain 355 (Kp355) expressing OPS_KpO2. Next, T-cell epitopes were fused at the end of the CNP(G33D), and animal experiments showed that fusion of the M51 peptide induced high antibody titers, consistent with the levels of the original nanovaccine, CNP-OPS_KpO2. Hence, we provide an effective approach for the high-yield production of K. pneumoniae bioconjugate nanovaccines and guidance for uncovering glycosylation mechanisms and refining glycosylation systems. Full article

The efficacy of anti-viral T-cell vaccines may greatly depend on their ability to generate high-magnitude responses targeting a broad range of different epitopes. Recently, we created the HIV T-cell immunogen HTI, designed to generate T-cell responses to protein fragments more frequently targeted by HIV controllers. In the present study, we aim to maximize the breadth and magnitude of the T-cell responses generated by HTI by combining different vaccine vectors expressing HTI. We evaluated the ability to induce strong and broad T-cell responses to the HTI immunogen through prime vaccination with DNA plasmid (D) or Chimpanzee Adenovirus Ox1 (ChAdOx1; C) vectors, followed by a Modified Virus Ankara (MVA; M) vaccine boost (DDD, DDDM, C, and CM). HTI-specific T-cell responses after vaccination were measured by IFN-γ-ELISpot assays in two inbred mice strains (C57BL/6 and BALB/c). CM was the schedule triggering the highest magnitude of the response in both mice strains. However, this effect was not reflected in an increase in the breadth of the response but rather in an increase in the magnitude of the response to specific immunodominant epitopes. Immunodominance profiles in the two mouse strains were different, with a clear dominance of T-cell responses to a Pol-derived peptide pool after CM vaccination in C57BL/6. Responses to CM vaccination were also maintained at higher magnitudes over time (13 weeks) compared to other vaccination regimens. Thus, while a ChAdOx1 prime combined with MVA booster vaccination generated stronger and more sustained T-cell responses compared to three DNA vaccinations, the ChAdOx1 primed responses were more narrowly targeted. In conclusion, our findings suggest that the choice of vaccine vectors and prime-boost regimens plays a crucial role in determining the strength, duration, breadth, and focus of T-cell responses, providing further guidance for selecting vaccination strategies. Full article