Carriers For Epitopes Research Articles

Glycomics of cervicovaginal fluid from women at risk of preterm birth reveals immuno-regulatory epitopes that are hallmarks of cancer and viral glycosylation

During pregnancy the immune system needs to maintain immune tolerance of the foetus while also responding to infection, which can cause premature activation of the inflammatory pathways leading to the onset of labour and preterm birth. The vaginal microbiome is an important modifier of preterm birth risk, with Lactobacillus dominance during pregnancy associated with term delivery while high microbial diversity is associated with an increased risk of preterm birth. Glycans on glycoproteins along the lower female reproductive tract are fundamental to microbiota-host interactions and the mediation of inflammatory responses. However, the specific glycan epitopes involved in these processes are not well understood. To address this, we conducted glycomic analyses of cervicovaginal fluid (CVF) from 36 pregnant women at high risk of preterm birth and 4 non-pregnant women. Our analysis of N- and O-glycans revealed a rich CVF glycome. While O-glycans were shown to be the main carriers of ABO blood group epitopes, the main features of N-glycans were the presence of abundant paucimannose and high mannose glycans, and a remarkable diversity of complex bi-, tri-, and tetra-antennary glycans decorated with fucose and sialic acid. We identified immuno-regulatory epitopes, such as Lewis antigens, and found that fucosylation was negatively correlated to pro-inflammatory factors, such as IL-1β, MMP-8, C3a and C5a, while glycans with only sialylated antennae were mainly positively correlated to those. Similarly, paucimannose glycans showed a positive correlation to pro-inflammatory factors. We revealed a high abundance of glycans which have previously been identified as hallmarks of cancer and viral glycosylation, such as Man8 and Man9 high mannose glycans. Although each pregnant woman had a unique glycomic profile, longitudinal studies showed that the main glycosylation features were consistent throughout pregnancy in women who delivered at term, whereas women who experienced extreme preterm birth exhibited sharp changes in the CVF glycome shortly before delivery. These findings shed light on the processes underlying the role of glycosylation in maintaining a healthy vaginal microbiome and associated host immune responses. In addition, these discoveries facilitate our understanding of the lower female reproductive tract which has broad implications for women’s health.

Precise Epitope Organization with Self‐adjuvant Framework Nucleic Acid for Efficient COVID‐19 Peptide Vaccine Construction

AbstractPeptide vaccines have advantages in easy fabrication and high safety, but their effectiveness is hampered by the poor immunogenicity of the epitopes themselves. Herein, we constructed a series of framework nucleic acids (FNAs) with regulated rigidity and size to precisely organize epitopes in order to reveal the influence of epitope spacing and carrier rigidity on the efficiency of peptide vaccines. We found that assembling epitopes on rigid tetrahedral FNAs (tFNAs) with the appropriate size could efficiently enhance their immunogenicity. Further, by integrating epitopes from SARS‐CoV‐2 on preferred tFNAs, we constructed a COVID‐19 peptide vaccine which could induce high titers of IgG against the receptor binding domain (RBD) of SARS‐CoV‐2 spike protein and increase the ratio of memory B and T cells in mice. Considering the good biocompatibility of tFNAs, our research provides a new idea for developing efficient peptide vaccines against viruses and possibly other diseases.

Precise Epitope Organization with Self-adjuvant Framework Nucleic Acid for Efficient COVID-19 Peptide Vaccine Construction.

Peptide vaccines have advantages in easy fabrication and high safety, but their effectiveness is hampered by the poor immunogenicity of the epitopes themselves. Herein, we constructed a series of framework nucleic acids (FNAs) with regulated rigidity and size to precisely organize epitopes in order to reveal the influence of epitope spacing and carrier rigidity on the efficiency of peptide vaccines. We found that assembling epitopes on rigid tetrahedral FNAs (tFNAs) with the appropriate size could efficiently enhance their immunogenicity. Further, by integrating epitopes from SARS-CoV-2 on preferred tFNAs, we constructed a COVID-19 peptide vaccine which could induce high titers of IgG against the receptor binding domain (RBD) of SARS-CoV-2 spike protein and increase the ratio of memory B and T cells in mice. Considering the good biocompatibility of tFNAs, our research provides a new idea for developing efficient peptide vaccines against viruses and possibly other diseases.

Production and characterization of chimeric SARS-CoV-2 antigens based on the capsid protein of cowpea chlorotic mottle virus

The COVID-19 pandemic has highlighted the need for new vaccine platforms to rapidly develop solutions against emerging pathogens. In particular, some plant viruses offer several advantages for developing subunit vaccines, such as high expression rates in E. coli, high immunogenicity and safety, and absence of pre-immunity that could interfere with the vaccine's efficacy. Cowpea chlorotic mottle virus (CCMV) is a model system that has been extensively characterized, with key advantages for its use as an epitope carrier. In the present study, three relevant epitopes from the SARS-CoV-2 Spike protein were genetically inserted into the CCMV CP and expressed in E. coli cultures, resulting in the CCMV1, CCMV2, and CCMV3 chimeras. The recombinant CP mutants were purified from the formed inclusion bodies and refolded, and their immunogenicity as a subunit vaccine was assessed in BALB/c mice. The three mutants are immunogenic as they induce high IgG antibody titers that recognize the recombinant full-length S protein. This study supports the application of CCMV CP as an attractive carrier for the clinical evaluation of vaccine candidates against SARS-CoV-2. Furthermore, it suggests that VLPs assembled from these chimeric proteins could result in antigens with better immunogenicity.

Plant Virus Nanoparticles for Anti-cancer Therapy.

Plant virus nanoparticles (VNPs) are inexpensive to produce, safe, biodegradable and efficacious as treatments. The applications of r plant virus nanoparticles range from epitope carriers for vaccines to agents in cancer immunotherapy. Both VNPs and virus-like particles (VLPs) are highly immunogenic and are readily phagocytosed by antigen presenting cells (APCs), which in turn elicit antigen processing and display of pathogenic epitopes on their surfaces. Since the VLPs are composed of multiple copies of their respective capsid proteins, they present repetitive multivalent scaffolds which aid in antigen presentation. Therefore, the VLPs prove to be highly suitable platforms for delivery and presentation of antigenic epitopes, resulting in induction of more robust immune response compared to those of their soluble counterparts. Since the tumor microenvironment poses the challenge of self-antigen tolerance, VLPs are preferrable platforms for delivery and display of self-antigens as well as otherwise weakly immunogenic antigens. These properties, in addition to their diminutive size, enable the VLPs to deliver vaccines to the draining lymph nodes in addition to promoting APC interactions. Furthermore, many plant viral VLPs possess inherent adjuvant properties dispensing with the requirement of additional adjuvants to stimulate immune activity. Some of the highly immunogenic VLPs elicit innate immune activity, which in turn instigate adaptive immunity in tumor micro-environments. Plant viral VLPs are nontoxic, inherently stable, and capable of being mass-produced as well as being modified with antigens and drugs, therefore providing an attractive option for eliciting anti-tumor immunity. The following review explores the use of plant viruses as epitope carrying nanoparticles and as a novel tools in cancer immunotherapy.

Epitope-coated polymer particles elicit neutralising antibodies against Plasmodium falciparum sporozoites

The current Malaria RTS,S vaccine is based on virus-like particles (VLPs) comprising the NANP repetitive epitopes from the cicumsporozoite protein (CSP) of Plasmodium falciparum. This vaccine has limited efficacy, only preventing severe disease in about 30% of vaccinated individuals. A more efficacious vaccine is urgently needed to combat malaria. Here we developed a particulate malaria vaccine based on the same CSP epitopes but using biopolymer particles (BPs) as an antigen carrier system. Specific B- and T-cell epitope-coated BPs were assembled in vivo inside an engineered endotoxin-free mutant of Escherichia coli. A high-yield production process leading to ~27% BP vaccine weight over biomass was established. The epitope-coated BPs were purified and their composition, i.e., the polymer core and epitope identity, was confirmed. Epitope-coated BPs were used alongside soluble peptide epitopes and empty BPs to vaccinate sheep. Epitope-coated BPs showed enhanced immunogenicity by inducing anti-NANP antibody titre of EC50 > 150,000 that were at least 20 times higher than induced by the soluble peptides. We concluded that the additional T-cell epitope was not required as it did not enhance immunogenicity when compared with the B-cell epitope-coated BPs. Antibodies specifically bound to the surface of Plasmodium falciparum sporozoites and efficiently inhibited sporozoite motility and traversal of human hepatocytes. This study demonstrated the utility of biologically self-assembled epitope-coated BPs as an epitope carrier for inclusion in next-generation malaria vaccines.

Enhanced epitope immunoreactivity of the dominant epitope of Toxoplasma gondii fused at the "N terminus" of HPV16L1

For improving epitope immunogenicity and achieving the co-immunization, late protein 1 (L1) of HPV type 16 (HPV16L1) was selected as the vector to carry the dominant epitope of Toxoplasma gondii because of the shared common population between Toxoplasma gondii and human papillomavirus (HPV). RSepitope-HPV16L1 (RSepitope fused at the "N-terminus" of HPV16L1) and HPV16L1-RSepitope (RSepitope fused at the "C-terminus" of HPV16L1) chimeras were constructed. After transfection of COS-7 cells with the recombinants, Western blot, RT-PCR, and immunofluorescence experiments confirmed that RSepitope-HPV16L1 could successfully express the corresponding mRNA and protein of RSepitope and HPV16L1, but the HPV16L1-RSepitope construct could not. A "prime-boost" immunization program was applied in mice to further evaluate the immune response elicited by the constructs, and the RSepitope-HPV16L1 immunization group produced the most significantly increased humoral and cellular immune responses (the highest RSepitope-specific IgG antibody level and the highest IFN-γ production, respectively), in which both elevated Th1 and Th2 immune responses were obtained. Moreover, the advantage of HPV16L1 as an epitope carrier was remarkable for RSepitope-HPV16L1, which induced a more prominent immunological response than RSepitope alone (without fusion with HPV16L1). Our research indicated that the N-terminus of HPV16L1 could be a better insertion site for enhancing target epitope immunogenicity, and our study offers a design for epitope vaccine of reasonable combination.

NOVEL APPROACH FOR DESIGNING ROTAVIRUS VACCINE CANDIDATE BASED ON TWO PLANT VIRUSES

Rotavirus А (genus Rotavirus, family Reoviridae) is still the main cause of viral gastroenteritis in children under 5 years. Existing attenuated vaccines have serious disadvantages, including the risk of potential reversion to pathogenic form, and side effects, the most dangerous of which is intussusception. Moreover, they occurred to be less effective in developing countries, where the most rotavirus-associated deaths are recorded. The development of an effective recombinant rotavirus A vaccine is an actual assignment; herewith the selection of effective and safe adjuvant is the key point for that. Plant viruses are very promising for innovative vaccine designing; they possess high immunostimulating properties, safe for humans and mammals and can serve as a carrier for pathogens’ epitopes. Here we suggest an approach for rotavirus A vaccine development that involves two plant viruses: Alternanthera mosaic virus (AltMV) and Tobacco mosaic virus (TMV) as simultaneously epitope carriers and adjuvants. Spherical particles (SPs) generating by the heating of tobacco mosaic virus were used as an adjuvant and platform for presentation of obtained in our previous study chimeric recombinant protein ER6, which is an AltMV coat protein (CP) fused with the epitope RV14 (RLSFQLMRPPNMTP) of rotavirus A antigen VP6. Epitope RV14 are able to induce protective immune response and is conservative for the majority of rotavirus A strains therefore its usage gives hope to the successful overcoming of one of the main difficulties in rotavirus A vaccine development: wide serological diversity. In present work, effective adsorption of ER6 on the SPs surface leading to the SPs-ER6 complex formation without loss of ER6 antigenic specificity was demonstrated. Two immune antisera with specificity to RV14 epitope within ER6 were obtained. The first serum was obtained via anti-ER6 sera depletion with AltMV CP and recombinant AltMV CP (AltMV rCP), which was expressed in Escherichia coli but did not contain RV14 sequence. The second serum was obtained by a direct immunization with synthetic peptide RLSFQLMRPPNMTP. These sera were utilized for studying RV14 within SPs-ER6 complexes. By means of immunofluorescent microscopy, SPs-ER6 complexes were demonstrated to interact with both depleted serum and anti-RV14 serum. Therefore, rotavirus epitope was confirmed to keep its ability to interact with antibodies within obtained complexes. Considering unique adjuvant properties of spherical particles and characteristics of selected epitope obtained SPs-ER6 complexes can be thought as a promising component for recombinant rotavirus A vaccine. Moreover, it can be hoped that the suggested in present work approach, involving the usage of TMV SPs as a platform and adjuvant for chimeric AltMV CP, containing pathogen’s epitope, will be useful not only for rotavirus A vaccine development but for designing of vaccines against other pathogens of humans or farm animals.

НОВЫЙ ПОДХОД К РАЗРАБОТКЕ КАНДИДАТНОЙ ВАКЦИНЫ ПРОТИВ РОТАВИРУСА НА ОСНОВЕ ДВУХ ВИРУСОВ РАСТЕНИЙ

Rotavirus А (genus Rotavirus, family Reoviridae) is still the main cause of viral gastroenteritis in children under 5 years. Existing attenuated vaccines have serious disadvantages, including the risk of potential reversion to pathogenic form, and side effects, the most dangerous of which is intussusception. Moreover, they occurred to be less effective in developing countries, where the most rotavirus-associated deaths are recorded. The development of an effective recombinant rotavirus A vaccine is an actual assignment; herewith the selection of effective and safe adjuvant is the key point for that. Plant viruses are very promising for innovative vaccine designing; they possess high immunostimulating properties, safe for humans and mammals and can serve as a carrier for pathogens’ epitopes. Here we suggest an approach for rotavirus A vaccine development that involves two plant viruses: Alternanthera mosaic virus (AltMV) and Tobacco mosaic virus (TMV) as simultaneously epitope carriers and adjuvants. Spherical particles (SPs) generating by the heating of tobacco mosaic virus were used as an adjuvant and platform for presentation of obtained in our previous study chimeric recombinant protein ER6, which is an AltMV coat protein (CP) fused with the epitope RV14 (RLSFQLMRPPNMTP) of rotavirus A antigen VP6. Epitope RV14 are able to induce protective immune response and is conservative for the majority of rotavirus A strains therefore its usage gives hope to the successful overcoming of one of the main difficulties in rotavirus A vaccine development: wide serological diversity. In present work, effective adsorption of ER6 on the SPs surface leading to the SPs-ER6 complex formation without loss of ER6 antigenic specificity was demonstrated. Two immune antisera with specificity to RV14 epitope within ER6 were obtained. The first serum was obtained via anti-ER6 sera depletion with AltMV CP and recombinant AltMV CP (AltMV rCP), which was expressed in Escherichia coli but did not contain RV14 sequence. The second serum was obtained by a direct immunization with synthetic peptide RLSFQLMRPPNMTP. These sera were utilized for studying RV14 within SPs-ER6 complexes. By means of immunofluorescent microscopy, SPs-ER6 complexes were demonstrated to interact with both depleted serum and anti-RV14 serum. Therefore, rotavirus epitope was confirmed to keep its ability to interact with antibodies within obtained complexes. Considering unique adjuvant properties of spherical particles and characteristics of selected epitope obtained SPs-ER6 complexes can be thought as a promising component for recombinant rotavirus A vaccine. Moreover, it can be hoped that the suggested in present work approach, involving the usage of TMV SPs as a platform and adjuvant for chimeric AltMV CP, containing pathogen’s epitope, will be useful not only for rotavirus A vaccine development but for designing of vaccines against other pathogens of humans or farm animals.

Heterovalent Glycodendrimers as Epitope Carriers for Antitumor Synthetic Vaccines.

The large majority of TACA‐based (TACA=Tumor‐Associated Carbohydrate Antigens) antitumor vaccines target only one carbohydrate antigen, thereby often resulting in the incomplete destruction of cancer cells. However, the morphological heterogeneity of the tumor glycocalix, which is in constant evolution during malignant transformation, is a crucial point to consider in the design of vaccine candidates. In this paper, an efficient synthetic strategy based on orthogonal chemoselective ligations to prepare fully synthetic glycosylated cyclopeptide scaffolds grafted with both Tn and TF antigen analogues is reported. To evaluate their ability to be recognized as tumor antigens, direct interaction ELISA assays have been performed with the anti‐Tn monoclonal antibody 9A7. Although both heterovalent structures showed binding capacities with 9A7, the presence of the second TF epitope did not interfere with the recognition of Tn except in one epitope arrangement. This heterovalent glycosylated structure thus represents an attractive epitope carrier to be further functionalized with T‐cell peptide epitopes.

Cellular Uptake Mechanism of Cationic Branched Polypeptides with Poly[l-Lys] Backbone.

Cationic macromolecular carriers can be effective carriers for small molecular compounds, drugs, epitopes, or nucleic acids. Polylysine-based polymeric branched polypeptides have been systematically studied on the level of cells and organisms as well. In the present study, we report our findings on the cellular uptake characteristics of nine structurally related polylysine-based polypeptides with cationic side chains composed of (i) single amino acid (poly[Lys(Xi)], XiK) or (ii) oligo[dl-alanine] (poly[Lys(dl-Alam)], AK) or (iii) oligo[dl-alanine] with an additional amino acid (X) at the terminal position (poly[Lys(Xi-dl-Alam)] (XAK)) or (iv) at the position next to the polylysine backbone (poly[Lys(dl-Alam-Xi)] (AXK)). In vitro cytotoxicity and cellular uptake were characterized on HT-29 human colon carcinoma and HepG2 human hepatocarcinoma cell lines. Data indicate that the polycationic polypeptides studied are essentially nontoxic in the concentration range studied, and their uptake is very much dependent on the side chain structure (length, identity of amino acid X, and distance between the terminal positive charges) and also on the cell lines. Our findings in uptake inhibition studies suggest that predominantly macropinocytosis and caveole/lipid raft mediated endocytosis are involved. The efficacy of their internalization is markedly influenced by the hydrophobicity and charge properties of the amino acid X. Interestingly, the uptake properties of the these polypeptides show certain similarities to the entry pathways of several cell penetrating peptides.

A Rationally Designed TNF-α Epitope-Scaffold Immunogen Induces Sustained Antibody Response and Alleviates Collagen-Induced Arthritis in Mice

The TNF-α biological inhibitors have significantly improved the clinical outcomes of many autoimmune diseases, in particular rheumatoid arthritis. However, the practical uses are limited due to high costs and the risk of anti-drug antibody responses. Attempts to develop anti-TNF-α vaccines have generated encouraging data in animal models, however, data from clinical trials have not met expectations. In present study, we designed a TNF-α epitope-scaffold immunogen DTNF7 using the transmembrane domain of diphtheria toxin, named DTT as a scaffold. Molecular dynamics simulation shows that the grafted TNF-α epitope is entirely surface-exposed and presented in a native-like conformation while the rigid helical structure of DTT is minimally perturbed, thereby rendering the immunogen highly stable. Immunization of mice with alum formulated DTNF7 induced humoral responses against native TNF-α, and the antibody titer was sustained for more than 6 months, which supports a role of the universal CD4 T cell epitopes of DTT in breaking self-immune tolerance. In a mouse model of rheumatoid arthritis, DTNF7-alum vaccination markedly delayed the onset of collagen-induced arthritis, and reduced incidence as well as clinical score. DTT is presumed safe as an epitope carrier because a catalytic inactive mutant of diphtheria toxin, CRM197 has good clinical safety records as an active vaccine component. Taken all together, we show that DTT-based epitope vaccine is a promising strategy for prevention and treatment of autoimmune diseases.

Replication-Competent Foamy Virus Vaccine Vectors as Novel Epitope Scaffolds for Immunotherapy.

The use of whole viruses as antigen scaffolds is a recent development in vaccination that improves immunogenicity without the need for additional adjuvants. Previous studies highlighted the potential of foamy viruses (FVs) in prophylactic vaccination and gene therapy. Replication-competent FVs can trigger immune signaling and integrate into the host genome, resulting in persistent antigen expression and a robust immune response. Here, we explored feline foamy virus (FFV) proteins as scaffolds for therapeutic B and T cell epitope delivery in vitro. Infection- and cancer-related B and T cell epitopes were grafted into FFV Gag, Env, or Bet by residue replacement, either at sites of high local sequence homology between the epitope and the host protein or in regions known to tolerate sequence alterations. Modified proviruses were evaluated in vitro for protein steady state levels, particle release, and virus titer in permissive cells. Modification of Gag and Env was mostly detrimental to their function. As anticipated, modification of Bet had no impact on virion release and affected virus titers of only some recombinants. Further evaluation of Bet as an epitope carrier was performed using T cell epitopes from the model antigen chicken ovalbumin (OVA), human tyrosinase-related protein 2 (TRP-2), and oncoprotein E7 of human papillomavirus type 16 (HPV16E7). Transfection of murine cells with constructs encoding Bet-epitope chimeric proteins led to efficient MHC-I-restricted epitope presentation as confirmed by interferon-gamma enzyme-linked immunospot assays using epitope-specific cytotoxic T lymphocyte (CTL) lines. FFV infection-mediated transduction of cells with epitope-carrying Bet also induced T-cell responses, albeit with reduced efficacy, in a process independent from the presence of free peptides. We show that primate FV Bet is also a promising T cell epitope carrier for clinical translation. The data demonstrate the utility of replication-competent and -attenuated FVs as antigen carriers in immunotherapy.

Construction of polyomavirus-derived pseudotype virus-like particles displaying a functionally active neutralizing antibody against hepatitis B virus surface antigen.

BackgroundVirus-like particles (VLPs) can be efficiently produced by heterologous expression of viral structural proteins in yeast. Due to their repetitive structure, VLPs are extensively used for protein engineering and generation of chimeric VLPs with inserted foreign epitopes. Hamster polyomavirus VP1 represents a promising epitope carrier. However, insertion of large sized protein sequences may interfere with its self-assembly competence. The co-expression of polyomavirus capsid protein VP1 with minor capsid protein VP2 or its fusion protein may result in pseudotype VLPs where an intact VP1 protein mediates VLP formation. In the current study, the capacity of VP1 protein to self-assemble to VLPs and interact with the modified VP2 protein has been exploited to generate pseudotype VLPs displaying large-sized antibody molecules.ResultsPolyomavirus-derived pseudotype VLPs harbouring a surface-exposed functionally active neutralizing antibody specific to hepatitis B virus (HBV) surface antigen (HBsAg) have been generated. The pseudotype VLPs consisting of an intact hamster polyomavirus (HaPyV) major capsid protein VP1 and minor capsid protein VP2 fused with the anti-HBsAg molecule were efficiently produced in yeast Saccharomyces cerevisiae and purified by density-gradient centrifugation. Formation of VLPs was confirmed by electron microscopy. Two types of pseudotype VLPs were generated harbouring either the single-chain fragment variable (scFv) or Fc-engineered scFv on the VLP surface. The antigen-binding activity of the purified pseudotype VLPs was evaluated by ELISA and virus-neutralization assay on HBV-susceptible primary hepatocytes from Tupaia belangeri. Both types of the pseudotype VLPs were functionally active and showed a potent HBV-neutralizing activity comparable to that of the parental monoclonal antibody. The VP2-fused scFv molecules were incorporated into the VLPs with higher efficiency as compared to the VP2-fused Fc-scFv. However, the pseudotype VLPs with displayed VP2-fused Fc-scFv molecule showed higher antigen-binding activity and HBV-neutralizing capacity that might be explained by a better accessibility of the Fc-engineered scFv of the VLP surface.ConclusionsPolyomavirus-derived pseudotype VLPs harbouring multiple functionally active antibody molecules with virus-neutralizing capability may represent a novel platform for developing therapeutic tools with a potential application for post-exposure or therapeutic treatment of viral infections.

The Quest for an HIV-1 Vaccine Adjuvant: Bacterial Toxins as New Potential Platforms.

While tremendous efforts are undergoing towards finding an effective HIV-1 vaccine, the search for an HIV-1 vaccine adjuvant lags behind and is understudied. More recently, however, efforts have focused on testing adjuvant formulations that can boost the immune response and generate broadly neutralizing antibodies to HIV-1 ENV (gp160). Despite this, there remain a number of challenges towards achieving this goal. These include safety of adjuvant formulations; stability of the incorporated antigens; maintenance of ENV immunogenicity; optimal inoculation sites; the effective combination of adjuvants; stability of ENV neutralizing epitopes in some adjuvant formulations; mucosal immunity; and long-term maintenance of the immune response. A new class of adjuvants for HIV-1 proteins is suggested to overcome many of the limitations of some other adjuvants. Type 1 (LT-I) and type 2 (LT-II) human E. coli enterotoxins (HLTs) and their non-toxic B-subunits derivatives are strong systemic and mucosal adjuvants and effective carriers for other proteins and epitopes. Their stable molecular structure in the presence of fused proteins and epitopes, and their ability to target surface receptors on antigen presenting cells make them ideal for the delivery of HIV-1 ENV or HIV other proteins. Importantly, unlike some other adjuvants, HLTs and derivatives have well-defined modes of immune system activation. The challenges in finding optimal HIV-1 vaccine adjuvant formulation and the important properties of HLTs are discussed.

Development of novel DNA vaccine for VEGF in murine cancer model

We developed DNA vaccine for vascular endothelial growth factor (VEGF), which may provide the therapeutic option instead of anti-VEGF antibody, bevacizumab. Plasmid containing VEGF mini-gene was constructed in the insertion of B-cell epitope of Hepatitis B core protein [HBc-VEGF], which was an epitope carrier. High titer of anti-VEGF antibody was observed in BALB/c mice which were intramuscularly immunized with HBc-VEGF by electropolator. In mice inoculated with colon 26 cells, tumor volume and microvessel density was decreased in HBc-VEGF with a significant prolonged survival. Co-treatment of purified IgG from immunized mice with HBc-VEGF showed in vitro neutralizing activity for VEGF-induced ERK phosphorylation and tube formation in cultured endothelial cells. Furthermore, intravitreally injection of this purified IgG reduced the neovessel formation in the mouse oxygen-induced retinopathy and laser-induced choroidal neovascularization models. These results first provided that DNA vaccine against VEGF possessed the anti-angiogenic effect, leading to prolonged survival in mouse cancer model.

Structure-based design and experimental engineering of a plant virus nanoparticle for the presentation of immunogenic epitopes and as a drug carrier

Biomaterials research for the discovery of new generation nanoparticles is one of the most active areas of nanotechnoloy. In the search of nature-made nanometer-sized objects, plant virus particles appear as symmetrically defined entities that can be formed by protein self-assembly. In particular, in the field of plant virology, there is plenty of literature available describing the exploitation of plant viral cages to produce safe vaccine vehicles and nanoparticles for drug delivery. In this context, we have investigated on the use of the artichoke mottled crinkle virus (AMCV) capsid both as a carrier of immunogenic epitopes and for the delivery of anticancer molecules. A dual approach that combines both in silico tools and experimental virology was applied for the rational design of immunologically active chimeric virus-like particles (VLPs) carrying immunogenic peptides. The atomic structures of wild type (wt) and chimeric VLPs were obtained by homology modeling. The effects of insertion of the HIV-1 2F5 neutralizing epitope on the structural stability of chimeric VLPs were predicted and assessed by detailed inspection of the nanoparticle intersubunit interactions at atomic level. Wt and chimeric VLPs, exposing on their surface the 2F5 epitope, were successfully produced in plants. In addition, we demonstrated that AMCV capsids could also function as drug delivery vehicles able to load the chemotherapeutic drug doxorubicin. To our knowledge, this is the first systematic predictive and empirical research addressing the question of how this icosahedral virus can be used for the production of both VLPs and viral nanoparticles for biomedical applications.

Inhibition of Neointima Formation through DNA Vaccination for Apolipoprotein(a): A New Therapeutic Strategy for Lipoprotein(a)

Lipoprotein(a) [Lp(a)] is an unique lipoprotein consisting of the glycoprotein apolipoprotein(a) [apo(a)] in low-density lipoprotein. Although Lp(a) is a well-known independent risk factor for cardiovascular disease; however, there is no drugs to decrease plasma Lp(a) level. Thus, to inhibit the biological activity of Lp(a), we developed DNA vaccine for apo(a) by the targeting to the selected 12 hydrophilic amino acids in the kringle-4 type 2 domain of apo(a). Hepatitis B virus core protein was used as an epitope carrier to enhance the immunogenicity. Intramuscular immunization with apo(a) vaccine resulted in the significant inhibition of neointima formation in carotid artery ligation model using Lp(a) transgenic mice, associated with anti-apo(a) antibody and decrease in vascular Lp(a) deposition. Overall, this study provided the first evidence that the pro-atherosclerotic actions of Lp(a) could be prevented by DNA vaccine directed against apo(a), suggesting a novel therapeutic strategy to treat cardiovascular diseases related to high Lp(a).

Effects of smoking on the clinical phenotype and response to treatment in rheumatoid arthritis

Smoking is the most studied and widely accepted environmental risk factor for the development of seropositive rheumatoid arthritis, especially in subjects with genetic susceptibility (shared epitope carriers). However, the effect of smoking on aspects of rheumatoid arthritis, such as disease presentation, inflammatory activity, disability and joint damage (radiographic progression), is not clear. Recent data strongly suggest a poor response to methotrexate and TNF antagonists in smokers. We review and analyze the current literature on the relationship between smoking and the clinical phenotype and outcomes of rheumatoid arthritis.

Chemo-enzymatic synthesis of functionalized oligomers of N-acetyllactosamine glycan derivatives and their immobilization on biomaterial surfaces

► Six novel functionalized β- d -GlcNAc derivatives as acceptor substrate of human β1,4 galactosyltransferase 1. ► Step-wise and one-pot enzymatic synthesis of poly- N -acetyllactosamine (poly-LacNAc) oligomers by combination of glycosyltransferases. ► Immobilization of poly-LacNAc oligomers as ligands of the fungal lectin CGL2. Poly- N -acetyllactosamines (poly-LacNAc, [−3Gal(β1–4)GlcNAc(β1−] n ) are terminal glycan structures present in glycoproteins and glycolipids. Their biological functions as ligands for galectins and as carriers of glycan epitopes are well documented. In the present paper we have characterized six novel functionalized β- d -GlcNAc derivatives, including aglyca of varying hydrophobicity and molecular weight, as substrates for recombinant human β1,4 galactosyltransferase 1 (β4GalT-1). The sugar derivatives carry short or long amino- or azide-terminated linker molecules for further modification or immobilization. The linker chemistry had an impact on enzyme kinetics and enzymatic syntheses of N -acetyllactosamine derivatives (LacNAc, Gal(β1–4)GlcNAc(β1-R). The combination of β4GalT-1 with bacterial β1,3- N -acetylglucosaminyltransferase (β3GlcNAc-T) resulted in the preparative syntheses of LacNAc oligomers with up to three LacNAc repeating units. All products were characterized by NMR and MS. The obtained LacNAc glycans were immobilized onto microtiter plates and their efficiency of binding of fungal galectin CGL2 was determined.