Sublingual Immunization With an RSV G Glycoprotein Fragment Primes IL-17-Mediated Immunopathology Upon Respiratory Syncytial Virus Infection.

Respiratory syncytial virus (RSV) is the leading cause of serious respiratory tract disease but there is no licensed RSV vaccine. Immunopathological mechanisms have long been suspected as operating in the development of severe RSV disease and have hampered the development of safe and effective vaccines. Here, we show that unlike intranasal immunization, sublingual immunization with RSV glycoprotein fragment containing the central conserved region (Gcf) primes the host for severe disease upon RSV challenge. This increased pathology does not require replication by the challenge virus and is associated with massive infiltration of inflammatory cells, extensive cell death, and excessive mucus production in the airway and lungs. This exacerbated RSV disease primed by sublingual Gcf immunization is distinct from the immunopathology by G-expressing vaccinia virus or formalin-inactivated RSV, and preceded by prominent IL-17 production. IL-17 deficiency abolished the enhanced disease. Our results suggest a novel mechanism of RSV vaccine-induced immunopathology by IL-17, and highlights the importance of vaccination site.

Development of Safe and Non-Self-Immunogenic Mucosal Adjuvant by Recombinant Fusion of Cholera Toxin A1 Subunit with Protein Transduction Domain.

Potential use of cholera toxin (CT) as a mucosal vaccine adjuvant has been documented in a variety of animal models. However, native CT is highly toxic to be used as a mucosal adjuvant in humans. Here, we demonstrate a new approach to generate a mucosal adjuvant by replacing the B subunit of CT with HIV-1 Tat protein transduction domain (PTD), which efficiently delivers fusion proteins into the cell cytoplasm by unspecific binding to cell surface. We compared the adjuvanticity and toxicity of Tat PTD-CTA1-Tat PTD (TCTA1T) with those of CT. Our results indicate that intranasal (i.n.) delivery of ovalbumin (OVA) with TCTA1T significantly augments the OVA-specific systemic and mucosal antibody responses to levels comparable to those seen with CT adjuvant. Moreover, in vivo cytotoxic T lymphocyte activity elicited by TCTA1T was significantly higher than that elicited by a mutant TCTA1T (TmCTA1T) lacking ADP-ribosyltransferase function. In addition, coadministration of influenza M2 protein with TCTA1T conferred near complete protection against lethal influenza virus challenge. Importantly, TCTA1T, in contrast to CT, did not induce serum IgG antibody responses to itself and was shown to be nontoxic. These results suggest that TCTA1T may be a safe and effective adjuvant when given by mucosal routes.

AXL-dependent infection of human fetal endothelial cells distinguishes Zika virus from other pathogenic flaviviruses.

Although a causal relationship between Zika virus (ZIKV) and microcephaly has been established, it remains unclear why ZIKV, but not other pathogenic flaviviruses, causes congenital defects. Here we show that when viruses are produced in mammalian cells, ZIKV, but not the closely related dengue virus (DENV) or West Nile virus (WNV), can efficiently infect key placental barrier cells that directly contact the fetal bloodstream. We show that AXL, a receptor tyrosine kinase, is the primary ZIKV entry cofactor on human umbilical vein endothelial cells (HUVECs), and that ZIKV uses AXL with much greater efficiency than does DENV or WNV. Consistent with this observation, only ZIKV, but not WNV or DENV, bound the AXL ligand Gas6. In comparison, when DENV and WNV were produced in insect cells, they also infected HUVECs in an AXL-dependent manner. Our data suggest that ZIKV, when produced from mammalian cells, infects fetal endothelial cells much more efficiently than other pathogenic flaviviruses because it binds Gas6 more avidly, which in turn facilitates its interaction with AXL.

Induction of long-term immunity against respiratory syncytial virus glycoprotein by an osmotic polymeric nanocarrier.

Respiratory syncytial virus (RSV) is one of the most common causes of viral deaths in infants worldwide, yet no effective vaccines are available. Here, we report an osmotically active polysaccharide-based polysorbitol transporter (PST) prepared from sorbitol diacrylate and low-molecular-weight polyethylenimine (PEI) showing a potent, yet safe, adjuvant activity and acting as an effective delivery tool for RSV glycoprotein (RGp) antigen. PST showed no toxicity in vitro or in vivo, unlike PEI and the well-known experimental mucosal adjuvant cholera toxin (CT). PST formed nano-sized complexes with RGp by simple mixing, without affecting antigenic stability. The complexes exhibited negative surface charges that made them highly efficient in the selective activation of phagocytic cells and enhancement of phagocytic uptake. This resulted in an improved cytokine production and in the significant augmentation of RGp-specific antibody production, which persisted for over 200 days. Interestingly, PST/RGp enhanced phagocytic uptake owing to the osmotic property of PST and its negative zeta potential, suggesting that PST could selectively stimulate phagocytic cells, thereby facilitating a long-lived antigen-specific immune response, which was presumably further enhanced by the polysaccharide properties of PST.

Development of safe and effective RSV vaccine by modified CD4 epitope in G protein core fragment (Gcf).

Respiratory syncytial virus (RSV) is a major cause of respiratory tract infection in infants and young children worldwide, but currently no safe and effective vaccine is available. The RSV G glycoprotein (RSVG), a major attachment protein, is an important target for the induction of protective immune responses during RSV infection. However, it has been thought that a CD4+ T cell epitope (a.a. 183-195) within RSVG is associated with pathogenic pulmonary eosinophilia. To develop safe and effective RSV vaccine using RSV G protein core fragment (Gcf), several Gcf variants resulting from modification to CD4+ T cell epitope were constructed. Mice were immunized with each variant Gcf, and the levels of RSV-specific serum IgG were measured. At day 4 post-challenge with RSV subtype A or B, lung viral titers and pulmonary eosinophilia were determined and changes in body weight were monitored. With wild type Gcf derived from RSV A2 (wtAGcf), although RSV A subtype-specific immune responses were induced, vaccine-enhanced disease characterized by excessive pulmonary eosinophil recruitment and body weight loss were evident, whereas wtGcf from RSV B1 (wtBGcf) induced RSV B subtype-specific immune responses without the signs of vaccine-enhanced disease. Mice immunized with Th-mGcf, a fusion protein consisting CD4+ T cell epitope from RSV F (F51-66) conjugated to mGcf that contains alanine substitutions at a.a. position 185 and 188, showed higher levels of RSV-specific IgG response than mice immunized with mGcf. Both wtAGcf and Th-mGcf provided complete protection against RSV A2 and partial protection against RSV B. Importantly, mice immunized with Th-mGcf did not develop vaccine-enhanced disease following RSV challenge. Immunization of Th-mGcf provided protection against RSV infection without the symptom of vaccine-enhanced disease. Our study provides a novel strategy to develop a safe and effective mucosal RSV vaccine by manipulating the CD4+ T cell epitope within RSV G protein.

Supplementation of oil-based inactivated H9N2 vaccine with M2e antigen enhances resistance against heterologous H9N2 avian influenza virus infection.

Avian influenza virus (AIV) subtype H9N2 has been evolving rapidly and vaccine escape variants have been reported to cause circulation of infections and economic losses. In the present study, we developed and evaluated ectodomain of the AIV matrix 2 (M2e) protein as a supplementing antigen for oil-based inactivated H9N2 vaccine to increase resistance against vaccine escape variants. AIV H9N2 M2e antigen was expressed in Escherichia coli and supplemented to inactivated H9N2 oil emulsion vaccine. Specific pathogen-free chickens received a single injection of inactivated H9N2 oil emulsion vaccines with or without M2e supplementation. At three weeks post vaccination, hemagglutination inhibition tests and enzyme-linked immunosorbent assays were performed to determine serological immune responses. Challenge study using a vaccine escape H9N2 variant was performed to evaluate the efficacy of M2e supplementation. M2e antigen supplemented in oil emulsion vaccine was highly immunogenic, and a single M2e-supplemented vaccination reduced challenge virus replication and shedding more effectively than non-supplemented vaccination.

Sublingual immunization with recombinant adenovirus encoding SARS-CoV spike protein induces systemic and mucosal immunity without redirection of the virus to the brain.

BACKGROUND: Sublingual (s.l.) administration of soluble protein antigens, inactivated viruses, or virus-like particles has been shown to induce broad immune responses in mucosal and extra-mucosal tissues. Recombinant replication-defective adenovirus vectors (rADVs) infect mucosa surface and therefore can serve as a mucosal antigen delivery vehicle. In this study we examined whether s.l. immunization with rADV encoding spike protein (S) (rADV-S) of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) induces protective immunity against SARS-CoV and could serve as a safe mucosal route for delivery of rADV. RESULTS: Here, we show that s.l. administration of rADV-S induced serum SARS-CoV neutralizing and airway IgA antibodies in mice. These antibody responses are comparable to those induced by intranasal (i.n.) administration. In addition, s.l. immunization induced antigen-specific CD8+ T cell responses in the lungs that are superior to those induced by intramuscular immunization. Importantly, unlike i.n. administration, s.l. immunization with rADV did not redirect the rADV vector to the olfactory bulb. CONCLUSION: Our study indicates that s.l. immunization with rADV-S is safe and effective in induction of a broad spectrum of immune responses and presumably protection against infection with SARS-CoV.

Dual role of respiratory syncytial virus glycoprotein fragment as a mucosal immunogen and chemotactic adjuvant.

Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract disease in infancy and early childhood. Despite its importance as a pathogen, there is no licensed vaccine to prevent RSV infection. The G glycoprotein of RSV, a major attachment protein, is a potentially important target for protective antiviral immune responses and has been shown to exhibit chemotactic activity through CX3C mimicry. Here, we show that sublingual or intranasal immunization of a purified G protein fragment of amino acids from 131 to 230, designated Gcf, induces strong serum IgG and mucosal IgA responses. Interestingly, these antibody responses could be elicited by Gcf even in the absence of any adjuvant, indicating a novel self-adjuvanting property of our vaccine candidate. Gcf exhibited potent chemotactic activity in in vitro cell migration assay and cysteine residues are necessary for chemotactic activity and self-adjuvanticity of Gcf in vivo. Mucosal immunization with Gcf also provides protection against RSV challenge without any significant lung eosinophilia or vaccine-induced weight loss. Together, our data demonstrate that mucosal administration of Gcf vaccine elicits beneficial protective immunity and represents a promising vaccine regimen preventing RSV infection.

Evaluation of protective efficacy of respiratory syncytial virus vaccine against A and B subgroup human isolates in Korea.

Human respiratory syncytial virus (HRSV) is a significant cause of upper and lower respiratory tract illness mainly in infants and young children worldwide. HRSV is divided into two subgroups, HRSV-A and HRSV-B, based on sequence variation within the G gene. Despite its importance as a respiratory pathogen, there is currently no safe and effective vaccine for HRSV. In this study, we have detected and identified the HRSV by RT-PCR from nasopharyngeal aspirates of Korean pediatric patients. Interestingly, all HRSV-B isolates exhibited unique deletion of 6 nucleotides and duplication of 60 nucleotides in the G gene. We successfully amplified two isolates ('KR/A/09-8' belonging to HRSV-A and 'KR/B/10-12' to HRSV-B) on large-scale, and evaluated the cross-protective efficacy of our recombinant adenovirus-based HRSV vaccine candidate, rAd/3xG, by challenging the immunized mice with these isolates. The single intranasal immunization with rAd/3xG protected the mice completely from KR/A/09-8 infection and partially from KR/B/10-12 infection. Our study contributes to the understanding of the genetic characteristics and distribution of subgroups in the seasonal HRSV epidemics in Korea and, for the first time, to the evaluation of the cross-protective efficacy of RSV vaccine against HRSV-A and -B field-isolates.

Intranasal immunization with plasmid DNA encoding spike protein of SARS-coronavirus/polyethylenimine nanoparticles elicits antigen-specific humoral and cellular immune responses.

BACKGROUND: Immunization with the spike protein (S) of severe acute respiratory syndrome (SARS)-coronavirus (CoV) in mice is known to produce neutralizing antibodies and to prevent the infection caused by SARS-CoV. Polyethylenimine 25K (PEI) is a cationic polymer which effectively delivers the plasmid DNA. RESULTS: In the present study, the immune responses of BALB/c mice immunized via intranasal (i.n.) route with SARS DNA vaccine (pci-S) in a PEI/pci-S complex form have been examined. The size of the PEI/pci-S nanoparticles appeared to be around 194.7 ± 99.3 nm, and the expression of the S mRNA and protein was confirmed in vitro. The mice immunized with i.n. PEI/pci-S nanoparticles produced significantly (P < 0.05) higher S-specific IgG1 in the sera and mucosal secretory IgA in the lung wash than those in mice treated with pci-S alone. Compared to those in mice challenged with pci-S alone, the number of B220+ cells found in PEI/pci-S vaccinated mice was elevated. Co-stimulatory molecules (CD80 and CD86) and class II major histocompatibility complex molecules (I-Ad) were increased on CD11c+ dendritic cells in cervical lymph node from the mice after PEI/pci-S vaccination. The percentage of IFN-γ-, TNF-α- and IL-2-producing cells were higher in PEI/pci-S vaccinated mice than in control mice. CONCLUSION: These results showed that intranasal immunization with PEI/pci-S nanoparticles induce antigen specific humoral and cellular immune responses.

Tumor angiogenesis promoted by ex vivo differentiated endothelial progenitor cells is effectively inhibited by an angiogenesis inhibitor, TK1-2.

Neovascularization plays a critical role in the growth and metastatic spread of tumors and involves recruitment of circulating endothelial progenitor cells (EPC) from bone marrow as well as sprouting of preexisting endothelial cells. In this study, we examined if EPCs could promote tumor angiogenesis and would be an effective cellular target for an angiogenesis inhibitor, the recombinant kringle domain of tissue-type plasminogen activator (TK1-2). When TK1-2 was applied in the ex vivo culture of EPCs isolated from human cord blood, TK1-2 inhibited adhesive differentiation of mononuclear EPCs into endothelial-like cells. In addition, it inhibited the migration of ex vivo cultivated EPCs and also inhibited their adhesion to fibronectin matrix or endothelial cell monolayer. When A549 cancer cells were coimplanted along with ex vivo cultivated EPCs s.c. in nude mice, the tumor growth was increased. However, the tumor growth and the vascular density of tumor tissues increased by coimplanted EPCs were decreased upon TK1-2 treatment. Accordingly, TK1-2 treatment reduced the remaining number of EPCs in tumor tissues and their incorporation into the host vascular channels. In addition, overall expression levels of vascular endothelial growth factor (VEGF) and von Willebrand factor in tumor tissues were decreased upon TK1-2 treatment. Interestingly, strong VEGF expression by implanted EPCs was decreased by TK1-2. Finally, we confirmed in vitro that TK1-2 inhibited VEGF secretion of EPCs. TK1-2 also inhibited endothelial cell proliferation and migration induced by the conditioned medium of EPCs. Therefore, we concluded that EPCs, as well as mature endothelial cells, could be an important target of TK1-2.

The recombinant kringle domain of urokinase plasminogen activator inhibits in vivo malignant glioma growth.

In a previous report, the recombinant kringle domain (UK1) of the urokinase type plasminogen activator (uPA) showed antiangiogenic activity. Here, we investigated in vivo antitumor effects of the UK1 of human uPA employing a brain tumor model. The systemic administration of UK1 purified from pichia expression (10 and 50 mg/kg/day intraperitoneally for 25 days) led to suppress the growth of a U87 human glioma xenograft, implanted into the brains of male BALB/cSlc nude mice, by 35% and 80%, respectively. In the immunohistochemical analysis, the tumors treated with UK1 showed decreased vascularity and expression of angiogenesis-related factors including vascular endothelial growth factor (VEGF), angiogenin, alpha-smooth muscle actin, von Willebrand's factor, and CD31 (PECAM-1 [Platelet endothelial cell adhesion molecule-1]), and increased apoptosis. UKl inhibited the in vitro proliferation and tube formation of VEGF-stimulated endothelial cells but not the proliferation of glioma cells. These results suggest that UK1 inhibits the malignant glioma growth by suppression of angiogenesis.

Potent anti-tumor and prolonged survival effects of E. coli-derived non-glycosylated kringle domain of tissue-type plasminogen activator.

The two-kringle domain of tissue-type plasminogen activator (TK1-2) has been identified as a novel angiogenesis inhibitor. In the previous study, purified Pichia-derived TK1-2 has been shown to suppress in vivo growth of human lung and colon cancer cells. Here, we demonstrate that E. coli-derived non-glycosylated TK1-2 suppresses tumor growth more potently than Pichia-derived TK1-2 and prolongs the survival of tumor bearing mice. The recombinant TK1-2 prepared through E. coli expression, His-tag affinity chromatography and in vitro refolding was injected intraperitoneally once daily into nude mice 7 days after subcutaneous implantation with PC14 lung cancer cells (n=10). Measurement of tumor volumes indicated that low-dose TK1-2 treatment (10 mg/kg) suppressed tumor growth by approximately 85.2% (p<0.01), while high-dose TK1-2 treatment (50 mg/kg) even more potently inhibited tumor growth (>93.8%) (p<0.005). Treatment of TK1-2 also prolonged the survival of tumor-bearing mice in a dose-dependent fashion. In an independent HCT116 xenograft model, E. coli-derived TK1-2 was more effective in suppressing tumor growth than Pichia-derived TK1-2. Immunohistochemical analysis of tumor tissue also revealed that the expression of VEGF, SMA-alpha, TNF-alpha and angiogenin was less positive in the E. coli-derived TK1-2-treated group than in the Pichia-derived TK1-2-treated group. These results suggest that E. coli-derived refolded, non-glycosylated TK1-2 can be used more effectively as an anti-cancer agent.

The kringle domain of tissue-type plasminogen activator inhibits in vivo tumor growth.

The two-kringle domain of tissue-type plasminogen activator (t-PA) has previously been shown to contain anti-angiogenesis activity. In this study, we explored the potential in vivo anti-tumor effects of the recombinant kringle domain (TK1-2) of human t-PA. Anti-tumor effects of purified Pichia-driven TK1-2 were examined in nude mice models by subcutaneous implantation of human lung (A-549) and colon (DLD-1, HCT-116) cancer cell lines. Mice bearing the tumors were injected with PBS or purified TK1-2 (30 mg/kg) i.p. every day for 22 days. TK1-2 treatment suppressed the A-549, DLD-1, and HCT-116 tumor growth by 85.3%, 52.4%, and 62.5%, respectively. Immunohistological examination of the tumor tissues showed that TK1-2 treatment decreased the vessel density and also the expression of angiogenesis-related factors including angiogenin, VEGF, alpha-SMA, vWF, and TNF-alpha, and increased the apoptotic fraction of cells. TK1-2 neither inhibited in vitro growth of these cancer cells nor affected t-PA-mediated fibrin clot lysis. These results suggest that TK1-2 inhibits the tumor growth by suppression of angiogenesis without interfering with fibrinolysis.