Yanbing Ma

Immunization against multidrug-resistant Acinetobacter baumannii effectively protects mice in both pneumonia and sepsis models.

Objective Acinetobacter baumannii is considered the prototypical example of a multi- or pan- drug-resistant bacterium. It has been increasingly implicated as a major cause of nosocomial and community-associated infections. This study proposed to evaluate the efficacy of immunological approaches to prevent and treat A. baumannii infections. Methods Mice were immunized with outer membrane vesicles (OMVs) prepared from a clinically isolated multidrug-resistant strain of A. baumannii. Pneumonia and sepsis models were used to evaluate the efficacy of active and passive immunization with OMVs. The probable effective mechanisms and the protective potential of clonally distinct clinical isolates were investigated in vitro using an opsonophagocytic assay. Results Intramuscular immunization with OMVs rapidly produced high levels of OMV-specific IgG antibodies, and subsequent intranasal challenge with A. baumannii elicited mucosal IgA and IgG responses. Both active and passive immunization protected the mice from challenges with homologue bacteria in a sepsis model. Bacterial burden in bronchoalveolar lavage fluids (BALF), lung, and spleen, inflammatory cell infiltration in BALF and lung, and inflammatory cytokine accumulation in BALF was significantly suppressed in the pneumonia model by both active and passive immunization strategies. The antisera from immunized mice presented with significant opsonophagocytic activities in a dose-dependent manner against not only homologous strains but also five of the other six clonally distinct clinical isolates. Conclusions Utilizing immunological characteristics of outer membrane proteins to elevate protective immunity and circumvent complex multidrug-resistance mechanisms might be a viable approach to effectively control A. baumannii infections.

Immunization with a 22-kDa outer membrane protein elicits protective immunity to multidrug-resistant Acinetobacter baumannii

A. baumannii infections are becoming more and more serious health issues with rapid emerging of multidrug and extremely drug resistant strains, and therefore, there is an urgent need for the development of nonantibiotic-based intervention strategies. This study aimed at identifying whether an outer membrane protein with molecular weight of about 22 kDa (Omp22) holds the potentials to be an efficient vaccine candidate and combat A. baumannii infection. Omp22 which has a molecule length of 217 amino acids kept more than 95% conservation in totally 851 reported A. baumannii strains. Recombinant Omp22 efficiently elicited high titers of specific IgG in mice. Both active and passive immunizations of Omp22 increased the survival rates of mice, suppressed the bacterial burdens in the organs and peripheral blood, and reduced the levels of serum inflammatory cytokines and chemokines. Opsonophagocytosis assays showed in vitro that Omp22 antiserum had highly efficient bactericidal activities on clonally distinct clinical A. baumannii isolates, which were partly complements-dependent and opsonophagocytic killing effects. Additionally, administration with as high as 500 μg of Omp22 didn’t cause obvious pathological changes in mice. In conclusion, Omp22 is a novel conserved and probably safe antigen for developing effective vaccines or antisera to control A. baumannii infections.

Employing Escherichia coli-derived outer membrane vesicles as an antigen delivery platform elicits protective immunity against Acinetobacter baumannii infection.

Outer membrane vesicles (OMVs) have proven to be highly immunogenic and induced an immune response against bacterial infection in human clinics and animal models. We sought to investigate whether engineered OMVs can be a feasible antigen-delivery platform for efficiently inducing specific antibody responses. In this study, Omp22 (an outer membrane protein of A. baumannii) was displayed on E. coli DH5α-derived OMVs (Omp22-OMVs) using recombinant gene technology. The morphological features of Omp22-OMVs were similar to those of wild-type OMVs (wtOMVs). Immunization with Omp22-OMVs induced high titers of Omp22-specific antibodies. In a murine sepsis model, Omp22-OMV immunization significantly protected mice from lethal challenge with a clinically isolated A. baumannii strain, which was evidenced by the increased survival rate of the mice, the reduced bacterial burdens in the lung, spleen, liver, kidney, and blood, and the suppressed serum levels of inflammatory cytokines. In vitro opsonophagocytosis assays showed that antiserum collected from Omp22-OMV-immunized mice had bactericidal activity against clinical isolates, which was partly specific antibody-dependent. These results strongly indicated that engineered OMVs could display a whole heterologous protein (~22 kDa) on the surface and effectively induce specific antibody responses, and thus OMVs have the potential to be a feasible vaccine platform.

HPV-16 E6 and E7 protein T cell epitopes prediction analysis based on distributions of HLA-A loci across populations: an in silico approach.

Human papillomavirus type 16 (HPV-16) is the most prevalent virus in human cervical cancers, as it is present in more than half of all cases. Many studies have found continued expression of E6 and E7 proteins in the majority of cervical cancer cases, but not in normal tissues. These results indicated that the E6 and E7 proteins could be ideal candidate therapeutic vaccines against HPV-16 infection and cervical cancer. Using the Immune Epitope Database Analysis Resource, cytotoxic T lymphocyte (CTL) epitopes of the HPV-16 E6 and E7 proteins were predicted according to worldwide frequency distributions of HLA-A alleles (HLA-A*01:01, -A*02:01, -A*02:06, -A*03:01, -A*11:01, -A*24:02, -A*26:01, -A*31:01 and -A*33:03). Our results predicted a total of 81 epitopes of HPV-16 E6 (n=59) and E7 (n=22). Epitope cluster analysis showed that among the 20 clusters of HPV-16 E6, cluster 3 contained the most epitopes (10 epitopes), which was represented by HLA-A*31:01 and -A*33:03. Of the 10 clusters of HPV-16 E7, cluster 3 contained the most epitopes (5 epitopes), which was represented by HLA-A*01:01 and -A*26:01. Our results indicated that the combination of epitopes FAFRDLCIVYR₅₂₋₆₂ of E6 (HLA-A*02:06, HLA-A*31:01, and HLA-A*33:03), PYAVCDKCLKF₆₆₋₇₆ of E6 (HLA-A*11:01 and HLA-A*24:02), HGDTPTLHEY₂₋₁₁ of E7 (HLA-A*01:01 and HLA-A*26:01), and YMLDLQPETT₁₁₋₂₀ of E7 (HLA-A*02:01) could vaccinate >50% of all individuals worldwide. Our results propose CTL epitopes or combinations of them predicted in current study for candidate therapeutic vaccines to effectively control HPV-16 infection and development of cervical cancer.

OmpW is a potential target for eliciting protective immunity against Acinetobacter baumannii infections

Acinetobacter baumannii (A. baumannii) is an important conditioned pathogen that causes nosocomial and community-associated infections. In this study, we sought to investigate whether outer membrane protein W (OmpW) is a potential target for eliciting protective immunity against A. baumannii infections. Mice immunized with the fusion protein thioredoxin-OmpW generated strong OmpW-specific IgG responses. In a sepsis model, both active and passive immunizations against OmpW effectively protected mice from A. baumannii infections. This protection was demonstrated by a significantly improved survival rate, reduced bacterial burdens within organs, and the suppressed accumulation of inflammatory cytokines and chemokines in sera. Opsonophagocytic assays with murine macrophage RAW264.7 cells indicated that the bactericidal effects of the antisera derived from the immunized mice are mediated synergistically by specific antibodies and complement components. The antisera presented significant opsonophagocytic activities against homologous strains and clonally distinct clinical isolates in vitro. Protein data analysis showed that the sequence of OmpW, which has a molecule length of 183 amino acids, is more than 91% conserved in reported A. baumannii strains. In conclusion, we identified OmpW as a highly immunogenic and conserved protein as a valuable antigen candidate for the development of an effective vaccine or the preparation of antisera to control A. baumannii infections.

Virus-like particles presenting interleukin-33 molecules: Immunization characteristics and potentials of blocking IL-33/ST2 pathway in allergic airway inflammation

We sought to develop an IL-33 vaccine and evaluate its efficacy in a mouse model of asthma. The full-length molecules of putative mature IL-33 were inserted into the immunodominant epitope region of hepatitis B core antigen using gene recombination techniques. The expressed chimeric protein presented as virus-like particles (VLPs) under observation using an electron microscopy. To investigate immunization characteristics of the VLPs, mice were immunized by using different doses, adjuvants, and routes. The VLPs induced sustained and high titers of IL-33-specific IgG and IgA even without the use of a conventional adjuvant, and the lowered ratio of IgG1/IgG2a in vaccinated mice indicated a shift from Th2 to Th1-like responses. To assess the vaccine effects on blocking the signaling of IL-33/ST2 pathway, mice receiving 3 vaccinations subjected to intraperitoneal sensitization and intranasal challenge with ovalbumin (OVA). Control animals received carrier or PBS in place of the vaccine. Immunization with the VLPs significantly suppressed inflammatory cell number and IL-33 level in BALF. OVA -induced goblet cell hyperplasia and lung tissue inflammatory cell infiltration were significantly suppressed in vaccinated mice. Our data indicate that IL-33 molecule-based vaccine, which may block IL-33/ST2 signaling pathway on a persistent basis, holds potential for treatment of asthma and, by extension, other diseases where overexpressed IL-33 plays a pivotal role in pathogenesis.

Chimeric HBcAg virus-like particles presenting a HPV 16 E7 epitope significantly suppressed tumor progression through preventive or therapeutic immunization in a TC-1-grafted mouse model

Background Therapeutic human papillomavirus (HPV) vaccines are currently being developed. However, no therapeutic efficacy has been achieved in clinical trials for the treatment of cervical intraepithelial neoplasia or cancer. One of the important issues in increasing vaccine efficacy is determining the best way to enhance tumor antigen-specific cellular immune responses. This study aimed to explore the virus-like particles (VLPs) of hepatitis B core antigen (HBcAg) as potential therapeutic vaccine carriers and to assess its immunological characteristics. Methods Chimeric VLPs presenting a HPV 16 cytotoxic T lymphocytes epitope E749–57 (amino acid 49–57 of the E7 protein) were prepared using recombinant genes. C57BL/6 mice were immunized with VLPs and grafted with tumor cells TC-1 which is an E7-expressing tumorigenic cell line. The dynamic tumor growth was monitored and anti-tumor immune responses were investigated. Results Using a preventive strategy, immunization with VLPs resulted in nearly complete suppression of tumor growth. In treatment studies, VLP immunization significantly suppressed the tumor progression in mice carrying 2–3 mm tumors and in those bearing even larger tumors with diameters up to 8–9 mm. The VLP structure was shown to be important to induce vigorous antitumor immunity and effects. In immunized mice, enhanced E749–57-specific cellular immune responses were evidenced by increased interferon (IFN)-γ expression and decreased interleukin (IL)-4 expression in splenic lymphocytes, as well as an elevated number of effector cells expressing IFN-γ in response to the in vitro stimulation of the specific peptide E749–57. In addition, effective immune memory after VLP immunization was maintained for at least 16 weeks, preventing significant tumor growth after subsequent TC-1 challenge. Conclusion While VLPs were highly immunogenic in stimulating humoral immunity, our results strongly indicated that VLPs, such as HBcAg particles, might also be potent therapeutic vaccine carriers to elicit robust cellular immune responses, even in the immunosuppressive microenvironment of a tumor.

Diverse expression levels of two codon-optimized genes that encode human papilloma virus type 16 major protein L1 in Hansenula polymorpha.

AbstractTwo versions of an optimized gene that encodes human papilloma virus type 16 major protein L1 were designed according to the codon usage frequency of Pichia pastoris. Y16 was highly expressed in both P. pastoris and Hansenula polymorpha. M16 expression was as efficient as that of Y16 in P. pastoris, but merely detectable in H. polymorpha even though transcription levels of M16 and Y16 were similar. H. polymorpha had a unique codon usage frequency that contains many more rare codons than Saccharomyces cerevisiae or P. pastoris. These findings indicate that even codon-optimized genes that are expressed well in S. cerevisiae and P. pastoris may be inefficiently expressed in H. polymorpha; thus rare codons must be avoided when universal optimized gene versions are designed to facilitate expression in a variety of yeast expression systems, especially H. polymorpha is involved.

Immunization against TGF-β1 reduces collagen deposition but increases sustained inflammation in a murine asthma model

ABSTRACT Transforming growth factor (TGF)-β1 is involved in the processes of airway inflammation and remodeling; however, its reported roles in asthma pathogenesis are controversial. We sought both to investigate the effects of active immunization targeting TGF-β1 on allergen-induced airway inflammatory responses and to evaluate its possible application for asthma treatment. BALB/c mice were immunized with a virus-like-particle (VLP) vaccine presenting a TGF-β1 peptide. For the preventive intervention of acute allergic airway inflammation, immunization was conducted before sensitization and challenges with ovalbumin (OVA), and for the therapeutic treatment of chronic inflammatory responses, immunization was initiated after inflammatory responses were established. Preventive immunization with VLPs led to increased proinflammatory IL-4, IL-13, and IL-33 levels in the bronchoalveolar lavage fluids (BALF) with no significant effects on lung tissue inflammation and airway goblet cell hyperplasia. Therapeutic treatment showed that at 24 h after the fourth 2-day challenge with OVA following 2 intraperitoneal sensitizations, airway subepithelial collagen deposition was significantly ameliorated in vaccinated mice, whereas the lung histology and cytokine profile in the BALF were not changed. In contrast, after a 4-week recovery from the last OVA challenge, the vaccinated mices collagen deposition remained reduced, but they sustained lung-tissue inflammation and goblet-cell hyperplasia; elevated IL-13, TNF, and IFN-γ levels in the BALF; and increased airway resistance, tissue resistance, and tissue elastance. In a conclusion, the role of TGF-β1 is complicated in allergic airway inflammatory responses. It is important to make a careful assessment in accordance with specific disease conditions when targeting TGF-β1 for a therapeutic purpose.

Transfection reagent Lipofectamine triggers type I interferon signaling activation in macrophages

The commercial transfection reagent Lipofectamine has been widely used for cytoplasmic delivery of nucleic acids and for cytosolic engagement with intracellular innate immune sensors to trigger type I interferon (IFN) production. However, the effect of Lipofectamine alone on type I IFN response has not been studied in detail. Here, we show that Lipofectamine induced type I IFN signaling in both RAW 264.7 macrophage‐like cells and primary bone marrow‐derived macrophages. Type I IFN induction was dependent on interferon regulatory factor (IRF)3 and IRF7 and partially required the toll/interleukin‐1 receptor‐domain‐containing adapter‐inducing interferon‐β. In contrast, the transfection reagent Xfect did not activate type I IFN signaling. Our study highlights the potential confounding experimental interpretation when using Lipofectamine‐based transfection for delivering intracellular ligands and provides important insights into lipid signaling in innate immune responses.