Expression, purification, and characterization of the Necator americanus aspartic protease-1 (Na-APR-1 (M74)) antigen, a component of the bivalent human hookworm vaccine.

PubMed

Seid, Christopher A; Curti, Elena; Jones, R Mark; Hudspeth, Elissa; Rezende, Wanderson; Pollet, Jeroen; Center, Lori; Versteeg, Leroy; Pritchard, Sonya; Musiychuk, Konstantin; Yusibov, Vidadi; Hotez, Peter J; Bottazzi, Maria Elena

2015-01-01

Over 400 million people living in the world's poorest developing nations are infected with hookworms, mostly of the genus Necator americanus. A bivalent human hookworm vaccine composed of the Necator americanus Glutathione S-Transferase-1 (Na-GST-1) and the Necator americanus Aspartic Protease-1 (Na-APR-1 (M74)) is currently under development by the Sabin Vaccine Institute Product Development Partnership (Sabin PDP). Both monovalent vaccines are currently in Phase 1 trials. Both Na-GST-1 and Na-APR-1 antigens are expressed as recombinant proteins. While Na-GST-1 was found to express with high yields in Pichia pastoris, the level of expression of Na-APR-1 in this host was too low to be suitable for a manufacturing process. When the tobacco plant Nicotiana benthamiana was evaluated as an expression system, acceptable levels of solubility, yield, and stability were attained. Observed expression levels of Na-APR-1 (M74) using this system are ∼300 mg/kg. Here we describe the achievements and obstacles encountered during process development as well as characterization and stability of the purified Na-APR-1 (M74) protein and formulated vaccine. The expression, purification and analysis of purified Na-APR-1 (M74) protein obtained from representative 5 kg reproducibility runs performed to qualify the Na-APR-1 (M74) production process is also presented. This process has been successfully transferred to a pilot plant and a 50 kg scale manufacturing campaign under current Good Manufacturing Practice (cGMP) has been performed. The 50 kg run has provided a sufficient amount of protein to support the ongoing hookworm vaccine development program of the Sabin PDP.

Lysosome-associated membrane glycoprotein (LAMP) – preliminary study on a hidden antigen target for vaccination against schistosomiasis

PubMed Central

Nawaratna, Sujeevi S. K.; Gobert, Geoffrey N.; Willis, Charlene; Mulvenna, Jason; Hofmann, Andreas; McManus, Donald P.; Jones, Malcolm K.

2015-01-01

Our previously reported gene atlasing of schistosome tissues revealed transcripts that were highly enriched in the digestive tract of Schistosoma mansoni. From these, we selected two candidates, Sm-LAMP and Sm-NPC2 for testing as vaccine targets. The two molecules were selected on the basis of relatively high expression in the gastrodermis, their potentially important biological function, divergence from homologous molecules of the host and possible apical membrane expression in the gastrodermis. Bacterially expressed recombinant peptides corresponding to regions excluding trans-membrane domains of the selected vaccine targets were used in blinded vaccine trials in CBA mice using alum-CpG as adjuvant. Vaccine trials using the recombinant insoluble Sm-LAMP protein showed 16–25% significant reduction in total worm burden. Faecal egg count reduction was 52% and 60% in two trials, respectively, with similar results for the solubly expressed protein. Liver egg burden was reduced significantly (20% and 38%) with an insoluble recombinant Sm-LAMP in two trials, but not with the soluble recombinant form. Parasite fecundity was not affected by either Sm-LAMP protein preparations in the trials. It is concluded that Sm-LAMP may provide limited protection towards S. mansoni infections but could be used in combination with other vaccine candidates, to provide more comprehensive protection. PMID:26472258

Design of Fusion Proteins for Efficient and Soluble Production of Immunogenic Ebola Virus Glycoprotein in Escherichia coli.

PubMed

Ji, Yang; Lu, Yuan; Yan, Yishu; Liu, Xinxin; Su, Nan; Zhang, Chong; Bi, Shengli; Xing, Xin-Hui

2018-03-03

The Ebola hemorrhagic fever caused by Ebola virus is an extremely dangerous disease, and effective therapeutic agents are still lacking. Platforms for the efficient production of vaccines are crucial to ensure quick response against an Ebola virus outbreak. Ebola virus glycoprotein (EbolaGP) on the virion surface is responsible for membrane binding and virus entry, thus becoming the key target for vaccine development. However, heterologous expression of this protein still faces engineering challenges such as low production levels and insoluble aggregation. Here, the authors design and compare various fusion strategies, attaching great importance to the solubility-enhancing effect, and tag removal process. It is found that a C-terminal intein-based tag greatly enhances the solubility of EbolaGP and allows one-step chromatographic purification of the untagged EbolaGP through thiol-catalyzed self-cleavage. The purified untagged EbolaGP alone or with Freund's adjuvant are highly immunogenic, as confirmed in a mouse model. Consequently, the present study puts forward a new strategy for the efficient and soluble expression of untagged immunogenic EbolaGP. The intein-based protein fusion approach may be of importance for the large-scale production of Ebola virus subunit vaccine. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

HER-2 Pulsed Dendritic Cell Vaccine Can Eliminate HER-2 Expression and Impact DCIS

PubMed Central

Sharma, Anupama; Koldovsky, Ursula; Xu, Shuwen; Mick, Rosemarie; Roses, Robert; Fitzpatrick, Elizabeth; Weinstein, Susan; Nisenbaum, Harvey; Levine, Bruce L; Fox, Kevin; Zhang, Paul; Koski, Gary; Czerniecki, Brian J

2011-01-01

Background HER-2/neu over-expression plays a critical role in breast cancer development and its expression in ductal carcinoma in situ (DCIS) is associated with development of invasive breast cancer. A vaccine targeting HER-2/neu expression in DCIS may initiate immunity against invasive cancer. Methods A HER-2/neu dendritic cell (DC) vaccine was administered to 27 patients with HER-2/neu over-expressing DCIS. The HER-2/neu vaccine was administered prior to surgical resection and pre- and post-vaccination analysis was conducted to assess clinical results. Results At surgery, 5 of 27 (18.5%) vaccinated subjects had no evidence of remaining disease, while among 22 subjects with residual DCIS, HER-2/neu expression was eradicated in 11 (50%). When comparing ERneg with ERpos DCIS lesions, vaccination was more effective in hormone-independent DCIS. Following vaccination, no residual DCIS was found in 40% of ERneg subjects compared to 5.9% in ERpos subject. Sustained HER-2/neu expression was found in 10% of ERneg subjects compared to 47.1% in ERpos subjects (p=0.04). Post-vaccination phenotypes were significantly different between ERpos and ERneg subjects (p=0.01), with 7 of 16 (43.8%) initially presenting with ERpos HER-2/neupos Luminal B phenotype finishing with the ERpos HER-2/neuneg Luminal A phenotype, and 3 of 6 (50%) with the ERneg HER-2/neupos phenotype changing to the ERneg HER-2/neuneg phenotype. Conclusions Results suggest vaccination against HER-2/neu is safe, well-tolerated and induces decline and or eradication of HER-2/neu expression. These findings warrant further exploration of HER-2/neu vaccination in estrogen-independent breast cancer and highlight the need to target additional tumor associated antigens and pathways. PMID:22252842

Intradermal Vaccination With Adjuvanted Ebola Virus Soluble Glycoprotein Subunit Vaccine by Microneedle Patches Protects Mice Against Lethal Ebola Virus Challenge.

PubMed

Liu, Ying; Ye, Ling; Lin, Fang; Gomaa, Yasmine; Flyer, David; Carrion, Ricardo; Patterson, Jean L; Prausnitz, Mark R; Smith, Gale; Glenn, Gregory; Wu, Hua; Compans, Richard W; Yang, Chinglai

2018-06-08

In this study, we investigated immune responses induced by purified Ebola virus (EBOV) soluble glycoprotein (sGP) subunit vaccines via intradermal immunization with microneedle (MN) patches in comparison with intramuscular (IM) injection in mice. Our results showed that MN delivery of EBOV sGP was superior to IM injection in eliciting higher levels and longer lasting antibody responses against EBOV sGP and GP antigens. Moreover, sGP-specific immune responses induced by MN or IM immunizations were effectively augmented by formulating sGP with a saponin-based adjuvant, and they were shown to confer complete protection of mice against lethal mouse-adapted EBOV (MA-EBOV) challenge. In comparison, mice that received sGP without adjuvant by MN or IM immunizations succumbed to lethal MA-EBOV challenge. These results show that immunization with EBOV sGP subunit vaccines with adjuvant by MN patches, which have been shown to provide improved safety and thermal stability, is a promising approach to protect against EBOV infection.

Potentiation of anthrax vaccines using protective antigen-expressing viral replicon vectors.

PubMed

Wang, Hai-Chao; An, Huai-Jie; Yu, Yun-Zhou; Xu, Qing

2015-02-01

DNA vaccines require improvement for human use because they are generally weak stimulators of the immune system in humans. The efficacy of DNA vaccines can be improved using a viral replicon as vector to administer antigen of pathogen. In this study, we comprehensively evaluated the conventional non-viral DNA, viral replicon DNA or viral replicon particles (VRP) vaccines encoding different forms of anthrax protective antigen (PA) for specific immunity and protective potency against anthrax. Our current results clearly suggested that these viral replicon DNA or VRP vaccines derived from Semliki Forest virus (SFV) induced stronger PA-specific immune responses than the conventional non-viral DNA vaccines when encoding the same antigen forms, which resulted in potent protection against challenge with the Bacillus anthracis strain A16R. Additionally, the naked PA-expressing SFV replicon DNA or VRP vaccines without the need for high doses or demanding particular delivery regimens elicited robust immune responses and afforded completely protective potencies, which indicated the potential of the SFV replicon as vector of anthrax vaccines for use in clinical application. Therefore, our results suggest that these PA-expressing SFV replicon DNA or VRP vaccines may be suitable as candidate vaccines against anthrax. Copyright © 2015 Elsevier B.V. All rights reserved.

Vaccine-induced modulation of gene expression in turbot peritoneal cells. A microarray approach.

PubMed

Fontenla, Francisco; Blanco-Abad, Verónica; Pardo, Belén G; Folgueira, Iria; Noia, Manuel; Gómez-Tato, Antonio; Martínez, Paulino; Leiro, José M; Lamas, Jesús

2016-07-01

We used a microarray approach to examine changes in gene expression in turbot peritoneal cells after injection of the fish with vaccines containing the ciliate parasite Philasterides dicentrarchi as antigen and one of the following adjuvants: chitosan-PVMMA microspheres, Freund́s complete adjuvant, aluminium hydroxide gel or Matrix-Q (Isconova, Sweden). We identified 374 genes that were differentially expressed in all groups of fish. Forty-two genes related to tight junctions and focal adhesions and/or actin cytoskeleton were differentially expressed in free peritoneal cells. The profound changes in gene expression related to cell adherence and cytoskeleton may be associated with cell migration and also with the formation of cell-vaccine masses and their attachment to the peritoneal wall. Thirty-five genes related to apoptosis were differentially expressed. Although most of the proteins coded by these genes have a proapoptotic effect, others are antiapoptotic, indicating that both types of signals occur in peritoneal leukocytes of vaccinated fish. Interestingly, many of the genes related to lymphocytes and lymphocyte activity were downregulated in the groups injected with vaccine. We also observed decreased expression of genes related to antigen presentation, suggesting that macrophages (which were abundant in the peritoneal cavity after vaccination) did not express these during the early inflammatory response in the peritoneal cavity. Finally, several genes that participate in the inflammatory response were differentially expressed, and most participated in resolution of inflammation, indicating that an M2 macrophage response is generated in the peritoneal cavity of fish one day post vaccination. Copyright © 2016 Elsevier Ltd. All rights reserved.

Tandem SUMO fusion vectors for improving soluble protein expression and purification.

PubMed

Guerrero, Fernando; Ciragan, Annika; Iwaï, Hideo

2015-12-01

Availability of highly purified proteins in quantity is crucial for detailed biochemical and structural investigations. Fusion tags are versatile tools to facilitate efficient protein purification and to improve soluble overexpression of proteins. Various purification and fusion tags have been widely used for overexpression in Escherichia coli. However, these tags might interfere with biological functions and/or structural investigations of the protein of interest. Therefore, an additional purification step to remove fusion tags by proteolytic digestion might be required. Here, we describe a set of new vectors in which yeast SUMO (SMT3) was used as the highly specific recognition sequence of ubiquitin-like protease 1, together with other commonly used solubility enhancing proteins, such as glutathione S-transferase, maltose binding protein, thioredoxin and trigger factor for optimizing soluble expression of protein of interest. This tandem SUMO (T-SUMO) fusion system was tested for soluble expression of the C-terminal domain of TonB from different organisms and for the antiviral protein scytovirin. Copyright © 2015 Elsevier Inc. All rights reserved.

Recombinant vaccines against T. gondii: comparison between homologous and heterologous vaccination protocols using two viral vectors expressing SAG1.

PubMed

Mendes, Érica Araújo; Fonseca, Flavio G; Casério, Bárbara M; Colina, Janaína P; Gazzinelli, Ricardo Tostes; Caetano, Braulia C

2013-01-01

The use of recombinant viral vectors expressing T. gondii antigens is a safe and efficient approach to induce immune response against the parasite and a valuable tool for vaccine development. We have previously protected mice from toxoplasmosis by immunizing the animals with an adenovirus expressing the protein SAG1 (AdSAG1) of T. gondii. We are now looking for ways to improve the vaccination strategy and enhance protection. One limitation of homologous vaccinations (sequential doses of the same vector) is induction of anti-vector immune response that blocks cell transduction, restricts transgene expression and, consequently, compromises the overall outcome of vaccination. One way to avert the effects of anti-vector response is to use different viruses in prime and boost (heterologous vaccination). Bearing this in mind, we generated a modified Vaccinia Virus Ankara encoding SAG1 (MVASAG1), to be tested as boost agent after prime with AdSAG1. Although minor differences were observed in the magnitude of the anti-SAG1 immune response induced by each vaccination protocol, the heterologous immunization with AdSAG1 followed by MVASAG1 resulted in improved capacity to control brain cyst formation in a model of chronic toxoplasmosis in C57BL/6 mice.

Efficacy of a potential trivalent vaccine based on Hc fragments of botulinum toxins A, B, and E produced in a cell-free expression system.

PubMed

Zichel, R; Mimran, A; Keren, A; Barnea, A; Steinberger-Levy, I; Marcus, D; Turgeman, A; Reuveny, S

2010-05-01

Botulinum toxins produced by the anaerobic bacterium Clostridium botulinum are the most potent biological toxins in nature. Traditionally, people at risk are immunized with a formaldehyde-inactivated toxin complex. Second generation vaccines are based on the recombinant carboxy-terminal heavy-chain (Hc) fragment of the neurotoxin. However, the materialization of this approach is challenging, mainly due to the high AT content of clostridial genes. Herein, we present an alternative strategy in which the native genes encoding Hc proteins of botulinum toxins A, B, and E were used to express the recombinant Hc fragments in a cell-free expression system. We used the unique property of this open system to introduce different combinations of chaperone systems, protein disulfide isomerase (PDI), and reducing/oxidizing environments directly to the expression reaction. Optimized expression conditions led to increased production of soluble Hc protein, which was successfully scaled up using a continuous exchange (CE) cell-free system. Hc proteins were produced at a concentration of more than 1 mg/ml and purified by one-step Ni(+) affinity chromatography. Mice immunized with three injections containing 5 microg of any of the in vitro-expressed, alum-absorbed, Hc vaccines generated a serum enzyme-linked immunosorbent assay (ELISA) titer of 10(5) against the native toxin complex, which enabled protection against a high-dose toxin challenge (10(3) to 10(6) mouse 50% lethal dose [MsLD(50)]). Finally, immunization with a trivalent HcA, HcB, and HcE vaccine protected mice against the corresponding trivalent 10(5) MsLD(50) toxin challenge. Our results together with the latest developments in scalability of the in vitro protein expression systems offer alternative routes for the preparation of botulinum vaccine.

Engineering and expression of a human rotavirus candidate vaccine in Nicotiana benthamiana.

PubMed

Pêra, Francisco F P G; Mutepfa, David L R; Khan, Ayesha M; Els, Johann H; Mbewana, Sandiswa; van Dijk, Alberdina A A; Rybicki, Edward P; Hitzeroth, Inga I

2015-12-02

Human rotaviruses are the main cause of severe gastroenteritis in children and are responsible for over 500 000 deaths annually. There are two live rotavirus vaccines currently available, one based on human rotavirus serotype G1P[8], and the other a G1-G4 P[8] pentavalent vaccine. However, the recent emergence of the G9 and other novel rotavirus serotypes in Africa and Asia has prompted fears that current vaccines might not be fully effective against these new varieties. We report an effort to develop an affordable candidate rotavirus vaccine against the new emerging G9P[6] (RVA/Human-wt/ZAF/GR10924/1999/G9P[6]) strain. The vaccine is based on virus-like particles which are both highly immunogenic and safe. The vaccine candidate was produced in Nicotiana benthamiana by transient expression, as plants allow rapid production of antigens at lower costs, without the risk of contamination by animal pathogens. Western blot analysis of plant extracts confirmed the successful expression of two rotavirus capsid proteins, VP2 and VP6. These proteins assembled into VLPs resembling native rotavirus particles when analysed by transmission electron microscopy (TEM). Expression of the rotavirus glycoprotein VP7 and the spike protein VP4 was also tried. However, VP7 expression caused plant wilting during the course of the time trial and expression could never be detected for either protein. We therefore created three fusion proteins adding the antigenic part of VP4 (VP8*) to VP6 in an attempt to produce more appropriately immunogenic particles. Fusion protein expression in tobacco plants was detected by western blot using anti-VP6 and anti-VP4 antibodies, but no regular particles were observed by TEM, even when co-expressed with VP2. Our results suggest that the rotavirus proteins produced in N. benthamiana are candidates for a subunit vaccine specifically for the G9P[6] rotavirus strain. This could be more effective in developing countries, thereby possibly providing a higher

Design and Development of Stable, Water-soluble, Human Toll-like Receptor 2-Specific, Monoacyl Lipopeptides as Candidate Vaccine Adjuvants

PubMed Central

Salunke, Deepak B.; Connelly, Seth W.; Shukla, Nikunj M.; Hermanson, Alec R.; Fox, Lauren M.; David, Sunil A.

2013-01-01

Antigens in modern subunit vaccines are largely soluble and poorly immunogenic proteins inducing relatively short-lived immune responses. Appropriate adjuvants initiate early innate immune responses, amplifying subsequent adaptive immune responses. Agonists of TLR2 are devoid of significant pro-inflammatory activity in ex vivo human blood models, and yet potently adjuvantic, suggesting that this chemotype may be a safe and effective adjuvant. Our earlier work on the monoacyl lipopeptide class of TLR2 agonists led to the design of a highly potent lead, but with negligible aqueous solubility, necessitating the reintroduction of aqueous solubility. We explored several strategies of introducing ionizable groups on the lipopeptide, as well as the systematic evaluation of chemically stable bioisosteres of the ester-linked palmitoyl group. These studies have led to a fully optimized, chemically stable, and highly water-soluble, human TLR2-specific agonist, which was found to have an excellent safety profile and displayed prominent adjuvantic activities in rabbit models. PMID:23795818

[Expression and Preliminary Research on the Soluble Domain of EV-D68 3A Protein].

PubMed

Li, Ting; Kong, Jia; Yu, Xiao-fang; Han, Xue

2015-11-01

To understand the structure of the soluble region of Enterovirus 68 3A protein, we construct a prokaryotic expression vector expressing the soluble region of EV-D68 3A protein, and identify the forms of expression product after purification. The EV-D68 3A(1-61) gene was amplified by PCR and then cloned into the expression vector pET-28a-His-SUMO. The recombinant plasmid was transformed into Escherichia coli BL21 induced by IPTG to express the fusion protein His-SUMO-3A(1-61). The recombinant protein was purified by Ni-NTA Agarose and cleaved by ULP Protease to remove His-SUMO tag. After that, the target protein 3A(1-61) was purified by a series of purification methods such as Ni-NTA, anion exchange chromatography and gel filtration chromato- graphy. Chemical cross-linking reaction assay was taken to determine the multiple polymerization state of the 3A soluble region. A prokaryotic expression vector pET28a-His-SUMO-3A(1-61) expressing the solution region of EV-D68 3A was successfully constructed and plenty of highly pure target proteins were obtained by multiple purification steps . The total protein amount was about 5 mg obtained from 1L Escherichia coli BL21 with purity > 95%. At the same time, those results determined the homomultimer form of soluble 3A construct. These data demonstrated that the expression and purification system of the soluble region of 3A were successfully set up and provide some basic konwledge for the research about 3A crystal structure and the development of antiviral drugs targeted at 3A to block viral replication.

Genetically Engineered Poxviruses for Recombinant Gene Expression, Vaccination, and Safety

NASA Astrophysics Data System (ADS)

Moss, Bernard

1996-10-01

Vaccinia virus, no longer required for immunization against smallpox, now serves as a unique vector for expressing genes within the cytoplasm of mammalian cells. As a research tool, recombinant vaccinia viruses are used to synthesize and analyze the structure--function relationships of proteins, determine the targets of humoral and cell-mediated immunity, and investigate the types of immune response needed for protection against specific infectious diseases and cancer. The vaccine potential of recombinant vaccinia virus has been realized in the form of an effective oral wild-life rabies vaccine, although no product for humans has been licensed. A genetically altered vaccinia virus that is unable to replicate in mammalian cells and produces diminished cytopathic effects retains the capacity for high-level gene expression and immunogenicity while promising exceptional safety for laboratory workers and potential vaccine recipients.

Brucellosis vaccines for livestock.

PubMed

Goodwin, Zakia I; Pascual, David W

2016-11-15

Brucellosis is a livestock disease responsible for fetal loss due to abortions. Worldwide, this disease has profound economic and social impact by reducing the ability of livestock producers to provide an adequate supply of disease-free meat and dairy products. In addition to its presence in domesticated animals, brucellosis is harbored in a number of wildlife species creating new disease reservoirs, which adds to the difficulty of eradicating this disease. Broad and consistent use of the available vaccines would contribute in reducing the incidence of brucellosis. Unfortunately, this practice is not common. In addition, the current brucellosis vaccines cannot provide sterilizing immunity, and in certain circumstances, vaccinated livestock are not protected against co-mingling Brucella-infected wildlife. Given that these vaccines are inadequate for conferring complete protection for some vaccinated livestock, alternatives are being sought, and these include genetic modifications of current vaccines or their reformulations. Alternatively, many groups have sought to develop new vaccines. Subunit vaccines, delivered as a combination of soluble vaccine plus adjuvant or the heterologous expression of Brucella epitopes by different vaccine vectors are currently being tested. New live attenuated Brucella vaccines are also being developed and tested in their natural hosts. Yet, what is rarely considered is the route of vaccination which could improve vaccine efficacy. Since Brucella infections are mostly transmitted mucosally, mucosal delivery of a vaccine has the potential of eliciting a more robust protective immune response for improved efficacy. Hence, this review will examine these questions and provide the status of new vaccines for livestock brucellosis. Copyright © 2016 Elsevier B.V. All rights reserved.

Expression and Purification of Soluble STAT5b/STAT3 Proteins for SH2 Domain Binding Assay.

PubMed

Asai, Akira; Takakuma, Kazuyuki

2017-01-01

When a large hydrophobic full-length protein is expressed in bacteria, it is often challenging to obtain recombinant proteins in the soluble fraction. One way to overcome this challenge is expression of deletion mutants that have improved solubility while maintaining biological activity. In this chapter, we describe a protocol for expression of truncated forms of STAT5b and STAT3 proteins that are soluble and retain SH2-mediated activity for phospho-Tyr peptide recognition.

Expression and purification of soluble porcine CTLA-4 in yeast Pichia pastoris

PubMed Central

Peraino, Jaclyn; Zhang, Huiping; Hermanrud, Christina E.; Li, Guoying; Sachs, David H.; Huang, Christene A.; Wang, Zhirui

2012-01-01

Co-stimulation blockade can be used to modulate the immune response for induction of organ transplantation tolerance, treatment of autoimmune disease as well as cancer treatment. Cytotoxic T-Lymphocyte Antigen-4 (CTLA-4), also known as CD152, is an important co-stimulatory molecule which serves as a negative regulator for T cell proliferation and differentiation. CTLA-4/CD28-CD80/CD86 pathway is a critical co-stimulatory pathway for adaptive immune response. T cell activation through the T cell receptor and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for CD80 and CD86. MGH MHC-defined miniature swine provide a unique large animal model useful for preclinical studies of transplantation tolerance and immune regulation. In this study, we have expressed the codon-optimized soluble porcine CTLA-4 in the yeast Pichia pastoris system. The secreted porcine CTLA-4 was captured using Ni-Sepharose 6 fast flow resin and further purified using strong anion exchange resin Poros 50HQ. Glycosylation analysis using PNGase F demonstrated the N-linked glycosylation on Pichia pastoris expressed soluble porcine CTLA-4. To improve the expression level and facilitate the downstream purification we mutated the two potential N-linked glycosylation sites with non-polarized alanines by site-directed mutagenesis. Removal of the two N-glycosylation sites significantly improved the production level from ~2 mg/L to ~8 mg/L. Biotinylated glycosylated and non-N-glycosylated soluble porcine CTLA-4 both bind to a porcine CD80-expressing B-cell lymphoma cell line (KD = 13 nM) and competitively inhibit the binding of an anti-CD80 monoclonal antibody. The availability of soluble porcine CTLA-4, especially the non-N-glycosylated CTLA-4, will provide a very valuable tool for assessing co-stimulatory blockade treatment for translational studies in the clinically relevant porcine model. PMID:22326797

Increased expression of cytokines, soluble cytokine receptors, soluble apoptosis ligand and apoptosis in dengue.

PubMed

Arias, Julia; Valero, Nereida; Mosquera, Jesús; Montiel, Milagros; Reyes, Eduardo; Larreal, Yraima; Alvarez-Mon, Melchor

2014-03-01

Several studies have been performed to determine biomarkers that define the risk factors to developing severe forms of dengue. In this study, the levels of TNF-α, IL-6, IL-1, IL-17, soluble interleukin-1 receptor like 1 protein (sST2), soluble TNF-related apoptosis-inducing ligand (sTRAIL), IL-12 and soluble receptors for TNF (sTNF-RI and sTNF-RII) were determined by ELISA in dengue patients and monocyte/macrophage cultures. Dengue was classified as dengue without warning symptoms (DNWS), with warning symptoms (DWWS) and severe dengue (SD). High values of IL-6, sTNFRI, sTNFRII and sST2 were observed in DWWS and/or SD and IL-12 and sTRAIL in DNWS. TNF-α and IL-17 were increased not associated to the disease severity. High production of TNF-α, IL-1β, IL-12, IL-17, sST2 and sTRAIL and apoptosis expression were observed in dengue monocyte/macrophage cultures. This study shows that beneficial or deleterious biomarkers can be present in dengue regardless the disease severity and that monocytes may be in part the source of studied molecules. Copyright © 2014 Elsevier Inc. All rights reserved.

Chaperone-Assisted Soluble Expression of a Humanized Anti-EGFR ScFv Antibody in E. Coli

PubMed Central

Veisi, Kamal; Farajnia, Safar; Zarghami, Nosratollah; Khoram Khorshid, Hamid Reza; Samadi, Nasser; Ahdi Khosroshahi, Shiva; Zarei Jaliani, Hossein

2015-01-01

Purpose: Formation of inclusion bodies is a considerable obstacle threatening the advantages of E. coli expression system to serve as the most common and easiest system in recombinant protein production. To solve this problem, several strategies have been proposed among which application of molecular chaperones is of remarkable consideration. The aim of this study was to evaluate the effects of molecular chaperones on soluble expression of aggregation-prone humanized single chain antibody. Methods: To increase the solubility of a humanized single chain antibody (hscFv), different chaperone plasmids including PG-tf2 (GroES- GroEL- tig), ptf16 (tig) and pGro7 (GroES- GroEL) were co-expressed in BL21 cells containing pET-22b- hscFv construct. The solubility of recombinant hscFv was analyzed by SDS-PAGE. After purification of soluble hscFv by Ni-NTA column, the biological activity and cytotoxicity of the recombinant protein were tested by ELISA and MTT assay, respectively. Results: SDS-PAGE analysis of the hscFv revealed that chaperone utility remarkably increased (up to 50%) the solubility of the protein. ELISA test and MTT assay analyses also confirmed the biological activity of the gained hscFv in reaction with A431 cells (OD value: 2.6) and inhibition of their proliferation, respectively. Conclusion: The results of this study revealed that co-expression of chaperones with hscFv leads to remarkable increase in the solubility of the recombinant hscFv, which could be of great consideration for large scale production of recombinant single chain antibodies. PMID:26793607

Different applications of virus-like particles in biology and medicine: Vaccination and delivery systems.

PubMed

Shirbaghaee, Zeinab; Bolhassani, Azam

2016-03-01

Virus-like particles (VLPs) mimic the whole construct of virus particles devoid of viral genome as used in subunit vaccine design. VLPs can elicit efficient protective immunity as direct immunogens compared to soluble antigens co-administered with adjuvants in several booster injections. Up to now, several prokaryotic and eukaryotic systems such as insect, yeast, plant, and E. coli were used to express recombinant proteins, especially for VLP production. Recent studies are also generating VLPs in plants using different transient expression vectors for edible vaccines. VLPs and viral particles have been applied for different functions such as gene therapy, vaccination, nanotechnology, and diagnostics. Herein, we describe VLP production in different systems as well as its applications in biology and medicine. © 2015 Wiley Periodicals, Inc.

Improved soluble expression and characterization of the Hc domain of Clostridium botulinum neurotoxin serotype A in Escherichia coli by using a PCR-synthesized gene and a Trx co-expression strain.

PubMed

Chen, Rongchang; Shi, Jing; Cai, Kun; Tu, Wei; Hou, Xiaojun; Liu, Hao; Xiao, Le; Wang, Qin; Tang, Yunming; Wang, Hui

2010-05-01

Botulinum neurotoxin serotype A (BoNT/A) is an extremely potent bacterial protein toxin. The Hc fragment of BoNT/A (AHc) was shown to be non-toxic, antigenic, and capable of eliciting a protective immunity in animals challenged with homologous BoNT. In this study, we synthesized AHc gene by using T4 DNA ligase and PCR. The AHc was expressed at a high level in Escherichia coli successfully. Because of using the Trx co-expression strain, the expressed AHc is in a soluble and active form. The yield of the purified AHc was about 70mg/L, and its purity was up to 90% through one-step affinity chromatography. The AHc was positively identified by the antibodies raised against BoNT/A using immunological-dot-blot and Western blot assays. AHc was shown to bind with gangliosides and elicit immunity against BoNT/A, indicating that the expressed and purified AHc protein retains a functionally active conformation. Furthermore, the purified AHc has a strong immunogenicity and can be used as a potential subunit candidate vaccine for botulinum toxin serotype A. Copyright (c) 2009 Elsevier Inc. All rights reserved.

Soluble cysteine-rich tick saliva proteins Salp15 and Iric-1 from E. coli

PubMed Central

Kolb, Philipp; Vorreiter, Jolanta; Habicht, Jüri; Bentrop, Detlef; Wallich, Reinhard; Nassal, Michael

2014-01-01

Ticks transmit numerous pathogens, including borreliae, which cause Lyme disease. Tick saliva contains a complex mix of anti-host defense factors, including the immunosuppressive cysteine-rich secretory glycoprotein Salp15 from Ixodes scapularis ticks and orthologs like Iric-1 from Ixodesricinus. All tick-borne microbes benefit from the immunosuppression at the tick bite site; in addition, borreliae exploit the binding of Salp15 to their outer surface protein C (OspC) for enhanced transmission. Hence, Salp15 proteins are attractive targets for anti-tick vaccines that also target borreliae. However, recombinant Salp proteins are not accessible in sufficient quantity for either vaccine manufacturing or for structural characterization. As an alternative to low-yield eukaryotic systems, we investigated cytoplasmic expression in Escherichia coli, even though this would not result in glycosylation. His-tagged Salp15 was efficiently expressed but insoluble. Among the various solubility-enhancing protein tags tested, DsbA was superior, yielding milligram amounts of soluble, monomeric Salp15 and Iric-1 fusions. Easily accessible mutants enabled epitope mapping of two monoclonal antibodies that, importantly, cross-react with glycosylated Salp15, and revealed interaction sites with OspC. Free Salp15 and Iric-1 from protease-cleavable fusions, despite limited solubility, allowed the recording of 1H–15N 2D NMR spectra, suggesting partial folding of the wild-type proteins but not of Cys-free variants. Fusion to the NMR-compatible GB1 domain sufficiently enhanced solubility to reveal first secondary structure elements in 13C/15N double-labeled Iric-1. Together, E. coli expression of appropriately fused Salp15 proteins may be highly valuable for the molecular characterization of the function and eventually the 3D structure of these medically relevant tick proteins. PMID:25628987

Soluble cysteine-rich tick saliva proteins Salp15 and Iric-1 from E. coli.

PubMed

Kolb, Philipp; Vorreiter, Jolanta; Habicht, Jüri; Bentrop, Detlef; Wallich, Reinhard; Nassal, Michael

2015-01-01

Ticks transmit numerous pathogens, including borreliae, which cause Lyme disease. Tick saliva contains a complex mix of anti-host defense factors, including the immunosuppressive cysteine-rich secretory glycoprotein Salp15 from Ixodes scapularis ticks and orthologs like Iric-1 from Ixodes ricinus. All tick-borne microbes benefit from the immunosuppression at the tick bite site; in addition, borreliae exploit the binding of Salp15 to their outer surface protein C (OspC) for enhanced transmission. Hence, Salp15 proteins are attractive targets for anti-tick vaccines that also target borreliae. However, recombinant Salp proteins are not accessible in sufficient quantity for either vaccine manufacturing or for structural characterization. As an alternative to low-yield eukaryotic systems, we investigated cytoplasmic expression in Escherichia coli, even though this would not result in glycosylation. His-tagged Salp15 was efficiently expressed but insoluble. Among the various solubility-enhancing protein tags tested, DsbA was superior, yielding milligram amounts of soluble, monomeric Salp15 and Iric-1 fusions. Easily accessible mutants enabled epitope mapping of two monoclonal antibodies that, importantly, cross-react with glycosylated Salp15, and revealed interaction sites with OspC. Free Salp15 and Iric-1 from protease-cleavable fusions, despite limited solubility, allowed the recording of (1)H-(15)N 2D NMR spectra, suggesting partial folding of the wild-type proteins but not of Cys-free variants. Fusion to the NMR-compatible GB1 domain sufficiently enhanced solubility to reveal first secondary structure elements in (13)C/(15)N double-labeled Iric-1. Together, E. coli expression of appropriately fused Salp15 proteins may be highly valuable for the molecular characterization of the function and eventually the 3D structure of these medically relevant tick proteins.

Aging-dependent alterations in gene expression and a mitochondrial signature of responsiveness to human influenza vaccination.

PubMed

Thakar, Juilee; Mohanty, Subhasis; West, A Phillip; Joshi, Samit R; Ueda, Ikuyo; Wilson, Jean; Meng, Hailong; Blevins, Tamara P; Tsang, Sui; Trentalange, Mark; Siconolfi, Barbara; Park, Koonam; Gill, Thomas M; Belshe, Robert B; Kaech, Susan M; Shadel, Gerald S; Kleinstein, Steven H; Shaw, Albert C

2015-01-01

To elucidate gene expression pathways underlying age-associated impairment in influenza vaccine response, we screened young (age 21-30) and older (age≥65) adults receiving influenza vaccine in two consecutive seasons and identified those with strong or absent response to vaccine, including a subset of older adults meeting criteria for frailty. PBMCs obtained prior to vaccination (Day 0) and at day 2 or 4, day 7 and day 28 post-vaccine were subjected to gene expression microarray analysis. We defined a response signature and also detected induction of a type I interferon response at day 2 and a plasma cell signature at day 7 post-vaccine in young responders. The response signature was dysregulated in older adults, with the plasma cell signature induced at day 2, and was never induced in frail subjects (who were all non-responders). We also identified a mitochondrial signature in young vaccine responders containing genes mediating mitochondrial biogenesis and oxidative phosphorylation that was consistent in two different vaccine seasons and verified by analyses of mitochondrial content and protein expression. These results represent the first genome-wide transcriptional profiling analysis of age-associated dynamics following influenza vaccination, and implicate changes in mitochondrial biogenesis and function as a critical factor in human vaccine responsiveness.

High-level expression of soluble recombinant proteins in Escherichia coli using an HE-maltotriose-binding protein fusion tag.

PubMed

Han, Yingqian; Guo, Wanying; Su, Bingqian; Guo, Yujie; Wang, Jiang; Chu, Beibei; Yang, Guoyu

2018-02-01

Recombinant proteins are commonly expressed in prokaryotic expression systems for large-scale production. The use of genetically engineered affinity and solubility enhancing fusion proteins has increased greatly in recent years, and there now exists a considerable repertoire of these that can be used to enhance the expression, stability, solubility, folding, and purification of their fusion partner. Here, a modified histidine tag (HE) used as an affinity tag was employed together with a truncated maltotriose-binding protein (MBP; consisting of residues 59-433) from Pyrococcus furiosus as a solubility enhancing tag accompanying a tobacco etch virus protease-recognition site for protein expression and purification in Escherichia coli. Various proteins tagged at the N-terminus with HE-MBP(Pyr) were expressed in E. coli BL21(DE3) cells to determine expression and solubility relative to those tagged with His6-MBP or His6-MBP(Pyr). Furthermore, four HE-MBP(Pyr)-fused proteins were purified by immobilized metal affinity chromatography to assess the affinity of HE with immobilized Ni 2+ . Our results showed that HE-MBP(Pyr) represents an attractive fusion protein allowing high levels of soluble expression and purification of recombinant protein in E. coli. Copyright © 2017 Elsevier Inc. All rights reserved.

Effective non-denaturing purification method for improving the solubility of recombinant actin-binding proteins produced by bacterial expression.

PubMed

Chung, Jeong Min; Lee, Sangmin; Jung, Hyun Suk

2017-05-01

Bacterial expression is commonly used to produce recombinant and truncated mutant eukaryotic proteins. However, heterologous protein expression may render synthesized proteins insoluble. The conventional method used to express a poorly soluble protein, which involves denaturation and refolding, is time-consuming and inefficient. There are several non-denaturing approaches that can increase the solubility of recombinant proteins that include using different bacterial cell strains, altering the time of induction, lowering the incubation temperature, and employing different detergents for purification. In this study, we compared several non-denaturing protocols to express and purify two insoluble 34 kDa actin-bundling protein mutants. The solubility of the mutant proteins was not affected by any of the approaches except for treatment with the detergent sarkosyl. These results indicate that sarkosyl can effectively improve the solubility of insoluble proteins during bacterial expression. Copyright © 2016. Published by Elsevier Inc.

The immune response induced by DNA vaccine expressing nfa1 gene against Naegleria fowleri.

PubMed

Kim, Jong-Hyun; Lee, Sang-Hee; Sohn, Hae-Jin; Lee, Jinyoung; Chwae, Yong-Joon; Park, Sun; Kim, Kyongmin; Shin, Ho-Joon

2012-12-01

The pathogenic free-living amoeba, Naegleria fowleri, causes fatal primary amoebic meningoencephalitis in experimental animals and in humans. The nfa1 gene that was cloned from N. fowleri is located on pseudopodia, especially amoebic food cups and plays an important role in the pathogenesis of N. fowleri. In this study, we constructed and characterized retroviral vector and lentiviral vector systems for nfa1 DNA vaccination in mice. We constructed the retroviral vector (pQCXIN) and the lentiviral vector (pCDH) cloned with the egfp-nfa1 gene. The expression of nfa1 gene in Chinese hamster ovary cell and human primary nasal epithelial cell transfected with the pQCXIN/egfp-nfa1 vector or pCDH/egfp-nfa1 vector was observed by fluorescent microscopy and Western blotting analysis. Our viral vector systems effectively delivered the nfa1 gene to the target cells and expressed the Nfa1 protein within the target cells. To evaluate immune responses of nfa1-vaccinated mice, BALB/c mice were intranasally vaccinated with viral particles of each retro- or lentiviral vector expressing nfa1 gene. DNA vaccination using viral vectors expressing nfa1 significantly stimulated the production of Nfa1-specific IgG subclass, as well as IgG levels. In particular, both levels of IgG2a (Th1) and IgG1 (Th2) were significantly increased in mice vaccinated with viral vectors. These results show the nfa1-vaccination induce efficiently Th1 type, as well as Th2 type immune responses. This is the first report to construct viral vector systems and to evaluate immune responses as DNA vaccination in N. fowleri infection. Furthermore, these results suggest that nfal vaccination may be an effective method for treatment of N. fowleri infection.

[Tobacco--a highly efficient producer of vaccines].

PubMed

Budzianowski, Jaromir

2010-01-01

Along with the depreciation of tobacco as a source of nicotine-containing commercial products, the increase of its appreciation as a potential producer of recombinant therapeutical proteins can be observed. Two species of tobacco--Nicotiana tabacum L. and N. benthamiana are easily grown by well established methods of field or green-house cultivation or cell culture, yield high biomass and soluble protein content, can be easily transformed by several methods and are not food for humans or feed for animals. Expression of foreign proteins, including vaccines, can be achieved in those plants either through stable transformation of nuclear or plastid (chloroplast) genomes or by transient transformation using infection with plant virus or bacteria--Agrobacterium tumefaciens (agroinfiltration). The most advanced mode of agrofiltration termed magnifection, which combines benefits of virus and Agrobacterium and depends on using Agrobacterium with viral pro-vectors, enables high-yield and rapid expression of therapeutical proteins, even in a few days, and can be employed on an industrial scale. Expression of many antigenic proteins, which may serve as antiviral, antibacterial, antiprotozoan and anticancer vaccines, and additionally a few autoantigens designed for the treatment of autoimunogenic diseases, like diabetes, have been achieved in tobacco. To date, a vaccine against Newcastle virus disease in poultry produced by tobacco cell culture has been approved for commercial application and several other vaccines are in advanced stage of development. The possibility of a high-level production of vaccines in tobacco against pandemic influenza or anthrax and plague due to a bioterroristic attack, as well as of individualised anticancer vaccines against non-Hodgkin's lymphoma (NHL) in a much shorter period of time than by traditional methods became realistic and hence caused increased interest in tobacco as a high-efficient producer of vaccines not only of specialistic

A novel expression vector for the improved solubility of recombinant scorpion venom in Escherichia coli

DOE Office of Scientific and Technical Information (OSTI.GOV)

Han, Tianqing; Ming, Hongyan; Deng, Lili

Recombinant scorpion anti-excitation peptide (rANEP) has previously been expressed using the pET32a system and purified via affinity chromatography. However, rANEP is expressed in BL21(DE3) cells as an inclusion body, and the affinity tag can not be removed. To overcome this problem, we used a variety of protein, DsbA, MBP, TrxA, intein, and affinity tags in fusion and co-expression to achieve soluble and functional rANEP without any affinity tag. In the pCIT-ANEP expression vector, the highest soluble expression level was approximately 90% of the total cellular proteins in E. coli, and the rANEP was cleaved by the intein protein and subsequently purifiedmore » to obtain rANEP, which had the same activity as the natural ANEP. The purity of rANEP obtained using this method was over 95%, with a quantity of 5.1 mg from of purified rANEP from 1 L of culture. This method could expand the application of the soluble expression of disulfide-rich peptides in E. coli.« less

Non-conventional expression systems for the production of vaccine proteins and immunotherapeutic molecules

PubMed Central

Legastelois, Isabelle; Buffin, Sophie; Peubez, Isabelle; Mignon, Charlotte; Sodoyer, Régis; Werle, Bettina

2017-01-01

ABSTRACT The increasing demand for recombinant vaccine antigens or immunotherapeutic molecules calls into question the universality of current protein expression systems. Vaccine production can require relatively low amounts of expressed materials, but represents an extremely diverse category consisting of different target antigens with marked structural differences. In contrast, monoclonal antibodies, by definition share key molecular characteristics and require a production system capable of very large outputs, which drives the quest for highly efficient and cost-effective systems. In discussing expression systems, the primary assumption is that a universal production platform for vaccines and immunotherapeutics will unlikely exist. This review provides an overview of the evolution of traditional expression systems, including mammalian cells, yeast and E.coli, but also alternative systems such as other bacteria than E. coli, transgenic animals, insect cells, plants and microalgae, Tetrahymena thermophila, Leishmania tarentolae, filamentous fungi, cell free systems, and the incorporation of non-natural amino acids. PMID:27905833

Bacterial expression of human kynurenine 3-monooxygenase: Solubility, activity, purification☆

PubMed Central

Wilson, K.; Mole, D.J.; Binnie, M.; Homer, N.Z.M.; Zheng, X.; Yard, B.A.; Iredale, J.P.; Auer, M.; Webster, S.P.

2014-01-01

Kynurenine 3-monooxygenase (KMO) is an enzyme central to the kynurenine pathway of tryptophan metabolism. KMO has been implicated as a therapeutic target in several disease states, including Huntington’s disease. Recombinant human KMO protein production is challenging due to the presence of transmembrane domains, which localise KMO to the outer mitochondrial membrane and render KMO insoluble in many in vitro expression systems. Efficient bacterial expression of human KMO would accelerate drug development of KMO inhibitors but until now this has not been achieved. Here we report the first successful bacterial (Escherichia coli) expression of active FLAG™-tagged human KMO enzyme expressed in the soluble fraction and progress towards its purification. PMID:24316190

Comprehensive gene expression profiling following DNA vaccination of rainbow trout against infectious hematopoietic necrosis virus

USGS Publications Warehouse

Purcell, Maureen K.; Nichols, Krista M.; Winton, James R.; Kurath, Gael; Thorgaard, Gary H.; Wheeler, Paul; Hansen, John D.; Herwig, Russell P.; Park, Linda K.

2006-01-01

The DNA vaccine based on the glycoprotein gene of Infectious hematopoietic necrosis virus induces a non-specific anti-viral immune response and long-term specific immunity against IHNV. This study characterized gene expression responses associated with the early anti-viral response. Homozygous rainbow trout were injected intra-muscularly (I.M.) with vector DNA or the IHNV DNA vaccine. Gene expression in muscle tissue (I.M. site) was evaluated using a 16,008 feature salmon cDNA microarray. Eighty different genes were significantly modulated in the vector DNA group while 910 genes were modulated in the IHNV DNA vaccinate group relative to control group. Quantitative reverse-transcriptase PCR was used to examine expression of selected immune genes at the I.M. site and in other secondary tissues. In the localized response (I.M. site), the magnitudes of gene expression changes were much greater in the vaccinate group relative to the vector DNA group for the majority of genes analyzed. At secondary systemic sites (e.g. gill, kidney and spleen), type I IFN-related genes were up-regulated in only the IHNV DNA vaccinated group. The results presented here suggest that the IHNV DNA vaccine induces up-regulation of the type I IFN system across multiple tissues, which is the functional basis of early anti-viral immunity.

Bacterial expression of human kynurenine 3-monooxygenase: solubility, activity, purification.

PubMed

Wilson, K; Mole, D J; Binnie, M; Homer, N Z M; Zheng, X; Yard, B A; Iredale, J P; Auer, M; Webster, S P

2014-03-01

Kynurenine 3-monooxygenase (KMO) is an enzyme central to the kynurenine pathway of tryptophan metabolism. KMO has been implicated as a therapeutic target in several disease states, including Huntington's disease. Recombinant human KMO protein production is challenging due to the presence of transmembrane domains, which localise KMO to the outer mitochondrial membrane and render KMO insoluble in many in vitro expression systems. Efficient bacterial expression of human KMO would accelerate drug development of KMO inhibitors but until now this has not been achieved. Here we report the first successful bacterial (Escherichia coli) expression of active FLAG™-tagged human KMO enzyme expressed in the soluble fraction and progress towards its purification. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

Soluble Fibre Meal Challenge Reduces Airway Inflammation and Expression of GPR43 and GPR41 in Asthma.

PubMed

Halnes, Isabel; Baines, Katherine J; Berthon, Bronwyn S; MacDonald-Wicks, Lesley K; Gibson, Peter G; Wood, Lisa G

2017-01-10

Short chain fatty acids (SCFAs) are produced following the fermentation of soluble fibre by gut bacteria. In animal models, both dietary fibre and SCFAs have demonstrated anti-inflammatory effects via the activation of free fatty acid receptors, such as G protein-coupled receptor 41 and 43 (GPR41 and GPR43). This pilot study examined the acute effect of a single dose of soluble fibre on airway inflammation-including changes in gene expression of free fatty acid receptors-in asthma. Adults with stable asthma consumed a soluble fibre meal ( n = 17) containing 3.5 g inulin and probiotics, or a control meal ( n = 12) of simple carbohydrates. Exhaled nitric oxide (eNO) was measured and induced sputum was collected at 0 and 4 h for differential cell counts, measurement of interleukin-8 (IL-8) protein concentration, and GPR41 and GPR43 gene expression. At 4 h after meal consumption, airway inflammation biomarkers, including sputum total cell count, neutrophils, macrophages, lymphocytes, sputum IL-8, and eNO significantly decreased compared to baseline in the soluble fibre group only. This corresponded with upregulated GPR41 and GPR43 sputum gene expression and improved lung function in the soluble fibre group alone. Soluble fibre has acute anti-inflammatory effects in asthmatic airways. Long-term effects of soluble fibre as an anti-inflammatory therapy in asthma warrants further investigation.

Performance of LBSap Vaccine after Intradermal Challenge with L. infantum and Saliva of Lu. longipalpis: Immunogenicity and Parasitological Evaluation

PubMed Central

Roatt, Bruno Mendes; Aguiar-Soares, Rodrigo Dian de Oliveira; Vitoriano-Souza, Juliana; Coura-Vital, Wendel; Braga, Samuel Leôncio; Corrêa-Oliveira, Rodrigo; Martins-Filho, Olindo Assis; Teixeira-Carvalho, Andréa; de Lana, Marta; Gontijo, Nelder Figueiredo; Marques, Marcos José; Giunchetti, Rodolfo Cordeiro; Reis, Alexandre Barbosa

2012-01-01

In the last decade, the search for new vaccines against canine visceral leishmaniasis has intensified. However, the pattern related to immune protection during long periods after experimental infection in vaccine trials is still not fully understood. Herein, we investigated the immunogenicity and parasitological levels after intradermal challenge with Leishmania infantum plus salivary gland extract in dogs immunized with a vaccine composed of L. braziliensis antigens plus saponin as an adjuvant (LBSap vaccine). The LBSap vaccine elicited higher levels of total anti-Leishmania IgG as well as both IgG1 and IgG2. Furthermore, dogs vaccinated had increased levels of lymphocytes, particularly circulating B cells (CD21+) and both CD4+ and CD8+ T lymphocytes. LBSap also elicited an intense in vitro cell proliferation associated with higher levels of CD4+ T lymphocytes specific for vaccine soluble antigen and soluble lysate of L. infantum antigen even 885 days after experimental challenge. Furthermore, LBSap vaccinated dogs presented high IFN-γ and low IL-10 and TGF-β1 expression in spleen with significant reduction of parasite load in this tissue. Overall, our results validate the potential of LBSap vaccine to protect against L. infantum experimental infection and strongly support further evaluation of efficiency of LBSap against CVL in natural infection conditions. PMID:23189161

TCR hypervariable regions expressed by T cells that respond to effective tumor vaccines.

PubMed

Jordan, Kimberly R; Buhrman, Jonathan D; Sprague, Jonathan; Moore, Brandon L; Gao, Dexiang; Kappler, John W; Slansky, Jill E

2012-10-01

A major goal of immunotherapy for cancer is the activation of T cell responses against tumor-associated antigens (TAAs). One important strategy for improving antitumor immunity is vaccination with peptide variants of TAAs. Understanding the mechanisms underlying the expansion of T cells that respond to the native tumor antigen is an important step in developing effective peptide-variant vaccines. Using an immunogenic mouse colon cancer model, we compare the binding properties and the TCR genes expressed by T cells elicited by peptide variants that elicit variable antitumor immunity directly ex vivo. The steady-state affinity of the natural tumor antigen for the T cells responding to effective peptide vaccines was higher relative to ineffective peptides, consistent with their improved function. Ex vivo analysis showed that T cells responding to the effective peptides expressed a CDR3β motif, which was also shared by T cells responding to the natural antigen and not those responding to the less effective peptide vaccines. Importantly, these data demonstrate that peptide vaccines can expand T cells that naturally respond to tumor antigens, resulting in more effective antitumor immunity. Future immunotherapies may require similar stringent analysis of the responding T cells to select optimal peptides as vaccine candidates.

Plant-expressed Fc-fusion protein tetravalent dengue vaccine with inherent adjuvant properties.

PubMed

Kim, Mi Young; Copland, Alastair; Nayak, Kaustuv; Chandele, Anmol; Ahmed, Muhammad S; Zhang, Qibo; Diogo, Gil R; Paul, Matthew J; Hofmann, Sven; Yang, Moon-Sik; Jang, Yong-Suk; Ma, Julian K-C; Reljic, Rajko

2017-12-09

Dengue is a major global disease requiring improved treatment and prevention strategies. The recently licensed Sanofi Pasteur Dengvaxia vaccine does not protect children under the age of nine, and additional vaccine strategies are thus needed to halt this expanding global epidemic. Here, we employed a molecular engineering approach and plant expression to produce a humanized and highly immunogenic poly-immunoglobulin G scaffold (PIGS) fused to the consensus dengue envelope protein III domain (cEDIII). The immunogenicity of this IgG Fc receptor-targeted vaccine candidate was demonstrated in transgenic mice expressing human FcγRI/CD64, by induction of neutralizing antibodies and evidence of cell-mediated immunity. Furthermore, these molecules were able to prime immune cells from human adenoid/tonsillar tissue ex vivo as evidenced by antigen-specific CD4 + and CD8 + T-cell proliferation, IFN-γ and antibody production. The purified polymeric fraction of dengue PIGS (D-PIGS) induced stronger immune activation than the monomeric form, suggesting a more efficient interaction with the low-affinity Fcγ receptors on antigen-presenting cells. These results show that the plant-expressed D-PIGS have the potential for translation towards a safe and easily scalable single antigen-based tetravalent dengue vaccine. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Efficient osmolyte-based procedure to increase expression level and solubility of infectious hematopoietic necrosis virus (IHNV) nucleoprotein in E. coli.

PubMed

Mohammadinezhad, Rezvan; Farahmand, Hamid; Jalali, Seyed Amir Hossein; Mirvaghefi, Alireza

2018-05-01

The nucleoprotein of infectious hematopoietic necrosis virus (IHNV) is considered as the main target antigen for detection of IHNV infection in salmonid fish. This study aimed at improving the expression and solubility of IHNV nucleoprotein (IHNV-NP) in E. coli expression system. The effects of several expression strategies including host strain type, protein expression temperature, heat-shock treatment prior to protein induction, and additives in the growth medium and in the cell lysis buffer were examined. Results showed that bacterial strain type had a great impact on protein expression level, whereas it was not effective in preventing protein aggregation. Production of soluble IHNV-NP was proportionally increased with decreased incubation temperature. Heat-shock treatment prior to protein induction did not change the percent of solubility. For cells grown at low temperature, the presence of additives in the lysis buffer enhanced the solubility of IHNV-NP up to 24%. The highest yield of soluble protein was obtained via incorporation of osmolytes in the growth medium of cells exposed to a mild salt stress, in the following order: sucrose > sorbitol > glycerol > glycine. Soluble protein obtained by the optimized condition was efficiently purified in high yield and successfully detected by two monoclonal antibodies in a sandwich ELISA. Taken together, a combination of proper host strain, low-temperature expression, and timely application of osmolytes in the growth medium provided sufficient quantities of soluble recombinant IHNV-NP that has the potential to be used for diagnostic purposes.

TLR expression and NK cell activation after human yellow fever vaccination.

PubMed

Neves, Patrícia Cristina da Costa; Matos, Denise Cristina de Souza; Marcovistz, Rugimar; Galler, Ricardo

2009-09-18

The yellow fever vaccine is very effective with a single injection conferring protection for at least 10 years. Recent evidence suggests that the innate immune cells activated through Toll-like receptors (TLRs), are critical determinants of the robustness of the adaptive response. Therefore, we investigated the NK cell status in eight healthy volunteers after vaccination with YF 17DD virus. Shortly after vaccination, we observed increased expression of TLR-3 and TLR-9 in NK cells and markers such as CD69, HLA-DP-DQ-DR, CD38 and CD16. The up-regulation of CD69 was positively correlated with the presence of TLRs throughout the post-vaccination period and the circulating IFN-gamma was significantly augmented. These results suggest that TLRs may play an important role in NK cell activation during the immune response to vaccination, indicating a potential role for NK cells in helping the development of long-lasting protective memory.

Regulation of fibroblast Fas expression by soluble and mechanical pro-fibrotic stimuli.

PubMed

Dodi, Amos E; Ajayi, Iyabode O; Chang, Christine; Beard, Meghan; Ashley, Shanna L; Huang, Steven K; Thannickal, Victor J; Tschumperlin, Daniel J; Sisson, Thomas H; Horowitz, Jeffrey C

2018-05-10

Fibroblast apoptosis is a critical component of normal repair and the acquisition of an apoptosis-resistant phenotype contributes to the pathogenesis of fibrotic repair. Fibroblasts from fibrotic lungs of humans and mice demonstrate resistance to apoptosis induced by Fas-ligand and prior studies have shown that susceptibility to apoptosis is enhanced when Fas (CD95) expression is increased in these cells. Moreover, prior work shows that Fas expression in fibrotic lung fibroblasts is reduced by epigenetic silencing of the Fas promoter. However, the mechanisms by which microenvironmental stimuli such as TGF-β1 and substrate stiffness affect fibroblast Fas expression are not well understood. Primary normal human lung fibroblasts (IMR-90) were cultured on tissue culture plastic or on polyacrylamide hydrogels with Young's moduli to recapitulate the compliance of normal (400 Pa) or fibrotic (6400 Pa) lung tissue and treated with or without TGF-β1 (10 ng/mL) in the presence or absence of protein kinase inhibitors and/or inflammatory cytokines. Expression of Fas was assessed by quantitative real time RT-PCR, ELISA and Western blotting. Soluble Fas (sFas) was measured in conditioned media by ELISA. Apoptosis was assessed using the Cell Death Detection Kit and by Western blotting for cleaved PARP. Fas expression and susceptibility to apoptosis was diminished in fibroblasts cultured on 6400 Pa substrates compared to 400 Pa substrates. TGF-β1 reduced Fas mRNA and protein in a time- and dose-dependent manner dependent on focal adhesion kinase (FAK). Surprisingly, TGF-β1 did not significantly alter cell-surface Fas expression, but did stimulate secretion of sFas. Finally, enhanced Fas expression and increased susceptibility to apoptosis was induced by combined treatment with TNF-α/IFN-γ and was not inhibited by TGF-β1. Soluble and matrix-mediated pro-fibrotic stimuli promote fibroblast resistance to apoptosis by decreasing Fas transcription while stimulating soluble

Dose-dependent immunogenicity of a soluble Neospora caninum tachyzoite-extract vaccine formulated with a soy lecithin/β-glucan adjuvant in cattle.

PubMed

Mansilla, F C; Czepluch, W; Malacari, D A; Hecker, Y P; Bucafusco, D; Franco-Mahecha, O L; Moore, D P; Capozzo, A V

2013-10-18

Mice immunized with a soluble extract of Neospora caninum tachyzoites (sNcAg) formulated with Providean-AVEC, an aqueous soy-based adjuvant, are fully protected from N. caninum multiplication. Here we evaluated the dose-dependent immunogenicity of this vaccine formulation in cattle. Cattle (N=3 per group) were immunized with two applications (30 days apart) of formulations containing Providean-AVEC and different payloads of sNcAg (100, 50 and 10 μg), that were five to fifty times lower than the only reported study using this same antigen in cattle. Kinetics and magnitude of the vaccine-induced immune responses were dose-dependent. Cattle immunized with 100 μg-sNcAg elicited high-avidity specific antibodies 3 weeks after the primary vaccination while those that received 50 μg of antigen had maximum levels of specific high-avidity antibodies 5 days after the day 30 boost. Vaccination with 10 μg of sNcAg induced comparable antibody responses after 2 weeks post re-vaccination. IgG1 was the predominant isotype in all vaccinated animals. Maximum systemic IFN-γ levels were measured in cattle immunized with 50 and 100 μg-sNcAg (14 ± 2.8 ng/ml). CD4(+)-T cells from vaccinated animals proliferated after sNcAg stimulation in vitro, producing IFN-γ. Recall IFN-γ responses mediated by CD4(+)-T cells were detected up to 140 days post vaccination. Formulations containing Providean-AVEC and 50 μg of sNcAg stimulated broad cellular and humoral immune responses against N. caninum in cattle. The profile and magnitude of the immune response elicited by this vaccine can be modified by the antigen-dose and vaccination schedule. This is the first dose-response study performed in cattle using sNcAg as antigen. Copyright © 2013 Elsevier B.V. All rights reserved.

Vaccines to combat river blindness: expression, selection and formulation of vaccines against infection with Onchocerca volvulus in a mouse model

PubMed Central

Hess, Jessica A.; Zhan, Bin; Bonne-Année, Sandra; Deckman, Jessica M.; Bottazzi, Maria Elena; Hotez, Peter J.; Klei, Thomas R.; Lustigman, Sara; Abraham, David

2014-01-01

Human onchocerciasis is a neglected tropical disease caused by Onchocerca volvulus and an important cause of blindness and chronic disability in the developing world. Although mass drug administration of ivermectin has had a profound effect on control of the disease, additional tools are critically needed including the need for a vaccine against onchocerciasis. The objectives of the present study were to: (i) select antigens with known vaccine pedigrees as components of a vaccine; (ii) produce the selected vaccine antigens under controlled conditions, using two expression systems and in one laboratory and (iii) evaluate their vaccine efficacy using a single immunization protocol in mice. In addition, we tested the hypothesis that joining protective antigens as a fusion protein or in combination, into a multivalent vaccine, would improve the ability of the vaccine to induce protective immunity. Out of eight vaccine candidates tested in this study, Ov-103, Ov-RAL-2 and Ov-CPI-2M were shown to reproducibly induce protective immunity when administered individually, as fusion proteins or in combination. Although there was no increase in the level of protective immunity induced by combining the antigens into one vaccine, these antigens remain strong candidates for inclusion in a vaccine to control onchocerciasis in humans. PMID:24907553

Multiplexed quantitation of protein expression and phosphorylation based on functionalized soluble nanopolymers

PubMed Central

Pan, Li; Iliuk, Anton; Yu, Shuai; Geahlen, Robert L.; Tao, W. Andy

2012-01-01

We report here for the first time the multiplexed quantitation of phosphorylation and protein expression based on a functionalized soluble nanopolymer. The soluble nanopolymer, pIMAGO, is functionalized with Ti (IV) ions for chelating phosphoproteins in high specificity, and with infrared fluorescent tags for direct, multiplexed assays. The nanopolymer allows for direct competition for epitopes on proteins of interest, thus facilitating simultaneous detection of phosphorylation by pIMAGO and total protein amount by protein antibody in the same well of microplates. The new strategy has a great potential to measure cell signaling events by clearly distinguishing actual phosphorylation signals from protein expression changes, thus providing a powerful tool to accurately profile cellular signal transduction in healthy and disease cells. We anticipate broad applications of this new strategy in monitoring cellular signaling pathways and discovering new signaling events. PMID:23088311

Increase in DNA vaccine efficacy by virosome delivery and co-expression of a cytolytic protein.

PubMed

Gargett, Tessa; Grubor-Bauk, Branka; Miller, Darren; Garrod, Tamsin; Yu, Stanley; Wesselingh, Steve; Suhrbier, Andreas; Gowans, Eric J

2014-06-01

The potential of DNA vaccines has not been realised due to suboptimal delivery, poor antigen expression and the lack of localised inflammation, essential for antigen presentation and an effective immune response to the immunogen. Initially, we examined the delivery of a DNA vaccine encoding a model antigen, luciferase (LUC), to the respiratory tract of mice by encapsulation in a virosome. Virosomes that incorporated influenza virus haemagglutinin effectively delivered DNA to cells in the mouse respiratory tract and resulted in antigen expression and systemic and mucosal immune responses to the immunogen after an intranasal (IN) prime/intradermal (ID) boost regimen, whereas a multidose ID regimen only generated systemic immunity. We also examined systemic immune responses to LUC after ID vaccination with a DNA vaccine, which also encoded one of the several cytolytic or toxic proteins. Although the herpes simplex virus thymidine kinase, in the presence of the prodrug, ganciclovir, resulted in cell death, this failed to increase the humoral or cell-mediated immune responses. In contrast, the co-expression of LUC with the rotavirus non-structural protein 4 (NSP4) protein or a mutant form of mouse perforin, proteins which are directly cytolytic, resulted in increased LUC-specific humoral and cell-mediated immunity. On the other hand, co-expression of LUC with diphtheria toxin subunit A or overexpression of perforin or NSP4 resulted in a lower level of immunity. In summary, the efficacy of DNA vaccines can be improved by targeted IN delivery of DNA or by the induction of cell death in vaccine-targeted cells after ID delivery.

Modeling for influenza vaccines and adjuvants profile for safety prediction system using gene expression profiling and statistical tools

PubMed Central

Sasaki, Eita; Momose, Haruka; Hiradate, Yuki; Furuhata, Keiko; Takai, Mamiko; Asanuma, Hideki; Ishii, Ken J.

2018-01-01

Historically, vaccine safety assessments have been conducted by animal testing (e.g., quality control tests and adjuvant development). However, classical evaluation methods do not provide sufficient information to make treatment decisions. We previously identified biomarker genes as novel safety markers. Here, we developed a practical safety assessment system used to evaluate the intramuscular, intraperitoneal, and nasal inoculation routes to provide robust and comprehensive safety data. Influenza vaccines were used as model vaccines. A toxicity reference vaccine (RE) and poly I:C-adjuvanted hemagglutinin split vaccine were used as toxicity controls, while a non-adjuvanted hemagglutinin split vaccine and AddaVax (squalene-based oil-in-water nano-emulsion with a formulation similar to MF59)-adjuvanted hemagglutinin split vaccine were used as safety controls. Body weight changes, number of white blood cells, and lung biomarker gene expression profiles were determined in mice. In addition, vaccines were inoculated into mice by three different administration routes. Logistic regression analyses were carried out to determine the expression changes of each biomarker. The results showed that the regression equations clearly classified each vaccine according to its toxic potential and inoculation amount by biomarker expression levels. Interestingly, lung biomarker expression was nearly equivalent for the various inoculation routes. The results of the present safety evaluation were confirmed by the approximation rate for the toxicity control. This method may contribute to toxicity evaluation such as quality control tests and adjuvant development. PMID:29408882

Recombinant rabies virus expressing dog GM-CSF is an efficacious oral rabies vaccine for dogs.

PubMed

Zhou, Ming; Wang, Lei; Zhou, Songqin; Wang, Zhao; Ruan, Juncheng; Tang, Lijun; Jia, Ziming; Cui, Min; Zhao, Ling; Fu, Zhen F

2015-11-17

Developing efficacious oral rabies vaccines is an important step to increase immunization coverage for stray dogs, which are not accessible for parenteral vaccination. Our previous studies have demonstrated that recombinant rabies virus (RABV) expressing cytokines/chemokines induces robust protective immune responses after oral immunization in mice by recruiting and activating dendritic cells (DCs) and B cells. To develop an effective oral rabies vaccine for dogs, a recombinant attenuated RABV expressing dog GM-CSF, designated as LBNSE-dGM-CSF was constructed and used for oral vaccination in a dog model. Significantly more DCs or B cells were activated in the peripheral blood of dogs vaccinated orally with LBNSE-dGM-CSF than those vaccinated with the parent virus LBNSE, particularly at 3 days post immunization (dpi). As a result, significantly higher levels of virus neutralizing antibodies (VNAs) were detected in dogs immunized with LBNSE-dGM-CSF than with the parent virus. All the immunized dogs were protected against a lethal challenge with 4500 MICLD50 of wild-type RABV SXTYD01. LBNSE-dGM-CSF was found to replicate mainly in the tonsils after oral vaccination as detected by nested RT-PCR and immunohistochemistry. Taken together, our results indicate that LBNSE-dGM-CSF could be a promising oral rabies vaccine candidate for dogs.

Pre- and post-exposure safety and efficacy of attenuated rabies virus vaccines are enhanced by their expression of IFNγ

DOE Office of Scientific and Technical Information (OSTI.GOV)

Barkhouse, Darryll A.; Center for Neurovirology 1020 Locust St., Jefferson Alumni Hall, Room 454, Philadelphia, PA 19107; Faber, Milosz

Consistent with evidence of a strong correlation between interferon gamma (IFNγ) production and rabies virus (RABV) clearance from the CNS, we recently demonstrated that engineering a pathogenic RABV to express IFNγ highly attenuates the virus. Reasoning that IFNγ expression by RABV vaccines would enhance their safety and efficacy, we reverse-engineered two proven vaccine vectors, GAS and GASGAS, to express murine IFNγ. Mortality and morbidity were monitored during suckling mice infection, immunize/challenge experiments and mixed intracranial infections. We demonstrate that GASγ and GASγGAS are significantly attenuated in suckling mice compared to the GASGAS vaccine. GASγ better protects mice from lethal DRV4more » RABV infection in both pre- and post-exposure experiments compared to GASGAS. Finally, GASγGAS reduces post-infection neurological sequelae, compared to control, during mixed intracranial infection with DRV4. These data show IFNγ expression by a vaccine vector can enhance its safety while increasing its efficacy as pre- and post-exposure treatment. - Highlights: • IFNγ expression improves attenuated rabies virus safety and immunogenicity. • IFNγ expression is safer and more immunogenic than doubling glycoprotein expression. • Co-infection with IFNγ-expressing RABV prevents wild-type rabies virus lethality. • Vaccine safety and efficacy is additive for IFNγ and double glycoprotein expression.« less

Advancing a multivalent ‘Pan-anthelmintic’ vaccine against soil-transmitted nematode infections

PubMed Central

Zhan, Bin; Beaumier, Coreen M; Briggs, Neima; Jones, Kathryn M; Keegan, Brian P; Bottazzi, Maria Elena; Hotez, Peter J

2014-01-01

Ascaris lumbricoides The Sabin Vaccine Institute Product Development Partnership is developing a Pan-anthelmintic vaccine that simultaneously targets the major soil-transmitted nematode infections, in other words, ascariasis, trichuriasis and hookworm infection. The approach builds off the current bivalent Human Hookworm Vaccine now in clinical development and would ultimately add both a larval Ascaris lumbricoides antigen and an adult-stage Trichuris trichiura antigen from the parasite stichosome. Each selected antigen would partially reproduce the protective immunity afforded by UV-attenuated Ascaris eggs and Trichuris stichosome extracts, respectively. Final antigen selection will apply a ranking system that includes the evaluation of expression yields and solubility, feasibility of process development and the absence of circulating antigen-specific IgE among populations living in helminth-endemic regions. Here we describe a five year roadmap for the antigen discovery, feasibility and antigen selection, which will ultimately lead to the scale-up expression, process development, manufacture, good laboratory practices toxicology and preclinical evaluation, ultimately leading to Phase 1 clinical testing. PMID:24392641

Expression and prognostic value of soluble CD97 and its ligand CD55 in intrahepatic cholangiocarcinoma.

PubMed

Meng, Ze-Wu; Liu, Min-Chao; Hong, Hai-Jie; Du, Qiang; Chen, Yan-Ling

2017-03-01

The incidence rate of intrahepatic cholangiocarcinoma is rising, and treatment options are limited. Therefore, new biological markers of intrahepatic cholangiocarcinoma are needed. Immunohistochemistry and enzyme-linked immunosorbent assay were applied to analyze the expressions of CD97, CD55, and soluble CD97 in 71 patients with intrahepatic cholangiocarcinoma and 10 patients with hepatolithiasis. CD97 and CD55 were not expressed in hepatolithiatic tissues, but positive expression was observed in 76.1% (54/71) and 70.4% (50/71) of intrahepatic cholangiocarcinoma patients. The univariate analyses indicated that the positive expressions of CD97 and CD55 were related to short intrahepatic cholangiocarcinoma survival of patients (both p = 0.001). Furthermore, CD97 and CD55 expressions and biliary soluble CD97 levels were significantly associated with histological grade (p = 0.004, 0.002, and 0.012, respectively), lymph node metastases (p = 0.020, 0.038, and 0.001, respectively), and venous invasion (p = 0.003, 0.002, and 0.001, respectively). The multivariate analyses indicated that lymph node metastases (hazard ratio: 2.407, p = 0.003), positive CD55 expression (hazard ratio: 4.096, p = 0.003), and biliary soluble CD97 levels (hazard ratio: 2.434, p = 0.002) were independent risk factors for the intrahepatic cholangiocarcinoma survival. The receiver operating characteristic (ROC) curve analysis indicated that when the cutoff values of biliary soluble CD97 were 1.15 U/mL, the diagnostic value for predicting lymph node metastasis had a sensitivity of 87.5% and a specificity of 51.3%. For intrahepatic cholangiocarcinoma patient death within 60 months at a cutoff value of 0.940 U/mL, the diagnostic value sensitivity was 89.3% and the specificity was 93.3%. Biliary soluble CD97 may be a new biological marker for early diagnosis, prediction of lymph node metastasis and poor prognosis, and discovery of a therapeutic target.

A plant based protective antigen [PA(dIV)] vaccine expressed in chloroplasts demonstrates protective immunity in mice against anthrax.

PubMed

Gorantala, Jyotsna; Grover, Sonam; Goel, Divya; Rahi, Amit; Jayadev Magani, Sri Krishna; Chandra, Subhash; Bhatnagar, Rakesh

2011-06-15

The currently available anthrax vaccines are limited by being incompletely characterized, potentially reactogenic and have an expanded dosage schedule. Plant based vaccines offer safe alternative for vaccine production. In the present study, we expressed domain IV of Bacillus anthracis protective antigen gene [PA(dIV)] in planta (by nuclear agrobacterium and chloroplast transformation) and E. coli [rPA(dIV)]. The presence of transgene and the expression of PA(dIV) in planta was confirmed by molecular analysis. Expression levels up to 5.3% of total soluble protein (TSP) were obtained with AT rich (71.8% AT content) PA(dIV) gene in transplastomic plants while 0.8% of TSP was obtained in nuclear transformants. Further, we investigated the protective response of plant and E. coli derived PA(dIV) in mice by intraperitoneal (i.p.) and oral immunizations with or without adjuvant. Antibody titers of >10(4) were induced upon i.p. and oral immunizations with plant derived PA(dIV) and oral immunization with E. coli derived PA(dIV). Intraperitoneal injections with adjuvanted E. coli derived PA(dIV), generated highest antibody titers of >10(5). All the immunized groups demonstrated predominant IgG1 titers over IgG2a indicating a polarized Th2 type response. We also evaluated the mucosal antibody response in orally immunized groups. When fecal extracts were analyzed, low sIgA titer was demonstrated in adjuvanted plant and E. coli derived PA(dIV) groups. Further, PA(dIV) antisera enhanced B. anthracis spore uptake by macrophages in vitro and also demonstrated an anti-germinating effect suggesting a potent role at mucosal surfaces. The antibodies from various groups were efficient in neutralizing the lethal toxin in vitro. When mice were challenged with B. anthracis, mice immunized with adjuvanted plant PA(dIV) imparted 60% and 40% protection while E. coli derived PA(dIV) conferred 100% and 80% protection upon i.p. and oral immunizations. Thus, our study is the first attempt in

Distribution and expression in vitro and in vivo of DNA vaccine against lymphocystis disease virus in Japanese flounder ( Paralichthys olivaceus)

NASA Astrophysics Data System (ADS)

Zheng, Fengrong; Sun, Xiuqin; Liu, Hongzhan; Wu, Xingan; Zhong, Nan; Wang, Bo; Zhou, Guodong

2010-01-01

Lymphocystis disease, caused by the lymphocystis disease virus (LCDV), is a significant worldwide problem in fish industry causing substantial economic losses. In this study, we aimed to develop the DNA vaccine against LCDV, using DNA vaccination technology. We evaluated plasmid pEGFP-N2-LCDV1.3 kb as a DNA vaccine candidate. The plasmid DNA was transiently expressed after liposome transfection into the eukaryotic COS 7 cell line. The distribution and expression of the DNA vaccine (pEGFP-N2-LCDV1.3kb) were also analyzed in tissues of the vaccinated Japanese flounder by PCR, RT-PCR and fluorescent microscopy. Results from PCR analysis indicated that the vaccine-containing plasmids were distributed in injected muscle, the muscle opposite the injection site, the hind intestine, gill, spleen, head, kidney and liver, 6 and 25 days after vaccination. The vaccine plasmids disappeared 100 d post-vaccination. Fluorescent microscopy revealed green fluorescence in the injected muscle, the muscle opposite the injection site, the hind intestine, gill, spleen, head, kidney and liver of fish 48 h post-vaccination, green fluorescence did not appear in the control treated tissue. Green fluorescence became weak at 60 days post-vaccination. RT-PCR analysis indicated that the mcp gene was expressed in all tested tissues of vaccinated fish 6-50 days post-vaccination. These results demonstrate that the antigen encoded by the DNA vaccine is distributed and expressed in all of the tissues analyzed in the vaccinated fish. The antigen would therefore potentially initiate a specific immune response. the plasmid DNA was injected into Japanese flounder ( Paralichthys olivaceus) intramuscularly and antibodies against LCDV were evaluated. The results indicate that the plasmid encoded DNA vaccine could induce an immune response to LCDV and would therefore offer immune protection against LCD. Further studies are required for the development and application of this promising DNA vaccine.

A CRISPR/Cas9 and Cre/Lox system-based express vaccine development strategy against re-emerging Pseudorabies virus.

PubMed

Liang, Xun; Sun, Leqiang; Yu, Teng; Pan, Yongfei; Wang, Dongdong; Hu, Xueying; Fu, Zhenfang; He, Qigai; Cao, Gang

2016-01-18

Virus evolves rapidly to escape vaccine-induced immunity, posing a desperate demand for efficient vaccine development biotechnologies. Here we present an express vaccine development strategy based on CRISPR/Cas9 and Cre/Lox system against re-emerging Pseudorabies virus, which caused the recent devastating swine pseudorabies outbreak in China. By CRISPR/Cas9 system, the virulent genes of the newly isolated strain were simultaneously substituted by marker genes, which were subsequently excised using Cre/Lox system for vaccine safety concern. Notably, single cell FACS technology was applied to further promote virus purification efficiency. The combination of these state-of-art technologies greatly accelerated vaccine development. Finally, vaccination and challenge experiments proved this vaccine candidate's protective efficacy in pigs and the promise to control current pseudorabies outbreak. This is, to our knowledge, the first successful vaccine development based on gene edit technologies, demonstrating these technologies leap from laboratory to industry. It may pave the way for future express antiviral vaccine development.

A CRISPR/Cas9 and Cre/Lox system-based express vaccine development strategy against re-emerging Pseudorabies virus

PubMed Central

Liang, Xun; Sun, Leqiang; Yu, Teng; Pan, Yongfei; Wang, Dongdong; Hu, Xueying; Fu, Zhenfang; He, Qigai; Cao, Gang

2016-01-01

Virus evolves rapidly to escape vaccine-induced immunity, posing a desperate demand for efficient vaccine development biotechnologies. Here we present an express vaccine development strategy based on CRISPR/Cas9 and Cre/Lox system against re-emerging Pseudorabies virus, which caused the recent devastating swine pseudorabies outbreak in China. By CRISPR/Cas9 system, the virulent genes of the newly isolated strain were simultaneously substituted by marker genes, which were subsequently excised using Cre/Lox system for vaccine safety concern. Notably, single cell FACS technology was applied to further promote virus purification efficiency. The combination of these state-of-art technologies greatly accelerated vaccine development. Finally, vaccination and challenge experiments proved this vaccine candidate’s protective efficacy in pigs and the promise to control current pseudorabies outbreak. This is, to our knowledge, the first successful vaccine development based on gene edit technologies, demonstrating these technologies leap from laboratory to industry. It may pave the way for future express antiviral vaccine development. PMID:26777545

Protective immunity induced by the vaccination of recombinant Proteus mirabilis OmpA expressed in Pichia pastoris.

PubMed

Zhang, Yongbing; Yang, Shifa; Dai, Xiumei; Liu, Liping; Jiang, Xiaodong; Shao, Mingxu; Chi, Shanshan; Wang, Chuanwen; Yu, Cuilian; Wei, Kai; Zhu, Ruiliang

2015-01-01

Proteus mirabilis (P. mirabilis) is a zoonotic pathogen that has recently presented a rising infection rate in the poultry industry. To develop an effective vaccine to protect chickens against P. mirabilis infection, OmpA, one of the major outer membrane proteins of P. mirabilis, was expressed in Pichia pastoris. The concentration of the expressed recombinant OmpA protein reached 8.0μg/mL after induction for 96h with 1.0% methanol in the culture. In addition, OmpA protein was confirmed by SDS-PAGE and Western blot analysis using the antibody against Escherichia coli-expressed OmpA protein. Taishan Pinus massoniana pollen polysaccharide, a known plant-derived adjuvant, was mixed into the recombinant OmpA protein to prepare the OmpA subunit vaccine. We then subcutaneously inoculated this vaccine into chickens to examine the immunoprotective effects. ELISA analysis indicated that an excellent antibody response against OmpA was elicited in the vaccinated chickens. Moreover, a high protection rate of 80.0% was observed in the vaccinated group, which was subsequently challenged with P. mirabilis. The results suggest that the eukaryotic P. mirabilis OmpA was an ideal candidate protein for developing an effective subunit vaccine against P. mirabilis infection. Copyright © 2014 Elsevier Inc. All rights reserved.

Vaccines to combat river blindness: expression, selection and formulation of vaccines against infection with Onchocerca volvulus in a mouse model.

PubMed

Hess, Jessica A; Zhan, Bin; Bonne-Année, Sandra; Deckman, Jessica M; Bottazzi, Maria Elena; Hotez, Peter J; Klei, Thomas R; Lustigman, Sara; Abraham, David

2014-08-01

Human onchocerciasis is a neglected tropical disease caused by Onchocerca volvulus and an important cause of blindness and chronic disability in the developing world. Although mass drug administration of ivermectin has had a profound effect on control of the disease, additional tools are critically needed including the need for a vaccine against onchocerciasis. The objectives of the present study were to: (i) select antigens with known vaccine pedigrees as components of a vaccine; (ii) produce the selected vaccine antigens under controlled conditions, using two expression systems and in one laboratory and (iii) evaluate their vaccine efficacy using a single immunisation protocol in mice. In addition, we tested the hypothesis that joining protective antigens as a fusion protein or in combination, into a multivalent vaccine, would improve the ability of the vaccine to induce protective immunity. Out of eight vaccine candidates tested in this study, Ov-103, Ov-RAL-2 and Ov-CPI-2M were shown to reproducibly induce protective immunity when administered individually, as fusion proteins or in combination. Although there was no increase in the level of protective immunity induced by combining the antigens into one vaccine, these antigens remain strong candidates for inclusion in a vaccine to control onchocerciasis in humans. Copyright © 2014 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Vaccination with Recombinant Parainfluenza Virus 5 Expressing Neuraminidase Protects against Homologous and Heterologous Influenza Virus Challenge

PubMed Central

Mooney, Alaina J.; Gabbard, Jon D.; Li, Zhuo; Dlugolenski, Daniel A.; Johnson, Scott K.

2017-01-01

ABSTRACT Seasonal human influenza virus continues to cause morbidity and mortality annually, and highly pathogenic avian influenza (HPAI) viruses along with other emerging influenza viruses continue to pose pandemic threats. Vaccination is considered the most effective measure for controlling influenza; however, current strategies rely on a precise vaccine match with currently circulating virus strains for efficacy, requiring constant surveillance and regular development of matched vaccines. Current vaccines focus on eliciting specific antibody responses against the hemagglutinin (HA) surface glycoprotein; however, the diversity of HAs across species and antigenic drift of circulating strains enable the evasion of virus-inhibiting antibody responses, resulting in vaccine failure. The neuraminidase (NA) surface glycoprotein, while diverse, has a conserved enzymatic site and presents an appealing target for priming broadly effective antibody responses. Here we show that vaccination with parainfluenza virus 5 (PIV5), a promising live viral vector expressing NA from avian (H5N1) or pandemic (H1N1) influenza virus, elicited NA-specific antibody and T cell responses, which conferred protection against homologous and heterologous influenza virus challenges. Vaccination with PIV5-N1 NA provided cross-protection against challenge with a heterosubtypic (H3N2) virus. Experiments using antibody transfer indicate that antibodies to NA have an important role in protection. These findings indicate that PIV5 expressing NA may be effective as a broadly protective vaccine against seasonal influenza and emerging pandemic threats. IMPORTANCE Seasonal influenza viruses cause considerable morbidity and mortality annually, while emerging viruses pose potential pandemic threats. Currently licensed influenza virus vaccines rely on the antigenic match of hemagglutinin (HA) for vaccine strain selection, and most vaccines rely on HA inhibition titers to determine efficacy, despite the growing

Vaccination with Recombinant Parainfluenza Virus 5 Expressing Neuraminidase Protects against Homologous and Heterologous Influenza Virus Challenge.

PubMed

Mooney, Alaina J; Gabbard, Jon D; Li, Zhuo; Dlugolenski, Daniel A; Johnson, Scott K; Tripp, Ralph A; He, Biao; Tompkins, S Mark

2017-12-01

Seasonal human influenza virus continues to cause morbidity and mortality annually, and highly pathogenic avian influenza (HPAI) viruses along with other emerging influenza viruses continue to pose pandemic threats. Vaccination is considered the most effective measure for controlling influenza; however, current strategies rely on a precise vaccine match with currently circulating virus strains for efficacy, requiring constant surveillance and regular development of matched vaccines. Current vaccines focus on eliciting specific antibody responses against the hemagglutinin (HA) surface glycoprotein; however, the diversity of HAs across species and antigenic drift of circulating strains enable the evasion of virus-inhibiting antibody responses, resulting in vaccine failure. The neuraminidase (NA) surface glycoprotein, while diverse, has a conserved enzymatic site and presents an appealing target for priming broadly effective antibody responses. Here we show that vaccination with parainfluenza virus 5 (PIV5), a promising live viral vector expressing NA from avian (H5N1) or pandemic (H1N1) influenza virus, elicited NA-specific antibody and T cell responses, which conferred protection against homologous and heterologous influenza virus challenges. Vaccination with PIV5-N1 NA provided cross-protection against challenge with a heterosubtypic (H3N2) virus. Experiments using antibody transfer indicate that antibodies to NA have an important role in protection. These findings indicate that PIV5 expressing NA may be effective as a broadly protective vaccine against seasonal influenza and emerging pandemic threats. IMPORTANCE Seasonal influenza viruses cause considerable morbidity and mortality annually, while emerging viruses pose potential pandemic threats. Currently licensed influenza virus vaccines rely on the antigenic match of hemagglutinin (HA) for vaccine strain selection, and most vaccines rely on HA inhibition titers to determine efficacy, despite the growing

Leaf-Encapsulated Vaccines: Agroinfiltration and Transient Expression of the Antigen Staphylococcal Endotoxin B in Radish Leaves

PubMed Central

Liu, Pei-Feng; Wang, Yanhan; Ulrich, Robert G.; Simmons, Christopher W.; VanderGheynst, Jean S.; Gallo, Richard L.

2018-01-01

Transgene introgression is a major concern associated with transgenic plant-based vaccines. Agroinfiltration can be used to selectively transform nonreproductive organs and avoid introgression. Here, we introduce a new vaccine modality in which Staphylococcal enterotoxin B (SEB) genes are agroinfiltrated into radishes (Raphanw sativus L.), resulting in transient expression and accumulation of SEB in planta. This approach can simultaneously express multiple antigens in a single leaf. Furthermore, the potential of high-throughput vaccine production was demonstrated by simultaneously agroinfiltrating multiple radish leaves using a multichannel pipette. The expression of SEB was detectable in two leaf cell types (epidermal and guard cells) in agroinfiltrated leaves. ICR mice intranasally immunized with homogenized leaves agroinfiltrated with SEB elicited detectable antibody to SEB and displayed protection against SEB-induced interferon-gamma (IFN-γ) production. The concept of encapsulating antigens in leaves rather than purifying them for immunization may facilitate rapid vaccine production during an epidemic disease. PMID:29577048

Cloning and Expression of Genes for Dengue Virus Type-2 Encoded-Antigens for Rapid Diagnosis and Vaccine Development

DTIC Science & Technology

1988-10-31

00 0 Cloning and Expression of Genes for Dengue Virus (Type-2 Encoded-Antigens for Rapid ODiagnosis and Vaccine DevelopmentN| ANNUAL PROGRESS REPORT...11. TITLE (include Security Classification) Cloning and Expression of Genes f or Dengue Virus Type 2 Fncoded Antigens for Rapid Diagnosis and Vaccine ...epidemics in Central and South Americas and the Caribbean is a cause of major concern. An effective vaccine is not available to protect individuals

DNA vaccine expressing herpes simplex virus 1 glycoprotein C and D protects mice against herpes simplex keratitis

PubMed Central

Dong, Li-Li; Tang, Ru; Zhai, Yu-Jia; Malla, Tejsu; Hu, Kai

2017-01-01

AIM To investigate whether DNA vaccine encoding herpes simplex virus 1 (HSV-1) glycoprotein C (gC) and glycoprotein D (gD) will achieve better protective effect against herpes simplex keratitis (HSK) than DNA vaccine encoding gD alone. METHODS DNA vaccine expressing gD or gC combined gD (gD.gC) were constructed and carried by chitosan nanoparticle. The expression of fusion protein gD and gC were detected in DNA/nanoparticle transfected 293T cells by Western-blot. For immunization, mice were inoculated with DNA/nanoparticle for 3 times with 2wk interval, and two weeks after the final immunization, the specific immune responses and clinical degrees of primary HSK were evaluated. RESULTS Fusion protein gD.gC could be expressed successfully in cultured 293T cells. And, pRSC-gC.gD-IL21 DNA/chitosan nanoparticle could effectively elicit strongest humoral and cellular immune response in primary HSK mice evidenced by higher levels of specific neutralizing antibody and sIgA production, enhanced cytotoxicities of splenocytes and nature killer cells (NK), when compared with those of gD alone or mocked vaccine immunized mice. As a result, gC-based vaccine immunized mice showed least HSK disease. CONCLUSION gC-based DNA vaccine could effectively prevent the progress of primary HSK, suggesting that this DNA vaccine could be a promising vaccine for HSK treatment in the future. PMID:29181304

Broadly protective anti-hemagglutinin stalk antibodies induced by live attenuated influenza vaccine expressing chimeric hemagglutinin.

PubMed

Isakova-Sivak, Irina; Korenkov, Daniil; Smolonogina, Tatiana; Kotomina, Tatiana; Donina, Svetlana; Matyushenko, Victoria; Mezhenskaya, Daria; Krammer, Florian; Rudenko, Larisa

2018-05-01

The development of influenza vaccines that can provide broad protection against all drifted seasonal virus variants, zoonotic infections and emerging pandemic strains, has been a priority for two decades. Here we propose a strategy of inducing broadly-reactive anti-stalk antibody by sequential immunizations with live attenuated influenza vaccines (LAIVs) expressing chimeric HAs (cHAs). These vaccines are designed to contain identical hemagglutinin stalk domains from H1N1 virus but antigenically unrelated globular head domains from avian influenza virus subtypes H5, H8 and H9. Mouse experiments demonstrated enhanced cross-protection of cHA-containing LAIVs compared to the relevant vaccine viruses expressing natural HAs, and this enhanced protection was driven by stalk-HA-reactive IgG antibodies. The establishment of fully functional cross-protective immunity after two doses of cHA LAIV vaccination in naïve animals suggests that a similar effect might be expected after a single cHA LAIV dose in primed individuals, or after two to three doses in naïve children. Copyright © 2018 Elsevier Inc. All rights reserved.

A replication-deficient rabies virus vaccine expressing Ebola virus glycoprotein is highly attenuated for neurovirulence

DOE Office of Scientific and Technical Information (OSTI.GOV)

Papaneri, Amy B.; Wirblich, Christoph; Cann, Jennifer A.

We are developing inactivated and live-attenuated rabies virus (RABV) vaccines expressing Ebola virus (EBOV) glycoprotein for use in humans and endangered wildlife, respectively. Here, we further characterize the pathogenesis of the live-attenuated RABV/EBOV vaccine candidates in mice in an effort to define their growth properties and potential for safety. RABV vaccines expressing GP (RV-GP) or a replication-deficient derivative with a deletion of the RABV G gene (RV{Delta}G-GP) are both avirulent after intracerebral inoculation of adult mice. Furthermore, RV{Delta}G-GP is completely avirulent upon intracerebral inoculation of suckling mice unlike parental RABV vaccine or RV-GP. Analysis of RV{Delta}G-GP in the brain bymore » quantitative PCR, determination of virus titer, and immunohistochemistry indicated greatly restricted virus replication. In summary, our findings indicate that RV-GP retains the attenuation phenotype of the live-attenuated RABV vaccine, and RV{Delta}G-GP would appear to be an even safer alternative for use in wildlife or consideration for human use.« less

Benzoate-mediated changes on expression profile of soluble proteins in Serratia sp. DS001.

PubMed

Pandeeti, E V P; Chinnaboina, M R; Siddavattam, D

2009-05-01

To assess differences in protein expression profile associated with shift in carbon source from succinate to benzoate in Serratia sp. DS001 using a proteomics approach. A basic proteome map was generated for the soluble proteins extracted from Serratia sp. DS001 grown in succinate and benzoate. The differently and differentially expressed proteins were identified using ImageMaster 2D Platinum software (GE Healthcare). The identity of the proteins was determined by employing MS or MS/MS. Important enzymes such as Catechol 1,2 dioxygenase and transcriptional regulators that belong to the LysR superfamily were identified. Nearly 70 proteins were found to be differentially expressed when benzoate was used as carbon source. Based on the protein identity and degradation products generated from benzoate it is found that ortho pathway is operational in Serratia sp. DS001. Expression profile of the soluble proteins associated with shift in carbon source was mapped. The study also elucidates degradation pathway of benzoate in Serratia sp. DS001 by correlating the proteomics data with the catabolites of benzoate.

Evaluation of detergents for the soluble expression of alpha-helical and beta-barrel-type integral membrane proteins by a preparative scale individual cell-free expression system.

PubMed

Klammt, Christian; Schwarz, Daniel; Fendler, Klaus; Haase, Winfried; Dötsch, Volker; Bernhard, Frank

2005-12-01

Cell-free expression has become a highly promising tool for the fast and efficient production of integral membrane proteins. The proteins can be produced as precipitates that solubilize in mild detergents usually without any prior denaturation steps. Alternatively, membrane proteins can be synthesized in a soluble form by adding detergents to the cell-free system. However, the effects of a representative variety of detergents on the production, solubility and activity of a wider range of membrane proteins upon cell-free expression are currently unknown. We therefore analyzed the cell-free expression of three structurally very different membrane proteins, namely the bacterial alpha-helical multidrug transporter, EmrE, the beta-barrel nucleoside transporter, Tsx, and the porcine vasopressin receptor of the eukaryotic superfamily of G-protein coupled receptors. All three membrane proteins could be produced in amounts of several mg per one ml of reaction mixture. In general, the detergent 1-myristoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] was found to be most effective for the resolubilization of membrane protein precipitates, while long chain polyoxyethylene-alkyl-ethers proved to be most suitable for the soluble expression of all three types of membrane proteins. The yield of soluble expressed membrane protein remained relatively stable above a certain threshold concentration of the detergents. We report, for the first time, the high-level cell-free expression of a beta-barrel type membrane protein in a functional form. Structural and functional variations of the analyzed membrane proteins are evident that correspond with the mode of expression and that depend on the supplied detergent.

Vector Development for the Expression of Foreign Proteins in the Vaccine Strain Brucella abortus S19

PubMed Central

Comerci, Diego J.; Pollevick, Guido D.; Vigliocco, Ana M.; Frasch, Alberto C. C.; Ugalde, Rodolfo A.

1998-01-01

A vector for the expression of foreign antigens in the vaccine strain Brucella abortus S19 was developed by using a DNA fragment containing the regulatory sequences and the signal peptide of the Brucella bcsp31 gene. This fragment was cloned in broad-host-range plasmid pBBR4MCS, resulting in plasmid pBEV. As a reporter protein, a repetitive antigen of Trypanosoma cruzi was used. The recombinant fusion protein is stably expressed and secreted into the Brucella periplasmic space, inducing a good antibody response against the T. cruzi antigen. The expression of the repetitive antigen in Brucella neither altered its growth pattern nor generated a toxic or lethal effect during experimental infection. The application of this strategy for the generation of live recombinant vaccines and the tagging of B. abortus S19 vaccine is discussed. This is the first time that a recombinant protein has been expressed in the periplasm of brucellae. PMID:9673273

Cross reactivity of serum antibody responses elicited by DNA vaccines expressing HA antigens from H1N1 subtype influenza vaccines in the past 30 years.

PubMed

Almansour, Iman; Chen, Huaiqing; Wang, Shixia; Lu, Shan

2013-10-01

In the past three decades, ten H1 subtype influenza vaccines have been recommended for global seasonal flu vaccination. Some of them were used only for one year before being replaced by another H1 flu vaccine while others may be used for up to seven years. While the selection of a new seasonal flu vaccine was based on the escape of a new emerging virus that was not effectively protected by the existing flu formulation, there is limited information on the magnitude and breadth of cross reactivity among H1 subtype virus circulation over a long period. In the current study, HA-expressing DNA vaccines were constructed to express individual HA antigens from H1 subtype vaccines used in the past 30 y. Rabbits naïve to HA antibody responses were immunized with these HA DNA vaccines and the cross reactivity of these sera against HA antigen and related H1 viruses in the same period was studied. Our data indicate that the level of cross reactivity was different for different viral isolates and the key mutations responsible for the cross reactivity may involve only a limited number of residues. Our results provide useful information for the development of improved seasonal vaccines than can achieve broad protection against viruses within the same H1 subtype.

Cloning and expression of Clostridium perfringens type D vaccine strain epsilon toxin gene in E. coli as a recombinant vaccine candidate.

PubMed

Aziminia, Parastoo; Pilehchian-Langroudi, Reza; Esmaeilnia, Kasra

2016-08-01

Clostridium perfringens, a Gram-positive obligate anaerobic bacterium, is able to form resistant spores which are widely distributed in the environment. C. perfringens is subdivided into five types A to E based on its four major alpha, beta, epsilon and iota toxins. The aim of the present study was cloning and expression of C. perfringens type D vaccine strain epsilon toxin gene. Genomic DNA was extracted and the epsilon toxin gene was amplified using Pfu DNA polymerase. The PCR product was cloned into pJET1.2/blunt cloning vector. The recombinant vector (pJETε) was sequenced using universal primers. At the next step epsilon toxin gene was subcloned into pET22b(+) expression vector and transformed into E. coli Rosetta (DE3) host strain. The recombinant protein has been expressed in E. coli Rosetta (DE3) cells after subcloning of C. perfringens etx gene (1008 bp) into the expression vector. We concluded that E. coli Rosetta strain was suitable for the expression of recombinant C. perfringens epsilon toxin protein from pET22ε expression vector. This recombinant cell can be used for further research on recombinant vaccine development.

The Corn Smut ('Huitlacoche') as a New Platform for Oral Vaccines.

PubMed

Juárez-Montiel, Margarita; Romero-Maldonado, Andrea; Monreal-Escalante, Elizabeth; Becerra-Flora, Alicia; Korban, Schuyler S; Rosales-Mendoza, Sergio; Jiménez-Bremont, Juan Francisco

2015-01-01

The development of new alternative platforms for subunit vaccine production is a priority in the biomedical field. In this study, Ustilago maydis, the causal agent of common corn smut or 'huitlacoche'has been genetically engineered to assess expression and immunogenicity of the B subunit of the cholera toxin (CTB), a relevant immunomodulatory agent in vaccinology. An oligomeric CTB recombinant protein was expressed in corn smut galls at levels of up to 1.3 mg g-1 dry weight (0.8% of the total soluble protein). Mice orally immunized with 'huitlacoche'-derived CTB showed significant humoral responses that were well-correlated with protection against challenge with the cholera toxin (CT). These findings demonstrate the feasibility of using edible corn smut as a safe, effective, and low-cost platform for production and delivery of a subunit oral vaccine. The implications of this platform in the area of molecular pharming are discussed.

Association of the expression of Th cytokines with peripheral CD4 and CD8 lymphocyte subsets after vaccination with FMD vaccine in Holstein young sires.

PubMed

Yang, Ling; Liu, Zhichao; Li, Jianbin; He, Kaili; Kong, Lingna; Guo, Runqing; Liu, Wenjiao; Gao, Yundong; Zhong, Jifeng

2018-05-25

High immune response (HIR) cows have a balanced and robust host defense and lower disease incidence, and immune response is more important to consider for selecting young sires than for selecting cows. The protective immune response against foot-and-mouth disease (FMD) virus infection is T-cell-independent in an animal experimental model. However, there is no convenient method to select young sires with a HIR to FMD virus. In this study, 39 healthy Holstein young sires were vaccinated with the trivalent (A, O and Asia 1) FMD vaccine, and T-lymphocyte subsets in peripheral blood lymphocytes (PBLs) were detected using flow cytometric analysis before and after vaccination. The expression of interferon-gamma (IFN-γ), interleukin-2 (IL-2), IL-4, and IL-6 mRNA in PBLs was analyzed after stimulation by lipopolysaccharide (LPS) or Concanavalin A (ConA) after vaccination. According to the percentage of CD4 + lymphocyte and CD4/CD8 ratio after vaccination for selecting the HIR young sires, the results showed that the percentages of CD3 + , CD4 + , CD3 + CD4 + lymphocytes and the CD4/CD8 ratio in the HIR group were higher compared to those in the medium immune response (MIR) and low immune response (LIR) groups before vaccination. Additionally, the percentage of CD4 + lymphocytes and the CD4/CD8 ratio after vaccination were positively associated with the expression level of IFN-γ mRNA in the PBLs after stimulation by LPS. In conclusion, the in vitro expression level of IFN-γ mRNA in the PBLs stimulated by LPS may serve as a parameter for selecting young sires with a HIR to FMD virus. Copyright © 2018 Elsevier Ltd. All rights reserved.

Mutual enhancement of IL-2 and IL-7 on DNA vaccine immunogenicity mainly involves regulations on their receptor expression and receptor-expressing lymphocyte generation.

PubMed

Zhang, Yonghong; Liang, Shuang; Li, Xiujin; Wang, Liyue; Zhang, Jianlou; Xu, Jian; Huo, Shanshan; Cao, Xuebin; Zhong, Zhenyu; Zhong, Fei

2015-07-09

Our previous study showed that IL-2 and IL-7 could mutually enhance the immunogenicity of canine parvovirus VP2 DNA vaccine, although the underlying mechanism remained unknown. Here, we used the OVA gene as a DNA vaccine in a mouse model to test their enhancement on DNA vaccine immunogenicity and to explore the molecular mechanism. Results showed that both IL-2 and IL-7 genes significantly increased the immunogenicity of OVA DNA vaccine in mice. Co-administration of IL-2 and IL-7 genes with OVA DNA significantly increased OVA-specific antibody titers, T cell proliferation and IFN-γ production compared with IL-2 or IL-7 alone, confirming that IL-2 and IL-7 mutually enhanced DNA vaccine immunogenicity. Mechanistically, we have shown that IL-2 significantly stimulated generation of IL-7 receptor-expressing lymphocytes, and that IL-7 significantly induced IL-2 receptor expression. These results contribute to an explanation of the mechanism of the mutual effects of IL-2 and IL-7 on enhancing DNA vaccine immunogenicity and provided a basis for further investigation on their mutual effects on adjuvant activity and immune regulation. Copyright © 2015 Elsevier Ltd. All rights reserved.

Cell-free production of a functional oligomeric form of a Chlamydia major outer-membrane protein (MOMP) for vaccine development

DOE PAGES

He, Wei; Felderman, Martina; Evans, Angela C.; ...

2017-07-24

Chlamydia is a prevalent sexually transmitted disease that infects more than 100 million people worldwide. Although most individuals infected with Chlamydia trachomatis are initially asymptomatic, symptoms can arise if left undiagnosed. Long-term infection can result in debilitating conditions such as pelvic inflammatory disease, infertility, and blindness. Chlamydia infection, therefore, constitutes a significant public health threat, underscoring the need for a Chlamydia-specific vaccine. Chlamydia strains express a major outer-membrane protein (MOMP) that has been shown to be an effective vaccine antigen. However, approaches to produce a functional recombinant MOMP protein for vaccine development are limited by poor solubility, low yield, andmore » protein misfolding. For this study, we used an Escherichia coli-based cell-free system to express a MOMP protein from the mouse-specific species Chlamydia muridarum (MoPn-MOMP or mMOMP). The codon-optimized mMOMP gene was co-translated with Δ49apolipoprotein A1 (Δ49ApoA1), a truncated version of mouse ApoA1 in which the N-terminal 49 amino acids were removed. This co-translation process produced mMOMP supported within a telodendrimer nanolipoprotein particle (mMOMP–tNLP). The cell-free expressed mMOMP–tNLPs contain mMOMP multimers similar to the native MOMP protein. This cell-free process produced on average 1.5 mg of purified, water-soluble mMOMP–tNLP complex in a 1-ml cell-free reaction. The mMOMP–tNLP particle also accommodated the co-localization of CpG oligodeoxynucleotide 1826, a single-stranded synthetic DNA adjuvant, eliciting an enhanced humoral immune response in vaccinated mice. Using our mMOMP–tNLP formulation, we demonstrate a unique approach to solubilizing and administering membrane-bound proteins for future vaccine development. This method can be applied to other previously difficult-to-obtain antigens while maintaining full functionality and immunogenicity.« less

Cell-free production of a functional oligomeric form of a Chlamydia major outer-membrane protein (MOMP) for vaccine development

DOE Office of Scientific and Technical Information (OSTI.GOV)

He, Wei; Felderman, Martina; Evans, Angela C.

Chlamydia is a prevalent sexually transmitted disease that infects more than 100 million people worldwide. Although most individuals infected with Chlamydia trachomatis are initially asymptomatic, symptoms can arise if left undiagnosed. Long-term infection can result in debilitating conditions such as pelvic inflammatory disease, infertility, and blindness. Chlamydia infection, therefore, constitutes a significant public health threat, underscoring the need for a Chlamydia-specific vaccine. Chlamydia strains express a major outer-membrane protein (MOMP) that has been shown to be an effective vaccine antigen. However, approaches to produce a functional recombinant MOMP protein for vaccine development are limited by poor solubility, low yield, andmore » protein misfolding. For this study, we used an Escherichia coli-based cell-free system to express a MOMP protein from the mouse-specific species Chlamydia muridarum (MoPn-MOMP or mMOMP). The codon-optimized mMOMP gene was co-translated with Δ49apolipoprotein A1 (Δ49ApoA1), a truncated version of mouse ApoA1 in which the N-terminal 49 amino acids were removed. This co-translation process produced mMOMP supported within a telodendrimer nanolipoprotein particle (mMOMP–tNLP). The cell-free expressed mMOMP–tNLPs contain mMOMP multimers similar to the native MOMP protein. This cell-free process produced on average 1.5 mg of purified, water-soluble mMOMP–tNLP complex in a 1-ml cell-free reaction. The mMOMP–tNLP particle also accommodated the co-localization of CpG oligodeoxynucleotide 1826, a single-stranded synthetic DNA adjuvant, eliciting an enhanced humoral immune response in vaccinated mice. Using our mMOMP–tNLP formulation, we demonstrate a unique approach to solubilizing and administering membrane-bound proteins for future vaccine development. This method can be applied to other previously difficult-to-obtain antigens while maintaining full functionality and immunogenicity.« less

Smallpox subunit vaccine produced in planta confers protection in mice

PubMed Central

Golovkin, Maxim; Spitsin, Sergei; Andrianov, Vyacheslav; Smirnov, Yuriy; Xiao, Yuhong; Pogrebnyak, Natalia; Markley, Karen; Brodzik, Robert; Gleba, Yuri; Isaacs, Stuart N.; Koprowski, Hilary

2007-01-01

We report here the in planta production of the recombinant vaccinia virus B5 antigenic domain (pB5), an attractive component of a subunit vaccine against smallpox. The antigenic domain was expressed by using efficient transient and constitutive plant expression systems and tested by various immunization routes in two animal models. Whereas oral administration in mice or the minipig with collard-derived insoluble pB5 did not generate an anti-B5 immune response, intranasal administration of soluble pB5 led to a rise of B5-specific immunoglobulins, and parenteral immunization led to a strong anti-B5 immune response in both mice and the minipig. Mice immunized i.m. with pB5 generated an antibody response that reduced virus spread in vitro and conferred protection from challenge with a lethal dose of vaccinia virus. These results indicate the feasibility of producing safe and inexpensive subunit vaccines by using plant production systems. PMID:17428917

Dendritic Cell Immaturity during Infancy Restricts the Capacity To Express Vaccine-Specific T-Cell Memory

PubMed Central

Upham, John W.; Rate, Angela; Rowe, Julie; Kusel, Merci; Sly, Peter D.; Holt, Patrick G.

2006-01-01

The capacity of the immune system in infants to develop stable T-cell memory in response to vaccination is attenuated, and the mechanism(s) underlying this developmental deficiency in humans is poorly understood. The present study focuses on the capacity for expression of in vitro recall responses to tetanus and diphtheria antigens in lymphocytes from 12-month-old infants vaccinated during the first 6 months of life. We demonstrate that supplementation of infant lymphocytes with “matured” dendritic cells (DC) cultured from autologous CD14+ precursors unmasks previously covert cellular immunity in the form of Th2-skewed cytokine production. Supplementation of adult lymphocytes with comparable prematured autologous DC also boosted vaccine-specific T-cell memory expression, but in contrast to the case for the infants, these cytokine responses were heavily Th1 skewed. Compared to adults, infants had significantly fewer circulating myeloid DC (P < 0.0001) and plasmacytoid DC (P < 0.0001) as a proportion of peripheral blood mononuclear cells. These findings suggest that deficiencies in the numbers of antigen-presenting cells and their functional competence at 12 months of age limit the capacity to express effector memory responses and are potentially a key factor in reduced vaccine responsiveness in infants. PMID:16428758

Preferential expression and immunogenicity of HIV-1 Tat fusion protein expressed in tomato plant.

PubMed

Cueno, Marni E; Hibi, Yurina; Karamatsu, Katsuo; Yasutomi, Yasuhiro; Imai, Kenichi; Laurena, Antonio C; Okamoto, Takashi

2010-10-01

HIV-1 Tat plays a major role in viral replication and is essential for AIDS development making it an ideal vaccine target providing that both humoral and cellular immune responses are induced. Plant-based antigen production, due to its cheaper cost, appears ideal for vaccine production. In this study, we created a plant-optimized tat and mutant (Cys30Ala/Lys41Ala) tat (mtat) gene and ligated each into a pBI121 expression vector with a stop codon and a gusA gene positioned immediately downstream. The vector construct was bombarded into tomato leaf calli and allowed to develop. We thus generated recombinant tomato plants preferentially expressing a Tat-GUS fusion protein over a Tat-only protein. In addition, plants bombarded with either tat or mtat genes showed no phenotypic difference and produced 2-4 microg Tat-GUS fusion protein per milligram soluble plant protein. Furthermore, tomato extracts intradermally inoculated into mice were found to induce a humoral and, most importantly, cellular immunity.

[Study on solubility of Chinese herbal compound by solubility parameter].

PubMed

Wu, Dezhi; Chen, Lihua; Wang, Sen; Zhu, Weifeng; Guan, Yongmei

2010-02-01

To demonstrate the solubility of Chinese herbal compound with solubility parameters. The solubility parameters of Liangfu effective components and Liangfu compound were determined by inverse gas chromatograph (IGC) and group contribution. Hansen ball was plotting by HSPiP, which could be used to investigate the solubility of Liangfu effective components and Liangfu compound in different solvents. And the results were verified by approximate solubility. Liangfu effective components and Liangfu compound could be dissolved in chloroform, ethyl acetate, acetone, octanol and ether, and were slightly soluble in glycerol, methanol, ethanol and propanediol, but could not be dissolved in water. They were all liposoluble, and the results were the same as the test results of the approximate solubility. The solubility of Chinese herbal compound can be expressed by solubility parameters, and it is accurate, convenient and visual.

Recent advances in recombinant protein-based malaria vaccines

PubMed Central

Draper, Simon J.; Angov, Evelina; Horii, Toshihiro; Miller, Louis H.; Srinivasan, Prakash; Theisen, Michael; Biswas, Sumi

2015-01-01

Plasmodium parasites are the causative agent of human malaria, and the development of a highly effective vaccine against infection, disease and transmission remains a key priority. It is widely established that multiple stages of the parasite's complex lifecycle within the human host and mosquito vector are susceptible to vaccine-induced antibodies. The mainstay approach to antibody induction by subunit vaccination has been the delivery of protein antigen formulated in adjuvant. Extensive efforts have been made in this endeavor with respect to malaria vaccine development, especially with regard to target antigen discovery, protein expression platforms, adjuvant testing, and development of soluble and virus-like particle (VLP) delivery platforms. The breadth of approaches to protein-based vaccines is continuing to expand as innovative new concepts in next-generation subunit design are explored, with the prospects for the development of a highly effective multi-component/multi-stage/multi-antigen formulation seeming ever more likely. This review will focus on recent progress in protein vaccine design, development and/or clinical testing for a number of leading malaria antigens from the sporozoite-, merozoite- and sexual-stages of the parasite's lifecycle–including PfCelTOS, PfMSP1, PfAMA1, PfRH5, PfSERA5, PfGLURP, PfMSP3, Pfs48/45 and Pfs25. Future prospects and challenges for the development, production, human delivery and assessment of protein-based malaria vaccines are discussed. PMID:26458807

Recent advances in recombinant protein-based malaria vaccines.

PubMed

Draper, Simon J; Angov, Evelina; Horii, Toshihiro; Miller, Louis H; Srinivasan, Prakash; Theisen, Michael; Biswas, Sumi

2015-12-22

Plasmodium parasites are the causative agent of human malaria, and the development of a highly effective vaccine against infection, disease and transmission remains a key priority. It is widely established that multiple stages of the parasite's complex lifecycle within the human host and mosquito vector are susceptible to vaccine-induced antibodies. The mainstay approach to antibody induction by subunit vaccination has been the delivery of protein antigen formulated in adjuvant. Extensive efforts have been made in this endeavor with respect to malaria vaccine development, especially with regard to target antigen discovery, protein expression platforms, adjuvant testing, and development of soluble and virus-like particle (VLP) delivery platforms. The breadth of approaches to protein-based vaccines is continuing to expand as innovative new concepts in next-generation subunit design are explored, with the prospects for the development of a highly effective multi-component/multi-stage/multi-antigen formulation seeming ever more likely. This review will focus on recent progress in protein vaccine design, development and/or clinical testing for a number of leading malaria antigens from the sporozoite-, merozoite- and sexual-stages of the parasite's lifecycle-including PfCelTOS, PfMSP1, PfAMA1, PfRH5, PfSERA5, PfGLURP, PfMSP3, Pfs48/45 and Pfs25. Future prospects and challenges for the development, production, human delivery and assessment of protein-based malaria vaccines are discussed. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

A Lactococcus lactis BFE920 feed vaccine expressing a fusion protein composed of the OmpA and FlgD antigens from Edwardsiella tarda was significantly better at protecting olive flounder (Paralichthys olivaceus) from edwardsiellosis than single antigen vaccines.

PubMed

Beck, Bo Ram; Lee, Soon Ho; Kim, Daniel; Park, Ji Hye; Lee, Hyun Kyung; Kwon, San-Sung; Lee, Kwan Hee; Lee, Jae Il; Song, Seong Kyu

2017-09-01

Edwardsiellosis is a major fish disease that causes a significant economic damage in the aquaculture industry. Here, we assessed vaccine efficacy after feeding oral vaccines to olive flounder (Paralichthys olivaceus), either L. lactis BFE920 expressing Edwardsiella tarda outer membrane protein A (OmpA), flagellar hook protein D (FlgD), or a fusion antigen of the two. Feed vaccination was done twice with a one-week interval. Fish were fed regular feed adsorbed with the vaccines. Feed vaccination was given over the course of one week to maximize the interaction between the feed vaccines and the fish intestine. Flounder fed the vaccine containing the fusion antigen had significantly elevated levels T cell genes (CD4-1, CD4-2, and CD8α), type 1 helper T cell (Th1) subset indicator genes (T-bet and IFN-γ), and antigen-specific antibodies compared to the groups fed the single antigen-expressing vaccines. Furthermore, the superiority of the fusion vaccine was also observed in survival rates when fish were challenged with E. tarda: OmpA-FlgD-expressing vaccine (82.5% survival); FlgD-vaccine (55.0%); OmpA-vaccine (50%); WT L. lactis BFE920 (37.5%); Ctrl (10%). In addition, vaccine-fed fish exhibited increased weight gain (∼20%) and a decreased feed conversion ratio (∼20%) during the four week vaccination period. Flounder fed the FlgD-expressing vaccine, either the single or the fusion form, had significantly increased expression of TLR5M, IL-1β, and IL-12p40, suggesting that the FlgD may be a ligand of olive flounder TLR5M receptor or closely related to the TLR5M pathway. In conclusion, the present study demonstrated that olive flounder fed L. lactis BFE920 expressing a fusion antigen composed of E. tarda OmpA and FlgD showed a strong protective effect against edwardsiellosis indicating this may be developed as an E. tarda feed vaccine. Copyright © 2017 Elsevier Ltd. All rights reserved.

A Novel Virus-Like Particle Based Vaccine Platform Displaying the Placental Malaria Antigen VAR2CSA.

PubMed

Thrane, Susan; Janitzek, Christoph M; Agerbæk, Mette Ø; Ditlev, Sisse B; Resende, Mafalda; Nielsen, Morten A; Theander, Thor G; Salanti, Ali; Sander, Adam F

2015-01-01

Placental malaria caused by Plasmodium falciparum is a major cause of mortality and severe morbidity. Clinical testing of a soluble protein-based vaccine containing the parasite ligand, VAR2CSA, has been initiated. VAR2CSA binds to the human receptor chondroitin sulphate A (CSA) and is responsible for sequestration of Plasmodium falciparum infected erythrocytes in the placenta. It is imperative that a vaccine against malaria in pregnancy, if administered to women before they become pregnant, can induce a strong and long lasting immune response. While most soluble protein-based vaccines have failed during clinical testing, virus-like particle (VLP) based vaccines (e.g., the licensed human papillomavirus vaccines) have demonstrated high efficacy, suggesting that the spatial assembly of the vaccine antigen is a critical parameter for inducing an optimal long-lasting protective immune response. We have developed a VLP vaccine display platform by identifying regions of the HPV16 L1 coat protein where a biotin acceptor site (AviTagTM) can be inserted without compromising VLP-assembly. Subsequent biotinylation of Avi-L1 VLPs allow us to anchor monovalent streptavidin (mSA)-fused proteins to the biotin, thereby obtaining a dense and repetitive VLP-display of the vaccine antigen. The mSA-VAR2CSA antigen was delivered on the Avi-L1 VLP platform and tested in C57BL/6 mice in comparison to two soluble protein-based vaccines consisting of naked VAR2CSA and mSA-VAR2CSA. The mSA-VAR2CSA Avi-L1 VLP and soluble mSA-VAR2CSA vaccines induced higher antibody titers than the soluble naked VAR2CSA vaccine after three immunizations. The VAR2CSA Avi-L1 VLP vaccine induced statistically significantly higher endpoint titres compared to the soluble mSA-VAR2CSA vaccine, after 1st and 2nd immunization; however, this difference was not statistically significant after 3rd immunization. Importantly, the VLP-VAR2CSA induced antibodies were functional in inhibiting the binding of parasites to CSA

An Adenovirus Vaccine Expressing Ebola Virus Variant Makona Glycoprotein Is Efficacious in Guinea Pigs and Nonhuman Primates.

PubMed

Wu, Shipo; Kroeker, Andrea; Wong, Gary; He, Shihua; Hou, Lihua; Audet, Jonathan; Wei, Haiyan; Zhang, Zhe; Fernando, Lisa; Soule, Geoff; Tran, Kaylie; Bi, Shengli; Zhu, Tao; Yu, Xuefeng; Chen, Wei; Qiu, Xiangguo

2016-10-15

A licensed vaccine against Ebola virus (EBOV) remains unavailable, despite >11 000 deaths from the 2014-2016 outbreak of EBOV disease in West Africa. Past studies have shown that recombinant vaccine viruses expressing EBOV glycoprotein (GP) are able to protect nonhuman primates (NHPs) from a lethal EBOV challenge. However, these vaccines express the viral GP-based EBOV variants found in Central Africa, which has 97.3% amino acid homology to the Makona variant found in West Africa. Our previous study showed that a recombinant adenovirus serotype 5 (Ad5)-vectored vaccine expressing the Makona EBOV GP (MakGP) was safe and immunogenic during clinical trials in China, but it is unknown whether the vaccine protects against EBOV infection. Here, we demonstrate that guinea pigs immunized with Ad5-MakGP developed robust humoral responses and were protected against exposure to guinea pig-adapted EBOV. Ad5-MakGP also elicited specific B- and T-cell immunity in NHPs and conferred 100% protection when animals were challenged 4 weeks after immunization. These results support further clinical development of this candidate and highlight the utility of Ad5-MakGP as a prophylactic measure in future outbreaks of EBOV disease. © Crown copyright 2016.

Soluble expression and one-step purification of recombinant mouse interferon-λ3 in Escherichia coli.

PubMed

Wang, Y Q; Zhou, M; Zeng, L M; Gao, Q Y; Yuan, X L; Li, Y; Li, M C

2015-02-01

Interferon (IFN)-λ3, a member of the type III IFN family, is a pleiotropic cytokine that exhibits potent antiproliferative, antiviral, and immunoregulatory activities. For further functional study of IFN-λ3, we developed an efficient procedure that includes cloning, expression, and purification to obtain relatively large quantity of mouse IFN-λ3 fusion protein. The mature IFN-λ3 protein-coding region was cloned into the prokaryotic expression vector pET-44. IFN-λ3 contains a hexahistidine tag at its C-terminus. We used Ni(2+)-nitrilotriacetic acid agarose-affinity chromatography to purify the expressed soluble protein. The purified IFN-λ3 inhibited significantly IL-13 production in stimulated RAW264.7 macrophages. Our findings show that the production of soluble IFN-λ3 proteins by the pET-44 vector in Escherichia coli is a good alternative for the production of native IFN-λ3 and could be useful for the production of other IFN proteins.

Vaccine development against the Taenia solium parasite: the role of recombinant protein expression in Escherichia coli.

PubMed

Gauci, Charles; Jayashi, César; Lightowlers, Marshall W

2013-01-01

Taenia solium is a zoonotic parasite that causes cysticercosis. The parasite is a major cause of human disease in impoverished communities where it is transmitted to humans from pigs which act as intermediate hosts. Vaccination of pigs to prevent transmission of T. solium to humans is an approach that has been investigated to control the disease. A recombinant vaccine antigen, TSOL18, has been remarkably successful at reducing infection of pigs with T. solium in several experimental challenge trials. The vaccine has been shown to eliminate transmission of naturally acquired T. solium in a field trial conducted in Africa. We recently reported that the vaccine was also effective in a field trial conducted in Peru. The TSOL18 recombinant antigen for each of these trials has been produced by expression in Escherichia coli. Here we discuss research that has been undertaken on the TSOL18 antigen and related antigens with a focus on improved methods of preparation of recombinant TSOL18 and optimized expression in Escherichia coli.

In Vitro Evaluation of a Soluble Leishmania Promastigote Surface Antigen as a Potential Vaccine Candidate against Human Leishmaniasis

PubMed Central

Bahi-Jaber, Narges; Petitdidier, Elodie; Markikou-Ouni, Wafa; Aoun, Karim; Moreno, Javier; Carrillo, Eugenia; Salotra, Poonam; Kaushal, Himanshu; Negi, Narender Singh; Arevalo, Jorge; Falconi-Agapito, Francesca; Privat, Angela; Cruz, Maria; Pagniez, Julie; Papierok, Gérard-Marie; Rhouma, Faten Bel Haj; Torres, Pilar; Lemesre, Jean-Loup; Chenik, Mehdi; Meddeb-Garnaoui, Amel

2014-01-01

PSA (Promastigote Surface Antigen) belongs to a family of membrane-bound and secreted proteins present in several Leishmania (L.) species. PSA is recognized by human Th1 cells and provides a high degree of protection in vaccinated mice. We evaluated humoral and cellular immune responses induced by a L. amazonensis PSA protein (LaPSA-38S) produced in a L. tarentolae expression system. This was done in individuals cured of cutaneous leishmaniasis due to L. major (CCLm) or L. braziliensis (CCLb) or visceral leishmaniasis due to L. donovani (CVLd) and in healthy individuals. Healthy individuals were subdivided into immune (HHR-Lm and HHR-Li: Healthy High Responders living in an endemic area for L. major or L. infantum infection) or non immune/naive individuals (HLR: Healthy Low Responders), depending on whether they produce high or low levels of IFN-γ in response to Leishmania soluble antigen. Low levels of total IgG antibodies to LaPSA-38S were detected in sera from the studied groups. Interestingly, LaPSA-38S induced specific and significant levels of IFN-γ, granzyme B and IL-10 in CCLm, HHR-Lm and HHR-Li groups, with HHR-Li group producing TNF-α in more. No significant cytokine response was observed in individuals immune to L. braziliensis or L. donovani infection. Phenotypic analysis showed a significant increase in CD4+ T cells producing IFN-γ after LaPSA-38S stimulation, in CCLm. A high positive correlation was observed between the percentage of IFN-γ-producing CD4+ T cells and the released IFN-γ. We showed that the LaPSA-38S protein was able to induce a mixed Th1 and Th2/Treg cytokine response in individuals with immunity to L. major or L. infantum infection indicating that it may be exploited as a vaccine candidate. We also showed, to our knowledge for the first time, the capacity of Leishmania PSA protein to induce granzyme B production in humans with immunity to L. major and L. infantum infection. PMID:24786587

A chimeric alphavirus replicon particle vaccine expressing the hemagglutinin and fusion proteins protects juvenile and infant rhesus macaques from measles.

PubMed

Pan, Chien-Hsiung; Greer, Catherine E; Hauer, Debra; Legg, Harold S; Lee, Eun-Young; Bergen, M Jeff; Lau, Brandyn; Adams, Robert J; Polo, John M; Griffin, Diane E

2010-04-01

Measles remains a major cause of child mortality, in part due to an inability to vaccinate young infants with the current live attenuated virus vaccine (LAV). To explore new approaches to infant vaccination, chimeric Venezuelan equine encephalitis/Sindbis virus (VEE/SIN) replicon particles were used to express the hemagglutinin (H) and fusion (F) proteins of measles virus (MV). Juvenile rhesus macaques vaccinated intradermally with a single dose of VEE/SIN expressing H or H and F proteins (VEE/SIN-H or VEE/SIN-H+F, respectively) developed high titers of MV-specific neutralizing antibody and gamma-interferon (IFN-gamma)-producing T cells. Infant macaques vaccinated with two doses of VEE/SIN-H+F also developed neutralizing antibody and IFN-gamma-producing T cells. Control animals were vaccinated with LAV or with a formalin-inactivated measles vaccine (FIMV). Neutralizing antibody remained above the protective level for more than 1 year after vaccination with VEE/SIN-H, VEE/SIN-H+F, or LAV. When challenged with wild-type MV 12 to 17 months after vaccination, all vaccinated juvenile and infant monkeys vaccinated with VEE/SIN-H, VEE/SIN-H+F, and LAV were protected from rash and viremia, while FIMV-vaccinated monkeys were not. Antibody was boosted by challenge in all groups. T-cell responses to challenge were biphasic, with peaks at 7 to 25 days and at 90 to 110 days in all groups, except for the LAV group. Recrudescent T-cell activity coincided with the presence of MV RNA in peripheral blood mononuclear cells. We conclude that VEE/SIN expressing H or H and F induces durable immune responses that protect from measles and offers a promising new approach for measles vaccination. The viral and immunological factors associated with long-term control of MV replication require further investigation.

Influence of haplotypes, gene expression and soluble levels of L-selectin on the risk of acute coronary syndrome.

PubMed

Sandoval-Pinto, Elena; Padilla-Gutiérrez, Jorge Ramón; Hernández-Bello, Jorge; Martínez-Fernández, Diana Emilia; Valdés-Alvarado, Emmanuel; Muñoz-Valle, José Francisco; Flores-Salinas, H E; Valle, Yeminia

2017-08-20

L-selectin gene (SELL) is a candidate gene for the development of acute coronary syndrome (ACS) that contributes to endothelial dysfunction. The -642C>T (rs2205849) and 725C>T (rs2229569) polymorphisms have been associated with changes in gene expression, ligand affinity and increased risk of cardiovascular disease. The aim of this study was to investigate the association between the haplotypes constructed with the -642C>T and 725C>T polymorphisms of the SELL gene, the expression levels of its mRNA and the serum levels of soluble L-selectin with ACS. We recruited 615 individuals of Mexican origin matched by age, including 342 patients with ACS and 273 individuals without personal history of ischemic cardiopathy as control group (CG). Genotyping was performed by PCR-RFLP. The qPCR technique was used to analyze the expression of mRNA using TaqMan® UPL probes. The levels of soluble L-selectin were measured with ELISA. The allele variants in both polymorphisms were over-represented in the CG compared to the ACS (OR range: 0.371-0.716, p<0.006). The CT and TT haplotypes had a protective effect against the development of ACS (OR=0.401, p<0.0001; OR=0.628, p<0.0001, respectively). SELL expression was 3.076 times higher in the ACS group compared to CG (p<0.001). The levels of soluble L-selectin were similar between ACS and CG. Both polymorphisms had no effect on mRNA expression and soluble protein levels. The polymorphisms -642C>T and 725C>T of the SELL gene are protective factors against the development of ACS. There is an increased gene expression of L-selectin in ACS compared to CG in the population of Western Mexico. Copyright © 2017 Elsevier B.V. All rights reserved.

Periscope: quantitative prediction of soluble protein expression in the periplasm of Escherichia coli

NASA Astrophysics Data System (ADS)

Chang, Catherine Ching Han; Li, Chen; Webb, Geoffrey I.; Tey, Bengti; Song, Jiangning; Ramanan, Ramakrishnan Nagasundara

2016-03-01

Periplasmic expression of soluble proteins in Escherichia coli not only offers a much-simplified downstream purification process, but also enhances the probability of obtaining correctly folded and biologically active proteins. Different combinations of signal peptides and target proteins lead to different soluble protein expression levels, ranging from negligible to several grams per litre. Accurate algorithms for rational selection of promising candidates can serve as a powerful tool to complement with current trial-and-error approaches. Accordingly, proteomics studies can be conducted with greater efficiency and cost-effectiveness. Here, we developed a predictor with a two-stage architecture, to predict the real-valued expression level of target protein in the periplasm. The output of the first-stage support vector machine (SVM) classifier determines which second-stage support vector regression (SVR) classifier to be used. When tested on an independent test dataset, the predictor achieved an overall prediction accuracy of 78% and a Pearson’s correlation coefficient (PCC) of 0.77. We further illustrate the relative importance of various features with respect to different models. The results indicate that the occurrence of dipeptide glutamine and aspartic acid is the most important feature for the classification model. Finally, we provide access to the implemented predictor through the Periscope webserver, freely accessible at http://lightning.med.monash.edu/periscope/.

A Highly Immunogenic and Protective Middle East Respiratory Syndrome Coronavirus Vaccine Based on a Recombinant Measles Virus Vaccine Platform

PubMed Central

Malczyk, Anna H.; Kupke, Alexandra; Prüfer, Steffen; Scheuplein, Vivian A.; Hutzler, Stefan; Kreuz, Dorothea; Beissert, Tim; Bauer, Stefanie; Hubich-Rau, Stefanie; Tondera, Christiane; Eldin, Hosam Shams; Schmidt, Jörg; Vergara-Alert, Júlia; Süzer, Yasemin; Seifried, Janna; Hanschmann, Kay-Martin; Kalinke, Ulrich; Herold, Susanne; Sahin, Ugur; Cichutek, Klaus; Waibler, Zoe; Eickmann, Markus; Becker, Stephan

2015-01-01

ABSTRACT In 2012, the first cases of infection with the Middle East respiratory syndrome coronavirus (MERS-CoV) were identified. Since then, more than 1,000 cases of MERS-CoV infection have been confirmed; infection is typically associated with considerable morbidity and, in approximately 30% of cases, mortality. Currently, there is no protective vaccine available. Replication-competent recombinant measles virus (MV) expressing foreign antigens constitutes a promising tool to induce protective immunity against corresponding pathogens. Therefore, we generated MVs expressing the spike glycoprotein of MERS-CoV in its full-length (MERS-S) or a truncated, soluble variant of MERS-S (MERS-solS). The genes encoding MERS-S and MERS-solS were cloned into the vaccine strain MVvac2 genome, and the respective viruses were rescued (MVvac2-CoV-S and MVvac2-CoV-solS). These recombinant MVs were amplified and characterized at passages 3 and 10. The replication of MVvac2-CoV-S in Vero cells turned out to be comparable to that of the control virus MVvac2-GFP (encoding green fluorescent protein), while titers of MVvac2-CoV-solS were impaired approximately 3-fold. The genomic stability and expression of the inserted antigens were confirmed via sequencing of viral cDNA and immunoblot analysis. In vivo, immunization of type I interferon receptor-deficient (IFNAR−/−)-CD46Ge mice with 2 × 105 50% tissue culture infective doses of MVvac2-CoV-S(H) or MVvac2-CoV-solS(H) in a prime-boost regimen induced robust levels of both MV- and MERS-CoV-neutralizing antibodies. Additionally, induction of specific T cells was demonstrated by T cell proliferation, antigen-specific T cell cytotoxicity, and gamma interferon secretion after stimulation of splenocytes with MERS-CoV-S presented by murine dendritic cells. MERS-CoV challenge experiments indicated the protective capacity of these immune responses in vaccinated mice. IMPORTANCE Although MERS-CoV has not yet acquired extensive distribution

Field evaluation of poultry red mite (Dermanyssus gallinae) native and recombinant prototype vaccines.

PubMed

Bartley, Kathryn; Turnbull, Frank; Wright, Harry W; Huntley, John F; Palarea-Albaladejo, Javier; Nath, Mintu; Nisbet, Alasdair J

2017-09-15

Vaccination is a desirable emerging strategy to combat poultry red mite (PRM), Dermanyssus gallinae. We performed trials, in laying hens in a commercial-style cage facility, to test the vaccine efficacy of a native preparation of soluble mite extract (SME) and of a recombinant antigen cocktail vaccine containing bacterially-expressed versions of the immunogenic SME proteins Deg-SRP-1, Deg-VIT-1 and Deg-PUF-1. Hens (n=384 per group) were injected with either vaccine or adjuvant only (control group) at 12 and 17 weeks of age and then challenged with PRM 10days later. PRM counts were monitored and, at the termination of the challenge period (17 weeks post challenge), average PRM counts in cages containing birds vaccinated with SME were reduced by 78% (p<0.001), compared with those in the adjuvant-only control group. When the trial was repeated using the recombinant antigen cocktail vaccine, no statistically significant differences in mean PRM numbers were observed in cages containing vaccinated or adjuvant-only immunised birds. The roles of antigen-specific antibody levels and duration in providing vaccine-induced and exposure-related protective immunity are discussed. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Cytokine Signaling in Splenic Leukocytes from Vaccinated and Non-Vaccinated Chickens after Intravenous Infection with Salmonella Enteritidis

PubMed Central

Matulova, Marta; Stepanova, Hana; Sisak, Frantisek; Havlickova, Hana; Faldynova, Marcela; Kyrova, Kamila; Volf, Jiri; Rychlik, Ivan

2012-01-01

In order to design a new Salmonella enterica vaccine, one needs to understand how naive and immune chickens interact differently when exposed to S. enterica. In this study we therefore determined the immune response of vaccinated and non-vaccinated chickens after intravenous infection with Salmonella enterica serovar Enteritidis (S. Enteritidis). Using flow cytometry we showed that 4 days post infection (DPI), counts of CD4 and B-lymphocytes did not change, CD8 and γδ T-lymphocytes decreased and macrophages and heterophils increased in the spleen. When vaccinated and non-vaccinated chickens were compared, only macrophages and heterophils were found in significantly higher counts in the spleens of the non-vaccinated chickens. The non-vaccinated chickens also expressed higher anti-LPS antibodies than the vaccinated chickens. The expression of interleukin (IL)1β, IL6, IL8, IL18, LITAF, IFNγ and iNOS did not exhibit any clear pattern in the cells sorted from the spleens of vaccinated or non-vaccinated chickens. Only IL17 and IL22 showed a differential expression in the CD4 T-lymphocytes of the vaccinated and non-vaccinated chickens at 4 DPI, both being expressed at a higher level in the non-vaccinated chickens. Due to a similar IFNγ expression in the CD4 T-lymphocytes in both the vaccinated and non-vaccinated chickens, and a variable IL17 expression oscillating around IFNγ expression levels, the IL17∶IFNγ ratio in CD4 T-lymphocytes was found to be central for the outcome of the immune response. When IL17 was expressed at higher levels than IFNγ in the non-vaccinated chickens, the Th17 immune response with a higher macrophage and heterophil infiltration in the spleen dominated. However, when the expression of IL17 was lower than that of IFNγ as in the vaccinated chickens, the Th1 response with a higher resistance to S. Enteritidis infection dominated. PMID:22384225

Macromolecular systems for vaccine delivery.

PubMed

MuŽíková, G; Laga, R

2016-10-20

Vaccines have helped considerably in eliminating some life-threatening infectious diseases in past two hundred years. Recently, human medicine has focused on vaccination against some of the world's most common infectious diseases (AIDS, malaria, tuberculosis, etc.), and vaccination is also gaining popularity in the treatment of cancer or autoimmune diseases. The major limitation of current vaccines lies in their poor ability to generate a sufficient level of protective antibodies and T cell responses against diseases such as HIV, malaria, tuberculosis and cancers. Among the promising vaccination systems that could improve the potency of weakly immunogenic vaccines belong macromolecular carriers (water soluble polymers, polymer particels, micelles, gels etc.) conjugated with antigens and immunistumulatory molecules. The size, architecture, and the composition of the high molecular-weight carrier can significantly improve the vaccine efficiency. This review includes the most recently developed (bio)polymer-based vaccines reported in the literature.

Ki-67 expression reveals strong, transient influenza specific CD4 T cell responses after adult vaccination.

PubMed

Li, Xi; Miao, Hongyu; Henn, Alicia; Topham, David J; Wu, Hulin; Zand, Martin S; Mosmann, Tim R

2012-06-29

Although previous studies have found minimal changes in CD4 T cell responses after vaccination of adults with trivalent inactivated influenza vaccine, daily sampling and monitoring of the proliferation marker Ki-67 have now been used to reveal that a substantial fraction of influenza-specific CD4 T cells respond to vaccination. At 4-6 days after vaccination, there is a sharp rise in the numbers of Ki-67-expressing PBMC that produce IFNγ, IL-2 and/or TNFα in vitro in response to influenza vaccine or peptide. Ki-67(+) cell numbers then decline rapidly, and 10 days after vaccination, both Ki-67(+) and overall influenza-specific cell numbers are similar to pre-vaccination levels. These results provide a tool for assessing the quality and quantity of CD4 T cell responses to different influenza vaccines, and raise the possibility that the anti-influenza T cell memory response may be qualitatively altered by vaccination, even if the overall memory cell numbers do not change significantly. Copyright © 2012. Published by Elsevier Ltd.

Improving solubility of Shewanella oneidensis MR-1 and Clostridium thermocellum JW-20 proteins expressed into Esherichia coli.

PubMed

Kataeva, Irina; Chang, Jessie; Xu, Hao; Luan, Chi-Hao; Zhou, Jizhong; Uversky, Vladimir N; Lin, Dawei; Horanyi, Peter; Liu, Z J; Ljungdahl, Lars G; Rose, John; Luo, Ming; Wang, Bi-Cheng

2005-01-01

Low solubility of proteins overexpressed in E. coli is a frequent problem in high-throughput structural genomics. To improve solubility of proteins from mesophilic Shewanella oneidensis MR-1 and thermophilic Clostridium thermocellum JW20, an approach was attempted that included a fusion of the target protein to a maltose-binding protein (MBP) and a decrease of induction temperature. The MBP was selected as the most efficient solubilizing carrier when compared to a glutathione S-transferase and a Nus A protein. A tobacco etch virus (TEV) protease recognition site was introduced between fused proteins using a double polymerase-chain reaction and four primers. In this way, 79 S. oneidensis proteins have been expressed in one case with an N-terminal 30-residue tag and in another case as a fusion protein with MBP. A foreign tag might significantly affect the properties of the target polypeptide. At 37 degrees C and 18 degrees C induction temperatures, only 5 and 17 tagged proteins were soluble, respectively. In fusion with MBP 4, 34, and 38 proteins were soluble upon induction at 37 degrees, 28 degrees, and 18 degrees C, respectively. The MBP is assumed to increase stability and solubility of a target protein by changing both the mechanism and the cooperativity of folding/unfolding. The 66 C. thermocellum proteins were expressed as fusion proteins with MBP. Induction at 37 degrees, 28 degrees, and 18 degrees C produced 34, 57, and 60 soluble proteins, respectively. The higher solubility of C. thermocellum proteins in comparison with the S. oneidensis proteins under similar conditions of induction correlates with the thermophilicity of the host. The two-factor Wilkinson-Harrison statistical model was used to identify soluble and insoluble proteins. Theoretical and experimental data showed good agreement for S. oneidensis proteins; however, the model failed to identify soluble/insoluble Clostridium proteins. A suggestion has been made that the Wilkinson-Harrison model is

Soluble triggering receptor expressed on myeloid cells 1 and the diagnosis of sepsis.

PubMed

Barati, Mitra; Bashar, Farshid Rahimi; Shahrami, Reza; Zadeh, Mohammad Hossein Jarrah; Taher, Mahshid Talebi; Nojomi, Marzieh

2010-06-01

Early diagnosis and assessment of the systemic inflammatory response to infection are difficult with usual markers (fever, leukocytosis, C-reactive protein [CRP]). Triggering receptor expressed on myeloid cells-1 (TREM-1) expression on phagocytes is up-regulated by microbial products. We studied the ability of soluble TREM-1 (sTREM-1) to identify patients with sepsis. Plasma samples were obtained on intensive care unit admission from patients with systemic inflammatory response syndrome for sTREM-1 measurement. Soluble TREM-1, CRP concentrations and erythrocyte sedimentation rate (ESR) were higher in the sepsis group (n = 52) than in the non-infectious systemic inflammatory response syndrome group (n = 43; P = .00, .02, and .001, respectively). Soluble TREM-1, CRP concentrations, white blood cell count and ESR were higher in the sepsis group than in the non SIRS group (n = 37; P = .04, .00, .01, and .00, respectively). In a receiver-operating characteristic curve analysis, ESR, CRP and sTREM-1 had an area under the curve larger than 0.65 (P = .00), in distinguishing between septic and non-infectious SIRS patients. CRP, ESR, sTREM-1 had a sensitivity of 60%, 70% and 70% and a specificity of 60%, 69% and, 60% respectively in diagnosing infection in SIRS. C-reactive protein and ESR performed better than sTREM-1 and white blood cell count in diagnosing infection. Copyright (c) 2010. Published by Elsevier Inc.

Avirulent Marek’s Disease Virus Type 1 Strain 814 Vectored Vaccine Expressing Avian Influenza (AI) Virus H5 Haemagglutinin Induced Better Protection Than Turkey Herpesvirus Vectored AI Vaccine

PubMed Central

Cui, Xianlan; Zhao, Yan; Shi, Xingming; Li, Qiaoling; Yan, Shuai; Gao, Ming; Wang, Mei; Liu, Changjun; Wang, Yunfeng

2013-01-01

Background Herpesvirus of turkey (HVT) as a vector to express the haemagglutinin (HA) of avian influenza virus (AIV) H5 was developed and its protection against lethal Marek’s disease virus (MDV) and highly pathogenic AIV (HPAIV) challenges was evaluated previously. It is well-known that avirulemt MDV type 1 vaccines are more effective than HVT in prevention of lethal MDV infection. To further increase protective efficacy against HPAIV and lethal MDV, a recombinant MDV type 1 strain 814 was developed to express HA gene of HPAIV H5N1. Methodology/Principal Findings A recombinant MDV-1 strain 814 expressing HA gene of HPAIV H5N1 virus A/goose/Guangdong/3/96 at the US2 site (rMDV-HA) was developed under the control of a human CMV immediate-early promoter. The HA expression in the rMDV-HA was tested by immunofluorescence and Western blot analyses, and in vitro and in vivo growth properties of rMDV-HA were also analyzed. Furthermore, we evaluated and compared the protective immunity of rMDV-HA and previously constructed rHVT-HA against HPAIV and lethal MDV. Vaccination of chickens with rMDV-HA induced 80% protection against HPAIV, which was better than the protection rate by rHVT-HA (66.7%). In the animal study with MDV challenge, chickens immunized with rMDV-HA were completely protected against virulent MDV strain J-1 whereas rHVT-HA only induced 80% protection with the same challenge dose. Conclusions/Significance The rMDV-HA vaccine was more effective than rHVT-HA vaccine for protection against lethal MDV and HPAIV challenges. Therefore, avirulent MDV type 1 vaccine is a better vector than HVT for development of a recombinant live virus vaccine against virulent MDV and HPAIV in poultry. PMID:23301062

Enhanced Neutralizing Antibody Response Induced by Respiratory Syncytial Virus Prefusion F Protein Expressed by a Vaccine Candidate

PubMed Central

Liang, Bo; Surman, Sonja; Amaro-Carambot, Emerito; Kabatova, Barbora; Mackow, Natalie; Lingemann, Matthias; Yang, Lijuan; McLellan, Jason S.; Graham, Barney S.; Kwong, Peter D.; Schaap-Nutt, Anne; Collins, Peter L.

2015-01-01

ABSTRACT Respiratory syncytial virus (RSV) and human parainfluenza virus type 3 (HPIV3) are the first and second leading viral agents of severe respiratory tract disease in infants and young children worldwide. Vaccines are not available, and an RSV vaccine is particularly needed. A live attenuated chimeric recombinant bovine/human PIV3 (rB/HPIV3) vector expressing the RSV fusion (F) glycoprotein from an added gene has been under development as a bivalent vaccine against RSV and HPIV3. Previous clinical evaluation of this vaccine candidate suggested that increased genetic stability and immunogenicity of the RSV F insert were needed. This was investigated in the present study. RSV F expression was enhanced 5-fold by codon optimization and by modifying the amino acid sequence to be identical to that of an early passage of the original clinical isolate. This conferred a hypofusogenic phenotype that presumably reflects the original isolate. We then compared vectors expressing stabilized prefusion and postfusion versions of RSV F. In a hamster model, prefusion F induced increased quantity and quality of RSV-neutralizing serum antibodies and increased protection against wild-type (wt) RSV challenge. In contrast, a vector expressing the postfusion F was less immunogenic and protective. The genetic stability of the RSV F insert was high and was not affected by enhanced expression or the prefusion or postfusion conformation of RSV F. These studies provide an improved version of the previously well-tolerated rB/HPIV3-RSV F vaccine candidate that induces a superior RSV-neutralizing serum antibody response. IMPORTANCE Respiratory syncytial virus (RSV) and human parainfluenza virus type 3 (HPIV3) are two major causes of pediatric pneumonia and bronchiolitis. The rB/HPIV3 vector expressing RSV F protein is a candidate bivalent live vaccine against HPIV3 and RSV. Previous clinical evaluation indicated the need to increase the immunogenicity and genetic stability of the RSV F

The Corn Smut (‘Huitlacoche’) as a New Platform for Oral Vaccines

PubMed Central

Juárez-Montiel, Margarita; Romero-Maldonado, Andrea; Monreal-Escalante, Elizabeth; Becerra-Flora, Alicia; Korban, Schuyler S.; Rosales-Mendoza, Sergio; Jiménez-Bremont, Juan Francisco

2015-01-01

The development of new alternative platforms for subunit vaccine production is a priority in the biomedical field. In this study, Ustilago maydis, the causal agent of common corn smut or ‘huitlacoche’has been genetically engineered to assess expression and immunogenicity of the B subunit of the cholera toxin (CTB), a relevant immunomodulatory agent in vaccinology. An oligomeric CTB recombinant protein was expressed in corn smut galls at levels of up to 1.3 mg g-1 dry weight (0.8% of the total soluble protein). Mice orally immunized with ‘huitlacoche’-derived CTB showed significant humoral responses that were well-correlated with protection against challenge with the cholera toxin (CT). These findings demonstrate the feasibility of using edible corn smut as a safe, effective, and low-cost platform for production and delivery of a subunit oral vaccine. The implications of this platform in the area of molecular pharming are discussed. PMID:26207365

A lentiviral vaccine expressing KMP11-HASPB fusion protein increases immune response to Leishmania major in BALB/C.

PubMed

Mortazavidehkordi, Nahid; Fallah, Ali; Abdollahi, Abbas; Kia, Vahid; Khanahmad, Hossein; Najafabadi, Zahra Ghayour; Hashemi, Nooshin; Estiri, Bahareh; Roudbari, Zahra; Najafi, Ali; Farjadfar, Akbar; Hejazi, Seyed Hossein

2018-05-29

Hydrophilic acylated surface protein B (HASPB) is an immunogenic Leishmania-specific protein that antibodies are produced against it in the sera of Leishmania-infected individuals. Kinetoplastid membrane protein 11 (KMP11) is another Leishmania antigen and considered as the suitable candidate for vaccine development Leishmaniasis. It is a highly conserved surface protein expressed in both promastigotes and amastigotes. In this study, KMP11 and HASPB coding sequences were cloned into a pCDH-cGFP lentiviral vector as a fusion protein to be used as a DNA vaccine against L. major. The KMP11-HASPB fusion protein was successfully expressed as evidenced by RT-PCR and Western blot assays. The effect of the vaccine was determined by evaluating the level of IFN-γ, IL-10, IgG1, and IgG2a performed using ELISA as well as determining the parasite load after challenge with L. major in vaccinated mice. The results revealed that IFN-γ, IL-10, IgG1, and IgG2a significantly increased after vaccination using KMP11-HASPB-expressing lentiviruses in BALB/c mice. It is noteworthy that the level of IFN-γ and IgG2a was higher than that of IL-10 and IgG1, respectively, which indicates the activation Th1 cells, macrophages, and cellular immunity. Moreover, the parasite load in the spleen and lymph node of vaccinated mice after challenge was significantly lower than that of controls.

Coregulation of soybean vegetative storage protein gene expression by methyl jasmonate and soluble sugars.

PubMed

Mason, H S; Dewald, D B; Creelman, R A; Mullet, J E

1992-03-01

The soybean vegetative storage protein genes vspA and vspB are highly expressed in developing leaves, stems, flowers, and pods as compared with roots, seeds, and mature leaves and stems. In this paper, we report that physiological levels of methyl jasmonate (MeJA) and soluble sugars synergistically stimulate accumulation of vsp mRNAs. Treatment of excised mature soybean (Glycine max Merr. cv Williams) leaves with 0.2 molar sucrose and 10 micromolar MeJA caused a large accumulation of vsp mRNAs, whereas little accumulation occurred when these compounds were supplied separately. In soybean cell suspension cultures, the synergistic effect of sucrose and MeJA on the accumulation of vspB mRNA was maximal at 58 millimolar sucrose and was observed with fructose or glucose substituted for sucrose. In dark-grown soybean seedlings, the highest levels of vsp mRNAs occurred in the hypocotyl hook, which also contained high levels of MeJA and soluble sugars. Lower levels of vsp mRNAs, MeJA, and soluble sugars were found in the cotyledons, roots, and nongrowing regions of the stem. Wounding of mature soybean leaves induced a large accumulation of vsp mRNAs when wounded plants were incubated in the light. Wounded plants kept in the dark or illuminated plants sprayed with dichlorophenyldimethylurea, an inhibitor of photosynthetic electron transport, showed a greatly reduced accumulation of vsp mRNAs. The time courses for the accumulation of vsp mRNAs induced by wounding or sucrose/MeJA treatment were similar. These results strongly suggest that vsp expression is coregulated by endogenous levels of MeJA (or jasmonic acid) and soluble carbohydrate during normal vegetative development and in wounded leaves.

In vivo gene expression and the adaptive response: from pathogenesis to vaccines and antimicrobials.

PubMed Central

Heithoff, D M; Sinsheimer, R L; Low, D A; Mahan, M J

2000-01-01

Microbial pathogens possess a repertoire of virulence determinants that each make unique contributions to fitness during infection. Analysis of these in vivo-expressed functions reveals the biology of the infection process, encompassing the bacterial infection strategies and the host ecological and environmental retaliatory strategies designed to combat them (e.g. thermal, osmotic, oxygen, nutrient and acid stress). Many of the bacterial virulence functions that contribute to a successful infection are normally only expressed during infection. A genetic approach was used to isolate mutants that ectopically expressed many of these functions in a laboratory setting. Lack of DNA adenine methylase (Dam) in Salmonella typhimurium abolishes the preferential expression of many bacterial virulence genes in host tissues. Dam- Salmonella were proficient in colonization of mucosal sites but were defective in colonization of deeper tissue sites. Additionally, Dam- mutants were totally avirulent and effective as live vaccines against murine typhoid fever. Since dam is highly conserved in many pathogenic bacteria that cause significant morbidity and mortality worldwide, Dams are potentially excellent targets for both vaccines and antimicrobials. PMID:10874736

Listeria-vectored vaccine expressing the Mycobacterium tuberculosis 30 kDa major secretory protein via the constitutively active prfA* regulon boosts BCG efficacy against tuberculosis.

PubMed

Jia, Qingmei; Dillon, Barbara Jane; Masleša-Galić, Saša; Horwitz, Marcus A

2017-06-19

A potent vaccine against tuberculosis, one of the world's deadliest diseases, is needed to enhance the immunity of people worldwide, most of whom have been vaccinated with the partially effective BCG vaccine. Here we investigate novel live attenuated recombinant Listeria monocytogenes (rLm) vaccines expressing the Mycobacterium tuberculosis (Mtb) 30 kDa major secretory protein (r30/Ag85B) (rLm30) as heterologous booster vaccines in animals primed with BCG. Using three attenuated Lm vectors, rLm Δ actA (LmI), rLm Δ actA Δ inlB (LmII), and rLm Δ actA Δ inlB prfA * (LmIII), we constructed five rLm30 vaccine candidates expressing the r30 linked in-frame to the Lm Listeriolycin O signal sequence and driven by the hly promoter (h30) or linked in-frame to the ActA N-terminus and driven by the actA promoter (a30). All five rLm30 vaccines secreted r30 in broth and macrophages; while rLm expressing r30 via a constitutively active prfA * regulon (rLmIII/a30) expressed the greatest amount of r30 in broth culture, all five rLm vaccines expressed equivalent amounts of r30 in infected macrophages. In comparative studies, boosting BCG-immunized mice with rLmIII/a30 induced the strongest antigen-specific T-cell responses, including splenic and lung polyfunctional CD4+ T-cells expressing the three cytokines of interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), and interleukin-2 (IL-2) ( P < 0.001) and splenic and lung CD8+ T-cells expressing IFN-γ ( P < 0.0001). In mice and guinea pigs, rLmIII/a30 and rLmI/h30 vaccines were generally more potent booster vaccines than r30 in adjuvant and a recombinant adenovirus vaccine expressing r30. In a setting in which BCG alone was highly immunoprotective, boosting mice with rLmIII/a30, the most potent of the vaccines, significantly enhanced protection against aerosolized Mtb ( P <0.01). Copyright © 2017 American Society for Microbiology.

A Bivalent Typhoid Live Vector Vaccine Expressing both Chromosome- and Plasmid-Encoded Yersinia pestis Antigens Fully Protects against Murine Lethal Pulmonary Plague Infection

PubMed Central

Wang, Jin Yuan; Carrasco, Jose A.; Lloyd, Scott A.; Mellado-Sanchez, Gabriela; Diaz-McNair, Jovita; Franco, Olga; Buskirk, Amanda D.; Nataro, James P.; Pasetti, Marcela F.

2014-01-01

Live attenuated bacteria hold great promise as multivalent mucosal vaccines against a variety of pathogens. A major challenge of this approach has been the successful delivery of sufficient amounts of vaccine antigens to adequately prime the immune system without overattenuating the live vaccine. Here we used a live attenuated Salmonella enterica serovar Typhi strain to create a bivalent mucosal plague vaccine that produces both the protective F1 capsular antigen of Yersinia pestis and the LcrV protein required for secretion of virulence effector proteins. To reduce the metabolic burden associated with the coexpression of F1 and LcrV within the live vector, we balanced expression of both antigens by combining plasmid-based expression of F1 with chromosomal expression of LcrV from three independent loci. The immunogenicity and protective efficacy of this novel vaccine were assessed in mice by using a heterologous prime-boost immunization strategy and compared to those of a conventional strain in which F1 and LcrV were expressed from a single low-copy-number plasmid. The serum antibody responses to lipopolysaccharide (LPS) induced by the optimized bivalent vaccine were indistinguishable from those elicited by the parent strain, suggesting an adequate immunogenic capacity maintained through preservation of bacterial fitness; in contrast, LPS titers were 10-fold lower in mice immunized with the conventional vaccine strain. Importantly, mice receiving the optimized bivalent vaccine were fully protected against lethal pulmonary challenge. These results demonstrate the feasibility of distributing foreign antigen expression across both chromosomal and plasmid locations within a single vaccine organism for induction of protective immunity. PMID:25332120

Pre-clinical evaluation of a replication-competent recombinant adenovirus serotype 4 vaccine expressing influenza H5 hemagglutinin.

PubMed

Alexander, Jeff; Ward, Simone; Mendy, Jason; Manayani, Darly J; Farness, Peggy; Avanzini, Jenny B; Guenther, Ben; Garduno, Fermin; Jow, Lily; Snarsky, Victoria; Ishioka, Glenn; Dong, Xin; Vang, Lo; Newman, Mark J; Mayall, Tim

2012-01-01

Influenza virus remains a significant health and social concern in part because of newly emerging strains, such as avian H5N1 virus. We have developed a prototype H5N1 vaccine using a recombinant, replication-competent Adenovirus serotype 4 (Ad4) vector, derived from the U.S. military Ad4 vaccine strain, to express the hemagglutinin (HA) gene from A/Vietnam/1194/2004 influenza virus (Ad4-H5-Vtn). Our hypothesis is that a mucosally-delivered replicating Ad4-H5-Vtn recombinant vector will be safe and induce protective immunity against H5N1 influenza virus infection and disease pathogenesis. The Ad4-H5-Vtn vaccine was designed with a partial deletion of the E3 region of Ad4 to accommodate the influenza HA gene. Replication and growth kinetics of the vaccine virus in multiple human cell lines indicated that the vaccine virus is attenuated relative to the wild type virus. Expression of the HA transgene in infected cells was documented by flow cytometry, western blot analysis and induction of HA-specific antibody and cellular immune responses in mice. Of particular note, mice immunized intranasally with the Ad4-H5-Vtn vaccine were protected against lethal H5N1 reassortant viral challenge even in the presence of pre-existing immunity to the Ad4 wild type virus. Several non-clinical attributes of this vaccine including safety, induction of HA-specific humoral and cellular immunity, and efficacy were demonstrated using an animal model to support Phase 1 clinical trial evaluation of this new vaccine.

Parvovirus-like particles as vaccine vectors.

PubMed

Casal, J I; Rueda, P; Hurtado, A

1999-09-01

A wide array of systems have been developed to improve "classic" vaccines. The use of small polypeptides able to elicit potent antibody and cytotoxic responses seems to have enormous potential in the design of safer vaccines. While peptide coupling to large soluble proteins such as keyhole limpet hemocyanin is the current method of choice for eliciting antibody responses and insertion in live viruses for cytotoxic T-lymphocyte responses, alternative cheaper and/or safer methods will clearly be required in the future. Virus-like particles constitute very immunogenic molecules that allow for covalent coupling of the epitopes of interest in a simple way. In this article, we detail the methodology employed for the preparation of efficient virus vectors as delivery systems. We used parvovirus as the model for the design of new vaccine vectors. Recently parvovirus-like particles have been engineered to express foreign polypeptides in certain positions, resulting in the production of large quantities of highly immunogenic peptides, and to induce strong antibody, helper-T-cell, and cytotoxic T-lymphocyte responses. We discuss the different alternatives and the necessary steps to carry out this process, placing special emphasis on the flow of decisions that need to be made during the project. Copyright 1999 Academic Press.

Improving the Expression and Purification of Soluble, Recombinant Native-Like HIV-1 Envelope Glycoprotein Trimers by Targeted Sequence Changes

PubMed Central

Ringe, Rajesh P.; Ozorowski, Gabriel; Yasmeen, Anila; Cupo, Albert; Cruz Portillo, Victor M.; Pugach, Pavel; Golabek, Michael; Rantalainen, Kimmo; Holden, Lauren G.; Cottrell, Christopher A.; Wilson, Ian A.; Sanders, Rogier W.; Ward, Andrew B.; Klasse, P. J.

2017-01-01

ABSTRACT Soluble, recombinant native-like envelope glycoprotein (Env) trimers of various human immunodeficiency virus type 1 (HIV-1) genotypes are being developed for structural studies and as vaccine candidates aimed at the induction of broadly neutralizing antibodies (bNAbs). The prototypic design is designated SOSIP.664, but many HIV-1 env genes do not yield fully native-like trimers efficiently. One such env gene is CZA97.012 from a neutralization-resistant (tier 2) clade C virus. As appropriately purified, native-like CZA97.012 SOSIP.664 trimers induce autologous neutralizing antibodies (NAbs) efficiently in immunized rabbits, we sought to improve the efficiency with which they can be produced and to better understand the limitations to the original design. By using structure- and antigenicity-guided mutagenesis strategies focused on the V2 and V3 regions and the gp120-gp41 interface, we developed the CZA97 SOSIP.v4.2-M6.IT construct. Fully native-like, stable trimers that display multiple bNAb epitopes could be expressed from this construct in a stable CHO cell line and purified at an acceptable yield using either a PGT145 or a 2G12 bNAb affinity column. We also show that similar mutagenesis strategies can be used to improve the yields and properties of SOSIP.664 trimers of the DU422, 426c, and 92UG037 genotypes. IMPORTANCE Recombinant trimeric proteins based on HIV-1 env genes are being developed for future vaccine trials in humans. A feature of these proteins is their mimicry of the envelope glycoprotein (Env) structure on virus particles that is targeted by neutralizing antibodies, i.e., antibodies that prevent cells from becoming infected. The vaccine concept under exploration is that recombinant trimers may be able to elicit virus-neutralizing antibodies when delivered as immunogens. Because HIV-1 is extremely variable, a practical vaccine may need to incorporate Env trimers derived from multiple different virus sequences. Accordingly, we need to

Soluble expression of an amebic cysteine protease in the cytoplasm of Escherichia coli SHuffle Express cells and purification of active enzyme.

PubMed

Jalomo-Khayrova, Ekaterina; Mares, Rosa E; Muñoz, Patricia L A; Meléndez-López, Samuel G; Rivero, Ignacio A; Ramos, Marco A

2018-04-03

Recombinant production of amebic cysteine proteases using Escherichia coli cells as the bacterial system has become a challenging effort, with protein insolubility being the most common issue. Since many of these enzymes need a native conformation stabilized by disulfide bonds, an elaborate process of oxidative folding is usually demanded to get a functional protein. The cytoplasm of E. coli SHuffle Express cells owns an enhanced ability to properly fold proteins with disulfide bonds. Because of this cellular feature, it was possible to assume that this strain represents a reliable expression system and worthwhile been considered as an efficient bacterial host for the recombinant production of amebic cysteine proteases. Using E. coli SHuffle Express cells as the bacterial system, we efficiently produce soluble recombinant EhCP1protein. Enzymatic and inhibition analyses revealed that it exhibits proper catalytic abilities, proceeds effectively over the substrate (following an apparent Michaelis-Menten kinetics), and displays a typical inhibition profile. We report the first feasibility study of the recombinant production of amebic cysteine proteases using E. coli SHuffle Express as the bacterial host. We present a simple protocol for the recombinant expression and purification of fully soluble and active EhCP1 enzyme. We confirm the suitability of recombinant EhCP1 as a therapeutic target. We propose an approachable bacterial system for the recombinant production of amebic proteins, particularly for those with a need for proper oxidative folding.

Enhanced Neutralizing Antibody Response Induced by Respiratory Syncytial Virus Prefusion F Protein Expressed by a Vaccine Candidate.

PubMed

Liang, Bo; Surman, Sonja; Amaro-Carambot, Emerito; Kabatova, Barbora; Mackow, Natalie; Lingemann, Matthias; Yang, Lijuan; McLellan, Jason S; Graham, Barney S; Kwong, Peter D; Schaap-Nutt, Anne; Collins, Peter L; Munir, Shirin

2015-09-01

Respiratory syncytial virus (RSV) and human parainfluenza virus type 3 (HPIV3) are the first and second leading viral agents of severe respiratory tract disease in infants and young children worldwide. Vaccines are not available, and an RSV vaccine is particularly needed. A live attenuated chimeric recombinant bovine/human PIV3 (rB/HPIV3) vector expressing the RSV fusion (F) glycoprotein from an added gene has been under development as a bivalent vaccine against RSV and HPIV3. Previous clinical evaluation of this vaccine candidate suggested that increased genetic stability and immunogenicity of the RSV F insert were needed. This was investigated in the present study. RSV F expression was enhanced 5-fold by codon optimization and by modifying the amino acid sequence to be identical to that of an early passage of the original clinical isolate. This conferred a hypofusogenic phenotype that presumably reflects the original isolate. We then compared vectors expressing stabilized prefusion and postfusion versions of RSV F. In a hamster model, prefusion F induced increased quantity and quality of RSV-neutralizing serum antibodies and increased protection against wild-type (wt) RSV challenge. In contrast, a vector expressing the postfusion F was less immunogenic and protective. The genetic stability of the RSV F insert was high and was not affected by enhanced expression or the prefusion or postfusion conformation of RSV F. These studies provide an improved version of the previously well-tolerated rB/HPIV3-RSV F vaccine candidate that induces a superior RSV-neutralizing serum antibody response. Respiratory syncytial virus (RSV) and human parainfluenza virus type 3 (HPIV3) are two major causes of pediatric pneumonia and bronchiolitis. The rB/HPIV3 vector expressing RSV F protein is a candidate bivalent live vaccine against HPIV3 and RSV. Previous clinical evaluation indicated the need to increase the immunogenicity and genetic stability of the RSV F insert. Here, we

Prime-boost bacillus Calmette-Guérin vaccination with lentivirus-vectored and DNA-based vaccines expressing antigens Ag85B and Rv3425 improves protective efficacy against Mycobacterium tuberculosis in mice.

PubMed

Xu, Ying; Yang, Enzhuo; Wang, Jianguang; Li, Rui; Li, Guanghua; Liu, Guoyuan; Song, Na; Huang, Qi; Kong, Cong; Wang, Honghai

2014-10-01

To prevent the global spread of tuberculosis (TB), more effective vaccines and vaccination strategies are urgently needed. As a result of the success of bacillus Calmette-Guérin (BCG) in protecting children against miliary and meningeal TB, the majority of individuals will have been vaccinated with BCG; hence, boosting BCG-primed immunity will probably be a key component of future vaccine strategies. In this study, we compared the ability of DNA-, protein- and lentiviral vector-based vaccines that express the antigens Ag85B and Rv3425 to boost the effects of BCG in the context of immunity and protection against Mycobacterium tuberculosis in C57BL/6 mice. Our results demonstrated that prime-boost BCG vaccination with a lentiviral vector expressing the antigens Ag85B and Rv3425 significantly enhanced immune responses, including T helper type 1 and CD8(+) cytotoxic T lymphocyte responses, compared with DNA- and protein-based vaccines. However, lentivirus-vectored and DNA-based vaccines greatly improved the protective efficacy of BCG against M. tuberculosis, as indicated by a lack of weight loss and significantly reduced bacterial loads and histological damage in the lung. Our study suggests that the use of lentiviral or DNA vaccines containing the antigens Ag85B and Rv3425 to boost BCG is a good choice for the rational design of an efficient vaccination strategy against TB. © 2014 John Wiley & Sons Ltd.

An inducible expression system for high-level expression of recombinant proteins in slow growing mycobacteria.

PubMed

Leotta, Lisa; Spratt, Joanne M; Kong, Carlyn U; Triccas, James A

2015-09-01

A novel protein expression vector utilising the inducible hspX promoter of Mycobacterium tuberculosis was constructed and evaluated in this study. High-level induction of three mycobacterial antigens, comprising up to 9% of bacterial sonicate, was demonstrated in recombinant Mycobacterium bovis BCG when grown under low-oxygen tension, which serves to enhance hspX promoter activity. Recombinant proteins were efficiently purified from bacterial lysates in a soluble form by virtue of a C-terminal 6-histidine tag. Purification of the immunodominant M. tuberculosis Ag85B antigen using this system resulted in a recombinant protein that stimulated significant IFN-γ release from Ag85B-reactive T cells generated after vaccination of mice with an Ag85B-expressing vaccine. Further, the M. tuberculosis L-alanine dehydrogenase (Ald) protein purified from recombinant BCG displayed strong enzymatic activity in recombinant form. This study demonstrated that high levels of native-like recombinant mycobacterial proteins can be produced in mycobacterial hosts, and this may aid the analysis of mycobacterial protein function and the development of new treatments. Copyright © 2015 Elsevier B.V. All rights reserved.

[Construction and eukaryotic expression of PVAX1-hPV58mE6E7fcGB composite gene vaccine].

PubMed

Wang, He; Yu, Jiyun; Li, Li

2013-10-01

To construct and express a composite gene vaccine for human papillomavirus 58(HPV58)-associated cervical cancer, we inserted HPV58mE6E7 fusion gene into pCI-Fc-GPI eukaryotic expression vector, constructing a recombinant plasmid named pCI-sig-HPV58mE6E7-Fc-GPI. Then we further inserted fragment of sig-HPV58mE6E7Fc-GPI into the novel vaccine vector PVAX1-IRES-GM/B7, constructing PVAX1-HPV58mE6E7FcGB composite gene vaccine. PVAX1-HPV58mE6E7FcGB vaccine was successfully constructed and identified by restriction endonuclease and sequencing analysis. Eukaryotic expression of fusion antigen sig-HPV58mE6E7-Fc-GPI and molecular ad-juvant GM-CSF and B7. 1 were proved to be realized at the same time by flow cytometry and immunofluorescence. So PVAX1-HPV58mE6E7FcGB can be taken as a candidate of therapeutic vaccine for HPV58-associated tumors and their precancerous transformations.

Bile salts-containing vesicles: promising pharmaceutical carriers for oral delivery of poorly water-soluble drugs and peptide/protein-based therapeutics or vaccines.

PubMed

Aburahma, Mona Hassan

2016-07-01

Most of the new drugs, biological therapeutics (proteins/peptides) and vaccines have poor performance after oral administration due to poor solubility or degradation in the gastrointestinal tract (GIT). Though, vesicular carriers exemplified by liposomes or niosomes can protect the entrapped agent to a certain extent from degradation. Nevertheless, the harsh GIT environment exemplified by low pH, presence of bile salts and enzymes limits their capabilities by destabilizing them. In response to that, more resistant bile salts-containing vesicles (BS-vesicles) were developed by inclusion of bile salts into lipid bilayers constructs. The effectiveness of orally administrated BS-vesicles in improving the performance of vesicles has been demonstrated in researches. Yet, these attempts did not gain considerable attention. This is the first review that provides a comprehensive overview of utilizing BS-vesicles as a promising pharmaceutical carrier with a special focus on their successful applications in oral delivery of therapeutic macromolecules and vaccines. Insights on the possible mechanisms by which BS-vesicles improve the oral bioavailability of the encapsulated drug or immunological response of entrapped vaccine are explained. In addition, methods adopted to prepare and characterize BS-vesicles are described. Finally, the gap in the scientific researches tackling BS-vesicles that needs to be addressed is highlighted.

Pre-Clinical Development of a Recombinant, Replication-Competent Adenovirus Serotype 4 Vector Vaccine Expressing HIV-1 Envelope 1086 Clade C

PubMed Central

Alexander, Jeff; Mendy, Jason; Vang, Lo; Avanzini, Jenny B.; Garduno, Fermin; Manayani, Darly J.; Ishioka, Glenn; Farness, Peggy; Ping, Li-Hua; Swanstrom, Ronald; Parks, Robert; Liao, Hua-Xin; Haynes, Barton F.; Montefiori, David C.; LaBranche, Celia; Smith, Jonathan; Gurwith, Marc; Mayall, Tim

2013-01-01

Background There is a well-acknowledged need for an effective AIDS vaccine that protects against HIV-1 infection or limits in vivo viral replication. The objective of these studies is to develop a replication-competent, vaccine vector based on the adenovirus serotype 4 (Ad4) virus expressing HIV-1 envelope (Env) 1086 clade C glycoprotein. Ad4 recombinant vectors expressing Env gp160 (Ad4Env160), Env gp140 (Ad4Env140), and Env gp120 (Ad4Env120) were evaluated. Methods The recombinant Ad4 vectors were generated with a full deletion of the E3 region of Ad4 to accommodate the env gene sequences. The vaccine candidates were assessed in vitro following infection of A549 cells for Env-specific protein expression and for posttranslational transport to the cell surface as monitored by the binding of broadly neutralizing antibodies (bNAbs). The capacity of the Ad4Env vaccines to induce humoral immunity was evaluated in rabbits for Env gp140 and V1V2-specific binding antibodies, and HIV-1 pseudovirus neutralization. Mice immunized with the Ad4Env160 vaccine were assessed for IFNγ T cell responses specific for overlapping Env peptide sets. Results Robust Env protein expression was confirmed by western blot analysis and recognition of cell surface Env gp160 by multiple bNAbs. Ad4Env vaccines induced humoral immune responses in rabbits that recognized Env 1086 gp140 and V1V2 polypeptide sequences derived from 1086 clade C, A244 clade AE, and gp70 V1V2 CASE A2 clade B fusion protein. The immune sera efficiently neutralized tier 1 clade C pseudovirus MW965.26 and neutralized the homologous and heterologous tier 2 pseudoviruses to a lesser extent. Env-specific T cell responses were also induced in mice following Ad4Env160 vector immunization. Conclusions The Ad4Env vaccine vectors express high levels of Env glycoprotein and induce both Env-specific humoral and cellular immunity thus supporting further development of this new Ad4 HIV-1 Env vaccine platform in Phase 1 clinical

Construction and characterization of human rotavirus recombinant VP8* subunit parenteral vaccine candidates.

PubMed

Wen, Xiaobo; Cao, Dianjun; Jones, Ronald W; Li, Jianping; Szu, Shousun; Hoshino, Yasutaka

2012-09-21

Two currently licensed live oral rotavirus vaccines (Rotarix® and RotaTeq®) are highly efficacious against severe rotavirus diarrhea. However, the efficacy of such vaccines in selected low-income African and Asian countries is much lower than that in middle or high-income countries. Additionally, these two vaccines have recently been associated with rare case of intussusception in vaccinated infants. We developed a novel recombinant subunit parenteral rotavirus vaccine which may be more effective in low-income countries and also avert the potential problem of intussusception. Truncated recombinant VP8* (ΔVP8*) protein of human rotavirus strain Wa P[8], DS-1 P[4] or 1076 P[6] expressed in Escherichia coli was highly soluble and was generated in high yield. Guinea pigs hyperimmunized intramuscularly with each of the ΔVP8* proteins (i.e., P[8], P[4] or P[6]) developed high levels of homotypic as well as variable levels of heterotypic neutralizing antibodies. Moreover, the selected ΔVP8* proteins when administered to mice at a clinically relevant dosage, route and schedule, elicited high levels of serum anti-VP8* IgG and/or neutralizing antibodies. Our data indicated that the ΔVP8* proteins may be a plausible additional candidate as new parenteral rotavirus vaccines. Published by Elsevier Ltd.

Construction and Characterization of Human Rotavirus Recombinant VP8* Subunit Parenteral Vaccine Candidates

PubMed Central

Wen, Xiaobo; Cao, Dianjun; Jones, Ronald W.; Li, Jianping; Szu, Shousun; Hoshino, Yasutaka

2012-01-01

Two currently licensed live oral rotavirus vaccines (Rotarix® and RotaTeq®) are highly efficacious against severe rotavirus diarrhea. However, the efficacy of such vaccines in selected low-income African and Asian countries is much lower than that in middle or high-income countries. Additionally, these two vaccines have recently been associated with rare case of intussusception in vaccinated infants. We developed a novel recombinant subunit parenteral rotavirus vaccine which may be more effective in low-income countries and also avert the potential problem of intussusception. Truncated recombinant VP8* (ΔVP8*) protein of human rotavirus strain Wa P[8], DS-1 P[4] or 1076 P[6] expressed in E. coli was highly soluble and was generated in high yield. Guinea pigs hyperimmunized intramuscularly with each of the ΔVP8* proteins (i.e., (P[8], P[4] or P[6]) developed high levels of homotypic as well as variable levels of heterotypic neutralizing antibodies. Moreover, the selected ΔVP8* proteins when administered to mice at a clinically relevant dosage, route and schedule, elicited high levels of serum anti-VP8* IgG and/or neutralizing antibodies. Our data indicated that the ΔVP8* proteins may be a plausible additional candidate as new parenteral rotavirus vaccines. PMID:22885016

Vaccination with lentiviral vector expressing the nfa1 gene confers a protective immune response to mice infected with Naegleria fowleri.

PubMed

Kim, Jong-Hyun; Sohn, Hae-Jin; Lee, Jinyoung; Yang, Hee-Jong; Chwae, Yong-Joon; Kim, Kyongmin; Park, Sun; Shin, Ho-Joon

2013-07-01

Naegleria fowleri, a pathogenic free-living amoeba, causes fatal primary amoebic meningoencephalitis (PAM) in humans and animals. The nfa1 gene (360 bp), cloned from a cDNA library of N. fowleri, produces a 13.1-kDa recombinant protein which is located on pseudopodia, particularly the food cup structure. The nfa1 gene plays an important role in the pathogenesis of N. fowleri infection. To examine the effect of nfa1 DNA vaccination against N. fowleri infection, we constructed a lentiviral vector (pCDH) expressing the nfa1 gene. For the in vivo mouse study, BALB/c mice were intranasally vaccinated with viral particles of a viral vector expressing the nfa1 gene. To evaluate the effect of vaccination and immune responses of mice, we analyzed the IgG levels (IgG, IgG1, and IgG2a), cytokine induction (interleukin-4 [IL-4] and gamma interferon [IFN-γ]), and survival rates of mice that developed PAM. The levels of both IgG and IgG subclasses (IgG1 and IgG2a) in vaccinated mice were significantly increased. The cytokine analysis showed that vaccinated mice exhibited greater IL-4 and IFN-γ production than the other control groups, suggesting a Th1/Th2 mixed-type immune response. In vaccinated mice, high levels of Nfa1-specific IgG antibodies continued until 12 weeks postvaccination. The mice vaccinated with viral vector expressing the nfa1 gene also exhibited significantly higher survival rates (90%) after challenge with N. fowleri trophozoites. Finally, the nfa1 vaccination effectively induced protective immunity by humoral and cellular immune responses in N. fowleri-infected mice. These results suggest that DNA vaccination using a viral vector may be a potential tool against N. fowleri infection.

Vaccination with Lentiviral Vector Expressing the nfa1 Gene Confers a Protective Immune Response to Mice Infected with Naegleria fowleri

PubMed Central

Kim, Jong-Hyun; Sohn, Hae-Jin; Lee, Jinyoung; Yang, Hee-Jong; Chwae, Yong-Joon; Kim, Kyongmin; Park, Sun

2013-01-01

Naegleria fowleri, a pathogenic free-living amoeba, causes fatal primary amoebic meningoencephalitis (PAM) in humans and animals. The nfa1 gene (360 bp), cloned from a cDNA library of N. fowleri, produces a 13.1-kDa recombinant protein which is located on pseudopodia, particularly the food cup structure. The nfa1 gene plays an important role in the pathogenesis of N. fowleri infection. To examine the effect of nfa1 DNA vaccination against N. fowleri infection, we constructed a lentiviral vector (pCDH) expressing the nfa1 gene. For the in vivo mouse study, BALB/c mice were intranasally vaccinated with viral particles of a viral vector expressing the nfa1 gene. To evaluate the effect of vaccination and immune responses of mice, we analyzed the IgG levels (IgG, IgG1, and IgG2a), cytokine induction (interleukin-4 [IL-4] and gamma interferon [IFN-γ]), and survival rates of mice that developed PAM. The levels of both IgG and IgG subclasses (IgG1 and IgG2a) in vaccinated mice were significantly increased. The cytokine analysis showed that vaccinated mice exhibited greater IL-4 and IFN-γ production than the other control groups, suggesting a Th1/Th2 mixed-type immune response. In vaccinated mice, high levels of Nfa1-specific IgG antibodies continued until 12 weeks postvaccination. The mice vaccinated with viral vector expressing the nfa1 gene also exhibited significantly higher survival rates (90%) after challenge with N. fowleri trophozoites. Finally, the nfa1 vaccination effectively induced protective immunity by humoral and cellular immune responses in N. fowleri-infected mice. These results suggest that DNA vaccination using a viral vector may be a potential tool against N. fowleri infection. PMID:23677321

Respiratory syncytial virus subunit vaccine based on a recombinant fusion protein expressed transiently in mammalian cells.

PubMed

Nallet, Sophie; Amacker, Mario; Westerfeld, Nicole; Baldi, Lucia; König, Iwo; Hacker, David L; Zaborosch, Christiane; Zurbriggen, Rinaldo; Wurm, Florian M

2009-10-30

Although respiratory syncytial virus (RSV) causes severe lower respiratory tract infection in infants and adults at risk, no RSV vaccine is currently available. In this report, efforts toward the generation of an RSV subunit vaccine using recombinant RSV fusion protein (rRSV-F) are described. The recombinant protein was produced by transient gene expression (TGE) in suspension-adapted human embryonic kidney cells (HEK-293E) in 4 L orbitally shaken bioreactors. It was then purified and formulated in immunostimulating reconstituted influenza virosomes (IRIVs). The candidate vaccine induced anti-RSV-F neutralizing antibodies in mice, and challenge studies in cotton rats are ongoing. If successful in preclinical and clinical trials, this will be the first recombinant subunit vaccine produced by large-scale TGE in mammalian cells.

Enhancement of solubility, purification and inclusion-bodies-refolding of an active pectin lyase from Penicillium occitanis expressed in Escherichia coli.

PubMed

Hadj Sassi, Azza; Trigui-Lahiani, Hèla; Abdeljalil, Salma; Gargouri, Ali

2017-02-01

Pectin lyase (pnl) is the only pectinase able to hydrolyze directly the highly methylated pectin without liberating the toxic methanol and without disturbing ester content responsible for specific aroma of juices. The cDNA of Penicillium occitanis pnl (mature form) was cloned into pET-21a as expression vector and over-expressed into Esherichia coli. Most of recombinant pnl was expressed as inclusion bodies. Pnl activity was confirmed by colorimetric assay. To enhance the solubility yield of the expressed pnl, the effects of induction temperature, host strain and expression level were optimized. Maximal production of functional pnl was obtained after induction by 0.4mM IPTG at 30°C and 150rpm for 16h. Interestingly, the use of Origami host strain, having an oxidized cytoplasm favoring disulfide bonds formation required for the active conformation of the enzyme, has significantly improved the yield of the soluble active form of recombinant pnl. This pnl was successfully purified through a single step purification using His-Trap affinity column chromatography. This work is the first to report pnl expression into Origami strain. Alternatively, the inclusion bodies were isolated, denatured by high concentration of urea and gradually refolded by successive dialysis, leading to their transformation into soluble and active form. Copyright © 2016 Elsevier B.V. All rights reserved.

Cloning and expression of soluble truncated variants of Borrelia OspA, OspB and Vmp7

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dunn, J.J.; Barbour, A.G.

1996-11-05

A method is provided for preparing soluble recombinant variations of Borrelia lipoproteins such as Borrelia burgdorferi outer surface protein A (OspA) and outer surface protein B (OspB), and B. hermsii variable major protein 7 (Vmp7). The method includes synthesizing a set of oligonucleotide primers, amplifying the template DNA utilizing the PCR, purifying the amplification products, cloning the amplification products into a suitable expression vector, transforming a suitable host utilizing the cloned expression vector, cultivating the transformed host for protein production and subsequently isolating and purifying the resulting protein. Also provided are soluble, recombinant variations of Borrelia burgdorferi outer surface proteinmore » A (OspA), outer surface protein B (OspB), and B. hermsii variable major protein 7 (Vmp7). The expression vectors harboring DNA encoding the recombinant variations, pET9-OspA, pET9-OspB and pET9-Vmp7, as well as the E. coli host BL21(DE3)/pLysS transformed with each of these vectors, are also disclosed. 38 figs.« less

Cloning and expression of soluble truncated variants of Borrelia OspA, OspB and Vmp7

DOEpatents

Dunn, John J.; Barbour, Alan G.

1996-11-05

A method is provided herein for preparing soluble recombinant variations of Borrelia lipoproteins such as Borrelia burgdorferi outer surface protein A (OspA) and outer surface protein B (OspB), and B. hermsii variable major protein 7 (Vmp7). The method includes synthesizing a set of oligonucleotide primers, amplifying the template DNA utilizing the PCR, purifying the amplification products, cloning the amplification products into a suitable expression vector, transforming a suitable host utilizing the cloned expression vector, cultivating the transformed host for protein production and subsequently isolating and purifying the resulting protein. Also provided are soluble, recombinant variations of Borrelia burgdorferi outer surface protein A (OspA), outer surface protein B (OspB), and B. hermsii variable major protein 7 (Vmp7). The expression vectors harboring DNA encoding the recombinant variations, pET9-OspA, pET9-OspB and pET9-Vmp7, as well as the E. coli host BL21(DE3)/pLysS transformed with each of these vectors, are also disclosed.

Cloning and expression of soluble truncated variants of Borrelia OspA, OspB and Vmp7

DOEpatents

Dunn, J.J.; Barbour, A.G.

1996-11-05

A method is provided for preparing soluble recombinant variations of Borrelia lipoproteins such as Borrelia burgdorferi outer surface protein A (OspA) and outer surface protein B (OspB), and B. hermsii variable major protein 7 (Vmp7). The method includes synthesizing a set of oligonucleotide primers, amplifying the template DNA utilizing the PCR, purifying the amplification products, cloning the amplification products into a suitable expression vector, transforming a suitable host utilizing the cloned expression vector, cultivating the transformed host for protein production and subsequently isolating and purifying the resulting protein. Also provided are soluble, recombinant variations of Borrelia burgdorferi outer surface protein A (OspA), outer surface protein B (OspB), and B. hermsii variable major protein 7 (Vmp7). The expression vectors harboring DNA encoding the recombinant variations, pET9-OspA, pET9-OspB and pET9-Vmp7, as well as the E. coli host BL21(DE3)/pLysS transformed with each of these vectors, are also disclosed. 38 figs.

Co-expression of tetanus toxin fragment C in Escherichia coli with thioredoxin and its evaluation as an effective subunit vaccine candidate.

PubMed

Yu, Yun-Zhou; Gong, Zheng-Wei; Ma, Yao; Zhang, Shu-Ming; Zhu, Heng-Qi; Wang, Wen-Bing; Du, Yun; Wang, Shuang; Yu, Wei-Yuan; Sun, Zhi-Wei

2011-08-11

The receptor-binding domain of tetanus toxin (THc), which mediates the binding of the toxin to the nerve cells, is a candidate subunit vaccine against tetanus. In this study one synthetic gene encoding the THc was constructed and highly expressed in Escherichia coli by co-expression with thioredoxin (Trx). The purified THc-vaccinated mice were completely protected against an active toxin challenge in mouse models of disease and the potency of two doses of THc was comparable to that of three doses of toxoid vaccine. And a solid-phase assay showed that the anti-THc sera inhibited the binding of THc or toxoid to the ganglioside GT1b as the anti-tetanus toxoid sera. Furthermore, mice were vaccinated once or twice at four different dosages of THc and a dose-response was observed in both the antibody titer and protective efficacy with increasing dosage of THc and number of vaccinations. The data presented in the report showed that the recombinant THc expressed in E. coli is efficacious in protecting mice against challenge with tetanus toxin suggesting that the THc protein may be developed into a human subunit vaccine candidate designed for the prevention of tetanus. Copyright © 2011 Elsevier Ltd. All rights reserved.

Distinct Immunogenicity and Efficacy of Poxvirus-Based Vaccine Candidates against Ebola Virus Expressing GP and VP40 Proteins.

PubMed

Lázaro-Frías, Adrián; Gómez-Medina, Sergio; Sánchez-Sampedro, Lucas; Ljungberg, Karl; Ustav, Mart; Liljeström, Peter; Muñoz-Fontela, César; Esteban, Mariano; García-Arriaza, Juan

2018-06-01

Zaire and Sudan ebolavirus species cause a severe disease in humans and nonhuman primates (NHPs) characterized by a high mortality rate. There are no licensed therapies or vaccines against Ebola virus disease (EVD), and the recent 2013 to 2016 outbreak in West Africa highlighted the need for EVD-specific medical countermeasures. Here, we generated and characterized head-to-head the immunogenicity and efficacy of five vaccine candidates against Zaire ebolavirus (EBOV) and Sudan ebolavirus (SUDV) based on the highly attenuated poxvirus vector modified vaccinia virus Ankara (MVA) expressing either the virus glycoprotein (GP) or GP together with the virus protein 40 (VP40) forming virus-like particles (VLPs). In a human monocytic cell line, the different MVA vectors (termed MVA-EBOVs and MVA-SUDVs) triggered robust innate immune responses, with production of beta interferon (IFN-β), proinflammatory cytokines, and chemokines. Additionally, several innate immune cells, such as dendritic cells, neutrophils, and natural killer cells, were differentially recruited in the peritoneal cavity of mice inoculated with MVA-EBOVs. After immunization of mice with a homologous prime/boost protocol (MVA/MVA), total IgG antibodies against GP or VP40 from Zaire and Sudan ebolavirus were differentially induced by these vectors, which were mainly of the IgG1 and IgG3 isotypes. Remarkably, an MVA-EBOV construct coexpressing GP and VP40 protected chimeric mice challenged with EBOV to a greater extent than a vector expressing GP alone. These results support the consideration of MVA-EBOVs and MVA-SUDVs expressing GP and VP40 and producing VLPs as best-in-class potential vaccine candidates against EBOV and SUDV. IMPORTANCE EBOV and SUDV cause a severe hemorrhagic fever affecting humans and NHPs. Since their discovery in 1976, they have caused several sporadic epidemics, with the recent outbreak in West Africa from 2013 to 2016 being the largest and most severe, with more than 11,000 deaths

Flying vaccinator; a transgenic mosquito delivers a Leishmania vaccine via blood feeding.

PubMed

Yamamoto, D S; Nagumo, H; Yoshida, S

2010-06-01

'Flying vaccinator' is the concept of using genetically engineered hematophagous insects to deliver vaccines. Here we show the generation of a transgenic anopheline mosquito that expresses the Leishmania vaccine candidate, SP15, fused to monomeric red fluorescent protein (mDsRed) in its salivary glands. Importantly, mice bitten repeatedly by the transgenic mosquitoes raised anti-SP15 antibodies, indicating delivery of SP15 via blood feeding with its immunogenicity intact. Thus, this technology makes possible the generation of transgenic mosquitoes that match the original concept of a 'flying vaccinator'. However, medical safety issues and concerns about informed consent mitigate the use of the 'flying vaccinator' as a method to deliver vaccines. We propose that this expression system could be applied to elucidate saliva-malaria sporozoite interactions.

A soluble form of Siglec-9 provides an antitumor benefit against mammary tumor cells expressing MUC1 in transgenic mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tomioka, Yukiko, E-mail: ytomi@muses.tottori-u.ac.jp; Avian Zoonosis Research Center, Faculty of Agriculture, Tottori University, Tottori 680-8553; Morimatsu, Masami, E-mail: mmorimat@vetmed.hokudai.ac.jp

Highlights: • Tumor-associated antigen MUC1 binds to Siglec-9. • Soluble Siglec-9 reduced proliferation of MUC1-positive tumor in transgenic mice. • Soluble Siglec-9 and MUC1 on tumor cells were colocalized in transgenic mice. • MUC1 expression on tumor cells were reduced in soluble Siglec-9 transgenic mice. - Abstract: Tumor-associated MUC1 binds to Siglec-9, which is expected to mediate tumor cell growth and negative immunomodulation. We hypothesized that a soluble form of Siglec-9 (sSiglec-9) competitively inhibits a binding of MUC1 to its receptor molecules like human Siglec-9, leading to provide antitumor benefit against MUC1-expressing tumor, and generated transgenic mouse lines expressing sSiglec-9more » (sSiglec-9 Tg). When mammary tumor cells expressing MUC1 were intraperitoneally transplanted into sSiglec-9 Tg, tumor proliferation was slower with the lower histological malignancy as compared with non-transgenic mice. The sSiglec-9 was detected in the ascites caused by the tumor in the sSiglec-9 Tg, and sSiglec-9 and MUC1 were often colocalized on surfaces of the tumor cells. PCNA immunohistochemistry also revealed the reduced proliferation of the tumor cells in sSiglec-9 Tg. In sSiglec-9 Tg with remarkable suppression of tumor proliferation, MUC1 expressions were tend to be reduced. In the ascites of sSiglec-9 Tg bearing the tumor, T cells were uniformly infiltrated, whereas aggregations of degenerative T cells were often observed in the non-transgenic mice. These results suggest that sSiglec-9 has an antitumor benefit against MUC1-expressing tumor in the transgenic mice, which may avoid the negative immunomodulation and/or suppress tumor-associated MUC1 downstream signal transduction, and subsequent tumor proliferation.« less

Co-expression of Interleukin-15 Enhances the Protective Immune Responses Induced by Immunization with a Murine Malaria MVA-Based Vaccine Encoding the Circumsporozoite Protein.

PubMed

Parra, Marcela; Liu, Xia; Derrick, Steven C; Yang, Amy; Molina-Cruz, Alvaro; Barillas-Mury, Carolina; Zheng, Hong; Thao Pham, Phuong; Sedegah, Martha; Belmonte, Arnel; Litilit, Dianne D; Waldmann, Thomas A; Kumar, Sanjai; Morris, Sheldon L; Perera, Liyanage P

2015-01-01

Malaria remains a major global public health problem with an estimated 200 million cases detected in 2012. Although the most advanced candidate malaria vaccine (RTS,S) has shown promise in clinical trials, its modest efficacy and durability have created uncertainty about the impact of RTS,S immunization (when used alone) on global malaria transmission. Here we describe the development and characterization of a novel modified vaccinia virus Ankara (MVA)-based malaria vaccine which co-expresses the Plasmodium yoelii circumsporozoite protein (CSP) and IL-15. Vaccination/challenge studies showed that C57BL/6 mice immunized with the MVA-CSP/IL15 vaccine were protected significantly better against a P. yoelii 17XNL sporozoite challenge than either mice immunized with an MVA vaccine expressing only CSP or naïve controls. Importantly, the levels of total anti-CSP IgG were elevated about 100-fold for the MVA-CSP/IL15 immunized group compared to mice immunized with the MVA-CSP construct that does not express IL-15. Among the IgG subtypes, the IL-15 expressing MVA-CSP vaccine induced levels of IgG1 (8 fold) and IgG2b (80 fold) higher than the MVA-CSP construct. The significantly enhanced humoral responses and protection detected after immunization with the MVA-CSP/IL15 vaccine suggest that this IL-15 expressing MVA construct could be considered in the development of future malaria immunization strategies.

A Comparison of Red Fluorescent Proteins to Model DNA Vaccine Expression by Whole Animal In Vivo Imaging.

PubMed

Kinnear, Ekaterina; Caproni, Lisa J; Tregoning, John S

2015-01-01

DNA vaccines can be manufactured cheaply, easily and rapidly and have performed well in pre-clinical animal studies. However, clinical trials have so far been disappointing, failing to evoke a strong immune response, possibly due to poor antigen expression. To improve antigen expression, improved technology to monitor DNA vaccine transfection efficiency is required. In the current study, we compared plasmid encoded tdTomato, mCherry, Katushka, tdKatushka2 and luciferase as reporter proteins for whole animal in vivo imaging. The intramuscular, subcutaneous and tattooing routes were compared and electroporation was used to enhance expression. We observed that overall, fluorescent proteins were not a good tool to assess expression from DNA plasmids, with a highly heterogeneous response between animals. Of the proteins used, intramuscular delivery of DNA encoding either tdTomato or luciferase gave the clearest signal, with some Katushka and tdKatushka2 signal observed. Subcutaneous delivery was weakly visible and nothing was observed following DNA tattooing. DNA encoding haemagglutinin was used to determine whether immune responses mirrored visible expression levels. A protective immune response against H1N1 influenza was induced by all routes, even after a single dose of DNA, though qualitative differences were observed, with tattooing leading to high antibody responses and subcutaneous DNA leading to high CD8 responses. We conclude that of the reporter proteins used, expression from DNA plasmids can best be assessed using tdTomato or luciferase. But, the disconnect between visible expression level and immunogenicity suggests that in vivo whole animal imaging of fluorescent proteins has limited utility for predicting DNA vaccine efficacy.

Coxsackievirus B3 vaccines: use as an expression vector for prevention of myocarditis.

PubMed

Henke, Andreas; Jarasch, Nadine; Wutzler, Peter

2008-12-01

Coxsackievirus B3 (CVB3), a member of the Picornaviridae family, is considered to be one of the most important infectious agents to cause virus-induced myocarditis. Despite improvements in studying virus pathology, structure and molecular biology, as well as the diagnosis of this disease, there is still no virus-specific drug or vaccine in clinical use. During the last 20 years many investigations have been performed to develop classic and modern immunization techniques against CVB3-induced heart disease. One promising approach among others includes the insertion of coding sequences of cytokines into the viral genome. The application of an IFN-gamma-expressing recombinant coxsackievirus vector is especially efficient against CVB3-induced myocarditis. Beside direct IFN-gamma-mediated antiviral effects, the local and simultaneous expression of IFN-gamma by the virus itself activates the immune system in a strong and long-lasting manner, which protects animals completely against subsequent lethal infections independently of the age of the immunized individual and the route of vaccine administration.

Immunogenicity of recombinant measles vaccine expressing fusion protein of respiratory syncytial virus in cynomolgus monkeys.

PubMed

Sawada, Akihito; Yunomae, Kiyokazu; Nakayama, Tetsuo

2018-02-01

Respiratory syncytial virus (RSV) is a common cause of respiratory infections in infants. Effective vaccines are currently being sought, but no vaccine is thus far available. In our previous study, recombinant AIK-C measles vaccine expressing the RSV fusion protein (MVAIK/RSV/F) was developed and protective immunity against RSV demonstrated in cotton rats. In the present study, the immunogenicity and protective effects were investigated in three cynomolgus monkeys immunized with MVAIK/RSV/F. Neutralizing test antibodies against RSV were detected and no infectious virus was recovered from the lungs of monkeys immunized with MVAIK/RSV/F after challenge. MVAIK/RSV/F has the potential to inhibit RSV infection. © 2018 The Societies and John Wiley & Sons Australia, Ltd.

Active immunotherapy for mouse breast cancer with irradiated whole-cell vaccine expressing VEGFR2.

PubMed

Yan, Heng-Xiu; Cheng, Ping; Wei, Hai-Yan; Shen, Guo-Bo; Fu, Li-Xin; Ni, Jie; Wu, Yang; Wei, Yu-Quan

2013-04-01

As tumor-associated antigens are not well characterized for the majority of human tumors, polyvalent vaccines prepared with whole-tumor antigens are an attractive approach for tumor vaccination. Vascular endothelial growth factor receptor-2 (VEGFR2), as a model antigen with which to explore the feasibility of immunotherapy, has shown great promise as a tumor vaccine. However, the efficacy of immunotherapy is often not ideal when used alone. In this study, we explored the therapeutic efficacy of an irradiated AdVEGFR2-infected cell vaccine-based immunotherapy in the weakly immunogenic and highly metastatic 4T1 murine mammary cancer model. An adenovirus encoding the VEGFR2 gene (AdVEGFR2) was constructed. Lethally irradiated, virus-infected 4T1 cells were used as vaccines. Vaccination with lethally irradiated AdVEGFR2-infected 4T1 cells inhibited subsequent tumor growth and pulmonary metastasis compared with challenge inoculations. Angiogenesis was inhibited, and the number of CD8+ T lymphocytes was increased within the tumors. Antitumor activity was also caused by the adoptive transfer of isolated spleen lymphocytes. In vitro, the expression of HMGB1 and HSP70 in the AdVEGFR2‑infected 4T1 cells was increased, and was involved in the activation of tumor antigen-specific T-cell immunity. Our results indicate that the immunotherapy based on irradiated AdVEGFR2-infected whole-cancer cell vaccines may be a potentially effective strategy for 4T1 cancer treatment.

Immune and histopathological responses in animals vaccinated with recombinant vaccinia viruses that express individual genes of human respiratory syncytial virus.

PubMed

Stott, E J; Taylor, G; Ball, L A; Anderson, K; Young, K K; King, A M; Wertz, G W

1987-12-01

Previous reports have established that vaccinia virus (VV) recombinants expressing G, F, or N protein of respiratory syncytial (RS) virus protect small animals against intranasal challenge with live RS virus. This work demonstrates that a variety of parameters affect the protection induced by recombinant viruses. The route of vaccination, the subtype of challenge virus, and the species used influenced the antibody titers and extent of protection. During these studies, observations were also made on the subclass of antibody generated, and pulmonary histopathological changes induced by challenge after vaccination were noted. The effect of route of inoculation on host response was examined by vaccinating mice intranasally, intraperitoneally, or by scarification with a recombinant VV expressing the RS virus G glycoprotein. Intranasal vaccination induced 25-fold-higher titers of antibody to RS virus in the lung than the intraperitoneal route did, but both routes resulted in complete suppression of virus replication after intranasal challenge 21 days after vaccination. Scarification was a less effective method of vaccination. The antibody induced by recombinant VV in mice was mostly immunoglobulin G2a (IgG2a) with some IgG2b. No antibody to RS virus was detected in the IgA, IgM, IgG1, or IgG3 subclass irrespective of the vaccination route. The G and F glycoproteins were shown to elicit similar subclasses of antibody. However, animals vaccinated with the G and F vectors differed strikingly in their response to challenge by heterologous virus. Mice or cotton rats vaccinated with recombinant VV carrying the G gene of RS virus were protected against challenge only with homologous subtype A virus. Vaccination with a recombinant VV expressing the F glycoprotein induced protection against both homologous and heterologous subtype B virus challenge. The protection induced in mice was greater than that detected in cotton rats, indicating that the host may also affect immunity

Global gene expression in channel catfish after vaccination with an attenuated Edwardsiella ictaluri

USDA-ARS?s Scientific Manuscript database

To understand the global gene expression in channel catfish after immersion vaccination with an attenuated Edwardsiella ictaluri (AquaVac ESCTM), microarray analysis of 65,182 UniGene transcripts were performed. With a filter of false-discovery rate less than 0.05 and fold change greater than 2, a t...

The effect of eukaryotic expression vectors and adjuvants on DNA vaccines in chickens using an avian influenza model.

PubMed

Suarez, D L; Schultz-Cherry, S

2000-01-01

Vaccination of poultry with naked plasmid DNA has been successfully demonstrated with several different poultry pathogens, but the technology needs to be further developed before it can be practically implemented. Many different methods can conceivably enhance the efficacy of DNA vaccines, and this report examines the use of different eukaryotic expression vectors with different promoters and different adjuvants to express the influenza hemagglutinin protein. Four different promoters in five different plasmids were used to express the hemagglutinin protein of an H5 avian influenza virus, including two different immediate early cytomegaloviruses (CMVs), Rous sarcoma virus, chicken actin, and simian virus 40 promoters. All five constructs expressed detectable hemagglutinin protein in cell culture, but the pCI-neo HA plasmid with the CMV promoter provided the best response in chickens when vaccinated intramuscularly at 1 day of age on the basis of antibody titer and survivability after challenge with a highly pathogenic avian influenza virus at 6 wk postinoculation. A beneficial response was observed in birds boostered at 3 wk of age, in birds given larger amounts of DNA, and with the use of multiple injection sites to administer the vaccine. With the use of the pCI-neo construct, the effects of different adjuvants designed to increase the uptake of plasmid DNA, including 25% sucrose, diethylaminoethyl dextran, calcium phosphate, polybrene, and two different cationic liposomes, were examined. Both liposomes tested enhanced antibody titers as compared with the positive controls, but the other chemical adjuvants decreased the antibody response as compared with the control chickens that received just the plasmid alone. The results observed are promising for continued studies, but continued improvements in vaccine response and reduced costs are necessary before the technology can be commercially developed.

Haematopoietic depletion in vaccine-induced neonatal pancytopenia depends on both the titre and specificity of alloantibody and levels of MHC I expression.

PubMed

Bell, Charlotte R; MacHugh, Niall D; Connelley, Timothy K; Degnan, Kathryn; Morrison, W Ivan

2015-07-09

Bovine Neonatal Pancytopenia (BNP) is a disease of calves characterised by haematopoietic depletion, mediated by ingestion of alloantibodies in colostrum. It has been linked epidemiologically to vaccination of the dams of affected calves with a particular vaccine (Pregsure) containing a novel adjuvant. Evidence suggests that BNP-alloantibodies are directed against MHC I molecules, induced by contaminant bovine cellular material from Madin-Darby Bovine Kidney (MDBK) cells used in the vaccine's production. We aimed to investigate the specificity of BNP-alloantibody for bovine MHC I alleles, particularly those expressed by MDBK cells, and whether depletion of particular cell types is due to differential MHC I expression levels. A complement-mediated cytotoxicity assay was used to assess functional serum alloantibody titres in BNP-dams, Pregsure-vaccinated dams with healthy calves, cows vaccinated with an alternative product and unvaccinated controls. Alloantibody specificity was investigated using transfected mouse lines expressing the individual MHC I alleles identified from MDBK cells and MHC I-defined bovine leukocyte lines. All BNP-dams and 50% of Pregsure-vaccinated cows were shown to have MDBK-MHC I specific alloantibodies, which cross-reacted to varying degrees with other MHC I genotypes. MHC I expression levels on different blood cell types, assessed by flow cytometry, were found to correlate with levels of alloantibody-mediated damage in vitro and in vivo. Alloantibody-killed bone marrow cells were shown to express higher levels of MHC I than undamaged cells. The results provide evidence that MHC I-specific alloantibodies play a dominant role in the pathogenesis of BNP. Haematopoietic depletion was shown to be dependent on the titre and specificity of alloantibody produced by individual cows and the density of surface MHC I expression by different cell types. Collectively, the results support the hypothesis that MHC I molecules originating from MDBK cells

Avian Paramyxovirus Type-3 as a Vaccine Vector: Identification of a Genome Location for High Level Expression of a Foreign Gene.

PubMed

Yoshida, Asuka; Samal, Siba K

2017-01-01

Avian paramyxovirus serotype 3 (APMV-3) causes infection in a wide variety of avian species, but it does not cause apparent diseases in chickens. On the contrary, APMV-1, also known as Newcastle disease virus (NDV), can cause severe disease in chickens. Currently, natural low virulence strains of NDV are used as live-attenuated vaccines throughout the world. NDV is also being evaluated as a vaccine vector against poultry pathogens. However, due to routine vaccination programs, chickens often possess pre-existing antibodies against NDV, which may cause the chickens to be less sensitive to recombinant NDV vaccines expressing antigens of other avian pathogens. Therefore, it may be possible for an APMV-3 vector vaccine to circumvent this issue. In this study, we determined the optimal insertion site in the genome of APMV-3 for high level expression of a foreign gene. We generated recombinant APMV-3 viruses expressing the green fluorescent protein (GFP) by inserting the GFP gene at five different intergenic regions in the genome. The levels of GFP transcription and translation were evaluated. Interestingly, the levels of GFP transcription and translation did not follow the 3'-to-5' attenuation mechanism of non-segmented, negative-sense RNA viruses. The insertion of GFP gene into the P-M gene junction resulted in higher level of expression of GFP than when the gene was inserted into the upstream N-P gene junction. Unlike NDV, insertion of GFP did not attenuate the growth efficiency of AMPV-3. Thus, APMV-3 could be a more useful vaccine vector for avian pathogens than NDV.

Using rabies virus vaccine strain SRV9 as viral vector to express exogenous gene.

PubMed

Wang, Hualei; Jin, Hongli; Feng, Na; Zheng, Xuexing; Li, Ling; Qi, Yinglin; Liang, Meng; Zhao, Yongkun; Wang, Tiecheng; Gao, Yuwei; Tu, Changchun; Jin, Ningyi; Yang, Songtao; Xia, Xianzhu

2015-04-01

Rabies virus (RABV) can cause a fatal neurological disease in human and animals, and vaccines were generally applied for the immunoprophylaxis of rabies. Here, a recombinant viral vector carrying the exogenous gene expression component between phosphoprotein (P) and matrix protein (M) genes of RABV was constructed based on the vaccine strain SRV9 used in China. To develop a reverse genetic system, the full-length cDNA plasmids of SRV9 were constructed using the eukaryotic expression vector pCI or pcDNA3.1(+). However, recovery efficiency based on the pcDNA3.1 vector was significantly higher than that of the pCI vector. The exogenous gene expression component PE-PS-BsiWI-PmeI or PS-BsiWI-PmeI-PE was introduced in different locations between the P and M genes of SRV9. When the enhanced green fluorescent protein (eGFP) was used as a reporter gene, both locations could rescue recombinant RABV (rRABV) expressing eGFP with high efficiency. Characterization of rRABV expressing eGFP in vitro revealed that its growth was similar to that of the parental virus. Animal experiments showed that rRABV expressing eGFP could replicate and express eGFP in the brains of suckling mice. Furthermore, rRABV of SRV9 was nonpathogenic for 3-week-old mice and could be cleared from the central nervous system at 5 days post-inoculation. Our results showed that the recombinant SRV9 virus could be used as a useful viral vector for exogenous gene expression.

Co-expression of HIV-1 virus-like particles and granulocyte-macrophage colony stimulating factor by GEO-D03 DNA vaccine.

PubMed

Hellerstein, Michael; Xu, Yongxian; Marino, Tracie; Lu, Shan; Yi, Hong; Wright, Elizabeth R; Robinson, Harriet L

2012-11-01

Here, we report on GEO-D03, a DNA vaccine that co-expresses non-infectious HIV-1 virus-like particles (VLPs) and the human cytokine, granulocyte-macrophage colony-stimulating factor (GM-CSF). The virus-like particles display the native gp160 form of the HIV-1 Envelope glycoprotein (Env) and are designed to elicit antibody against the natural form of Env on virus and virus-infected cells. The DNA-expressed HIV Gag, Pol and Env proteins also have the potential to elicit virus-specific CD4 and CD8 T cells. The purpose of the co-expressed GM-CSF is to target a cytokine that recruits, expands and differentiates macrophages and dendritic cells to the site of VLP expression. The GEO-D03 DNA vaccine is currently entered into human trials as a prime for a recombinant modified vaccinia Ankara (MVA) boost. In preclinical studies in macaques using an SIV prototype vaccine, this vaccination regimen elicited both anti-viral T cells and antibody, and provided 70% protection against acquisition during 12 weekly rectal exposures with a heterologous SIV. Higher avidity of the Env-specific Ab for the native form of the Env in the challenge virus correlated with lower likelihood of SIV infection.

Effects of Environmental Factors on Soluble Expression of a Humanized Anti-TNF-α scFv Antibody in Escherichia coli

PubMed Central

Sina, Mohammad; Farajzadeh, Davoud; Dastmalchi, Siavoush

2015-01-01

Purpose: The bacterial cultivation conditions for obtaining anti-TNF-α single chain variable fragment (scFv) antibody as the soluble product in E. coli was investigated. Methods: To avoid the production of inclusion bodies, the effects of lactose, IPTG, incubation time, temperature, shaking protocol, medium additives (Mg+2, sucrose), pH, osmotic and heat shocks were examined. Samples from bacterial growth conditions with promising results of soluble expression of GST-hD2 scFv were affinity purified and quantified by SDS-PAGE and image processing for further evaluation. Results: The results showed that cultivation in LB medium under induction by low concentrations of lactose and incubation at 10 °C led to partial solubilization of the expressed anti-TNF-α scFv (GST-hD2). Other variables which showed promising increase in soluble expression of GST-hD2 were osmotic shock and addition of magnesium chloride. Furthermore, addition of sucrose to medium suppressed the expression of scFv completely. The other finding was that the addition of sorbitol decreased the growth rate of bacteria. Conclusion: It can be concluded that low cultivation temperature in the presence of low amount of inducer under a long incubation time or addition of magnesium chloride are the most effective environmental factors studied for obtaining the maximum solubilization of GST-hD2 recombinant protein. PMID:26819916

Effects of Environmental Factors on Soluble Expression of a Humanized Anti-TNF-α scFv Antibody in Escherichia coli.

PubMed

Sina, Mohammad; Farajzadeh, Davoud; Dastmalchi, Siavoush

2015-11-01

The bacterial cultivation conditions for obtaining anti-TNF-α single chain variable fragment (scFv) antibody as the soluble product in E. coli was investigated. To avoid the production of inclusion bodies, the effects of lactose, IPTG, incubation time, temperature, shaking protocol, medium additives (Mg+2, sucrose), pH, osmotic and heat shocks were examined. Samples from bacterial growth conditions with promising results of soluble expression of GST-hD2 scFv were affinity purified and quantified by SDS-PAGE and image processing for further evaluation. The results showed that cultivation in LB medium under induction by low concentrations of lactose and incubation at 10 °C led to partial solubilization of the expressed anti-TNF-α scFv (GST-hD2). Other variables which showed promising increase in soluble expression of GST-hD2 were osmotic shock and addition of magnesium chloride. Furthermore, addition of sucrose to medium suppressed the expression of scFv completely. The other finding was that the addition of sorbitol decreased the growth rate of bacteria. It can be concluded that low cultivation temperature in the presence of low amount of inducer under a long incubation time or addition of magnesium chloride are the most effective environmental factors studied for obtaining the maximum solubilization of GST-hD2 recombinant protein.

Coregulation of Soybean Vegetative Storage Protein Gene Expression by Methyl Jasmonate and Soluble Sugars 1

PubMed Central

Mason, Hugh S.; DeWald, Daryll B.; Creelman, Robert A.; Mullet, John E.

1992-01-01

The soybean vegetative storage protein genes vspA and vspB are highly expressed in developing leaves, stems, flowers, and pods as compared with roots, seeds, and mature leaves and stems. In this paper, we report that physiological levels of methyl jasmonate (MeJA) and soluble sugars synergistically stimulate accumulation of vsp mRNAs. Treatment of excised mature soybean (Glycine max Merr. cv Williams) leaves with 0.2 molar sucrose and 10 micromolar MeJA caused a large accumulation of vsp mRNAs, whereas little accumulation occurred when these compounds were supplied separately. In soybean cell suspension cultures, the synergistic effect of sucrose and MeJA on the accumulation of vspB mRNA was maximal at 58 millimolar sucrose and was observed with fructose or glucose substituted for sucrose. In dark-grown soybean seedlings, the highest levels of vsp mRNAs occurred in the hypocotyl hook, which also contained high levels of MeJA and soluble sugars. Lower levels of vsp mRNAs, MeJA, and soluble sugars were found in the cotyledons, roots, and nongrowing regions of the stem. Wounding of mature soybean leaves induced a large accumulation of vsp mRNAs when wounded plants were incubated in the light. Wounded plants kept in the dark or illuminated plants sprayed with dichlorophenyldimethylurea, an inhibitor of photosynthetic electron transport, showed a greatly reduced accumulation of vsp mRNAs. The time courses for the accumulation of vsp mRNAs induced by wounding or sucrose/MeJA treatment were similar. These results strongly suggest that vsp expression is coregulated by endogenous levels of MeJA (or jasmonic acid) and soluble carbohydrate during normal vegetative development and in wounded leaves. ImagesFigure 1Figure 4Figure 5 PMID:16668757

Immunogenicity of adenovirus vaccines expressing the PCV2 capsid protein in pigs.

PubMed

Li, Delong; Du, Qian; Wu, Bin; Li, Juejun; Chang, Lingling; Zhao, Xiaomin; Huang, Yong; Tong, Dewen

2017-08-24

Porcine circovirus type 2 (PCV2) is the main pathogen of porcine circovirus associated disease (PCVAD), causing great economic losses in pig industry. In previous study, we constructed adenovirus vector vaccines expressing PCV2 Cap either modified with Intron A and WPRE, or CD40L and GMCSF, and evaluated all of these vaccines in mice and in pigs. Although Ad-A-C-W and Ad-CD40L-Cap-GMCSF could induce stronger immune responses than Ad-Cap, neither of them was better than commercial inactivated vaccine PCV2 SH-strain. In this study, secretory recombinant adenoviruses (Ad-A-spCap-W and Ad-A-spCD40L-spCap-spGMCSF-W) and non-secretory recombinant adenovirus Ad-A-CD40L-Cap-GMCSF-W were constructed, and identified by western blot and confocal laser microscope observation. The results of ELISA and VN showed that humoral immune responses induced by Ad-A-spCap-W and Ad-A-CD40L-Cap-GMCSF-W were not significantly different from SH-strain, but Ad-A-spCD40L-spCap-spGMCSF-W could induce significantly higher humoral immune response than SH-strain. Lymphocytes proliferative and cytokines releasing levels of Ad-A-spCap-W and Ad-A-CD40L-Cap-GMCSF-W were not significantly different from SH-strain, but Ad-A-spCD40L-spCap-spGMCSF-W was significantly higher than SH-strain. PCV2-challenge experiment showed that virus loads were significantly reduced in Ad-A-spCD40L-spCap-spGMCSF-W vaccinated group, and no obviously clinical and microscopic lesions were observed in Ad-A-spCD40L-spCap-spGMCSF-W vaccinated group. Altogether, these results demonstrate that recombinant adenovirus vaccine Ad-A-spCD40L-spCap-spGMCSF-W induces stronger immune responses and provides better protection than commercial inactivated vaccine PCV2 SH-strain, and suggest that Ad-A-spCD40L-spCap-spGMCSF-W could be a potential vaccine candidate against PCVAD. Copyright © 2017 Elsevier Ltd. All rights reserved.

Protection against California 2002 NDV strain afforded by adenovirus vectored vaccine expressing Fusion or Hemagglutination-neuraminidase genes

USDA-ARS?s Scientific Manuscript database

Vectored vaccines expressing the combination of the hemagglutinin-neuraminidase (HN) and fusion (F) genes generally have better clinical protection against Newcastle disease virus (NDV) than when either the F and HN genes are expressed alone. Interestingly, the protection induced by F is usually bet...

Siderophore vaccine conjugates protect against uropathogenic Escherichia coli urinary tract infection

PubMed Central

Mike, Laura A.; Smith, Sara N.; Sumner, Christopher A.; Eaton, Kathryn A.; Mobley, Harry L. T.

2016-01-01

Uropathogenic Escherichia coli (UPEC) is the primary cause of uncomplicated urinary tract infections (UTIs). Whereas most infections are isolated cases, 1 in 40 women experience recurrent UTIs. The rise in antibiotic resistance has complicated the management of chronic UTIs and necessitates new preventative strategies. Currently, no UTI vaccines are approved for use in the United States, and the development of a highly effective vaccine remains elusive. Here, we have pursued a strategy for eliciting protective immunity by vaccinating with small molecules required for pathogenesis, rather than proteins or peptides. Small iron-chelating molecules called siderophores were selected as antigens to vaccinate against UTI for this vaccine strategy. These pathogen-associated stealth siderophores evade host immune defenses and enhance bacterial virulence. Previous animal studies revealed that vaccination with siderophore receptor proteins protects against UTI. The poor solubility of these integral outer-membrane proteins in aqueous solutions limits their practical utility. Because their cognate siderophores are water soluble, we hypothesized that these bacterial-derived small molecules are prime vaccine candidates. To test this hypothesis, we immunized mice with siderophores conjugated to an immunogenic carrier protein. The siderophore–protein conjugates elicited an adaptive immune response that targeted bacterial stealth siderophores and protected against UTI. Our study has identified additional antigens suitable for a multicomponent UTI vaccine and highlights the potential use of bacterial-derived small molecules as antigens in vaccine therapies. PMID:27821778

Siderophore vaccine conjugates protect against uropathogenic Escherichia coli urinary tract infection.

PubMed

Mike, Laura A; Smith, Sara N; Sumner, Christopher A; Eaton, Kathryn A; Mobley, Harry L T

2016-11-22

Uropathogenic Escherichia coli (UPEC) is the primary cause of uncomplicated urinary tract infections (UTIs). Whereas most infections are isolated cases, 1 in 40 women experience recurrent UTIs. The rise in antibiotic resistance has complicated the management of chronic UTIs and necessitates new preventative strategies. Currently, no UTI vaccines are approved for use in the United States, and the development of a highly effective vaccine remains elusive. Here, we have pursued a strategy for eliciting protective immunity by vaccinating with small molecules required for pathogenesis, rather than proteins or peptides. Small iron-chelating molecules called siderophores were selected as antigens to vaccinate against UTI for this vaccine strategy. These pathogen-associated stealth siderophores evade host immune defenses and enhance bacterial virulence. Previous animal studies revealed that vaccination with siderophore receptor proteins protects against UTI. The poor solubility of these integral outer-membrane proteins in aqueous solutions limits their practical utility. Because their cognate siderophores are water soluble, we hypothesized that these bacterial-derived small molecules are prime vaccine candidates. To test this hypothesis, we immunized mice with siderophores conjugated to an immunogenic carrier protein. The siderophore-protein conjugates elicited an adaptive immune response that targeted bacterial stealth siderophores and protected against UTI. Our study has identified additional antigens suitable for a multicomponent UTI vaccine and highlights the potential use of bacterial-derived small molecules as antigens in vaccine therapies.

Analysis of the Expression of Peptide–Major Histocompatibility Complexes Using High Affinity Soluble Divalent T Cell Receptors

PubMed Central

O'Herrin, Sean M.; Lebowitz, Michael S.; Bieler, Joan G.; al-Ramadi, Basel K.; Utz, Ursula; Bothwell, Alfred L.M.; Schneck, Jonathan P.

1997-01-01

Understanding the regulation of cell surface expression of specific peptide–major histocompatibility complex (MHC) complexes is hindered by the lack of direct quantitative analyses of specific peptide–MHC complexes. We have developed a direct quantitative biochemical approach by engineering soluble divalent T cell receptor analogues (TCR–Ig) that have high affinity for their cognate peptide–MHC ligands. The generality of this approach was demonstrated by specific staining of peptide-pulsed cells with two different TCR–Ig complexes: one specific for the murine alloantigen 2C, and one specific for a viral peptide from human T lymphocyte virus–1 presented by human histocompatibility leukocyte antigens–A2. Further, using 2C TCR– Ig, a more detailed analysis of the interaction with cognate peptide–MHC complexes revealed several interesting findings. Soluble divalent 2C TCR–Ig detected significant changes in the level of specific antigenic–peptide MHC cell surface expression in cells treated with γ-interferon (γ-IFN). Interestingly, the effects of γ-IFN on expression of specific peptide–MHC complexes recognized by 2C TCR–Ig were distinct from its effects on total H-2 Ld expression; thus, lower doses of γ-IFN were required to increase expression of cell surface class I MHC complexes than were required for upregulation of expression of specific peptide–MHC complexes. Analysis of the binding of 2C TCR–Ig for specific peptide–MHC ligands unexpectedly revealed that the affinity of the 2C TCR–Ig for the naturally occurring alloreactive, putatively, negatively selecting, complex, dEV-8–H-2 Kbm3, is very low, weaker than 71 μM. The affinity of the 2C TCR for the other naturally occurring, negatively selecting, alloreactive complex, p2Ca–H-2 Ld, is ∼1000-fold higher. Thus, negatively selecting peptide–MHC complexes do not necessarily have intrinsically high affinity for cognate TCR. These results, uniquely revealed by this analysis

Combat pneumococcal infections: adhesins as candidates for protein-based vaccine development.

PubMed

Gamez, Gustavo; Hammerschmidt, Sven

2012-03-01

Streptococcus pneumoniae (pneumococcus) is an asymptomatic colonizer of the upper respiratory tract in humans. However, these apparently harmless bacteria have also a high virulence potential and are known as the etiologic agent of respiratory and life-threatening invasive diseases. Dissemination of pneumococci from the nasopharynx into the lungs or bloodstream leads to community-acquired pneumonia, septicaemia and meningitis. Traditionally, pneumococcal diseases are treated with antibiotics and prevented with polysaccharide-based vaccines. However, due to the dramatic increase in antibiotic resistance and limitations of the current available vaccines, the burden of diseases remains high. Thus, combating pneumococcal transmission and infections has emphasized the need for a new generation of protein-based vaccines. Interactions of pneumococci with soluble host proteins or cellular receptors are crucial for adherence, colonization, transmigration of host barriers and immune evasion. Therefore, surface-exposed proteins involved in these pathogenic processes and virtually expressed by all pneumococcal strains and serotypes are the prime potential targets for an immunogenic and highly protective pneumococcal-derived carrier protein of a vaccine. In this review, we will address the state of the art in deciphering, i). the conservation, distribution and pathogenic role of recently discovered pneumococcal adhesins in colonization and invasive diseases, ii). the interactions of these virulence factors with host-proteins and receptors, iii). the subversion of the host immune and cellular responses, and iv). the potential of pneumococcal adhesins as vaccine candidates.

Sequential infection with common pathogens promotes human-like immune gene expression and altered vaccine response

PubMed Central

Reese, Tiffany A.; Bi, Kevin; Kambal, Amal; Filali-Mouhim, Ali; Beura, Lalit K.; Bürger, Matheus C.; Pulendran, Bali; Sekaly, Rafick; Jameson, Stephen C.; Masopust, David; Haining, W. Nicholas; Virgin, Herbert W.

2016-01-01

Summary Immune responses differ between laboratory mice and humans. Chronic infection with viruses and parasites are common in humans, but are absent in laboratory mice, and thus represent potential contributors to inter-species differences in immunity. To test this, we sequentially infected laboratory mice with herpesviruses, influenza, and an intestinal helminth, and compared their blood immune signatures to mock-infected mice before and after vaccination against Yellow Fever Virus (YFV-17D). Sequential infection altered pre- and post-vaccination gene expression, cytokines, and antibodies in blood. Sequential pathogen exposure induced gene signatures that recapitulated those seen in blood from pet store-raised versus laboratory mice, and adult versus cord blood in humans. Therefore basal and vaccine-induced murine immune responses are altered by infection with agents common outside of barrier facilities. This raises the possibility that we can improve mouse models of vaccination and immunity by selective microbial exposure of laboratory animals to mimic that of humans. PMID:27107939

Comparison of gene expression of Toll-like receptors and cytokines between Piau and Commercial line (Landrace×Large White crossbred) pigs vaccinated against Pasteurella multocida type D.

PubMed

Sousa, Katiene Régia Silva; Ribeiro, André Mauric Frossard; Dantas, Waleska de Melo Ferreira; Oliveira, Leandro Licursi de; Gasparino, Eliane; Guimarães, Simone Eliza Facioni

2017-10-01

We aimed to compare Toll-like receptors (TLR) and cytokines expression in local Piau breed and a Commercial line (Landrace×Large White crossbred) pigs in response to vaccination against Pasteurella multocida type D. Seronegative gilts for Pasteurella multocida type D and Mycoplasma hyopneumoniae were used, from which peripheral blood mononuclear cells (PBMC) were collected in four time points (T0, T1, T2 and T3; before and after each vaccination dose). For bronchoalveolar lavage fluid cells (BALF), we set groups of vaccinated and unvaccinated animals for both genetic groups. Gene expression was evaluated on PBMC and BALF. In PBMC, when we analyzed time points within breeds, significant differences in expression for TLRs and cytokines, except TGFβ, were observed for Commercial animals. For the Piau pigs, only TGFβ showed differential expression. Comparing the expression among genetic groups, the Commercial pigs showed higher expression for TLRs after first vaccination dose, while for IL2, IL6, IL12 and IL13, higher expression was also observed in T3 and IL8 and IL10, in T1 and T3. Still comparing the breeds, the crossbred animals showed higher expression for TNFα in T1 and T2, while for TGFβ only in T2. For gene expression in BALF, vaccinated Commercial pigs showed higher expression of TLR6, TLR10, IL6, IL8, IL10, TNFα and TGFβ genes than vaccinated Piau pigs. The Commercial line pigs showed higher sensitivity to vaccination, while in local Piau breed lower responsiveness, which may partly explain genetic variability in immune response and will let us better understand the tolerance/susceptibility for pasteurellosis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Differential miRNA expression in B cells is associated with inter-individual differences in humoral immune response to measles vaccination.

PubMed

Haralambieva, Iana H; Kennedy, Richard B; Simon, Whitney L; Goergen, Krista M; Grill, Diane E; Ovsyannikova, Inna G; Poland, Gregory A

2018-01-01

MicroRNAs are important mediators of post-transcriptional regulation of gene expression through RNA degradation and translational repression, and are emerging biomarkers of immune system activation/response after vaccination. We performed Next Generation Sequencing (mRNA-Seq) of intracellular miRNAs in measles virus-stimulated B and CD4+ T cells from high and low antibody responders to measles vaccine. Negative binomial generalized estimating equation (GEE) models were used for miRNA assessment and the DIANA tool was used for gene/target prediction and pathway enrichment analysis. We identified a set of B cell-specific miRNAs (e.g., miR-151a-5p, miR-223, miR-29, miR-15a-5p, miR-199a-3p, miR-103a, and miR-15a/16 cluster) and biological processes/pathways, including regulation of adherens junction proteins, Fc-receptor signaling pathway, phosphatidylinositol-mediated signaling pathway, growth factor signaling pathway/pathways, transcriptional regulation, apoptosis and virus-related processes, significantly associated with neutralizing antibody titers after measles vaccination. No CD4+ T cell-specific miRNA expression differences between high and low antibody responders were found. Our study demonstrates that miRNA expression directly or indirectly influences humoral immunity to measles vaccination and suggests that B cell-specific miRNAs may serve as useful predictive biomarkers of vaccine humoral immune response.

Avian Paramyxovirus Type-3 as a Vaccine Vector: Identification of a Genome Location for High Level Expression of a Foreign Gene

PubMed Central

Yoshida, Asuka; Samal, Siba K.

2017-01-01

Avian paramyxovirus serotype 3 (APMV-3) causes infection in a wide variety of avian species, but it does not cause apparent diseases in chickens. On the contrary, APMV-1, also known as Newcastle disease virus (NDV), can cause severe disease in chickens. Currently, natural low virulence strains of NDV are used as live-attenuated vaccines throughout the world. NDV is also being evaluated as a vaccine vector against poultry pathogens. However, due to routine vaccination programs, chickens often possess pre-existing antibodies against NDV, which may cause the chickens to be less sensitive to recombinant NDV vaccines expressing antigens of other avian pathogens. Therefore, it may be possible for an APMV-3 vector vaccine to circumvent this issue. In this study, we determined the optimal insertion site in the genome of APMV-3 for high level expression of a foreign gene. We generated recombinant APMV-3 viruses expressing the green fluorescent protein (GFP) by inserting the GFP gene at five different intergenic regions in the genome. The levels of GFP transcription and translation were evaluated. Interestingly, the levels of GFP transcription and translation did not follow the 3′-to-5′ attenuation mechanism of non-segmented, negative-sense RNA viruses. The insertion of GFP gene into the P-M gene junction resulted in higher level of expression of GFP than when the gene was inserted into the upstream N-P gene junction. Unlike NDV, insertion of GFP did not attenuate the growth efficiency of AMPV-3. Thus, APMV-3 could be a more useful vaccine vector for avian pathogens than NDV. PMID:28473820

Chimeric epitope vaccine against Leptospira interrogans infection and induced specific immunity in guinea pigs.

PubMed

Lin, Xu'ai; Xiao, Guohui; Luo, Dongjiao; Kong, Liangliang; Chen, Xu; Sun, Dexter; Yan, Jie

2016-10-14

Leptospirosis is an important reemerging zoonosis, with more than half a million cases reported annually, and is caused by pathogenic Leptospira species. Development of a universal vaccine is one of the major strategic goals to overcome the disease burden of leptospirosis. In this study, a chimeric multi-epitope protein-based vaccine was designed and tested for its potency to induce a specific immune response and provide protection against L. interrogans infection. The protein, containing four repeats of six T- and B-cell combined epitopes from the leptospiral outer membrane proteins, OmpL1, LipL32 and LipL21, was expressed and purified. Western blot analysis showed that the recombinant protein (named r4R) mainly expressed in a soluble pattern, and reacted with antibodies raised in rabbit against heat-killed Leptospira and in guinea pigs against the r4R vaccine. Microscopic agglutination tests showed that r4R antisera was immunological cross-reactive with a range of Chinese standard reference strains of Leptospira belonging to different serogroups. In guinea pigs, the r4R vaccine induced a Th1-biased immune response, as reflected by the IgG2a/IgG1 ratio and cytokine production of stimulated splenocytes derived from immunized animals. Finally, r4R-immunized guinea pigs showed increased survival of lethal Leptospira challenges compared with PBS-immunized animals and tissue damage and leptospiral colonization of the kidney were reduced. The multi-epitope chimeric r4R protein is a promising antigen for the development of a universal cross-reactive vaccine against leptospirosis.

Differential miRNA Expression in the Liver of Balb/c Mice Protected by Vaccination during Crisis of Plasmodium chabaudi Blood-Stage Malaria

PubMed Central

Dkhil, Mohamed A.; Al-Quraishy, Saleh A.; Abdel-Baki, Abdel-Azeem S.; Delic, Denis; Wunderlich, Frank

2017-01-01

MicroRNAs are increasingly recognized as epigenetic regulators for outcome of diverse infectious diseases and vaccination efficacy, but little information referring to this exists for malaria. This study investigates possible effects of both protective vaccination and P. chabaudi malaria on the miRNome of the liver as an effector against blood-stage malaria using miRNA microarrays and quantitative PCR. Plasmodium chabaudi blood-stage malaria takes a lethal outcome in female Balb/c mice, but a self-healing course after immunization with a non-infectious blood-stage vaccine. The liver robustly expresses 71 miRNA species at varying levels, among which 65 miRNA species respond to malaria evidenced as steadily increasing or decreasing expressions reaching highest or lowest levels toward the end of the crisis phase on day 11 p.i. in lethal malaria. Protective vaccination does not affect constitutive miRNA expression, but leads to significant (p < 0.05) changes in the expression of 41 miRNA species, however evidenced only during crisis. In vaccination-induced self-healing infections, 18 miRNA-species are up- and 14 miRNA-species are down-regulated by more than 50% during crisis in relation to non-vaccinated mice. Vaccination-induced self-healing and survival of otherwise lethal infections of P. chabaudi activate epigenetic miRNA-regulated remodeling processes in the liver manifesting themselves during crisis. Especially, liver regeneration is accelerated as suggested by upregulation of let-7a-5p, let-7b-5p, let-7c-5p, let-7d-5p, let-7f-5p, let-7g-5p, let-7i-5p, miR-26a, miR-122-5p, miR30a, miR27a, and mir-29a, whereas the up-regulated expression of miR-142-3p by more than 100% is compatible with the view of enhanced hepatic erythropoiesis, possibly at expense of megakaryopoiesis, during crisis of P. chabaudi blood-stage malaria. PMID:28123381

Targeting the Intratumoral Dendritic Cells by the Oncolytic Adenoviral Vaccine Expressing RANTES Elicits Potent Antitumor Immunity

PubMed Central

Lapteva, Natalia; Aldrich, Melissa; Weksberg, David; Rollins, Lisa; Goltsova, Tatiana; Chen, Si-Yi; Huang, Xue F.

2014-01-01

Summary Dendritic cells (DCs) are professional antigen (Ag)-presenting cells capable of inducing immune responses to tumor Ags and, therefore, play a central role in the induction of antitumor immunity. There is a large amount of evidence, however, about paucity of tumor-associated DCs and that DCs’ immunogenic functions are suppressed in a tumor environment. Here we describe a potent in situ vaccine targeting tumoral DCs in vivo. This vaccine comprised of an oncolytic adenovirus expressing RANTES (regulated upon activation, normally T expressed, and presumably secreted) (Ad-RANTES-E1A), enhanced tumor infiltration, and maturation of Ag-presenting cells in vivo. In this study, we show that intratumoral vaccinations with Ad-RANTES-E1A induced significant primary tumor growth regression and blocked metastasis formation in JC and E.G-7 murine tumor models. This vaccine recruited DCs, macrophages, natural killer cells, and CD8+ T cells to the tumor site, and thus enhanced Ag-specific cytotoxic T lymphocyte responses and natural killer cell responses. DCs purified from the Ad-RANTES-E1A–treated E.G-7 tumors secreted significantly higher levels of interferon-γ and interleukin-12, as compared with control groups and more efficiently enhanced CD8+ T-cell response. This in situ immunization strategy could be a potent antitumor immunotherapy approach for aggressive established tumors. PMID:19238013

Vaccine protection of chickens against antigenically diverse H5 highly pathogenic avian influenza isolates with a live HVT vector vaccine expressing the influenza hemagglutinin gene derived from a clade 2.2 avian influenza virus.

PubMed

Kapczynski, Darrell R; Esaki, Motoyuki; Dorsey, Kristi M; Jiang, Haijun; Jackwood, Mark; Moraes, Mauro; Gardin, Yannick

2015-02-25

Vaccination is an important tool in the protection of poultry against avian influenza (AI). For field use, the overwhelming majority of AI vaccines produced are inactivated whole virus formulated into an oil emulsion. However, recombinant vectored vaccines are gaining use for their ability to induce protection against heterologous isolates and ability to overcome maternal antibody interference. In these studies, we compared protection of chickens provided by a turkey herpesvirus (HVT) vector vaccine expressing the hemagglutinin (HA) gene from a clade 2.2 H5N1 strain (A/swan/Hungary/4999/2006) against homologous H5N1 as well as heterologous H5N1 and H5N2 highly pathogenic (HP) AI challenge. The results demonstrated all vaccinated birds were protected from clinical signs of disease and mortality following homologous challenge. In addition, oral and cloacal swabs taken from challenged birds demonstrated that vaccinated birds had lower incidence and titers of viral shedding compared to sham-vaccinated birds. Following heterologous H5N1 or H5N2 HPAI challenge, 80-95% of birds receiving the HVT vector AI vaccine at day of age survived challenge with fewer birds shedding virus after challenge than sham vaccinated birds. In vitro cytotoxicity analysis demonstrated that splenic T lymphocytes from HVT-vector-AI vaccinated chickens recognized MHC-matched target cells infected with H5, as well as H6, H7, or H9 AI virus. Taken together, these studies provide support for the use of HVT vector vaccines expressing HA to protect poultry against multiple lineages of HPAI, and that both humoral and cellular immunity induced by live vaccines likely contributes to protection. Published by Elsevier Ltd.

Topical herpes simplex virus 2 (HSV-2) vaccination with human papillomavirus vectors expressing gB/gD ectodomains induces genital-tissue-resident memory CD8+ T cells and reduces genital disease and viral shedding after HSV-2 challenge.

PubMed

Çuburu, Nicolas; Wang, Kening; Goodman, Kyle N; Pang, Yuk Ying; Thompson, Cynthia D; Lowy, Douglas R; Cohen, Jeffrey I; Schiller, John T

2015-01-01

No herpes simplex virus 2 (HSV-2) vaccine has been licensed for use in humans. HSV-2 glycoproteins B (gB) and D (gD) are targets of neutralizing antibodies and T cells, but clinical trials involving intramuscular (i.m.) injection of HSV-2 gB and gD in adjuvants have not been effective. Here we evaluated intravaginal (ivag) genetic immunization of C57BL/6 mice with a replication-defective human papillomavirus pseudovirus (HPV PsV) expressing HSV-2 gB (HPV-gB) or gD (HPV-gD) constructs to target different subcellular compartments. HPV PsV expressing a secreted ectodomain of gB (gBsec) or gD (gDsec), but not PsV expressing a cytoplasmic or membrane-bound form, induced circulating and intravaginal-tissue-resident memory CD8(+) T cells that were able to secrete gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) as well as moderate levels of serum HSV neutralizing antibodies. Combined immunization with HPV-gBsec and HPV-gDsec (HPV-gBsec/gDsec) vaccines conferred longer survival after vaginal challenge with HSV-2 than immunization with HPV-gBsec or HPV-gDsec alone. HPV-gBsec/gDsec ivag vaccination was associated with a reduced severity of genital lesions and lower levels of viral shedding in the genital tract after HSV-2 challenge. In contrast, intramuscular vaccination with a soluble truncated gD protein (gD2t) in alum and monophosphoryl lipid A (MPL) elicited high neutralizing antibody titers and improved survival but did not reduce genital lesions and viral shedding. Vaccination combining ivag HPV-gBsec/gDsec and i.m. gD2t-alum-MPL improved survival and reduced genital lesions and viral shedding. Finally, high levels of circulating HSV-2-specific CD8(+) T cells, but not serum antibodies, correlated with reduced viral shedding. Taken together, our data underscore the potential of HPV PsV as a platform for a topical mucosal vaccine to control local manifestations of primary HSV-2 infection. Genital herpes is a highly prevalent chronic disease caused by

Topical Herpes Simplex Virus 2 (HSV-2) Vaccination with Human Papillomavirus Vectors Expressing gB/gD Ectodomains Induces Genital-Tissue-Resident Memory CD8+ T Cells and Reduces Genital Disease and Viral Shedding after HSV-2 Challenge

PubMed Central

Çuburu, Nicolas; Wang, Kening; Goodman, Kyle N.; Pang, Yuk Ying; Thompson, Cynthia D.; Lowy, Douglas R.; Cohen, Jeffrey I.

2014-01-01

ABSTRACT No herpes simplex virus 2 (HSV-2) vaccine has been licensed for use in humans. HSV-2 glycoproteins B (gB) and D (gD) are targets of neutralizing antibodies and T cells, but clinical trials involving intramuscular (i.m.) injection of HSV-2 gB and gD in adjuvants have not been effective. Here we evaluated intravaginal (ivag) genetic immunization of C57BL/6 mice with a replication-defective human papillomavirus pseudovirus (HPV PsV) expressing HSV-2 gB (HPV-gB) or gD (HPV-gD) constructs to target different subcellular compartments. HPV PsV expressing a secreted ectodomain of gB (gBsec) or gD (gDsec), but not PsV expressing a cytoplasmic or membrane-bound form, induced circulating and intravaginal-tissue-resident memory CD8+ T cells that were able to secrete gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) as well as moderate levels of serum HSV neutralizing antibodies. Combined immunization with HPV-gBsec and HPV-gDsec (HPV-gBsec/gDsec) vaccines conferred longer survival after vaginal challenge with HSV-2 than immunization with HPV-gBsec or HPV-gDsec alone. HPV-gBsec/gDsec ivag vaccination was associated with a reduced severity of genital lesions and lower levels of viral shedding in the genital tract after HSV-2 challenge. In contrast, intramuscular vaccination with a soluble truncated gD protein (gD2t) in alum and monophosphoryl lipid A (MPL) elicited high neutralizing antibody titers and improved survival but did not reduce genital lesions and viral shedding. Vaccination combining ivag HPV-gBsec/gDsec and i.m. gD2t-alum-MPL improved survival and reduced genital lesions and viral shedding. Finally, high levels of circulating HSV-2-specific CD8+ T cells, but not serum antibodies, correlated with reduced viral shedding. Taken together, our data underscore the potential of HPV PsV as a platform for a topical mucosal vaccine to control local manifestations of primary HSV-2 infection. IMPORTANCE Genital herpes is a highly prevalent chronic

Monocytes Phenotype and Cytokine Production in Human Immunodeficiency Virus-1 Infected Patients Receiving a Modified Vaccinia Ankara-Based HIV-1 Vaccine: Relationship to CD300 Molecules Expression.

PubMed

Vitallé, Joana; Zenarruzabeitia, Olatz; Terrén, Iñigo; Plana, Montserrat; Guardo, Alberto C; Leal, Lorna; Peña, José; García, Felipe; Borrego, Francisco

2017-01-01

A modified vaccinia Ankara-based HIV-1 vaccine clade B (MVA-B) has been tested for safety and immunogenicity in low-risk human immunodeficiency virus (HIV)-uninfected individuals and as a therapeutic vaccine in HIV-1-infected individuals on combined antiretroviral therapy (cART). As a therapeutic vaccine, MVA-B was safe and broadly immunogenic; however, patients still showed a viral rebound upon treatment interruption. Monocytes are an important part of the viral reservoir and several studies suggest that they are partly responsible for the chronic inflammation observed in cART-treated HIV-infected people. The CD300 family of receptors has an important role in several diseases, including viral infections. Monocytes express CD300a, c, e, and f molecules and lipopolysaccharide (LPS) and other stimuli regulate their expression. However, the expression and function of CD300 receptors on monocytes in HIV infection is still unknown. In this work, we investigated for the first time the expression of CD300 molecules and the cytokine production in response to LPS on monocytes from HIV-1-infected patients before and after vaccination with MVA-B. Our results showed that CD300 receptors expression on monocytes from HIV-1-infected patients correlates with markers of HIV infection progression and immune inflammation. Specifically, we observed a positive correlation between the expression of CD300e and CD300f receptors on monocytes with the number of CD4+ T cells of HIV-1-infected patients before vaccination. We also saw a positive correlation between the expression of the inhibitory receptor CD300f and the expression of CD163 on monocytes from HIV-1-infected individuals before and after vaccination. In addition, monocytes exhibited a higher cytokine production in response to LPS after vaccination, almost at the same levels of monocytes from healthy donors. Furthermore, we also described a correlation in the expression of CD300e and CD300f receptors with TNF-α production in

Blood-stage malaria of Plasmodium chabaudi induces differential Tlr expression in the liver of susceptible and vaccination-protected Balb/c mice.

PubMed

Al-Quraishy, Saleh; Dkhil, Mohamed A; Alomar, Suliman; Abdel-Baki, Abdel Azeem S; Delic, Denis; Wunderlich, Frank; Araúzo-Bravo, Marcos J

2016-05-01

Protective vaccination induces self-healing of otherwise lethal blood-stage infections of Plasmodium chabaudi malaria. Here, we investigate mRNA expression patterns of all 12 members of the Toll-like receptor (Tlr) gene family in the liver, a major effector organ against blood-stage malaria, during lethal and vaccination-induced self-healing infections of P. chabaudi in female Balb/c mice. Gene expression microarrays reveal that all 12 Tlr genes are constitutively expressed, though at varying levels, and specifically respond to infection. Protective vaccination does not affect constitutive expression of any of the 12 Tlr genes but leads to differential expression (p < 0.05) of seven Tlrs (1, 2, 4, 7, 8, 12, and 13) in response to malaria. Quantitative PCR substantiates differential expression at p < 0.01. There is an increased expression of Tlr2 by approximately five-fold on day 1 post-infection (p.i.) and Tlr1 by approximately threefold on day 4 p.i.. At peak parasitemia on day 8 p.i., none of the 12 Tlrs display any differential expression. After peak parasitemia, towards the end of the crisis phase on day 11 p.i., expression of Tlrs 1, 4, and 12 is increased by approximately four-, two-, and three-fold, respectively, and that of Tlr7 is decreased by approximately two-fold. Collectively, our data suggest that though all 12 members of the Tlr gene family are specifically responsive to malaria in the liver, not only Tlr2 at the early stage of infection but also the Tlrs 1, 4, 7, and 12 towards the end of crisis phase are critical for vaccination-induced resolution and survival of otherwise lethal blood-stage malaria.

Magnolol Nanoparticles Exhibit Improved Water Solubility and Suppress TNF-α-Induced VCAM-1 Expression in Endothelial Cells.

PubMed

Lee, Chiang-Wen; Hu, Stephen Chu-Sung; Yen, Feng-Lin; Hsu, Lee-Fen; Lee, I-Ta; Lin, Zih-Chan; Tsai, Ming-Horng; Huang, Chieh-Liang; Liang, Chan-Jung; Chiang, Yao-Chang

2017-03-01

The expression of the adhesion molecule vascular cell adhesion molecule-1 (VCAM-1) on endothelial cells enables the attachment of leukocytes to the endothelium, which may lead to inflammation and the development of atherosclerosis. Magnolol is a major bioactive compound derived from the plant species Magnolia officinalis. In this study, we synthesized a novel nanoparticle formulation of magnolol to improve its water solubility and physicochemical properties, evaluated its effects on TNF-α-induced VCAM-1 expression in endothelial cells, and determined the signal transduction pathways involved. Our findings demonstrated that the magnolol nanoparticle system showed great improvements in physicochemical properties and water solubility owing to a reduction in particle size, transformation from a crystalline to amorphous structure, and the formation of hydrogen bonds with the nanoparticle carriers. In terms of its biological actions, magnolol nanoparticles attenuated TNF-α-induced VCAM-1 protein expression, promoter activity, and mRNA expression in endothelial cells in vitro. This was found to be mediated by the ERK, AKT, and NF-κB signaling pathways. In addition, magnolol nanoparticles inhibited TNF-α-induced leukocyte adhesion to endothelial cells, and suppressed TNF-α-induced VCAM-1 expression in the aortic endothelium of mice. In summary, since magnolol nanoparticles inhibit endothelial VCAM-1 expression and leukocyte adhesion to endothelial cells, this novel drug formulation may be a potentially useful therapeutic formulation to prevent the development of atherosclerosis and inflammatory diseases.

Recombinant Measles AIK-C Vaccine Strain Expressing the prM-E Antigen of Japanese Encephalitis Virus.

PubMed

Higuchi, Akira; Toriniwa, Hiroko; Komiya, Tomoyoshi; Nakayama, Tetsuo

2016-01-01

An inactivated Japanese encephalitis virus (JEV) vaccine, which induces neutralizing antibodies, has been used for many years in Japan. In the present study, the JEV prM-E protein gene was cloned, inserted at the P/M junction of measles AIK-C cDNA, and an infectious virus was recovered. The JEV E protein was expressed in B95a cells infected with the recombinant virus. Cotton rats were inoculated with recombinant virus. Measles PA antibodies were detected three weeks after immunization. Neutralizing antibodies against JEV developed one week after inoculation, and EIA antibodies were detected three weeks after immunization. The measles AIK-C-based recombinant virus simultaneously induced measles and JEV immune responses, and may be a candidate for infant vaccines. Therefore, the present strategy of recombinant viruses based on a measles vaccine vector would be applicable to the platform for vaccine development.

The Web-Based DNA Vaccine Database DNAVaxDB and Its Usage for Rational DNA Vaccine Design.

PubMed

Racz, Rebecca; He, Yongqun

2016-01-01

A DNA vaccine is a vaccine that uses a mammalian expression vector to express one or more protein antigens and is administered in vivo to induce an adaptive immune response. Since the 1990s, a significant amount of research has been performed on DNA vaccines and the mechanisms behind them. To meet the needs of the DNA vaccine research community, we created DNAVaxDB ( http://www.violinet.org/dnavaxdb ), the first Web-based database and analysis resource of experimentally verified DNA vaccines. All the data in DNAVaxDB, which includes plasmids, antigens, vaccines, and sources, is manually curated and experimentally verified. This chapter goes over the detail of DNAVaxDB system and shows how the DNA vaccine database, combined with the Vaxign vaccine design tool, can be used for rational design of a DNA vaccine against a pathogen, such as Mycobacterium bovis.

Staphylococcus aureus Phenol-Soluble Modulins Impair Interleukin Expression in Bovine Mammary Epithelial Cells

PubMed Central

Deplanche, Martine; Alekseeva, Ludmila; Semenovskaya, Ksenia; Fu, Chih-Lung; Dessauge, Frederic; Finot, Laurence; Petzl, Wolfram; Zerbe, Holm; Le Loir, Yves; Rainard, Pascal; Smith, David G. E.; Germon, Pierre; Otto, Michael

2016-01-01

The role of the recently described interleukin-32 (IL-32) in Staphylococcus aureus-induced mastitis, an inflammation of the mammary gland, is unclear. We determined expression of IL-32, IL-6, and IL-8 in S. aureus- and Escherichia coli-infected bovine mammary gland epithelial cells. Using live bacteria, we found that in S. aureus-infected cells, induction of IL-6 and IL-8 expression was less pronounced than in E. coli-infected cells. Notably, IL-32 expression was decreased in S. aureus-infected cells, while it was increased in E. coli-infected cells. We identified the staphylococcal phenol-soluble modulin (PSM) peptides as key contributors to these effects, as IL-32, IL-6, and IL-8 expression by epithelial cells exposed to psm mutant strains was significantly increased compared to that in cells exposed to the isogenic S. aureus wild-type strain, indicating that PSMs inhibit the production of these interleukins. The use of genetically complemented strains confirmed this observation. Inasmuch as the decreased expression of IL-32, which is involved in dendritic cell maturation, impairs immune responses, our results support a PSM-dependent mechanism that allows for the development of chronic S. aureus-related mastitis. PMID:27001539

Differential miRNA expression in B cells is associated with inter-individual differences in humoral immune response to measles vaccination

PubMed Central

Haralambieva, Iana H.; Kennedy, Richard B.; Simon, Whitney L.; Goergen, Krista M.; Grill, Diane E.; Ovsyannikova, Inna G.

2018-01-01

Background MicroRNAs are important mediators of post-transcriptional regulation of gene expression through RNA degradation and translational repression, and are emerging biomarkers of immune system activation/response after vaccination. Methods We performed Next Generation Sequencing (mRNA-Seq) of intracellular miRNAs in measles virus-stimulated B and CD4+ T cells from high and low antibody responders to measles vaccine. Negative binomial generalized estimating equation (GEE) models were used for miRNA assessment and the DIANA tool was used for gene/target prediction and pathway enrichment analysis. Results We identified a set of B cell-specific miRNAs (e.g., miR-151a-5p, miR-223, miR-29, miR-15a-5p, miR-199a-3p, miR-103a, and miR-15a/16 cluster) and biological processes/pathways, including regulation of adherens junction proteins, Fc-receptor signaling pathway, phosphatidylinositol-mediated signaling pathway, growth factor signaling pathway/pathways, transcriptional regulation, apoptosis and virus-related processes, significantly associated with neutralizing antibody titers after measles vaccination. No CD4+ T cell-specific miRNA expression differences between high and low antibody responders were found. Conclusion Our study demonstrates that miRNA expression directly or indirectly influences humoral immunity to measles vaccination and suggests that B cell-specific miRNAs may serve as useful predictive biomarkers of vaccine humoral immune response. PMID:29381765

Simultaneously achieve soluble expression and biomimetic immobilization of Candida antarctica lipase B by introducing polyamine tags.

PubMed

Zhou, Xiaoxue; Han, Yu; Lv, Zheng; Tian, Xuemei; Li, Han; Xie, Panpan; Zheng, Liangyu

2017-05-10

Polyamine tags fused in Candida antarctica lipase B (CalB) can help achieve high soluble expression of CalB in E. coli and can directly mediate silicification, which leads to rapid formation of a CalB-silica particle complex through a one-step approach. After optimization experiments, the fused lipase CalB tagged with 6-histidine at the N terminal and 10-lysine at the C terminal (6His-CalB-10Lys) is effectively expressed with high solubility (0.1mg/mL) and specific activity (10.1U/mg), and easily cross-linked in silica particles with a high immobilization efficiency of 96.8% and activity recovery of 81.5%. The immobilized lipase 6His-CalB-10Lys exhibits excellent performance at broad temperature ranges, high thermal and storage stabilities, and superior reusability. Michaelis-Menten kinetics indicates that the affinity and enantioselectivity of the free and immobilized 6His-CalB-10Lys toward the substrate are better than that of commercial Novozym 435 in enantioselective resolution of (S)-N-(2-ethyl-6-methylphenyl) alanine ((S)-NEMPA). The strategies described in this paper are useful for the facile expression and construction of diverse enzyme systems with high efficiency and excellent recyclability. Copyright © 2017 Elsevier B.V. All rights reserved.

Development of a multi-epitope peptide vaccine inducing robust T cell responses against brucellosis using immunoinformatics based approaches.

PubMed

Saadi, Mahdiye; Karkhah, Ahmad; Nouri, Hamid Reza

2017-07-01

Current investigations have demonstrated that a multi-epitope peptide vaccine targeting multiple antigens could be considered as an ideal approach for prevention and treatment of brucellosis. According to the latest findings, the most effective immunogenic antigens of brucella to induce immune responses are included Omp31, BP26, BLS, DnaK and L7-L12. Therefore, in the present study, an in silico approach was used to design a novel multi-epitope vaccine to elicit a desirable immune response against brucellosis. First, five novel T-cell epitopes were selected from Omp31, BP26, BLS, DnaK and L7-L12 proteins using different servers. In addition, helper epitopes selected from Tetanus toxin fragment C (TTFrC) were applied to induce CD4+ helper T lymphocytes (HTLs) responses. Selected epitopes were fused together by GPGPG linkers to facilitate the immune processing and epitope presentation. Moreover, cholera toxin B (CTB) was linked to N terminal of vaccine construct as an adjuvant by using EAAAK linker. A multi-epitope vaccine was designed based on predicted epitopes which was 377 amino acid residues in length. Then, the physico-chemical properties, secondary and tertiary structures, stability, intrinsic protein disorder, solubility and allergenicity of this multi-epitope vaccine were assessed using immunoinformatics tools and servers. Based on obtained results, a soluble, and non-allergic protein with 40.59kDa molecular weight was constructed. Expasy ProtParam classified this chimeric protein as a stable protein and also 89.8% residues of constructed vaccine were located in favored regions of the Ramachandran plot. Furthermore, this multi-epitope peptide vaccine was able to strongly induce T cell and B-cell mediated immune responses. In conclusion, immunoinformatics analysis indicated that this multi-epitope peptide vaccine can be effectively expressed and potentially be used for prophylactic or therapeutic usages against brucellosis. Copyright © 2017 Elsevier B.V. All

Membrane and envelope virus proteins co-expressed as lysosome associated membrane protein (LAMP) fused antigens: a potential tool to develop DNA vaccines against flaviviruses.

PubMed

Dhalia, Rafael; Maciel, Milton; Cruz, Fábia S P; Viana, Isabelle F T; Palma, Mariana L; August, Thomas; Marques, Ernesto T A

2009-12-01

Vaccination is the most practical and cost-effective strategy to prevent the majority of the flavivirus infection to which there is an available vaccine. However, vaccines based on attenuated virus can potentially promote collateral side effects and even rare fatal reactions. Given this scenario, the development of alternative vaccination strategies such as DNA-based vaccines encoding specific flavivirus sequences are being considered. Endogenous cytoplasmic antigens, characteristically plasmid DNA-vaccine encoded, are mainly presented to the immune system through Major Histocompatibility Complex class I - MHC I molecules. The MHC I presentation via is mostly associated with a cellular cytotoxic response and often do not elicit a satisfactory humoral response. One of the main strategies to target DNA-encoded antigens to the MHC II compartment is expressing the antigen within the Lysosome-Associated Membrane Protein (LAMP). The flavivirus envelope protein is recognized as the major virus surface protein and the main target for neutralizing antibodies. Different groups have demonstrated that co-expression of flavivirus membrane and envelope proteins in mammalian cells, fused with the carboxyl-terminal of LAMP, is able to induce satisfactory levels of neutralizing antibodies. Here we reviewed the use of the envelope flavivirus protein co-expression strategy as LAMP chimeras with the aim of developing DNA vaccines for dengue, West Nile and yellow fever viruses.

High-dimensional gene expression profiling studies in high and low responders to primary smallpox vaccination.

PubMed

Haralambieva, Iana H; Oberg, Ann L; Dhiman, Neelam; Ovsyannikova, Inna G; Kennedy, Richard B; Grill, Diane E; Jacobson, Robert M; Poland, Gregory A

2012-11-15

The mechanisms underlying smallpox vaccine-induced variations in immune responses are not well understood, but are of considerable interest to a deeper understanding of poxvirus immunity and correlates of protection. We assessed transcriptional messenger RNA expression changes in 197 recipients of primary smallpox vaccination representing the extremes of humoral and cellular immune responses. The 20 most significant differentially expressed genes include a tumor necrosis factor-receptor superfamily member, an interferon (IFN) gene, a chemokine gene, zinc finger protein genes, nuclear factors, and histones (P ≤ 1.06E(-20), q ≤ 2.64E(-17)). A pathway analysis identified 4 enriched pathways with cytokine production by the T-helper 17 subset of CD4+ T cells being the most significant pathway (P = 3.42E(-05)). Two pathways (antiviral actions of IFNs, P = 8.95E(-05); and IFN-α/β signaling pathway, P = 2.92E(-04)), integral to innate immunity, were enriched when comparing high with low antibody responders (false discovery rate, < 0.05). Genes related to immune function and transcription (TLR8, P = .0002; DAPP1, P = .0003; LAMP3, P = 9.96E(-05); NR4A2, P ≤ .0002; EGR3, P = 4.52E(-05)), and other genes with a possible impact on immunity (LNPEP, P = 3.72E(-05); CAPRIN1, P = .0001; XRN1, P = .0001), were found to be expressed differentially in high versus low antibody responders. We identified novel and known immunity-related genes and pathways that may account for differences in immune response to smallpox vaccination.

Oral administration of a Salmonella enterica-based vaccine expressing Bacillus anthracis protective antigen confers protection against aerosolized B. anthracis.

PubMed

Stokes, Margaret G M; Titball, Richard W; Neeson, Brendan N; Galen, James E; Walker, Nicola J; Stagg, Anthony J; Jenner, Dominic C; Thwaite, Joanne E; Nataro, James P; Baillie, Leslie W J; Atkins, Helen S

2007-04-01

Bacillus anthracis is the causative agent of anthrax, a disease that affects wildlife, livestock, and humans. Protection against anthrax is primarily afforded by immunity to the B. anthracis protective antigen (PA), particularly PA domains 4 and 1. To further the development of an orally delivered human vaccine for mass vaccination against anthrax, we produced Salmonella enterica serovar Typhimurium expressing full-length PA, PA domains 1 and 4, or PA domain 4 using codon-optimized PA DNA fused to the S. enterica serovar Typhi ClyA and under the control of the ompC promoter. Oral immunization of A/J mice with Salmonella expressing full-length PA protected five of six mice against a challenge with 10(5) CFU of aerosolized B. anthracis STI spores, whereas Salmonella expressing PA domains 1 and 4 provided only 25% protection (two of eight mice), and Salmonella expressing PA domain 4 or a Salmonella-only control afforded no measurable protection. However, a purified recombinant fusion protein of domains 1 and 4 provided 100% protection, and purified recombinant 4 provided protection in three of eight immunized mice. Thus, we demonstrate for the first time the efficacy of an oral S. enterica-based vaccine against aerosolized B. anthracis spores.

A Novel Vaccine Delivery Model of the Apicomplexan Eimeria tenella Expressing Eimeria maxima Antigen Protects Chickens against Infection of the Two Parasites

PubMed Central

Tang, Xinming; Liu, Xianyong; Yin, Guangwen; Suo, Jingxia; Tao, Geru; Zhang, Sixin; Suo, Xun

2018-01-01

Vaccine delivery is critical in antigen discovery and vaccine efficacy and safety. The diversity of infectious diseases in humans and livestock has required the development of varied delivery vehicles to target different pathogens. In livestock animals, previous strategies for the development of coccidiosis vaccines have encountered several hurdles, limiting the development of multiple species vaccine formulations. Here, we describe a novel vaccine delivery system using transgenic Eimeria tenella expressing immunodominant antigens of Eimeria maxima. In this delivery system, the immune mapped protein 1 of E. maxima (EmIMP1) was delivered by the closely related species of E. tenella to the host immune system during the whole endogenous life cycle. The overexpression of the exogenous antigen did not interfere with the reproduction and immunogenicity of transgenic Eimeria. After immunization with the transgenic parasite, we detected EmIMP1’s and E. maxima oocyst antigens’ specific humoral and cellular immune responses. In particular, we observed partial protection of chickens immunized with transgenic E. tenella against subsequent E. maxima infections. Our results demonstrate that the transgenic Eimeria parasite is an ideal coccidia antigen delivery vehicle and represents a new type of coccidiosis vaccines. In addition, this model could potentially be used in the development of malaria live sporozoite vaccines, in which antigens from different strains can be expressed in the vaccine strain. PMID:29375584

A Novel Vaccine Delivery Model of the Apicomplexan Eimeria tenella Expressing Eimeria maxima Antigen Protects Chickens against Infection of the Two Parasites.

PubMed

Tang, Xinming; Liu, Xianyong; Yin, Guangwen; Suo, Jingxia; Tao, Geru; Zhang, Sixin; Suo, Xun

2017-01-01

Vaccine delivery is critical in antigen discovery and vaccine efficacy and safety. The diversity of infectious diseases in humans and livestock has required the development of varied delivery vehicles to target different pathogens. In livestock animals, previous strategies for the development of coccidiosis vaccines have encountered several hurdles, limiting the development of multiple species vaccine formulations. Here, we describe a novel vaccine delivery system using transgenic Eimeria tenella expressing immunodominant antigens of Eimeria maxima . In this delivery system, the immune mapped protein 1 of E. maxima (EmIMP1) was delivered by the closely related species of E. tenella to the host immune system during the whole endogenous life cycle. The overexpression of the exogenous antigen did not interfere with the reproduction and immunogenicity of transgenic Eimeria . After immunization with the transgenic parasite, we detected EmIMP1's and E. maxima oocyst antigens' specific humoral and cellular immune responses. In particular, we observed partial protection of chickens immunized with transgenic E. tenella against subsequent E. maxima infections. Our results demonstrate that the transgenic Eimeria parasite is an ideal coccidia antigen delivery vehicle and represents a new type of coccidiosis vaccines. In addition, this model could potentially be used in the development of malaria live sporozoite vaccines, in which antigens from different strains can be expressed in the vaccine strain.

Identifying gnostic predictors of the vaccine response.

PubMed

Haining, W Nicholas; Pulendran, Bali

2012-06-01

Molecular predictors of the response to vaccination could transform vaccine development. They would allow larger numbers of vaccine candidates to be rapidly screened, shortening the development time for new vaccines. Gene-expression based predictors of vaccine response have shown early promise. However, a limitation of gene-expression based predictors is that they often fail to reveal the mechanistic basis of their ability to classify response. Linking predictive signatures to the function of their component genes would advance basic understanding of vaccine immunity and also improve the robustness of vaccine prediction. New analytic tools now allow more biological meaning to be extracted from predictive signatures. Functional genomic approaches to perturb gene expression in mammalian cells permit the function of predictive genes to be surveyed in highly parallel experiments. The challenge for vaccinologists is therefore to use these tools to embed mechanistic insights into predictors of vaccine response. Copyright © 2012 Elsevier Ltd. All rights reserved.

Virus-Vectored Influenza Virus Vaccines

PubMed Central

Tripp, Ralph A.; Tompkins, S. Mark

2014-01-01

Despite the availability of an inactivated vaccine that has been licensed for >50 years, the influenza virus continues to cause morbidity and mortality worldwide. Constant evolution of circulating influenza virus strains and the emergence of new strains diminishes the effectiveness of annual vaccines that rely on a match with circulating influenza strains. Thus, there is a continued need for new, efficacious vaccines conferring cross-clade protection to avoid the need for biannual reformulation of seasonal influenza vaccines. Recombinant virus-vectored vaccines are an appealing alternative to classical inactivated vaccines because virus vectors enable native expression of influenza antigens, even from virulent influenza viruses, while expressed in the context of the vector that can improve immunogenicity. In addition, a vectored vaccine often enables delivery of the vaccine to sites of inductive immunity such as the respiratory tract enabling protection from influenza virus infection. Moreover, the ability to readily manipulate virus vectors to produce novel influenza vaccines may provide the quickest path toward a universal vaccine protecting against all influenza viruses. This review will discuss experimental virus-vectored vaccines for use in humans, comparing them to licensed vaccines and the hurdles faced for licensure of these next-generation influenza virus vaccines. PMID:25105278

Oral Delivery of Probiotics Expressing Dendritic Cell-Targeting Peptide Fused with Porcine Epidemic Diarrhea Virus COE Antigen: A Promising Vaccine Strategy against PEDV.

PubMed

Wang, Xiaona; Wang, Li; Huang, Xuewei; Ma, Sunting; Yu, Meiling; Shi, Wen; Qiao, Xinyuan; Tang, Lijie; Xu, Yigang; Li, Yijing

2017-10-25

Porcine epidemic diarrhea virus (PEDV), an enteric coronavirus, is the causative agent of porcine epidemic diarrhea (PED) that damages intestinal epithelial cells and results in severe diarrhea and dehydration in neonatal suckling pigs with up to 100% mortality. The oral vaccine route is reported as a promising approach for inducing protective immunity against PEDV invasion. Furthermore, dendritic cells (DCs), professional antigen-presenting cells, link humoral and cellular immune responses for homeostasis of the intestinal immune environment. In this study, in order to explore an efficient oral vaccine against PEDV infection, a mucosal DC-targeting oral vaccine was developed using Lactobacillus casei to deliver the DC-targeting peptide (DCpep) fused with the PEDV core neutralizing epitope (COE) antigen. This probiotic vaccine could efficiently elicit secretory immunoglobulin A (SIgA)-based mucosal and immunoglobulin G (IgG)-based humoral immune responses via oral vaccination in vivo. Significant differences ( p < 0.05) in the immune response levels were observed between probiotics expressing the COE-DCpep fusion protein and COE antigen alone, suggesting better immune efficiency of the probiotics vaccine expressing the DC-targeting peptide fused with PEDV COE antigen. This mucosal DC-targeting oral vaccine delivery effectively enhances vaccine antigen delivery efficiency, providing a useful strategy to induce efficient immune responses against PEDV infection.

Use of a Recombinant Gamma-2 Herpesvirus Vaccine Vector against Dengue Virus in Rhesus Monkeys.

PubMed

Bischof, Georg F; Magnani, Diogo M; Ricciardi, Michael; Shin, Young C; Domingues, Aline; Bailey, Varian K; Gonzalez-Nieto, Lucas; Rakasz, Eva G; Watkins, David I; Desrosiers, Ronald C

2017-08-15

Research on vaccine approaches that can provide long-term protection against dengue virus infection is needed. Here we describe the construction, immunogenicity, and preliminary information on the protective capacity of recombinant, replication-competent rhesus monkey rhadinovirus (RRV), a persisting herpesvirus. One RRV construct expressed nonstructural protein 5 (NS5), while a second recombinant expressed a soluble variant of the E protein (E85) of dengue virus 2 (DENV2). Four rhesus macaques received a single vaccination with a mixture of both recombinant RRVs and were subsequently challenged 19 weeks later with 1 × 10 5 PFU of DENV2. During the vaccine phase, plasma of all vaccinated monkeys showed neutralizing activity against DENV2. Cellular immune responses against NS5 were also elicited, as evidenced by major histocompatibility complex class I (MHC-I) tetramer staining in the one vaccinated monkey that was Mamu-A*01 positive. Unlike two of two unvaccinated controls, two of the four vaccinated monkeys showed no detectable viral RNA sequences in plasma after challenge. One of these two monkeys also showed no anamnestic increases in antibody levels following challenge and thus appeared to be protected against the acquisition of DENV2 following high-dose challenge. Continued study will be needed to evaluate the performance of herpesviral and other persisting vectors for achieving long-term protection against dengue virus infection. IMPORTANCE Continuing studies of vaccine approaches against dengue virus (DENV) infection are warranted, particularly ones that may provide long-term immunity against all four serotypes. Here we investigated whether recombinant rhesus monkey rhadinovirus (RRV) could be used as a vaccine against DENV2 infection in rhesus monkeys. Upon vaccination, all animals generated antibodies capable of neutralizing DENV2. Two of four vaccinated monkeys showed no detectable viral RNA after subsequent high-dose DENV2 challenge at 19 weeks

Live attenuated measles vaccine expressing HIV-1 Gag virus like particles covered with gp160DELTAV1V2 is strongly immunogenic

DOE Office of Scientific and Technical Information (OSTI.GOV)

Guerbois, Mathilde; Moris, Arnaud; Combredet, Chantal

Although a live attenuated HIV vaccine is not currently considered for safety reasons, a strategy inducing both T cells and neutralizing antibodies to native assembled HIV-1 particles expressed by a replicating virus might mimic the advantageous characteristics of live attenuated vaccine. To this aim, we generated a live attenuated recombinant measles vaccine expressing HIV-1 Gag virus-like particles (VLPs) covered with gp160DELTAV1V2 Env protein. The measles-HIV virus replicated efficiently in cell culture and induced the intense budding of HIV particles covered with Env. In mice sensitive to MV infection, this recombinant vaccine stimulated high levels of cellular and humoral immunity tomore » both MV and HIV with neutralizing activity. The measles-HIV virus infected human professional antigen-presenting cells, such as dendritic cells and B cells, and induced efficient presentation of HIV-1 epitopes and subsequent activation of human HIV-1 Gag-specific T cell clones. This candidate vaccine will be next tested in non-human primates. As a pediatric vaccine, it might protect children and adolescents simultaneously from measles and HIV.« less

Live attenuated rubella vectors expressing SIV and HIV vaccine antigens replicate and elicit durable immune responses in rhesus macaques

PubMed Central

2013-01-01

Background Live attenuated viruses are among our most potent and effective vaccines. For human immunodeficiency virus, however, a live attenuated strain could present substantial safety concerns. We have used the live attenuated rubella vaccine strain RA27/3 as a vector to express SIV and HIV vaccine antigens because its safety and immunogenicity have been demonstrated in millions of children. One dose protects for life against rubella infection. In previous studies, rubella vectors replicated to high titers in cell culture while stably expressing SIV and HIV antigens. Their viability in vivo, however, as well as immunogenicity and antibody persistence, were unknown. Results This paper reports the first successful trial of rubella vectors in rhesus macaques, in combination with DNA vaccines in a prime and boost strategy. The vectors grew robustly in vivo, and the protein inserts were highly immunogenic. Antibody titers elicited by the SIV Gag vector were greater than or equal to those elicited by natural SIV infection. The antibodies were long lasting, and they were boosted by a second dose of replication-competent rubella vectors given six months later, indicating the induction of memory B cells. Conclusions Rubella vectors can serve as a vaccine platform for safe delivery and expression of SIV and HIV antigens. By presenting these antigens in the context of an acute infection, at a high level and for a prolonged duration, these vectors can stimulate a strong and persistent immune response, including maturation of memory B cells. Rhesus macaques will provide an ideal animal model for demonstrating immunogenicity of novel vectors and protection against SIV or SHIV challenge. PMID:24041113

Identifying gnostic predictors of the vaccine response

PubMed Central

Haining, W. Nicholas; Pulendran, Bali

2012-01-01

Molecular predictors of the response to vaccination could transform vaccine development. They would allow larger numbers of vaccine candidates to be rapidly screened, shortening the development time for new vaccines. Gene-expression based predictors of vaccine response have shown early promise. However, a limitation of gene-expression based predictors is that they often fail to reveal the mechanistic basis for their ability to classify response. Linking predictive signatures to the function of their component genes would advance basic understanding of vaccine immunity and also improve the robustness of outcome classification. New analytic tools now allow more biological meaning to be extracted from predictive signatures. Functional genomic approaches to perturb gene expression in mammalian cells permit the function of predictive genes to be surveyed in highly parallel experiments. The challenge for vaccinologists is therefore to use these tools to embed mechanistic insights into predictors of vaccine response. PMID:22633886

Protective Vaccination against Blood-Stage Malaria of Plasmodium chabaudi: Differential Gene Expression in the Liver of Balb/c Mice toward the End of Crisis Phase

PubMed Central

Al-Quraishy, Saleh A.; Dkhil, Mohamed A.; Abdel-Baki, Abdel-Azeem A.; Delic, Denis; Wunderlich, Frank

2016-01-01

Protective vaccination induces self-healing of otherwise fatal blood-stage malaria of Plasmodium chabaudi in female Balb/c mice. To trace processes critically involved in self-healing, the liver, an effector against blood-stage malaria, is analyzed for possible changes of its transcriptome in vaccination-protected in comparison to non-protected mice toward the end of the crisis phase. Gene expression microarray analyses reveal that vaccination does not affect constitutive expression of mRNA and lincRNA. However, malaria induces significant (p < 0.01) differences in hepatic gene and lincRNA expression in vaccination-protected vs. non-vaccinated mice toward the end of crisis phase. In vaccination-protected mice, infections induce up-regulations of 276 genes and 40 lincRNAs and down-regulations of 200 genes and 43 lincRNAs, respectively, by >3-fold as compared to the corresponding constitutive expressions. Massive up-regulations, partly by >100-fold, are found for genes as RhD, Add2, Ank1, Ermap, and Slc4a, which encode proteins of erythrocytic surface membranes, and as Gata1 and Gfi1b, which encode transcription factors involved in erythrocytic development. Also, Cldn13 previously predicted to be expressed on erythroblast surfaces is up-regulated by >200-fold, though claudins are known as main constituents of tight junctions acting as paracellular barriers between epithelial cells. Other genes are up-regulated by <100- and >10-fold, which can be subgrouped in genes encoding proteins known to be involved in mitosis, in cell cycle regulation, and in DNA repair. Our data suggest that protective vaccination enables the liver to respond to P. chabaudi infections with accelerated regeneration and extramedullary erythropoiesis during crisis, which contributes to survival of otherwise lethal blood-stage malaria. PMID:27471498

Quantitative expression profiling of immune response genes in rainbow trout following infectious haematopoietic necrosis virus (IHNV) infection or DNA vaccination

USGS Publications Warehouse

Purcell, Maureen K.; Kurath, Gael; Garver, Kyle A.; Herwig, Russell P.; Winton, James R.

2004-01-01

Infectious haematopoietic necrosis virus (IHNV) is a well-studied virus of salmonid fishes. A highly efficacious DNA vaccine has been developed against this virus and studies have demonstrated that this vaccine induces both an early and transient non-specific anti-viral phase as well as long-term specific protection. The mechanisms of the early anti-viral phase are not known, but previous studies noted changes in Mx gene expression, suggesting a role for type I interferon. This study used quantitative real-time reverse transcriptase PCR methodology to compare expression changes over time of a number of cytokine or cytokine-related genes in the spleen of rainbow trout following injection with poly I:C, live IHNV, the IHNV DNA vaccine or a control plasmid encoding the non-antigenic luciferase gene. The target genes included Mx-1, viral haemorrhagic septicaemia virus induced gene 8 (Vig-8), TNF-α1, TNF-α2, IL-1β1, IL-8, TGF-β1 and Hsp70. Poly I:C stimulation induced several genes but the strongest and significant response was observed in the Mx-1 and Vig-8 genes. The live IHN virus induced a significant response in all genes examined except TGF-β1. The control plasmid construct and the IHNV DNA vaccine marginally induced a number of genes, but the main difference between these two groups was a statistically significant induction of the Mx-1 and Vig-8 genes by the IHNV vaccine only. The gene expression profiles elicited by the live virus and the IHNV DNA vaccine differed in a number of aspects but this study confirms the clear role for a type I interferon-like response in early anti-viral defence.

Self-Amplifying mRNA Vaccines Expressing Multiple Conserved Influenza Antigens Confer Protection against Homologous and Heterosubtypic Viral Challenge

PubMed Central

Magini, Diletta; Giovani, Cinzia; Mangiavacchi, Simona; Maccari, Silvia; Cecchi, Raffaella; Ulmer, Jeffrey B.; De Gregorio, Ennio; Geall, Andrew J.; Brazzoli, Michela; Bertholet, Sylvie

2016-01-01

Current hemagglutinin (HA)-based seasonal influenza vaccines induce vaccine strain-specific neutralizing antibodies that usually fail to provide protection against mismatched circulating viruses. Inclusion in the vaccine of highly conserved internal proteins such as the nucleoprotein (NP) and the matrix protein 1 (M1) was shown previously to increase vaccine efficacy by eliciting cross-reactive T-cells. However, appropriate delivery systems are required for efficient priming of T-cell responses. In this study, we demonstrated that administration of novel self-amplifying mRNA (SAM®) vectors expressing influenza NP (SAM(NP)), M1 (SAM(M1)), and NP and M1 (SAM(M1-NP)) delivered with lipid nanoparticles (LNP) induced robust polyfunctional CD4 T helper 1 cells, while NP-containing SAM also induced cytotoxic CD8 T cells. Robust expansions of central memory (TCM) and effector memory (TEM) CD4 and CD8 T cells were also measured. An enhanced recruitment of NP-specific cytotoxic CD8 T cells was observed in the lungs of SAM(NP)-immunized mice after influenza infection that paralleled with reduced lung viral titers and pathology, and increased survival after homologous and heterosubtypic influenza challenge. Finally, we demonstrated for the first time that the co-administration of RNA (SAM(M1-NP)) and protein (monovalent inactivated influenza vaccine (MIIV)) was feasible, induced simultaneously NP-, M1- and HA-specific T cells and HA-specific neutralizing antibodies, and enhanced MIIV efficacy against a heterologous challenge. In conclusion, systemic administration of SAM vectors expressing conserved internal influenza antigens induced protective immune responses in mice, supporting the SAM® platform as another promising strategy for the development of broad-spectrum universal influenza vaccines. PMID:27525409

A Recombinant Raccoon Poxvirus Vaccine Expressing both Yersinia pestis F1 and Truncated V Antigens Protects Animals against Lethal Plague

PubMed Central

Rocke, Tonie E.; Kingstad-Bakke, Brock; Berlier, Willy; Osorio, Jorge E.

2014-01-01

In previous studies, we demonstrated in mice and prairie dogs that simultaneous administration of two recombinant raccoon poxviruses (rRCN) expressing Yersinia pestis antigens (F1 and V307—a truncated version of the V protein) provided superior protection against plague challenge compared to individual single antigen constructs. To reduce costs of vaccine production and facilitate implementation of a sylvatic plague vaccine (SPV) control program for prairie dogs, a dual antigen construct is more desirable. Here we report the construction and characterization of a novel RCN-vectored vaccine that simultaneously expresses both F1 and V307 antigens. This dual antigen vaccine provided similar levels of protection against plague in both mice and prairie dogs as compared to simultaneous administration of the two single antigen constructs and was also shown to protect mice against an F1 negative strain of Y. pestis. The equivalent safety, immunogenicity and efficacy profile of the dual RCN-F1/V307 construct warrants further evaluation in field efficacy studies in sylvatic plague endemic areas. PMID:26344891

A recombinant raccoon poxvirus vaccine expressing both Yersinia pestis F1 and truncated V antigens protects animals against lethal plague.

USGS Publications Warehouse

Rocke, Tonie E.; Kingstad-Bakke, B; Berlier, W; Osorio, J.E.

2014-01-01

In previous studies, we demonstrated in mice and prairie dogs that simultaneous administration of two recombinant raccoon poxviruses (rRCN) expressing Yersinia pestis antigens (F1 and V307-a truncated version of the V protein) provided superior protection against plague challenge compared to individual single antigen constructs. To reduce costs of vaccine production and facilitate implementation of a sylvatic plague vaccine (SPV) control program for prairie dogs, a dual antigen construct is more desirable. Here we report the construction and characterization of a novel RCN-vectored vaccine that simultaneously expresses both F1 and V307 antigens. This dual antigen vaccine provided similar levels of protection against plague in both mice and prairie dogs as compared to simultaneous administration of the two single antigen constructs and was also shown to protect mice against an F1 negative strain of Y. pestis.. The equivalent safety, immunogenicity and efficacy profile of the dual RCN-F1/V307 construct warrants further evaluation in field efficacy studies in sylvatic plague endemic areas.

Use of a current varicella vaccine as a live polyvalent vaccine vector.

PubMed

Murakami, Kouki; Mori, Yasuko

2016-01-04

Varicella-zoster virus (VZV) is the causative agent of varicella and zoster. The varicella vaccine was developed to control VZV infection in children. The currently available Oka vaccine strain is the only live varicella vaccine approved by the World Health Organization. We previously cloned the complete genome of the Oka vaccine strain into a bacterial artificial chromosome vector and then successfully reconstituted the virus. We then used this system to generate a recombinant Oka vaccine virus expressing mumps virus gene(s). The new recombinant vaccine may be an effective polyvalent live vaccine that provides protection against both varicella and mumps viruses. In this review, we discussed about possibility of polyvalent live vaccine(s) using varicella vaccine based on our recent studies. Copyright © 2014 Elsevier Ltd. All rights reserved.

Full-length soluble CD147 promotes MMP-2 expression and is a potential serological marker in detection of hepatocellular carcinoma.

PubMed

Wu, Jiao; Hao, Zhi-Wei; Zhao, You-Xu; Yang, Xiang-Min; Tang, Hao; Zhang, Xin; Song, Fei; Sun, Xiu-Xuan; Wang, Bin; Nan, Gang; Chen, Zhi-Nan; Bian, Huijie

2014-07-04

As a surface glycoprotein, CD147 is capable of stimulating the production of matrix metalloproteinases (MMPs) from neighboring fibroblasts. The aim of the present study is to explore the role of soluble CD147 on MMPs secretion from hepatocellular carcinoma (HCC) cells, and to investigate the diagnostic value of serum soluble CD147 in the HCC detection. We identified the form of soluble CD147 in cell culture supernate of HCC cells and serum of patients with HCC, and explored the role of soluble CD147 on MMPs secretion. Serum CD147 levels were detected by the enzyme-linked immunosorbent assay, and the value of soluble CD147 as a marker in HCC detection was analyzed. Full length soluble CD147 was presented in the culture medium of HCC cells and serum of patients with HCC. The extracellular domain of soluble CD147 promoted the expression of CD147 and MMP-2 from HCC cells. Knockdown of CD147 markedly diminished the up-regulation of CD147 and MMP-2 which induced by soluble CD147. Soluble CD147 activated ERK, FAK, and PI3K/Akt pathways, leading to the up-regulation of MMP-2. The level of soluble CD147 in serum of patients with HCC was significantly elevated compared with healthy individuals (P < 0.001). Soluble CD147 levels were found to be associated with HCC tumor size (P = 0.007) and Child-Pugh grade (P = 0.007). Moreover, soluble CD147 showed a better performance in distinguishing HCC compared with alpha-fetoprotein. The extracellular domain of soluble CD147 enhances the secretion of MMP-2 from HCC cells, requiring the cooperation of membrane CD147 and activation of ERK, FAK, and PI3K/Akt signaling. The measurement of soluble CD147 may offer a useful approach in diagnosis of HCC.

Vaccination for Disease

NASA Astrophysics Data System (ADS)

Oehen, Stephan; Hengartner, Hans; Zinkernagel, Rolf M.

1991-01-01

Recombinant virus vaccines that express a limited number of epitopes are currently being developed to prevent disease by changing the relative balance between viral spread and the immune response. Some circumstances, however, were found in infections with a noncytopathic virus in which vaccination caused disease; sensitive parameters included the genetic background of the host, the time or dose of infection, and the constituents of the vaccine. Thus, immunopathologic damage by T cells may be an unwanted consequence of vaccination with the new types of peptide or recombinant vaccines that are being investigated for the human immunodeficiency viruses and other pathogens.

System-Wide Associations between DNA-Methylation, Gene Expression, and Humoral Immune Response to Influenza Vaccination.

PubMed

Zimmermann, Michael T; Oberg, Ann L; Grill, Diane E; Ovsyannikova, Inna G; Haralambieva, Iana H; Kennedy, Richard B; Poland, Gregory A

2016-01-01

Failure to achieve a protected state after influenza vaccination is poorly understood but occurs commonly among aged populations experiencing greater immunosenescence. In order to better understand immune response in the elderly, we studied epigenetic and transcriptomic profiles and humoral immune response outcomes in 50-74 year old healthy participants. Associations between DNA methylation and gene expression reveal a system-wide regulation of immune-relevant functions, likely playing a role in regulating a participant's propensity to respond to vaccination. Our findings show that sites of methylation regulation associated with humoral response to vaccination impact known cellular differentiation signaling and antigen presentation pathways. We performed our analysis using per-site and regionally average methylation levels, in addition to continuous or dichotomized outcome measures. The genes and molecular functions implicated by each analysis were compared, highlighting different aspects of the biologic mechanisms of immune response affected by differential methylation. Both cis-acting (within the gene or promoter) and trans-acting (enhancers and transcription factor binding sites) sites show significant associations with measures of humoral immunity. Specifically, we identified a group of CpGs that, when coordinately hypo-methylated, are associated with lower humoral immune response, and methylated with higher response. Additionally, CpGs that individually predict humoral immune responses are enriched for polycomb-group and FOXP2 transcription factor binding sites. The most robust associations implicate differential methylation affecting gene expression levels of genes with known roles in immunity (e.g. HLA-B and HLA-DQB2) and immunosenescence. We believe our data and analysis strategy highlight new and interesting epigenetic trends affecting humoral response to vaccination against influenza; one of the most common and impactful viral pathogens.

Vaccination directed against the human endogenous retrovirus-K (HERV-K) gag protein slows HERV-K gag expressing cell growth in a murine model system.

PubMed

Kraus, Benjamin; Fischer, Katrin; Sliva, Katja; Schnierle, Barbara S

2014-03-26

Human endogenous retroviruses (HERVs) are remnants of ancestral infections and chromosomally integrated in all cells of an individual, are transmitted only vertically and are defective in viral replication. However enhanced expression of HERV-K accompanied by the emergence of anti-HERV-K-directed immune responses has been observed inter-alia in HIV-infected individuals and tumor patients. Therefore HERV-K might serve as a tumor-specific antigen or even as a constant target for the development of an HIV vaccine. To verify our hypothesis, we tested the immunogenicity of HERV-K Gag by using a recombinant vaccinia virus (MVA-HKcon) expressing the HERV-K Gag protein and established an animal model to test its vaccination efficacy. Murine renal carcinoma cells (Renca) were genetically altered to express E. coli beta-galactosidase (RLZ cells) and the HERV-K Gag protein (RLZ-HKGag cells). Subcutaneous application of RLZ-HKGag cells into syngenic BALB/c mice resulted in the formation of local tumors in MVA vaccinated mice. MVA-HKcon vaccination reduced the tumor growth. Furthermore, intravenous injection of RLZ-HKGag cells led to the formation of pulmonary metastases. Vaccination of tumor-bearing mice with MVA-HKcon drastically reduced the number of pulmonary RLZ-HKGag tumor nodules compared to vaccination with wild-type MVA. The data demonstrate that HERV-K Gag is a useful target for vaccine development and might offer new treatment opportunities for cancer patients.

Staphylococcus aureus Phenol-Soluble Modulins Impair Interleukin Expression in Bovine Mammary Epithelial Cells.

PubMed

Deplanche, Martine; Alekseeva, Ludmila; Semenovskaya, Ksenia; Fu, Chih-Lung; Dessauge, Frederic; Finot, Laurence; Petzl, Wolfram; Zerbe, Holm; Le Loir, Yves; Rainard, Pascal; Smith, David G E; Germon, Pierre; Otto, Michael; Berkova, Nadia

2016-06-01

The role of the recently described interleukin-32 (IL-32) in Staphylococcus aureus-induced mastitis, an inflammation of the mammary gland, is unclear. We determined expression of IL-32, IL-6, and IL-8 in S. aureus- and Escherichia coli-infected bovine mammary gland epithelial cells. Using live bacteria, we found that in S. aureus-infected cells, induction of IL-6 and IL-8 expression was less pronounced than in E. coli-infected cells. Notably, IL-32 expression was decreased in S. aureus-infected cells, while it was increased in E. coli-infected cells. We identified the staphylococcal phenol-soluble modulin (PSM) peptides as key contributors to these effects, as IL-32, IL-6, and IL-8 expression by epithelial cells exposed to psm mutant strains was significantly increased compared to that in cells exposed to the isogenic S. aureus wild-type strain, indicating that PSMs inhibit the production of these interleukins. The use of genetically complemented strains confirmed this observation. Inasmuch as the decreased expression of IL-32, which is involved in dendritic cell maturation, impairs immune responses, our results support a PSM-dependent mechanism that allows for the development of chronic S. aureus-related mastitis. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Efficacy and effectiveness of an rVSV-vectored vaccine expressing Ebola surface glycoprotein: interim results from the Guinea ring vaccination cluster-randomised trial.

PubMed

Henao-Restrepo, Ana Maria; Longini, Ira M; Egger, Matthias; Dean, Natalie E; Edmunds, W John; Camacho, Anton; Carroll, Miles W; Doumbia, Moussa; Draguez, Bertrand; Duraffour, Sophie; Enwere, Godwin; Grais, Rebecca; Gunther, Stephan; Hossmann, Stefanie; Kondé, Mandy Kader; Kone, Souleymane; Kuisma, Eeva; Levine, Myron M; Mandal, Sema; Norheim, Gunnstein; Riveros, Ximena; Soumah, Aboubacar; Trelle, Sven; Vicari, Andrea S; Watson, Conall H; Kéïta, Sakoba; Kieny, Marie Paule; Røttingen, John-Arne

2015-08-29

A recombinant, replication-competent vesicular stomatitis virus-based vaccine expressing a surface glycoprotein of Zaire Ebolavirus (rVSV-ZEBOV) is a promising Ebola vaccine candidate. We report the results of an interim analysis of a trial of rVSV-ZEBOV in Guinea, west Africa. For this open-label, cluster-randomised ring vaccination trial, suspected cases of Ebola virus disease in Basse-Guinée (Guinea, west Africa) were independently ascertained by Ebola response teams as part of a national surveillance system. After laboratory confirmation of a new case, clusters of all contacts and contacts of contacts were defined and randomly allocated 1:1 to immediate vaccination or delayed (21 days later) vaccination with rVSV-ZEBOV (one dose of 2 × 10(7) plaque-forming units, administered intramuscularly in the deltoid muscle). Adults (age ≥18 years) who were not pregnant or breastfeeding were eligible for vaccination. Block randomisation was used, with randomly varying blocks, stratified by location (urban vs rural) and size of rings (≤20 vs >20 individuals). The study is open label and masking of participants and field teams to the time of vaccination is not possible, but Ebola response teams and laboratory workers were unaware of allocation to immediate or delayed vaccination. Taking into account the incubation period of the virus of about 10 days, the prespecified primary outcome was laboratory-confirmed Ebola virus disease with onset of symptoms at least 10 days after randomisation. The primary analysis was per protocol and compared the incidence of Ebola virus disease in eligible and vaccinated individuals in immediate vaccination clusters with the incidence in eligible individuals in delayed vaccination clusters. This trial is registered with the Pan African Clinical Trials Registry, number PACTR201503001057193. Between April 1, 2015, and July 20, 2015, 90 clusters, with a total population of 7651 people were included in the planned interim analysis. 48 of

Newcastle disease virus vectored vaccines as bivalent or antigen delivery vaccines

PubMed Central

2017-01-01

Recent advances in reverse genetics techniques make it possible to manipulate the genome of RNA viruses such as Newcastle disease virus (NDV). Several NDV vaccine strains have been used as vaccine vectors in poultry, mammals, and humans to express antigens of different pathogens. The safety, immunogenicity, and protective efficacy of these NDV-vectored vaccines have been evaluated in pre-clinical and clinical studies. The vaccines are safe in mammals, humans, and poultry. Bivalent NDV-vectored vaccines against pathogens of economic importance to the poultry industry have been developed. These bivalent vaccines confer solid protective immunity against NDV and other foreign antigens. In most cases, NDV-vectored vaccines induce strong local and systemic immune responses against the target foreign antigen. This review summarizes the development of NDV-vectored vaccines and their potential use as a base for designing other effective vaccines for veterinary and human use. PMID:28775971

Expression and immunogenicity of an Escherichia coli K99 fimbriae subunit antigen in soybean.

PubMed

Piller, Kenneth J; Clemente, Thomas E; Jun, Sang Mu; Petty, Cynthia C; Sato, Shirley; Pascual, David W; Bost, Kenneth L

2005-09-01

Enterotoxigenic Escherichia coli (ETEC) cause acute diarrhea in humans and farm animals, and can be fatal if the host is left untreated. As a potential alternative to traditional needle vaccination of cattle, we investigated the feasibility of expressing the major K99 fimbrial subunit, FanC, in soybean (Glycine max) for use as an edible subunit vaccine. As a first step in this developmental process, a synthetic version of fanC was optimized for expression in the cytosol and transferred to soybean via Agrobacterium-mediated transformation. Western analysis of T(0) events revealed the presence of a peptide with the expected mobility for FanC in transgenic protein extracts, and immunofluorescense confirmed localization to the cytosol. Two T(0) lines, which accumulated FanC to levels near 0.5% of total soluble protein, were chosen for further molecular characterization in the T(1) and T(2) generations. Mice immunized intraperitoneally with protein extract derived from transgenic leaves expressing synthetic FanC developed significant antibody titers against bacterially derived FanC and produced antigen-specific CD4(+) T lymphocytes, demonstrating the ability of transgenic FanC to function as an immunogen. These experiments are the first to demonstrate the expression and immunogenicity of a model subunit antigen in the soybean system, and mark the first steps toward the development of a K99 edible vaccine to protect against ETEC.

Elicitation of strong immune responses by a DNA vaccine expressing a secreted form of hepatitis C virus envelope protein E2 in murine and porcine animal models

PubMed Central

Li, Yi-Ping; Kang, Hye Na; Babiuk, Lorne A; Liu, Qiang

2006-01-01

AIM: To characterize the immunogenicity of a hepatitis C virus (HCV) E2 DNA vaccine alone or with a protein vaccine boost in murine and porcine animal models. METHODS: A DNA vaccine expressing a secreted form of HCV E2 protein was constructed and used to vaccinate mice and piglets with or without boosting with a recombinant E2 protein vaccine formulated with CpG ODN and 10% Emulsigen. The immunogenicity of HCV E2 vaccines was analyzed by ELISA for antibody responses, MTT assay for lymphocyte proliferation, ELISPOT for the number of interferon-γ secreting cells, and cytotoxic T lymphocyte assays. RESULTS: Intradermal injection of E2 DNA vaccine induced strong Th1-like immune responses in mice. In piglets, E2 DNA vaccine elicited moderate and more balanced immune responses. A DNA vaccine prime and protein boost vaccination strategy induced significantly higher E2-specific antibody levels and shifted the immune response towards Th2-like ones in piglets. CONCLUSION: A DNA vaccine expressing a secreted form of HCV E2 protein elicited E2-specific immune responses in mice and piglets. Recombinant E2 protein vaccination following DNA immunization significantly increased the antibody response in piglets. These HCV E2 vaccines may represent promising hepatitis C vaccine candidates for further investigations. PMID:17131474

A practical approach to pancreatic cancer immunotherapy using resected tumor lysate vaccines processed to express α-gal epitopes

PubMed Central

Miyoshi, Eiji; Eguchi, Hidetoshi; Nagano, Hiroaki; Matsunami, Katsuyoshi; Nagaoka, Satoshi; Yamada, Daisaku; Asaoka, Tadafumi; Noda, Takehiro; Wada, Hiroshi; Kawamoto, Koichi; Goto, Kunihito; Taniyama, Kiyomi; Mori, Masaki; Doki, Yuichiro

2017-01-01

Objectives Single-agent immunotherapy is ineffective against poorly immunogenic cancers, including pancreatic ductal adenocarcinoma (PDAC). The aims of this study were to demonstrate the feasibility of production of novel autologous tumor lysate vaccines from resected PDAC tumors, and verify vaccine safety and efficacy. Methods Fresh surgically resected tumors obtained from human patients were processed to enzymatically synthesize α-gal epitopes on the carbohydrate chains of membrane glycoproteins. Processed membranes were analyzed for the expression of α-gal epitopes and the binding of anti-Gal, and vaccine efficacy was assessed in vitro and in vivo. Results Effective synthesis of α-gal epitopes was demonstrated after processing of PDAC tumor lysates from 10 different patients, and tumor lysates readily bound an anti-Gal monoclonal antibody. α-gal(+) PDAC tumor lysate vaccines elicited strong antibody production against multiple tumor-associated antigens and activated multiple tumor-specific T cells. The lysate vaccines stimulated a robust immune response in animal models, resulting in tumor suppression and a significant improvement in survival without any adverse events. Conclusions Our data suggest that α-gal(+) PDAC tumor lysate vaccination may be a practical and effective new immunotherapeutic approach for treating pancreatic cancer. PMID:29077749

A practical approach to pancreatic cancer immunotherapy using resected tumor lysate vaccines processed to express α-gal epitopes.

PubMed

Furukawa, Kenta; Tanemura, Masahiro; Miyoshi, Eiji; Eguchi, Hidetoshi; Nagano, Hiroaki; Matsunami, Katsuyoshi; Nagaoka, Satoshi; Yamada, Daisaku; Asaoka, Tadafumi; Noda, Takehiro; Wada, Hiroshi; Kawamoto, Koichi; Goto, Kunihito; Taniyama, Kiyomi; Mori, Masaki; Doki, Yuichiro

2017-01-01

Single-agent immunotherapy is ineffective against poorly immunogenic cancers, including pancreatic ductal adenocarcinoma (PDAC). The aims of this study were to demonstrate the feasibility of production of novel autologous tumor lysate vaccines from resected PDAC tumors, and verify vaccine safety and efficacy. Fresh surgically resected tumors obtained from human patients were processed to enzymatically synthesize α-gal epitopes on the carbohydrate chains of membrane glycoproteins. Processed membranes were analyzed for the expression of α-gal epitopes and the binding of anti-Gal, and vaccine efficacy was assessed in vitro and in vivo. Effective synthesis of α-gal epitopes was demonstrated after processing of PDAC tumor lysates from 10 different patients, and tumor lysates readily bound an anti-Gal monoclonal antibody. α-gal(+) PDAC tumor lysate vaccines elicited strong antibody production against multiple tumor-associated antigens and activated multiple tumor-specific T cells. The lysate vaccines stimulated a robust immune response in animal models, resulting in tumor suppression and a significant improvement in survival without any adverse events. Our data suggest that α-gal(+) PDAC tumor lysate vaccination may be a practical and effective new immunotherapeutic approach for treating pancreatic cancer.

Vaccination with poly(D,L-lactide-co-glycolide) nanoparticles loaded with soluble Leishmania antigens and modified with a TNFα-mimicking peptide or monophosphoryl lipid A confers protection against experimental visceral leishmaniasis.

PubMed

Margaroni, Maritsa; Agallou, Maria; Athanasiou, Evita; Kammona, Olga; Kiparissides, Costas; Gaitanaki, Catherine; Karagouni, Evdokia

2017-01-01

Visceral leishmaniasis (VL) persists as a major public health problem, and since the existing chemotherapy is far from satisfactory, development of an effective vaccine emerges as the most appropriate strategy for confronting VL. The development of an effective vaccine relies on the selection of the appropriate antigen and also the right adjuvant and/or delivery vehicle. In the present study, the protective efficacy of poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles (NPs), which were surface-modified with a TNFα-mimicking eight-amino-acid peptide (p8) and further functionalized by encapsulating soluble Leishmania infantum antigens (sLiAg) and monophosphoryl lipid A (MPLA), a TLR4 ligand, was evaluated against challenge with L. infantum parasites in BALB/c mice. Vaccination with these multifunctionalized PLGA nanoformulations conferred significant protection against parasite infection in vaccinated mice. In particular, vaccination with PLGA-sLiAg-MPLA or p8-PLGA-sLiAg NPs resulted in almost complete elimination of the parasite in the spleen for up to 4 months post-challenge. Parasite burden reduction was accompanied by antigen-specific humoral and cellular immune responses. Specifically, injection with PLGA-sLiAg-MPLA raised exclusively anti-sLiAg IgG1 antibodies post-vaccination, while in p8-PLGA-sLiAg-vaccinated mice, no antibody production was detected. However, 4 months post-challenge, in mice vaccinated with all the multifunctionalized NPs, antibody class switching towards IgG2a subtype was observed. The study of cellular immune responses revealed the increased proliferation capacity of spleen cells against sLiAg, consisting of IFNγ-producing CD4 + and CD8 + T cells. Importantly, the activation of CD8 + T cells was exclusively attributed to vaccination with PLGA NPs surface-modified with the p8 peptide. Moreover, characterization of cytokine production in vaccinated-infected mice revealed that protection was accompanied by significant increase of IFN�

Established human papillomavirus type 16-expressing tumors are effectively eradicated following vaccination with long peptides.

PubMed

Zwaveling, Sander; Ferreira Mota, Sandra C; Nouta, Jan; Johnson, Mark; Lipford, Grayson B; Offringa, Rienk; van der Burg, Sjoerd H; Melief, Cornelis J M

2002-07-01

Peptide-based vaccines aimed at the induction of effective T cell responses against established cancers have so far only met with limited clinical success and clearly need to be improved. In a preclinical model of human papillomavirus (HPV)16-induced cervical cancer we show that prime-boost vaccinations with the HPV16-derived 35 amino-acid long peptide E7(43-77), containing both a CTL epitope and a Th epitope, resulted in the induction of far more robust E7-specific CD8(+) T cell responses than vaccinations with the minimal CTL epitope only. We demonstrate that two distinct mechanisms are responsible for this effect. First, vaccinations with the long peptide lead to the generation of E7-specific CD4(+) Th cells. The level of the induced E7-specific CD8(+) T cell response proved to be dependent on the interactions of these Th cells with professional APC. Second, we demonstrate that vaccination with the long peptide and dendritic cell-activating agents resulted in a superior induction of E7-specific CD8(+) T cells, even when T cell help was excluded. This suggests that, due to its size, the long peptide was preferably endocytosed, processed, and presented by professional APCs. Moreover, the efficacy of this superior HPV-specific T cell induction was demonstrated in therapeutic prime-boost vaccinations in which the long peptide admixed with the dendritic cell-activating adjuvant oligodeoxynucleotide-CpG resulted in the eradication of large, established HPV16-expressing tumors. Because the vaccine types used in this study are easy to prepare under good manufacturing practice conditions and are safe to administer to humans, these data provide important information for future clinical trials.

Optimization of HIV-1 Envelope DNA Vaccine Candidates within Three Different Animal Models, Guinea Pigs, Rabbits and Cynomolgus Macaques.

PubMed

Borggren, Marie; Vinner, Lasse; Andresen, Betina Skovgaard; Grevstad, Berit; Repits, Johanna; Melchers, Mark; Elvang, Tara Laura; Sanders, Rogier W; Martinon, Frédéric; Dereuddre-Bosquet, Nathalie; Bowles, Emma Joanne; Stewart-Jones, Guillaume; Biswas, Priscilla; Scarlatti, Gabriella; Jansson, Marianne; Heyndrickx, Leo; Grand, Roger Le; Fomsgaard, Anders

2013-07-19

HIV-1 DNA vaccines have many advantageous features. Evaluation of HIV-1 vaccine candidates often starts in small animal models before macaque and human trials. Here, we selected and optimized DNA vaccine candidates through systematic testing in rabbits for the induction of broadly neutralizing antibodies (bNAb). We compared three different animal models: guinea pigs, rabbits and cynomolgus macaques. Envelope genes from the prototype isolate HIV-1 Bx08 and two elite neutralizers were included. Codon-optimized genes, encoded secreted gp140 or membrane bound gp150, were modified for expression of stabilized soluble trimer gene products, and delivered individually or mixed. Specific IgG after repeated i.d. inoculations with electroporation confirmed in vivo expression and immunogenicity. Evaluations of rabbits and guinea pigs displayed similar results. The superior DNA construct in rabbits was a trivalent mix of non-modified codon-optimized gp140 envelope genes. Despite NAb responses with some potency and breadth in guinea pigs and rabbits, the DNA vaccinated macaques displayed less bNAb activity. It was concluded that a trivalent mix of non-modified gp140 genes from rationally selected clinical isolates was, in this study, the best option to induce high and broad NAb in the rabbit model, but this optimization does not directly translate into similar responses in cynomolgus macaques.

Optimization of HIV-1 Envelope DNA Vaccine Candidates within Three Different Animal Models, Guinea Pigs, Rabbits and Cynomolgus Macaques

PubMed Central

Borggren, Marie; Vinner, Lasse; Andresen, Betina Skovgaard; Grevstad, Berit; Repits, Johanna; Melchers, Mark; Elvang, Tara Laura; Sanders, Rogier W; Martinon, Frédéric; Dereuddre-Bosquet, Nathalie; Bowles, Emma Joanne; Stewart-Jones, Guillaume; Biswas, Priscilla; Scarlatti, Gabriella; Jansson, Marianne; Heyndrickx, Leo; Le Grand, Roger; Fomsgaard, Anders

2013-01-01

HIV-1 DNA vaccines have many advantageous features. Evaluation of HIV-1 vaccine candidates often starts in small animal models before macaque and human trials. Here, we selected and optimized DNA vaccine candidates through systematic testing in rabbits for the induction of broadly neutralizing antibodies (bNAb). We compared three different animal models: guinea pigs, rabbits and cynomolgus macaques. Envelope genes from the prototype isolate HIV-1 Bx08 and two elite neutralizers were included. Codon-optimized genes, encoded secreted gp140 or membrane bound gp150, were modified for expression of stabilized soluble trimer gene products, and delivered individually or mixed. Specific IgG after repeated i.d. inoculations with electroporation confirmed in vivo expression and immunogenicity. Evaluations of rabbits and guinea pigs displayed similar results. The superior DNA construct in rabbits was a trivalent mix of non-modified codon-optimized gp140 envelope genes. Despite NAb responses with some potency and breadth in guinea pigs and rabbits, the DNA vaccinated macaques displayed less bNAb activity. It was concluded that a trivalent mix of non-modified gp140 genes from rationally selected clinical isolates was, in this study, the best option to induce high and broad NAb in the rabbit model, but this optimization does not directly translate into similar responses in cynomolgus macaques. PMID:26344115

Immune Responses Induced by Gene Gun or Intramuscular Injection of DNA Vaccines That Express Immunogenic Regions of the Serine Repeat Antigen from Plasmodium falciparum

PubMed Central

Belperron, Alexia A.; Feltquate, David; Fox, Barbara A.; Horii, Toshihiro; Bzik, David J.

1999-01-01

The liver- and blood-stage-expressed serine repeat antigen (SERA) of Plasmodium falciparum is a candidate protein for a human malaria vaccine. We compared the immune responses induced in mice immunized with SERA-expressing plasmid DNA vaccines delivered by intramuscular (i.m.) injection or delivered intradermally by Gene Gun immunization. Mice were immunized with a pcdna3 plasmid encoding the entire 47-kDa domain of SERA (amino acids 17 to 382) or the N-terminal domain (amino acids 17 to 110) of SERA. Minimal antibody responses were detected following DNA vaccination with the N-terminal domain of SERA, suggesting that the N-terminal domain alone is not highly immunogenic by this route of vaccine delivery. Immunization of mice by Gene Gun delivery of the 47-kDa domain of SERA elicited a significantly higher serum antibody titer to the antigen than immunization of mice by i.m. injection with the same plasmid did. The predominant isotype subclass of the antibodies elicited to the SERA protein following i.m. and Gene Gun immunizations with SERA plasmid DNA was immunoglobulin G1. Coimmunization of mice with SERA plasmid DNA and a plasmid expressing the hepatitis B surface antigen (pCMV-s) by the i.m. route resulted in higher anti-SERA titers than those generated in mice immunized with the SERA DNA plasmid alone. Vaccination with DNA may provide a viable alternative or may be used in conjunction with protein-based subunit vaccines to maximize the efficacy of a human malaria vaccine that includes immunogenic regions of the SERA protein. PMID:10496891

Oral vaccination with salmonella simultaneously expressing Yersinia pestis F1 and V antigens protects against bubonic and pneumonic plague.

PubMed

Yang, Xinghong; Hinnebusch, B Joseph; Trunkle, Theresa; Bosio, Catharine M; Suo, Zhiyong; Tighe, Mike; Harmsen, Ann; Becker, Todd; Crist, Kathryn; Walters, Nancy; Avci, Recep; Pascual, David W

2007-01-15

The gut provides a large area for immunization enabling the development of mucosal and systemic Ab responses. To test whether the protective Ags to Yersinia pestis can be orally delivered, the Y. pestis caf1 operon, encoding the F1-Ag and virulence Ag (V-Ag) were cloned into attenuated Salmonella vaccine vectors. F1-Ag expression was controlled under a promoter from the caf1 operon; two different promoters (P), PtetA in pV3, PphoP in pV4, as well as a chimera of the two in pV55 were tested. F1-Ag was amply expressed; the chimera in the pV55 showed the best V-Ag expression. Oral immunization with Salmonella-F1 elicited elevated secretory (S)-IgA and serum IgG titers, and Salmonella-V-Ag(pV55) elicited much greater S-IgA and serum IgG Ab titers than Salmonella-V-Ag(pV3) or Salmonella-V-Ag(pV4). Hence, a new Salmonella vaccine, Salmonella-(F1+V)Ags, made with a single plasmid containing the caf1 operon and the chimeric promoter for V-Ag allowed the simultaneous expression of F1 capsule and V-Ag. Salmonella-(F1+V)Ags elicited elevated Ab titers similar to their monotypic derivatives. For bubonic plague, mice dosed with Salmonella-(F1+V)Ags and Salmonella-F1-Ag showed similar efficacy (>83% survival) against approximately 1000 LD(50) Y. pestis. For pneumonic plague, immunized mice required immunity to both F1- and V-Ags because the mice vaccinated with Salmonella-(F1+V)Ags protected against 100 LD(50) Y. pestis. These results show that a single Salmonella vaccine can deliver both F1- and V-Ags to effect both systemic and mucosal immune protection against Y. pestis.

Immune responses and protective efficacy of the gene vaccine expressing Ag85B and ESAT6 fusion protein from Mycobacterium tuberculosis.

PubMed

Chang-hong, Shi; Xiao-wu, Wang; Hai, Zhang; Ting-fen, Zhang; Li-Mei, Wang; Zhi-kai, Xu

2008-04-01

Genetic immunity is a new promising approach for the development of novel tuberculosis vaccines. In this study, it is shown that DNA vaccines expressing the fusion protein of antigen 85B (Ag85B) and early secreted antigenic target 6-kDa antigen (ESAT6) can induce high levels of specific IgG2a antibody subtype in the mice. With the prolongation of postimmunization time, the levels of IgG2a antibody decrease gradually. Although a high-level specific IgG2a antibody subtype is also elicited by classical BCG, the ratio of antibody subtypes IgG2a to IgG1 changes 4 weeks after immunization, and IgG1 is gradually shifted to the main antibody subtype. DNA vaccines also elicit cellular immunity as shown by specific spleen lymphocytes proliferation to Ag85B or ESAT6 protein and the production of high levels of IFN-gamma and IL-2, which is similar to that elicited by BCG. Vaccination of mice with DNA vaccines expressing the fusion protein Ag85B-ESAT6 results in a significant level of protection against the subsequent high-dose challenge with virulent Mycobacterium tuberculosis (MTB) H37Rv. Dramatic reduction in the number of MTB colony-forming units in the spleens and lungs is observed. Pathological examination showed that recombinant plasmid and BCG groups have only minor damage and organizational structures that are kept relatively complete, while in the control group, spleens and lungs are damaged seriously. Therefore, although the reducing degree of mycobacterial loads in the organ of mice immunized with recombinant plasmid is not more than that of BCG, through the analysis of pathological changes, we may conclude that the protective effect provided by DNA vaccine expressing the Ag85B-ESAT6 fusion protein is equivalent to that afforded by the classical BCG.

Refolding of the recombinant protein Sm29, a step toward the production of the vaccine candidate against schistosomiasis.

PubMed

Chura-Chambi, Rosa M; Nakajima, Erika; de Carvalho, Roberta R; Miyasato, Patricia A; Oliveira, Sergio C; Morganti, Ligia; Martins, Elizabeth A L

2013-12-01

Schistosomiasis is an important parasitic disease, with about 240 million people infected worldwide. Humans and animals can be infected, imposing an enormous social and economic burden. The only drug available for chemotherapy, praziquantel, does not control reinfections, and an efficient vaccine for prophylaxis is still missing. However, the tegumental protein Sm29 of Schistosoma mansoni was shown to be a promising antigen to compose an anti-schistosomiasis vaccine. Though, recombinant Sm29 is expressed in Escherichia coli as insoluble inclusion bodies requiring an efficient process of refolding, thus, hampering its production in large scale. We present in this work studies to refold the recombinant Sm29 using high hydrostatic pressure, a mild condition to dissociate aggregated proteins, leading to refolding on a soluble conformation. Our studies resulted in high yield of rSm29 (73%) as a stably soluble and structured protein. The refolded antigen presented protective effect against S. mansoni development in immunized mice. We concluded that the refolding process by application of high hydrostatic pressure succeeded, and the procedure can be scaled-up, allowing industrial production of Sm29. Copyright © 2013 Elsevier B.V. All rights reserved.

Development and immunogenicity of recombinant GapA(+) Mycoplasma gallisepticum vaccine strain ts-11 expressing infectious bronchitis virus-S1 glycoprotein and chicken interleukin-6.

PubMed

Shil, Pollob K; Kanci, Anna; Browning, Glenn F; Markham, Philip F

2011-04-12

Mycoplasma gallisepticum (MG) is a major pathogen of poultry that causes chronic respiratory disease in chickens and infectious sinusitis in turkeys. A live attenuated vaccine, ts-11, has been used for the control of MG in several countries. The efficacy of this vaccine is highly dose dependent and the flock antibody response is weak. To improve the functionality of the vaccine and investigate its potential as a delivery vector for foreign antigens and immunomodulatory proteins, we developed a derivative of ts-11 expressing infectious bronchitis virus-S1 glycoprotein (IBV-S1) and releasing chicken interleukin-6 into the extracellular milieu (MG ts-11 C3 (+CS)) using a transposon-based delivery vector. Following administration of MG ts-11 C3 (+CS) to chickens by eye-drop, an antibody response to MG and IBV-S1, as determined by the rapid serum agglutination test (RSA) and Western blotting, respectively, could be detected. Birds inoculated with the recombinant vaccine had significantly enhanced weight gain and were partially protected against damage by pathogenic IBV. These results indicate that the ChIL-6 released by MG ts-11 C3 (+CS) may have had a non-specific effect on growth rate. They also suggest that ts-11 is a promising vaccine vector, capable of delivering heterologous protective antigens, and may also provide non-specific benefits when engineered to express immunomodulatory proteins. With some improvements in the expression system, it could be used to induce a targeted immune response against specific mucosal pathogens, and co-expression of several antigens would allow development of a novel multivalent vaccine. Copyright © 2011 Elsevier Ltd. All rights reserved.

Towards a universal vaccine for avian influenza: Protective efficacy of modified Vaccinia virus Ankara and Adenovirus vaccines expressing conserved influenza antigens in chickens challenged with low pathogenic avian influenza virus

PubMed Central

Boyd, Amy C.; Ruiz-Hernandez, Raul; Peroval, Marylene Y.; Carson, Connor; Balkissoon, Devanand; Staines, Karen; Turner, Alison V.; Hill, Adrian V.S.; Gilbert, Sarah C.; Butter, Colin

2013-01-01

Current vaccines targeting surface proteins can drive antigenic variation resulting either in the emergence of more highly pathogenic viruses or of antigenically distinct viruses that escape control by vaccination and thereby persist in the host population. Influenza vaccines typically target the highly mutable surface proteins and do not provide protection against heterologous challenge. Vaccines which induce immune responses against conserved influenza epitopes may confer protection against heterologous challenge. We report here the results of vaccination with recombinant modified Vaccinia virus Ankara (MVA) and Adenovirus (Ad) expressing a fusion construct of nucleoprotein and matrix protein (NP + M1). Prime and boost vaccination regimes were trialled in different ages of chicken and were found to be safe and immunogenic. Interferon-γ (IFN-γ) ELISpot was used to assess the cellular immune response post secondary vaccination. In ovo Ad prime followed by a 4 week post hatch MVA boost was identified as the most immunogenic regime in one outbred and two inbred lines of chicken. Following vaccination, one inbred line (C15I) was challenged with low pathogenic avian influenza (LPAI) H7N7 (A/Turkey/England/1977). Birds receiving a primary vaccination with Ad-NP + M1 and a secondary vaccination with MVA-NP + M1 exhibited reduced cloacal shedding as measured by plaque assay at 7 days post infection compared with birds vaccinated with recombinant viruses containing irrelevant antigen. This preliminary indication of efficacy demonstrates proof of concept in birds; induction of T cell responses in chickens by viral vectors containing internal influenza antigens may be a productive strategy for the development of vaccines to induce heterologous protection against influenza in poultry. PMID:23200938

Full-length soluble CD147 promotes MMP-2 expression and is a potential serological marker in detection of hepatocellular carcinoma

PubMed Central

2014-01-01

Background As a surface glycoprotein, CD147 is capable of stimulating the production of matrix metalloproteinases (MMPs) from neighboring fibroblasts. The aim of the present study is to explore the role of soluble CD147 on MMPs secretion from hepatocellular carcinoma (HCC) cells, and to investigate the diagnostic value of serum soluble CD147 in the HCC detection. Methods We identified the form of soluble CD147 in cell culture supernate of HCC cells and serum of patients with HCC, and explored the role of soluble CD147 on MMPs secretion. Serum CD147 levels were detected by the enzyme-linked immunosorbent assay, and the value of soluble CD147 as a marker in HCC detection was analyzed. Results Full length soluble CD147 was presented in the culture medium of HCC cells and serum of patients with HCC. The extracellular domain of soluble CD147 promoted the expression of CD147 and MMP-2 from HCC cells. Knockdown of CD147 markedly diminished the up-regulation of CD147 and MMP-2 which induced by soluble CD147. Soluble CD147 activated ERK, FAK, and PI3K/Akt pathways, leading to the up-regulation of MMP-2. The level of soluble CD147 in serum of patients with HCC was significantly elevated compared with healthy individuals (P < 0.001). Soluble CD147 levels were found to be associated with HCC tumor size (P = 0.007) and Child-Pugh grade (P = 0.007). Moreover, soluble CD147 showed a better performance in distinguishing HCC compared with alpha-fetoprotein. Conclusions The extracellular domain of soluble CD147 enhances the secretion of MMP-2 from HCC cells, requiring the cooperation of membrane CD147 and activation of ERK, FAK, and PI3K/Akt signaling. The measurement of soluble CD147 may offer a useful approach in diagnosis of HCC. PMID:24996644

Immunogenicity of a meningococcal native outer membrane vesicle vaccine with attenuated endotoxin and over-expressed factor H binding protein in infant rhesus monkeys

PubMed Central

Koeberling, Oliver; Seubert, Anja; Santos, George; Colaprico, Annalisa; Ugozzoli, Mildred; Donnelly, John; Granoff, Dan M.

2011-01-01

We previously investigated immunogenicity of meningococcal native outer membrane vesicle (NOMV) vaccines prepared from recombinant strains with attenuated endotoxin (ΔLpxL1) and over-expressed factor H binding protein (fHbp) in a mouse model. The vaccines elicited broad serum bactericidal antibody responses. While human toll-like receptor 4 (TLR-4) is mainly stimulated by wildtype meningococcal endotoxin, mouse TLR-4 is stimulated by both the wildtype and mutant endotoxin. An adjuvant effect in mice of the mutant endotoxin would be expected to be much less in humans, and may have contributed to the broad mouse bactericidal responses. Here we show that as previously reported for humans, rhesus primate peripheral blood mononuclear cells incubated with a NOMV vaccine from ΔLpxL1 recombinant strains had lower proinflammatory cytokine responses than with a control wildtype NOMV vaccine. The cytokine responses to the mutant vaccine were similar to those elicited by a detergent-treated, wildtype outer membrane vesicle vaccine that had been safely administered to humans. Monkeys (N=4) were immunized beginning at ages 2 to 3 months with three doses of a NOMV vaccine prepared from ΔLpxL1 recombinant strains with over-expressed fHbp in the variant 1 and 2 groups. The mutant NOMV vaccine elicited serum bactericidal titers ≥1:4 against all 10 genetically diverse strains tested, including 9 with heterologous PorA to those in the vaccine. Negative-control animals had serum bactericidal titers <1:4. Thus, the mutant NOMV vaccine elicited broadly protective serum antibodies in a non-human infant primate model that is more relevant for predicting human antibody responses than mice. PMID:21571025

Phosphate Solubilization and Gene Expression of Phosphate-Solubilizing Bacterium Burkholderia multivorans WS-FJ9 under Different Levels of Soluble Phosphate.

PubMed

Zeng, Qingwei; Wu, Xiaoqin; Wang, Jiangchuan; Ding, Xiaolei

2017-04-28

Phosphate-solubilizing bacteria (PSB) have the ability to dissolve insoluble phosphate and enhance soil fertility. However, the growth and mineral phosphate solubilization of PSB could be affected by exogenous soluble phosphate and the mechanism has not been fully understood. In the present study, the growth and mineral phosphate-solubilizing characteristics of PSB strain Burkholderia multivorans WS-FJ9 were investigated at six levels of exogenous soluble phosphate (0, 0.5, 1, 5, 10, and 20 mM). The WS-FJ9 strain showed better growth at high levels of soluble phosphate. The phosphate-solubilizing activity of WS-FJ9 was reduced as the soluble phosphate concentration increased, as well as the production of pyruvic acid. Transcriptome profiling of WS-FJ9 at three levels of exogenous soluble phosphate (0, 5, and 20 mM) identified 446 differentially expressed genes, among which 44 genes were continuously up-regulated when soluble phosphate concentration was increased and 81 genes were continuously down-regulated. Some genes related to cell growth were continuously up-regulated, which would account for the better growth of WS-FJ9 at high levels of soluble phosphate. Genes involved in glucose metabolism, including glycerate kinase, 2-oxoglutarate dehydrogenase, and sugar ABC-type transporter, were continuously down-regulated, which indicates that metabolic channeling of glucose towards the phosphorylative pathway was negatively regulated by soluble phosphate. These findings represent an important first step in understanding the molecular mechanisms of soluble phosphate effects on the growth and mineral phosphate solubilization of PSB.

Expression of accessory colonization factor subunit A (ACFA) of Vibrio cholerae and ACFA fused to cholera toxin B subunit in transgenic tomato (Solanum lycopersicum).

PubMed

Sharma, Manoj Kumar; Jani, Dewal; Thungapathra, M; Gautam, J K; Meena, L S; Singh, Yogendra; Ghosh, Amit; Tyagi, Akhilesh Kumar; Sharma, Arun Kumar

2008-05-20

In earlier study from our group, cholera toxin B subunit had been expressed in tomato for developing a plant-based vaccine against cholera. In the present investigation, gene for accessory colonization factor (acf) subunit A, earlier reported to be essential for efficient colonization in the intestine, has been expressed in Escherichia coli as well as tomato plants. Gene encoding for a chimeric protein having a fusion of cholera toxin B subunit and accessory colonization factor A was also expressed in tomato to generate more potent combinatorial antigen. CaMV35S promoter with a duplicated enhancer sequence was used for expression of these genes in tomato. Integration of transgenes into tomato genome was confirmed by PCR and Southern hybridization. Expression of the genes was confirmed at transcript and protein levels. Accessory colonization factor A and cholera toxin B subunit fused to this protein accumulated up to 0.25% and 0.08% of total soluble protein, respectively, in the fruits of transgenic plants. Whereas protein purified from E. coli, in combination with cholera toxin B subunit can be used for development of conventional subunit vaccine, tomato fruits expressing these proteins can be used together with tomato plants expressing cholera toxin B subunit for development of oral vaccine against cholera.

Framework for the rapid optimization of soluble protein expression in Escherichia coli combining microscale experiments and statistical experimental design.

PubMed

Islam, R S; Tisi, D; Levy, M S; Lye, G J

2007-01-01

A major bottleneck in drug discovery is the production of soluble human recombinant protein in sufficient quantities for analysis. This problem is compounded by the complex relationship between protein yield and the large number of variables which affect it. Here, we describe a generic framework for the rapid identification and optimization of factors affecting soluble protein yield in microwell plate fermentations as a prelude to the predictive and reliable scaleup of optimized culture conditions. Recombinant expression of firefly luciferase in Escherichia coli was used as a model system. Two rounds of statistical design of experiments (DoE) were employed to first screen (D-optimal design) and then optimize (central composite face design) the yield of soluble protein. Biological variables from the initial screening experiments included medium type and growth and induction conditions. To provide insight into the impact of the engineering environment on cell growth and expression, plate geometry, shaking speed, and liquid fill volume were included as factors since these strongly influence oxygen transfer into the wells. Compared to standard reference conditions, both the screening and optimization designs gave up to 3-fold increases in the soluble protein yield, i.e., a 9-fold increase overall. In general the highest protein yields were obtained when cells were induced at a relatively low biomass concentration and then allowed to grow slowly up to a high final biomass concentration, >8 g.L-1. Consideration and analysis of the model results showed 6 of the original 10 variables to be important at the screening stage and 3 after optimization. The latter included the microwell plate shaking speeds pre- and postinduction, indicating the importance of oxygen transfer into the microwells and identifying this as a critical parameter for subsequent scale translation studies. The optimization process, also known as response surface methodology (RSM), predicted there to be a

High-Yield Expression of M2e Peptide of Avian Influenza Virus H5N1 in Transgenic Duckweed Plants.

PubMed

Firsov, Aleksey; Tarasenko, Irina; Mitiouchkina, Tatiana; Ismailova, Natalya; Shaloiko, Lyubov; Vainstein, Alexander; Dolgov, Sergey

2015-07-01

Avian influenza is a major viral disease in poultry. Antigenic variation of this virus hinders vaccine development. However, the extracellular domain of the virus-encoded M2 protein (peptide M2e) is nearly invariant in all influenza A strains, enabling the development of a broad-range vaccine against them. Antigen expression in transgenic plants is becoming a popular alternative to classical expression methods. Here we expressed M2e from avian influenza virus A/chicken/Kurgan/5/2005(H5N1) in nuclear-transformed duckweed plants for further development of avian influenza vaccine. The N-terminal fragment of M2, including M2e, was selected for expression. The M2e DNA sequence fused in-frame to the 5' end of β-glucuronidase was cloned into pBI121 under the control of CaMV 35S promoter. The resulting plasmid was successfully used for duckweed transformation, and western analysis with anti-β-glucuronidase and anti-M2e antibodies confirmed accumulation of the target protein (M130) in 17 independent transgenic lines. Quantitative ELISA of crude protein extracts from these lines showed M130-β-glucuronidase accumulation ranging from 0.09-0.97 mg/g FW (0.12-1.96 % of total soluble protein), equivalent to yields of up to 40 μg M2e/g plant FW. This relatively high yield holds promise for the development of a duckweed-based expression system to produce an edible vaccine against avian influenza.

High-Dimensional Gene Expression Profiling Studies in High and Low Responders to Primary Smallpox Vaccination

PubMed Central

Haralambieva, Iana H.; Oberg, Ann L.; Dhiman, Neelam; Ovsyannikova, Inna G.; Kennedy, Richard B.; Grill, Diane E.; Jacobson, Robert M.; Poland, Gregory A.

2012-01-01

Background. The mechanisms underlying smallpox vaccine-induced variations in immune responses are not well understood, but are of considerable interest to a deeper understanding of poxvirus immunity and correlates of protection. Methods. We assessed transcriptional messenger RNA expression changes in 197 recipients of primary smallpox vaccination representing the extremes of humoral and cellular immune responses. Results. The 20 most significant differentially expressed genes include a tumor necrosis factor–receptor superfamily member, an interferon (IFN) gene, a chemokine gene, zinc finger protein genes, nuclear factors, and histones (P ≤ 1.06E−20, q ≤ 2.64E−17). A pathway analysis identified 4 enriched pathways with cytokine production by the T-helper 17 subset of CD4+ T cells being the most significant pathway (P = 3.42E−05). Two pathways (antiviral actions of IFNs, P = 8.95E−05; and IFN-α/β signaling pathway, P = 2.92E−04), integral to innate immunity, were enriched when comparing high with low antibody responders (false discovery rate, < 0.05). Genes related to immune function and transcription (TLR8, P = .0002; DAPP1, P = .0003; LAMP3, P = 9.96E−05; NR4A2, P ≤ .0002; EGR3, P = 4.52E−05), and other genes with a possible impact on immunity (LNPEP, P = 3.72E−05; CAPRIN1, P = .0001; XRN1, P = .0001), were found to be expressed differentially in high versus low antibody responders. Conclusion. We identified novel and known immunity-related genes and pathways that may account for differences in immune response to smallpox vaccination. PMID:22949304

Optimized transitory ectopic expression of promastigote surface antigen protein in Nicotiana benthamiana, a potential anti-leishmaniasis vaccine candidate.

PubMed

Lacombe, Séverine; Bangratz, Martine; Brizard, Jean-Paul; Petitdidier, Elodie; Pagniez, Julie; Sérémé, Drissa; Lemesre, Jean-Loup; Brugidou, Christophe

2018-01-01

In recent years, plants have been shown to be an efficient alternative expression system for high-value pharmaceuticals such as vaccines. However, constitutive expression of recombinant protein remains uncertain on their level of production and biological activity. To overcome these problems, transitory expression systems have been developed. Here, a series of experiments were performed to determine the most effective conditions to enhance vaccine antigen transient accumulation in Nicotiana benthamiana leaves using the promastigote surface antigen (PSA) from the parasitic protozoan Leishmania infantum. This protein has been previously identified as the major antigen of a licensed canine anti-leishmaniasis vaccine. The classical prokaryote Escherichia coli biosystem failed in accumulating PSA. Consequently, the standard plant system based on N. benthamiana has been optimized for the production of putatively active PSA. First, the RNA silencing defense mechanism set up by the plant against PSA ectopic expression was abolished by using three viral suppressors acting at different steps of the RNA silencing pathway. Then, we demonstrated that the signal peptide at the N-terminal side of the PSA is required for its accumulation. The PSA ER signaling and retention with the PSA signal peptide and the KDEL motif, respectively were optimized to significantly increase its accumulation. Finally, we demonstrate that the production of recombinant PSA in N. benthamiana leaves allows the conservation of its immunogenic property. These approaches demonstrate that based on these optimizations, plant based systems can be used to effectively produce the biological active PSA protein. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Recombinant adenovirus expressing the haemagglutinin of Peste des petits ruminants virus (PPRV) protects goats against challenge with pathogenic virus; a DIVA vaccine for PPR.

PubMed

Herbert, Rebecca; Baron, Jana; Batten, Carrie; Baron, Michael; Taylor, Geraldine

2014-02-26

Peste des petits ruminants virus (PPRV) is a morbillivirus that can cause severe disease in sheep and goats, characterised by pyrexia, pneumo-enteritis, and gastritis. The socio-economic burden of the disease is increasing in underdeveloped countries, with poor livestock keepers being affected the most. Current vaccines consist of cell-culture attenuated strains of PPRV, which induce a similar antibody profile to that induced by natural infection. Generation of a vaccine that enables differentiation of infected from vaccinated animals (DIVA) would benefit PPR control and eradication programmes, particularly in the later stages of an eradication campaign and for countries where the disease is not endemic. In order to create a vaccine that would enable infected animals to be distinguished from vaccinated ones (DIVA vaccine), we have evaluated the immunogenicity of recombinant fowlpox (FP) and replication-defective recombinant human adenovirus 5 (Ad), expressing PPRV F and H proteins, in goats. The Ad constructs induced higher levels of virus-specific and neutralising antibodies, and primed greater numbers of CD8+ T cells than the FP-vectored vaccines. Importantly, a single dose of Ad-H, with or without the addition of Ad expressing ovine granulocyte macrophage colony-stimulating factor and/or ovine interleukin-2, not only induced strong antibody and cell-mediated immunity but also completely protected goats against challenge with virulent PPRV, 4 months after vaccination. Replication-defective Ad-H therefore offers the possibility of an effective DIVA vaccine.

Development of a vaccine against Streptococcus agalactiae in fish based on truncated cell wall surface anchor proteins.

PubMed

Liu, H; Zhang, S; Shen, Z; Ren, G; Liu, L; Ma, Y; Zhang, Y; Wang, W

2016-10-08

Streptococcus agalactiae is an important fish pathogen and a leading cause of major economic losses to the aquaculture industry worldwide. In the present study, the two truncated recombinant proteins of cell wall surface anchor family of S agalactiae, CWSAP465 and CWSAP1035, were expressed in Escherichia coli, and their immunogenicity and efficacy against the bacterium were evaluated in tilapia and turbot. The results showed that the prokaryotic expression of the two constructs, p32a-CWSAP465 and p32a-CWSAP1035, gave rise to a high yield of soluble proteins with good immunogenicity. The immunisation-challenge study revealed that tilapia and turbot immunised with recombinant truncated proteins produced high levels of antibodies with a peak at four weeks after immunisation and were protected from a challenge by a virulent S agalactiae at a dose of 1×10 9 colony forming units/ml. The recombinant truncated proteins had higher efficacy than the whole-cell inactivated vaccine. Therefore, the study demonstrated that CWSAP465 and CWSAP1035 are two viable vaccine candidates against S agalactiae in fish. British Veterinary Association.

Protective immunity against tularemia provided by an adenovirus-vectored vaccine expressing Tul4 of Francisella tularensis.

PubMed

Kaur, Ravinder; Chen, Shan; Arévalo, Maria T; Xu, Qingfu; Chen, Yanping; Zeng, Mingtao

2012-03-01

Francisella tularensis, a category A bioterrorism agent, is a highly infectious organism that is passed on via skin contact and inhalation routes. A live attenuated vaccine strain (LVS) has been developed, but it has not been licensed for public use by the FDA due to safety concerns. Thus, there exists a need for a safer and improved vaccine. In this study, we have constructed a replication-incompetent adenovirus, Ad/opt-Tul4, carrying a codon-optimized gene for expression of a membrane protein, Tul4, of F. tularensis LVS. Its ability to protect against lethal challenge and its immunogenicity were evaluated in a murine model. An intramuscular injection of a single dose (1 × 10(7) PFU) of Ad/opt-Tul4 elicited a robust Tul4-specific antibody response. Assays suggest a Th1-driven response. A single dose elicited 20% protection against challenge with 100 × 50% lethal dose (LD(50)) F. tularensis LVS; two additional booster shots resulted in 60% protection. In comparison, three doses of 5 μg recombinant Tul4 protein did not elicit significant protection against challenge. Therefore, the Ad/opt-Tul4 vaccine was more effective than the protein vaccine, and protection was dose dependent. Compared to LVS, the protection rate is lower, but an adenovirus-vectored vaccine may be more attractive due to its enhanced safety profile and mucosal route of delivery. Furthermore, simple genetic modification of the vaccine may potentially produce antibodies protective against a fully virulent strain of F. tularensis. Our data support the development and further research of an adenovirus-vectored vaccine against Tul4 of F. tularensis LVS.

Characterisation of a live Salmonella vaccine stably expressing the Mycobacterium tuberculosis Ag85B–ESAT6 fusion protein

PubMed Central

Hall, Lindsay J.; Clare, Simon; Pickard, Derek; Clark, Simon O.; Kelly, Dominic L.F.; Ghany, Moataz Abd El; Hale, Christine; Dietrich, Jes; Andersen, Peter; Marsh, Philip D.; Dougan, Gordon

2009-01-01

A recombinant Salmonella enterica serovar Typhimurium (S. Typhimurium) vaccine strain was constructed that stably expressed the Mycobacterium tuberculosis fusion antigen Ag85B–ESAT6 from the chromosome. Live oral vaccination of mice with the Salmonella/Ag85B–ESAT6 strain generated a potent anti-Ag85B–ESAT6 TH1 response with high antibody titres with a IgG2a-bias and significant IFN-γ production lasting over a 120-day period. When mice primed with the Salmonella/Ag85B–ESAT6 vaccine were mucosally boosted with the Ag85B–ESAT6 antigen and adjuvant the IFN-γ responses increased markedly. To determine the protective efficacy of this vaccine strain, guinea pigs were immunised and followed for a 30-week period after aerosol challenge with M. tuberculosis. The heterologous prime-boost strategy of live Salmonella vaccine followed by a systemic boost of antigen and adjuvant reduced the levels of M. tuberculosis bacteria in the lungs and spleen to the same extent as BCG. Additionally, this vaccination regimen was observed to be statistically equivalent in terms of protection to immunisation with BCG. Thus, live oral priming with the recombinant Salmonella/Ag85B–ESAT6 and boosting with Ag85B–ESAT6 plus the adjuvant LTK63 represents an effective mucosal vaccination regimen. PMID:19755145

Facilitating protein solubility by use of peptide extensions

DOEpatents

Freimuth, Paul I; Zhang, Yian-Biao; Howitt, Jason

2013-09-17

Expression vectors for expression of a protein or polypeptide of interest as a fusion product composed of the protein or polypeptide of interest fused at one terminus to a solubility enhancing peptide extension are provided. Sequences encoding the peptide extensions are provided. The invention further comprises antibodies which bind specifically to one or more of the solubility enhancing peptide extensions.

Soluble Human Cytomegalovirus gH/gL/pUL128-131 Pentameric Complex, but Not gH/gL, Inhibits Viral Entry to Epithelial Cells and Presents Dominant Native Neutralizing Epitopes.

PubMed

Loughney, John W; Rustandi, Richard R; Wang, Dai; Troutman, Matthew C; Dick, Lawrence W; Li, Guanghua; Liu, Zhong; Li, Fengsheng; Freed, Daniel C; Price, Colleen E; Hoang, Van M; Culp, Timothy D; DePhillips, Pete A; Fu, Tong-Ming; Ha, Sha

2015-06-26

Congenital infection of human cytomegalovirus (HCMV) is one of the leading causes of nongenetic birth defects, and development of a prophylactic vaccine against HCMV is of high priority for public health. The gH/gL/pUL128-131 pentameric complex mediates HCMV entry into endothelial and epithelial cells, and it is a major target for neutralizing antibody responses. To better understand the mechanism by which antibodies interact with the epitopes of the gH/gL/pUL128-131 pentameric complex resulting in viral neutralization, we expressed and purified soluble gH/gL/pUL128-131 pentameric complex and gH/gL from Chinese hamster ovary cells to >95% purity. The soluble gH/gL, which exists predominantly as (gH/gL)2 homodimer with a molecular mass of 220 kDa in solution, has a stoichiometry of 1:1 and a pI of 6.0-6.5. The pentameric complex has a molecular mass of 160 kDa, a stoichiometry of 1:1:1:1:1, and a pI of 7.4-8.1. The soluble pentameric complex, but not gH/gL, adsorbs 76% of neutralizing activities in HCMV human hyperimmune globulin, consistent with earlier reports that the most potent neutralizing epitopes for blocking epithelial infection are unique to the pentameric complex. Functionally, the soluble pentameric complex, but not gH/gL, blocks viral entry to epithelial cells in culture. Our results highlight the importance of the gH/gL/pUL128-131 pentameric complex in HCMV vaccine design and emphasize the necessity to monitor the integrity of the pentameric complex during the vaccine manufacturing process. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

The history of vaccination against cytomegalovirus.

PubMed

Plotkin, Stanley

2015-06-01

Cytomegalovirus vaccine development started in the 1970s with attenuated strains. In the 1980s, one of the strains was shown to be safe and effective in renal transplant patients. Then, attention switched to glycoprotein gB, which was shown to give moderate but transient protection against acquisition of the virus by women. The identification of the pp65 tegument protein as the principal target of cellular immune responses resulted in new approaches, particularly DNA, plasmids to protect hematogenous stem cell recipients. The subsequent discovery of the pentameric protein complex that generates most neutralizing antibodies led to efforts to incorporate that complex into vaccines. At this point, there are many candidate CMV vaccines, including live recombinants, replication-defective virus, DNA plasmids, soluble pentameric proteins, peptides, virus-like particles and vectored envelope proteins.

Mycobacterium leprae Activates Toll-Like Receptor-4 Signaling and Expression on Macrophages Depending on Previous Bacillus Calmette-Guerin Vaccination

PubMed Central

Polycarpou, Anastasia; Holland, Martin J.; Karageorgiou, Ioannis; Eddaoudi, Ayad; Walker, Stephen L.; Willcocks, Sam; Lockwood, Diana N. J.

2016-01-01

Toll-like receptor (TLR)-1 and TLR2 have been shown to be receptors for Mycobacterium leprae (M. leprae), yet it is unclear whether M. leprae can signal through alternative TLRs. Other mycobacterial species possess ligands for TLR4 and genetic association studies in human populations suggest that people with TLR4 polymorphisms may be protected against leprosy. Using human embryonic kidney (HEK)-293 cells co-transfected with TLR4, we demonstrate that M. leprae activates TLR4. We used human macrophages to show that M. leprae stimulation of cytokine production is diminished if pre-treated with TLR4 neutralizing antibody. TLR4 protein expression was up-regulated on macrophages derived from non-bacillus Calmette-Guerin (BCG) vaccinated healthy volunteers after incubation with M. leprae, whereas it was down-regulated in macrophages derived from BCG-vaccinated donors. Finally, pre-treatment of macrophages derived from BCG-naive donors with BCG reversed the effect of M. leprae on TLR4 expression. This may be a newly described phenomenon by which BCG vaccination stimulates “non-specific” protection to the human immune system. PMID:27458573

Mycobacterium leprae Activates Toll-Like Receptor-4 Signaling and Expression on Macrophages Depending on Previous Bacillus Calmette-Guerin Vaccination.

PubMed

Polycarpou, Anastasia; Holland, Martin J; Karageorgiou, Ioannis; Eddaoudi, Ayad; Walker, Stephen L; Willcocks, Sam; Lockwood, Diana N J

2016-01-01

Toll-like receptor (TLR)-1 and TLR2 have been shown to be receptors for Mycobacterium leprae (M. leprae), yet it is unclear whether M. leprae can signal through alternative TLRs. Other mycobacterial species possess ligands for TLR4 and genetic association studies in human populations suggest that people with TLR4 polymorphisms may be protected against leprosy. Using human embryonic kidney (HEK)-293 cells co-transfected with TLR4, we demonstrate that M. leprae activates TLR4. We used human macrophages to show that M. leprae stimulation of cytokine production is diminished if pre-treated with TLR4 neutralizing antibody. TLR4 protein expression was up-regulated on macrophages derived from non-bacillus Calmette-Guerin (BCG) vaccinated healthy volunteers after incubation with M. leprae, whereas it was down-regulated in macrophages derived from BCG-vaccinated donors. Finally, pre-treatment of macrophages derived from BCG-naive donors with BCG reversed the effect of M. leprae on TLR4 expression. This may be a newly described phenomenon by which BCG vaccination stimulates "non-specific" protection to the human immune system.

Stable Chromosomal Expression of Shigella flexneri 2a and 3a O-Antigens in the Live Salmonella Oral Vaccine Vector Ty21a

PubMed Central

Osorio, Manuel; Takeda, Kazuyo; Stibitz, Scott; Kopecko, Dennis J.

2017-01-01

ABSTRACT We have been exploring the use of the live attenuated Salmonella enterica serovar Typhi Ty21a vaccine strain as a versatile oral vaccine vector for the expression and delivery of multiple foreign antigens, including Shigella O-antigens. In this study, we separately cloned genes necessary for the biosynthesis of the Shigella flexneri serotype 2a and 3a O-antigens, which have been shown to provide broad cross-protection to multiple disease-predominant S. flexneri serotypes. The cloned S. flexneri 2a rfb operon, along with bgt and gtrII, contained on the SfII bacteriophage, was sufficient in Ty21a to express the heterologous S. flexneri 2a O-antigen containing the 3,4 antigenic determinants. Further, this rfb operon, along with gtrA, gtrB, and gtrX contained on the Sfx bacteriophage and oac contained on the Sf6 bacteriophage, was sufficient to express S. flexneri 3a O-antigen containing the 6, 7, and 8 antigenic determinants. Ty21a, with these plasmid-carried or chromosomally inserted genes, demonstrated simultaneous and stable expression of homologous S. Typhi O-antigen plus the heterologous S. flexneri O-antigen. Candidate Ty21a vaccine strains expressing heterologous S. flexneri 2a or 3a lipopolysaccharide (LPS) elicited significant serum antibody responses against both homologous S. Typhi and heterologous Shigella LPS and protected mice against virulent S. flexneri 2a or 3a challenges. These new S. flexneri 2a and 3a O-antigen-expressing Ty21a vaccine strains, together with our previously constructed Ty21a strains expressing Shigella sonnei or Shigella dysenteriae 1 O-antigens, have the potential to be used together for simultaneous protection against the predominant causes of shigellosis worldwide as well as against typhoid fever. PMID:29046309

Distribution of immunoglobulin G (IgG) and A (IgA) subclasses following Q fever vaccination with soluble phase I Coxiella burnetii extract.

PubMed

Camacho, M T; Outschoorn, I; Kovácová, E; Téllez, A

2000-03-06

High levels of IgG1, IgG3 and IgA2 antibodies have been observed in patients with Q fever following Coxiella burnetii infection. This IgG subclass distribution is more typical of viral and autoimmune diseases than of bacterial infections. It seemed, therefore, of interest to carry out a prospective study of the distribution of immunoglobulin subclasses after vaccination with phase I C. burnetii tricloroacetic soluble extracts to detect possible differences with respect to natural infection. The antibody response found in vaccinees was mainly restricted to the IgG1, IgG2 and IgA1 subclasses. These findings confirm differences in isotype distribution when compared to those of patients with acute or chronic Coxiella infections and opens an area of interest with respect to the role of IgA subclasses.

Understanding vaccine hesitancy around vaccines and vaccination from a global perspective: a systematic review of published literature, 2007-2012.

PubMed

Larson, Heidi J; Jarrett, Caitlin; Eckersberger, Elisabeth; Smith, David M D; Paterson, Pauline

2014-04-17

Vaccine "hesitancy" is an emerging term in the literature and discourse on vaccine decision-making and determinants of vaccine acceptance. It recognizes a continuum between the domains of vaccine acceptance and vaccine refusal and de-polarizes previous characterization of individuals and groups as either anti-vaccine or pro-vaccine. The primary aims of this systematic review are to: 1) identify research on vaccine hesitancy; 2) identify determinants of vaccine hesitancy in different settings including its context-specific causes, its expression and its impact; and 3) inform the development of a model for assessing determinants of vaccine hesitancy in different settings as proposed by the Strategic Advisory Group of Experts Working Group (SAGE WG) for dealing with vaccine hesitancy. A broad search strategy, built to capture multiple dimensions of public trust, confidence and hesitancy around vaccines, was applied across multiple databases. Peer-reviewed studies were selected for inclusion if they focused on childhood vaccines [≤ 7 years of age], used multivariate analyses, and were published between January 2007 and November 2012. Our results show a variety of factors as being associated with vaccine hesitancy but they do not allow for a complete classification and confirmation of their independent and relative strength of influence. Determinants of vaccine hesitancy are complex and context-specific - varying across time, place and vaccines. Copyright © 2014 Elsevier Ltd. All rights reserved.

Increased efficacy of an adenovirus-vectored foot-and-mouth disease capsid subunit vaccine expressing nonstructural protein 2B is associated with a specific T cell response

USDA-ARS?s Scientific Manuscript database

We previously demonstrated that an adenovirus-based FMDV serotype A24 subunit vaccine, Ad5-A24, expressed under the control of a cytomegalovirus promoter (CMV) can protect swine and bovines against homologous challenge, but swine vaccinated with an Ad5-vectored FMDV O1 Campos vaccine, Ad5-O1Campos (...

Good manufacturing practices production of a purification-free oral cholera vaccine expressed in transgenic rice plants.

PubMed

Kashima, Koji; Yuki, Yoshikazu; Mejima, Mio; Kurokawa, Shiho; Suzuki, Yuji; Minakawa, Satomi; Takeyama, Natsumi; Fukuyama, Yoshiko; Azegami, Tatsuhiko; Tanimoto, Takeshi; Kuroda, Masaharu; Tamura, Minoru; Gomi, Yasuyuki; Kiyono, Hiroshi

2016-03-01

The first Good Manufacturing Practices production of a purification-free rice-based oral cholera vaccine (MucoRice-CTB) from transgenic plants in a closed cultivation system yielded a product meeting regulatory requirements. Despite our knowledge of their advantages, plant-based vaccines remain unavailable for human use in both developing and industrialized countries. A leading, practical obstacle to their widespread use is producing plant-based vaccines that meet governmental regulatory requirements. Here, we report the first production according to current Good Manufacturing Practices of a rice-based vaccine, the cholera vaccine MucoRice-CTB, at an academic institution. To this end, we established specifications and methods for the master seed bank (MSB) of MucoRice-CTB, which was previously generated as a selection-marker-free line, evaluated its propagation, and given that the stored seeds must be renewed periodically. The production of MucoRice-CTB incorporated a closed hydroponic system for cultivating the transgenic plants, to minimize variations in expression and quality during vaccine manufacture. This type of molecular farming factory can be operated year-round, generating three harvests annually, and is cost- and production-effective. Rice was polished to a ratio of 95 % and then powdered to produce the MucoRice-CTB drug substance, and the identity, potency, and safety of the MucoRice-CTB product met pre-established release requirements. The formulation of MucoRice-CTB made by fine-powdering of drug substance and packaged in an aluminum pouch is being evaluated in a physician-initiated phase I study.

Expression of Plasmodium falciparum Circumsporozoite Proteins in Escherichia coli for Potential Use in a Human Malaria Vaccine

NASA Astrophysics Data System (ADS)

Young, James F.; Hockmeyer, Wayne T.; Gross, Mitchell; Ripley Ballou, W.; Wirtz, Robert A.; Trosper, James H.; Beaudoin, Richard L.; Hollingdale, Michael R.; Miller, Louis H.; Diggs, Carter L.; Rosenberg, Martin

1985-05-01

The circumsporozoite (CS) protein of the human malaria parasite Plasmodium falciparum may be the most promising target for the development of a malaria vaccine. In this study, proteins composed of 16, 32, or 48 tandem copies of a tetrapeptide repeating sequence found in the CS protein were efficiently expressed in the bacterium Escherichia coli. When injected into mice, these recombinant products resulted in the production of high titers of antibodies that reacted with the authentic CS protein on live sporozoites and blocked sporozoite invasion of human hepatoma cells in vitro. These CS protein derivatives are therefore candidates for a human malaria vaccine.

Optimizing expression of the pregnancy malaria vaccine candidate, VAR2CSA in Pichia pastoris.

PubMed

Avril, Marion; Hathaway, Marianne J; Cartwright, Megan M; Gose, Severin O; Narum, David L; Smith, Joseph D

2009-06-29

VAR2CSA is the main candidate for a vaccine against pregnancy-associated malaria, but vaccine development is complicated by the large size and complex disulfide bonding pattern of the protein. Recent X-ray crystallographic information suggests that domain boundaries of VAR2CSA Duffy binding-like (DBL) domains may be larger than previously predicted and include two additional cysteine residues. This study investigated whether longer constructs would improve VAR2CSA recombinant protein secretion from Pichia pastoris and if domain boundaries were applicable across different VAR2CSA alleles. VAR2CSA sequences were bioinformatically analysed to identify the predicted C11 and C12 cysteine residues at the C-termini of DBL domains and revised N- and C-termimal domain boundaries were predicted in VAR2CSA. Multiple construct boundaries were systematically evaluated for protein secretion in P. pastoris and secreted proteins were tested as immunogens. From a total of 42 different VAR2CSA constructs, 15 proteins (36%) were secreted. Longer construct boundaries, including the predicted C11 and C12 cysteine residues, generally improved expression of poorly or non-secreted domains and permitted expression of all six VAR2CSA DBL domains. However, protein secretion was still highly empiric and affected by subtle differences in domain boundaries and allelic variation between VAR2CSA sequences. Eleven of the secreted proteins were used to immunize rabbits. Antibodies reacted with CSA-binding infected erythrocytes, indicating that P. pastoris recombinant proteins possessed native protein epitopes. These findings strengthen emerging data for a revision of DBL domain boundaries in var-encoded proteins and may facilitate pregnancy malaria vaccine development.

Optimizing expression of the pregnancy malaria vaccine candidate, VAR2CSA in Pichia pastoris

PubMed Central

Avril, Marion; Hathaway, Marianne J; Cartwright, Megan M; Gose, Severin O; Narum, David L; Smith, Joseph D

2009-01-01

Background VAR2CSA is the main candidate for a vaccine against pregnancy-associated malaria, but vaccine development is complicated by the large size and complex disulfide bonding pattern of the protein. Recent X-ray crystallographic information suggests that domain boundaries of VAR2CSA Duffy binding-like (DBL) domains may be larger than previously predicted and include two additional cysteine residues. This study investigated whether longer constructs would improve VAR2CSA recombinant protein secretion from Pichia pastoris and if domain boundaries were applicable across different VAR2CSA alleles. Methods VAR2CSA sequences were bioinformatically analysed to identify the predicted C11 and C12 cysteine residues at the C-termini of DBL domains and revised N- and C-termimal domain boundaries were predicted in VAR2CSA. Multiple construct boundaries were systematically evaluated for protein secretion in P. pastoris and secreted proteins were tested as immunogens. Results From a total of 42 different VAR2CSA constructs, 15 proteins (36%) were secreted. Longer construct boundaries, including the predicted C11 and C12 cysteine residues, generally improved expression of poorly or non-secreted domains and permitted expression of all six VAR2CSA DBL domains. However, protein secretion was still highly empiric and affected by subtle differences in domain boundaries and allelic variation between VAR2CSA sequences. Eleven of the secreted proteins were used to immunize rabbits. Antibodies reacted with CSA-binding infected erythrocytes, indicating that P. pastoris recombinant proteins possessed native protein epitopes. Conclusion These findings strengthen emerging data for a revision of DBL domain boundaries in var-encoded proteins and may facilitate pregnancy malaria vaccine development. PMID:19563628

Full-length soluble urokinase plasminogen activator receptor down-modulates nephrin expression in podocytes

PubMed Central

Alfano, Massimo; Cinque, Paola; Giusti, Guido; Proietti, Silvia; Nebuloni, Manuela; Danese, Silvio; D’Alessio, Silvia; Genua, Marco; Portale, Federica; Lo Porto, Manuela; Singhal, Pravin C.; Rastaldi, Maria Pia; Saleem, Moin A.; Mavilio, Domenico; Mikulak, Joanna

2015-01-01

Increased plasma level of soluble urokinase-type plasminogen activator receptor (suPAR) was associated recently with focal segmental glomerulosclerosis (FSGS). In addition, different clinical studies observed increased concentration of suPAR in various glomerular diseases and in other human pathologies with nephrotic syndromes such as HIV and Hantavirus infection, diabetes and cardiovascular disorders. Here, we show that suPAR induces nephrin down-modulation in human podocytes. This phenomenon is mediated only by full-length suPAR, is time-and dose-dependent and is associated with the suppression of Wilms’ tumor 1 (WT-1) transcription factor expression. Moreover, an antagonist of αvβ3 integrin RGDfv blocked suPAR-induced suppression of nephrin. These in vitro data were confirmed in an in vivo uPAR knock out Plaur−/− mice model by demonstrating that the infusion of suPAR inhibits expression of nephrin and WT-1 in podocytes and induces proteinuria. This study unveiled that interaction of full-length suPAR with αvβ3 integrin expressed on podocytes results in down-modulation of nephrin that may affect kidney functionality in different human pathologies characterized by increased concentration of suPAR. PMID:26380915

Efficacy of chimeric DNA vaccines encoding Eimeria tenella 5401 and chicken IFN-γ or IL-2 against coccidiosis in chickens.

PubMed

Song, Xiaokai; Huang, Xinmei; Yan, Ruofeng; Xu, Lixin; Li, Xiangrui

2015-09-01

Chimeric DNA vaccines encoding Eimeria tenella (E. tenella) surface antigen 5401 were constructed and their efficacies against E. tenella challenge were studied. The open reading frame (ORF) of 5401 was cloned into the prokaryotic expression vector pGEX-4T2 to express the recombinant protein and the expressed recombinant protein was identified by Western blot. The ORF of 5401 and chicken cytokine gene IFN-γ or IL-2 were cloned into the eukaryotic expression vector pVAX1 consecutively to construct DNA vaccines pVAX-5401-IFN-γ, pVAX-5401-IL-2 and pVAX-5401. The expression of aim genes in vivo was detected by reverse transcription-polymerase chain reaction and Western blot. Fourteen-day-old chickens were inoculated twice at an interval of 7 days with 100 µg of plasmids pVAX-5401, pVAX-5401-IFN-γ and pVAX-5401-IL-2 or 200 µg of recombinant 5401 protein by leg intramuscular injection, respectively. Seven days after the second inoculation, all chickens except the unchallenged control group were challenged orally with 5 × 10(4) sporulated oocysts of E. tenella. Seven days after challenge, all chickens were weighted and slaughtered to determine the effects of immunization. The results showed the recombinant protein was about 90 kDa and reacted with antiserum against soluble sporozoites. The animal experiment showed that all the DNA vaccines pVAX-5401, pVAX-5401-IFN-γ or pVAX-5401-IL-2 and the recombinant 5401 protein could obviously alleviate body weight loss and cecal lesions as compared with non-vaccinated challenged control and empty vector pVAX1control. Furthermore, pVAX-5401-IFN-γ or pVAX-5401-IL-2 induced anti-coccidial index (ACI) of 180.01 or 177.24 which were significantly higher than that of pVAX-5401. The results suggested that 5401 was an effective candidate antigen for vaccine. This finding also suggested that chicken IFN-γ or IL-2 could effectively improve the efficacies of DNA vaccines against avian coccidiosis. Copyright © 2015 Elsevier

Rice-based mucosal vaccine as a global strategy for cold-chain- and needle-free vaccination

PubMed Central

Nochi, Tomonori; Takagi, Hidenori; Yuki, Yoshikazu; Yang, Lijun; Masumura, Takehiro; Mejima, Mio; Nakanishi, Ushio; Matsumura, Akiko; Uozumi, Akihiro; Hiroi, Takachika; Morita, Shigeto; Tanaka, Kunisuke; Takaiwa, Fumio; Kiyono, Hiroshi

2007-01-01

Capable of inducing antigen-specific immune responses in both systemic and mucosal compartments without the use of syringe and needle, mucosal vaccination is considered ideal for the global control of infectious diseases. In this study, we developed a rice-based oral vaccine expressing cholera toxin B subunit (CTB) under the control of the endosperm-specific expression promoter 2.3-kb glutelin GluB-1 with codon usage optimization for expression in rice seed. An average of 30 μg of CTB per seed was stored in the protein bodies, which are storage organelles in rice. When mucosally fed, rice seeds expressing CTB were taken up by the M cells covering the Peyer's patches and induced CTB-specific serum IgG and mucosal IgA antibodies with neutralizing activity. When expressed in rice, CTB was protected from pepsin digestion in vitro. Rice-expressed CTB also remained stable and thus maintained immunogenicity at room temperature for >1.5 years, meaning that antigen-specific mucosal immune responses were induced at much lower doses than were necessary with purified recombinant CTB. Because they require neither refrigeration (cold-chain management) nor a needle, these rice-based mucosal vaccines offer a highly practical and cost-effective strategy for orally vaccinating large populations against mucosal infections, including those that may result from an act of bioterrorism. PMID:17573530

A modified vaccinia Ankara vaccine vector expressing a mosaic H5 hemagglutinin reduces viral shedding in rhesus macaques.

PubMed

Florek, Nicholas W; Kamlangdee, Attapon; Mutschler, James P; Kingstad-Bakke, Brock; Schultz-Darken, Nancy; Broman, Karl W; Osorio, Jorge E; Friedrich, Thomas C

2017-01-01

The rapid antigenic evolution of influenza viruses requires frequent vaccine reformulations. Due to the economic burden of continuous vaccine reformulation and the threat of new pandemics, there is intense interest in developing vaccines capable of eliciting broadly cross-reactive immunity to influenza viruses. We recently constructed a "mosaic" hemagglutinin (HA) based on subtype 5 HA (H5) and designed to stimulate cellular and humoral immunity to multiple influenza virus subtypes. Modified vaccinia Ankara (MVA) expressing this H5 mosaic (MVA-H5M) protected mice against multiple homosubtypic H5N1 strains and a heterosubtypic H1N1 virus. To assess its potential as a human vaccine we evaluated the ability of MVA-H5M to provide heterosubtypic immunity to influenza viruses in a non-human primate model. Rhesus macaques received an initial dose of either MVA-H5M or plasmid DNA encoding H5M, followed by a boost of MVA-H5M, and then were challenged, together with naïve controls, with the heterosubtypic virus A/California/04/2009 (H1N1pdm). Macaques receiving either vaccine regimen cleared H1N1pdm challenge faster than naïve controls. Vaccination with H5M elicited antibodies that bound H1N1pdm HA, but did not neutralize the H1N1pdm challenge virus. Plasma from vaccinated macaques activated NK cells in the presence of H1N1pdm HA, suggesting that vaccination elicited cross-reactive antibodies capable of mediating antibody-dependent cell-mediated cytotoxicity (ADCC). Although HA-specific T cell responses to the MVA-H5M vaccine were weak, responses after challenge were stronger in vaccinated macaques than in control animals. Together these data suggest that mosaic HA antigens may provide a means for inducing broadly cross-reactive immunity to influenza viruses.

A modified vaccinia Ankara vaccine vector expressing a mosaic H5 hemagglutinin reduces viral shedding in rhesus macaques

PubMed Central

Mutschler, James P.; Kingstad-Bakke, Brock; Schultz-Darken, Nancy; Broman, Karl W.; Osorio, Jorge E.

2017-01-01

The rapid antigenic evolution of influenza viruses requires frequent vaccine reformulations. Due to the economic burden of continuous vaccine reformulation and the threat of new pandemics, there is intense interest in developing vaccines capable of eliciting broadly cross-reactive immunity to influenza viruses. We recently constructed a “mosaic” hemagglutinin (HA) based on subtype 5 HA (H5) and designed to stimulate cellular and humoral immunity to multiple influenza virus subtypes. Modified vaccinia Ankara (MVA) expressing this H5 mosaic (MVA-H5M) protected mice against multiple homosubtypic H5N1 strains and a heterosubtypic H1N1 virus. To assess its potential as a human vaccine we evaluated the ability of MVA-H5M to provide heterosubtypic immunity to influenza viruses in a non-human primate model. Rhesus macaques received an initial dose of either MVA-H5M or plasmid DNA encoding H5M, followed by a boost of MVA-H5M, and then were challenged, together with naïve controls, with the heterosubtypic virus A/California/04/2009 (H1N1pdm). Macaques receiving either vaccine regimen cleared H1N1pdm challenge faster than naïve controls. Vaccination with H5M elicited antibodies that bound H1N1pdm HA, but did not neutralize the H1N1pdm challenge virus. Plasma from vaccinated macaques activated NK cells in the presence of H1N1pdm HA, suggesting that vaccination elicited cross-reactive antibodies capable of mediating antibody-dependent cell-mediated cytotoxicity (ADCC). Although HA-specific T cell responses to the MVA-H5M vaccine were weak, responses after challenge were stronger in vaccinated macaques than in control animals. Together these data suggest that mosaic HA antigens may provide a means for inducing broadly cross-reactive immunity to influenza viruses. PMID:28771513

Enhancing immunogenicity and transmission-blocking activity of malaria vaccines by fusing Pfs25 to IMX313 multimerization technology.

PubMed

Li, Yuanyuan; Leneghan, Darren B; Miura, Kazutoyo; Nikolaeva, Daria; Brian, Iona J; Dicks, Matthew D J; Fyfe, Alex J; Zakutansky, Sarah E; de Cassan, Simone; Long, Carole A; Draper, Simon J; Hill, Adrian V S; Hill, Fergal; Biswas, Sumi

2016-01-08

Transmission-blocking vaccines (TBV) target the sexual-stages of the malaria parasite in the mosquito midgut and are widely considered to be an essential tool for malaria elimination. High-titer functional antibodies are required against target antigens to achieve effective transmission-blocking activity. We have fused Pfs25, the leading malaria TBV candidate antigen to IMX313, a molecular adjuvant and expressed it both in ChAd63 and MVA viral vectors and as a secreted protein-nanoparticle. Pfs25-IMX313 expressed from viral vectors or as a protein-nanoparticle is significantly more immunogenic and gives significantly better transmission-reducing activity than monomeric Pfs25. In addition, we demonstrate that the Pfs25-IMX313 protein-nanoparticle leads to a qualitatively improved antibody response in comparison to soluble Pfs25, as well as to significantly higher germinal centre (GC) responses. These results demonstrate that antigen multimerization using IMX313 is a very promising strategy to enhance antibody responses against Pfs25, and that Pfs25-IMX313 is a highly promising TBV candidate vaccine.

Safety and High Level Efficacy of the Combination Malaria Vaccine Regimen of RTS,S/AS01B With Chimpanzee Adenovirus 63 and Modified Vaccinia Ankara Vectored Vaccines Expressing ME-TRAP

PubMed Central

Rampling, Tommy; Ewer, Katie J.; Bowyer, Georgina; Bliss, Carly M.; Edwards, Nick J.; Wright, Danny; Payne, Ruth O.; Venkatraman, Navin; de Barra, Eoghan; Snudden, Claudia M.; Poulton, Ian D.; de Graaf, Hans; Sukhtankar, Priya; Roberts, Rachel; Ivinson, Karen; Weltzin, Rich; Rajkumar, Bebi-Yassin; Wille-Reece, Ulrike; Lee, Cynthia K.; Ockenhouse, Christian F.; Sinden, Robert E.; Gerry, Stephen; Lawrie, Alison M.; Vekemans, Johan; Morelle, Danielle; Lievens, Marc; Ballou, Ripley W.; Cooke, Graham S.; Faust, Saul N.; Gilbert, Sarah; Hill, Adrian V. S.

2016-01-01

Background. The need for a highly efficacious vaccine against Plasmodium falciparum remains pressing. In this controlled human malaria infection (CHMI) study, we assessed the safety, efficacy and immunogenicity of a schedule combining 2 distinct vaccine types in a staggered immunization regimen: one inducing high-titer antibodies to circumsporozoite protein (RTS,S/AS01B) and the other inducing potent T-cell responses to thrombospondin-related adhesion protein (TRAP) by using a viral vector. Method. Thirty-seven healthy malaria-naive adults were vaccinated with either a chimpanzee adenovirus 63 and modified vaccinia virus Ankara–vectored vaccine expressing a multiepitope string fused to TRAP and 3 doses of RTS,S/AS01B (group 1; n = 20) or 3 doses of RTS,S/AS01B alone (group 2; n = 17). CHMI was delivered by mosquito bites to 33 vaccinated subjects at week 12 after the first vaccination and to 6 unvaccinated controls. Results. No suspected unexpected serious adverse reactions or severe adverse events related to vaccination were reported. Protective vaccine efficacy was observed in 14 of 17 subjects (82.4%) in group 1 and 12 of 16 subjects (75%) in group 2. All control subjects received a diagnosis of blood-stage malaria parasite infection. Both vaccination regimens were immunogenic. Fourteen protected subjects underwent repeat CHMI 6 months after initial CHMI; 7 of 8 (87.5%) in group 1 and 5 of 6 (83.3%) in group 2 remained protected. Conclusions. The high level of sterile efficacy observed in this trial is encouraging for further evaluation of combination approaches using these vaccine types. Clinical Trials Registration. NCT01883609. PMID:27307573

Safety and High Level Efficacy of the Combination Malaria Vaccine Regimen of RTS,S/AS01B With Chimpanzee Adenovirus 63 and Modified Vaccinia Ankara Vectored Vaccines Expressing ME-TRAP.

PubMed

Rampling, Tommy; Ewer, Katie J; Bowyer, Georgina; Bliss, Carly M; Edwards, Nick J; Wright, Danny; Payne, Ruth O; Venkatraman, Navin; de Barra, Eoghan; Snudden, Claudia M; Poulton, Ian D; de Graaf, Hans; Sukhtankar, Priya; Roberts, Rachel; Ivinson, Karen; Weltzin, Rich; Rajkumar, Bebi-Yassin; Wille-Reece, Ulrike; Lee, Cynthia K; Ockenhouse, Christian F; Sinden, Robert E; Gerry, Stephen; Lawrie, Alison M; Vekemans, Johan; Morelle, Danielle; Lievens, Marc; Ballou, Ripley W; Cooke, Graham S; Faust, Saul N; Gilbert, Sarah; Hill, Adrian V S

2016-09-01

The need for a highly efficacious vaccine against Plasmodium falciparum remains pressing. In this controlled human malaria infection (CHMI) study, we assessed the safety, efficacy and immunogenicity of a schedule combining 2 distinct vaccine types in a staggered immunization regimen: one inducing high-titer antibodies to circumsporozoite protein (RTS,S/AS01B) and the other inducing potent T-cell responses to thrombospondin-related adhesion protein (TRAP) by using a viral vector. Thirty-seven healthy malaria-naive adults were vaccinated with either a chimpanzee adenovirus 63 and modified vaccinia virus Ankara-vectored vaccine expressing a multiepitope string fused to TRAP and 3 doses of RTS,S/AS01B (group 1; n = 20) or 3 doses of RTS,S/AS01B alone (group 2; n = 17). CHMI was delivered by mosquito bites to 33 vaccinated subjects at week 12 after the first vaccination and to 6 unvaccinated controls. No suspected unexpected serious adverse reactions or severe adverse events related to vaccination were reported. Protective vaccine efficacy was observed in 14 of 17 subjects (82.4%) in group 1 and 12 of 16 subjects (75%) in group 2. All control subjects received a diagnosis of blood-stage malaria parasite infection. Both vaccination regimens were immunogenic. Fourteen protected subjects underwent repeat CHMI 6 months after initial CHMI; 7 of 8 (87.5%) in group 1 and 5 of 6 (83.3%) in group 2 remained protected. The high level of sterile efficacy observed in this trial is encouraging for further evaluation of combination approaches using these vaccine types. NCT01883609. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America.

Two novel heat-soluble protein families abundantly expressed in an anhydrobiotic tardigrade.

PubMed

Yamaguchi, Ayami; Tanaka, Sae; Yamaguchi, Shiho; Kuwahara, Hirokazu; Takamura, Chizuko; Imajoh-Ohmi, Shinobu; Horikawa, Daiki D; Toyoda, Atsushi; Katayama, Toshiaki; Arakawa, Kazuharu; Fujiyama, Asao; Kubo, Takeo; Kunieda, Takekazu

2012-01-01

Tardigrades are able to tolerate almost complete dehydration by reversibly switching to an ametabolic state. This ability is called anhydrobiosis. In the anhydrobiotic state, tardigrades can withstand various extreme environments including space, but their molecular basis remains largely unknown. Late embryogenesis abundant (LEA) proteins are heat-soluble proteins and can prevent protein-aggregation in dehydrated conditions in other anhydrobiotic organisms, but their relevance to tardigrade anhydrobiosis is not clarified. In this study, we focused on the heat-soluble property characteristic of LEA proteins and conducted heat-soluble proteomics using an anhydrobiotic tardigrade. Our heat-soluble proteomics identified five abundant heat-soluble proteins. All of them showed no sequence similarity with LEA proteins and formed two novel protein families with distinct subcellular localizations. We named them Cytoplasmic Abundant Heat Soluble (CAHS) and Secretory Abundant Heat Soluble (SAHS) protein families, according to their localization. Both protein families were conserved among tardigrades, but not found in other phyla. Although CAHS protein was intrinsically unstructured and SAHS protein was rich in β-structure in the hydrated condition, proteins in both families changed their conformation to an α-helical structure in water-deficient conditions as LEA proteins do. Two conserved repeats of 19-mer motifs in CAHS proteins were capable to form amphiphilic stripes in α-helices, suggesting their roles as molecular shield in water-deficient condition, though charge distribution pattern in α-helices were different between CAHS and LEA proteins. Tardigrades might have evolved novel protein families with a heat-soluble property and this study revealed a novel repertoire of major heat-soluble proteins in these anhydrobiotic animals.

A Crimean-Congo hemorrhagic fever (CCHF) viral vaccine expressing nucleoprotein is immunogenic but fails to confer protection against lethal disease.

PubMed

Dowall, S D; Buttigieg, K R; Findlay-Wilson, S J D; Rayner, E; Pearson, G; Miloszewska, A; Graham, V A; Carroll, M W; Hewson, R

2016-01-01

Crimean-Congo Hemorrhagic Fever (CCHF) is a severe tick-borne disease, endemic in many countries in Africa, the Middle East, Eastern Europe and Asia. Between 15-70% of reported cases are fatal with no approved vaccine available. In the present study, the attenuated poxvirus vector, Modified Vaccinia virus Ankara, was used to develop a recombinant candidate vaccine expressing the CCHF virus nucleoprotein. Cellular and humoral immunogenicity was confirmed in 2 mouse strains, including type I interferon receptor knockout mice, which are susceptible to CCHF disease. Despite the immune responses generated post-immunisation, the vaccine failed to protect animals from lethal disease in a challenge model.

A Crimean-Congo hemorrhagic fever (CCHF) viral vaccine expressing nucleoprotein is immunogenic but fails to confer protection against lethal disease

PubMed Central

Dowall, SD; Buttigieg, KR; Findlay-Wilson, SJD; Rayner, E; Pearson, G; Miloszewska, A; Graham, VA; Carroll, MW; Hewson, R

2016-01-01

Crimean-Congo Hemorrhagic Fever (CCHF) is a severe tick-borne disease, endemic in many countries in Africa, the Middle East, Eastern Europe and Asia. Between 15–70% of reported cases are fatal with no approved vaccine available. In the present study, the attenuated poxvirus vector, Modified Vaccinia virus Ankara, was used to develop a recombinant candidate vaccine expressing the CCHF virus nucleoprotein. Cellular and humoral immunogenicity was confirmed in 2 mouse strains, including type I interferon receptor knockout mice, which are susceptible to CCHF disease. Despite the immune responses generated post-immunisation, the vaccine failed to protect animals from lethal disease in a challenge model. PMID:26309231

Purification and immunogenicity of hemagglutinin from highly pathogenic avian influenza virus H5N1 expressed in Nicotiana benthamiana

PubMed Central

Pua, Teen-Lee; Chan, Xiao Ying; Loh, Hwei-San; Omar, Abdul Rahman; Yusibov, Vidadi; Musiychuk, Konstantin; Hall, Alexandra C.; Coffin, Megan V.; Shoji, Yoko; Chichester, Jessica A.; Bi, Hong; Streatfield, Stephen J.

2017-01-01

ABSTRACT Highly pathogenic avian influenza (HPAI) H5N1 is an ongoing global health concern due to its severe sporadic outbreaks in Asia, Africa and Europe, which poses a potential pandemic threat. The development of safe and cost-effective vaccine candidates for HPAI is considered the best strategy for managing the disease and addressing the pandemic preparedness. The most potential vaccine candidate is the antigenic determinant of influenza A virus, hemagglutinin (HA). The present research was aimed at developing optimized expression in Nicotiana benthamiana and protein purification process for HA from the Malaysian isolate of H5N1 as a vaccine antigen for HPAI H5N1. Expression of HA from the Malaysian isolate of HPAI in N. benthamiana was confirmed, and more soluble protein was expressed as truncated HA, the HA1 domain over the entire ectodomain of HA. Two different purification processes were evaluated for efficiency in terms of purity and yield. Due to the reduced yield, protein degradation and length of the 3-column purification process, the 2-column method was chosen for target purification. Purified HA1 was found immunogenic in mice inducing H5 HA-specific IgG and a hemagglutination inhibition antibody. This paper offers an alternative production system of a vaccine candidate against a locally circulating HPAI, which has a regional significance. PMID:27929750

Selecting soluble/foldable protein domains through single-gene or genomic ORF filtering: structure of the head domain of Burkholderia pseudomallei antigen BPSL2063.

PubMed

Gourlay, Louise J; Peano, Clelia; Deantonio, Cecilia; Perletti, Lucia; Pietrelli, Alessandro; Villa, Riccardo; Matterazzo, Elena; Lassaux, Patricia; Santoro, Claudio; Puccio, Simone; Sblattero, Daniele; Bolognesi, Martino

2015-11-01

The 1.8 Å resolution crystal structure of a conserved domain of the potential Burkholderia pseudomallei antigen and trimeric autotransporter BPSL2063 is presented as a structural vaccinology target for melioidosis vaccine development. Since BPSL2063 (1090 amino acids) hosts only one conserved domain, and the expression/purification of the full-length protein proved to be problematic, a domain-filtering library was generated using β-lactamase as a reporter gene to select further BPSL2063 domains. As a result, two domains (D1 and D2) were identified and produced in soluble form in Escherichia coli. Furthermore, as a general tool, a genomic open reading frame-filtering library from the B. pseudomallei genome was also constructed to facilitate the selection of domain boundaries from the entire ORFeome. Such an approach allowed the selection of three potential protein antigens that were also produced in soluble form. The results imply the further development of ORF-filtering methods as a tool in protein-based research to improve the selection and production of soluble proteins or domains for downstream applications such as X-ray crystallography.

Enhancing solubility of deoxyxylulose phosphate pathway enzymes for microbial isoprenoid production

PubMed Central

2012-01-01

Background Recombinant proteins are routinely overexpressed in metabolic engineering. It is well known that some over-expressed heterologous recombinant enzymes are insoluble with little or no enzymatic activity. This study examined the solubility of over-expressed homologous enzymes of the deoxyxylulose phosphate pathway (DXP) and the impact of inclusion body formation on metabolic engineering of microbes. Results Four enzymes of this pathway (DXS, ISPG, ISPH and ISPA), but not all, were highly insoluble, regardless of the expression systems used. Insoluble dxs (the committed enzyme of DXP pathway) was found to be inactive. Expressions of fusion tags did not significantly improve the solubility of dxs. However, hypertonic media containing sorbitol, an osmolyte, successfully doubled the solubility of dxs, with the concomitant improvement in microbial production of the metabolite, DXP. Similarly, sorbitol significantly improved the production of soluble and functional ERG12, the committed enzyme in the mevalonate pathway. Conclusion This study demonstrated the unanticipated findings that some over-expressed homologous enzymes of the DXP pathway were highly insoluble, forming inclusion bodies, which affected metabolite formation. Sorbitol was found to increase both the solubility and function of some of these over-expressed enzymes, a strategy to increase the production of secondary metabolites. PMID:23148661

Associations Between Exposure to and Expression of Negative Opinions About Human Papillomavirus Vaccines on Social Media: An Observational Study.

PubMed

Dunn, Adam G; Leask, Julie; Zhou, Xujuan; Mandl, Kenneth D; Coiera, Enrico

2015-06-10

Groups and individuals that seek to negatively influence public opinion about the safety and value of vaccination are active in online and social media and may influence decision making within some communities. We sought to measure whether exposure to negative opinions about human papillomavirus (HPV) vaccines in Twitter communities is associated with the subsequent expression of negative opinions by explicitly measuring potential information exposure over the social structure of Twitter communities. We hypothesized that prior exposure to opinions rejecting the safety or value of HPV vaccines would be associated with an increased risk of posting similar opinions and tested this hypothesis by analyzing temporal sequences of messages posted on Twitter (tweets). The study design was a retrospective analysis of tweets related to HPV vaccines and the social connections between users. Between October 2013 and April 2014, we collected 83,551 English-language tweets that included terms related to HPV vaccines and the 957,865 social connections among 30,621 users posting or reposting the tweets. Tweets were classified as expressing negative or neutral/positive opinions using a machine learning classifier previously trained on a manually labeled sample. During the 6-month period, 25.13% (20,994/83,551) of tweets were classified as negative; among the 30,621 users that tweeted about HPV vaccines, 9046 (29.54%) were exposed to a majority of negative tweets. The likelihood of a user posting a negative tweet after exposure to a majority of negative opinions was 37.78% (2780/7361) compared to 10.92% (1234/11,296) for users who were exposed to a majority of positive and neutral tweets corresponding to a relative risk of 3.46 (95% CI 3.25-3.67, P<.001). The heterogeneous community structure on Twitter appears to skew the information to which users are exposed in relation to HPV vaccines. We found that among users that tweeted about HPV vaccines, those who were more often exposed to

Associations Between Exposure to and Expression of Negative Opinions About Human Papillomavirus Vaccines on Social Media: An Observational Study

PubMed Central

Leask, Julie; Zhou, Xujuan; Mandl, Kenneth D; Coiera, Enrico

2015-01-01

Background Groups and individuals that seek to negatively influence public opinion about the safety and value of vaccination are active in online and social media and may influence decision making within some communities. Objective We sought to measure whether exposure to negative opinions about human papillomavirus (HPV) vaccines in Twitter communities is associated with the subsequent expression of negative opinions by explicitly measuring potential information exposure over the social structure of Twitter communities. Methods We hypothesized that prior exposure to opinions rejecting the safety or value of HPV vaccines would be associated with an increased risk of posting similar opinions and tested this hypothesis by analyzing temporal sequences of messages posted on Twitter (tweets). The study design was a retrospective analysis of tweets related to HPV vaccines and the social connections between users. Between October 2013 and April 2014, we collected 83,551 English-language tweets that included terms related to HPV vaccines and the 957,865 social connections among 30,621 users posting or reposting the tweets. Tweets were classified as expressing negative or neutral/positive opinions using a machine learning classifier previously trained on a manually labeled sample. Results During the 6-month period, 25.13% (20,994/83,551) of tweets were classified as negative; among the 30,621 users that tweeted about HPV vaccines, 9046 (29.54%) were exposed to a majority of negative tweets. The likelihood of a user posting a negative tweet after exposure to a majority of negative opinions was 37.78% (2780/7361) compared to 10.92% (1234/11,296) for users who were exposed to a majority of positive and neutral tweets corresponding to a relative risk of 3.46 (95% CI 3.25-3.67, P<.001). Conclusions The heterogeneous community structure on Twitter appears to skew the information to which users are exposed in relation to HPV vaccines. We found that among users that tweeted about

Stronger T Cell Immunogenicity of Ovalbumin Expressed Intracellularly in Gram-Negative than in Gram-Positive Bacteria

PubMed Central

Martner, Anna; Östman, Sofia; Lundin, Samuel; Rask, Carola; Björnsson, Viktor; Telemo, Esbjörn; Collins, L. Vincent; Axelsson, Lars; Wold, Agnes E.

2013-01-01

This study aimed to clarify whether Gram-positive (G+) and Gram-negative (G−) bacteria affect antigen-presenting cells differently and thereby influence the immunogenicity of proteins they express. Lactobacilli, lactococci and Escherichia coli strains were transformed with plasmids conferring intracellular ovalbumin (OVA) production. Murine splenic antigen presenting cells (APCs) were pulsed with washed and UV-inactivated OVA-producing bacteria, control bacteria, or soluble OVA. The ability of the APCs to activate OVA-specific DO11.10 CD4+ T cells was assessed by measurments of T cell proliferation and cytokine (IFN-γ, IL-13, IL-17, IL-10) production. OVA expressed within E. coli was strongly immunogenic, since 500 times higher concentrations of soluble OVA were needed to achieve a similar level of OVA-specific T cell proliferation. Furthermore, T cells responding to soluble OVA produced mainly IL-13, while T cells responding to E. coli-expressed OVA produced high levels of both IFN-γ and IL-13. Compared to E. coli, G+ lactobacilli and lactococci were poor inducers of OVA-specific T cell proliferation and cytokine production, despite efficient intracellular expression and production of OVA and despite being efficiently phagocytosed. These results demonstrate a pronounced difference in immunogenicity of intracellular antigens in G+ and G− bacteria and may be relevant for the use of bacterial carriers in vaccine development. PMID:23741469

Rhesus macaque and mouse models for down-selecting circumsporozoite protein based malaria vaccines differ significantly in immunogenicity and functional outcomes.

PubMed

Phares, Timothy W; May, Anthony D; Genito, Christopher J; Hoyt, Nathan A; Khan, Farhat A; Porter, Michael D; DeBot, Margot; Waters, Norman C; Saudan, Philippe; Dutta, Sheetij

2017-03-13

Non-human primates, such as the rhesus macaques, are the preferred model for down-selecting human malaria vaccine formulations, but the rhesus model is expensive and does not allow for direct efficacy testing of human malaria vaccines. Transgenic rodent parasites expressing genes of human Plasmodium are now routinely used for efficacy studies of human malaria vaccines. Mice have however rarely predicted success in human malaria trials and there is scepticism whether mouse studies alone are sufficient to move a vaccine candidate into the clinic. A comparison of immunogenicity, fine-specificity and functional activity of two Alum-adjuvanted Plasmodium falciparum circumsporozoite protein (CSP)-based vaccines was conducted in mouse and rhesus models. One vaccine was a soluble recombinant protein (CSP) and the other was the same CSP covalently conjugated to the Qβ phage particle (Qβ-CSP). Mice showed different kinetics of antibody responses and different sensitivity to the NANP-repeat and N-terminal epitopes as compared to rhesus. While mice failed to discern differences between the protective efficacy of CSP versus Qβ-CSP vaccine following direct challenge with transgenic Plasmodium berghei parasites, rhesus serum from the Qβ-CSP-vaccinated animals induced higher in vivo sporozoite neutralization activity. Despite some immunologic parallels between models, these data demonstrate that differences between the immune responses induced in the two models risk conflicting decisions regarding potential vaccine utility in humans. In combination with historical observations, the data presented here suggest that although murine models may be useful for some purposes, non-human primate models may be more likely to predict the human response to investigational vaccines.

High-yield soluble expression, purification and characterization of human steroidogenic acute regulatory protein (StAR) fused to a cleavable Maltose-Binding Protein (MBP).

PubMed

Sluchanko, Nikolai N; Tugaeva, Kristina V; Faletrov, Yaroslav V; Levitsky, Dmitrii I

2016-03-01

Steroidogenic acute regulatory protein (StAR) is responsible for the rapid delivery of cholesterol to mitochondria where the lipid serves as a source for steroid hormones biosynthesis in adrenals and gonads. Despite many successful investigations, current understanding of the mechanism of StAR action is far from being completely clear. StAR was mostly obtained using denaturation/renaturation or in minor quantities in a soluble form at decreased temperatures that, presumably, limited the possibilities for its consequent detailed exploration. In our hands, existing StAR expression constructs could be bacterially expressed almost exclusively as insoluble forms, even upon decreased expression temperatures and in specific strains of Escherichia coli, and isolated protein tended to aggregate and was difficult to handle. To maximize the yield of soluble protein, optimized StAR sequence encompassing functional domain STARD1 (residues 66-285) was fused to the C-terminus of His-tagged Maltose-Binding Protein (MBP) with the possibility to cleave off the whole tag by 3C protease. The developed protocol of expression and purification comprising of a combination of subtractive immobilized metal affinity chromatography (IMAC) and size-exclusion chromatography allowed us to obtain up to 25 mg/1 L culture of completely soluble StAR protein, which was (i) homogenous according to SDS-PAGE, (ii) gave a single symmetrical peak on a gel-filtration, (iii) showed the characteristic CD spectrum and (iv) pH-dependent ability to bind a fluorescently-labeled cholesterol analogue. We conclude that our strategy provides fully soluble and native StAR protein which in future could be efficiently used for biotechnology and drug discovery aimed at modulation of steroids production. Copyright © 2015 Elsevier Inc. All rights reserved.

Recombinant human adenovirus-5 expressing capsid proteins of Indian vaccine strains of foot-and-mouth disease virus elicits effective antibody response in cattle.

PubMed

Sreenivasa, B P; Mohapatra, J K; Pauszek, S J; Koster, M; Dhanya, V C; Tamil Selvan, R P; Hosamani, M; Saravanan, P; Basagoudanavar, Suresh H; de Los Santos, T; Venkataramanan, R; Rodriguez, L L; Grubman, M J

2017-05-01

Recombinant adenovirus-5 vectored foot-and-mouth disease constructs (Ad5- FMD) were made for three Indian vaccine virus serotypes O, A and Asia 1. Constructs co-expressing foot-and- mouth disease virus (FMDV) capsid and viral 3C protease sequences, were evaluated for their ability to induce a neutralizing antibody response in indigenous cattle (Bos indicus). Purified Ad5-FMD viruses were inoculated in cattle as monovalent (5×10 9 pfu/animal) or trivalent (5×10 9 pfu/animal per serotype) vaccines. Animals vaccinated with monovalent Ad5-FMD vaccines were boosted 63days later with the same dose. After primary immunization, virus neutralization tests (VNT) showed seroconversion in 83, 67 and 33% of animals vaccinated with Ad5-FMD O, A and Asia 1, respectively. Booster immunization elicited seroconversion in all of the animals (100%) in the monovalent groups. When used in a trivalent form, the Ad5-FMD vaccine induced neutralizing antibodies in only 33, 50 and 16% of animals against serotypes O, A and Asia 1, respectively on primo-vaccination, and titers were significantly lower than when the same vectors were used in monovalent form. Neutralizing antibody titers differed by serotype for both Ad5-FMD monovalent and trivalent vaccines, with Asia 1 serotype inducing the lowest titers. Antibody response to Ad5 vector in immunized cattle was also assessed by VNT. It appeared that the vector immunity did not impact the recall responses to expressed FMDV antigens on booster immunization. In summary, the study suggested that the recombinant Ad5-FMD vaccine has a potential use in monovalent form, while its application in multivalent form is not currently encouraging. Copyright © 2017 Elsevier B.V. All rights reserved.

Synthetic Self-Adjuvanting Glycopeptide Cancer Vaccines

NASA Astrophysics Data System (ADS)

Payne, Richard; McDonald, David; Byrne, Scott

2015-10-01

Due to changes in glycosyltransferase expression during tumorigenesis, the glycoproteins of cancer cells often carry highly truncated carbohydrate chains compared to those on healthy cells. These glycans are known as tumor-associated carbohydrate antigens, and are prime targets for use in vaccines for the prevention and treatment of cancer. Herein, we review the state-of-the-art in targeting the immune system towards tumor-associated glycopeptide antigens via synthetic self adjuvanting vaccines, in which the antigenic and adjuvanting moieties of the vaccines are present in the same molecule. The majority of the self-adjuvanting glycopeptide cancer vaccines reported to date employ antigens from mucin 1, a protein which is highly over-expressed and aberrantly glycosylated in many forms of cancer. The adjuvants used in these vaccines predominantly include lipopeptide- or lipoamino acid-based TLR2 agonists, although studies investigating stimulation of TLR9 and TLR4 are also discussed. Most of these adjuvants are highly lipophilic, and, upon conjugation to antigenic peptides, provide amphiphilic vaccine molecules. The amphiphilic nature of these vaccine constructs can lead to the formation of higher-order structures by vaccines in solution, which are likely to be important for their efficacy in vivo.

Evaluation and Selection of Appropriate Reference Genes for Real-Time Quantitative PCR Analysis of Gene Expression in Nile Tilapia (Oreochromis niloticus) during Vaccination and Infection

PubMed Central

Wang, Erlong; Wang, Kaiyu; Chen, Defang; Wang, Jun; He, Yang; Long, Bo; Yang, Lei; Yang, Qian; Geng, Yi; Huang, Xiaoli; Ouyang, Ping; Lai, Weimin

2015-01-01

qPCR as a powerful and attractive methodology has been widely applied to aquaculture researches for gene expression analyses. However, the suitable reference selection is critical for normalizing target genes expression in qPCR. In the present study, six commonly used endogenous controls were selected as candidate reference genes to evaluate and analyze their expression levels, stabilities and normalization to immune-related gene IgM expression during vaccination and infection in spleen of tilapia with RefFinder and GeNorm programs. The results showed that all of these candidate reference genes exhibited transcriptional variations to some extent at different periods. Among them, EF1A was the most stable reference with RefFinder, followed by 18S rRNA, ACTB, UBCE, TUBA and GAPDH respectively and the optimal number of reference genes for IgM normalization under different experiment sets was two with GeNorm. Meanwhile, combination the Cq (quantification cycle) value and the recommended comprehensive ranking of reference genes, EF1A and ACTB, the two optimal reference genes, were used together as reference genes for accurate analysis of immune-related gene expression during vaccination and infection in Nile tilapia with qPCR. Moreover, the highest IgM expression level was at two weeks post-vaccination when normalized to EF1A, 18S rRNA, ACTB, and EF1A together with ACTB compared to one week post-vaccination before normalizing, which was also consistent with the IgM antibody titers detection by ELISA. PMID:25941937

Expression, purification and characterization of soluble red rooster laforin as a fusion protein in Escherichia coli.

PubMed

Brewer, M Kathryn; Husodo, Satrio; Dukhande, Vikas V; Johnson, Mary Beth; Gentry, Matthew S

2014-04-02

The gene that encodes laforin, a dual-specificity phosphatase with a carbohydrate-binding module, is mutated in Lafora disease (LD). LD is an autosomal recessive, fatal progressive myoclonus epilepsy characterized by the intracellular buildup of insoluble, hyperphosphorylated glycogen-like particles, called Lafora bodies. Laforin dephosphorylates glycogen and other glucans in vitro, but the structural basis of its activity remains unknown. Recombinant human laforin when expressed in and purified from E. coli is largely insoluble and prone to aggregation and precipitation. Identification of a laforin ortholog that is more soluble and stable in vitro would circumvent this issue. In this study, we cloned multiple laforin orthologs, established a purification scheme for each, and tested their solubility and stability. Gallus gallus (Gg) laforin is more stable in vitro than human laforin, Gg-laforin is largely monomeric, and it possesses carbohydrate binding and phosphatase activity similar to human laforin. Gg-laforin is more soluble and stable than human laforin in vitro, and possesses similar activity as a glucan phosphatase. Therefore, it can be used to model human laforin in structure-function studies. We have established a protocol for purifying recombinant Gg-laforin in sufficient quantity for crystallographic and other biophysical analyses, in order to better understand the function of laforin and define the molecular mechanisms of Lafora disease.

IL-4 and IL-13 mediated down-regulation of CD8 expression levels can dampen anti-viral CD8⁺ T cell avidity following HIV-1 recombinant pox viral vaccination.

PubMed

Wijesundara, Danushka K; Jackson, Ronald J; Tscharke, David C; Ranasinghe, Charani

2013-09-23

We have shown that mucosal HIV-1 recombinant pox viral vaccination can induce high, avidity HIV-specific CD8(+) T cells with reduced interleukin (IL)-4 and IL-13 expression compared to, systemic vaccine delivery. In the current study how these cytokines act to regulate anti-viral CD8(+) T, cell avidity following HIV-1 recombinant pox viral prime-boost vaccination was investigated. Out of a panel of T cell avidity markers tested, only CD8 expression levels were found to be enhanced on, KdGag197-205 (HIV)-specific CD8(+) T cells obtained from IL-13(-/-), IL-4(-/-) and signal transducer and, activator of transcription of 6 (STAT6)(-/-) mice compared to wild-type (WT) controls following, vaccination. Elevated CD8 expression levels in this instance also correlated with polyfunctionality, (interferon (IFN)-γ, tumour necorsis factor (TNF)-α and IL-2 production) and the avidity of HIVspecific CD8(+) T cells. Furthermore, mucosal vaccination and vaccination with the novel adjuvanted IL-13 inhibitor (i.e. IL-13Rα2) vaccines significantly enhanced CD8 expression levels on HIV-specific CD8(+), T cells, which correlated with avidity. Using anti-CD8 antibodies that blocked CD8 availability on CD8(+), T cells, it was established that CD8 played an important role in increasing HIV-specific CD8(+) T cell avidity and polyfunctionality in IL-4(-/-), IL-13(-/-) and STAT6(-/-) mice compared to WT controls, following vaccination. Collectively, our data demonstrate that IL-4 and IL-13 dampen CD8 expression levels on anti-viral CD8(+) T cells, which can down-regulate anti-viral CD8(+) T cell avidity and, polyfunctionality following HIV-1 recombinant pox viral vaccination. These findings can be exploited to, design more efficacious vaccines not only against HIV-1, but many chronic infections where high, avidity CD8(+) T cells help protection. Copyright © 2013 Elsevier Ltd. All rights reserved.

DNA vaccines

NASA Astrophysics Data System (ADS)

Gregersen, Jens-Peter

2001-12-01

Immunization by genes encoding immunogens, rather than with the immunogen itself, has opened up new possibilities for vaccine research and development and offers chances for new applications and indications for future vaccines. The underlying mechanisms of antigen processing, immune presentation and regulation of immune responses raise high expectations for new and more effective prophylactic or therapeutic vaccines, particularly for vaccines against chronic or persistent infectious diseases and tumors. Our current knowledge and experience of DNA vaccination is summarized and critically reviewed with particular attention to basic immunological mechanisms, the construction of plasmids, screening for protective immunogens to be encoded by these plasmids, modes of application, pharmacokinetics, safety and immunotoxicological aspects. DNA vaccines have the potential to accelerate the research phase of new vaccines and to improve the chances of success, since finding new immunogens with the desired properties is at least technically less demanding than for conventional vaccines. However, on the way to innovative vaccine products, several hurdles have to be overcome. The efficacy of DNA vaccines in humans appears to be much less than indicated by early studies in mice. Open questions remain concerning the persistence and distribution of inoculated plasmid DNA in vivo, its potential to express antigens inappropriately, or the potentially deleterious ability to insert genes into the host cell's genome. Furthermore, the possibility of inducing immunotolerance or autoimmune diseases also needs to be investigated more thoroughly, in order to arrive at a well-founded consensus, which justifies the widespread application of DNA vaccines in a healthy population.

Activity of glycated chitosan and other adjuvants to PDT vaccines

NASA Astrophysics Data System (ADS)

Korbelik, Mladen; Banáth, Judit; Čiplys, Evaldas; Szulc, Zdzislaw; Bielawska, Alicja; Chen, Wei R.

2015-03-01

Glycated chitosan (GC), a water soluble galactose-conjugated natural polysaccharide, has proven to be an effective immunoadjuvant for treatment of tumors based on laser thermal therapy. It was also shown to act as adjuvant for tumor therapy with high-intensity ultrasound and in situ photodynamic therapy (PDT). In the present study, GC was examined as potential adjuvant to PDT-generated cancer vaccine. Two other agents, pure calreticulin protein and acid ceramidase inhibitor LCL521, were also tested as prospective adjuvants for use in conjunction with PDT vaccines. Single treatment with GC, included with PDT vaccine cells suspension, improved the therapeutic efficacy when compared to vaccine alone. This attractive prospect of GC application remains to be carefully optimized and mechanistically elucidated. Both calreticulin and LCL521 proved also effective adjuvants when combined with PDT vaccine tumor treatment.

Soluble Expression of Human Leukemia Inhibitory Factor with Protein Disulfide Isomerase in Escherichia coli and Its Simple Purification

PubMed Central

Chong, Seon-Ha; Kim, Kyunhoo; Choi, Dong Kyu; Thi Vu, Thu Trang; Nguyen, Minh Tan; Jeong, Boram; Ryu, Han-Bong; Kim, Injune; Jang, Yeon Jin; Robinson, Robert Charles; Choe, Han

2013-01-01

Human leukemia inhibitory factor (hLIF) is a multifunctional cytokine that is essential for maintaining the pluripotency of embryonic stem cells. hLIF may be also be useful in aiding fertility through its effects on increasing the implantation rate of fertilized eggs. Thus these applications in biomedical research and clinical medicine create a high demand for bioactive hLIF. However, production of active hLIF is problematic since eukaryotic cells demonstrate limited expression and prokaryotic cells produce insoluble protein. Here, we have adopted a hybrid protein disulfide isomerase design to increase the solubility of hLIF in Escherichia coli. Low temperature expression of hLIF fused to the b'a' domain of protein disulfide isomerase (PDIb'a') increased the soluble expression in comparison to controls. A simple purification protocol for bioactive hLIF was established that includes removal of the PDIb'a' domain by cleavage by TEV protease. The resulting hLIF, which contains one extra glycine residue at the N-terminus, was highly pure and demonstrated endotoxin levels below 0.05 EU/μg. The presence of an intramolecular disulfide bond was identified using mass spectroscopy. This purified hLIF effectively maintained the pluripotency of a murine embryonic stem cell line. Thus we have developed an effective method to produce a pure bioactive version of hLIF in E. coli for use in biomedical research. PMID:24358310

Production of biopharmaceuticals and vaccines in plants via the chloroplast genome.

PubMed

Daniell, Henry

2006-10-01

Transgenic plants offer many advantages, including low cost of production (by elimination of fermenters), storage and transportation; heat stability; and absence of human pathogens. When therapeutic proteins are orally delivered, plant cells protect antigens in the stomach through bioencapsulation and eliminate the need for expensive purification and sterile injections, in addition to development of both systemic and mucosal immunity. Chloroplast genetic engineering offers several advantages, including high levels of transgene expression, transgene containment via maternal inheritance and multi-gene expression in a single transformation event. Hyper-expression of vaccine antigens against cholera, tetanus, anthrax, plague or canine parvovirus (4-31% of total soluble protein, tsp) in transgenic chloroplasts (leaves) or non-green plastids (carrots, tomato), as well as the availability of antibiotic-free selectable markers or the ability to excise selectable marker genes, facilitate oral delivery. Hyper-expression of several therapeutic proteins, including human serum albumin (11.1% tsp), somatotropin (7% tsp), interferon-gamma (6% tsp), anti-microbial peptide (21.5% tsp), facilitates efficient and economic purification. Also, the presence of chaperones and enzymes in chloroplasts facilitate assembly of complex multi-subunit proteins and correct folding of human blood proteins with proper disulfide bonds. Functionality of chloroplast-derived vaccine antigens and therapeutic proteins has been demonstrated by several assays, including the macrophage lysis assay, GM1-ganglioside binding assay, protection of HeLa cells or human lung carcinoma cells against encephalomyocarditis virus, systemic immune response, protection against pathogen challenge, and growth or inhibition of cell cultures. Thus, transgenic chloroplasts are ideal bioreactors for production of functional human and animal therapeutic proteins in an environmentally friendly manner.

Vaccination with poly(D,L-lactide-co-glycolide) nanoparticles loaded with soluble Leishmania antigens and modified with a TNFα-mimicking peptide or monophosphoryl lipid A confers protection against experimental visceral leishmaniasis

PubMed Central

Margaroni, Maritsa; Agallou, Maria; Athanasiou, Evita; Kammona, Olga; Kiparissides, Costas; Gaitanaki, Catherine; Karagouni, Evdokia

2017-01-01

Visceral leishmaniasis (VL) persists as a major public health problem, and since the existing chemotherapy is far from satisfactory, development of an effective vaccine emerges as the most appropriate strategy for confronting VL. The development of an effective vaccine relies on the selection of the appropriate antigen and also the right adjuvant and/or delivery vehicle. In the present study, the protective efficacy of poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles (NPs), which were surface-modified with a TNFα-mimicking eight-amino-acid peptide (p8) and further functionalized by encapsulating soluble Leishmania infantum antigens (sLiAg) and monophosphoryl lipid A (MPLA), a TLR4 ligand, was evaluated against challenge with L. infantum parasites in BALB/c mice. Vaccination with these multifunctionalized PLGA nanoformulations conferred significant protection against parasite infection in vaccinated mice. In particular, vaccination with PLGA-sLiAg-MPLA or p8-PLGA-sLiAg NPs resulted in almost complete elimination of the parasite in the spleen for up to 4 months post-challenge. Parasite burden reduction was accompanied by antigen-specific humoral and cellular immune responses. Specifically, injection with PLGA-sLiAg-MPLA raised exclusively anti-sLiAg IgG1 antibodies post-vaccination, while in p8-PLGA-sLiAg-vaccinated mice, no antibody production was detected. However, 4 months post-challenge, in mice vaccinated with all the multifunctionalized NPs, antibody class switching towards IgG2a subtype was observed. The study of cellular immune responses revealed the increased proliferation capacity of spleen cells against sLiAg, consisting of IFNγ-producing CD4+ and CD8+ T cells. Importantly, the activation of CD8+ T cells was exclusively attributed to vaccination with PLGA NPs surface-modified with the p8 peptide. Moreover, characterization of cytokine production in vaccinated–infected mice revealed that protection was accompanied by significant increase of IFNγ and

Generation of Newcastle Disease Virus (NDV) Recombinants Expressing the Infectious Laryngotracheitis Virus (ILTV) Glycoprotein gB or gD as Dual Vaccines.

PubMed

Zhao, Wei; Spatz, Stephen; Zsak, Laszlo; Yu, Qingzhong

2016-01-01

Infectious laryngotracheitis (ILT) is a highly contagious acute respiratory disease of chickens caused by infection with infectious laryngotracheitis virus (ILTV), a member of the family Herpesviridae. The current commercial ILT vaccines are either unsafe or ineffective. Therefore, there is a pressing need to develop safer and more efficacious vaccines. Newcastle disease (ND), caused by infection with Newcastle disease virus (NDV), a member of the family Paramyxoviridae, is one of the most serious infectious diseases of poultry. The NDV LaSota strain, a naturally occurring low-virulence NDV strain, has been routinely used as a live vaccine throughout the world. This chapter describes the generation of Newcastle disease virus (NDV) LaSota vaccine strain-based recombinant viruses expressing glycoprotein B (gB) or glycoprotein D (gD) of ILTV as dual vaccines against ND and ILT using reverse genetics technology.

DNA vaccines: roles against diseases

PubMed Central

Khan, Kishwar Hayat

2013-01-01

Vaccination is the most successful application of immunological principles to human health. Vaccine efficacy needs to be reviewed from time to time and its safety is an overriding consideration. DNA vaccines offer simple yet effective means of inducing broad-based immunity. These vaccines work by allowing the expression of the microbial antigen inside host cells that take up the plasmid. These vaccines function by generating the desired antigen inside the cells, with the advantage that this may facilitate presentation through the major histocompatibility complex. This review article is based on a literature survey and it describes the working and designing strategies of DNA vaccines. Advantages and disadvantages for this type of vaccines have also been explained, together with applications of DNA vaccines. DNA vaccines against cancer, tuberculosis, Edwardsiella tarda, HIV, anthrax, influenza, malaria, dengue, typhoid and other diseases were explored. PMID:24432284

Stable 293 T and CHO cell lines expressing cleaved, stable HIV-1 envelope glycoprotein trimers for structural and vaccine studies.

PubMed

Chung, Nancy P Y; Matthews, Katie; Kim, Helen J; Ketas, Thomas J; Golabek, Michael; de Los Reyes, Kevin; Korzun, Jacob; Yasmeen, Anila; Sanders, Rogier W; Klasse, Per Johan; Wilson, Ian A; Ward, Andrew B; Marozsan, Andre J; Moore, John P; Cupo, Albert

2014-04-25

Recombinant soluble, cleaved HIV-1 envelope glycoprotein SOSIP.664 gp140 trimers based on the subtype A BG505 sequence are being studied structurally and tested as immunogens in animals. For these trimers to become a vaccine candidate for human trials, they would need to be made in appropriate amounts at an acceptable quality. Accomplishing such tasks by transient transfection is likely to be challenging. The traditional way to express recombinant proteins in large amounts is via a permanent cell line, usually of mammalian origin. Making cell lines that produce BG505 SOSIP.664 trimers requires the co-expression of the Furin protease to ensure that the cleavage site between the gp120 and gp41 subunits is fully utilized. We designed a vector capable of expressing Env and Furin, and used it to create Stable 293 T and CHO Flp-In™ cell lines through site-specific recombination. Both lines produce high quality, cleaved trimers at yields of up to 12-15 mg per 1 × 109 cells. Trimer expression at such levels was maintained for up to 30 days (10 passages) after initial seeding and was consistently superior to what could be achieved by transient transfection. Electron microscopy studies confirm that the purified trimers have the same native-like appearance as those derived by transient transfection and used to generate high-resolution structures. They also have appropriate antigenic properties, including the presentation of the quaternary epitope for the broadly neutralizing antibody PGT145. The BG505 SOSIP.664 trimer-expressing cell lines yield proteins of an appropriate quality for structural studies and animal immunogenicity experiments. The methodology is suitable for making similar lines under Good Manufacturing Practice conditions, to produce trimers for human clinical trials. Moreover, any env gene can be incorporated into this vector system, allowing the manufacture of SOSIP trimers from multiple genotypes, either by transient transfection or from stable cell

Tandem truncated rotavirus VP8* subunit protein with T cell epitope as non-replicating parenteral vaccine is highly immunogenic.

PubMed

Wen, Xiaobo; Cao, Dianjun; Jones, Ronald W; Hoshino, Yasutaka; Yuan, Lijuan

2015-01-01

The two currently available live oral rotavirus vaccines, Rotarix(®) and RotaTeq(®), are highly efficacious in the developed countries. However, the efficacy of such vaccines in resource deprived countries in Africa and Southeast Asia is low. We reported previously that a bacterially-expressed rotavirus P2-P[8] ΔVP8* subunit vaccine candidate administered intramuscularly elicited high-titers of neutralizing antibodies in guinea pigs and mice and significantly shortened the duration of diarrhea in neonatal gnotobiotic pigs upon oral challenge with virulent human rotavirus Wa strain. To further improve its vaccine potential and provide wider coverage against rotavirus strains of global and regional epidemiologic importance, we constructed 2 tandem recombinant VP8* proteins, P2-P[8] ΔVP8*-P[8] ΔVP8* and P2-P[8] ΔVP8*-P[6] ΔVP8* based on Escherichia coli expression system. The two resulting recombinant tandem proteins were highly soluble and P2-P[8] ΔVP8*-P[8] ΔVP8* was generated with high yield. Moreover, guinea pigs immunized intramuscularly by 3 doses of the P2-P[8] ΔVP8*-P[8] ΔVP8* or P2-P[8] ΔVP8*-P[6] ΔVP8* vaccine with aluminum phosphate adjuvant developed high titers of homotypic and heterotypic neutralizing antibodies against human rotaviruses bearing G1-G4, G8, G9 and G12 with P[8], P[4] or P[6] combination. The results suggest that these 2 subunit vaccines in monovalent or bivalent formulation can provide antigenic coverage to almost all the rotavirus G (VP7) types and major P (VP4) types of global as well as regional epidemiologic importance.

Application of SGT1-Hsp90 chaperone complex for soluble expression of NOD1 LRR domain in E. coli

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hong, Tae-Joon; Hahn, Ji-Sook

NOD1 is an intracellular sensor of innate immunity which is related to a number of inflammatory diseases. NOD1 is known to be difficult to express and purify for structural and biochemical studies. Based on the fact that Hsp90 and its cochaperone SGT1 are necessary for the stabilization and activation of NOD1 in mammals, SGT1 was chosen as a fusion partner of the leucine-rich repeat (LRR) domain of NOD1 for its soluble expression in Escherichia coli. Fusion of human SGT1 (hSGT1) to NOD1 LRR significantly enhanced the solubility, and the fusion protein was stabilized by coexpression of mouse Hsp90α. The expressionmore » level of hSGT1-NOD1 LRR was further enhanced by supplementation of rare codon tRNAs and exchange of antibiotic marker genes. - Highlights: • The NOD1 LRR domain was solubilized by SGT1 fusion in E. coli. • The coexpression of HSP90 stabilized the SGT1-NOD1 LRR fusion protein. • Several optimizations could enhance the expression level of the fusion protein.« less

Circulating inflammatory monocytes contribute to impaired influenza vaccine responses in HIV-infected participants.

PubMed

George, Varghese K; Pallikkuth, Suresh; Pahwa, Rajendra; de Armas, Lesley R; Rinaldi, Stefano; Pan, Li; Pahwa, Savita

2018-06-19

Antibody responses are often impaired in old age and in HIV-positive (HIV+) infection despite virologic control with antiretroviral therapy but innate immunologic determinants are not well understood. Monocytes and natural killer cells were examined for relationships to age, HIV infection and influenza vaccine responses. Virologically suppressed HIV+ (n = 139) and HIV-negative (HIV-) (n = 137) participants classified by age as young (18-39 years), middle-aged (40-59 years) and old (≥60 years) were evaluated preinfluenza and postinfluenza vaccination. Prevaccination frequencies of inflammatory monocytes were highest in old HIV+ and HIV-, with old HIV+ exhibiting higher frequency of integrin CD11b on inflammatory monocytes that was correlated with age, expression of C-C chemokine receptor-2 (CCR2) and plasma soluble tumor necrosis factor receptor-1 (sTNFR1), with inverse correlation with postvaccination influenza H1N1 antibody titers. Higher frequencies of CD11b inflammatory monocytes (CD11b, >48.4%) compared with low frequencies of CD11b inflammatory monocytes (<15.8%) was associated with higher prevaccination frequencies of total and inflammatory monocytes and higher CCR2 MFI, higher plasma sTNFR1 and CXCL-10 with higher lipopolysaccharide stimulated expression of TNFα and IL-6, concomitant with lower postvaccination influenza antibody titers. In HIV+ CD11b expressers, the depletion of inflammatory monocytes from peripheral blood mononuclear cells resulted in enhanced antigen-specific CD4 T-cell proliferation. Immature CD56 natural killer cells were lower in young HIV+ compared with young HIV- participants. Perturbations of innate immunity and inflammation signified by high CD11b on inflammatory monocytes are exacerbated with aging in HIV+ and negatively impact immune function involved in Ab response to influenza vaccination.

Immunogenicity and efficacy in mice of an adenovirus-based bicistronic rotavirus vaccine expressing NSP4 and VP7.

PubMed

Xie, Li; Yan, Min; Wang, Xiaonan; Ye, Jing; Mi, Kai; Yan, Shanshan; Niu, Xianglian; Li, Hongjun; Sun, Maosheng

2015-12-02

NSP4 and VP7 are important functional proteins of rotavirus. Proper combination of viral gene expression is favorable to improving the protection effect of subunit vaccine. In the present study, We evaluated the immunogenicity and efficacy of the bicistronic recombinant adenovirus (rAd-NSP4-VP7) and two single-gene expressing adenoviruses (rAd-NSP4, rAd-VP7). The three adenovirus vaccines were used to immunize mice by intramuscular or intranasal administration. The data showed significant increases in serum antibodies, T lymphocyte subpopulations proliferation, and cytokine secretions of splenocyte in all immunized groups. However, the serum IgA and neutralizing antibody levels of the rAd-NSP4-VP7 or rAd-VP7 groups were significantly higher than those of the rAd-NSP4, while the splenocyte numbers of IFN-γ secretion in the rAd-NSP4-VP7 or rAd-NSP4 groups was greater than that of the rAd-VP7. Furthermore, the efficacy evaluation in a suckling mice model indicated that only rAd-NSP4-VP7 conferred significant protection against rotavirus shedding challenge. These results suggest that the co-expression of NSP4 and VP7 in an adenovirus vector induce both humoral and cell-mediated immune responses efficiently, and provide potential efficacy for protection against rotavirus disease. It is possible to represent an efficacious subunits vaccine strategy for control of rotavirus infection and transmission. Copyright © 2015 Elsevier B.V. All rights reserved.

Extending antigen release from particulate vaccines results in enhanced antitumor immune response.

PubMed

Kapadia, Chintan H; Tian, Shaomin; Perry, Jillian L; Sailer, David; Christopher Luft, J; DeSimone, Joseph M

2018-01-10

Tumor-specific CD8 + cytotoxic T lymphocytes (CTLs) play a critical role in an anti-tumor immune response. However, vaccination intended to elicit a potent CD8 + T cell responses employing tumor-associated peptide antigens, are typically ineffective due to poor immunogenicity. Previously, we engineered a polyethylene glycol (PEG) hydrogel-based subunit vaccine for the delivery of an antigenic peptide and CpG (adjuvant) to elicit potent CTLs. In this study, we further examined the effect of antigen release kinetics on their induced immune responses. A CD8 + T cell epitope peptide from OVA (CSIINFEKL) and CpG were co-conjugated to nanoparticles utilizing either a disulfide or a thioether linkage. Subsequent studies comparing peptide release rates as a function of linker, determined that the thioether linkage provided sustained release of peptide over 72h. Ability to control the release of peptide resulted in both higher and prolonged antigen presentation when compared to disulfide-linked peptide. Both NP vaccine formulations resulted in activation and maturation of bone marrow derived dendritic cells (BMDCs) and induced potent CD8 + T cell responses when compared to soluble antigen and soluble CpG. Immunization with either disulfide or thioether linked vaccine constructs effectively inhibited EG7-OVA tumor growth in mice, however only treatment with the thioether linked vaccine construct resulted in enhanced survival. Copyright © 2017. Published by Elsevier B.V.

[Antitumor effect of recombinant T7 phage vaccine expressing xenogenic vascular endothelial growth factor on Lewis lung cancer in mice].

PubMed

Li, Xiao-Hui; Tang, Liang; Liu, Dong; Sun, Hong-Mei; Zhou, Cai-Cun; Tan, Li-Song; Wang, Li-Ping; Zhang, Pei-De; Zhang, Shang-Quan

2006-10-01

Angiogenesis plays an important role in growth and metastasis of tumors. Vascular endothelial growth factor (VEGF) is considered as a fundamental regulator for angiogenesis. This study was to construct a recombinant T7 phage vaccine expressing xenogenic VEGF on the capsid, and test its inhibitory effect on Lewis lung cancer cells in mice. VEGF gene was cloned by reverse transcription-polymerase chain reaction (RT-PCR) from human lung cancer tissues, and inserted into phage using T7 Select10-3b kit to construct T7 Select10-3b_VEGF vaccine. The titer of prepared phage reached 1x10(13) pfu/ml. C57BL/6J mice were randomly divided into 3 groups: T7 Select10-3b_VEGF vaccine group (T7-VEGF), T7 phage (T7) group, normal saline (NS) group (10 mice/group). Each mouse was injected with Freundos adjuvant mixed with 1x10(12) pfu/200 microl T7 Select10-3b_VEGF, or T7, or normal saline once a week for 4 weeks. Lewis lung carcinoma model (LL/2) was established in C57BL/6J mice after 4-week immunization. Tumor growth and mouse's physical status were observed during immunization. Tumor weight and serum level of specific anti-VEGF antibody were measured by enzyme-linked immunosorbent assay (ELISA). Microvessel density (MVD) of tumors was detected by immunohistochemistry 14 days after the inoculation of tumor cells. Tumor weight of T7-VEGF vaccine group,T7 group, and NS group were (0.543+/-0.259)g, (0.982+/-0.359)g, (1.169+/-0.460)g, respectively. Tumor weight of T7-VEGF vaccine group was significantly lower than that of NS group (P<0.01). Serum anti-VEGF antibody level in T7-VEGF vaccine group was 1:1,000. MVD was significantly lower in T7-VEGF vaccine group than in NS group (8.5+/-0.8 vs 18.5+/-1.6, P<0.05). MVD in T7 group was 16.4+/-1.3. Recombinant T7 phage vaccine expressing xenogenic VEGF can break immunologic tolerance against self-VEGF and inhibit the growth of Lewis lung cancer cells.

Mucosal vaccination with recombinant poxvirus vaccines protects ferrets against symptomatic CDV infection.

PubMed

Welter, J; Taylor, J; Tartaglia, J; Paoletti, E; Stephensen, C B

1999-01-28

Canine distemper virus (CDV) infection of ferrets causes a disease characterized by fever, erythema, conjunctivitis and leukocytopenia, similar clinically to measles except for the fatal neurologic sequelae of CDV. We vaccinated juvenile ferrets twice at 4-week intervals by the intranasal or intraduodenal route with attenuated vaccinia (NYVAC) or canarypox virus (ALVAC) constructs containing the CDV hemagglutinin and fusion genes. Controls were vaccinated with the same vectors expressing rabies glycoprotein. Animals were challenged intranasally 4 weeks after the second vaccination with virulent CDV. Body weights, white blood cell (WBC) counts and temperatures were monitored and ferrets were observed daily for clinical signs of infection. WBCs were assayed for the presence of viral RNA by RT-PCR. Intranasally vaccinated animals survived challenge with no virologic or clinical evidence of infection. Vaccination by the intraduodenal route did not provide complete protection. All control animals developed typical distemper. Ferrets can be effectively protected against distemper by mucosal vaccination with poxvirus vaccines.

Differential regulation of polysaccharide-specific antibody responses to isolated polysaccharides, conjugate vaccines, and intact Gram-positive versus Gram-negative extracellular bacteria.

PubMed

Snapper, Clifford M

2016-06-24

Bacterial capsular polysaccharides are major virulence factors and are key targets in a number of licensed anti-bacterial vaccines. Their major characteristics are their large molecular weight and expression of repeating antigenic epitopes that mediate multivalent B cell receptor cross-linking. In addition, since the majority of these antigens cannot associate with MHC-II they fail to recruit CD4+ T cell help and are referred to as T cell-independent antigens. In this review I will discuss a series of studies from my laboratory that have underscored the importance of understanding polysaccharide-specific antibody responses within the context in which the PS is expressed (i.e. in isolation, as a component of conjugate vaccines, and expressed naturally by intact bacteria). We have shown that multivalent B cell receptor crosslinking, as mediated by polysaccharides, uniquely determines the qualitative response of the B cell to subsequent stimuli, but by itself is insufficient to induce antibody secretion or class switching. For these latter events to occur, second signals must act in concert with primary signals derived from the B cell receptor. The co-expression of polysaccharide and protein within intact bacteria promotes recruitment of CD4+ T cell help for the associated PS-specific IgG response, in contrast to isolated polysaccharides. Further, the particulate nature of extracellular bacteria confers properties to the polysaccharide-specific IgG response that makes it distinct immunologically from soluble conjugate vaccines. Finally, the underlying biochemical and/or structural differences that distinguish Gram-positive and Gram-negative bacteria appear to play critical roles in differentially regulating the associated polysaccharide-specific IgG responses to these groups of pathogens. These studies have a number of implications for the understanding and future design of polysaccharide-based vaccines. Published by Elsevier Ltd.

Construction and expression of immunogenic hybrid enterotoxigenic Escherichia coli CFA/I and CS2 colonization fimbriae for use in vaccines.

PubMed

Tobias, Joshua; Svennerholm, Ann-Mari; Holmgren, Jan; Lebens, Michael

2010-07-01

Enterotoxigenic Escherichia coli (ETEC) are an important cause of diarrheal morbidity in developing countries, especially in children and also of traveler's diarrhea. Colonization factors (CFs) of ETEC, like CFA/I and CS2 which are genetically and structurally related, play a substantial role in pathogenicity, and since intestinal-mucosal immune responses against CFs appear to be protective, much effort has focused on the development of a CF-based ETEC vaccine. We have constructed hybrid operons in which the major CS2 subunit-encoding cotA gene was inserted into the CFA/I operon, either replacing (hybrid I) or being added to the major CFA/I subunit-encoding cfaB gene (hybrid II). Using specific monoclonal antibodies against the major subunits of CFA/I and CS2, high levels of surface expression of both fimbrial subunits were shown in E. coli carrying the hybrid II operon. Oral immunization of mice with formalin-killed bacteria expressing hybrid II fimbriae induced strong CFA/I- and CS2-specific serum IgG + IgM and fecal IgA antibody responses, which were higher than those achieved by similar immunization with the reference strains. Bacteria expressing hybrid fimbriae are potential candidate strains in an oral-killed CF-ETEC vaccine, and the approach represents an attractive and novel means of producing a broad-spectrum ETEC vaccine.

[Recombinant Vp2 protein of infectious bursal disease virus AH1 strain expressed in insect cells: a vaccine candidate].

PubMed

Ouyang, Wei; Wang, Yongshan; Zhou, Yu; Zhang, Haibin; Tang, Yude

2010-05-01

Protective immune response of the available IBD vaccine is insufficient to fully protect against the prevailing strain of the infectious bursal disease virus (IBDV). Such a vaccination escape IBDV field isolate idenfied from Anhui province of China in December 2007, where IBD broke out at 2 weeks post vaccination. The IBDV vp2 gene was cloned into pFastBacHTA donor plasmid, followed by generation of the recombinant bacmid DNA pBac-VP2. The latter was used to transfect insect cell Sf9 with Lipofectamine to produce recombinant baculovirus vBac-VP2. The Sf9 cells infected with vBac-VP2 were stained positive against IBDV antibody using the indirect immunofluorescence assay (IFA), which was also confirmed by the detection of IBDV Vp2 protein in the infected Sf9 cells by IBDV sandwich ELISA. Western blotting revealed that the calculated protein of approximately 53 kDa was in the expressed in the insect cells. Moreover, virus-like particles (VLPs) and "inclusion body-like"structure in the infected Sf9 cells were observed under electron microscopy. We further developed an indirect ELISA for the detection of the IBDV antibodies, which was specific and sensitive. In addition, the lysates of vBac-VP2 infected cells was used to immunize 2-week-old SPF chickens, followed by challenging with the virulent IBDV, the survival rate was 30% at 14 days post primary immunization, however, the survival rate was 100% at 14 d after the booster vaccination. The ELISA antibody titers was up to 3.2 x 10(3) and neutralization antibody titer was 2536, significantly higher than those of one-shot vaccination, 8 x 10(2) and 1106, respectively. The immunized chickens did not show any clinical signs and histopathological changes of infection in 7-days trial time. The bursa/body-weight ratios were higher than those of the unimmunized control (P < 0.05). The virus-like-particle recombinant Vp2 protein expressed in insect cells promises to be a novel subunit vaccine and diagnostic reagent candidate

Current and future vaccines and vaccination strategies against infectious laryngotracheitis (ILT) respiratory disease of poultry.

PubMed

García, Maricarmen

2017-07-01

Infectious laryngotracheitis (ILT) is an economically important respiratory disease of poultry that affects the industry worldwide. Vaccination is the principal tool in the control of the disease. Two types of vaccines, live attenuated and recombinant viral vector, are commercially available. The first generation of GaHV-1 vaccines available since the early 1960's are live viruses, attenuated by continuous passages in cell culture or embryos. These vaccines significantly reduce mortalities and, in particular, the chicken embryo origin (CEO) vaccines have shown to limit outbreaks of the disease. However, the CEO vaccines can regain virulence and become the source of outbreaks. Recombinant viral vector vaccines, the second generation of GaHV-1 vaccines, were first introduced in the early 2000's. These are Fowl Pox virus (FPV) and Herpes virus of turkeys (HVT) vectors expressing one or multiple GaHV-1 immunogenic proteins. Recombinant viral vector vaccines are considered a much safer alternative because they do not regain virulence. In the face of challenge, they improve bird performance and ameliorate clinical signs of the disease but fail to reduce shedding of the challenge virus increasing the likelihood of outbreaks. At the moment, several new strategies are being evaluated to improve both live attenuated and viral vector vaccines. Potential new live vaccines attenuated by deletion of genes associated with virulence or by selection of CEO viral subpopulations that do not exhibit increased virulence upon passages in birds are being evaluated. Also new vector alternatives to express GaHV-1 glycoproteins in Newcastle diseases virus (NDV) or in modified very virulent (vv) serotype I Marek's disease virus (MDV) were developed and evaluated. Copyright © 2016 Elsevier B.V. All rights reserved.

Kunjin Virus Replicon-Based Vaccines Expressing Ebola Virus Glycoprotein GP Protect the Guinea Pig Against Lethal Ebola Virus Infection

PubMed Central

Reynard, O.; Mokhonov, V.; Mokhonova, E.; Leung, J.; Page, A.; Mateo, M.; Pyankova, O.; Georges-Courbot, M. C.; Raoul, H.; Khromykh, A. A.

2011-01-01

Pre- or postexposure treatments against the filoviral hemorrhagic fevers are currently not available for human use. We evaluated, in a guinea pig model, the immunogenic potential of Kunjin virus (KUN)–derived replicons as a vaccine candidate against Ebola virus (EBOV). Virus like particles (VLPs) containing KUN replicons expressing EBOV wild-type glycoprotein GP, membrane anchor-truncated GP (GP/Ctr), and mutated GP (D637L) with enhanced shedding capacity were generated and assayed for their protective efficacy. Immunization with KUN VLPs expressing full-length wild-type and D637L-mutated GPs but not membrane anchor–truncated GP induced dose-dependent protection against a challenge of a lethal dose of recombinant guinea pig-adapted EBOV. The surviving animals showed complete clearance of the virus. Our results demonstrate the potential for KUN replicon vectors as vaccine candidates against EBOV infection. PMID:21987742

Engineering new mycobacterial vaccine design for HIV–TB pediatric vaccine vectored by lysine auxotroph of BCG

PubMed Central

Saubi, Narcís; Gea-Mallorquí, Ester; Ferrer, Pau; Hurtado, Carmen; Sánchez-Úbeda, Sara; Eto, Yoshiki; Gatell, Josep M; Hanke, Tomáš; Joseph, Joan

2014-01-01

In this study, we have engineered a new mycobacterial vaccine design by using an antibiotic-free plasmid selection system. We assembled a novel Escherichia coli (E. coli)–mycobacterial shuttle plasmid p2auxo.HIVA, expressing the HIV-1 clade A immunogen HIVA. This shuttle vector employs an antibiotic resistance-free mechanism for plasmid selection and maintenance based on glycine complementation in E. coli and lysine complementation in mycobacteria. This plasmid was first transformed into glycine auxotroph of E. coli strain and subsequently transformed into lysine auxotroph of Mycobacterium bovis BCG strain to generate vaccine BCG.HIVA2auxo. We demonstrated that the episomal plasmid p2auxo.HIVA was stable in vivo over a 7-week period and genetically and phenotypically characterized the BCG.HIVA2auxo vaccine strain. The BCG.HIVA2auxo vaccine in combination with modified vaccinia virus Ankara (MVA). HIVA was safe and induced HIV-1 and Mycobacterium tuberculosis-specific interferon-γ-producing T-cell responses in adult BALB/c mice. Polyfunctional HIV-1-specific CD8+ T cells, which produce interferon-γ and tumor necrosis factor-α and express the degranulation marker CD107a, were induced. Thus, we engineered a novel, safer, good laboratory practice–compatible BCG-vectored vaccine using prototype immunogen HIVA. This antibiotic-free plasmid selection system based on “double” auxotrophic complementation might be a new mycobacterial vaccine platform to develop not only recombinant BCG-based vaccines expressing second generation of HIV-1 immunogens but also other major pediatric pathogens to prime protective response soon after birth. PMID:26015961

Post-translational intracellular trafficking determines the type of immune response elicited by DNA vaccines expressing Gag antigen of Human Immunodeficiency Virus Type 1 (HIV-1).

PubMed

Wallace, Aaron; West, Kim; Rothman, Alan L; Ennis, Francis A; Lu, Shan; Wang, Shixia

2013-10-01

In the current study, immune responses induced by Gag DNA vaccines with different designs were evaluated in Balb/C mice. The results demonstrated that the DNA vaccine with the full length wild type gag gene (Wt-Gag) mainly produced Gag antigens intracellularly and induced a higher level of cell-mediated immune (CMI) responses, as measured by IFN-gamma ELISPOT, intracellular cytokine staining (ICS), and cytotoxic T lymphocytes (CTL) assays against a dominant CD8(+) T cell epitope (AMQMLKETI). In contrast, the addition of a tissue plasminogen activator (tPA) leader sequence significantly improved overall Gag protein expression/secretion and Gag-specific antibody responses; however, Gag-specific CMI responses were decreased. The mutation of zinc-finger motif changed Gag protein expression patterns and reduced the ability to generate both CMI and antibody responses against Gag. These findings indicate that the structure and post-translational processing of antigens expressed by DNA vaccines play a critical role in eliciting optimal antibody or CMI responses.

Accurate quantitation for in vitro refolding of single domain antibody fragments expressed as inclusion bodies by referring the concomitant expression of a soluble form in the periplasms of Escherichia coli.

PubMed

Noguchi, Tomoaki; Nishida, Yuichi; Takizawa, Keiji; Cui, Yue; Tsutsumi, Koki; Hamada, Takashi; Nishi, Yoshisuke

2017-03-01

Single domain antibody fragments from two species, a camel V H H (PM1) and a shark V NAR (A6), were derived from inclusion bodies of E. coli and refolded in vitro following three refolding recipes for comparing refolding efficiencies: three-step cold dialysis refolding (TCDR), one-step hot dialysis refolding (OHDR), and one-step cold dialysis refolding (OCDR), as these fragments were expressed as 'a soluble form' either in cytoplasm or periplasm, but the amount were much less than those expressed as 'an insoluble form (inclusion body)' in cytoplasm and periplasm. In order to verify the refolding efficiencies from inclusion bodies correctly, proteins purified from periplasmic soluble fractions were used as reference samples. These samples showed far-UV spectra of a typical β-sheet-dominant structure in circular dichroism (CD) spectroscopy and so did the refolded samples as well. As the maximal magnitude of ellipticity in millidegrees (θ max ) observed at a given wave length was proportional to the concentrations of the respective reference samples, we could draw linear regression lines for the magnitudes vs. sample concentrations. By using these lines, we measured the concentrations for the refolded PM1 and A6 samples purified from solubilized cytoplasmic insoluble fractions. The refolding efficiency of PM1 was almost 50% following TCDR and 40% and 30% following OHDR and OCDR, respectively, whereas the value of A6 was around 30% following TCDR, and out of bound for quantitation following the other two recipes. The ELISA curves, which were derived from the refolded samples, coincided better with those obtained from the reference samples after converting the values from the protein-concentrations at recovery to the ones of refolded proteins using recovery ratios, indicating that such a correction gives better results for the accurate measure of the ELISA curves than those without correction. Our method require constructing a dual expression system, expressed both in

How advances in immunology provide insight into improving vaccine efficacy

PubMed Central

Slifka, Mark K.; Amanna, Ian

2014-01-01

Vaccines represent one of the most compelling examples of how biomedical research has improved society by saving lives and dramatically reducing the burden of infectious disease. Despite the importance of vaccinology, we are still in the early stages of understanding how the best vaccines work and how we can achieve better protective efficacy through improved vaccine design. Most successful vaccines have been developed empirically, but recent advances in immunology are beginning to shed new light on the mechanisms of vaccine-mediated protection and development of long-term immunity. Although natural infection will often elicit lifelong immunity, almost all current vaccines require booster vaccination in order to achieve durable protective humoral immune responses, regardless of whether the vaccine is based on infection with replicating live-attenuated vaccine strains of the specific pathogen or whether they are derived from immunization with inactivated, non-replicating vaccines or subunit vaccines. The form of the vaccine antigen (e.g., soluble or particulate/aggregate) appears to play an important role in determining immunogenicity and the interactions between dendritic cells, B cells and T cells in the germinal center are likely to dictate the magnitude and duration of protective immunity. By learning how to optimize these interactions, we may be able to elicit more effective and long-lived immunity with fewer vaccinations. PMID:24709587

Recombinant yellow fever vaccine virus 17D expressing simian immunodeficiency virus SIVmac239 gag induces SIV-specific CD8+ T-cell responses in rhesus macaques.

PubMed

Bonaldo, Myrna C; Martins, Mauricio A; Rudersdorf, Richard; Mudd, Philip A; Sacha, Jonah B; Piaskowski, Shari M; Costa Neves, Patrícia C; Veloso de Santana, Marlon G; Vojnov, Lara; Capuano, Saverio; Rakasz, Eva G; Wilson, Nancy A; Fulkerson, John; Sadoff, Jerald C; Watkins, David I; Galler, Ricardo

2010-04-01

Here we describe a novel vaccine vector for expressing human immunodeficiency virus (HIV) antigens. We show that recombinant attenuated yellow fever vaccine virus 17D expressing simian immunodeficiency virus SIVmac239 Gag sequences can be used as a vector to generate SIV-specific CD8(+) T-cell responses in the rhesus macaque. Priming with recombinant BCG expressing SIV antigens increased the frequency of these SIV-specific CD8(+) T-cell responses after recombinant YF17D boosting. These recombinant YF17D-induced SIV-specific CD8(+) T cells secreted several cytokines, were largely effector memory T cells, and suppressed viral replication in CD4(+) T cells.

Expression of chicken parvovirus VP2 in chicken embryo fibroblasts requires codon optimization for production of naked DNA and vectored meleagrid herpesvirus type 1 vaccines.

PubMed

Spatz, Stephen J; Volkening, Jeremy D; Mullis, Robert; Li, Fenglan; Mercado, John; Zsak, Laszlo

2013-10-01

Meleagrid herpesvirus type 1 (MeHV-1) is an ideal vector for the expression of antigens from pathogenic avian organisms in order to generate vaccines. Chicken parvovirus (ChPV) is a widespread infectious virus that causes serious disease in chickens. It is one of the etiological agents largely suspected in causing Runting Stunting Syndrome (RSS) in chickens. Initial attempts to express the wild-type gene encoding the capsid protein VP2 of ChPV by insertion into the thymidine kinase gene of MeHV-1 were unsuccessful. However, transient expression of a codon-optimized synthetic VP2 gene cloned into the bicistronic vector pIRES2-Ds-Red2, could be demonstrated by immunocytochemical staining of transfected chicken embryo fibroblasts (CEFs). Red fluorescence could also be detected in these transfected cells since the red fluorescent protein gene is downstream from the internal ribosome entry site (IRES). Strikingly, fluorescence could not be demonstrated in cells transiently transfected with the bicistronic vector containing the wild-type or non-codon-optimized VP2 gene. Immunocytochemical staining of these cells also failed to demonstrate expression of wild-type VP2, indicating that the lack of expression was at the RNA level and the VP2 protein was not toxic to CEFs. Chickens vaccinated with a DNA vaccine consisting of the bicistronic vector containing the codon-optimized VP2 elicited a humoral immune response as measured by a VP2-specific ELISA. This VP2 codon-optimized bicistronic cassette was rescued into the MeHV-1 genome generating a vectored vaccine against ChPV disease.

Survey of Australian inpatients on vaccination status and perceptions of influenza vaccination.

PubMed

Loke, Xin Yee; Tran, Winnie; Alderman, Christopher P

2012-08-01

patients described positive views about influenza vaccine efficacy and expressed willingness to receive the vaccine if recommended by their doctor. In this audit, vaccination status appeared to be age-dependent, with higher vaccination coverage among older patients. Those who perceived that the influenza vaccine is associated with many side effects were less likely to be vaccinated. Pharmacists may have a role in encouraging older adults to be vaccinated.

Human recombinant soluble guanylyl cyclase: expression, purification, and regulation

NASA Technical Reports Server (NTRS)

Lee, Y. C.; Martin, E.; Murad, F.

2000-01-01

The alpha1- and beta1-subunits of human soluble guanylate cyclase (sGC) were coexpressed in the Sf9 cells/baculovirus system. In addition to the native enzyme, constructs with hexahistidine tag at the amino and carboxyl termini of each subunit were coexpressed. This permitted the rapid and efficient purification of active recombinant enzyme on a nickel-affinity column. The enzyme has one heme per heterodimer and was readily activated with the NO donor sodium nitroprusside or 3-(5'-hydroxymethyl-2'furyl)-1-benzyl-indazole (YC-1). Sodium nitroprusside and YC-1 treatment potentiated each other in combination and demonstrated a remarkable 2,200-fold stimulation of the human recombinant sGC. The effects were inhibited with 1H-(1,2, 4)oxadiazole(4,3-a)quinoxalin-1one (ODQ). The kinetics of the recombinant enzyme with respect to GTP was examined. The products of the reaction, cGMP and pyrophosphate, inhibited the enzyme. The extent of inhibition by cGMP depended on the activation state of the enzyme, whereas inhibition by pyrophosphate was not affected by the enzyme state. Both reaction products displayed independent binding and cooperativity with respect to enzyme inhibition. The expression of large quantities of active enzyme will facilitate structural characterization of the protein.

Liposomes containing monophosphoryl lipid A and QS-21 serve as an effective adjuvant for soluble circumsporozoite protein malaria vaccine FMP013.

PubMed

Genito, Christopher J; Beck, Zoltan; Phares, Timothy W; Kalle, Fanta; Limbach, Keith J; Stefaniak, Maureen E; Patterson, Noelle B; Bergmann-Leitner, Elke S; Waters, Norman C; Matyas, Gary R; Alving, Carl R; Dutta, Sheetij

2017-07-05

Malaria caused by Plasmodium falciparum continues to threaten millions of people living in the tropical parts of the world. A vaccine that confers sterile and life-long protection remains elusive despite more than 30years of effort and resources invested in solving this problem. Antibodies to a malaria vaccine candidate circumsporozoite protein (CSP) can block invasion and can protect humans against malaria. We have manufactured the Falciparum Malaria Protein-013 (FMP013) vaccine based on the nearly full-length P. falciparum CSP 3D7 strain sequence. We report here immunogenicity and challenge data on FMP013 antigen in C57BL/6 mice formulated with two novel adjuvants of the Army Liposome Formulation (ALF) series and a commercially available adjuvant Montanide ISA 720 (Montanide) as a control. ALF is a liposomal adjuvant containing a synthetic monophosphoryl lipid A (3D-PHAD®). In our study, FMP013 was adjuvanted with ALF alone, ALF containing aluminum hydroxide (ALFA) or ALF containing QS-21 (ALFQ). Adjuvants ALF and ALFA induced similar antibody titers and protection against transgenic parasite challenge that were comparable to Montanide. ALFQ was superior to the other three adjuvants as it induced higher antibody titers with improved boosting after the third immunization, higher serum IgG2c titers, and enhanced protection. FMP013+ALFQ also augmented the numbers of splenic germinal center-derived activated B-cells and antibody secreting cells compared to Montanide. Further, FMP013+ALFQ induced antigen-specific IFN-γ ELISPOT activity, CD4 + T-cells and a T H 1-biased cytokine profile. These results demonstrate that soluble CSP can induce a potent and sterile protective immune response when formulated with the QS-21 containing adjuvant ALFQ. Comparative mouse immunogenicity data presented here were used as the progression criteria for an ongoing non-human primate study and a regulatory toxicology study in preparation for a controlled human malaria infection (CHMI

Yeast-expressed recombinant protein of the receptor-binding domain in SARS-CoV spike protein with deglycosylated forms as a SARS vaccine candidate

PubMed Central

Chen, Wen-Hsiang; Du, Lanying; Chag, Shivali M; Ma, Cuiqing; Tricoche, Nancy; Tao, Xinrong; Seid, Christopher A; Hudspeth, Elissa M; Lustigman, Sara; Tseng, Chien-Te K; Bottazzi, Maria Elena; Hotez, Peter J; Zhan, Bin; Jiang, Shibo

2014-01-01

Development of vaccines for preventing a future pandemic of severe acute respiratory syndrome (SARS) caused by SARS coronavirus (SARS-CoV) and for biodefense preparedness is urgently needed. Our previous studies have shown that a candidate SARS vaccine antigen consisting of the receptor-binding domain (RBD) of SARS-CoV spike protein can induce potent neutralizing antibody responses and protection against SARS-CoV challenge in vaccinated animals. To optimize expression conditions for scale-up production of the RBD vaccine candidate, we hypothesized that this could be potentially achieved by removing glycosylation sites in the RBD protein. In this study, we constructed two RBD protein variants: 1) RBD193-WT (193-aa, residues 318–510) and its deglycosylated forms (RBD193-N1, RBD193-N2, RBD193-N3); 2) RBD219-WT (219-aa, residues 318–536) and its deglycosylated forms (RBD219-N1, RBD219-N2, and RBD219-N3). All constructs were expressed as recombinant proteins in yeast. The purified recombinant proteins of these constructs were compared for their antigenicity, functionality and immunogenicity in mice using alum as the adjuvant. We found that RBD219-N1 exhibited high expression yield, and maintained its antigenicity and functionality. More importantly, RBD219-N1 induced significantly stronger RBD-specific antibody responses and a higher level of neutralizing antibodies in immunized mice than RBD193-WT, RBD193-N1, RBD193-N3, or RBD219-WT. These results suggest that RBD219-N1 could be selected as an optimal SARS vaccine candidate for further development. PMID:24355931

Yeast-expressed recombinant protein of the receptor-binding domain in SARS-CoV spike protein with deglycosylated forms as a SARS vaccine candidate.

PubMed

Chen, Wen-Hsiang; Du, Lanying; Chag, Shivali M; Ma, Cuiqing; Tricoche, Nancy; Tao, Xinrong; Seid, Christopher A; Hudspeth, Elissa M; Lustigman, Sara; Tseng, Chien-Te K; Bottazzi, Maria Elena; Hotez, Peter J; Zhan, Bin; Jiang, Shibo

2014-01-01

Development of vaccines for preventing a future pandemic of severe acute respiratory syndrome (SARS) caused by SARS coronavirus (SARS-CoV) and for biodefense preparedness is urgently needed. Our previous studies have shown that a candidate SARS vaccine antigen consisting of the receptor-binding domain (RBD) of SARS-CoV spike protein can induce potent neutralizing antibody responses and protection against SARS-CoV challenge in vaccinated animals. To optimize expression conditions for scale-up production of the RBD vaccine candidate, we hypothesized that this could be potentially achieved by removing glycosylation sites in the RBD protein. In this study, we constructed two RBD protein variants: 1) RBD193-WT (193-aa, residues 318-510) and its deglycosylated forms (RBD193-N1, RBD193-N2, RBD193-N3); 2) RBD219-WT (219-aa, residues 318-536) and its deglycosylated forms (RBD219-N1, RBD219-N2, and RBD219-N3). All constructs were expressed as recombinant proteins in yeast. The purified recombinant proteins of these constructs were compared for their antigenicity, functionality and immunogenicity in mice using alum as the adjuvant. We found that RBD219-N1 exhibited high expression yield, and maintained its antigenicity and functionality. More importantly, RBD219-N1 induced significantly stronger RBD-specific antibody responses and a higher level of neutralizing antibodies in immunized mice than RBD193-WT, RBD193-N1, RBD193-N3, or RBD219-WT. These results suggest that RBD219-N1 could be selected as an optimal SARS vaccine candidate for further development.

Vaccination of carp against SVCV with an oral DNA vaccine or an insect cells-based subunit vaccine.

PubMed

Embregts, C W E; Rigaudeau, D; Tacchi, L; Pijlman, G P; Kampers, L; Veselý, T; Pokorová, D; Boudinot, P; Wiegertjes, G F; Forlenza, M

2018-03-19

We recently reported on a successful vaccine for carp against SVCV based on the intramuscular injection of a DNA plasmid encoding the SVCV glycoprotein (SVCV-G). This shows that the intramuscular (i.m.) route of vaccination is suitable to trigger protective responses against SVCV, and that the SVCV G-protein is a suitable vaccine antigen. Yet, despite the general success of DNA vaccines, especially against fish rhabdoviruses, their practical implementation still faces legislative as well as consumer's acceptance concerns. Furthermore, the i.m. route of plasmid administration is not easily combined with most of the current vaccination regimes largely based on intraperitoneal or immersion vaccination. For this reason, in the current study we evaluated possible alternatives to a DNA-based i.m. injectable vaccine using the SVCV-G protein as the vaccine antigen. To this end, we tested two parallel approaches: the first based on the optimization of an alginate encapsulation method for oral delivery of DNA and protein antigens; the second based on the baculovirus recombinant expression of transmembrane SVCV-G protein in insect cells, administered as whole-cell subunit vaccine through the oral and injection route. In addition, in the case of the oral DNA vaccine, we also investigated the potential benefits of the mucosal adjuvants Escherichia coli lymphotoxin subunit B (LTB). Despite the use of various vaccine types, doses, regimes, and administration routes, no protection was observed, contrary to the full protection obtained with our reference i.m. DNA vaccine. The limited protection observed under the various conditions used in this study, the nature of the host, of the pathogen, the type of vaccine and encapsulation method, will therefore be discussed in details to provide an outlook for future vaccination strategies against SVCV. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Soluble HLA-G: Are They Clinically Relevant?

PubMed Central

Pistoia, Vito; Morandi, Fabio; Wang, Xinhui; Ferrone, Soldano

2007-01-01

HLA-G is a non-classical HLA-class Ib molecule with multiple immunoregulatory properties. Its main function in physiological conditions is to abrogate maternal NK cell activity against foetal tissue and to establish immune tolerance at maternal-foetal interface. HLA-G is expressed not only as a membrane bound molecule on the surface of cells, but also as a soluble moiety in body fluids. The major isoforms of HLA-G present in serum are soluble HLA-G1 and HLA-G5 which are generated by shedding or proteolytic cleavage of the membrane bound isoform and by secretion of a soluble isoform, respectively. Here we review the data about soluble HLA-G (sHLA-G) serum levels in different pathological conditions, including immune-mediated disorders, transplantation, and malignancies. In particular, we focus on sHLA-G expression and function in human neuroblastoma, a pediatric tumor, with special emphasis on a novel potential immuno escape mechanism utilized by NB to instruct monocytes to produce and release sHLA-G. Finally, the potential clinical relevance of sHLA-G serum levels is discussed. PMID:17825579

High-level expression, purification, crystallization and preliminary X-ray crystallographic studies of the receptor binding domain of botulinum neurotoxin serotype D

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Yanfeng; Gao, Xiaoli; Qin, Lin

2010-12-01

Botulinum neurotoxins (BoNTs) are highly toxic proteins for humans and can cause neuroparalytic disease botulism. Due to the limitations of production and manipulation of holoenzymes, expressing non-toxic heavy chain receptor binding domains (HCR) has become a common strategy for vaccine and antibody development. Meanwhile, large quantities and highly purified soluble proteins are required for research areas such as antibody maturation and structural biology. We present high level expression and purification of the BoNT serotype D HCR in E. coli using a codon-optimized cDNA. By varying expression conditions, especially at low temperature, the protein was expressed at a high level withmore » high solubility. About 150-200 mg protein was purified to >90% purity from 1 L cell culture. The recombinant D_HCR was crystallized and the crystals diffracted to 1.65 Å resolution. The crystals belong to space group P212121 with unit cell dimensions a = 60.8 Å, b = 89.7 Å, c = 93.9 Å. Preliminary crystallographic data analysis revealed one molecule in asymmetric unit.« less

A stable live bacterial vaccine.

PubMed

Kunda, Nitesh K; Wafula, Denis; Tram, Meilinn; Wu, Terry H; Muttil, Pavan

2016-06-01

Formulating vaccines into a dry form enhances its thermal stability. This is critical to prevent administering damaged and ineffective vaccines, and to reduce its final cost. A number of vaccines in the market as well as those being evaluated in the clinical setting are in a dry solid state; yet none of these vaccines have achieved long-term stability at high temperatures. We used spray-drying to formulate a recombinant live attenuated Listeria monocytogenes (Lm; expressing Francisella tularensis immune protective antigen pathogenicity island protein IglC) bacterial vaccine into a thermostable dry powder using various sugars and an amino acid. Lm powder vaccine showed minimal loss in viability when stored for more than a year at ambient room temperature (∼23°C) or for 180days at 40°C. High temperature viability was achieved by maintaining an inert atmosphere in the storage container and removing oxygen free radicals that damage bacterial membranes. Further, in vitro antigenicity was confirmed by infecting a dendritic cell line with cultures derived from spray dried Lm and detection of an intracellularly expressed protective antigen. A combination of stabilizing excipients, a cost effective one-step drying process, and appropriate storage conditions could provide a viable option for producing, storing and transporting heat-sensitive vaccines, especially in regions of the world that require them the most. Copyright © 2016 Elsevier B.V. All rights reserved.

Correlative Gene Expression to Protective Seroconversion in Rift Valley Fever Vaccinates.

PubMed

Laughlin, Richard C; Drake, Kenneth L; Morrill, John C; Adams, L Garry

2016-01-01

Rift Valley fever Virus (RVFV), a negative-stranded RNA virus, is the etiological agent of the vector-borne zoonotic disease, Rift Valley fever (RVF). In both humans and livestock, protective immunity can be achieved through vaccination. Earlier and more recent vaccine trials in cattle and sheep demonstrated a strong neutralizing antibody and total IgG response induced by the RVF vaccine, authentic recombinant MP-12 (arMP-12). From previous work, protective immunity in sheep and cattle vaccinates normally occurs from 7 to 21 days after inoculation with arMP-12. While the serology and protective response induced by arMP-12 has been studied, little attention has been paid to the underlying molecular and genetic events occurring prior to the serologic immune response. To address this, we isolated RNA from whole blood of vaccinated calves over a time course of 21 days before and after vaccination with arMP-12. The time course RNAs were sequenced by RNASeq and bioinformatically analyzed. Our results revealed time-dependent activation or repression of numerous gene ontologies and pathways related to the vaccine induced immune response and its regulation. Additional bioinformatic analyses identified a correlative relationship between specific host immune response genes and protective immunity prior to the detection of protective serum neutralizing antibody responses. These results contribute an important proof of concept for identifying molecular and genetic components underlying the immune response to RVF vaccination and protection prior to serologic detection.

Salmonella-based plague vaccines for bioterrorism.

PubMed

Calhoun, Leona Nicole; Kwon, Young-Min

2006-04-01

Yersinia pestis, the causative agent of plague, is an emerging threat as a means of bioterrorism. Accordingly, the Working Group on Civilian Biodefense, as well as the Centers for Disease Control and Prevention, has specified Y. pestis as a prime candidate for use in bioterrorism. As the threat of bioterrorism increases, so does the need for an effective vaccine against this potential agent. Experts agree that a stable, non-invasive vaccine would be necessary for the rapid large-scale immunization of a population following a bioterrorism attack. Thus far, live Salmonella-based oral vaccines show the most potential for this purpose. When delivered via a mucosal route, Salmonella-based plague vaccines show the ability to protect against the deadly pneumonic form of plague. Also, mass production, distribution, and administration are easier and less costly for attenuated Salmonella-based plague vaccines than for plague vaccines consisting of purified proteins. Most attenuated Salmonella-based plague vaccines have utilized a plasmid-based expression system to deliver plague antigen(s) to the mucosa. However, these systems are frequently associated with plasmid instability, an increased metabolic burden upon the vaccine strain, and highly undesirable antibiotic resistance genes. The future of Salmonella-based plague vaccines seems to lie in the use of chromosomally encoded plague antigens and the use of in vivo inducible promoters to drive their expression. This method of vaccine development has been proven to greatly increase the retention of foreign genes, and also eliminates the need for antibiotic resistance genes within Salmonella-based vaccines.

What maintains parental support for vaccination when challenged by anti-vaccination messages? A qualitative study.

PubMed

Leask, Julie; Chapman, Simon; Hawe, Penelope; Burgess, Margaret

2006-11-30

This study sought to explore how parents respond to competing media messages about vaccine safety. Six focus groups with mothers of infants were shown television vignettes of typical pro- and anti-vaccination claims. Thematic analysis of transcripts was undertaken. Mothers expressed surprise and concern about alleged vaccine risks but quickly reinstated their support for vaccination by deference to authority figures; type-casting immunisation opponents; and notions of anticipatory regret, good parenting and social responsibility. We conclude that personal experiences, value systems and level of trust in health professionals are fundamental to parental decision making about vaccination. Vaccination advocacy should increase the focus on matters of process such as maintaining trust and public confidence, particularly in health professionals. Stories about people affected by vaccine-preventable diseases need to re-enter the public discourse.

Immunogenicity of recombinant vaccinia virus vaccines co-expressing GP3/GP5 of European PRRSV and Cap protein of PCV2 in pigs.

PubMed

Han, Jicheng; Ma, Haibin; Cao, Liang; Jing, Jie; Xiao, Pengpeng; Sun, Wenchao; Xie, Changzhan; Wen, Shubo; Li, Yiquan; Tian, Mingyao; Lu, Huijun; Jin, Ningyi

2018-02-01

Porcine reproductive and respiratory syndrome (PRRS) is almost always caused by the North American strain of PRRS virus (PRRSV) in China; the European genotype of PRRSV has emerged in China. The mixed infection of PRRSV and Porcine circovirus type 2 virus (PCV2) are always found in pigs and PRRSV-augmented PCV2 replication and serious clinical symptoms. Current vaccines cannot protect mixed European PRRSV and PCV2 infections. Therefore, the development of a safe and effective new vaccine to prevent and control the mixed infection of European PRRSV and PCV2 is both urgent and necessary. In this study, we developed a recombinant vaccinia vaccine co-expressing the GP3 and GP5 proteins of European PRRSV and the ORF2 protein of PCV2 and evaluated the immunogenicity and its protective effects and its inactivated vaccine in pigs. The recombinant vaccinia vaccine and its inactivated vaccine both elicited significant humoral and cellular immune responses with a higher level of specific antibody responses and T-lymphocyte proliferation than the control group. Furthermore, the pigs inoculated with the recombinant vaccinia vaccine were completely protected against challenge with 10 5 TCID 50 of European PRRSV strain LV. These data suggest that the recombinant vaccinia vaccine is a potential candidate vaccine against European PRRSV and PCV2.

Ecdysone receptor isoform-B mediates soluble trehalase expression to regulate growth and development in the mirid bug, Apolygus lucorum (Meyer-Dür).

PubMed

Tan, Y-A; Xiao, L-B; Zhao, J; Xiao, Y-F; Sun, Y; Bai, L-X

2015-12-01

Ecdysone receptor (EcR) is the hormonal receptor of ecdysteroids and strictly regulates growth and development in insects. However, the action mechanism of EcR is not very clear. In this study, the cDNA of EcR isoform-B was cloned from Apolygus lucorum (AlEcR-B) and its expression profile was investigated. We reduced AlEcR-B mRNA expression using systemic RNA interference in vivo, and obtained knockdown specimens. Examination of these specimens indicated that AlEcR-B is required for nymphal survival, and that reduced expression is associated with longer development time and lower nymphal weight. To investigate the underlying molecular mechanism of the observed suppression effects, we selected trehalase for a detailed study. Transcript encoding soluble trehalase (AlTre-1) was up-regulated by 20-hydroxyecdysone and in agreement with the mRNA expression of AlEcR-B. The expression profile of AlTre-1, soluble trehalase activity and translated protein level in the midgut of surviving nymphs were down-regulated, compared with controls, after the knockdown expression of AlEcR-B. By contrast, membrane-bound trehalase activity, the related gene expression and translated protein level remained at their initial levels. However, trehalose content significantly increased and the glucose content significantly decreased under the same conditions. We propose that AlEcR-B controls normal carbohydrate metabolism by mediating the expression of AlTre-1 to regulate the growth and development in A. lucorum, which provide an extended information into the functions of AlEcR-B. © 2015 The Royal Entomological Society.

Pre-vaccination inflammation and B-cell signalling predict age-related hyporesponse to hepatitis B vaccination

PubMed Central

Fourati, Slim; Cristescu, Razvan; Loboda, Andrey; Talla, Aarthi; Filali, Ali; Railkar, Radha; Schaeffer, Andrea K.; Favre, David; Gagnon, Dominic; Peretz, Yoav; Wang, I-Ming; Beals, Chan R.; Casimiro, Danilo R.; Carayannopoulos, Leonidas N.; Sékaly, Rafick-Pierre

2016-01-01

Aging is associated with hyporesponse to vaccination, whose mechanisms remain unclear. In this study hepatitis B virus (HBV)-naive older adults received three vaccines, including one against HBV. Here we show, using transcriptional and cytometric profiling of whole blood collected before vaccination, that heightened expression of genes that augment B-cell responses and higher memory B-cell frequencies correlate with stronger responses to HBV vaccine. In contrast, higher levels of inflammatory response transcripts and increased frequencies of pro-inflammatory innate cells correlate with weaker responses to this vaccine. Increased numbers of erythrocytes and the haem-induced response also correlate with poor response to the HBV vaccine. A transcriptomics-based pre-vaccination predictor of response to HBV vaccine is built and validated in distinct sets of older adults. This moderately accurate (area under the curve≈65%) but robust signature is supported by flow cytometry and cytokine profiling. This study is the first that identifies baseline predictors and mechanisms of response to the HBV vaccine. PMID:26742691

Vaccination of horses with a recombinant modified vaccinia Ankara virus (MVA) expressing African horse sickness (AHS) virus major capsid protein VP2 provides complete clinical protection against challenge.

PubMed

Alberca, Berta; Bachanek-Bankowska, Katarzyna; Cabana, Marta; Calvo-Pinilla, Eva; Viaplana, Elisenda; Frost, Lorraine; Gubbins, Simon; Urniza, Alicia; Mertens, Peter; Castillo-Olivares, Javier

2014-06-17

African horse sickness virus (AHSV) is an arthropod-borne pathogen that infects all species of equidae and causes high mortality in horses. Previously, a recombinant modified vaccinia Ankara (MVA) virus expressing the protein VP2 of AHSV serotype 4 was shown to induce virus neutralising antibodies in horses and protected interferon alpha receptor gene knock-out mice (IFNAR -/-) against virulent AHSV challenge. This study builds on the previous work, examining the protective efficacy of MVA-VP2 vaccination in the natural host of AHSV infection. A study group of 4 horses was vaccinated twice with a recombinant MVA virus expressing the major capsid protein (VP2) of AHSV serotype 9. Vaccinated animals and a control group of unvaccinated horses were then challenged with a virulent strain of AHSV-9. The vaccinated animals were completely protected against clinical disease and also against viraemia as measured by standard end-point dilution assays. In contrast, all control horses presented viraemia after challenge and succumbed to the infection. These results demonstrate the potential of recombinant MVA viruses expressing the outer capsid VP2 of AHSV as a protective vaccine against AHSV infection in the field. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

Coadministration of Recombinant Adenovirus Expressing GM-CSF with Inactivated H5N1 Avian Influenza Vaccine Increased the Immune Responses and Protective Efficacy Against a Wild Bird Source of H5N1 Challenge.

PubMed

Wang, Xiangwei; Wang, Xinglong; Jia, Yanqing; Wang, Chongyang; Tang, Qiuxia; Han, Qingsong; Xiao, Sa; Yang, Zengqi

2017-10-01

Wild birds play a key role in the spread of avian influenza virus (AIV). There is a continual urgent requirement for AIV vaccines to address the ongoing genetic changes of AIV. In the current study, we trialed a novel AIV vaccine against the wild bird source of H5N1 type AIV with recombinant adenovirus expressing granulocyte monocyte colony-stimulating factor (GM-CSF) as an adjuvant. A total of 150-day-old commercial chicks, with AIV-maternal-derived antibody, were divided into 6 groups. The primary vaccination was performed at day 14 followed by a subsequent boosting and intramuscular challenge on day 28 and 42, respectively. Recombinant GM-CSF (rGM-CSF) expressed by adenovirus, named as rAd-GM-CSF, raised the hemagglutination inhibition (HI) titers (log 2 ) against AIV from 7.0 (vaccinate with inactivated vaccine alone) to 8.4 after booster immunization. Moreover, the rGM-CSF addition markedly increased the expression of interferon-γ, interleukin-4, and major histocompatibility complex-II in the lungs, compared with those immunized with inactivated vaccine alone on day 29, that is, 18 h post booster immunization. Following challenge, chicks inoculated with the inactivated AIV vaccine and rAd-GM-CSF together exhibited mild clinical signs and 62% survivals compared to 33% in the group immunized with inactivated AIV vaccine alone. Higher level of HI titers, immune related molecule expressions, and protection ratio demonstrates a good potential of rGM-CSF in improving humoral and cell mediated immune responses of inactivated AIV vaccines.

High levels of the soluble form of CD30 molecule in rheumatoid arthritis (RA) are expression of CD30+ T cell involvement in the inflamed joints.

PubMed Central

Gerli, R; Muscat, C; Bistoni, O; Falini, B; Tomassini, C; Agea, E; Tognellini, R; Biagini, P; Bertotto, A

1995-01-01

The CD30 is a surface molecule expressed by Th2-type lymphokine-producing T cells upon activation. CD30-expressing activated T cells release a soluble form of the molecule, which can be detectable both in vitro and in vivo. In the present study, high levels of soluble CD30 were found in peripheral blood and synovial fluid from patients with RA. However, CD30+ CD3+ cells, either CD4+ or CD8+, were significantly present in synovial fluid, but not in peripheral blood, of RA patients. Serum values of soluble CD30 were higher in active than inactive RA patients and directly correlated with rheumatoid factor serum titres. These data strongly support an involvement of CD30+ T cells in the immune processes of rheumatoid synovitis, and may suggest a relationship between Th2-type cytokine-secreting T cells and the pathological response in RA. PMID:8536371

[Benefit-risk assessment of vaccination strategies].

PubMed

Hanslik, Thomas; Boëlle, Pierre Yves

2007-04-01

This article summarises the various stages of the risk/benefit assessment of vaccination strategies. Establishing the awaited effectiveness of a vaccination strategy supposes to have an epidemiologic description of the disease to be prevented. The effectiveness of the vaccine strategy will be thus expressed in numbers of cases, hospitalizations or deaths avoided. The effectiveness can be direct, expressed as the reduction of the incidence of the infectious disease in the vaccinated subjects compared to unvaccinated subjects. It can also be indirect, the unvaccinated persons being protected by the suspension in circulation of the pathogenic agent, consecutive to the implementation of the vaccination campaign. The risks of vaccination related to the adverse effects detected during the clinical trials preceding marketing are well quantified, but other risks can occur after marketing: e.g., serious and unexpected adverse effects detected by vaccinovigilance systems, or risk of increase in the age of cases if the vaccination coverage is insufficient. The medico-economic evaluation forms a part of the risks/benefit assessment, by positioning the vaccine strategy comparatively with other interventions for health. Epidemiologic and vaccinovigilance informations must be updated very regularly, which underlines the need for having an operational and reliable real time monitoring system to accompany the vaccination strategies. Lastly, in the context of uncertainty which often accompanies the risks/benefit assessments, it is important that an adapted communication towards the public and the doctors is planned.

Wheat germ cell-free expression: Two detergents with a low critical micelle concentration allow for production of soluble HCV membrane proteins.

PubMed

Fogeron, Marie-Laure; Badillo, Aurélie; Jirasko, Vlastimil; Gouttenoire, Jérôme; Paul, David; Lancien, Loick; Moradpour, Darius; Bartenschlager, Ralf; Meier, Beat H; Penin, François; Böckmann, Anja

2015-01-01

Membrane proteins are notoriously difficult to express in a soluble form. Here, we use wheat germ cell-free expression in the presence of various detergents to produce the non-structural membrane proteins 2, 4B and 5A of the hepatitis C virus (HCV). We show that lauryl maltose neopentyl glycol (MNG-3) and dodecyl octaethylene glycol ether (C12E8) detergents can yield essentially soluble membrane proteins at detergent concentrations that do not inhibit the cell-free reaction. This finding can be explained by the low critical micelle concentration (CMC) of these detergents, which keeps the monomer concentrations low while at the same time providing the necessary excess of detergent concentration above CMC required for full target protein solubilization. We estimate that a tenfold excess of detergent micelles with respect to the protein concentration is sufficient for solubilization, a number that we propose as a guideline for detergent screening assays. Copyright © 2014 Elsevier Inc. All rights reserved.

Evaluation of a vectored equine herpesvirus type 1 (EHV-1) vaccine expressing H3 haemagglutinin in the protection of dogs against canine influenza.

PubMed

Rosas, Cristina; Van de Walle, Gerlinde R; Metzger, Stephan M; Hoelzer, Karin; Dubovi, Edward J; Kim, Sung G; Parrish, Colin R; Osterrieder, Nikolaus

2008-05-02

In 2004, canine influenza virus (CIV) was identified as a respiratory pathogen of dogs for the first time and found to be closely related to H3N8 equine influenza virus (EIV). We generated a recombinant vectored vaccine that expresses H3 of a recent isolate of EIV using equine herpesvirus type 1 (EHV-1) as the delivery vehicle. This EHV-1 vectored vaccine exhibited robust and stable EIV H3 expression and induced a strong influenza virus-specific response in both mice and dogs upon intranasal or subcutaneous administration. Furthermore, upon challenge with the recent CIV isolate A/canine/PA/10915-07, protection of vaccinated dogs could be demonstrated by a significant reduction in clinical sings, and, more importantly, by a significant reduction in virus shedding. We concluded that the EHV-1/H3 recombinant vector can be a valuable alternative for protection of dogs against clinical disease induced by CIV and can significantly reduce virus spread.

Evaluation of a vectored equine herpesvirus type 1 (EHV-1) vaccine expressing H3 haemagglutinin in the protection of dogs against canine influenza

PubMed Central

Rosas, Cristina; Van de Walle, Gerlinde R.; Metzger, Stephan M.; Hoelzer, Karin; Dubovi, Edward J.; Kim, Sung G.; Parrish, Colin R.; Osterrieder, Nikolaus

2008-01-01

In 2004, canine influenza virus (CIV) was identified as a respiratory pathogen of dogs for the first time and is closely related to H3N8 equine influenza virus (EIV). We generated a recombinant vectored vaccine that expresses H3 of a recent isolate of EIV using equine herpesvirus type 1 (EHV-1) as the delivery vehicle. This EHV-1 vectored vaccine exhibited robust and stable EIV H3 expression and induced a strong influenza virus-specific response in both mice and dogs upon intranasal or subcutaneous administration. Furthermore, upon challenge with the recent CIV isolate A/canine/PA/10915-07, protection of vaccinated dogs could be demonstrated by a significant reduction in clinical sings, and, more importantly, by a significant reduction in virus shedding. We concluded that the EHV-1/H3 recombinant vector can be a valuable alternative for protection of dogs against clinical disease induced by CIV and can significantly reduce spread. PMID:18407383

The need and challenges for development of an Epstein-Barr virus vaccine

PubMed Central

Cohen, Jeffrey I.; Mocarski, Edward S.; Raab-Traub, Nancy; Corey, Lawrence; Nabel, Gary J.

2012-01-01

Epstein-Barr virus (EBV) is the major cause of infectious mononucleosis and is associated with several malignancies including nasopharyngeal carcinoma, gastric carcinoma, Hodgkin lymphoma, Burkitt lymphoma, and lymphoma after organ or stem cell transplant. A candidate vaccine containing soluble EBV glycoprotein gp350 protected cottontop tamarins from EBV lymphoma after challenge with EBV. In the only phase 2 trial of an EBV vaccine in humans, soluble gp350 in alum and monophosphoryl lipid A adjuvant reduced the rate of infectious mononucleosis in EBV seronegative adults, but did not affect the rate of EBV infection. A peptide vaccine corresponding to EBV latency proteins has been tested in a small number of adults to prevent infectious mononucleosis. Some of the barriers to development of an EBV vaccine include (a) whether additional viral proteins in addition to gp350 would be more effective for preventing mononucleosis or EBV malignancies, (b) the difficulty of performing clinical trials to prevent EBV associated malignancies in the absence of good surrogate markers for tumor development, and the long period of time between primary EBV infection and development of many EBV tumors, (c) the lack of knowledge of immune correlates for protection against EBV infection and disease, (d) the limitations in animal models to study protection against EBV infection and disease, and (e) the need for additional information on the economic and societal burden of infectious mononucleosis to assess the cost-benefit of a prophylactic vaccine. PMID:23598481

Self-assembly of virus-like particles of porcine circovirus type 2 capsid protein expressed from Escherichia coli

PubMed Central

2010-01-01

Background Porcine circovirus 2 (PCV2) is a serious problem to the swine industry and can lead to significant negative impacts on profitability of pork production. Syndrome associated with PCV2 is known as porcine circovirus closely associated with post-weaning multisystemic wasting syndrome (PMWS). The capsid (Cap) protein of PCV2 is a major candidate antigen for development of recombinant vaccine and serological diagnostic method. The recombinant Cap protein has the ability to self-assemble into virus-like particles (VLPs) in vitro, it is particularly opportunity to develop the PV2 VLPs vaccine in Escherichia coli,(E.coli ), because where the cost of the vaccine must be weighed against the value of the vaccinated pig, when it was to extend use the VLPs vaccine of PCV2. Results In this report, a highly soluble Cap-tag protein expressed in E.coli was constructed with a p-SMK expression vector with a fusion tag of small ubiquitin-like modifiers (SUMO). The recombinant Cap was purified using Ni2+ affinity resins, whereas the tag was used to remove the SUMO protease. Simultaneously, the whole native Cap protein was able to self-assemble into VLPs in vitro when viewed under an electron microscope. The Cap-like particles had a size and shape that resembled the authentic Cap. The result could also be applied in the large-scale production of VLPs of PCV2 and could be used as a diagnostic antigen or a potential VLP vaccine against PCV2 infection in pigs. Conclusion we have, for the first time, utilized the SUMO fusion motif to successfully express the entire authentic Cap protein of PCV2 in E. coli. After the cleavage of the fusion motif, the nCap protein has the ability to self-assemble into VLPs, which can be used as as a potential vaccine to protect pigs from PCV2-infection. PMID:20646322

Construction and phase I clinical evaluation of the safety and immunogenicity of a candidate enterotoxigenic Escherichia coli vaccine strain expressing colonization factor antigen CFA/I.

PubMed

Turner, Arthur K; Beavis, Juliet C; Stephens, Jonathan C; Greenwood, Judith; Gewert, Cornelia; Thomas, Nicola; Deary, Alison; Casula, Gabriella; Daley, Alexandra; Kelly, Paul; Randall, Roger; Darsley, Michael J

2006-02-01

Oral delivery of toxin-negative derivatives of enterotoxigenic Escherichia coli (ETEC) that express colonization factor antigens (CFA) with deletions of the aroC, ompC, ompF, and toxin genes may be an effective approach to vaccination against ETEC-associated diarrhea. We describe the creation and characterization of an attenuated CFA/I-expressing ETEC vaccine candidate, ACAM2010, from a virulent isolate in which the heat-stable enterotoxin (ST) and CFA/I genes were closely linked and on the same virulence plasmid as the enteroaggregative E. coli heat-stable toxin (EAST1) gene. A new suicide vector (pJCB12) was constructed and used to delete the ST and EAST1 genes and to introduce defined deletion mutations into the aroC, ompC, and ompF chromosomal genes. A phase I trial, consisting of an open-label dose escalation phase in 18 adult outpatient volunteers followed by a placebo-controlled double-blind phase in an additional 31 volunteers, was conducted. The vaccine was administered in two formulations, fresh culture and frozen suspension. These were both well tolerated, with no evidence of significant adverse events related to vaccination. Immunoglobulin A (IgA) and IgG antibody-secreting cells specific for CFA/I were assayed by ELISPOT. Positive responses (greater than twofold increase) were seen in 27 of 37 (73%) subjects who received the highest dose level of vaccine (nominally 5 x 10(9) CFU). Twenty-nine of these volunteers were secreting culturable vaccine organisms at day 3 following vaccination; five were still positive on day 7, with a single isolation on day 13. This live attenuated bacterial vaccine is safe and immunogenic in healthy adult volunteers.

A novel, disruptive vaccination technology: self-adjuvanted RNActive(®) vaccines.

PubMed

Kallen, Karl-Josef; Heidenreich, Regina; Schnee, Margit; Petsch, Benjamin; Schlake, Thomas; Thess, Andreas; Baumhof, Patrick; Scheel, Birgit; Koch, Sven D; Fotin-Mleczek, Mariola

2013-10-01

Nucleotide based vaccines represent an enticing, novel approach to vaccination. We have developed a novel immunization technology, RNActive(®) vaccines, that have two important characteristics: mRNA molecules are used whose protein expression capacity has been enhanced by 4 to 5 orders of magnitude by modifications of the nucleotide sequence with the naturally occurring nucleotides A (adenosine), G (guanosine), C (cytosine), U (uridine) that do not affect the primary amino acid sequence. Second, they are complexed with protamine and thus activate the immune system by involvement of toll-like receptor (TLR) 7. Essentially, this bestows self-adjuvant activity on RNActive(®) vaccines. RNActive(®) vaccines induce strong, balanced immune responses comprising humoral and cellular responses, effector and memory responses as well as activation of important subpopulations of immune cells, such as Th1 and Th2 cells. Pre-germinal center and germinal center B cells were detected in human patients upon vaccination. RNActive(®) vaccines successfully protect against lethal challenges with a variety of different influenza strains in preclinical models. Anti-tumor activity was observed preclinically under therapeutic as well as prophylactic conditions. Initial clinical experiences suggest that the preclinical immunogenicity of RNActive(®) could be successfully translated to humans.

Immunotoxicity of aflatoxin B1: Impairment of the cell-mediated response to vaccine antigen and modulation of cytokine expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Meissonnier, Guylaine M.; Alltech-France, European Regulatory Department, F-92593 Levallois-Perret; Pinton, Philippe

Aflatoxin B1 (AFB1), a mycotoxin produced by Aspergillus flavus or A. parasiticus, is a frequent contaminant of food and feed. This toxin is hepatotoxic and immunotoxic. The present study analyzed in pigs the influence of AFB1 on humoral and cellular responses, and investigated whether the immunomodulation observed is produced through interference with cytokine expression. For 28 days, pigs were fed a control diet or a diet contaminated with 385, 867 or 1807 {mu}g pure AFB1/kg feed. At days 4 and 15, pigs were vaccinated with ovalbumin. AFB1 exposure, confirmed by an observed dose-response in blood aflatoxin-albumin adduct, had no majormore » effect on humoral immunity as measured by plasma concentrations of total IgA, IgG and IgM and of anti-ovalbumin IgG. Toxin exposure did not impair the mitogenic response of lymphocytes but delayed and decreased their specific proliferation in response to the vaccine antigen, suggesting impaired lymphocyte activation in pigs exposed to AFB1. The expression level of pro-inflammatory (TNF-{alpha}, IL-1{beta}, IL-6, IFN-{gamma}) and regulatory (IL-10) cytokines was assessed by real-time PCR in spleen. A significant up-regulation of all 5 cytokines was observed in spleen from pigs exposed to the highest dose of AFB1. In pigs exposed to the medium dose, IL-6 expression was increased and a trend towards increased IFN-{gamma} and IL-10 was observed. In addition we demonstrate that IL-6 impaired in vitro the antigenic- but not the mitogenic-induced proliferation of lymphocytes from control pigs vaccinated with ovalbumin. These results indicate that AFB1 dietary exposure decreases cell-mediated immunity while inducing an inflammatory response. These impairments in the immune response could participate in failure of vaccination protocols and increased susceptibility to infections described in pigs exposed to AFB1.« less

High-level soluble expression of a thermostable xylanase from thermophilic fungus Thermomyces lanuginosus in Escherichia coli via fusion with OsmY protein.

PubMed

Le, Yilin; Wang, Huilei

2014-07-01

A thermostable xylanase is encoded by xynA from fungus Thermomyces lanuginosus. The problem emerged from overexpression of xynA in Escherichia coli has been the formation of inclusion bodies. Here we describe the xynA was fused with the hyperosmotically inducible periplasmic protein of E. coli, OsmY. The fusion protein OsmY-xynA was expressed as almost all soluble form. The soluble expression level of fusion protein reached 98±6U/ml when cells containing pET-OsmY-xynA were expressed without IPTG induction at 37°C. The induction is probably due to auto-induction due to lactose in the medium (Studier (2005) [21]). The cells harboring pET-OsmY-xynA expressed an activity level about 24 times higher than that expressed from pET-20b-xynA. Xylanase activity was observed in the extracellular (36±1.3U/ml) and the periplasmic (42±4U/ml) when cells containing pET-OsmY-xynA were induced without IPTG addition. After the cold osmotic shock procedure followed by nickel affinity chromatography, the purified fusion protein showed a single band on SDS-PAGE gel with a molecular mass of 44kDa. The purified fusion enzyme exhibited the highest activity at 65°C and pH 6.0. Copyright © 2014 Elsevier Inc. All rights reserved.

Edible vaccines against veterinary parasitic diseases--current status and future prospects.

PubMed

Jacob, Siju S; Cherian, Susan; Sumithra, T G; Raina, O K; Sankar, M

2013-04-08

Protection of domestic animals against parasitic infections remains a major challenge in most of the developing countries, especially in the surge of drug resistant strains. In this circumstance vaccination seems to be the sole practical strategy to combat parasites. Most of the presently available live or killed parasitic vaccines possess many disadvantages. Thus, expression of parasitic antigens has seen a continued interest over the past few decades. However, only a limited success was achieved using bacterial, yeast, insect and mammalian expression systems. This is witnessed by an increasing number of reports on transgenic plant expression of previously reported and new antigens. Oral delivery of plant-made vaccines is particularly attractive due to their exceptional advantages. Moreover, the regulatory burden for veterinary vaccines is less compared to human vaccines. This led to an incredible investment in the field of transgenic plant vaccines for veterinary purpose. Plant based vaccine trials have been conducted to combat various significant parasitic diseases such as fasciolosis, schistosomosis, poultry coccidiosis, porcine cycticercosis and ascariosis. Besides, passive immunization by oral delivery of antibodies expressed in transgenic plants against poultry coccidiosis is an innovative strategy. These trials may pave way to the development of promising edible veterinary vaccines in the near future. As the existing data regarding edible parasitic vaccines are scattered, an attempt has been made to assemble the available literature. Copyright © 2013 Elsevier Ltd. All rights reserved.

Effects of insulin, triiodothyronine and fat soluble vitamins on adipocyte differentiation and LPL gene expression in the stromal-vascular cells of red sea bream, Pagrus major.

PubMed

Oku, Hiromi; Tokuda, Masaharu; Okumura, Takuji; Umino, Tetsuya

2006-07-01

Various kinds of hormones including insulin, triiodothyronine (T(3)) and fat-soluble vitamins have been proposed as mediators of adipocyte differentiation in mammals. To investigate the factors which are responsible for fish adipocyte differentiation, we developed a serum-free culture system of stromal-vascular cells of red sea bream adipose tissue and examined the effects of bovine insulin, T(3), and fat-soluble vitamins (all-trans retinoic acid, retinyl acetate and 1,25-dihydroxyvitamin D(3)) on the differentiation-linked expression of the lipoprotein lipase (LPL) gene. As assessed by the increase in LPL gene expression after 3 day cultivation, like in mammalian adipocytes, insulin enhanced the adipocyte differentiation in a concentration-dependent manner. During 2 week cultivation, bovine insulin promoted lipid accumulation in differentiating adipocytes concentration-dependently until the terminal differentiation. These results indicate that the differentiation of fish adipocytes is inducible by insulin alone. T(3) alone had no effect but enhanced the differentiation-linked LPL gene expression in the presence of insulin. Fat-soluble vitamins, unlike in mammalian adipocytes, did not show any significant effects. The method developed in this study should be of interest for the characterization of factors involved in fish adipocyte differentiation.

Safety of an Escherichia coli-expressed bivalent human papillomavirus (types 16 and 18) L1 virus-like particle vaccine: an open-label phase I clinical trial.

PubMed

Hu, Yue-Mei; Huang, Shou-Jie; Chu, Kai; Wu, Ting; Wang, Zhong-Ze; Yang, Chang-Lin; Cai, Jia-Ping; Jiang, Han-Min; Wang, Yi-Jun; Guo, Meng; Liu, Xiao-Hui; Huang, Hong-Jiang; Zhu, Feng-Cai; Zhang, Jun; Xia, Ning-Shao

2014-01-01

An Escherichia coli-expressed recombinant bivalent human papillomavirus (types 16 and 18) vaccine candidate has been shown to be safe and immunogenic in preclinical trials. The safety of this vaccine was analyzed in an open-label phase I clinical trial in Jiangsu province, China. Thirty-eight healthy women from 18 to 55 y of age were enrolled and vaccinated at 0, 1, and 6 mo. Adverse events that occurred within 30 d after each injection and serious adverse events that occurred throughout the study were recorded. In addition, blood parameters were tested before and after each injection. All but one woman received all 3 doses. Thirty-two (84.2%) of the participants reported adverse events, all adverse events of which were mild, of short duration and resolved spontaneously. No serious adverse events occurred during the study. Changes in blood parameters after each injection were random, mild, and not clinically significant. These preliminary results show that a new Escherichia coli-expressed recombinant HPV 16/18 bivalent vaccine is well tolerated in healthy women and support further immunogenicity and efficacy studies for this HPV vaccine candidate.

Assessment of the utility of the tomato fruit-specific E8 promoter for driving vaccine antigen expression.

PubMed

He, Zhu-Mei; Jiang, Xiao-Ling; Qi, Yu; Luo, Di-Qing

2008-06-01

To assess the utility of the tomato fruit-specific E8 gene's promoter for driving vaccine antigen expression in plant, the 2.2 kb and 1.1 kb E8 promoters were isolated and sequenced from Lycopersicon esculentum cv. Jinfeng #1. The 1.1 kb promoter was fused to vaccine antigen HBsAg M gene for the transfer to Nicotiana tabacum, and the CaMV 35S promoter was used for comparison. Cholera toxin B (ctb) gene under the control of the 1.1 kb promoter was transformed into both N. tabacum and L. esculentum. Southern blot hybridization confirmed the stable integration of the target genes into the tomato and tobacco genomes. ELISA assay showed that the expression product of HBsAg M gene under the control of the 1.1 kb E8 promoter could not be detected in transgenic tobacco tissues such as leaves, flowers, and seeds. In contrast, the expression of HBsAg M gene driven by CaMV 35S promoter could be detected in transgenic tobacco. ELISA assay for CTB proved that the 1.1 kb E8 promoter was able to direct the expression of exotic gene in ripe fruits of transgenic tomato, but expression was absent in leaf, flower, and unripe fruit of tomato, and CTB protein was not detected in transgenic tobacco tissues such as leaves, flowers, and seeds when the gene was under the control of the 1.1 kb E8 promoter. The results indicated that the E8 promoter acted not only in an organ-specific, but also in a species-specific fashion in plant transformation.

Blood-stage malaria vaccines: post-genome strategies for the identification of novel vaccine candidates.

PubMed

Ntege, Edward H; Takashima, Eizo; Morita, Masayuki; Nagaoka, Hikaru; Ishino, Tomoko; Tsuboi, Takafumi

2017-08-01

An efficacious malaria vaccine is necessary to advance the current control measures towards malaria elimination. To-date, only RTS,S/AS01, a leading pre-erythrocytic stage vaccine completed phase 3 trials, but with an efficacy of 28-36% in children, and 18-26% in infants, that waned over time. Blood-stage malaria vaccines protect against disease, and are considered effective targets for the logical design of next generation vaccines to improve the RTS,S field efficacy. Therefore, novel blood-stage vaccine candidate discovery efforts are critical, albeit with several challenges including, high polymorphisms in vaccine antigens, poor understanding of targets of naturally protective immunity, and difficulties in the expression of high AT-rich plasmodial proteins. Areas covered: PubMed ( www.ncbi.nlm.nih.gov/pubmed ) was searched to review the progress and future prospects of malaria vaccine research and development. We focused on post-genome vaccine candidate discovery, malaria vaccine development, sequence diversity, pre-clinical and clinical trials. Expert commentary: Post-genome high-throughput technologies using wheat germ cell-free protein synthesis technology and immuno-profiling with sera from malaria patients with clearly defined outcomes are highlighted to overcome current challenges of malaria vaccine candidate discovery.

Murine Antibody Responses to Cleaved Soluble HIV-1 Envelope Trimers Are Highly Restricted in Specificity

PubMed Central

Hu, Joyce K.; Crampton, Jordan C.; Cupo, Albert; Ketas, Thomas; van Gils, Marit J.; Sliepen, Kwinten; de Taeye, Steven W.; Sok, Devin; Ozorowski, Gabriel; Deresa, Isaiah; Stanfield, Robyn; Ward, Andrew B.; Burton, Dennis R.; Klasse, Per Johan; Sanders, Rogier W.; Moore, John P.

2015-01-01

ABSTRACT Generating neutralizing antibodies (nAbs) is a major goal of many current HIV-1 vaccine efforts. To be of practical value, these nAbs must be both potent and cross-reactive in order to be capable of preventing the transmission of the highly diverse and generally neutralization resistant (Tier-2) HIV-1 strains that are in circulation. The HIV-1 envelope glycoprotein (Env) spike is the only target for nAbs. To explore whether Tier-2 nAbs can be induced by Env proteins, we immunized conventional mice with soluble BG505 SOSIP.664 trimers that mimic the native Env spike. Here, we report that it is extremely difficult for murine B cells to recognize the Env epitopes necessary for inducing Tier-2 nAbs. Thus, while trimer-immunized mice raised Env-binding IgG Abs and had high-quality T follicular helper (Tfh) cell and germinal center (GC) responses, they did not make BG505.T332N nAbs. Epitope mapping studies showed that Ab responses in mice were specific to areas near the base of the soluble trimer. These areas are not well shielded by glycans and likely are occluded on virions, which is consistent with the lack of BG505.T332N nAbs. These data inform immunogen design and suggest that it is useful to obscure nonneutralizing epitopes presented on the base of soluble Env trimers and that the glycan shield of well-formed HIV Env trimers is virtually impenetrable for murine B cell receptors (BCRs). IMPORTANCE Human HIV vaccine efficacy trials have not generated meaningful neutralizing antibodies to circulating HIV strains. One possible hindrance has been the lack of immunogens that properly mimic the native conformation of the HIV envelope trimer protein. Here, we tested the first generation of soluble, native-like envelope trimer immunogens in a conventional mouse model. We attempted to generate neutralizing antibodies to neutralization-resistant circulating HIV strains. Various vaccine strategies failed to induce neutralizing antibodies to a neutralization

Estradiol rapidly inhibits soluble guanylyl cyclase expression in rat uterus

NASA Technical Reports Server (NTRS)

Krumenacker, J. S.; Hyder, S. M.; Murad, F.

2001-01-01

Previous reports that investigated the regulation of the NO/soluble guanylyl cyclase (sGC)/cGMP pathway by estrogenic compounds have focused primarily on the levels of NO, NO-producing enzymes, and cGMP in various tissues. In this study, we demonstrate that 17beta-estradiol (E2) regulates the alpha(1) and beta(1) subunits of the NO receptor, sGC, at the mRNA and protein levels in rat uterus. Using real-time quantitative PCR, we found that within 1 h of in vivo E2 administration to rats, sGC mRNA levels begin to diminish. After 3 h, there is a maximal diminution of sGC mRNA expression (sGC alpha(1) 10% and sGC beta(1) 33% of untreated). This effect was blocked by the estrogen receptor antagonist, ICI 182,780, indicating that estrogen receptor is required. The effect of E2 also was observed in vitro with incubations of uterine tissue, indicating that the response does not depend on the secondary release of other hormones or factors from other tissues. Puromycin did not block the effect, suggesting the effects occur because of preexisting factors in uterine tissues and do not require new protein synthesis. Using immunoblot analysis, we found that sGC protein levels also were reduced by E2 over a similar time course as the sGC mRNA. We conclude that sGC plays a vital role in the NO/sGC/cGMP regulatory pathway during conditions of elevated estrogen levels in the rat uterus as a result of the reduction of sGC expression.

Vaccines in development against West Nile virus.

PubMed

Brandler, Samantha; Tangy, Frederic

2013-09-30

West Nile encephalitis emerged in 1999 in the United States, then rapidly spread through the North American continent causing severe disease in human and horses. Since then, outbreaks appeared in Europe, and in 2012, the United States experienced a new severe outbreak reporting a total of 5,387 cases of West Nile virus (WNV) disease in humans, including 243 deaths. So far, no human vaccine is available to control new WNV outbreaks and to avoid worldwide spreading. In this review, we discuss the state-of-the-art of West Nile vaccine development and the potential of a novel safe and effective approach based on recombinant live attenuated measles virus (MV) vaccine. MV vaccine is a live attenuated negative-stranded RNA virus proven as one of the safest, most stable and effective human vaccines. We previously described a vector derived from the Schwarz MV vaccine strain that stably expresses antigens from emerging arboviruses, such as dengue, West Nile or chikungunya viruses, and is strongly immunogenic in animal models, even in the presence of MV pre-existing immunity. A single administration of a recombinant MV vaccine expressing the secreted form of WNV envelope glycoprotein elicited protective immunity in mice and non-human primates as early as two weeks after immunization, indicating its potential as a human vaccine.

Information Curation among Vaccine Cautious Parents: Web 2.0, Pinterest Thinking, and Pediatric Vaccination Choice.

PubMed

Sobo, Elisa J; Huhn, Arianna; Sannwald, Autumn; Thurman, Lori

2016-01-01

To learn about pediatric vaccine decision-making, we surveyed and interviewed US parents with at least one child kindergarten age or younger (N = 53). Through an anthropologically informed content analysis, we found that fully vaccinating parents (n = 33) mostly saw vaccination as routine. In contrast, selective and nonvaccinating parents (n = 20) exhibited the type of self-informed engagement that the health care system recommends. Selective vaccinators also expressed multiple, sometimes contradictory positions on vaccination that were keyed to individual children's biologies, child size, environmental hazards, specific diseases, and discrete vaccines. Rather than logical progressions, viewpoints were presented as assembled collections, reflecting contemporary information filtering and curation practices and the prevalence of collectively experienced and constructed digital "hive" narratives. Findings confirm the need for a noncategorical approach to intervention that accommodates the fluid, polyvalent nature of vaccine reasoning and the curatorial view selectively vaccinating parents take toward information while honoring their efforts at engaged healthcare consumption.

Plant-based vaccines against diarrheal diseases.

PubMed

Tacket, Carol O

2007-01-01

Every year 1.6 million deaths occur due to diarrhea related to unsafe water and inadequate sanitation-the vast majority in children under 5 years old. Safe and effective vaccines against enteric infections could contribute to control of these diseases. However, purification of protective antigens for inclusion in vaccines using traditional expression systems is expensive and unattractive to vaccine manufacturers who see the vaccine market as economically uninviting. Cost is one of the persistent barriers to deployment of new vaccines to populations that need them most urgently. Transgenic plant-derived vaccines offer a new strategy for development of safe, inexpensive vaccines against diarrheal diseases. In phase 1 clinical studies, these vaccines have been safe and immunogenic without the need for a buffer or vehicle other than the plant cell. This paper describes early clinical studies evaluating oral transgenic plant vaccines against enteric infections such as enterotoxigenic E. coli infection and norovirus.

Effects of Chicken Interferon Gamma on Newcastle Disease Virus Vaccine Immunogenicity

PubMed Central

Cardenas-Garcia, Stivalis; Dunwoody, Robert P.; Marcano, Valerie; Diel, Diego G.; Williams, Robert J.; Gogal, Robert M.; Brown, Corrie C.; Miller, Patti J.; Afonso, Claudio L.

2016-01-01

More effective vaccines are needed to control avian diseases. The use of chicken interferon gamma (chIFNγ) during vaccination is a potentially important but controversial approach that may improve the immune response to antigens. In the present study, three different systems to co-deliver chIFNγ with Newcastle disease virus (NDV) antigens were evaluated for their ability to enhance the avian immune response and their protective capacity upon challenge with virulent NDV. These systems consisted of: 1) a DNA vaccine expressing the Newcastle disease virus fusion (F) protein co-administered with a vector expressing the chIFNγ gene for in ovo and booster vaccination, 2) a recombinant Newcastle disease virus expressing the chIFNγ gene (rZJ1*L/IFNγ) used as a live vaccine delivered in ovo and into juvenile chickens, and 3) the same rZJ1*L/IFNγ virus used as an inactivated vaccine for juvenile chickens. Co-administration of chIFNγ with a DNA vaccine expressing the F protein resulted in higher levels of morbidity and mortality, and higher amounts of virulent virus shed after challenge when compared to the group that did not receive chIFNγ. The live vaccine system co-delivering chIFNγ did not enhanced post-vaccination antibody response, nor improved survival after hatch, when administered in ovo, and did not affect survival after challenge when administered to juvenile chickens. The low dose of the inactivated vaccine co-delivering active chIFNγ induced lower antibody titers than the groups that did not receive the cytokine. The high dose of this vaccine did not increase the antibody titers or antigen-specific memory response, and did not reduce the amount of challenge virus shed or mortality after challenge. In summary, regardless of the delivery system, chIFNγ, when administered simultaneously with the vaccine antigen, did not enhance Newcastle disease virus vaccine immunogenicity. PMID:27409587

Antibody responses to prime-boost vaccination with an HIV-1 gp145 envelope protein and chimpanzee adenovirus vectors expressing HIV-1 gp140.

PubMed

Emmer, Kristel L; Wieczorek, Lindsay; Tuyishime, Steven; Molnar, Sebastian; Polonis, Victoria R; Ertl, Hildegund C J

2016-10-23

Over 2 million individuals are infected with HIV type 1 (HIV-1) each year, yet an effective vaccine remains elusive. The most successful HIV-1 vaccine to date demonstrated 31% efficacy. Immune correlate analyses associated HIV-1 envelope (Env)-specific antibodies with protection, thus providing a path toward a more effective vaccine. We sought to test the antibody response from novel prime-boost vaccination with a chimpanzee-derived adenovirus (AdC) vector expressing a subtype C Env glycoprotein (gp)140 combined with either a serologically distinct AdC vector expressing gp140 of a different subtype C isolate or an alum-adjuvanted, partially trimeric gp145 from yet another subtype C isolate. Three different prime-boost regimens were tested in mice: AdC prime-protein boost, protein prime-AdC boost, and AdC prime-AdC boost. Each regimen was tested at two different doses of AdC vector in a total of six experimental groups. Sera were collected at various time points and evaluated by ELISA for Env-specific antibody binding, isotype, and avidity. Antibody functionality was assessed by pseudovirus neutralization assay. Priming with AdC followed by a protein boost or sequential immunizations with two AdC vectors induced HIV-1 Env-specific binding antibodies, including those to the variable region 2, whereas priming with protein followed by an AdC boost was relatively ineffective. Antibodies that cross-neutralized tier 1 HIV-1 from different subtypes were elicited with vaccine regimens that included immunizations with protein. Our study warrants further investigation of AdC vector and gp145 protein prime-boost vaccines and their ability to protect against acquisition in animal challenge studies.

Vaccines for the future: learning from human immunology

PubMed Central

De Gregorio, Ennio; Rappuoli, Rino

2012-01-01

Summary Conventional vaccines have been extremely successful in preventing infections by pathogens expressing relatively conserved antigens through antibody‐mediated effector mechanisms. Thanks to vaccination some diseases have been eradicated and mortality due to infectious diseases has been significantly reduced. However, there are still many infections that are not preventable with vaccination, which represent a major cause of mortality worldwide. Some of these infections are caused by pathogens with a high degree of antigen variability that cannot be controlled only by antibodies, but require a mix of humoral and cellular immune responses. Novel technologies for antigen discovery, expression and formulation allow now for the development of vaccines that can better cope with pathogen diversity and trigger multifunctional immune responses. In addition, the application of new genomic assays and systems biology approaches in human immunology can help to better identify vaccine correlates of protection. The availability of novel vaccine technologies, together with the knowledge of the distinct human immune responses that are required to prevent different types of infection, should help to rationally design effective vaccines where conventional approaches have failed. PMID:21880117

A low-invasive and effective transcutaneous immunization system using a novel dissolving microneedle array for soluble and particulate antigens.

PubMed

Matsuo, Kazuhiko; Yokota, Yayoi; Zhai, You; Quan, Ying-Shu; Kamiyama, Fumio; Mukai, Yohei; Okada, Naoki; Nakagawa, Shinsaku

2012-07-10

Transcutaneous immunization (TCI) is a promising needle-free, easy-to-use, and low-invasive vaccination method. The hydrogel patch-based TCI system induced immune responses against soluble antigens (Ags) like toxoids, but could not induce immune responses against particulate Ags. Here, as an effective TCI system against every form of Ag, we developed a dissolving microneedle array of three lengths (200, 300, or 800 μm) made of hyaluronate as a novel TCI device. Unlike conventional microneedles, the microneedles of our dissolving microneedle arrays dissolved in the skin after insertion. Each dissolving microneedle array effectively delivered both soluble and particulate Ags under the stratum corneum. TCI using these dissolving microneedle arrays induced effective immune responses in rats regardless of the Ag form that were comparable to conventional vaccination using subcutaneous immunization. In addition, application of these dissolving microneedle arrays caused only slight skin irritation. These findings suggest that our TCI system can simply, safely, and effectively improve protective immune responses for every vaccine Ag. Copyright © 2012 Elsevier B.V. All rights reserved.

Evaluation of vaccines in the EU TB Vaccine Cluster using a guinea pig aerosol infection model of tuberculosis.

PubMed

Williams, Ann; Hatch, Graham J; Clark, Simon O; Gooch, Karen E; Hatch, Kim A; Hall, Graham A; Huygen, Kris; Ottenhoff, Tom H M; Franken, Kees L M C; Andersen, Peter; Doherty, T Mark; Kaufmann, Stefan H E; Grode, Leander; Seiler, Peter; Martin, Carlos; Gicquel, Brigitte; Cole, Stewart T; Brodin, Priscille; Pym, Alexander S; Dalemans, Wilfried; Cohen, Joe; Lobet, Yves; Goonetilleke, Nilu; McShane, Helen; Hill, Adrian; Parish, Tanya; Smith, Debbie; Stoker, Neil G; Lowrie, Douglas B; Källenius, Gunilla; Svenson, Stefan; Pawlowski, Andrzej; Blake, Karen; Marsh, Philip D

2005-01-01

The TB Vaccine Cluster project funded by the EU Fifth Framework programme aims to provide novel vaccines against tuberculosis that are suitable for evaluation in humans. This paper describes the studies of the protective efficacy of vaccines in a guinea pig aerosol-infection model of primary tuberculosis. The objective was to conduct comparative evaluations of vaccines that had previously demonstrated efficacy in other animal models. Groups of 6 guinea pigs were immunized with vaccines provided by the relevant EU Vaccine Cluster partners. Survival over 17 or 26 weeks was used as the principal measure of vaccine efficacy following aerosol challenge with H37Rv. Counts of mycobacteria in lungs and spleens, and histopathological changes in the lungs, were also used to provide evidence of protection. A total of 24 vaccines were evaluated in 4 experiments each of a different design. A heterologous prime-boost strategy of DNA and MVA, each expressing Ag85A and a fusion protein of ESAT-6 and Ag85B in adjuvant, protected the guinea pigs to the same extent as BCG. Genetically modified BCG vaccines and boosted BCG strategies also protected guinea pigs to the same extent as BCG but not statistically significantly better. A relatively high aerosol-challenge dose and evaluation over a protracted time post-challenge allowed superior protection over BCG to be demonstrated by BCG boosted with MVA and fowl pox vectors expressing Ag85A.

Monocyte Chemotactic Protein 1 in Plasma from Soluble Leishmania Antigen-Stimulated Whole Blood as a Potential Biomarker of the Cellular Immune Response to Leishmania infantum

PubMed Central

Ibarra-Meneses, Ana V.; Sanchez, Carmen; Alvar, Jorge; Moreno, Javier; Carrillo, Eugenia

2017-01-01

New biomarkers are needed to identify asymptomatic Leishmania infection as well as immunity following vaccination or treatment. With the aim of finding a robust biomarker to assess an effective cellular immune response, monocyte chemotactic protein 1 (MCP-1) was examined in plasma from soluble Leishmania antigen (SLA)-stimulated whole blood collected from subjects living in a Leishmania infantum-endemic area. MCP-1, expressed 110 times more strongly than IL-2, identified 87.5% of asymptomatic subjects and verified some asymptomatic subjects close to the cutoff. MCP-1 was also significantly elevated in all patients cured of visceral leishmaniasis (VL), unlike IL-2, indicating the specific memory response generated against Leishmania. These results show MCP-1 to be a robust candidate biomarker of immunity that could be used as a marker of cure and to both select and follow the population in vaccine phase I–III human clinical trials with developed rapid, easy-to-use field tools. PMID:29033933

Comparing human papillomavirus vaccine concerns on Twitter: a cross-sectional study of users in Australia, Canada and the UK

PubMed Central

Shapiro, Gilla K; Surian, Didi; Dunn, Adam G; Perry, Ryan; Kelaher, Margaret

2017-01-01

Objective Opposition to human papillomavirus (HPV) vaccination is common on social media and has the potential to impact vaccine coverage. This study aims to conduct an international comparison of the proportions of tweets about HPV vaccines that express concerns, the types of concerns expressed and the social connections among users posting about HPV vaccines in Australia, Canada and the UK. Design Using a cross-sectional design, an international comparison of English language tweets about HPV vaccines and social connections among Twitter users posting about HPV vaccines between January 2014 and April 2016 was conducted. The Health Belief Model, one of the most widely used theories in health psychology, was used as the basis for coding the types of HPV vaccine concerns expressed on Twitter. Setting The content of tweets and the social connections between users who posted tweets about HPV vaccines from Australia, Canada and the UK. Population 16 789 Twitter users who posted 43 852 tweets about HPV vaccines. Main outcome measures The proportions of tweets expressing concern, the type of concern expressed and the proportions of local and international social connections between users. Results Tweets expressing concerns about HPV vaccines made up 14.9% of tweets in Canada, 19.4% in Australia and 22.6% in the UK. The types of concerns expressed were similar across the three countries, with concerns related to ‘perceived barriers’ being the most common. Users expressing concerns about HPV vaccines in each of the three countries had a relatively high proportion of international followers also expressing concerns. Conclusions The proportions and types of HPV vaccine concerns expressed on Twitter were similar across the three countries. Twitter users who mostly expressed concerns about HPV vaccines were better connected to international users who shared their concerns compared with users who did not express concerns about HPV vaccines. PMID:28982821

Expression of non-toxic mutant of Escherichia coli heat-labile enterotoxin in tobacco chloroplasts.

PubMed

Kang, Tae-Jin; Han, So-Chon; Kim, Mi-Young; Kim, Young-Sook; Yang, Moon-Sik

2004-11-01

Chloroplast transformation systems offer unique advantages in biotechnology, including high level of foreign gene expression, maternal inheritance, and polycistronic expression. We studied chloroplast expression of LTK63 (change Ser-->Lys at position 63 in the A subunit) which is the mutant of Escherichia coli heat-labile toxin. LTK63 is devoid of any toxic activity, but still retains its mucosal adjuvanticity. The LTK63 was cloned into chloroplast targeting vector and transformed to tobacco chloroplasts by particle bombardment. PCR and Southern blot analyses confirmed stable homologous recombination of the LTK63 gene into the chloroplast genome. The amount of LTK63 protein detected in tobacco chloroplasts was approximately 3.7% of the total soluble protein. The GM1-ganglioside binding assay confirmed that chloroplast-synthesized LTB of LTK63 binds to the intestinal membrane GM1-ganglioside receptor. Thus, the expression of LTK63 in chloroplasts provides a potential route toward the development of a plant-based edible vaccine for high expression system and environmentally friendly approach.

Uniqueness of Nash equilibrium in vaccination games.

PubMed

Bai, Fan

2016-12-01

One crucial condition for the uniqueness of Nash equilibrium set in vaccination games is that the attack ratio monotonically decreases as the vaccine coverage level increasing. We consider several deterministic vaccination models in homogeneous mixing population and in heterogeneous mixing population. Based on the final size relations obtained from the deterministic epidemic models, we prove that the attack ratios can be expressed in terms of the vaccine coverage levels, and also prove that the attack ratios are decreasing functions of vaccine coverage levels. Some thresholds are presented, which depend on the vaccine efficacy. It is proved that for vaccination games in homogeneous mixing population, there is a unique Nash equilibrium for each game.

Purification and biological characterization of soluble, recombinant mouse IFNβ expressed in insect cells.

PubMed

Stifter, Sebastian A; Gould, Jodee A; Mangan, Niamh E; Reid, Hugh H; Rossjohn, Jamie; Hertzog, Paul J; de Weerd, Nicole A

2014-02-01

Interferon β (IFNβ) is a member of the type I interferon family of cytokines widely recognised for their anti-viral, anti-proliferative and immunomodulatory properties. Recombinant, biologically active forms of this cytokine are used clinically for the treatment of multiple sclerosis and in laboratories to study the role of this cytokine in health and disease. Established methods for expression of IFNβ utilise either bacterial systems from which the insoluble recombinant proteins must be refolded, or mammalian expression systems in which large volumes of cell culture are required for recovery of acceptable yields. Utilising the baculovirus expression system and Trichoplusia ni (Cabbage Looper) BTI-TN-5B1-4 cell line, we report a reproducible method for production and purification of milligram/litre quantities of biologically active murine IFNβ. Due to the design of our construct and the eukaryotic nature of insect cells, the resulting soluble protein is secreted allowing purification of the Histidine-tagged natively-folded protein from the culture supernatant. The IFNβ purification method described is a two-step process employing immobilised metal-ion affinity chromatography (IMAC) and reverse-phase high performance liquid chromatography (RP-HPLC) that results in production of significantly more purified IFNβ than any other reported eukaryotic-based expression system. Recombinant murine IFNβ produced by this method was natively folded and demonstrated hallmark type I interferon biological effects including antiviral and anti-proliferative activities, and induced genes characteristic of IFNβ activity in vivo. Recombinant IFNβ also had specific activity levels exceeding that of the commercially available equivalent. Together, our findings provide a method for production of highly pure, biologically active murine IFNβ. Copyright © 2013 Elsevier Inc. All rights reserved.

Combined prime-boost vaccination against tick-borne encephalitis (TBE) using a recombinant vaccinia virus and a bacterial plasmid both expressing TBE virus non-structural NS1 protein

PubMed Central

Aleshin, SE; Timofeev, AV; Khoretonenko, MV; Zakharova, LG; Pashvykina, GV; Stephenson, JR; Shneider, AM; Altstein, AD

2005-01-01

Background Heterologous prime-boost immunization protocols using different gene expression systems have proven to be successful tools in protecting against various diseases in experimental animal models. The main reason for using this approach is to exploit the ability of expression cassettes to prime or boost the immune system in different ways during vaccination procedures. The purpose of the project was to study the ability of recombinant vaccinia virus (VV) and bacterial plasmid, both carrying the NS1 gene from tick-borne encephalitis (TBE) virus under the control of different promoters, to protect mice against lethal challenge using a heterologous prime-boost vaccination protocol. Results The heterologous prime-boost vaccination protocol, using a VV recombinant and bacterial plasmid, both containing the NS1 TBE virus protein gene under the control of different promoters, achieved a high level of protection in mice against lethal challenge with a highly pathogenic TBE virus strain. No signs of pronounced TBE infection were detected in the surviving animals. Conclusion Heterologous prime-boost vaccination protocols using recombinant VV and bacterial plasmids could be used for the development of flavivirus vaccines. PMID:16076390

Vaccination with recombinant Modified Vaccinia Ankara (MVA) viruses expressing single African horse sickness virus VP2 antigens induced cross-reactive virus neutralising antibodies (VNAb) in horses when administered in combination.

PubMed

Manning, Nicola Mary; Bachanek-Bankowska, Katarzyna; Mertens, Peter Paul Clement; Castillo-Olivares, Javier

2017-10-20

African horse sickness is a lethal viral disease of equids transmitted by biting midges of the Genus Culicoides. The disease is endemic to sub-Saharan Africa but outbreaks of high mortality and economic impact have occurred in the past in non-endemic regions of Africa, Asia and Southern Europe. Vaccination is critical for the control of this disease but only live attenuated vaccines are currently available. However, there are bio-safety concerns over the use of this type of vaccines, especially in non-endemic countries, and live attenuated vaccines do not have DIVA (Differentiation of Infected from Vaccinated Animals) capacity. In addition, large scale manufacturing of live attenuated vaccines of AHSV represents a significant environmental and health risk and level 3 bio-safety containment facilities are required for their production. A variety of different technologies have been investigated over the years to develop alternative AHSV vaccines, including the use of viral vaccine vectors such Modified Vaccinia Ankara virus (MVA). In previous studies we demonstrated that recombinant MVA expressing outer capsid protein AHSV-VP2 induced virus neutralising antibodies and protection against virulent challenge both in a mouse model and in the horse. However, AHSV-VP2 is antigenically variable and determines the existence of 9 different AHSV serotypes. Immunity against AHSV is serotype-specific and there is limited cross-reactivity between certain AHSV serotypes: 1 and 2, 3 and 7, 5 and 8, 6 and 9. In Africa, multiple serotypes circulate simultaneously and a polyvalent attenuated vaccine comprising different AHSV serotypes is used. We investigated the potential of a polyvalent AHSV vaccination strategy based on combinations of MVA-VP2 viruses each expressing a single VP2 antigen from a specific serotype. We showed that administration of 2 different recombinant MVA viruses, each expressing a single VP2 protein from AHSV serotype 4 or 9, denoted respectively as MVA-VP2

Plant-Based Vaccines Against Diarrheal Diseases

PubMed Central

Tacket, Carol O.

2007-01-01

Every year 1.6 million deaths occur due to diarrhea related to unsafe water and inadequate sanitation—the vast majority in children under 5 years old. Safe and effective vaccines against enteric infections could contribute to control of these diseases. However, purification of protective antigens for inclusion in vaccines using traditional expression systems is expensive and unattractive to vaccine manufacturers who see the vaccine market as economically uninviting. Cost is one of the persistent barriers to deployment of new vaccines to populations that need them most urgently. Transgenic plant-derived vaccines offer a new strategy for development of safe, inexpensive vaccines against diarrheal diseases. In phase 1 clinical studies, these vaccines have been safe and immunogenic without the need for a buffer or vehicle other than the plant cell. This paper describes early clinical studies evaluating oral transgenic plant vaccines against enteric infections such as enterotoxigenic E. coli infection and norovirus. PMID:18528491

Oral Vaccination Against Anthrax Using a Transgenic Plant Expressing Protective Antigen.

DTIC Science & Technology

1996-09-01

unpublished results). In Phase II, development of a tomato -based system for edible vaccine delivery will be carried out in parallel with experiments testing...Frederick, MD 21702-5012. AUTHORITY USAMRMC ltr, 4 Dec 2002 THIS PAGE IS UNCLASSIFIED AD CONTRACT NUMBER: DAMD17-95-C-5102 TITLE: Oral Vaccination ...COVERED I September 1996 Final - Phase I, 15 Auxg 95-14 Aug 96 4. TITLE AND 9UBTITLE 5. FUNDING NUMBERS Oral Vaccination Against Anthrax Using a Transgenic

Survey of rubella knowledge and acceptability of rubella vaccination among Brazilian adults prior to mass vaccination.

PubMed

Vieira, Juliana Costa; Carvalho, Marlene Tavares Barros de; Checchia, Ricardo L; Trombiere, Marcier; Flannery, Brendan

2011-10-01

Evaluate knowledge of rubella and acceptability of vaccination and identify sources of health information among brazilian adults to inform communication strategies for a national vaccination campaign to eliminate rubella and congenital rubella syndrome (CRS). From 5-8 July 2008 a qualitative telephone survey was conducted among a nonprobabilistic sample of brazilian adults 18 to 65 years of age (n = 1 023) from all five geographic regions of Brazil to measure knowledge of rubella and willingness to receive the vaccine and to identify sources of health information. Frequencies of responses were stratified by respondents' sex, age, education, and income. Although 69.9% of respondents said they knew what rubella was, actual knowledge of the disease was limited, with only 29.9% answering affirmatively when asked if they would recognize symptoms of rubella infection. Self-reported knowledge increased with increasing age, education, and income, and was higher among women than men. A total of 94.5% of the respondents expressed willingness to be vaccinated for rubella elimination. The most frequently mentioned sources of health information were television and doctors. Despite limited knowledge of rubella, brazilian adults expressed willingness to be vaccinated for disease elimination.

P and M gene junction is the optimal insertion site in Newcastle disease virus vaccine vector for foreign gene expression

USDA-ARS?s Scientific Manuscript database

Newcastle disease virus (NDV) has been developed as a vector for vaccine and gene therapy purposes. However, the optimal insertion site for foreign gene expression remained to be determined. In the present study, we inserted the green fluorescence protein (GFP) gene into five different intergenic ...

Venezuelan Equine Encephalitis Virus Replicon Particle Vaccine Protects Nonhuman Primates from Intramuscular and Aerosol Challenge with Ebolavirus

PubMed Central

Herbert, Andrew S.; Kuehne, Ana I.; Barth, James F.; Ortiz, Ramon A.; Nichols, Donald K.; Zak, Samantha E.; Stonier, Spencer W.; Muhammad, Majidat A.; Bakken, Russell R.; Prugar, Laura I.; Olinger, Gene G.; Groebner, Jennifer L.; Lee, John S.; Pratt, William D.; Custer, Max; Kamrud, Kurt I.; Smith, Jonathan F.; Hart, Mary Kate

2013-01-01

There are no vaccines or therapeutics currently approved for the prevention or treatment of ebolavirus infection. Previously, a replicon vaccine based on Venezuelan equine encephalitis virus (VEEV) demonstrated protective efficacy against Marburg virus in nonhuman primates. Here, we report the protective efficacy of Sudan virus (SUDV)- and Ebola virus (EBOV)-specific VEEV replicon particle (VRP) vaccines in nonhuman primates. VRP vaccines were developed to express the glycoprotein (GP) of either SUDV or EBOV. A single intramuscular vaccination of cynomolgus macaques with VRP expressing SUDV GP provided complete protection against intramuscular challenge with SUDV. Vaccination against SUDV and subsequent survival of SUDV challenge did not fully protect cynomolgus macaques against intramuscular EBOV back-challenge. However, a single simultaneous intramuscular vaccination with VRP expressing SUDV GP combined with VRP expressing EBOV GP did provide complete protection against intramuscular challenge with either SUDV or EBOV in cynomolgus macaques. Finally, intramuscular vaccination with VRP expressing SUDV GP completely protected cynomolgus macaques when challenged with aerosolized SUDV, although complete protection against aerosol challenge required two vaccinations with this vaccine. PMID:23408633

Venezuelan equine encephalitis virus replicon particle vaccine protects nonhuman primates from intramuscular and aerosol challenge with ebolavirus.

PubMed

Herbert, Andrew S; Kuehne, Ana I; Barth, James F; Ortiz, Ramon A; Nichols, Donald K; Zak, Samantha E; Stonier, Spencer W; Muhammad, Majidat A; Bakken, Russell R; Prugar, Laura I; Olinger, Gene G; Groebner, Jennifer L; Lee, John S; Pratt, William D; Custer, Max; Kamrud, Kurt I; Smith, Jonathan F; Hart, Mary Kate; Dye, John M

2013-05-01

There are no vaccines or therapeutics currently approved for the prevention or treatment of ebolavirus infection. Previously, a replicon vaccine based on Venezuelan equine encephalitis virus (VEEV) demonstrated protective efficacy against Marburg virus in nonhuman primates. Here, we report the protective efficacy of Sudan virus (SUDV)- and Ebola virus (EBOV)-specific VEEV replicon particle (VRP) vaccines in nonhuman primates. VRP vaccines were developed to express the glycoprotein (GP) of either SUDV or EBOV. A single intramuscular vaccination of cynomolgus macaques with VRP expressing SUDV GP provided complete protection against intramuscular challenge with SUDV. Vaccination against SUDV and subsequent survival of SUDV challenge did not fully protect cynomolgus macaques against intramuscular EBOV back-challenge. However, a single simultaneous intramuscular vaccination with VRP expressing SUDV GP combined with VRP expressing EBOV GP did provide complete protection against intramuscular challenge with either SUDV or EBOV in cynomolgus macaques. Finally, intramuscular vaccination with VRP expressing SUDV GP completely protected cynomolgus macaques when challenged with aerosolized SUDV, although complete protection against aerosol challenge required two vaccinations with this vaccine.

Novel Injectable Pentablock Copolymer Based Thermoresponsive Hydrogels for Sustained Release Vaccines.

PubMed

Bobbala, Sharan; Tamboli, Viral; McDowell, Arlene; Mitra, Ashim K; Hook, Sarah

2016-01-01

The need for multiple vaccinations to enhance the immunogenicity of subunit vaccines may be reduced by delivering the vaccine over an extended period of time. Here, we report two novel injectable pentablock copolymer based thermoresponsive hydrogels made of polyethyleneglycol-polycaprolactone-polylactide-polycaprolactone-polyethyleneglycol (PEG-PCL-PLA-PCL-PEG) with varying ratios of polycaprolactone (PCL) and polylactide (PLA), as single shot sustained release vaccines. Pentablock copolymer hydrogels were loaded with vaccine-encapsulated poly lactic-co-glycolic acid nanoparticles (PLGA-NP) or with the soluble vaccine components. Incorporation of PLGA-NP into the thermoresponsive hydrogels increased the complex viscosity of the gels, lowered the gelation temperature, and minimized the burst release of antigen and adjuvants. The two pentablock hydrogels stimulated both cellular and humoral responses. The addition of PLGA-NP to the hydrogels sustained immune responses for up to 49 days. The polymer with a higher ratio of PCL to PLA formed a more rigid gel, induced stronger immune responses, and stimulated effective anti-tumor responses in a prophylactic melanoma tumor model.

Water-Soluble Phosphinothiols for Traceless Staudinger Ligation and Integration with Expressed Protein Ligation

PubMed Central

Tam, Annie; Soellner, Matthew B.; Raines, Ronald T.

2010-01-01

The traceless Staudinger ligation is an effective means to synthesize an amide bond between two groups of otherwise orthogonal reactivity: a phosphinothioester and an azide. An important application of the Staudinger ligation is in the ligation of peptides at a variety of residues. Here, we demonstrate that the traceless Staudinger ligation can be achieved in water with a water-soluble reagent. Those reagents that provide a high yield of amide product discourage protonation of the nitrogen in the key iminophosphorane intermediate. The most efficacious reagent, bis(p-dimethylaminoethylphenyl)phosphinomethanethiol, mediates the rapid ligation of equimolar substrates in water. This reagent is also able to perform a transthioesterification reaction with the thioester intermediate formed during intein-mediated protein splicing. Hence, the traceless Staudinger ligation can be integrated with expressed protein ligation, extending the reach of modern protein chemistry. PMID:17713909

Soluble Alpha-APP (sAPPalpha) Regulates CDK5 Expression and Activity in Neurons

PubMed Central

Hartl, Daniela; Klatt, Stephan; Roch, Manfred; Konthur, Zoltan; Klose, Joachim; Willnow, Thomas E.; Rohe, Michael

2013-01-01

A growing body of evidence suggests a role for soluble alpha-amyloid precursor protein (sAPPalpha) in pathomechanisms of Alzheimer disease (AD). This cleavage product of APP was identified to have neurotrophic properties. However, it remained enigmatic what proteins, targeted by sAPPalpha, might be involved in such neuroprotective actions. Here, we used high-resolution two-dimensional polyacrylamide gel electrophoresis to analyze proteome changes downstream of sAPPalpha in neurons. We present evidence that sAPPalpha regulates expression and activity of CDK5, a kinase that plays an important role in AD pathology. We also identified the cytoprotective chaperone ORP150 to be induced by sAPPalpha as part of this protective response. Finally, we present functional evidence that the sAPPalpha receptor SORLA is essential to mediate such molecular functions of sAPPalpha in neurons. PMID:23776568

Secreted Expression of the Cap Gene of Porcine Circovirus Type 2 in Classical Swine Fever Virus C-Strain: Potential of C-Strain Used as a Vaccine Vector.

PubMed

Zhang, Lingkai; Li, Yongfeng; Xie, Libao; Wang, Xiao; Gao, Xulei; Sun, Yuan; Qiu, Hua-Ji

2017-10-16

Bivalent vaccines based on live attenuated viruses expressing a heterologous protein are an attractive strategy to address co-infections with various pathogens in the field. Considering the excellent efficacy and safety of the lapinized live attenuated vaccine C-strain (HCLV strain) of classical swine fever virus (CSFV), we proposed that C-strain has the potential as a viral vector for developing bivalent vaccines. To this end, we generated three recombinant viruses based on C-strain, one expressing the capsid ( Cap ) gene of porcine circovirus type 2 (PCV2) with the nuclear localization signal (NLS) (rHCLV-2ACap), and the other two expressing the PCV2 Cap gene without the NLS yet containing the signal peptide of the prolactin gene (rHCLV-pspCap) or that of the ubiquitin-specific peptidase gene (rHCLV-uspCap). All the recombinant viruses exhibited phenotypes similar to those of the parental virus and produced high-level anti-CSFV neutralizing antibodies (NAbs) in rabbits. Interestingly, rHCLV-uspCap and rHCLV-pspCap, but not rHCLV-2ACap, elicited detectable anti-Cap and -PCV2 NAbs in rabbits. Taken together, our data demonstrate that C-strain can be used as a viral vector to develop bivalent vaccines.

Secreted Expression of the Cap Gene of Porcine Circovirus Type 2 in Classical Swine Fever Virus C-Strain: Potential of C-Strain Used as a Vaccine Vector

PubMed Central

Zhang, Lingkai; Li, Yongfeng; Xie, Libao; Wang, Xiao; Gao, Xulei; Sun, Yuan; Qiu, Hua-Ji

2017-01-01

Bivalent vaccines based on live attenuated viruses expressing a heterologous protein are an attractive strategy to address co-infections with various pathogens in the field. Considering the excellent efficacy and safety of the lapinized live attenuated vaccine C-strain (HCLV strain) of classical swine fever virus (CSFV), we proposed that C-strain has the potential as a viral vector for developing bivalent vaccines. To this end, we generated three recombinant viruses based on C-strain, one expressing the capsid (Cap) gene of porcine circovirus type 2 (PCV2) with the nuclear localization signal (NLS) (rHCLV-2ACap), and the other two expressing the PCV2 Cap gene without the NLS yet containing the signal peptide of the prolactin gene (rHCLV-pspCap) or that of the ubiquitin-specific peptidase gene (rHCLV-uspCap). All the recombinant viruses exhibited phenotypes similar to those of the parental virus and produced high-level anti-CSFV neutralizing antibodies (NAbs) in rabbits. Interestingly, rHCLV-uspCap and rHCLV-pspCap, but not rHCLV-2ACap, elicited detectable anti-Cap and -PCV2 NAbs in rabbits. Taken together, our data demonstrate that C-strain can be used as a viral vector to develop bivalent vaccines. PMID:29035292

DNA vaccines co-expressing GP5 and M proteins of porcine reproductive and respiratory syndrome virus (PRRSV) display enhanced immunogenicity.

PubMed

Jiang, Yunbo; Xiao, Shaobo; Fang, Liurong; Yu, Xiaolan; Song, Yunfeng; Niu, Chuanshuang; Chen, Huanchun

2006-04-05

The two major membrane-associated proteins of porcine reproductive and respiratory syndrome virus (PRRSV), GP5 and M (encoded by ORF5 and ORF6 genes, respectively), are associated as disulfide-linked heterodimers (GP5/M) in the virus particle. In the present study, three different DNA vaccine constructs, expressing GP5 alone (pCI-ORF5), M alone (pCI-ORF6) or GP5 and M proteins simultaneously (pCI-ORF5/ORF6), were constructed. In vitro, the co-expressed GP5 and M proteins could form heterodimeric complexes in transfected cells and heterodimerization altered the subcellular localization of GP5. The immunogenicities of these DNA vaccine constructs were firstly investigated in a mouse model. Mice inoculated with pCI-ORF5/ORF6 developed PRRSV-specific neutralizing antibodies at 6 and 8 weeks after primary immunization. However, only some mice developed low levels of neutralizing antibodies in groups immunized with pCI-ORF5 or pCI-ORF6. The highest lymphocyte proliferation responses were also observed in mice immunized with pCI-ORF5/ORF6. Interestingly, significantly enhanced GP5-specific ELISA antibody could be detected in mice immunized with pCI-ORF5/ORF6 compared to mice immunized with pCI-ORF5. The immunogenicities of pCI-ORF5/ORF6 were further evaluated in piglets (the natural host) and all immunized piglets developed neutralizing antibodies at 10 weeks after primary immunization, whereas there was no detectable neutralizing antibodies in piglets immunized with pCI-ORF5. These results indicate that the formation of GP5/M heterodimers may be involved in post-translational modification and transport of GP5 and may play an important role in immune responses against PRRSV infection. More importantly, co-expression of GP5 and M protein in heterodimers can significantly improve the potency of DNA vaccination and could be used as a strategy to develop a new generation of vaccines against PRRSV.

Potent Immune Responses in Rhesus Macaques Induced by Nonviral Delivery of a Self-amplifying RNA Vaccine Expressing HIV Type 1 Envelope With a Cationic Nanoemulsion

PubMed Central

Bogers, Willy M.; Oostermeijer, Herman; Mooij, Petra; Koopman, Gerrit; Verschoor, Ernst J.; Davis, David; Ulmer, Jeffrey B.; Brito, Luis A.; Cu, Yen; Banerjee, Kaustuv; Otten, Gillis R.; Burke, Brian; Dey, Antu; Heeney, Jonathan L.; Shen, Xiaoying; Tomaras, Georgia D.; Labranche, Celia; Montefiori, David C.; Liao, Hua-Xin; Haynes, Barton; Geall, Andrew J.; Barnett, Susan W.

2015-01-01

Self-amplifying messenger RNA (mRNA) of positive-strand RNA viruses are effective vectors for in situ expression of vaccine antigens and have potential as a new vaccine technology platform well suited for global health applications. The SAM vaccine platform is based on a synthetic, self-amplifying mRNA delivered by a nonviral delivery system. The safety and immunogenicity of an HIV SAM vaccine encoding a clade C envelope glycoprotein formulated with a cationic nanoemulsion (CNE) delivery system was evaluated in rhesus macaques. The HIV SAM vaccine induced potent cellular immune responses that were greater in magnitude than those induced by self-amplifying mRNA packaged in a viral replicon particle (VRP) or by a recombinant HIV envelope protein formulated with MF59 adjuvant, anti-envelope binding (including anti-V1V2), and neutralizing antibody responses that exceeded those induced by the VRP vaccine. These studies provide the first evidence in nonhuman primates that HIV vaccination with a relatively low dose (50 µg) of formulated self-amplifying mRNA is safe and immunogenic. PMID:25234719

Modulation of systemic and mucosal immunity against an inactivated vaccine of Newcastle disease virus by oral co-administration of live attenuated Salmonella enterica serovar Typhimurium expressing chicken interleukin-18 and interferon-α

PubMed Central

RAHMAN, Md. Masudur; UYANGAA, Erdenebelig; HAN, Young Woo; HUR, Jin; PARK, Sang-Youel; LEE, John Hwa; KIM, Koanhoi; EO, Seong Kug

2014-01-01

Newcastle disease (ND) is a highly contagious disease of chickens causing significant economic losses worldwide. Due to limitations in the efficacy against currently circulating ND viruses, existing vaccination strategies require improvements, and incorporating immunomodulatory cytokines with existing vaccines might be a novel approach. Here, we investigated the systemic and mucosal immunomodulatory properties of oral co-administration of chicken interleukin-18 (chIL-18) and chicken interferon-α (chIFN-α) using attenuated Salmonella enterica serovar Typhimurium on an inactivated ND vaccine. Our results demonstrate that oral administration of S. enterica serovar Typhimurium expressing chIL-18 or chIFN-α provided enhanced systemic and mucosal immune responses, as determined by serum hemagglutination inhibition antibody and NDV Ag-specific IgG as well as NDV Ag-specific IgA in lung and duodenal lavages of chickens immunized with inactivated ND vaccine via the intramuscular or intranasal route. Notably, combined oral administration of S. enterica serovar Typhimurium expressing chIL-18 and chIFN-α significantly enhanced systemic and mucosal immunity in ND-vaccinated chickens, compared to single administration of S. enterica serovar Typhimurium expressing chIL-18 or chIFN-α. In addition, oral co-administration of S. enterica serovar Typhimurium expressing chIL-18 and chIFN-α provided enhanced NDV Ag-specific proliferation of peripheral blood mononuclear cells and Th1-biased cell-mediated immunity, compared to single administration of either construct. Therefore, our results provide valuable insight into the modulation of systemic and mucosal immunity by incorporation of immunomodulatory chIL-18 and chIFN-α using Salmonella vaccines into existing ND vaccines. PMID:25502364

Vaccines in Development against West Nile Virus

PubMed Central

Brandler, Samantha; Tangy, Frederic

2013-01-01

West Nile encephalitis emerged in 1999 in the United States, then rapidly spread through the North American continent causing severe disease in human and horses. Since then, outbreaks appeared in Europe, and in 2012, the United States experienced a new severe outbreak reporting a total of 5,387 cases of West Nile virus (WNV) disease in humans, including 243 deaths. So far, no human vaccine is available to control new WNV outbreaks and to avoid worldwide spreading. In this review, we discuss the state-of-the-art of West Nile vaccine development and the potential of a novel safe and effective approach based on recombinant live attenuated measles virus (MV) vaccine. MV vaccine is a live attenuated negative-stranded RNA virus proven as one of the safest, most stable and effective human vaccines. We previously described a vector derived from the Schwarz MV vaccine strain that stably expresses antigens from emerging arboviruses, such as dengue, West Nile or chikungunya viruses, and is strongly immunogenic in animal models, even in the presence of MV pre-existing immunity. A single administration of a recombinant MV vaccine expressing the secreted form of WNV envelope glycoprotein elicited protective immunity in mice and non-human primates as early as two weeks after immunization, indicating its potential as a human vaccine. PMID:24084235

HIV-1 vaccines

PubMed Central

Excler, Jean-Louis; Robb, Merlin L; Kim, Jerome H

2014-01-01

The development of a safe and effective preventive HIV-1 vaccine remains a public health priority. Despite scientific difficulties and disappointing results, HIV-1 vaccine clinical development has, for the first time, established proof-of-concept efficacy against HIV-1 acquisition and identified vaccine-associated immune correlates of risk. The correlate of risk analysis showed that IgG antibodies against the gp120 V2 loop correlated with decreased risk of HIV infection, while Env-specific IgA directly correlated with increased risk. The development of vaccine strategies such as improved envelope proteins formulated with potent adjuvants and DNA and vectors expressing mosaics, or conserved sequences, capable of eliciting greater breadth and depth of potentially relevant immune responses including neutralizing and non-neutralizing antibodies, CD4+ and CD8+ cell-mediated immune responses, mucosal immune responses, and immunological memory, is now proceeding quickly. Additional human efficacy trials combined with other prevention modalities along with sustained funding and international collaboration remain key to bring an HIV-1 vaccine to licensure. PMID:24637946

Preclinical development of BCG.HIVA2auxo.int, harboring an integrative expression vector, for a HIV-TB Pediatric vaccine. Enhancement of stability and specific HIV-1 T-cell immunity.

PubMed

Mahant, Aakash; Saubi, Narcís; Eto, Yoshiki; Guitart, Núria; Gatell, Josep Ma; Hanke, Tomáš; Joseph, Joan

2017-08-03

One of the critical issues that should be addressed in the development of a BCG-based HIV vaccine is genetic plasmid stability. Therefore, to address this issue we have considered using integrative vectors and the auxotrophic mutant of BCG complemented with a plasmid carrying a wild-type complementing gene. In this study, we have constructed an integrative E. coli-mycobacterial shuttle plasmid, p2auxo.HIVA int , expressing the HIV-1 clade A immunogen HIVA. This shuttle vector uses an antibiotic resistance-free mechanism for plasmid selection and maintenance. It was first transformed into a glycine auxotrophic E. coli strain and subsequently transformed into a lysine auxotrophic Mycobacterium bovis BCG strain to generate the vaccine BCG.HIVA 2auxo.int . Presence of the HIVA gene sequence and protein expression was confirmed. We demonstrated that the in vitro stability of the integrative plasmid p2auxo.HIVA int was increased 4-fold, as compared with the BCG strain harboring the episomal plasmid, and was genetically and phenotypically characterized. The BCG.HIVA 2auxo.int vaccine in combination with modified vaccinia virus Ankara (MVA).HIVA was found to be safe and induced HIV-1 and Mycobacterium tuberculosis-specific interferon-γ-producing T-cell responses in adult BALB/c mice. We have engineered a more stable and immunogenic BCG-vectored vaccine using the prototype immunogen HIVA. Thus, the use of integrative expression vectors and the antibiotic-free plasmid selection system based on "double" auxotrophic complementation are likely to improve the mycobacterial vaccine stability in vivo and immunogenicity to develop not only recombinant BCG-based vaccines expressing second generation of HIV-1 immunogens but also other major pediatric pathogens to prime protective responses shortly following birth.

New Strategies Toward Edible Vaccines: An Overview.

PubMed

Aryamvally, Anjali; Gunasekaran, Vignesh; Narenthiran, Keerthana Ragavi; Pasupathi, Rathinasabapathi

2016-04-11

With the ever growing population, advancements in edible vaccines and related technologies have seen a rise in popularity. Antigenic peptides incorporated into an edible part of a plant can be administered raw as a vaccine. While conventional vaccines have improved the quality of life by drastically reducing the onset of diseases, edible vaccines are able to perform the same with greater accessibility and at an affordable price. Low cost of production, ease of storage, transportation and administration are some of the many reasons behind the push for the development of edible vaccines. This article aims at giving an overview of the different plant systems used to produce vaccines in various experiments, as well as the merits and demerits of using that particular expression system. Further, the article elaborates on the problems faced in the production of edible vaccines and the measures adopted to surpass them. The major obstacle in the process is attaining a sufficiently large concentration of foreign antigen in the plant system. The article discusses various plant expression systems like banana, rice, alfalfa, mushroom, potato, tomato, pea, tobacco, and maize. When these were reviewed, it was found that the inability to produce the desired antigen concentration was one of the primary reasons why edible vaccines sometimes fail to generate the desired level of immune response in the recipient. We conclude with a promising solution to the problem by incorporating nano-technological advancements to the already existing protocols for edible vaccine development.

A simple and effective strategy for solving the problem of inclusion bodies in recombinant protein technology: His-tag deletions enhance soluble expression.

PubMed

Zhu, Shaozhou; Gong, Cuiyu; Ren, Lu; Li, Xingzhou; Song, Dawei; Zheng, Guojun

2013-01-01

The formation of inclusion bodies (IBs) in recombinant protein biotechnology has become one of the most frequent undesirable occurrences in both research and industrial applications. So far, the pET System is the most powerful system developed for the production of recombinant proteins when Escherichia coli is used as the microbial cell factory. Also, using fusion tags to facilitate detection and purification of the target protein is a commonly used tactic. However, there is still a large fraction of proteins that cannot be produced in E. coli in a soluble (and hence functional) form. Intensive research efforts have tried to address this issue, and numerous parameters have been modulated to avoid the formation of inclusion bodies. However, hardly anyone has noticed that adding fusion tags to the recombinant protein to facilitate purification is a key factor that affects the formation of inclusion bodies. To test this idea, the industrial biocatalysts uridine phosphorylase from Aeropyrum pernix K1 and (+)-γ-lactamase and (-)-γ-lactamase from Bradyrhizobium japonicum USDA 6 were expressed in E. coli by using the pET System and then examined. We found that using a histidine tag as a fusion partner for protein expression did affect the formation of inclusion bodies in these examples, suggesting that removing the fusion tag can promote the solubility of heterologous proteins. The production of soluble and highly active uridine phosphorylase, (+)-γ-lactamase, and (-)-γ-lactamase in our results shows that the traditional process needs to be reconsidered. Accordingly, a simple and efficient structure-based strategy for the production of valuable soluble recombinant proteins in E. coli is proposed.

Vaccine Platforms to Control Arenaviral Hemorrhagic Fevers.

PubMed

Carrion, Ricardo; Bredenbeek, Peter; Jiang, Xiaohong; Tretyakova, Irina; Pushko, Peter; Lukashevich, Igor S

2012-11-20

Arenaviruses are rodent-borne emerging human pathogens. Diseases caused by these viruses, e.g., Lassa fever (LF) in West Africa and South American hemorrhagic fevers (HFs), are serious public health problems in endemic areas. We have employed replication-competent and replication-deficient strategies to design vaccine candidates potentially targeting different groups "at risk". Our leader LF vaccine candidate, the live reassortant vaccine ML29, is safe and efficacious in all tested animal models including non-human primates. In this study we showed that treatment of fatally infected animals with ML29 two days after Lassa virus (LASV) challenge protected 80% of the treated animals. In endemic areas, where most of the target population is poor and many live far from health care facilities, a single-dose vaccination with ML29 would be ideal solution. Once there is an outbreak, a fast-acting vaccine or post-exposure prophylaxis would be best. The 2(nd) vaccine technology is based on Yellow Fever (YF) 17D vaccine. We designed YF17D-based recombinant viruses expressing LASV glycoproteins (GP) and showed protective efficacy of these recombinants. In the current study we developed a novel technology to clone LASV nucleocapsid within YF17D C gene. Low immunogenicity and stability of foreign inserts must be addressed to design successful LASV/YFV bivalent vaccines to control LF and YF in overlapping endemic areas of West Africa. The 3(rd) platform is based on the new generation of alphavirus replicon virus-like-particle vectors (VLPV). Using this technology we designed VLPV expressing LASV GP with enhanced immunogenicity and bivalent VLPV expressing cross-reactive GP of Junin virus (JUNV) and Machupo virus (MACV), causative agents of Argentinian and Bolivian HF, respectively. A prime-boost regimen required for VLPV immunization might be practical for medical providers, military, lab personnel, and visitors in endemic areas.

Japanese encephalitis virus replicon-based vaccine expressing enterovirus-71 epitope confers dual protection from lethal challenges.

PubMed

Huang, Yi-Ting; Liao, Jia-Teh; Yen, Li-Chen; Chang, Yung-Kun; Lin, Yi-Ling; Liao, Ching-Len

2015-09-11

To construct safer recombinant flavivirus vaccine, we exploited Japanese encephalitis virus (JEV) replicon-based platform to generate single-round infectious particles (SRIPs) that expressed heterologous neutralizing epitope SP70 derived from enterovirus-71 (EV71). Such pseudo-infectious virus particles, named SRIP-SP70, although are not genuine viable viruses, closely mimic live virus infection to elicit immune responses within one round of viral life cycle. We found that, besides gaining of full protection to thwart JEV lethal challenge, female outbred ICR mice, when were immunized with SRIP-SP70 by prime-boost protocol, could not only induce SP70-specific and IgG2a predominant antibodies but also provide their newborns certain degree of protection against EV71 lethal challenge. Our results therefore exemplify that this vaccination strategy could indeed confer an immunized host a dual protective immunity against subsequent lethal challenge from JEV or EV71.

Outer membrane vesicles as platform vaccine technology

PubMed Central

Stork, Michiel; van der Ley, Peter

2015-01-01