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A potential aquaculture vaccine vector: Evaluation of a double-gene attenuated Listeria monocytogenes in zebrafish ( Danio rerio )
Abstract
Although engineered Lm has strong adjuvant effects such as the delivery of antigens in antigen-presenting cells (APCs), it has not been evaluated if attenuated Lm has a potential to be used as an aquaculture vaccines vector. In this study, a double-gene attenuated Lm strain (Lm-actA/inlB) was constructed to evaluate the potential that using this attenuated strain to provide a delivery platform for aquatic vaccines. The attenuated Lm strain Lm-actA/inlB showed decreased invasion and apoptosis of HepG2 and Caco-2 host cells in in vitro infection models. Interestingly, the expression levels of the hly and plcB genes were significantly increased in Lm-actA/inlB compared with wild-type EGDe. The higher expression levels of the hly and plcB genes are indicative of greater advantages in terms of antigen presentation. To further explore the innate immune and adaptive immune response elicited by Lm-actA/inlB and its feasibility for use as a non-injection vaccine vector, we evaluated the expression of Toll-like receptor (TLR) signalling pathway-related gene markers, antigen presentation pathway-related gene markers and IgM via Lm-actA/inlB intraperitoneal injection and bath delivery. Transcription analysis indicated that the TLR signalling pathway might play a major role in the innate immune response and the major histocompatibility complex (MHC)-I and MHC-II antigen presentation pathways might also both exist in vertebrate fish. In conclusion, the live-attenuated Lm strain Lm-actA/inlB may be a potential platform for delivering aquatic vaccines against certain fish bacterial diseases commonly seen in the aquatic farm industry.
... As a vector for candidate vaccines, liveattenuated Lm has been studied in mammalian animal models and human clinical trial [9]. However, we found that Lm also might be a potential platform for delivering aquatic vaccines against certain fish bacterial diseases in our previous work [10], which provides new insights for understanding the multiple possibilities regarding the application of attenuated Lm. The vaccines used in fishery industry finally should be also transferred into human body. ...
... The data revealed that wild strain EGD-e had more efficient invasion and apoptosis than attenuated strain ΔactA/inlB in HepG2 hepatocytes and the Caco-2 epithelial cell line. However, no significant difference was observed between EGD-e and ΔactA/inlB in zebrafish challenge method [10]. ...
... The result from cellular level may explain why there is no significant difference in survival rate between EGD-e and ΔactA/inlB in zebrafish challenge method. In addition, our previous work also revealed a significant up-regulation of MHC-I and MHC-II pathways in zebrafish after attenuated Lm strain ΔactA/inlB challenged, suggesting that MHC-I and MHC-II antigen presentation pathways, which were first observed in mammals, might both exist in vertebrate fish [10]. Similarly, the expressions of CD4 and CD8a in ZF4 cells induced by EGD-e and ΔactA/ inlB, respectively, were measured by western blotting in this study. ...
... However, immune responses following immersion vaccination are generally less robust because of several formidable barriers including skin, cell and gastrointestinal tract (Hoare et al., 2017;Sudheesh and Cain, 2016). Therefore, efficient vectors could be a vital approach to delivery immersion vaccine into fish body and induce stronger immunoprotective against SVCV (Ding et al., 2017). ...
... Some investigators have reported that the IgM expression would be intensively increase in many tissues and organs from the second week after immunization and maintained almost one month, which is corresponding with our study (Tian et al., 2009). MHC-Ⅰ and MHC-Ⅱ are the markers reflecting the antigen presentation, the higher expression levels of MHC-Ⅰ and MHC-Ⅱ lead to increased advantages in terms of antigen presentation (Ding et al., 2017). The reason why SWCNTs could significantly enhance the cellular response and antigen presentation of our vaccine is possibly due to the increased numbers of cells (migration of head and leukocytes) participated in the process, in other words, SWCNTs make our DNA vaccine easier for attachment to specific target tissues and cells. ...
Spring viremia of carp virus (SVCV) has caused mass mortality in cyprinids, with case fatality rates of young fish up to 90%, resulting in enormous economic losses in the aquaculture industry. Immersion vaccination is considered as the most effective method for juvenile fish to combating disease, due to its convenience for mass vaccination and stress-free administration. However, immune responses following immersion vaccination are generally less robust and of shorter duration as those induced through intraperitoneal injection. Herein, to enhance the efficient of immersion vaccine, functionalized single-walled carbon nanotubes (SWCNTs) as carrier were used to manufacture immersion DNA vaccine system (SWCNTs-pEGFP-M) with chemical modification. Results showed that SWCNTs-pEGFP-M could enter into fish body via immersion administration and express antigen proteins in fish kidney and spleen. Moreover, stronger and longer duration immune responses (including serum antibody production and immune genes expression) can be induced in fish vaccinated with SWCNTs-pEGFP-M in comparison with those vaccinated with pEGFP-M alone. Notably, SWCNTs can increase the immune protective effect of naked DNA vaccine by ca. 23.8%. Altogether, this study demonstrates that SWCNTs as a promising DNA vaccine carrier might be used to vaccinate large-scale juvenile fish by bath administration approach, which can provide an outlook for future vaccination strategies against SVCV.
... Our previous work revealed that attenuated Lm could induce an immune response in zebrafish. Activation of TLR signaling pathways may play a major role in the innate immune response [13]. In addition, we found that attenuated Lm could protect zebrafish from the infection of Vibrio species. ...
Background
Attenuated Listeria monocytogenes (Lm) has been widely used as a vaccine vector in the prevention and treatment of pathogen infection and tumor diseases. In addition, previous studies have proved that the attenuated Lm can protect zebrafish from Vibrio infections, indicating that the attenuated Lm has a good application prospect in the field of aquatic vaccines. However, the limitation mainly lies in the lack of a set of well-characterized natural promoters for the expression of target antigens in attenuated Lm.
Results
In our study, candidate strong promoters were identified through RNA-seq analysis, and characterized in Lm through enhanced green fluorescent protein (EGFP). Nine native promoters that showed stronger activities than that of the known strong promoter P 36 under two tested temperatures (28 and 37 °C) were selected from the set, and P 29 with the highest activity was 24-fold greater than P 36 . Furthermore, we demonstrated that P 29 could initiate EGFP expression in ZF4 cells and zebrafish embryos.
Conclusions
This well-characterized promoter library can be used to fine-tune the expression of different proteins in Lm. The availability of a well-characterized promoter toolbox of Lm is essential for the analysis of yield increase for biotechnology applications.
... A vacinação é fundamental para o desenvolvimento de uma produção contínua e sustentável dos animais de produção, assim como uma medida essencial para a realização dos programas de controle e erradicação de diversas doenças dos animais domésticos. Devido as suas vantagens únicas, este modelo tem sido amplamente utilizado em experimentos de vacinação contra patógenos que causam prejuízos na aquicultura no Brasil e ao redor do mundo (BAILONE et al., 2020b), como contra Aeromonas hydrophila, bactéria causadora de grandes prejuízos na aquicultura mundial (GUO et al., 2018); Francisella noatunensis, bactéria causadora de doença granulomatosa em peixes de água doce e marinhos (LAGOS et al., 2017;BRUDAL et al., 2015); Flavobacterium columnare, bactéria intracelular causadora da columnariose (ZHANG et al., 2017); Streptococcus iniae, bactéria causadora da maioria das streptococoses em peixes (MEMBREBE et al., 2016); Edwardsiella tarda (GUO et al., 2015) e E. piscicida (BAO et al., 2019), importantes bactérias patógenas causadoras da edwardsiellosis; Vibrio anguillarum, bactéria causadora da vibriose (BAO et al., 2019;YE;WU;ZHANG, 2016;LIU et al., 2015;ZHANG et al., 2014); rabdovírus, vírus causador da septicemia viral hemorrágica, uma das mais importantes doenças virais em salmonídeos (KAVALIAUSKIS et al., 2016;NOVOA et al., 2006); Piscirickettsia salmonis, causadora da septicemia rickettsial de salmonídeos (TANDBERG et al., 2017); Listeria monocytogenes (DING et al., 2017), dentre outras doenças de importância econômica na piscicultura brasileira e mundial. ...
Zebrafish (Danio rerio) é um proeminente organismo utilizado como modelo para a avaliação da segurança e eficácia de novos compostos na saúde animal de maneira rápida e econômica. Considerando que, anualmente, mais de mil novas substâncias são introduzidas no mercado para o desenvolvimento de produtos como vacinas, medicamentos, aditivos alimentares e agroquímicos, o objetivo deste artigo é apresentar uma revisão do emprego do Zebrafish como modelo animal destinado a avaliação da eficácia e da biotoxidade de compostos em pesquisas em medicina veterinária. Diversos estudos confirmam que os perfis entre Zebrafish e mamíferos são surpreendentemente semelhantes, sendo que sua transparência, fertilização externa, pequeno porte e curto ciclo de vida permitem a avaliação direta in vivo e em tempo real dos (i) efeitos de compostos químicos no desenvolvimento animal, (II) eficácia de novas drogas no tratamento de doenças específicas, (iii) eficácia e segurança no desenvolvimento de vacinas contra doenças infecciosas, (iv) direcionamento de tratamento do câncer animal de forma direcionada e específica. Desta forma, a praticidade e eficiência deste modelo animal nas pesquisas pode acelerar o processo de desenvolvimento de novos compostos veterinários quando comparado com pesquisas realizadas em outros modelos animais, e com maior valor preditivo e informativo que pesquisas conduzidas com ensaios in vitro.
... For example, Rhabdovirus, which is one of the most important diseases in salmonids, is a virus that causes hemorrhagic viral septicemia [44,64]. Listeria monocytogenes [19,20]; Piscirickettsia salmonis which causes salmonid rickettsia sepsis (Tandberg et al. [83]); and in adjuvant test to improve the efficacy of vaccines [44], among others [82]. ...
Much of medical research relies on animal models to deepen knowledge of the causes of animal and human diseases, as well as to enable the development of innovative therapies. Despite rodents being the most widely used research model worldwide, in recent decades, the use of the zebrafish (Danio rerio) model has exponentially been adopted among the scientific community. This is because such a small tropical freshwater teleost fish has crucial genetic, anatomical and physiological homology with mammals. Therefore, zebrafish constitutes an excellent experimental model for behavioral, genetic and toxicological studies which unravels the mechanism of various human diseases. Furthermore, it serves well to test new therapeutic agents, such as the safety of new vaccines. The aim of this review was to provide a systematic literature review on the most recent studies carried out on the topic. It presents numerous advantages of this type of animal model in tests of efficacy and safety of both animal and human vaccines, thus highlighting gains in time and cost reduction of research and analyzes.
... The activation of innate immunity mediated by these cytokines could then condition the initiation of specific adaptive immune responses [53]. The expression of CD4 and MHC-II are the typical markers reflecting the exogenous antigen presentation, the higher expression levels of CD4 and MHC-II lead to increased advantages in terms of antigen presentation [54]. ...
Background:
Targeted delivery of virus-associated antigens to professional antigen-presenting cells (APCs) is considered as an efficient strategy to enhance the pyrophytic effect of vaccines against rhabdovirus disease.
Materials and methods:
In this study, we constructed a targeted carbon nanotubes-based vaccine deliver system (SWCNTs-MG) which can recognize the signature receptor (mannose) of APCs. An environmentally and economically important disease called spring viremia of carp (SVC) was studied as a model to evaluate the feasibility of single-walled carbon nanotubes (SWCNTs) conjugated with mannosylated antigen for rhabdovirus prevention.
Results:
Results showed that SWCNTs-MG could cross into fish body and present to internal immune-related tissues through gill, muscle and intestine within 6 h immersed vaccination. With further modification of mannose moiety, the obtained nanovaccine showed enhanced uptake by carp macrophages and immune-related tissues, which would then trigger strong immune responses against spring viremia of carp virus (SVCV) infection. Moreover, the survival rate of fish vaccinated with SWCNTs-MG (30 mg/L) was 63.5% after SVCV infection, whereas it was 0% for the control group.
Conclusion:
This study not only provide a theoretical basis and research template for the application of targeted nanovaccine system in aquatic animals, but also play an important role in supporting development of healthy aquaculture and ensuring the safety of aquatic products and ecology.
... For example, Rhabdovirus, which is one of the most important diseases in salmonids, is a virus that causes hemorrhagic viral septicemia [44,64]. Listeria monocytogenes [19,20]; Piscirickettsia salmonis which causes salmonid rickettsia sepsis (Tandberg et al. [83]); and in adjuvant test to improve the efficacy of vaccines [44], among others [82]. ...
Toxicity studies in mammals continue to be the most appropriate model for predicting risk in humans, but they tend to be expensive and time-consuming. In the aftermath of the genetic sequencing of zebrafish (Danio rerio), this species is highly genetically homologous to humans. The use of the zebrafish model to assess food toxicity is already a reality as it is capable of biological processes difficult to reproduce in vitro. Studies of complex mechanisms of absorption, distribution, metabolism, and excretion as well as cellular and tissue interactions are of great information value resulting in time, space and cost savings, when compared to studies with rodents. This review addresses the relevance of zebrafish model in food safety research, both in the use of ingredients and approved and generally recognized as safe food additives as well as for establishing levels of safe food contaminant residues present in the environment. Toxicological screening using the zebrafish model integrate the evaluation of teratogenicity, cardiotoxicity, hepatotoxicity, genotoxicity, neurotoxicity, endocrine toxicity, reproductive and behavioral aspects. These are important endpoints for food safety assessment, which take substantially less time than in mammalian tests. Furthermore, it serves well as a screening test follow-up for validating favorable results in murine models, hence accelerating the risk assessment process of products submitted for approval and registration, prioritizing safe compounds and reducing unnecessary costs in subsequent mammalian studies. In conclusion, the zebrafish model can be a useful tool for food safety tests; however, additional studies are needed to further validate this model for registration of new food ingredients and additives.
... Moreover, we found that Toll-like receptor (TLR) signaling pathway, MHC-I and MHC-II antigen presentation pathways were induced when zebrafish were challenged via intraperitoneal injection or bath delivery with attenuated Lm (EGDe-ΔactA/inlB). Our previous work provides new insights for understanding the multiple possibilities regarding the application of attenuated Lm [14]. ...
Live bacteria, including attenuated bacteria and probiotics, can be engineered to deliver target antigen to excite the host immune system. The preponderance of these live bacterial vaccine vectors is that they can stimulate durable humoral and cellular immunity. Moreover, delivery strategies of heterologous antigen in live bacterial promote the applications of new vaccine development. Genetic technologies are evolving, which potentiate the developing of heterologous antigen delivery systems, including bacterial surface display system, bacterial secretion system and balanced lethal vector system. Although the live bacterial vaccine vector is a powerful adjuvant, certain disadvantages, such as safety risk, must also be taken into account. In this review, we compare the development of representative live bacterial vectors, and summarize the main characterizations of the various delivery strategies of heterologous antigen in live vector vaccines.
The aquaculture sector predicts protein-rich meals by 2040 and has experienced significant economic shifts since
2000. However, challenges emanating from disease control measures, brood stock improvement, feed ad-
vancements, hatchery technology, and water quality management due to environmental fluctuations have been
taken as major causative agents for hindering the sector’s growth. For the past years, aquatic disease prevention
and control have principally depended on the use of various antibiotics, ecologically integrated control, other
immunoprophylaxis mechanisms, and chemical drugs, but the long-term use of chemicals such as antibiotics not
only escalates antibiotic-resistant bacteria and genes but also harms the fish and the environments, resulting in
drug residues in aquatic products, severely obstructing the growth of the aquaculture sector. The field of science
has opened new avenues in basic and applied research for creating and producing innovative and effective
vaccines and the enhancement of current vaccines to protect against numerous infectious diseases. Recent ad-
vances in vaccines and vaccinology could lead to novel vaccine candidates that can tackle fish diseases, including
parasitic organism agents, for which the current vaccinations are inadequate. In this review, we study and
evaluate the growing aquaculture production by focusing on the current knowledge, recent progress, and
prospects related to vaccinations and immunizations in the aquaculture industry and their effects on treating
bacterial and viral diseases. The subject matter covers a variety of vaccines, such as conventional inactivated and
attenuated vaccines as well as advanced vaccines, and examines their importance in real-world aquaculture
scenarios. To encourage enhanced importation of vaccines for aquaculture sustainability and profitability and
also help in dealing with challenges emanating from diseases, national and international scientific and policy
initiatives need to be informed about the fundamental understanding of vaccines.
Vibrio parahaemolyticus is considered to be one of the most threatening aquatic pathogens in aquaculture. Therefore, the development of an effective vaccine is crucial to prevent and control it. In this report, we identified the target antigen VP2309 with strong immunogenicity from four outer membrane proteins of V. parahaemolyticus. Furthermore, the live vector vaccine strain EGD-e△actA/inlB-VP2309 based on attenuated Listeria monocytogenes (Lm) was successfully constructed, consisting of secretory signal peptide of P60 protein, strong promoter Plmo2196 of Lm, and VP2309. It was able to express and secrete VP2309 efficiently. However, its expression retarded the growth of the host strain. The results of immune protection evaluation showed that both EGD-e△actA/inlB and EGD-e△actA/inlB-VP2309 strains reduced the mortality of zebrafish infected with V. parahaemolyticus. However, the vaccine strain carrying the VP2309 did not show better immune protection than the empty vector. Thus, we found that the attenuated Lm vector could offer a certain immune protection to zebrafish infected with V. parahaemolyticus, and is a potential vaccine vector that can be used in fish vaccine.
The disease caused by Micropterus salmoides rhabdovirus (MSRV) has brought substantial economic losses to the largemouth bass aquaculture industry in China. Vaccination was considered as a potential way to prevent and control this disease. As a kind of sustained and controlled release system, alginate and chitosan microspheres (SA‐CS) are widely used in the development of oral vaccination for fish. Here, we prepared a king of alginate‐chitosan composite microsphere to encapsulate the second segment of MSRV glycoprotein (G2 protein) and then evaluated the immune effect of the microsphere vaccine on largemouth bass. Largemouth bass were vaccinated via intragastric immunization by different treatments (PBS, SA‐CS, G2 and SA‐CS‐G2). The results showed that a stronger immune response including serum antibody levels, immune‐related physiological indexes (acid phosphatase, alkaline phosphatase, superoxide dismutase and total antioxidant capacity) and the expression of immune‐related gene (IgM、IL‐8、IL‐1β、CD4、TGF‐β、TNF‐α) can be induced obviously with SA‐CS‐G2 groups compared with G2 groups when fish were vaccinated. Furthermore, fish were injected with a lethal dose of MSRV after immunization for 28 days, and the highest relative percentage survival (54.8%) was observed in SA‐CS‐G2 group (40 μg per fish), which is significantly higher than that of G2 group (25.8%). This study showed that alginate‐chitosan microspheres as the vaccine carrier can effectively improve the immune effect of oral vaccination and induce better immune protection effect against MSRV infection.
The increasing resistance of Helicobacter pylori (H. pylori) to antibiotics has limited the efficacy of antibiotic therapy in the treatment of H. pylori-associated gastric diseases. The vaccine as an alternative method is becoming a safe and effective way to address this problem. In previous studies, live vector vaccines have proved to be effective in controlling H. pylori infection. Attenuated Listeria monocytogenes (L. monocytogenes) is a potential candidate vector applied in clinical trials, which can deliver foreign antigens and induce a broad immune response. To further explore the effectiveness of L. monocytogenes as a vaccine vector against H. pylori, attenuated L. monocytogenes-based vaccine EGDeΔactA/inlB(EGDeAB)-MECU was constructed to secrete a multi-epitope chimeric antigen (MECU) containing multiple B cell epitopes from H. pylori antigens. EGDeAB-MECU could secrete MECU stably. After immunized by gavage and intravenous injection, both EGDeAB and EGDeAB-MECU could significantly decrease gastric H. pylori colonization and induce a high level of specific antibodies against H. pylori. In conclusion, attenuated L. monocytogenes had an immunotherapeutic effect on H. pylori-infected mice, indicating its further development as a promising candidate vaccine vector for the H. pylori vaccine.
During the past decades, Vibrio spp. infections have been a serious problem that causes fish blight and very large economic losses, especially in China, Japan, Europe, and North America. Hitra disease caused by Vibrio salmonicida and winter ulcer disease caused by Vibrio viscosus have seriously affected cultured salmon in Norway and Iceland. In Asia, Vibriosis has caused huge losses in cultured grass carp and turbot of Chinese and cultured eels of Japan. Antibiotics and other chemical agents are the most common methods currently to prevent and control diseases but they have led to the emergence of drug-resistant strains and antibiotic residues. The vaccine is superior in high efficiency, safety, and convenience. It can also obtain greater economic benefits, which make it the most suitable method to control fish diseases. The most traditional method is inactivated vaccines with adjuvant, which is administered by intraperitoneal injection. With the development of molecular biology, a combination of specific pathogen components and new adjuvants can provide enhanced immune protection. This review provides the research progress of different types of Vibrio spp. vaccines, including inactivated vaccines, live attenuated vaccines, subunit vaccines, DNA vaccines, and live vector vaccines.
Vibrio harveyi causes vibriosis in various marine aquaculture fish species, especially when they are young. The infection subsequently leads to significant economic losses for aquaculture farms. Vaccination is recommended to control this disease. This study describes the efficacy of a live attenuated V. harveyi strain MVh_vhs (LAVh) as a vaccine candidate in controlling infection by wild‐type V. harveyi (WTVh) in Lates calcarifer. A total of 240 fingerlings were divided into four groups. Group 1 was not vaccinated and was not challenged, Group 2 was vaccinated with a formalin‐killed V. harveyi (FKVh), Group 3 was vaccinated with the LAVh before challenge and Group 4 was not vaccinated and was challenged. Bath vaccination was employed for one hour before the LAVh distribution was determined in the fish mucus, gill, liver, gut, kidney and spleen. The gills, livers, kidneys and skins were also sampled for gene expression analysis. To challenge the fish, skin abrasion was conducted before the fish were challenged by immersion with WTVh. The results revealed an extensive distribution of the LAVh in the liver and kidneys of the fish in Group 3 for the first 12 hr, resulting in mild lesions compared with Group 1. Similarly, there were significantly (p < .05) higher expressions of the Chemokine ligand 4 and major histocompatibility complex I genes in the skin and liver of the fish in Group 3 in comparison with other groups. Vaccination with LAVh resulted in a significantly high rate of survival (68%) of the fingerlings after being challenged with WTVh.
Glycoprotein (G) is the most common gene used in SVCV vaccine constructions. To identify the major immunogenicity determinant region of SVCV G gene, herein we truncated G gene to 4 parts (G-1, G-2, G-3 and G-4). Bioinformatics and the enzyme linked immunosorbent assay (ELISA) were used to identify the antigenicity of these 4 truncated G proteins. Immunological assays (serum antibody production, enzyme activity, immune genes expression and challenge test) were carried out to further identify the immunogenicity of the screened G protein in common carp. Moreover, to further verify the immune response of the screened G protein-based subunit vaccine, its protective effects on common carp against SVCV infection using single-walled carbon nanotubes (SWCNTs) as a carrier were evaluated. Results showed that G-3 protein could induce higher antibody titer than other truncated G proteins. Furthermore, carps vaccinated with G-3 and G (positive control) showed significant enhancement of immune response (serum antibody production, enzyme activity and immune related genes expression) when compared with control groups. Meanwhile, as a promising vaccine carrier, SWCNTs could significantly enhance the immune effect of naked subunit vaccine (G-3 and G). Notably, after SVCV challenge, there was no significant difference in immune protection between G-3 and G, nor between SWCNTs-G-3 and SWCNTs-G. These results so far suggest G-3 might be the potential antigen epitope of SVCV. This study lays a foundation for developing vaccine and immunodiagnostic techniques.
Live attenuated bacteria is a promising candidate vector for the delivery of vaccines in clinic trials. In the field of aquaculture industry, live vector vaccine also could provide long-term and effective protection against fish bacterial diseases. In our previous work, we demonstrated attenuated Listeria monocytogenes (Lm) had the potential to be an aquaculture vaccine vector in cellular level and zebrafish model. To further investigate the potential application of attenuated Lm in aquaculture vaccines, the outer membrane protein K (OmpK) from Vibrio parahaemolyticus (V. parahaemolyticus), as a conservative protective antigen, was fused to a new antigen-delivery system, and introduced into double-gene attenuated Lm strain (EGDe-ΔactA/inlB, Lmdd) to get live-vector vaccine strain Lmdd-OmpK. The strain Lmdd-OmpK showed the stable secrete efficacy of OmpK and was tested the cross-protective immunity against Vibrio species. After intraperitoneal administration in zebrafish, Lmdd and Lmdd-OmpK strain both improved the survival rates of zebrafish infected by V. parahaemolyticus, Vibrio alginolyticus (V. alginolyticus) and Vibrio anguillarum (V. anguillarum), respectively. In summary, attenuated Lm is able to protect zebrafish against Vibrio species challenge, illustrating its potential value for further aquaculture vaccines development.
Although vaccine is a prophylaxis measure for the contagious and fatal disease caused by spring viremia of carp virus (SVCV), until now, no commercial vaccine is available for worldwide use. To find more effective and safe means for the control of SVCV infection, here, an Escherichia coli DH5α bacterial ghost (DH5α-BG, non-living bacterial) based DNA vaccine delivery system (BG/pEG-G) was constructed. Carp were injected intramuscularly by different treatments (PBS, pEGFP, BG, pEG-G and BG/pEG-G), and the immunoprotective effect induced by these treatments were analyzed. We found a stronger and longer duration immune response (serum antibody production and immune-related genes expression) can be induced in fish vaccinated with BG/pEG-G in comparison with those vaccinated with pEG-G alone. Additionally, a good immune protective effect (relative percentage survival >81.48%) was observed in fish vaccinated with BG/pEG-G vaccine at the dose of 0.2 μg DNA/g of fish. Overall, this study for the first time demonstrates the ability of DH5α-BG as DNA vaccine carrier against SVCV infection in carps, suggesting a practical multivalent strategy for the control of aquatic disease.
The production performance, efficacy, and safety of two types of vaccines for infectious bursal disease virus (IBDV) were compared with in-ovo vaccination of Cobb 500 broiler chickens for gross and microscopic examination of the bursa of Fabricius, bursa/body weight (b/B) ratio, flow cytometry, and serologic response to Newcastle disease virus (NDV) vaccination. One vaccine was a recombinant HVT-IBD vector vaccine (HVT as for herpesvirus of turkeys) and the other was an intermediate plus live IBDV vaccine. A significant difference was detected at 21 d. Eight of 10 chickens that received the IBDV live vaccine had severe bursal lesions and a relatively low b/B ratio of 0.95, and an inhibited NDV vaccine response. On the other hand, the HVT-IBD vector vaccine resulted in mild bursal lesions and a b/B ratio of 1.89. Therefore, the live vaccine had lower safety than that of the HVT-IBD vector vaccine. To determine the protective efficacy, chickens were intraocularly challenged at 24 d. Eight of 10 chickens in the IBDV live vaccination group showed gross and histological lesions characterized by hemorrhage, cyst formation, lymphocytic depletion, and a decreased b/B ratio. In contrast, the HVT-IBD vector vaccinated chickens showed mild gross and histological lesions in three of 10 chickens with a b/B ratio of 1.36, which was similar to that of the unchallenged controls. Vaccinated chickens showed a significant increase in IBDV antibody titers, regardless of the type of vaccine used. In addition, significantly better broiler flock performance was observed with the HVT-IBD vector vaccine compared to that of the live vaccine. Our results revealed that the HVT-IBD vector vaccine could be used as an alternative vaccine to increase efficacy, and to have an improved safety profile compared with the IBDV live vaccine using in-ovo vaccination against the Korean very virulent IBDV in commercial broiler chickens.
Administered by intramuscular injection, a DNA vaccine (pIRF1A-G) containing the promoter regions upstream of the rainbow trout interferon regulatory factor 1A gene (IRF1A) driven the expression of the infectious hematopoietic necrosis virus (IHNV) glycoprotein (G) elicited protective immune responses in rainbow trout (Oncorhynchus mykiss). However, less laborious and cost-effective routes of DNA vaccine delivery are required to vaccinate large numbers of susceptible farmed fish. In this study, the pIRF1A-G vaccine was encapsulated into alginate microspheres and orally administered to rainbow trout. At 1, 3, 5, and 7 d post-vaccination, IHNV G transcripts were detected by quantitative real-time PCR in gills, spleen, kidney and intestinal tissues of vaccinated fish. This result suggested that the encapsulation of pIRF1A-G in alginate microparticles protected the DNA vaccine from degradation in the fish stomach and ensured vaccine early delivery to the hindgut, vaccine passage through the intestinal mucosa and its distribution thought internal and external organs of vaccinated fish. We also observed that the oral route required approximately 20-fold more plasmid DNA than the injection route to induce the expression of significant levels of IHNV G transcripts in kidney and spleen of vaccinated fish. Despite this limitation, increased IFN-1, TLR-7 and IgM gene expression was detected by qRT-PCR in kidney of vaccinated fish when a 10 μg dose of the oral pIRF1A-G vaccine was administered. In contrast, significant Mx-1, Vig-1, Vig-2, TLR-3 and TLR-8 gene expression was only detected when higher doses of pIRF1A-G (50 and 100 μg) were orally administered. The pIRF1A-G vaccine also induced the expression of several markers of the adaptive immune response (CD4, CD8, IgM and IgT) in kidney and spleen of immunized fish in a dose-dependent manner. When vaccinated fish were challenged by immersion with live IHNV, evidence of a dose-response effect of the oral vaccine could also be observed. Although the protective effects of the oral pIRF1A-G vaccine after a challenge with IHNV were partial, significant differences in cumulative percent mortalities among the orally vaccinated fish and the unvaccinated or empty-plasmid vaccinated fish were observed. Similar levels of protection were obtained after the intramuscular administration of 5 μg of pIRF1A-G or after the oral administration of a high dose of pIRF1A-G vaccine (100 μg); with 70 and 56 relative percent survival values, respectively. When fish were vaccinated with alginate microspheres containing high doses of the pIRF1A-G vaccine (50 or 100 μg), a significant increase in the production of anti-IHNV antibodies was detected in serum samples of the vaccinated fish compared with that in unvaccinated fish. At 10 days post-challenge, IHNV N gene expression was nearly undetectable in kidney and spleen of orally vaccinated fish which suggested that the vaccine effectively reduced the amount of virus in tissues of vaccinated fish that survived the challenge. In conclusion, our results demonstrated a significant increase in fish immune responses and resistance to an IHNV infection after the oral administration of increasing concentrations of a DNA vaccine against IHNV encapsulated into alginate microspheres.
Copyright © 2015. Published by Elsevier Ltd.
Listeria monocytogenes encodes a transcriptional activator PrfA to positively regulate the expression of virulence factors. We describe a strain M7 containing a PrfA*(G145S) that activates expression of virulence factors but with low pathogenicity. To uncover this contradictory relationship, we exchanged the prfA genes between strains EGDe and M7 (designated as EGDe-prfAM7 and M7-prfAEGDe). Constitutive activation of PrfA potentiated virulence of the pathogenic strain EGDe shown as increased adhesive and invasive abilities as well as enhanced cell-cell spread in cultured cell lines. However, the strain M7, though PrfA-activated, had significant defects in these virulence-related phenotypes and low pathogenicity in the murine infection model, as compared with EGDe or EGDe-PrfAM7 (PrfA*). To further uncover the possible mechanisms, we analyzed abundance and distributions of InlA, InlB, LLO and ActA proteins, all regulated by PrfA, in EGDe, M7 and their prfA mutants. Western blotting showed that the prfA-regulated genes of constitutively activated PrfA strains did overexpress in vitro, while different distributions were observed. In contrast to virulent strain EGDe-prfAM7, the majority of InlB in M7 were detected in the culture supernatants but not on the bacterial surface. We suppose that low virulence of strain M7 is due to its defects in infecting host cells possibly as a result of failed anchorage on the bacterial cells of surface proteins like InlB, a major protein involved in adhesion and invasion of pathogenic L. monocytogenes strains. Further research is warranted to address why InlB falls off the bacterial cells of this particular strain.
Listeria monocytogenes is a bacterial pathogen capable of causing severe infections in humans, often with fatal outcomes. Many different animal models exist to study L. monocytogenes pathogenicity, and we have investigated the chicken embryo as an infection model: What are the benefits and possible drawbacks? We have compared a defined wild-type strain with its isogenic strains lacking well-characterized virulence factors. Our results show that wild-type L. monocytogenes, already at a relatively low infection dose (~5 × 10² cfu), caused death of the chicken embryo within 36 h, in contrast to strains lacking the main transcriptional activator of virulence, PrfA, or the cytolysin LLO. Surprisingly, strains lacking the major adhesins InlA and InlB caused similar mortality as the wild-type strain. In conclusion, our results suggest that the chicken embryo is a practical model to study L. monocytogenes infections, especially when analyzing alternative virulence pathways independent of the InlA and InlB adhesins. However, the route of infection might be different from a human infection. The chicken embryo model and other Listeria infection models are discussed.
In the emerging field of active and specific cancer immunotherapy, strategies using live-attenuated bacterial vectors have matured in terms of academic and industrial development. Different bacterial species can be genetically engineered to deliver antigen to APCs with strong adjuvant effects due to their microbial origin. Proteic or DNA-encoding antigen delivery routes and natural bacterial tropisms might differ among species, permitting different applications. After many academic efforts to resolve safety and efficacy issues, some firms have recently engaged clinical trials with live Listeria or Salmonella spp. We describe here the main technological advances that allowed bacteria to become one of the most promising vectors in cancer immunotherapy.
The product of the spollM gene of Bacillus subtilis is required for complete septum migration and forespore engulfment during sporulation. To investigate whether expression of spollM is required in the forespore compartment of the sporangium, we have constructed a new integrational vector, pKSV7, which contains temperature-sensitive replication functions derived from pE194ts. The presence of the conditionally defective replication origin greatly stimulates plasmid excision when sporulation occurs at the permissive temperature. This facilities the use of a genetic technique employed by Illing et al [1] to distinguish genes whose expression must occur in the forespore from genes that may be expressed exclusively in the mother cell compartment. The results of the integration/excision experiments using pKSV7 support the conclusion that spollM must be expressed in the mother cell. The conditional integration analysis of porespore and mother cell fractions suggests that spollM is also expressed in the mother cell. The conditional integration vector pKSV7 replicates at high copy number in E coli and allows the identification of inserts in the polylinker cluster by disruption of α-complementation and thus should be useful other kinds of genetic manipulations in B subtilis.
The cell wall peptidoglycan of Gram-positive bacteria functions as a surface organelle for the transport and assembly of proteins that interact with the environment, in particular, the tissues of an infected host. Signal peptide-bearing precursor proteins are secreted across the plasma membrane of Gram-positive bacteria. Some precursors carry C-terminal sorting signals with unique sequence motifs that are cleaved by sortase enzymes and linked to the cell wall peptidoglycan of vegetative forms or spores. The sorting signals of pilin precursors are cleaved by pilus-specific sortases, which generate covalent bonds between proteins leading to the assembly of fimbrial structures. Other precursors harbour surface (S)-layer homology domains (SLH), which fold into a three-pronged spindle structure and bind secondary cell wall polysaccharides, thereby associating with the surface of specific Gram-positive microbes. Type VII secretion is a non-canonical secretion pathway for WXG100 family proteins in mycobacteria. Gram-positive bacteria also secrete WXG100 proteins and carry unique genes that either contribute to discrete steps in secretion or represent distinctive substrates for protein transport reactions.
Listeria monocytogenes has been exploited as a vaccine carrier based upon its ability to induce a strong cell-mediated immune response. At present,
the safety of live, attenuated L. monocytogenes vaccines in patients is being studied in clinical trials. L. monocytogenes is also an attractive vaccine vector for use in poultry; however, the pathogenicity and immunogenicity of this organism in
poultry remain to be fully elucidated. In this study, we investigated the pathogenicity and immunogenicity of an actA- and plcB-deficient L. monocytogenes strain, yzuLM4ΔactA/plcB, and its wild-type parent strain, yzuLM4, in an avian infection model. The results showed that the wild-type strain could
infect ISA brown chickens, causing serious tissue disruptions, including various degrees of degeneration, necrotic lesions,
and inflammatory cell infiltration in the liver, spleen, heart, and kidney. However, the mutant strain showed reduced virulence
in embryonated eggs compared with that of the parent strain (the 50% lethal dose [LD50] was 3 logs higher). The mutant strain also showed low virulence in chickens and was rapidly eliminated by the host. There
were no obvious pathological changes in tissue sections, but the mutant strain still retained the ability to stimulate high
levels of antibody against the protein listeriolysin O (LLO). Booster immunization with the mutant strain led to rapid bacterial
clearance from the livers and spleens of chickens challenged by the intramuscular route or the oral route. Collectively, our
data suggest that the wild-type serotype 1/2a L. monocytogenes strain can cause serious disease in chickens but the mutant strain with a deletion of the actA and plcB genes is less virulent but induces a strong immune response. This mutant strain of L. monocytogenes is therefore a promising candidate as a safe and effective vector for the delivery of heterologous antigens to prevent zoonosis
and infectious disease in poultry.
Neutralizing antibodies are thought to be required at mucosal surfaces to prevent human papillomavirus (HPV) transmission. However, the potential for cell-mediated immunity in mediating protection against HPV infection has not been well explored. We generated recombinant Listeria monocytogenes (Lm) constructs that secrete listeriolysin O (LLO) fused with overlapping N-terminal (LLO-L1(1-258)) or C-terminal (LLO-L1(238-474)) fragments of HPV type 16 major capsid protein L1 (HPV-16-L1). Oral immunization of mice with either construct induced IFN-gamma-producing CD8+ and CD4+ T cells in the spleen and in the Peyer's patches with the C-terminal construct. Oral immunization with both constructs resulted in diminished viral titers in the cervix and uterus of mice after intravaginal challenge with vaccinia virus expressing HPV-16-L1.
Listeria monocytogenes has been exploited previously as a vaccine vector for the delivery of heterologous proteins such as tumor-specific antigens
for active cancer immunotherapy. However, for effective use of live vector in clinics, safety is a major concern. In the present
study, we describe an irreversibly attenuated and highly immunogenic L. monocytogenes platform, the L. monocytogenes dal-, dat-, and actA-deleted strain that expresses the human prostate-specific antigen (PSA) using an antibiotic resistance marker-free plasmid
(the dal dat ΔactA 142 strain expressing PSA). Despite limited in vivo survival, the dal dat ΔactA 142 strain was able to elicit efficient immune responses required for tumor clearance. Our results showed that immunization
of mice with the dal dat ΔactA 142 strain caused the regression of the tumors established by the prostate adenocarcinoma cell line expressing PSA. An evaluation
of immunologic potency indicated that the dal dat ΔactA 142 strain elicits a high frequency of PSA-specific immune responses. Interestingly, immunization with the dal dat ΔactA 142 strain induced significant infiltration of PSA-specific T cells in the intratumoral milieu. Collectively, our data suggest
that the dal dat ΔactA 142 strain is a safe and potent vector for clinical use and that this platform may be further exploited as a potential candidate
to express other single or multiple antigens for cancer immunotherapy.
Listeria monocytogenes is a facultative intracellular bacterial pathogen that escapes from a phagosome and grows in the host cell cytosol. Escape
of the bacterium from the phagosome to the cytosol is mediated by the bacterial pore-forming protein listeriolysin O (LLO).
LLO has multiple mechanisms that optimize activity in the phagosome and minimize activity in the host cytosol. Mutants that
fail to compartmentalize LLO activity are cytotoxic and have reduced virulence. We sought to determine why cytotoxic bacteria
have attenuated virulence in the mouse model of listeriosis. In this study, we constructed a series of strains with mutations
in LLO and with various degrees of cytotoxicity. We found that the more cytotoxic the strain in cell culture, the less virulent
it was in mice. Induction of neutropenia increased the relative virulence of the cytotoxic strains 100-fold in the spleen
and 10-fold in the liver. The virulence defect was partially restored in neutropenic mice by adding gentamicin, an antibiotic
that kills extracellular bacteria. Additionally, L. monocytogenes grew more slowly in extracellular fluid (mouse serum) than within tissue culture cells. We concluded that L. monocytogenes controls the cytolytic activity of LLO to maintain its nutritionally rich intracellular niche and avoid extracellular defenses
of the host.
Vaccination with bacterial auxotrophs, particularly those with an interruption in the common pathway of aromatic amino-acid biosynthesis, known as the shikimate pathway, has been shown to be effective in the prevention of a variety of bacterial diseases. In order to evaluate this approach to vaccine development in the important marine pathogen Photobacterium damselae subsp. piscicida, the aroA gene of the shikimate pathway was identified from a P. damselae subsp. piscicida genomic library by complementation in an aroA mutant of Escherichia coli. The complementing plasmid was isolated and the nucleotide sequence of the P. damselae subsp. piscicida genomic insert was determined. Subsequent analysis of the DNA-sequence data demonstrated that the identified plasmid contained 3464 bp of P. damselae subsp. piscicida DNA, including the complete aroA gene. The sequence data was used to delete a 144 bp MscI fragment, and the kanamycin resistance gene (kan) from transposon Tn903 was ligated into the MscI site. This delta(aro)A::kan construct was sub-cloned into a suicide plasmid and transferred to a wild-type P. damselae subsp. piscicida by conjugation and allelic exchange. One selected mutant, LSU-P2, was confirmed phenotypically to require supplementation with aromatic metabolites for growth in minimal media, and was confirmed genotypically by PCR and DNA sequencing. Further, LSU-P2 was demonstrated to be avirulent in hybrid striped bass and to provide significant protection against disease following challenge with the wild-type strain.
The facultative intracellular bacterium Listeria monocytogenes is being developed as a cancer vaccine platform because of its ability to induce potent innate and adaptive immunity. For successful clinical application, it is essential to develop a Listeria platform strain that is safe yet retains the potency of vaccines based on wild-type bacteria. Here, we report the development of a recombinant live-attenuated vaccine platform strain that retains the potency of the fully virulent pathogen, combined with a >1,000-fold reduction in toxicity, as compared with wild-type Listeria. By selectively deleting two virulence factors, ActA (DeltaactA) and Internalin B (DeltainlB), the immunopotency of Listeria was maintained and its toxicity was diminished in vivo, largely by blocking the direct internalin B-mediated infection of nonphagocytic cells, such as hepatocytes, and the indirect ActA-mediated infection by cell-to-cell spread from adjacent phagocytic cells. In contrast, infection of phagocytic cells was not affected, leaving intact the ability of Listeria to stimulate innate immunity and to induce antigenspecific cellular responses. Listeria DeltaactA/DeltainlB-based vaccines were rapidly cleared from mice after immunization and induced potent and durable effector and memory T-cell responses with no measurable liver toxicity. Therapeutic vaccination of BALB/c mice bearing murine CT26 colon tumor lung metastases or palpable s.c. tumors (>100 mm(3)) with recombinant Listeria DeltaactA/DeltainlB expressing an endogenous tumor antigen resulted in breaking of self-tolerance and long-term survival. We propose that recombinant Listeria DeltaactA/DeltainlB expressing human tumor-associated antigens represents an attractive therapeutic strategy for further development and testing in human clinical trials.
The production of fish larvae is often hampered by high mortality rates, and it is believed that most of this economic loss due to infectious diseases is ca. 10% in Western European aquaculture sector. The development of strategies to control the pathogen load and immuno-prophylactic measures must be addressed further to realise the economic "potential" production of marine fish larvae and thus improve the overall production of adult fish. The innate defence includes both humoral and cellular defence mechanisms such as the complement system and the processes played by granulocytes and macrophages. A set of different substances such as beta-glucans, bacterial products, and plant constituents may directly initiate activation of the innate defence mechanisms acting on receptors and triggering intracellular gene activation that may result in production of anti-microbial molecules. These immunostimulants are often obtained from bacterial sources, brown or red algae and terrestrial fungi are also exploited as source of novel potentiating substances. The use of immunostimulants, as dietary supplements, can improve the innate defence of animals providing resistance to pathogens during periods of high stress, such as grading, reproduction, sea transfer and vaccination. The immunomodulation of larval fish has been proposed as a potential method for improving larval survival by increasing the innate responses of the developing animals until its adaptive immune response is sufficiently developed to mount an effective response to the pathogen. To this end it has been proposed that the delivery of immunostimulants as a dietary supplement to larval fish could be of considerable benefit in boosting the animals innate defences with little detriment to the developing animal. Conversely, there is a school of thought that raises the concern of immunomodulating a neotanous animal before its immune system is fully formed as this may adversely affect the development of a normal immune response.
Listeria monocytogenes can be used to deliver protein antigens or DNA and mRNA encoding such antigens directly into the cytosol of host cells because
of its intracellular lifestyle. In this study, we compare the in vivo efficiencies of activation of antigen-specific CD8 and
CD4 T cells when the antigen is secreted by L. monocytogenes or when antigen-encoding plasmid DNA or mRNA is released by self-destructing strains of L. monocytogenes. Infection of mice with self-destructing L. monocytogenes carriers delivering mRNA that encodes a nonsecreted form of ovalbumin (OVA) resulted in a significant OVA-specific CD8 T-cell
response. In contrast, infection with L. monocytogenes delivering OVA-encoding DNA failed to generate specific T cells. Secretion of OVA by the carrier bacteria yielded the strongest
immune response involving OVA-specific CD8 and CD4 T cells. In addition, we investigated the antigen delivery capacity of
a self-destructing, virulence-attenuated L. monocytogenes aroA/B mutant. In contrast to the wild-type strain, this mutant exhibited only marginal liver toxicity when high doses (5 × 107 CFU per animal administered intravenously) were used, and it was also able to deliver sufficient amounts of secreted OVA
into mice. Therefore, the results presented here could lay the groundwork for a rational combination of L. monocytogenes as an attenuated carrier for the delivery of protein and nucleic acid vaccines in novel vaccination strategies.
The usefulness of Salmonella vaccine vehicles is limited by the fact that control programmes relying on Salmonella bacteriology and serology cannot differentiate infected animals from vaccinated ones, an ability referred to as DIVA (differentiating infected from vaccinated animals). As a first step towards Salmonella-based DIVA vaccines, the ompA gene was deleted in live attenuated ΔphoP and ΔrpoS vaccine strains. The ompA gene is present in all Salmonella enterica serovars and it encodes an abundant, highly immunogenic outer membrane protein.
The double mutant ΔphoP ΔompA and ΔrpoS ΔompA strains showed similar virulence attenuation, safety and immunogenicity in a mouse model of infection as the parental ΔphoP and ΔrpoS strains. Sera from mice inoculated with the double mutant strains failed to recognise OmpA in Western blots of outer membrane extracts, whereas the protein was recognised by sera from mice inoculated with wild-type Salmonella or a mixture of double mutant and parental strains. These data suggest that OmpA can be a suitable negative marker for DIVA vaccines
Listeria monocytogenes is a facultative intracellular pathogen that causes gastroenteritis, meningitis, encephalitis and maternofetal infections. 20-30% of eubacterial ORFs are predicted to encode membrane proteins. The bacterial cytoplasmic membrane is a macromolecular structure, which plays a key role for the pathogenesis. Despite this, little knowledge exists regarding the function of cytoplasmic membrane proteins of Listeria during infection. Here, we investigated a predicted membrane protein of the pathogen L. monocytogenes, Lmo0412, of unknown function. Lmo0412 is only present in the Listeria genus and low conserved in the non-pathogenic species L. innocua. Bacterial fractionation and western blot analyses showed that Lmo0412 was only detectable in the membrane of L. monocytogenes EGDe during logarithmic growth phase. lmo0412 expression in L. monocytogenes was down-regulated during in vitro infection of JEG-3 epithelial cells. A L. monocytogenes mutant deficient in this membrane protein showed increased invasion of Caco-2 and NRK-49F host cells using in vitro infection models. Moreover, the lack of Lmo0412 in this deletion mutant increased the viable bacteria counts in the spleen and liver of mice compared to the wild type strain. Taken together, these data suggest a selective advantage conferred by the absence of Lmo0412 for the virulence of L. monocytogenes.
The intestinal mucosa promotes T cell responses that might be beneficial for effective mucosal vaccines. However, intestinal resident memory T (Trm) cell formation and function are poorly understood. We found that oral infection with Listeria monocytogenes induced a robust intestinal CD8 T cell response and blocking effector T cell migration showed that intestinal Trm cells were critical for secondary protection. Intestinal effector CD8 T cells were predominately composed of memory precursor effector cells (MPECs) that rapidly upregulated CD103, which was needed for T cell accumulation in the intestinal epithelium. CD103 expression, rapid MPEC formation, and maintenance in intestinal tissues were dependent on T cell intrinsic transforming growth factor β signals. Moreover, intestinal Trm cells generated after intranasal or intravenous infection were less robust and phenotypically distinct from Trm cells generated after oral infection, demonstrating the critical contribution of infection route for directing the generation of protective intestinal Trm cells.
Attenuated Listeria monocytogenes (LM) is a promising candidate vector for the delivery of cancer vaccines. After phagocytosis by antigen-presenting cells, this bacterium stimulates the major histocompatibility complex (MHC)-I and MHC-II pathways and induces the proliferation of antigen-specific T lymphocytes. A new strategy involving genetic modification of the replication-deficient LM strain ΔdalΔdat (Lmdd) to express and secrete human CD24 protein has been developed. CD24 is a hepatic cancer stem cell biomarker that is closely associated with apoptosis, metastasis and recurrence of hepatocellular carcinoma (HCC). After intravenous administration in mice, Lmdd-CD24 was distributed primarily in the spleen and liver and did not cause severe organ injury. Lmdd-CD24 effectively increased the number of interferon (IFN)-γ-producing CD8(+) T cells and IFN-γ secretion. Lmdd-CD24 also enhanced the number of IL-4- and IL-10-producing T helper 2 cells. The efficacy of the Lmdd-CD24 vaccine was further investigated against Hepa1-6-CD24 tumors, which were inguinally inoculated into mice. Lmdd-CD24 significantly reduced the tumor size in mice and increased their survival. Notably, a reduction of T regulatory cell (Treg) numbers and an enhancement of specific CD8(+) T-cell activity were observed in the tumor-infiltrating lymphocytes (TILs). These results suggest a potential application of the Lmdd-CD24 vaccine against HCC.Cellular & Molecular Immunology advance online publication, 3 February 2014; doi:10.1038/cmi.2013.64.
The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-DeltaDeltaCr) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-DeltaDeltaCr) method. In addition, we present the derivation and applications of two variations of the 2(-DeltaDeltaCr) method that may be useful in the analysis of real-time, quantitative PCR data. (C) 2001 Elsevier science.
The following aspects are described for each of the main bacterial diseases in which vaccination is employed: (i) the biochemical, antigenic and genetic heterogeneity of the etiological agents; (ii) their geographical distribution and host range; (iii) the effectiveness and problems of current commercial vaccines; and (iv) the new vaccination approaches using recombinant DNA technology or other strategies different to the classic bacterins. In addition, economic aspects and future trends related to fish vaccination are also addressed.
Despite the importance and success of vaccine immunization against bacterial diseases in fish, little is known about the molecular mechanisms of vaccine-induced immune protection in teleost fish. In this study, the live attenuated Edwardsiella tarda vaccine strain WED, which has been shown to evoke efficacious protection against edwardsiellosis and ascites diseases in fish, was extensively evaluated for multiple parameters in a 5-week immunization and challenge experiment in zebrafish. The parameters evaluated included the immunologic potency (relative percent survival, RPS), the specific IgM antibody titers and the expression profiles of multiple immune-related gene markers at multiple time points following immunization and challenge. During the 4-week immunization phase, the toll-like receptor (TLR) 5 signaling pathway, the MHC-I antigen processing pathway and cytotoxic T lymphocyte (CTL) responses were activated in succession. In contrast, the MHC-II antigen processing pathway and the markers of CD4+ T lymphocyte activation were down-regulated, and IgM transcription and specific IgM antibody titers were not significantly induced following immunization. During the 1-week challenge phase, the induction of MHC-I and CTL responses and the inhibition of MHC-II and CD4+ T cell responses were similarly observed in immunized zebrafish following challenge with wild E. tarda. With the 5-week immunization and challenge model, our data suggest the basic mechanism that underlying the long-lasting protective immunity elicited by WED in zebrafish. This mechanism involved the induction of the TLR-5 signaling pathway, the MHC-I antigen processing pathway and CTL effector function, and CTL function seems play a major role in the protection against E. tarda infection in zebrafish.
OmpK, one of the major outer-membrane proteins of Vibrio harveyi zj2008, was expressed in cells of the yeast Pichia pastoris GS115. After induction for 72 h in methanol-containing medium, the concentration of the expressed protein reached 2 μg mL−1. The recombinant protein was well recognized by antibody against Escherichia coli-expressed ompK in Western blotting analysis. Alginate microspheres containing the recombinant protein were prepared and fed to sea bass (Lateolabrax japonicus). ELISA analysis indicated that a significant antibody response against ompK was elicited in the vaccinated group. Challenge of vaccinated fish with V. harveyi zj2008 revealed that immunization provided protection against the pathogen. These results suggest that P. pastoris expressing ompK is a feasible means of generating an antigen that can protect marine fish against vibriosis. Thus, alginate microspheres encapsulating the antigen may be developed as an oral vaccine in the near future.
A sensory threshold can be defined generally as a stimulus intensity that produces a response in half of the trials. The definition of the population threshold is discussed. Five main classical statistical procedures for estimating thresholds are reviewed. They are the probit, the logistic, the Spearman-Karber, the moving average and the up-and-down procedures. Some new developments in statistical methods for estimating thresholds are outlined. The newly developed methods include the generalized probit and logistic models, the model based on the Beta-Binomial distribution, the trimmed Spearman-Karber method, the kernel method and the sigmoidally constrained maximum likelihood estimation method. The authors propose a new procedure based on the Beta-Binomial distribution for estimating population threshold.
Toll-like receptors (TLRs) are one of the key components of innate immunity. Among various types of TLRs, TLR5 is involved in recognizing bacterial flagellin and after binding, it triggers myeloid differentiation primary response gene 88 (MyD88)-dependent signaling pathway to induce pro-inflammatory cytokines. In this report, we analyzed the expression profile of TLR5 and its associated downstream signaling molecules like MyD88 and tumor necrosis factor (TNF) receptor-associated factor (TRAF) 6 in the Indian major carp (IMC), mrigal (Cirrhinus mrigala) which is highly commercially important fish species in the Indian subcontinent. Ontogeny analysis of TLR5, MyD88 and TRAF6 revealed constitutive expression of these genes in all embryonic developmental stages, and highlighted the importance of embryonic innate immune defense system in fish. Tissue specific expression analysis of these genes by quantitative real-time PCR (qRT-PCR) revealed their wide distribution in various organs and tissues; highest expression of TLR5 and MyD88 was in liver and TRAF6 was in kidney. Modulation of TLR5, MyD88 and TRAF6 gene expression, and the induction of interleukin (IL)-8 and TNF-α were analyzed in various organs by qRT-PCR following flagellin stimulation, and Aeromonas hydrophila and Edwardsiella tarda infection. In the treated fish, majority of the tested tissues exhibited significant induction of these genes, although with varied intensity among the tissues and with the types of treatments. Among the examined tissues, a significant relationship of TLR5 induction, MyD88 and TRAF6 up-regulation, and enhanced expression of IL-8 and TNF-α gene transcripts was observed in the blood and intestine of both flagellin stimulated and bacteria infected fish. These findings may indicate the involvement of TLR5 in inducing IL-8 and TNF-α, and suggest the important role of TLR5 in augmenting innate immunity in fish in response to pathogenic invasion. This study will enrich the information in understanding the innate immune mechanism in fish and may be helpful in developing preventive measures against infectious diseases in fish.
Listeriolysin O (LLO) is the major factor implicated in the escape of Listeria monocytogenes from the phagolysosome. It is the only representative of cholesterol-dependent cytolysins that exhibits pH-dependent activity. Despite intense studies of LLO pH-dependence, this feature of the toxin still remains incompletely explained. Here we used fluorescence and CD spectroscopy to show that the structure of LLO is not detectably affected by pH at room temperature. We observed slightly altered haemolytic and permeabilizing activities at different pH values, which we relate to reduced binding of LLO to the lipid membranes. However, alkaline pH and elevated temperatures caused rapid denaturation of LLO. Aggregates of the toxin were able to bind Congo red and Thioflavin T dyes and were visible under transmission electron microscopy as large, amorphous, micrometer-sized assemblies. The aggregates had the biophysical properties of amyloid. Analytical ultracentrifugation indicated dimerization of the protein in acidic conditions, which protects the protein against premature denaturation in the phagolysosome, where toxin activity takes place. We therefore suggest that LLO spontaneously aggregates at the neutral pH found in the host cell cytosol and that this is a major mechanism of LLO inactivation.
Structured digital abstract
The prototypic forms of teleost novel immune-type receptors (NITRs) consist of a variable (V) region, a unique V-like C2 (V/C2) domain, a transmembrane region and a cytoplasmic tail containing immunoreceptor tyrosine-based inhibition motifs (ITIMs). NITRs encode diversified V regions in large multigene families but do not undergo somatic rearrangement. Studies in four different bony fish model systems have identified a number of different organizational forms of NITRs. Specifically, NITR genes encode N-terminal ectodomains of the V-type but otherwise vary in the: total number of extracellular immunoglobulin domains, number and location of joining (J) region-like motifs, presence of transmembrane regions, presence of charged residues within transmembrane regions, presence of cytoplasmic tails, and/or distribution of ITIM(s) within the cytoplasmic tails. V region-containing NITRs constitute a far more complex family than recognized originally and currently include individual members that potentially function through inhibitory as well as activating mechanisms. The genomic organization of the NITR gene cluster as well as the structural diversity and overall architecture of the NITR proteins is reminiscent of genes encoded at the mammalian leukocyte receptor cluster (LRC); however, there presently is no functional evidence to support an orthologous relationship between NITR and LRC gene products. Comparisons of the predicted structures of the NITRs have identified several short regions of sequence identity and a novel cloning strategy has been devised that selects for secretory and transmembrane proteins that encode these short motifs. Using this approach, related genes termed immune-type receptors (ITRs) have been identified in cartilaginous fish. Taken together, these studies indicate that leukocyte regulatory receptors, including those that mediate natural killer function, might have emerged early in vertebrate evolution and that the NITR/ITR genes represent a new and potentially highly significant link between innate and adaptive immune responses.
Listeria monocytogenes vectors have shown promise for delivery of viral and tumor antigens in animals. We used two mutant vector strains deleted for actA/plcB (BMB72) and actA/inlB (BMB54), and engineered both strains to secrete a heterologous nucleoprotein antigen from the Influenza A virus. Strains were evaluated in vitro and in mice. Twenty-two healthy volunteers received single oral doses of either strain in a physiological study of safety, shedding, and immunogenicity. Volunteers were observed in the hospital for seven days and had daily blood cultures, routine safety blood tests (complete blood count with differential; hepatic and renal function), and fecal cultures; none had fever, positive blood cultures, prolonged shedding, or serious or unexpected events. Four of 12 volunteers who received the actA/plcB-deleted strain had minor, transient, asymptomatic serum transaminase elevations (maximum increase 1.4× upper normal). Six of six volunteers who received ≥4 × 10(9) colony forming units had detectable mucosal immune responses to listerial antigens, but not to the vectored influenza antigen. Approximately half the volunteers had modest interferon-γ ELISpot responses to a complex listerial antigen, but none had increases over their baseline responses to the influenza antigen. Comparison with prior work suggests that foreign antigen expression, and perhaps also freezing, may adversely affect the organisms' immunogenicity.
Displaying foreign antigens on the surface of attenuated or avirulent bacteria is an important strategy to develop live multivalent vector vaccines. In our previous work, several efficient surface display systems have been established based on outer membrane anchoring elements, which could successfully display heterologous proteins in attenuated Vibrio anguillarum. In this work, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from pathogenic Aeromonas hydrophila LSA34 was fused to seven display systems and introduced into attenuated V. anguillarum strain MVAV6203 (AV) to get seven GAPDH-display strains. The strain AV/pN-gapA showed the best display efficacy of GAPDH and was tested as the multivalent vaccine candidate. Further immune protection evaluation of AV/pN-gapA in turbot (Scophtalmus maximus) demonstrated that the attenuated V. anguillarum with surface-displayed GAPDH of A. hydrophila LSA34 effectively protected turbot from the infections of A. hydrophila and V. anguillarum and showed potential value for further multivalent vaccine development.
The outer membrane protein-OmpK has been considered as a vaccine candidate for the prevention of infections due to Vibrio harveyi, Vibrio alginolyticus and Vibrio parahaemolyticus in fish. Interestingly, the polyclonal antibody raised against the recombinant OmpK from V. harveyi strain EcGs020802 recognized the OmpK homologues from other strains of Vibrio species by immunoblotting. The ompK genes from 19 Vibrio strains including V. harveyi (11), V. alginolyticus (6) and V. parahaemolyticus (2) were then cloned and sequenced. Alignment analysis based on the amino acid sequences indicated that the OmpK from V. harveyi strain EcGs020802 had 71.7-99.2% of identities with those from V. harveyi, V. alginolyticus and V. parahaemolyticus. Western blot analysis revealed that the corresponding native proteins ranged between 28 and 31 kDa, consistent with predicated molecular weight of OmpK in Vibrio strains. Furthermore, the cross-protective property of recombinant OmpK was evaluated through challenge with heterogeneous virulent Vibrio strains in Orange-spotted groupers (Epinephelus coioides). Orange-spotted groupers vaccinated with recombinant OmpK were more tolerant of the infection by virulent Vibrio strains and their relative percentage survival (RPS) was correlative with the degree of the identity of deduced amino acid sequences of their OmpK. Taken together, the OmpK is a conserved protective antigen among tested Vibrio species and might be a potentially versatile vaccine candidate for the prevention of infections due to V. harveyi, V. alginolyticus and V. parahaemolyticus.
Mammalian immune response can be divided into innate and acquired immunity. Furthermore, much evidence has demonstrated that activation of innate immunity is a prerequisite to induction of acquired immunity. This paradigm shift has changed our thinking on the pathogenesis and treatment of infections, immune diseases, allergy, and cancers.
(Communicated by Tadamitsu KISHIMOTO, M.J.A.)
The outer membrane proteins of the fish pathogen, Vibrio harveyi, have a role in interaction between bacterium and host and are potential candidates for vaccine development. In this study, the gene encoding an outer membrane protein, OmpK, which serves as the receptor for broad-host-range vibriophage KVP40 in V. harveyi, was isolated and characterized. Then the OmpK gene coding for mature peptide was subcloned into prokaryotic expression vector pBV220 and transformed into Escherichia coli DH5 alpha strain. After temperature induction, a recombinant protein was detected about 28 kDa in molecular weight and accounted for 24.8% of total proteins of whole cell as estimated by SDS-PAGE and scanning analysis of gel image. Polyclonal antibodies were raised in rabbits against the purified protein and the reaction of the antibody was confirmed by western blotting using the purified protein and crude extract of V. harveyi. Orange-spotted groupers (Epinephelus coioides) vaccinated with recombinant OmpK produced specific antibodies, and were highly resistant to infection by virulent V. harveyi. These results indicate that the OmpK is an effective vaccine candidate against V. harveyi in Orange-spotted groupers.
The plcA gene of Listeria monocytogenes encodes a secreted phosphatidylinositol-specific phospholipase C (Pl-PLC). Recent studies have established that transposon mutations within plcA result in avirulence for mice and pleiotropic effects when examined in tissue-culture models of infection. Genetic analysis reveals that many of the effects of the transposon insertions are due to loss of readthrough transcription from plcA into the downstream gene prfA, which encodes an essential transcription factor of numerous L. monocytogenes virulence genes. Construction of an in-frame deletion within plcA had no effect on expression of prfA thus allowing direct assignment of a role of the Pl-PLC in pathogenesis. Pl-PLC was shown to play a significant role in mediating escape of L. monocytogenes from phagosomes of primary murine macrophages. Interestingly, this defect manifested itself in vivo in the liver but not in the spleen of infected mice.
As in all pathogenic bacteria, virulence of the facultative intracellular Listeria species is a multifactorial trait. The expression of the bacterial genes involved in the different steps of the infectious process--invasion, intracellular multiplication and spreading--is temporally and spatially controlled, thus ensuring the presence of the respective gene products at the right moment and place. So far, one network which is involved in the regulation of listerial virulence, the PrfA regulon, has been characterized rather well. The key element of this regulon, PrfA, belongs to the Crp/Fnr family of transcriptional regulators. Its synthesis and activity are influenced by a variety of physico-chemical signals outside and inside of eukaryotic host cells. The analysis of virulence gene expression in vivo, i.e. in infected host cells, indicates that yet uncharacterized bacterial factors other than PrfA, and possibly also host factors, modulate the expression of the PrfA regulon. Essentially nothing is known about the signal transduction pathways involved in the observed differential expression of virulence genes. Fermentable carbon sources seem to have a particular role in virulence gene regulation. In addition to the PrfA regulon, the Clp stress proteins have an impact on Listeria virulence. These two regulons interact with each other by an unknown mechanism.
When and how adaptive immunity emerged is one of the fundamental questions in immunology. Accumulated evidence suggests that the key components of adaptive immunity, rearranging receptor genes and the MHC, are unique to jawed vertebrates. Recent studies in protochordates, in particular, the draft genome sequence of the ascidian Ciona intestinalis, are providing important clues for understanding the origin of antigen receptors and the MHC. We discuss a group of newly identified protochordate genes along with some cold-blooded vertebrate genes, the ancestors of which might have provided key elements of antigen receptors. The organization of the proto-MHCs in protochordates provides convincing evidence that the MHC regions of jawed vertebrates emerged as a result of two rounds of chromosomal duplication.
Fish have developed protective strategies against monogeneans through immunological responses. In this study, immune adaptive response to parasites was analysed in the pompano Trachinotus marginatus infested by Bicotylophora trachinoti. Hosts were pre-treated with formalin and after 10 days assigned to one of the following experimental treatments: (1) fish infested with remaining eggs of B. trachinoti; (2) fish infested with remaining eggs of B. trachinoti and experimentally re-infested by exposure to T. marginatus heavily infested with B. trachinoti. Samples were collected at 0, 15, and 30 days. Gills were dissected to check the presence of B. trachinoti. Blood was collected for haematological and biochemical assays. Spleen and head-kidney were dissected for phagocytosis assay. The spleen-somatic index was also calculated. Re-infested fish showed a faster and higher parasite infestation than infested ones. The parasite mean abundance at 15 days was 24.86+/-13.32 and 11.67+/-8.57 for re-infested and infested fish, respectively. In both groups, hosts showed an immune adaptive response to parasite infestation that was marked by an increased number of leukocytes. Also, phagocytosis (%) in spleen and head-kidney cells was stimulated after parasite infestation (92.50+/-3.73 and 66.00+/-9.54, respectively), becoming later depressed (77.39+/-6.69 and 53.23+/-9.14, respectively). These results support the hypothesis that monogenean infestation induces a biphasic response of the non-specific defence mechanisms in the pompano T. marginatus. This response is marked by an initial stimulation followed by a later depression of the non-specific defence mechanisms.
Genes of five outer membrane proteins of Vibrio parahaemolyticus zj2003, including OmpW, OmpV, OmpK, OmpU and TolC, were cloned and expressed as N-terminal His(6)-tagged proteins in Escherichia coli. The recombinant fusion proteins were purified with nickel chelate affinity chromatography. To analyze the immunogenicity of these proteins, large yellow croaker (Pseudosciaena crocea) were immunized by intraperitoneal injection. Antibody response was assessed by method of enzyme-linked immunosorbent assay. Titres to all five recombinant proteins increased during 4 to 8 weeks post immunization, within the range of log 2 values of 5.75 to 10.8. Recorded relative survival percent (RPS) of the vaccinated groups varied from 80% to 90%, while 10 fish in control group all died. Western blot tests were undertaken with the serum of survival fish after experimental infection. Except for recombinant TolC, the other four recombinant proteins were recognized by the serum. It is indicated that four outer membrane proteins of V. parahaemolyticus zj2003, including OmpW, OmpV, OmpU and OmpK, are immunogenic during in vivo infection, which would be of some significance in developing efficient vaccine in aquaculture. This is the first report of successful vaccination against V. parahaemolyticus with purified recombinant outer membrane proteins.
The immunostimulatory characteristics and intracellular niche of Listeria monocytogenes make it uniquely suitable for use as a live bacterial vaccine vector. Preclinical results supporting this idea, and current strategies to induce beneficial cell-mediated immunity to both infectious diseases and cancer with this vector, are discussed in this review.
Listeria monocytogenes is a facultative intracellular bacterium that enters a variety of non-professional mammalian cells by triggered phagocytosis ("zipper mechanism") and replicates in the cytosol of the infected host cells. Therefore, it is a promising vaccine vector for the presentation of passenger antigens to the MHC class II and especially class I pathways. Here, we review recent progress made in our laboratory on the development of novel attenuated L. monocytogenes carrier strains for the delivery of heterologous antigens or antigen-encoding DNA and RNA to eukaryotic host cells. Based on the deletion of the chromosomal copy of the tryptophanyl-tRNA synthetase gene (trpS) and plasmid-based in trans complementation of the same, we were able to establish a balanced-lethal plasmid system in L. monocytogenes. Safety concerns in the antigen delivery in vivo were addressed by chromosomal deletion of genes in the basic branch of the aromatic amino acid pathway, resulting in safe, attenuated L. monocytogenes carrier strains. Furthermore, plasmid-based expression of a cytosolically expressed phage lysin resulted in a self-destructing carrier strain that has been successfully used for the delivery of antigens as well as antigen-encoding plasmid DNA and particularly mRNA, therefore overcoming bottlenecks that have been shown to exist for bacteria-mediated DNA delivery.