Immunogenicity of Japanese Encephalitis Virus Envelope Protein by Hyphantria cunea Nuclear Polyhedrosis Virus Vector in Guinea Pig

Japanese encephalitis virus (JEV) is an important pathogen causing febrile syndrome, encephalitis, and death. Envelop (E) glycoprotein is the major target of inducing neutralizing antibodies and protective immunity in host. In this study, E glycoprotein of JEV was expressed in Spodoptera frugiperd 9 cells as a fusion protein containing a gX signal sequence of pseudorabies virus. This purified HcE recombinant protein was evaluated for their immunogenicity and protective efficacy in guinea pig. The survival rates of guinea pig immunized with HcE protein was significantly increased over that of JE vaccine. This result indicates helpful information for developing a subunit vaccine against JEV.     

Effects of vitamin d treatment on skeletal muscle histology and ultrastructural changes in a rodent model

Vitamin D is well known for its role in maintaining calcium and phosphorus homeostasis and in promoting bone mineralization; however, more of its pleiotropic effects have been described recently. The aim of the present investigation was to study the effect of vitamin D treatment on skeletal muscles changes under different dietary conditions using an animal model. Four groups of C57BL/6J mice (n = 11 each) were maintained on either low fat diet (LFD) or high fat diet ??(HFD) with and without 1α,25-dihydroxyvitamin D3 (calcitriol) for 16 weeks. Animal weigh was recorded at baseline and then regular intervals, and at the end of the study, skeletal muscle tissues were harvested for the evaluation of the histopathological and ultrastructural changes. When control C57BL/6J mice were fed high-fat diet for 12 weeks, body weight gain was significantly increased compared with mice fed a LFD. (30.2% vs. 8.4%, p < 0.01). There was a significant gradual decrease in the weight of HFD fed mice that were treated with vitamin D as compared with a steady increase in the weights of controls (6.8% vs. 28.7%, p < 0.01). While the LFD group showed some ultrastructural changes, HDF fed on mice showed great muscle structural abnormalities. The whole sarcosome along with its membrane and cristae were severely damaged with scattered myocytes in HFD group. Furthermore, the mitochondria appeared weak and were on the verge of degenerations. The bands were diminished with loss of connections among myofibrils. These changes were attenuated in the HFD group treated with vitamin D with tissues have regained their normal structural appearance. The current findings indicate an important effect of vitamin D on skeletal muscle histology under HFD conditions.     

High Cell Density Process for Constitutive Production of a Recombinant Phytase in Thermotolerant Methylotrophic Yeast <Emphasis Type="Italic">Ogataea thermomethanolica</Emphasis> Using Table Sugar as Carbon Source

The yeast Ogataea thermomethanolica has recently emerged as a potential host for heterologous protein expression at elevated temperature. To evaluate the feasibility of O. thermomethanolica as heterologous host in large-scale fermentation, constitutive production of fungal phytase was investigated in fed-batch fermentation. The effect of different temperatures, substrate feeding strategies, and carbon sources on phytase production was investigated. It was found that O. thermomethanolica can grow in the temperature up to 40 °C and optimal at 34 °C. However, the maximum phytase production was observed at 30 °C and slightly decreased at 34 °C. The DOT stat control was the most efficient feeding strategy to obtain high cell density and avoid by-product formation. The table sugar can be used as an alternative substrate for phytase production in O. thermomethanolica. The highest phytase activity (134 U/mL) was obtained from table sugar at 34 °C which was 20-fold higher than batch culture (5.7 U/mL). At a higher cultivation temperature of 38 °C, table sugar can be used as a low-cost substrate for the production of phytase which was expressed with an acceptable yield (85 U/mL). Lastly, the results from this study reveal the industrial favorable benefits of employing O. thermomethanolica as a host for heterologous protein production.     

<Emphasis Type="Italic">Brugia malayi</Emphasis> Thioredoxin Peroxidase as a Potential Vaccine Candidate Antigen for Lymphatic Filariasis

Attempts were made to evaluate the protective efficacy of Brugia malayi thioredoxin peroxidase (BmTPX) in a mouse model. Mice immunized with a protein vaccine containing rBmTPX developed higher titres (1:5,000/1:10,000) of anti-BmTPX antibodies, compared with the mice immunized with the alum control. There was a higher level of cellular proliferative response in mice immunized with BmTPX compared with the alum control (p?<?0.05), which was associated with a Th2-type of response. In order to compare the prophylactic efficacy of BmTPX in natural infection, we evaluated the human immune responses to these antigens in endemic normals (EN) and infected individuals (microfilaraemic and chronic pathology). Results showed that EN subjects carry BmTPX-specific IgG1 and IgG3 circulating antibodies against natural exposure to filariasis. Peripheral blood mononuclear cells from EN subjects responded strongly to rBmTPX by proliferating, as well as by secreting interferon (IFN)-γ (Th1) and IL-5 (Th2), a mixed type of response to rBmTPX. In the case of infected individuals, there was no IFN-γ or IL-5 response. Thus, there was a clear dichotomy in the cytokine production by infected versus EN individuals. Our findings suggest that BmTPX may be a suitable antigen candidate for lymphatic filariasis, but a further study is still required.     

Vaccination with Klebsiella pneumoniae-derived extracellular vesicles protects against bacteria-induced lethality via both humoral and cellular immunity

The emergence of multidrug-resistant Klebsiella pneumoniae highlights the need to develop preventive measures to ameliorate Klebsiella infections. Bacteria-derived extracellular vesicles (EVs) are spherical nanometer-sized proteolipids enriched with outer membrane proteins. Gram-negative bacteria-derived EVs have gained interest for use as nonliving complex vaccines. In the present study, we evaluated whether K. pneumoniae-derived EVs confer protection against bacteria-induced lethality. K. pneumoniae-derived EVs isolated from in vitro bacterial culture supernatants induced innate immunity, including the upregulation of co-stimulatory molecule expression and proinflammatory mediator production. EV vaccination via the intraperitoneal route elicited EV-reactive antibodies and interferon-gamma-producing T-cell responses. Three vaccinations with the EVs prevented bacteria-induced lethality. As verified by sera and splenocytes adoptive transfer, the protective effect of EV vaccination was dependent on both humoral and cellular immunity. Taken together, these findings suggest that K. pneumoniae-derived EVs are a novel vaccine candidate against K. pneumoniae infections.     

Homologous and heterologous interactions between catalytic and regulatory subunits of <Emphasis Type="Italic">Escherichia coli</Emphasis> acetohydroxyacid synthase I and III

Homologous and heterologous interactions between acetohydroxyacid synthase (AHAS) I and III from E. coli have been studied by surface plasmon resonance (SPR). The catalytic and regulatory subunits association for AHAS I (K _D = 1.13 × 10⁻⁷ M) was stronger than that for AHAS III (K _D = 5.29 × 10⁻⁷ M). A strong heterologous association between regulatory and catalytic subunits and heterologous activation of catalytic subunits were observed. SPR results combined with enzyme kinetics indicate that the reconstituted heterologous enzymes had similar kinetic properties as homologous enzymes, implying that the regulatory subunit of AHAS I could be replaced by the regulatory subunit of AHAS III and vice versa. This work may be useful to further understandings of the mechanism of regulation of AHAS.     

Designing an efficient multi-epitope peptide vaccine against Vibrio cholerae via combined immunoinformatics and protein interaction based approaches

Cholera continues to be a major global health concern. Among different Vibrio cholerae strains, only O1 and O139 cause acute diarrheal diseases that are related to epidemic and pandemic outbreaks. The currently available cholera vaccines are mainly lived and attenuated vaccines consisting of V. cholerae virulence factors such as toxin-coregulated pili (TCP), outer membrane proteins (Omps), and nontoxic cholera toxin B subunit (CTB). Nowadays, there is a great interest in designing an efficient epitope vaccine against cholera. Epitope vaccines consisting of immunodominant epitopes and adjuvant molecules enhance the possibility of inciting potent protective immunity. In this study, V. cholerae protective antigens (OmpW, OmpU, TcpA and TcpF) and the CTB, which is broadly used as an immunostimulatory adjuvant, were analyzed using different bioinformatics and immunoinformatics tools. The common regions between promiscuous epitopes, binding to various HLA-II supertype alleles, and B-cell epitopes were defined based upon the aforementioned protective antigens. The ultimately selected epitopes and CTB adjuvant were fused together using proper GPGPG linkers to enhance vaccine immunogenicity. A three-dimensional model of the thus constructed vaccine was generated using I-TASSER. The model was structurally validated using the ProSA-web error-detection software and the Ramachandran plot. The validation results indicated that the initial 3D model needed refinement. Subsequently, a high-quality model obtained after various refinement cycles was used for defining conformational B-cell epitopes. Several linear and conformational B-cell epitopes were determined within the epitope vaccine, suggesting likely antibody triggering features of our designed vaccine. Next, molecular docking was performed between the 3D vaccine model and the tertiary structure of the toll like receptor 2 (TLR2). To gain further insight into the interaction between vaccine and TLR2, molecular dynamics simulation was performed, corroborating stable vaccine-TLR2 binding. In sum, the results suggest that our designed epitope vaccine could incite robust long-term protective immunity against V. cholera.     

Challenges in the Heterologous Production of Furanocoumarins in Escherichia coli

Coumarins and furanocoumarins are plant secondary metabolites with known biological activities. As they are present in low amounts in plants, their heterologous production emerged as a more sustainable and efficient approach to plant extraction. Although coumarins biosynthesis has been positively established, furanocoumarin biosynthesis has been far more challenging. This study aims to evaluate if Escherichia coli could be a suitable host for furanocoumarin biosynthesis. The biosynthetic pathway for coumarins biosynthesis in E. coli was effectively constructed, leading to the production of umbelliferone, esculetin and scopoletin (128.7, 17.6, and 15.7 µM, respectively, from tyrosine). However, it was not possible to complete the pathway with the enzymes that ultimately lead to furanocoumarins production. Prenyltransferase, psoralen synthase, and marmesin synthase did not show any activity when expressed in E. coli. Several strategies were tested to improve the enzymes solubility and activity with no success, including removing potential N-terminal transit peptides and expression of cytochrome P450 reductases, chaperones and/or enzymes to increase dimethylallylpyrophosphate availability. Considering the results herein obtained, E. coli does not seem to be an appropriate host to express these enzymes. However, new alternative microbial enzymes may be a suitable option for reconstituting the furanocoumarins pathway in E. coli. Nevertheless, until further microbial enzymes are identified, Saccharomyces cerevisiae may be considered a preferred host as it has already been proven to successfully express some of these plant enzymes.     

Production and characterization of monoclonal antibody for class-specific determination of O,O-dimethyl organophosphorus pesticides and effect of heterologous coating antigens on immunoassay sensitivity

A generic hapten (H1), 3-(4-Dimethoxyphosphorothioyloxy phenyl)propanoic acid, was synthesized to produce monoclonal antibodies (mAbs) for the determination of organophosphorus pesticides (OPs) in a class-specific manner. Six heterologous haptens were designed to study the effect of hapten heterology on immunoassay sensitivity. Several mice were immunized with this H1-BSA immunogen. Spleen cells of two immunized mice were fused with myeloma cells, and the resulting hybridomas were screened using H1-OVA. In a competitive indirect enzyme-linked immunosorbent assay (ELISA) format, three hybridoma cell lines (B4-C6, D12-B5, E5-H2) that produced mAbs with high selectivity and broad specificity were selected and expanded. The monoclonal antibody D12-B5 with higher titer was chosen for further study. In heterologous assay, the combination of D12-B5 and coating antigen H7-OVA constituted a particularly sensitive assay for competitive indirect ELISA and showed broad specificity for the determination of OPs, including parathion-methyl, chlorpyrifos-methyl, tolclofos-methyl, fenthion, malathion, fenitrothion. This combination resulted in the IC₅₀ of 0.58-10.47 µg/ml.     

13 C 183W-Kopplungen als sonde für metall—carbin-, —carbenund—alkyl-bindungen

Carbon-13 NMR data are reported for trimethyltin derivatives containing ER_n groups where E  C, Si, Ge, Sn, N, O and S including a series of cyclic amines with ring sizes from three to seven. Coupling constant values for the homologous series of fourth group derivatives give goood correlations with the electronegativity of E. The observation of the two-bond, ¦ ²J(¹¹⁹SnE¹³C) ¦ couplings only in the derivatives containing bulky R groups is rationalized by a bimolecular exchange of ER_n groups in the concentrated solutions studied.     

Genome Mining,Heterologous Expression,Antibacterial and Antioxidant Activities of Lipoamides and Amicoumacins from Compost-Associated Bacillus subtilis fmb60

Bacillus subtilis fmb60, which has broad-spectrum antimicrobial activities, was isolated from plant straw compost. A hybrid NRPS/PKS cluster was screened from the genome. Sixteen secondary metabolites produced by the gene cluster were isolated and identified using LC-HRMS and NMR. Three lipoamides D–F (1–3) and two amicoumacin derivatives, amicoumacins D, E (4, 5), were identified, and are reported here for the first time. Lipoamides D–F exhibited strong antibacterial activities against harmful foodborne bacteria, with the MIC ranging from 6.25 to 25 µg/mL. Amicoumacin E scavenged 38.8% of ABTS⁺ radicals at 1 mg/mL. Direct cloning and heterologous expression of the NRPS/PKS and ace gene cluster identified its importance for the biosynthesis of amicoumacins. This study demonstrated that there is a high potential for biocontrol utilization of B. subtilis fmb60, and genome mining for clusters of secondary metabolites of B. subtilis fmb60 has revealed a greater biosynthetic potential for the production of novel natural products than previously anticipated.     

Antibacterial Effects of Commiphora gileadensis Methanolic Extract on Wound Healing

Commiphora gileadensis (CG) is a small tree distributed throughout the Middle East. It was traditionally used in perfumes in countries in this area. In Saudi Arabia, it was used to treat wounds burns and as an antidote to scorpion stings. This study aimed to evaluate the antimicrobial activity and cutaneous wound healing efficiency of the CG extracts using microbiological tests, rate of wound contraction and histopathological changes. CG plant were extracted using the methanol extraction technique; then, the methanolic extract was characterized using liquid chromatography coupled with mass spectrometry (LC–MS). Afterwards, a six-millimetre (mm) excision wound was induced in 60 male Balb/c mice. Mice were classified into two classes; each class consisted of three groups of 10 mice. In the non-infected wound class, the group I was assigned as control and received normal saline. Group II received gentamicin treatment, and group III treated with CG-methanolic extract. In the Staphylococcus aureus-infected class, group IV received normal saline, and groups V and VI were treated with gentamicin and CG-methanolic extract, respectively. The colonization of infected wounds was determined using colony-forming units (CFUs), and the percentage of wound contraction was measured in all groups. Finally, the histopathologic semi-quantitative determination of wound healing was evaluated by inflammatory cell infiltration, the presence of collagen fibres and granulation tissue, and the grade of re-epithelization. Composition analysis of the methanolic extract confirmed the presence of a high amount of ceramide (69%) and, to a lesser extent, hexosylceramide (18%) and phosphatidylethanolamine (7%) of the total amount. Additionally, there was a statistically significant difference between the percentage of wound contraction in the CG-treated and control groups in both Staphylococcus aureus-infected and non-infected wounds (p < 0.01). The colonization of the infected wounds was lower in the group treated with CG than in the control group (p < 0.01). In both non-infected and infected wounds, the CG-treated group showed significant statistical differences in inflammatory cell infiltration, collagen fibres, re-epithelization and granulation tissue formation compared with the control group (p < 0.01). The CG extract possesses antibacterial and anti-inflammatory properties that induce wound healing.     

Chemical Composition and Variability of the Volatile Components of Myrciaria Species Growing in the Amazon Region

Myrciaria (Myrtaceae) species have been well investigated due to their chemical and biological relevance. The present work aimed to carry out the chemotaxonomic study of essential oils of the species M. dubia, M. floribunda, and M. tenella, sampled in the Brazilian Amazon and compare them with the volatile compositions from other Myrciaria species reported to Brazil and Colombia. The leaves of six Myrciaria specimens were collected (PA, Brazil) during the dry season, and their chemical compositions were analyzed by gas chromatography-mass spectrometer (GC-MS) and gas chromatography-flame ionization detector (GC-FID). The main compounds identified in the essential oils were monoterpenes with pinane and menthane skeletons, followed by sesquiterpenes with caryophyllane and cadinane skeletons. Among the sampled Myrciaria specimens, five chemical profiles were reported for the first time: profile I (M. dubia, α-pinene, 54.0–67.2%); profile II (M. floribunda, terpinolene 23.1%, α-phellandrene 17.7%, and γ-terpinene 8.7%); profile III (M. floribunda, γ-cadinene 17.5%, and an unidentified oxygenated sesquiterpene 15.0%); profile IV (M. tenella, E-caryophyllene 43.2%, and α-humulene 5.3%); and profile V (M. tenella, E-caryophyllene 19.1%, and caryophyllene oxide 41.1%). The Myrciaria chemical profiles showed significant variability in extraction methods, collection sites, plant parts, and genetic aspects.     

Identification of novel sesterterpenes by genome mining of phytopathogenic fungi Phoma and Colletotrichum sp.

Two homologous gene clusters for the biosynthesis of sesterterpenes betaestacins were identified from two phytopathogens, Phoma betae and Colletotrichum orbiculare. Heterologous expression of identified oxidation enzymes with previously-characterized PbTS1 (BtcA_Pb) resulted in the production of seven novel sesterterpenes. Although both strains possessed homologous enzymes, oxidation state of corresponding products were different from each other, suggesting that structural diversification of sesterterpene skeletons might be achieved by these homologous enzymes with different functions.     

Expression of Hybrid Peptide EF-1 in Pichia pastoris,Its Purification,and Antimicrobial Characterization

EF-1 is a novel peptide derived from two bacteriocins, plantaricin E and plantaricin F. It has a strong antibacterial activity against Escherichia coli and with negligible hemolytic effect on red blood cells. However, the chemical synthesis of EF-1 is limited by its high cost. In this study, we established a heterologous expression of EF-1 in Pichia pastoris. The transgenic strain successfully expressed hybrid EF-1 peptide, which had a molecular weight of ~5 kDa as expected. The recombinant EF-1 was purified by Ni²⁺ affinity chromatography and reversed-phase high performance liquid chromatography (RP-HPLC), which achieved a yield of 32.65 mg/L with a purity of 94.9%. The purified EF-1 exhibited strong antimicrobial and bactericidal activities against both Gram-positive and -negative bacteria. Furthermore, propidium iodide staining and scanning electron microscopy revealed that EF-1 can directly induce cell membrane permeabilization of E. coli. Therefore, the hybrid EF-1 not only preserves the individual properties of the parent peptides, but also acquires the ability to disrupt Gram-negative bacterial membrane. Meanwhile, such an expression system can reduce both the time and cost for large-scale peptide production, which ensures its potential application at the industrial level.     

Ethyl Acetate Extract from Artemisia argyi Prevents Liver Damage in ConA-Induced Immunological Liver Injury Mice via Bax/Bcl-2 and TLR4/MyD88/NF-κB Signaling Pathways

Background: Immunological liver injury (ILI) is a common liver disease and lacks potent drugs for treatment. Artemisia argyi Lévl. et Vant. (A. argyi), a medicinal and edible homologous plant usually used in diet therapy to cure various liver diseases, provides a great option for the prevention of ILI. Purpose: To investigate the effect that ethyl acetate extract of A. argyi (AaEA) on Concanavalin A (ConA)-induced ILI and the mechanism of regulating Bax/Bcl-2 and TLR4/MyD88/NF-κB signaling pathways. Methods: The chemical components of AaEA were studied by LC-MS. In animal experiments, the positive control group was administrated diammonium glycyrrhizinate (DIG, 100 mg/kg), while different doses of AaEA groups (AaEA-H, AaEA-M, AaEA-L) were pretreated with AaEA 2.00, 1.00, and 0.50 g/kg, respectively, by intragastric for seven days, once every day. Then, ConA (12.00 mg/kg) was used through tail intravenous injection to establish the ILI model. The blood samples and livers were collected to test the degree of liver dysfunction, inflammation, oxidative stress, histopathological changes, and cell apoptosis. Real-time PCR and Western blotting analysis were used to explain the mechanism of regulating Bax/Bcl-2 and TLR4/MyD88/NF-κB signaling pathways. Results: The way in which AaEA prevents liver damage in immunological liver injury (ILI) mice caused by ConA was investigated for the first time. Pretreatment with AaEA reduced the expression of ALT, AST, and inflammatory factors (TNF-α and IFN-γ). Meanwhile, AaEA also reduced MDA levels but upregulated the contents of IL-4, SOD, and GSH-px, alleviating oxidative stress induced by ILI. Western blotting and real-time PCR analysis demonstrated that AaEA could regulate the expression level and relative mRNA expression of key proteins on Bax/Bcl-2 and TLR4/MyD88/NF-κB signaling pathways. Finally, 504 components from AaEA were identified by LC-MS analysis, mainly including flavones, phenolic acids, and terpenoids with anti-inflammatory and liver protective activities, which highlights the potential of AaEA for diet treatment of ILI. Conclusion: AaEA can work against ConA-induced ILI in mice by regulating Bax/Bcl-2 and TLR4/MyD88/NF-κB signaling pathways, which has the potential to be a great strategy for the prevention of ILI.     

Spatial regulation of a common precursor from two distinct genes generates metabolite diversity

In secondary metabolite biosynthesis, core synthetic genes such as polyketide synthase genes usually encode proteins that generate various backbone precursors. These precursors are modified by other tailoring enzymes to yield a large variety of different secondary metabolites. The number of core synthesis genes in a given species correlates, therefore, with the number of types of secondary metabolites the organism can produce. In our study, heterologous expression of all the A. terreus NRPS-like genes showed that two NRPS-like proteins, encoded by atmelA and apvA, release the same natural product, aspulvinone E. In hyphae this compound is converted to aspulvinones whereas in conidia it is converted to melanin. The genes are expressed in different tissues and this spatial control is probably regulated by their own specific promoters. Comparative genomics indicates that atmelA and apvA might share a same ancestral gene and the gene apvA is located in a highly conserved region in Aspergillus species that contains genes coding for life-essential proteins. Our data reveal the first case in secondary metabolite biosynthesis in which the tissue specific production of a single compound directs it into two separate pathways, producing distinct compounds with different functions. Our data also reveal that a single trans-prenyltransferase, AbpB, prenylates two substrates, aspulvinones and butyrolactones, revealing that genes outside of contiguous secondary metabolism gene clusters can modify more than one compound thereby expanding metabolite diversity. Our study raises the possibility of incorporation of spatial, cell-type specificity in expression of secondary metabolites of biological interest and provides new insight into designing and reconstituting their biosynthetic pathways.     

Oxidative Stress and DNA Damage Effect of Dioscorea hispida Dennst. on Placental Tissues of Rats

Dioscorea hispida Dennst. locally known as “ubi gadung” has been used as a traditional remedy and source of carbohydrate among Malaysians. To assess the effect of Dioscorea hispida aqueous extract (DHAE) on the production of reactive oxygen species (ROS) and their effects on DNA damage in Sprague Dawley rat’s placental tissues, pregnant rats were randomly divided into four groups. The animals were orally treated with distilled water (negative control) and three different concentrations of DHAE (250, 500 and 1000 mg/kg body weight (BW)) from gestation day 6 until 20. The oxidative stress in placental tissues was evaluated at day 21 by measuring the level of ROS, superoxide dismutase (SOD) and lipid peroxidation biomarker, malondialdehyde (MDA) while comet assay was used for DNA damage. There was no significant production of ROS and SOD activities in all groups. Significant changes were observed in the MDA level at 1000 mg/kg BW DHAE. Comet assay revealed a significant increase (p < 0.05) of DNA damage on animals treated with 250 and 500 mg/kg BW DHAE but not at the highest concentration. It was postulated that the placental cells could have undergone necrosis which destroys all components including DNA. This occurrence simultaneously reduces the levels of DNA damage which can be represented by lower level of tail moments. This finding correlates with our histopathological examination where necrotic cells of spongiotrophoblast were observed in the basal zone of placental tissue. The high amount of hydrogen cyanide and other compounds in 1000 mg/kg BW DHAE could elevate the lipid peroxidation and directly induce cell necrosis which requires further investigation.     

A single HBsAg DNA vaccination in combination with electroporation elicits long-term antibody responses in sheep

Vaccines continue to be the most cost effective method to reduce the burden of disease in both human and animal health. However, there is a need to improve the duration of immunity following vaccination, since maintenance of protective levels of antibody in serum or the ability to rapidly respond upon re-exposure (memory) is critical if vaccines are to provide long-term protective immunity. The purpose of this experiment was to test the duration of antibody responses and the ability to generate anamnestic responses following a single immunization with a DNA vaccine encoding hepatitis B surface antigen (HBsAg) delivered by a variety of routes. Sheep immunized with the conventional HBsAg subunit vaccine (Engerix-B) as well as sheep immunized with a HBsAg DNA vaccine, combined with electroporation, generated significant antibody responses that were sustained for 25 weeks after primary immunization. At 25 weeks, all experimental groups received a secondary immunization with the HBsAg subunit vaccine. Sheep that received a primary DNA immunization, in combination with electroporation, mounted an anamnestic response similar to the cohort immunized with the HBsAg subunit vaccine. In contrast, animals immunized with DNA vaccines administered without electroporation elicited no detectable memory response. The presence of immune memory was significantly correlated with the induction of a prolonged primary immune response. Thus, a single DNA vaccination, in combination with electroporation, approached the efficacy of the commercial subunit vaccine in the maintenance of long-term protective serum antibody titres and immune memory.