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Japanese encephalitis virus (JEV) is the pathogen that causes Japanese encephalitis (JE) in humans and horses. Lethality of the virus was reported to be between 20–30%, of which, 30–50% of the JE survivors develop neurological and psychiatric sequelae. Attributed to the low effectiveness of current therapeutic approaches against JEV, vaccination remains the only effective approach to prevent the viral infection. Currently, live-attenuated and chimeric-live vaccines are widely used worldwide but these vaccines pose a risk of virulence restoration. Therefore, continuing development of JE vaccines with higher safety profiles and better protective efficacies is urgently needed. In this study, the Macrobrachium rosenbergii nodavirus (MrNV) capsid protein (CP) fused with the domain III of JEV envelope protein (JEV-DIII) was produced in Escherichia coli. The fusion protein (MrNV-CPJEV-DIII) assembled into virus-like particles (VLPs) with a diameter of approximately 18 nm. The BALB/c mice injected with the VLPs alone or in the presence of alum successfully elicited the production of anti-JEV-DIII antibody, with titers significantly higher than that in mice immunized with IMOJEV, a commercially available vaccine. Immunophenotyping showed that the MrNV-CPJEV-DIII supplemented with alum triggered proliferation of cytotoxic T-lymphocytes, macrophages, and natural killer (NK) cells. Additionally, cytokine profiles of the immunized mice revealed activities of cytotoxic T-lymphocytes, macrophages, and NK cells, indicating the activation of adaptive cellular and innate immune responses mediated by MrNV-CPJEV-DIII VLPs. Induction of innate, humoral, and cellular immune responses by the MrNV-CPJEV-DIII VLPs suggest that the chimeric protein is a promising JEV vaccine candidate.

Discovery of conserved antigens for universal influenza vaccines warrants solutions to a number of concerns pertinent to the currently licensed influenza vaccines, such as annual reformulation and mismatching with the circulating subtypes. The latter causes low vaccine efficacies, and hence leads to severe disease complications and high hospitalization rates among susceptible and immunocompromised individuals. A universal influenza vaccine ensures cross-protection against all influenza subtypes due to the presence of conserved epitopes that are found in the majority of, if not all, influenza types and subtypes, e.g., influenza matrix protein 2 ectodomain (M2e) and nucleoprotein (NP). Despite its relatively low immunogenicity, influenza M2e has been proven to induce humoral responses in human recipients. Influenza NP, on the other hand, promotes remarkable anti-influenza T-cell responses. Additionally, NP subunits are able to assemble into particles which can be further exploited as an adjuvant carrier for M2e peptide. Practically, the T-cell immunodominance of NP can be transferred to M2e when it is fused and expressed as a chimeric protein in heterologous hosts such as Escherichia coli without compromising the antigenicity. Given the ability of NP-M2e fusion protein in inducing cross-protective anti-influenza cell-mediated and humoral immunity, its potential as a universal influenza vaccine is therefore worth further exploration.

Hepatitis B is a major global health challenge. In the absence of an effective treatment for the disease, hepatitis B vaccines provide protection against the viral infection. However, some individuals do not have positive immune responses after being vaccinated with the hepatitis B vaccines available in the market. Thus, it is important to develop a more protective vaccine. Previously, we showed that hepatitis B virus (HBV) ‘a’ determinant (aD) displayed on the prawn nodavirus capsid (Nc) and expressed in Spodoptera frugiperda (Sf9) cells (namely, Nc-aD-Sf9) self-assembled into virus-like particles (VLPs). Immunisation of BALB/c mice with the Nc-aD-Sf9 VLPs showed significant induction of humoral, cellular and memory B-cell immunity. In the present study, the biophysical properties of the Nc-aD-Sf9 VLPs were studied using dynamic light scattering (DLS) and circular dichroism (CD) spectroscopy. Enzyme-linked immunosorbent assay (ELISA) was used to determine the antigenicity of the Nc-aD-Sf9 VLPs, and multiplex ELISA was employed to quantify the cytokine response induced by the VLPs administered intramuscularly into BALB/c mice (n = 8). CD spectroscopy of Nc-aD-Sf9 VLPs showed that the secondary structure of the VLPs predominantly consisted of beta (β)-sheets (44.8%), and they were thermally stable up to ~52 °C. ELISA revealed that the aD epitope of the VLPs was significantly antigenic to anti-HBV surface antigen (HBsAg) antibodies. In addition, multiplex ELISA of serum samples from the vaccinated mice showed a significant induction (p &lt, 0.001) of IFN-γ, IL-4, IL-5, IL-6, IL-10, and IL-12p70. This cytokine profile is indicative of natural killer cell, macrophage, dendritic cell and cytotoxic T-lymphocyte activities, which suggests a prophylactic innate and adaptive cellular immune response mediated by Nc-aD-Sf9 VLPs. Interestingly, Nc-aD-Sf9 induced a more robust release of the aforementioned cytokines than that of Nc-aD VLPs produced in Escherichia coli and a commercially used hepatitis B vaccine. Overall, Nc-aD-Sf9 VLPs are thermally stable and significantly antigenic, demonstrating their potential as an HBV vaccine candidate.

Foot-and-mouth disease (FMD) is a devastating livestock disease caused by foot-and-mouth disease virus (FMDV). Outbreaks of this disease in a country always result in conspicuous economic losses to livestock industry and subsequently lead to serious socioeconomic damages due to the immediate imposition of trade embargo. Rapid and accurate diagnoses are imperative to control this infectious virus. In the current review, enzyme-linked immunosorbent assay (ELISA)-based methods used in FMD diagnosis are extensively reviewed, particularly the sandwich, liquid-phase blocking, and solid-phase competition ELISA. The differentiation of infected animals from vaccinated animals using ELISA-based methods is also highlighted, in which the role of 3ABC polyprotein as a marker is reviewed intensively. Recently, more studies are focusing on the molecular diagnostic methods, which detect the viral nucleic acids based on reverse transcription-polymerase chain reaction (RT-PCR) and RT-loop-mediated isothermal amplification (RT-LAMP). These methods are generally more sensitive because of their ability to amplify a minute amount of the viral nucleic acids. In this digital era, the RT-PCR and RT-LAMP are progressing toward the mobile versions, aiming for on-site FMDV diagnosis. Apart from RT-PCR and RT-LAMP, another diagnostic assay specifically designed for on-site diagnosis is the lateral flow immunochromatographic test strips. These test strips have some distinct advantages over other diagnostic methods, whereby the assay often does not require the aid of an external device, which greatly lowers the cost per test. In addition, the on-site diagnostic test can be easily performed by untrained personnel including farmers, and the results can be obtained in a few minutes. Lastly, the use of FMDV diagnostic assays for progressive control of the disease is also discussed critically.

Chimeric virus-like particles (VLPs) have been widely exploited for various purposes including their use as vaccine candidates, particularly due to their ability to induce stronger immune responses than VLPs consisting of single viral proteins. In the present study, VLPs of the Macrobrachium rosenbergii nodavirus (MrNV) capsid protein (Nc) displaying the hepatitis B virus &ldquo, a&rdquo, determinant (aD) were produced in Spodoptera frugiperda (Sf9) insect cells. BALB/c mice immunised with the purified chimeric Nc-aD VLPs elicited a sustained titre of anti-aD antibody, which was significantly higher than that elicited by a commercially available hepatitis B vaccine and Escherichia coli-produced Nc-aD VLPs. Immunophenotyping showed that the Sf9-produced Nc-aD VLPs induced proliferation of cytotoxic T-lymphocytes and NK1.1 natural killer cells. Furthermore, enzyme-linked immunospot (ELISPOT)analysis showed the presence of antibody-secreting memory B cells in the mice splenocytes stimulated with the synthetic aD peptide. The significant humoral, natural killer cell and memory B cell immune responses induced by the Sf9-produced Nc-aD VLPs suggest that they present good prospects for use as a hepatitis B vaccine candidate.

Foot-and-mouth disease (FMD) is a major threat to the livestock industry worldwide. Despite constant surveillance and effective vaccination, the perpetual mutations of the foot-and-mouth disease virus (FMDV) pose a huge challenge to FMD diagnosis. The immunodominant region of the FMDV VP1 protein (residues 131–170) displayed on phage T7 has been used to detect anti-FMDV in bovine sera. In the present study, the functional epitope was further delineated using amino acid sequence alignment, homology modelling and phage display. Two highly conserved regions (VP1145–152 and VP1159–170) were identified among different FMDV serotypes. The coding regions of these two epitopes were fused separately to the T7 genome and displayed on the phage particles. Interestingly, chimeric phage displaying the VP1159–170 epitope demonstrated a higher antigenicity than that displaying the VP1131–170 epitope. By contrast, phage T7 displaying the VP1145–152 epitope did not react significantly with the anti-FMDV antibodies in vaccinated bovine sera. This study has successfully identified a smaller functional epitope, VP1159–170, located at the C-terminal end of the structural VP1 protein. The phage T7 displaying this shorter epitope is a promising diagnostic reagent to detect anti-FMDV antibodies in vaccinated animals.

White tail disease in the giant freshwater prawn Macrobrachium rosenbergii causes significant economic losses in shrimp farms and hatcheries and poses a threat to food-security in many developing countries. Outbreaks of Macrobrachium rosenbergii nodavirus (MrNV), the causative agent of white tail disease (WTD) are associated with up to 100% mortality rates. Recombinant expression of the capsid protein of MrNV in insect cells leads to the production of VLPs closely resembling the native virus. We have investigated the structure of MrNV VLPs by cryogenic electron microscopy, determining a structure of the viral capsid at 7 angstroms resolution. Our data show that MrNV VLPs package nucleic acids in a manner reminiscent of other known nodavirus structures. The structure of the capsid however shows striking differences from insect and fish infecting nodaviruses, which have been shown to assemble trimer-clustered T=3 icosahedral virus particles. MrNV particles have pronounced dimeric blade-shaped spikes extending up to 6 nm from the outer surface of the capsid shell. Our structural analysis supports the assertion that MrNV along with the related virus of marine shrimp Penaeus vannamei nodavirus (PvNV) may represent a new genus of the Nodaviridae.Author summaryMacrobrachium rosenbergii nodavirus (MrNV) is the causative agent of white tail disease (WTD) which leads to 100% mortality in shrimp-farms growing giant freshwater prawn (M. rosenbergii). MrNV is therefore a significant threat to food security and causes severe economic losses in developing countries such as Malaysia, Indonesia, Pakistan, Thailand and India. Here we have used electron microscopy to study the three-dimensional structure of MrNV, revealing that the viral capsid – the protein shell that encloses the viral genome, protecting it and transporting it from one host to the next – is differently organised to capsids produced by other viruses in the nodavirus family. The virus was found to have large blade-like spikes on its outer surface that are likely important in the early stages of infection, when the virus attaches to and enters a host cell.

Middle East respiratory syndrome (MERS) is a deadly viral respiratory disease caused by MERS-coronavirus (MERS-CoV) infection. To date, there is no specific treatment proven effective against this viral disease. In addition, no vaccine has been licensed to prevent MERS-CoV infection thus far. Therefore, our current review focuses on the most recent studies in search of an effective MERS vaccine. Overall, vaccine candidates against MERS-CoV are mainly based upon the viral spike (S) protein, due to its vital role in the viral infectivity, although several studies focused on other viral proteins such as the nucleocapsid (N) protein, envelope (E) protein, and non-structural protein 16 (NSP16) have also been reported. In general, the potential vaccine candidates can be classified into six types: viral vector-based vaccine, DNA vaccine, subunit vaccine, nanoparticle-based vaccine, inactivated-whole virus vaccine and live-attenuated vaccine, which are discussed in detail. Besides, the immune responses and potential antibody dependent enhancement of MERS-CoV infection are extensively reviewed. In addition, animal models used to study MERS-CoV and evaluate the vaccine candidates are discussed intensively.

The aquaculture of salmonid fishes is a multi-billion dollar industry with production over 3 million tons annually. However, infectious hematopoietic necrosis virus (IHNV), which infects and kills salmon and trout, significantly reduces the revenue of the salmon farming industry. Currently, there is no effective treatment for IHNV infected fishes; therefore, early detection and depopulation of the infected fishes remain the most common practices to contain the spread of IHNV. Apart from hygiene practices in aquaculture and isolation of infected fishes, loss of fishes due to IHNV infection can also be significantly reduced through vaccination programs. In the current review, some of the diagnostic methods for IHNV, spanning from clinical diagnosis to cell culture, serological and molecular methods are discussed in detail. In addition, some of the most significant candidate vaccines for IHNV are also extensively discussed, particularly the DNA vaccines.

Shigella-infected bacillary dysentery or commonly known as Shigellosis is a leading cause of morbidity and mortality worldwide. The gradual emergence of multidrug resistantShigellaspp. has triggered the search for alternatives to conventional antibiotics. Phage therapy could be one such suitable alternative, given its proven long term safety profile as well as the rapid expansion of phage therapy research. To be successful, phage therapy will need an adequate regulatory framework, effective strategies, the proper selection of appropriate phages, early solutions to overcome phage therapy limitations, the implementation of safety protocols, and finally improved public awareness. To achieve all these criteria and successfully apply phage therapy against multidrug resistant shigellosis, a comprehensive study is required. In fact, a variety of phage-based approaches and products including single phages, phage cocktails, mutated phages, genetically engineered phages, and combinations of phages with antibiotics have already been carried out to test the applications of phage therapy against multidrug resistantShigella.This review provides a broad survey of phage treatments from past to present, focusing on the history, applications, limitations and effective solutions related to, as well as the prospects for, the use of phage therapy against multidrug resistantShigellaspp. and other multidrug resistant bacterial pathogens.

Macrobrachium rosenbergii nodavirus (MrNV) is the causative agent of white tail disease (WTD) which seriously impedes the production of the giant freshwater prawn and has a major economic impact. MrNV contains two segmented RNA molecules, which encode the RNA dependent RNA polymerase (RdRp) and the capsid protein (MrNV-CP) containing 371 amino acid residues. MrNV-CP comprises of the Shell (S) and the Protruding (P) domains, ranging from amino acid residues 1–252 and 253–371, respectively. The P-domain assembles into dimeric protruding spikes, and it is believed to be involved in host cell attachment and internalization. In this study, the recombinant P-domain of MrNV-CP was successfully cloned and expressed in Escherichia coli, purified with an immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC) up to ~90% purity. Characterization of the purified recombinant P-domain with SEC revealed that it formed dimers, and dynamic light scattering (DLS) analysis demonstrated that the hydrodynamic diameter of the dimers was ~6 nm. Circular dichroism (CD) analysis showed that the P-domain contained 67.9% of beta-sheets, but without alpha-helical structures. This is in good agreement with the cryo-electron microscopic analysis of MrNV which demonstrated that the P-domain contains only beta-stranded structures. Our findings of this study provide essential information for the production of the P-domain of MrNV-CP that will aid future studies particularly studies that will shed light on anti-viral drug discovery and provide an understanding of virus-host interactions and the viral pathogenicity.

Epizootics of highly pathogenic avian influenza (HPAI) have resulted in the deaths of millions of birds leading to huge financial losses to the poultry industry worldwide. The roles of migratory wild birds in the harbouring, mutation, and transmission of avian influenza viruses (AIVs), and the lack of broad-spectrum prophylactic vaccines present imminent threats of a global panzootic. To prevent this, control measures that include effective AIV surveillance programmes, treatment regimens, and universal vaccines are being developed and analysed for their effectiveness. We reviewed the epidemiology of AIVs with regards to past avian influenza (AI) outbreaks in birds. The AIV surveillance programmes in wild and domestic birds, as well as their roles in AI control were also evaluated. We discussed the limitations of the currently used AI vaccines, which necessitated the development of a universal vaccine. We evaluated the current development of AI vaccines based upon virus-like particles (VLPs), particularly those displaying the matrix-2 ectodomain (M2e) peptide. Finally, we highlighted the prospects of these VLP vaccines as universal vaccines with the potential of preventing an AI panzootic.

The matrix (M) protein of Nipah virus (NiV) is a peripheral protein that plays a vital role in the envelopment of nucleocapsid protein and acts as a bridge between the viral surface and the nucleocapsid proteins. The M protein is also proven to play an important role in production of virus-like particles (VLPs) and is essential for assembly and budding of NiV particles. The recombinant M protein produced in Escherichia coli assembled into VLPs in the absence of the viral surface proteins. However, the E. coli produced VLPs are smaller than the native virus particles. Therefore, the aims of this study were to produce NiV M protein in Pichia pastoris, to examine the structure of the VLPs formed, and to assess the potential of the VLPs as a diagnostic reagent. The M protein was successfully expressed in P. pastoris and was detected with anti-myc antibody using Western blotting. The VLPs formed by the recombinant M protein were purified with sucrose density gradient ultracentrifugation, high-performance liquid chromatography (HPLC), and Immobilized Metal Affinity Chromatography (IMAC). Immunogold staining and transmission electron microscopy confirmed that the M protein assembled into VLPs as large as 200 nm. ELISA revealed that the NiV M protein produced in P. pastoris reacted strongly with positive NiV sera demonstrating its potential as a diagnostic reagent.

Macrobrachium rosenbergii nodavirus (MrNV) is a pathogen of freshwater prawns that poses a threat to food security and causes significant economic losses in the aquaculture industries of many developing nations. A detailed understanding of the MrNV virion structure will inform the development of strategies to control outbreaks. The MrNV capsid has also been engineered to display heterologous antigens, and thus knowledge of its atomic resolution structure will benefit efforts to develop tools based on this platform. Here, we present an atomic-resolution model of the MrNV capsid protein (CP), calculated by cryogenic electron microscopy (cryoEM) of MrNV virus-like particles (VLPs) produced in insect cells, and three-dimensional (3D) image reconstruction at 3.3 Å resolution. CryoEM of MrNV virions purified from infected freshwater prawn post-larvae yielded a 6.6 Å resolution structure, confirming the biological relevance of the VLP structure. Our data revealed that unlike other known nodavirus structures, which have been shown to assemble capsids having trimeric spikes, MrNV assembles a T = 3 capsid with dimeric spikes. We also found a number of surprising similarities between the MrNV capsid structure and that of the Tombusviridae: 1) an extensive network of N-terminal arms (NTAs) lines the capsid interior, forming long-range interactions to lace together asymmetric units; 2) the capsid shell is stabilised by 3 pairs of Ca2+ ions in each asymmetric unit; 3) the protruding spike domain exhibits a very similar fold to that seen in the spikes of the tombusviruses. These structural similarities raise questions concerning the taxonomic classification of MrNV., Macrobrachium rosenbergii nodavirus (MrNV) is an economically important pathogen of freshwater prawns. Determination of the structure of the MrNV virion by cryo-electron microscopy reveals a capsid protein that is highly divergent from other known nodaviruses but has a surprising resemblance to plant-infecting tombusviruses., Author summary The freshwater prawn Macrobrachium rosenbergii is widely cultivated for food. Production is threatened by Macrobrachium rosenbergii nodavirus (MrNV), the causative agent of white-tail disease. Outbreaks in hatcheries often result in mortality rates of up to 100% in larvae and post-larvae, leading to devastating economic losses and threatening food security. We describe the atomic structure of the MrNV capsid, solved by cryogenic electron microscopy and three-dimensional image reconstruction. Our analysis revealed surprising differences between the structure of MrNV and that of other known nodaviruses. Moreover, we observed several features in the MrNV capsid that have been previously described in virion structures of the plant-infecting tombusvirus family. Most notably, the MrNV capsid exhibits pronounced dimeric spikes on its surface, the topology of this region closely resembling tombusvirus capsid spikes. Known nodavirus structures have trimeric spikes and do not display the same protein fold. The MrNV capsid is stabilised by divalent cations and laced together by a network of N-terminal arms that line the interior of the virion. Our analysis raises questions about the taxonomic classification of MrNV as well as revealing the structure of the capsid of this important pathogen. These data have the potential to inform the development of future interventions to prevent white-tail disease.

Macrobrachium rosenbergii nodavirus (MrNv) causes white tail disease (WTD) in giant freshwater prawns, which leads to devastating economic losses in the aquaculture industry. Despite extensive research on MrNv, there is still no antiviral agent to treat WTD. Thus, the main aim of this study was to identify potential anti-MrNv molecules. A 12-mer phage-displayed peptide library was biopanned against the MrNv virus-like particle (VLP). After four rounds of biopanning, two dominant phages harbouring the amino acid sequences HTKQIPRHIYSA and VSRHQSWHPHDL were selected. An equilibrium binding assay in solution was performed to determine the relative dissociation constant ( K D r e l ) of the interaction between the MrNv VLP and the selected fusion phages. Phage-HTKQIPRHIYSA has a K D r e l value of 92.4±22.8 nM, and phage-VSRHQSWHPHDL has a K D r e l value of 12.7±3.8 nM. An in-cell elisa was used to determine the inhibitory effect of the synthetic peptides towards the entry of MrNv VLP into Spodoptera frugiperda (Sf9) cells. Peptides HTKQIPRHIYSA and VSRHQSWHPHDL inhibited the entry of the MrNv VLP into Sf9 cells with IC50 values of 30.4±3.6 and 26.5±8.8 µM, respectively. Combination of both peptides showed a significantly higher inhibitory effect with an IC50 of 4.9±0.4 µM. An MTT assay revealed that the viability of MrNv-infected cells increased to about 97 % in the presence of both peptides. A real-time RT-PCR assay showed that simultaneous application of both peptides significantly reduced the number of MrNv per infected cell, from 97±9 to 11±4. These peptides are lead compounds which can be further developed into potent anti-MrNv agents.

Virus-like particles composed of the core antigen of hepatitis B virus (HBcAg) have been shown to be an effective platform for the display of foreign epitopes in vaccine development. Heterologous sequences have been successfully inserted at both amino and carboxy termini as well as internally at the major immunodominant epitope. We used cryogenic electron microscopy (CryoEM) and three-dimensional image reconstruction to investigate the structure of VLPs assembled from an N-terminal extended HBcAg that contained a polyhistidine tag. The insert was seen to form a trimeric spike on the capsid surface that was poorly resolved, most likely owing to it being flexible. We hypothesise that the capacity of N-terminal inserts to form trimers may have application in the development of multivalent vaccines to trimeric antigens. Our analysis also highlights the value of tools for local resolution assessment in studies of partially disordered macromolecular assemblies by cryoEM.

Nodaviruses are small bipartite RNA viruses which belong to the family ofNodaviridae. They are categorized into alpha-nodavirus, which infects insects, and beta-nodavirus, which infects fishes. Another distinct group of nodavirus infects shrimps and prawns, which has been proposed to be categorized as gamma-nodavirus. Our current review focuses mainly on recent studies performed on nodaviruses. Nodavirus can be transmitted vertically and horizontally. Recent outbreaks have been reported in China, Indonesia, Singapore and India, affecting the aquaculture industry. It also decreased mullet stock in the Caspian Sea. Histopathology and transmission electron microscopy (TEM) are used to examine the presence of nodaviruses in infected fishes and prawns. For classification, virus isolation followed by nucleotide sequencing are required. In contrast to partial sequence identification, profiling the whole transcriptome using next generation sequencing (NGS) offers a more comprehensive comparison and characterization of the virus. For rapid diagnosis of nodavirus, assays targeting the viral RNA based on reverse-transcription PCR (RT-PCR) such as microfluidic chips, reverse-transcription loop-mediated isothermal amplification (RT-LAMP) and RT-LAMP coupled with lateral flow dipstick (RT-LAMP-LFD) have been developed. Besides viral RNA detections, diagnosis based on immunological assays such as enzyme-linked immunosorbent assay (ELISA), immunodot and Western blotting have also been reported. In addition, immune responses of fish and prawn are also discussed. Overall, in fish, innate immunity, cellular type I interferon immunity and humoral immunity cooperatively prevent nodavirus infections, whereas prawns and shrimps adopt different immune mechanisms against nodavirus infections, through upregulation of superoxide anion, prophenoloxidase, superoxide dismutase (SOD), crustin, peroxinectin, anti-lipopolysaccharides and heat shock proteins (HSP). Potential vaccines for fishes and prawns based on inactivated viruses, recombinant proteins or DNA, either delivered through injection, oral feeding or immersion, are also discussed in detail. Lastly, a comprehensive review on nodavirus virus-like particles (VLPs) is presented. In recent years, studies on prawn nodavirus are mainly focused onMacrobrachium rosenbergiinodavirus (MrNV). RecombinantMrNV VLPs have been produced in prokaryotic and eukaryotic expression systems. Their roles as a nucleic acid delivery vehicle, a platform for vaccine development, a molecular tool for mechanism study and in solving the structures ofMrNV are intensively discussed.

Cancers have killed millions of people in human history and are still posing a serious health problem worldwide. Therefore, there is an urgent need for developing preventive and therapeutic cancer vaccines. Among various cancer vaccine development platforms, virus-like particles (VLPs) offer several advantages. VLPs are multimeric nanostructures with morphology resembling that of native viruses and are mainly composed of surface structural proteins of viruses but are devoid of viral genetic materials rendering them neither infective nor replicative. In addition, they can be engineered to display multiple, highly ordered heterologous epitopes or peptides in order to optimize the antigenicity and immunogenicity of the displayed entities. Like native viruses, specific epitopes displayed on VLPs can be taken up, processed, and presented by antigen-presenting cells to elicit potent specific humoral and cell-mediated immune responses. Several studies also indicated that VLPs could overcome the immunosuppressive state of the tumor microenvironment and break self-tolerance to elicit strong cytotoxic lymphocyte activity, which is crucial for both virus clearance and destruction of cancerous cells. Collectively, these unique characteristics of VLPs make them optimal cancer vaccine candidates. This review discusses current progress in the development of VLP-based cancer vaccines and some potential drawbacks of VLPs in cancer vaccine development. Extracellular vesicles with close resembling to viral particles are also discussed and compared with VLPs as a platform in cancer vaccine developments.

Macrobrachium rosenbergii nodavirus (MrNv) poses a major threat to the prawn industry. Currently, no effective vaccine and treatment are available to prevent the spread of MrNv. Its infection mechanism and localisation in a host cell are also not well characterised. The MrNv capsid protein (MrNvc) produced in Escherichia coli self-assembled into virus-like particles (VLPs) resembling the native virus. Thus, fluorescein labelled MrNvc VLPs were employed as a model to study the virus entry and localisation in Spodoptera frugiperda, Sf9 cells. Through fluorescence microscopy and sub-cellular fractionation, the MrNvc was shown to enter Sf9 cells, and eventually arrived at the nucleus. The presence of MrNvc within the cytoplasm and nucleus of Sf9 cells was further confirmed by the Z-stack imaging. The presence of ammonium chloride (NH4Cl), genistein, methyl-β-cyclodextrin or chlorpromazine (CPZ) inhibited the entry of MrNvc into Sf9 cells, but cytochalasin D did not inhibit this process. This suggests that the internalisation of MrNvc VLPs is facilitated by caveolae- and clathrin-mediated endocytosis. The whole internalisation process of MrNvc VLPs into a Sf9 cell was recorded with live cell imaging. We have also identified a potential nuclear localisation signal (NLS) of MrNvc through deletion mutagenesis and verified by classical-NLS mapping. Overall, this study provides an insight into the journey of MrNvc VLPs in insect cells.

A modified phage-enzyme link immunosorbent assay (phage-ELISA) and a phage dot-blot assay specific for hepatitis B surface antigen (HBsAg) and its antibody were developed by using phage display technology. The phage-ELISA and phage dot-blot assays enabled to detect HBsAg and anti-HBsAg in human sera, and compatible to commercial detection kit. The fusion phages were immobilized onto microtiter plate wells and nitrocellulose membrane sheets, then blocked with 10% milk diluent, and added with human serum at dilution of 1:5000. The absorbance at 405 nm was determined once the colour changes formed. The same human serum also applied on the commercial diagnostic kit for comparison. The statistical analysis was carried out using ANOVA and T Test (LSD) for variable comparison between phage-ELISA and phage dot-blot assays. Based on these studies, the phage-ELISA was found to be more sensitive compared to phage dot-blot assay as the detection of HBsAg in human sera was about 80% as compared to 51.7% by using phage dot-blot assay. Meanwhile, the sensitivity for detection of anti-HBsAg by using phage-ELISA was slightly higher which showed about 83.3%. However, the sensitivity of the assay was dropped almost half when using phage dot-blot assay. Therefore, they are practical to be used as a reliable alternative way for the detection HBsAg and its antibody in human sera.

Foot-and-mouth disease (FMD) is a highly contagious epidemic disease threatening the cattle industry since the sixteenth century. In recent years, the development of diagnostic assays for FMD has benefited considerably from the advances of recombinant DNA technology. In this study, the immunodominant region of the capsid protein VP1 of the foot-and-mouth disease virus (FMDV) was fused to the T7 bacteriophage and expressed on the surface of the bacteriophage capsid protein. The recombinant protein of about 42 kDa was detected by the anti-T7 tag monoclonal antibody in Western blot analysis. Phage ELISA showed that both the vaccinated and positive infected bovine sera reacted significantly with the recombinant T7 particle. This study demonstrated the potential of the T7 phage displaying the VP1 epitope as a diagnostic reagent.

The core antigen (HBcAg) of hepatitis B virus (HBV) is one of the markers for the identification of the viral infection. The main purpose of this study was to develop a TaqMan real-time detection assay based on the concept of phage display mediated immuno-PCR (PD-IPCR) for the detection of HBcAg. PD-IPCR combines the advantages of immuno-PCR (IPCR) and phage display technology. IPCR integrates the versatility of enzyme-linked immunosorbent assay (ELISA) with the sensitivity and signal generation power of PCR. Whereas, phage display technology exploits the physical association between the displayed peptide and the encoding DNA within the same phage particle. In this study, a constrained peptide displayed on the surface of an M13 recombinant bacteriophage that interacts tightly with HBcAg was applied as a diagnostic reagent in IPCR. The phage displayed peptide and its encoding DNA can be used to replace monoclonal antibody (mAb) and chemically bound DNA, respectively. This method is able to detect as low as 10ng of HBcAg with 10(8)pfu/ml of the recombinant phage which is about 10,000 times more sensitive than the phage-ELISA. The PD-IPCR provides an alternative means for the detection of HBcAg in human serum samples.

Purification of virus-like particles (VLPs) in bind-and-elute mode has reached a bottleneck. Negative chromatography has emerged as the alternative solution; however, benchmark of negative chromatography media and their respective optimized conditions are absent. Hence, this study was carried out to compare the performance of different negative chromatography media for the purification of hepatitis B VLPs (HB-VLPs) from clarified Escherichia coli feedstock. The modified anion exchange media, core-shell adsorbents (InertShell and InertLayer 1000) and polymer grafted adsorbents (SQ) were compared. The results of chromatography from packed bed column of core-shell adsorbents showed that there is a trade-off between the purity and recovery of HB-VLPs in the flowthrough fraction due to the shell thickness. Atomic force microscopic analysis revealed funnel-shaped pore channels in the shell layer which may contribute to the entrapment of HB-VLPs. A longer residence time at a lower feed flow rate (0.5ml/min) improved slightly the HB-VLPs purity in all modified adsorbents, but the recovery in InertShell reduced substantially. The preheat-treatment is not recommended for the negative chromatography as the thermal-induced co-aggregation of HCPs and HB-VLPs would flow along with HB-VLPs and thus reduced the HB-VLPs purity in the flowthrough. Further reduction in the feedstock concentration enhanced the purity of HB-VLPs especially in InertLayer 1000 but reduced substantially the recovery of HB-VLPs. In general, the polymer grafted adsorbent, SQ, performed better than the core-shell adsorbents in handling a higher feedstock concentration.

The nucleocapsid (N) protein of Nipah virus (NiV) produced in a recombinant host can replace the use of inactivated virus as a diagnostic reagent because it is safer and affordable. The aim of this study was to express the N protein in Pichia pastoris. The N gene of NiV was cloned into the yeast expression vector, pPICZ B and expressed in P. pastoris. The recombinant N protein of NiV was purified using sucrose density gradient ultracentrifugation and was confirmed with Western blotting using rabbit anti-N antibody. The P. pastoris expressed N protein self-assembled into helical structures as large as 1.5 μm as shown in an electron micrograph. ELISA analysis performed with the swine sera obtained during the viral outbreak proved that the recombinant N protein to be highly antigenic. The NiV N protein produced in P. pastoris serves as an alternative to the recombinant N protein produced in Escherichia coli.

Macrobrachium rosenbergii nodavirus (MrNv) infects giant freshwater prawns and causes white tail disease (WTD). The coding region of the capsid protein of MrNv was amplified with RT-PCR and cloned into the pTrcHis2-TOPO vector. The recombinant plasmid was introduced into Escherichia coli and protein expression was induced with IPTG. SDS-PAGE showed that the recombinant protein containing the His-tag and myc epitope has a molecular mass of about 46 kDa and it was detected by the anti-His antibody in Western blotting. The protein was purified using immobilized metal affinity chromatography (IMAC) and transmission electron microscopic analysis revealed that the recombinant protein assembled into virus-like particles (VLPs) with a diameter of about 30±3 nm. The size of the particles was confirmed by dynamic light scattering. Nucleic acids were extracted from the VLPs and treatment with nucleases showed that they were mainly RNA molecules. This is the first report describing the production of MrNv capsid protein in bacteria and its assembly into VLPs.

The core antigen of the hepatitis B virus (HBcAg) has been used widely as a diagnostic reagent for the identification of the viral infection. However, purification using the conventional sucrose density gradient ultracentrifugation is time consuming and costly. To overcome this, HBcAg particles displaying His-tag on their surface were constructed and produced in Escherichia coli. The recombinant His-tagged HBcAgs were purified using immobilized metal affinity chromatography. Transmission electron microscopy and enzyme-linked immunosorbent assay (ELISA) revealed that the displayed His-tag did not impair the formation of the core particles and the antigenicity of HBcAg.

The nucleocapsid protein of Nipah virus produced in Escherichia coli assembled into herringbone-like particles. The amino- and carboxy-termini of the N protein were shortened progressively to define the minimum contiguous sequence involved in capsid assembly. The first 29 aa residues of the N protein are dispensable for capsid formation. The 128 carboxy-terminal residues do not play a role in the assembly of the herringbone-like particles. A region with amino acid residues 30–32 plays a crucial role in the formation of the capsid particle. Deletion of any of the four conserved hydrophobic regions in the N protein impaired capsid formation. Replacement of the central conserved regions with the respective sequences from the Newcastle disease virus restored capsid formation.

A panel of six monoclonal antibodies (mAbs) against the nucleocapsid (NP) protein of Newcastle disease virus (NDV) was produced by immunization of Balb/c mice with purified recombinant NP protein. Western Blot analysis showed that all the mAbs recognized linearized NP epitopes. Three different NP antigenic sites were identified using deleted truncated NP mutants purified from Escherichia coli. One of the antigenic sites was located at the C-terminal end (residues 441 to 489) of the NP protein. Two other antigenic sites were located within the N-terminal end (residues 26–121 and 122–375). This study demonstrates that the N- and C-terminal ends of the NP proteins are responsible in eliciting immune response, thus it is most likely that these ends are exposed on the NP.

Hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) plays a vital role in the viral infectivity, host immunity, and disease diagnosis. A portion of the HN gene encoding the ectodomain (nt 142-1739) was cloned and expressed in Escherichia coli yielding an insoluble HN protein and a soluble NusA-HN protein containing N-utilization substance A (NusA) fusion component. Both recombinant proteins were purified and used for immunization of chickens. The recombinant HN protein induced higher antibody titers as compared to the recombinant NusA-HN protein. These antibodies were able to react in immunoblot analysis with the corresponding recombinant proteins as well as with the HN protein of NDV.

Background Phage display is an alternative method for constructing and selecting antibodies with desired specificity towards an antigen. Objectives To construct a library of single chain variable fragment (ScFv) towards hepatitis B core antigen (HBcAg). To isolate a ScFv phage clone that interacts with HBcAg and to develop a phage-ELISA for detecting the antigen. Study design Mice were inoculated with HBcAg and RNA was extracted from their spleen cells. The genes encoding heavy (V H ) and light (V L ) chains were amplified, linked via PCR and cloned into a phagemid vector. Phage particles displaying ScFv were panned against HBcAg and a selected clone was characterized and employed as a diagnostic reagent for detecting HBcAg in serum samples. Results A phage clone that interacts with HBcAg was selected from the antibody library. The binding of the phage to HBcAg was inhibited by a cyclic peptide bearing the WSFFSNI sequence. A phage-ELISA was established using the recombinant phage and as low as 10 ng of HBcAg can be detected by the assay. Conclusion The ScFv displayed on the surface of filamentous phage is an alternative choice for diagnosis of HBcAg in serum samples.

Hepatitis B virus (HBV) infection remains a health problem globally despite the availability of effective vaccines. In the assembly of the infectious virion, both the preS and S regions of the HBV large surface antigen (L-HBsAg) interact synergistically with the viral core antigen (HBcAg). Peptides preS and S based on the L-HBsAg were demonstrated as potential inhibitors to block the viral assembly. Therefore, the objectives of this study were to determine the solution structures of these peptides and study their interactions with HBcAg. The solution structures of these peptides were solved using 1H, 13C, and 15N NMR spectroscopy. Peptide preS has several structured regions of β-turns at Ser7-Pro8-Pro9, Arg11-Thr12-Thr13 and Ser22-Thr23-Thr24 sequences whereas peptide S has only one structured region observed at Ser3-Asn4-His5. Both peptides contain bend-like structures surrounding the turn structures. Docking studies revealed that both peptides interacted with the immunodominant region of HBcAg located at the tip of the viral capsid spikes. Saturation Transfer Difference (STD) NMR experiments identified several aromatic residues in peptides preS and S that interact with HBcAg. This study provides insights into the contact regions of L-HBsAg and HBcAg at atomic resolution which can be used to design antiviral agents that inhibit HBV morphogenesis.

Nipah virus (NiV) causes fatal respiratory illness and encephalitis in humans and animals. The matrix (M) protein of NiV plays an important role in the viral assembly and budding process. Thus, an access to the NiV M protein is vital to the design of viral antigens as diagnostic reagents. In this study, recombinant DNA technology was successfully adopted in the cloning and expression of NiV M protein. A recombinant expression cassette (baculovirus expression vector) was used to encode an N-terminally His-tagged NiV M protein in insect cells. A time-course study demonstrated that the highest yield of recombinant M protein (400-500 μg) was expressed from 107 infected cells 3 days after infection. A single-step purification method based on metal ion affinity chromatography was established to purify the NiV M protein, which successfully yielded a purity level of 95.67% and a purification factor of 3.39. The Western blotting and enzyme-linked immunosorbent assay (ELISA) showed that the purified recombinant M protein (48 kDa) was antigenic and reacted strongly with the serum of a NiV infected pig.

Hepatitis B virus (HBV) has infected billions of people worldwide, and currently about 370 million people serve as chronic HBV carriers. Chronic infection by HBV may result in severe liver damage, which may eventually progress to liver cirrhosis and liver cancer. To date, an effective treatment for chronic HBV infection has yet to be established. Microorganisms have been used widely to combat HBV. Since the 1980s, recombinant HBV vaccines based upon HBV surface antigen (HBsAg) produced in yeasts have been used to prevent HBV infection. Large HBsAg (L-HBsAg) has also been produced in yeasts, which is believed to be a solution to nonresponders of the commercial vaccines. Apart from yeasts, bacteria, particularly Escherichia coli, have been extensively exploited for the production of HBV vaccines. Although the HBsAg produced in E. coli has many limitations as a vaccine, the potential of bacteria-produced HBV core antigen (HBcAg) as a therapeutic vaccine is promising. In addition, bacteriophages which can be used to display foreign epitopes and encapsidate foreign DNA make them excellent tools for developing multicomponent and DNA vaccines. Current recombinant vaccines, although considerably effective, are facing a big challenge to compete with rapid viral mutations, thus justifying a continuous need in the development of HBV vaccines.

Heat precipitation procedure has been regularly incorporated as a selective purification step in various thermostable proteins expressed in different hosts. This method is efficient in precipitation of most of the host proteins and also deactivates various host proteases that can be harmful to the desired gene products. In this study, introduction of heat treatment procedure in the purification of hepatitis B core antigen (HBcAg) produced in Escherichia coli has been investigated. Thermal treatment of the cell homogenate at 60 degrees C for 30 min prior to subsequent clarification steps has resulted in 1.4 times and 18% higher in purity and recovery yield, respectively, compared to the non-heat-treated cell homogenate. In direct capture of HBcAg by using anion-exchangers from unclarified feedstock, pre-conditioning the feedstock by heat treatment at 60 degrees C for 45 min has increased the recovery yield of HBcAg by 2.9-fold and 42% in purity compared to that treated for 10 min. Enzyme-linked immunosorbent assay (ELISA) analysis showed that the antigenicity of the core particles was not affected by the heat treatment process.

Hepatitis B virus (HBV) is a deadly pathogen that has killed countless people worldwide.Saccharomyces cerevisiae-derived HBV vaccines based upon hepatitis B surface antigen (HBsAg) is highly effective. However, the emergence of vaccine escape mutants due to mutations on the HBsAg and polymerase genes has produced a continuous need for the development of new HBV vaccines. In this study, the “a” determinant within HBsAg was displayed on the recombinant capsid protein ofMacrobrachium rosenbergiinodavirus (MrNV), which can be purified easily in a single step through immobilized metal affinity chromatography (IMAC). The purified protein self-assembled into virus-like particles (VLPs) when observed under a transmission electron microscope (TEM). Immunization of BALB/c mice with this chimeric protein induced specific antibodies against the “a” determinant. In addition, it induced significantly more natural killer and cytotoxic T cells, as well as an increase in interferon gamma (IFN-γ) secretion, which are vital for virus clearance. Collectively, these findings demonstrated that the MrNV capsid protein is a potential carrier for the HBV “a” determinant, which can be further extended to display other foreign epitopes. This paper is the first to report the application of MrNV VLPs as a novel platform to display foreign epitopes.

The nucleocapsids of hepatitis B virus (HBV) are made of 180 or 240 subunits of core proteins or known as core antigens (HBcAg). A fusion bacteriophage bearing the WSFFSNI sequence that interacts tightly to HBcAg was employed as a diagnostic reagent for the detection of the antigen using the phage-enzyme-linked immunosorbent (phage-ELISA), dot blot and immunoprecipitation assays. The results from phage-ELISA and dot blot assay showed that as low as 10 ng of HBcAg can be detected optimally by 1.0x10(12) pfu/ml fusion M13 bacteriophage. The sensitivity of the dot blot assay corresponds with that of the phage-ELISA. HBcAg in HBV positive serum samples can also be detected using the fusion phage via the phage-ELISA and phage-dot blot assay. The phage cross-linked to cyanogen bromide (CNBr) activated agarose can also be used to precipitate HBcAg in bacterial lysate. The optimum amount of phage needed for cross-linking to 1 g of agarose is about 7.0x10(6) pfu/ml which could also precipitate purified and unpurified HBcAg in bacterial lysate. This study demonstrates the potential of fusion bacteriophage bearing the sequence WSFFSNI as a diagnostic reagent and a ligand for the detection and purification of HBcAg respectively.

A method for serological diagnosis of Nipah virus (NiV) is described. DNA encoding truncated G protein of NiV was cloned into the pFastBac HT vector, and the fusion protein to His-tag was expressed in insect cells by recombinant baculovirus. The resulting His-G recombinant fusion protein was purified by affinity chromatography and used as the coating antigen for serological testing by indirect enzyme-linked immunosorbant assay (ELISA). When tested against a panel of swine serum samples, the recombinant G protein-based ELISA successfully discriminated all 40 samples previously determined to be serum neutralizing test (SNT) positive from 11 SNT negatives samples. The data show that the recombinant G protein exhibits the antigenic epitopes and conformation necessary for specific antigen-antibody recognition. The main advantage of the recombinant G protein-based NiV ELISA compared to an ELISA using whole virus antigen is the use of a single antigenic protein instead of inactivated whole virus which is required to be prepared under high risk and cost. This test is suitable for routine diagnosis of NiV and also for epidemiological surveys as it allows highly reliable testing of a large number of sera rapidly.

A random peptide library of heptamers displayed on the surface of M13 bacteriophage was used to identify specific epitopes of antibodies in pooled sera of swine naturally infected by Nipah virus. The selected heptapeptides were aligned with protein sequences of Nipah virus and several putative epitopes were identified within the nucleocapsid protein. A total of 41 of 60 (68%) selected phage clones had inserts resembling a region with the sequence SNRTQGE, located at the C-terminal end (amino acids 503-509) of the nucleocapsid protein. The binding of antibodies in the swine and human antisera to the phage clone was inhibited by a synthetic peptide corresponding to this region. Epitopes identified by phage display are consistent with the predicted antigenic sites for the Nipah virus nucleocapsid protein. The selected phage clone used as a coating antigen discriminated swine and human Nipah virus sera-positive from sera-negative samples exhibiting characteristics, which might be attractive for diagnostic tests.

The capsular polysaccharide Vi antigen (ViCPS) is an essential virulence factor and also a protective antigen of Salmonella enterica serovar Typhi. A random 12-mer phage-displayed peptide library was used to identify mimotopes (epitope analogues) of this antigen by panning against a ViCPS-specific monoclonal antibody (MAb) ATVi. Approximately 75% of the phage clones selected in the fourth round carried the peptide sequence TSHHDSHGLHRV, and the rest of the clones harbored ENHSPVNIAHKL and other related sequences. These two sequences were also obtained in a similar panning process by using pooled sera from patients with a confirmed diagnosis of typhoid fever, suggesting they mimic immunodominant epitopes of ViCPS antigens. Binding of MAb ATVi to the mimotopes was specifically blocked by ViCPS, indicating that they interact with the same binding site (paratope) of the MAb. Data and reagents generated in this study have important implications for the development of peptide-base diagnostic tests and peptide vaccines and may also provide a better understanding of the pathogenesis of typhoid fever.

The nucleocapsid protein (NP) of Newcastle disease virus expressed in E. coli assembled as ring- and herringbone-like particles. In order to identify the contiguous NP sequence essential for assembly of these particles, 11 N- or C-terminally deleted NP mutants were constructed and their ability to self-assemble was tested. The results indicate that a large part of the NP N-terminal end, encompassing amino acids 1 to 375, is required for proper folding to form a herringbone-like structure. In contrast, the C-terminal end covering amino acids 376 to 489 was dispensable for the formation of herringbone-like particles. A region located between amino acids 375 to 439 may play a role in regulating the length of the herringbone-like particles. Mutants with amino acid deletions further from the C-terminal end (84, 98, 109 and 114 amino acids) tended to form longer particles compared to mutants with shorter deletions (25 and 49 amino acids).

The coding region of the nucleocapsid (N) gene was amplified from the viral RNA and inserted into the bacterial expression vector, pTrcHis2, for intracellular expression in three Escherichia coli strains: TOP 10, BL 21 and SG 935. The N protein was expressed as a fusion protein containing the myc epitope and His-tag at its C-terminal end. The amount of the fusion protein expressed in strain SG 935 was significantly higher than the other two strains, and was detected by the anti-myc antibody, anti-His and swine anti-NiV serum. Hence, the N(fus) protein produced in E. coli could serve as an alternative antigen for the detection of anti-NiV in swine.

White tail disease (WTD) kills prawn larvae and causes drastic losses to the freshwater prawn (Macrobrachium rosenbergii) industry. The main causative agent of WTD is Macrobrachium rosenbergii nodavirus (MrNV). The N-terminal end of the MrNV capsid protein is very rich in positively charged amino acids and is postulated to interact with RNA molecules. N-terminal and internal deletion mutagenesis revealed that the RNA-binding region is located at positions 20–29, where 80 % of amino acids are positively charged. Substitution of all these positively charged residues with alanine abolished the RNA binding. Mutants without the RNA-binding region still assembled into virus-like particles, suggesting that this region is not a part of the capsid assembly domain. This paper is, to the best of our knowledge, the first to report the specific RNA-binding region of MrNV capsid protein.

Summary The nucleotide sequence of the haemagglutinin‐neuraminidase (HN) glycoprotein gene of Newcastle disease virus (NDV) variant strain V4(UPM) was determined by direct genomic RNA sequencing and confirmed by cycle sequencing. The gene comprises 1996 nucleotides encoding a 615 amino acid protein of size 67.4 kDa. The nucleotide and amino acid sequences of this strain were compared with those of the parent strain V4(QUE). There are 16 nucleotide substitutions on V4(UPM), eight of which are silent mutations and another eliminated a potential Asn‐linked glycosylation site in V4(UPM). In addition, an Arg (403) residue was shown to be absent in the variant strain. This deletion is thought to be significant because of its location in a highly conserved region of the HN protein.

The nucleotide sequence of the haemagglutinin-neuraminidase (HN) gene of Newcastle disease virus (NDV) viscerotropic-velogenic strain AF2240 was determined by direct RNA sequencing and by sequencing RT-PCR products. It encodes a single open reading frame of 581 amino acids with a calculated Mr of 63.8 kDa. The predicted sequence contains five asparagine glycosylation sites. Comparison of the AF2240 HN protein sequence with 13 other previously published sequences showed 88% homology. This HN protein is unique because it lacked the Arg 403 residue which is present in all of the other strains and cannot be grouped under the proposed three size classes of HN proteins in NDV.

The C-terminal domain of Nipah virus (NiV) nucleocapsid protein (NP₄₀₁₋₅₃₂) was inserted at the N-terminus and the immunodominant loop of hepatitis B core antigen (HBc). The stability of NP₄₀₁₋₅₃₂ increased tremendously when displayed on the HBc particles. These particles reacted specifically with the swine anti-NiV and the human anti-HBc antisera.

BACKGROUND: Colibacillosis is one of the main causes of economic loss in the poultry industry worldwide. Although antibiotics have been used to control this infection, the emergence of antibiotic-resistant bacteria poses a threat to animal and human health. Phage therapy has been reported as one of the potential alternative methods to control bacterial infections. However, efficient phage therapy is highly dependent on the characteristics of the phage isolated. In the present study the characteristics of a lytic phage, OEC1, which was found to be effective against the causative agent of colibacillosis in chickens in a previous in vivo study, are reported. RESULTS: Examination by transmission electron microscopy revealed that OEC1 is a DNA phage belonging to the Podoviridae family. OEC1 showed an optimum multiplicity of infection of 0.1-1. The latent period of OEC1 was 25 min, with a burst size of 200 particles per infected cell. Under the experimental conditions the maximum adsorption rate for OEC1 was 99.9% within 8 min. OEC1 demonstrated an optimum phage lytic activity at pH 6-9 and 25–41 °C. CONCLUSION: These characteristics can serve as a guideline for selection of effective candidates for phage therapy, in this case for collibacillosis control in chickens. Copyright © 2012 Society of Chemical Industry

Hepatitis B is a major public health problem worldwide which may lead to chronic liver diseases, cirrhosis and hepatocellular carcinoma. An interaction between hepatitis B virus (HBV) envelope protein, particularly the PreS1 region, and a specific cell surface receptor is believed to be the initial step of HBV infection through attachment to hepatocytes. In order to develop a gene delivery system, bacteriophage T7 was modified genetically to display polypeptides of the PreS1 region. A recombinant T7 phage displaying amino acids 60-108 of the PreS1 region (PreS1(60-108)) was demonstrated to be most effective in transfecting HepG2 cells in a dose- and time-dependant manner. The phage genome was recovered from the cell lysate and confirmed by PCR whereas the infectious form of the internalized phage was measured by a plaque-forming assay. The internalized phage exhibited the appearance of green fluorescent dots when examined by immunofluorescence microscopy. Surface modification, particularly by displaying the PreS1(60-108) enhanced phage uptake, resulting in more efficient in vitro gene transfer. The ability of the recombinant phage to transfect HepG2 cells demonstrates the potential of the phage display system as a gene therapy for liver cancer.

Polyclonal sera from typhoid patients and a monoclonal antibody, mAb ATVi, which recognizes the capsular polysaccharide Vi antigen (ViCPS), were used to select for peptides that mimic the ViCPS by using a phage-displayed random 12-mer peptide library. Two major common mimotopes selected from the library carried the amino acid sequences TSHHDSHGLHRV and ENHSPVNIAHKL. Enzyme-linked immunosorbent assays (ELISAs) showed that these peptides carry mimotopes to ViCPS. Phage clones that contained the 12-mer peptides were also tested against pooled/individual typhoid patients' sera and found to have 3 to 5 times higher binding compared to normal sera. By using Phage-ELISA assays, the derived synthetic peptides, TSHHDSHGLHRV and ENHSPVNIAHKL, were tested against a monoclonal antibody mAb ATVi and over 2-fold difference in binding was found between these peptides and a control unrelated peptide, CTLTTKLYC. Inhibition of the mAb's binding to ViCPS indicated that the synthetic peptides successfully competed with the capsular polysaccharide for antibody binding.