Search

Vaccines against inclusion body hepatitis in chickens are complicated by the involvement of antigenically diverse fowl adenovirus types. Though immunization with fiber protein confers robust protection, type specificity of fiber antibodies is an obstacle for the desired broad coverage. In this study, we utilized information on multiple linear epitopes predicted in the Fowl Aviadenovirus E (FAdV-E) fiber head (knob) to develop chimeric fibers with an exchange between two serotypes' sequences, each containing proposed epitopes. Two consecutive segments pertaining to amino acid positions 1 to 441 and 442 to 525/523 in the fibers of FAdV-8a and -8b, types of Fowl Aviadenovirus E that cause inclusion body hepatitis, were swapped reciprocally to result in novel chimeras, crecFib-8a/8b and crecFib-8b/8a. crecFib was indistinguishable from monospecific recombinant fibers in its eactivity with different FAdV antisera in Western blotting. However, contrary to the results for monospecific fibers, crecFib induced cross-neutralizing antibodies against both serotypes in chickens. This demonstrates three nonidentical epitopes in the FAdV-E fiber, the conserved epitope detected in Western blotting and at least two epitopes participating in neutralization, being type specific and located opposite residue position 441-442. Furthermore, we supply conformational evidence for a site in the fiber knob with accessibility critical for neutralization. With such an extended neutralization spectrum compared to those of individual fibers, crecFib was anticipated to fulfill and even extend the mechanistic basis of fiber-mediated protection toward bivalent coverage. Accordingly, crecFib, administered as a single-antigen component, protected chickens simultaneously against challenge with FAdV-8a or -8b, demonstrated by up-to-complete resistance to clinical disease, prevention of target organ-related changes, and significant reduction of viral load. IMPORTANCE The control of inclusion body hepatitis, a disease of economic importance for chicken production worldwide, is complicated by an etiology involving multiple divergent fowl adenovirus types. The fiber protein is principally efficacious in inducing neutralizing and protective antibodies in vaccinated chickens; however, it faces limitations due to its intrinsic type specificity for neutralization. In this study, based on an in silico -guided prediction of multiple epitopes in the fowl adenovirus fiber head's loops, we designed chimeric proteins, swapping N- and C-distal fiber portions, each containing putative epitopes, between divergent types FAdV-8a and -8b. In in vitro and in vivo studies, the chimeric fiber displayed extended properties compared to those of individual monotype-specific fibers, allowing the number, distribution, functionality, and conformational bearings of epitopes of the fowl adenovirus fiber to be characterized in more detail. Importantly, the chimeric fiber induced cross-neutralizing antibodies and protective responses in chickens against infections by both serotypes, promoting the advancement of broadly protective subunit vaccination strategies against FAdV.

Human noroviruses are the dominant causative agent of acute viral gastroenteritis worldwide. During the winter of 2014-2015, genotype GII.17 cluster IIIb strains emerged as the leading cause of norovirus infection in Asia and later spread to other parts of the world. It is speculated that mutation at blockade epitopes may have resulted in virus escape from herd immunity, leading to the emergence of GII.17 cluster IIIb variants. Here, we identify a GII.17 cluster IIIb-specific blockade epitope by monoclonal antibody (mAb)-based epitope mapping. Four mAbs (designated as M1 to M4) were generated from mice immunized with virus-like particle (VLP) of a GII.17 cluster IIIb strain. Among them, M1 and M3 reacted specifically with the cluster IIIb VLP but not with the VLPs from clusters II or IIIa. Moreover, M1 and M3 dose-dependently blocked cluster IIIb VLP binding with its ligand, histo-blood group antigens (HBGAs). Epitope mapping revealed that M1 and M3 recognized the same highly exposed epitope consisting of residues 293-296 and 299 in the capsid protein VP1. Sequence alignment showed that the M1/M3 epitope sequence is highly variable among different GII.17 clusters whereas it is identical for cluster IIIIb strains. These data define a dominant blockade epitope of GII.17 norovirus and provide evidence that blockade epitope evolution contributes to the emergence of GII.17 cluster IIIb strains.

Aims: Human papillomavirus (HPV) L1, L2 and E7 proteins were used as target antigens for development of preventive and therapeutic vaccines. Moreover, linkage of antigens to heat shock proteins (HSPs) could enhance the potency of vaccines. Curcumin and nanocurcumin compounds were suggested as the chemopreventive and chemotherapeutic agents against cancer. In this study, two multiepitope DNA and peptide-based vaccine constructs (L1-L2-E7 and HSP70-L1-L2-E7) were used along with curcumin and nanocurcumin to evaluate immune responses, and protective/therapeutic effects in tumor mouse model., Main Methods: At first, the multiepitope L1-L2-E7 and HSP70-L1-L2-E7 fusion genes were subcloned in eukaryotic and prokaryotic expression vectors. The recombinant multiepitope peptides were generated in E. coli strain. Then, the cytotoxic effects of curcumin and nanocurcumin were evaluated on HEK-293 T non-cancerous and C3 cancerous cells. Finally, mice vaccination was performed using different regimens. Curcumin and nanocurcumin compounds were administered alone or along with different vaccine constructs., Key Findings: Our data indicated that the use of nanocurcumin along with the multiepitope HSP70-L1-L2-E7 vaccine construct could completely protect mice against HPV-related C3 tumor cells, and eradicate tumors in a therapeutic test. Furthermore, nanocurcumin showed higher protection than curcumin alone. Generally, curcumin and nanocurcumin compounds could reduce tumor growth synergistically with the multiepitope vaccine constructs, but they did not influence the immune responses in different regimens., Significance: These data demonstrated that the designed multiepitope vaccine constructs along with curcumin and nanocurcumin can be used as a promising method for HPV vaccine development., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Coxsackievirus B3 (CVB3) -induced viral myocarditis is a common clinical cardiovascular disease without effective available vaccine. In this study, we tried to potentiate the immunoprotection efficacy of our previous CVB3 -specific VP1 protein vaccine by introducing a streptococcal protein G-derived, draining lymph nodes (dLNs)-targeting albumin-binding domain (ABD) peptide. We found that compared with the original VP1 vaccine, ABD-fused VP1 (ABD-VP1) vaccine gained the new ability to efficiently bind murine albumin both in vitro and in vivo , possessed a much longer serum half-life in serum and exhibited more abundance in the dLNs after immunization. Accordingly, ABD-VP1 immunization not only significantly facilitated the enrichment and maturation of dendritic cells (DCs), induced higher percentages of IFN -γ + CD8 + cells in the dLNs, but also robustly promoted VP1-induced T cell proliferation and cytotoxic T lymphocyte (CTL) responses in the spleens. More importantly, ABD-VP1 also elicited higher percentages of protective CD44 hi CD62L hi memory T cells in dLNs and spleens. Consequently, obvious protective effect against viral myocarditis was conferred by ABD-VP1 vaccine compared to the VP1 vaccine, reflected by the less body weight loss, improved cardiac function, alleviated cardiac histomorphological changes and an increased 28-day survival rate. Our results indicated that the ABD might be a promising immune-enhancing regime for vaccine design and development., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Gao, Yue and Xiong.)

Porcine circovirus type 2 (PCV2) causes many diseases in weaned piglets, leading to serious economic losses to the pig industry. This study investigated the immune response following oral administration of Lactobacillus casei ATCC393 ( L. casei 393 ) expressing PCV2 capsid protein (Cap) fusion with the Escherichia   coli heat-labile toxin B subunit (LTB) in mice. Recombinant L. casei strains were constructed using plasmids pPG611.1 and pPG612.1. The expression and localization of proteins from recombinant pPG611.1-Cap-LTB (pPG-1-Cap-LTB)/ L. casei 393 and pPG612.1-Cap-LTB (pPG-2-Cap-LTB)/ L. casei 393 were detected. All recombinant strains were found to be immunogenic by oral administration in mice and developed mucosal and systemic immune responses against PCV2. The titers of specific antibodies in mice administered pPG-2-Cap-LTB/ L. casei 393 were higher than those in mice administered pPG-1-Cap-LTB/ L. casei 393 in serum and the mucosal samples. The mucosal immune response was not only limited to the gastrointestinal tract but was also generated in other mucosal parts. Thus, the application of recombinant L. casei could aid in vaccine development for PCV2.

White tail disease (WTD) is a disease of Macrobrachium rosenbergii caused by Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) with the potential to devastate the aquaculture industry. The present study aimed to explore the possible protection of M. rosenbergii against the disease by oral administration of bacterially expressed recombinant capsid proteins of MrNV and XSV. Juvenile M. rosenbergii were fed with the feed coated with inactivated bacteria encapsulated expressed recombinant viral proteins either individually or in combination for 7 days. Challenge studies using WTD causing agents were carried out after 3 (group I), 10 (group II) and 20 (group III) days post-feeding of viral proteins. Recombinant capsid protein of MrNV showed better protection when compared to other treatments with relative per cent survival of 62.5% (group I), 57.9% (group II) and 39.5% (group III). Treatment controls of groups I, II and III showed 100%, 95% and 95% mortality, respectively. The study demonstrates that oral administration of recombinant capsid proteins of MrNV and XSV provides effective protection against WTD in freshwater prawn., (© 2020 John Wiley & Sons Ltd.)

Persistent infection with high-risk mucosal human papillomavirus (HPV) types has much association with the development of cervical cancer. The major capsid protein L1 has been confirmed to be a major candidate antigen for the development of vaccines. Here, the HPV18 L1 protein was successfully expressed and purified, then nine anti-HPV18 L1 monoclonal antibodies were prepared. Four neutralizing monoclonal antibodies (NmAbs) were identified by using hemagglutination inhibition assay and pseudovirus based neutralization assay. The results of Dot-ELISA, Western blot and indirect immunofluorescence assay showed that the neutralizing antibodies could cross-react with HPV16/18/45/31/33/58/35/39 L1. The mimotopes on HPV18/45 L1 proteins were identified and analyzed by using both phage display and Bioinformatics tool. The B cell epitopes 43-54 aa and 116-126 aa of HPV18 L1 protein, the B cell epitope 381-389 aa of HPV45 L1 protein, and the mimotopes epitope of HPV45 L1 protein were identified by peptide-ELISA and competitive ELISA. The results of PyMOL and Pepitope server analysis indicated that epitopes recognized by NmAbs 7F4, 5A6, 3G11, and 2F5 are located on the surface of L1 VLPs. The results of this study enriched the library of HPV neutralizing antibodies, revealed the mechanism of antibody neutralization, might open new perspectives on the antibody-antigen reaction and have important implications for the development of novel HPV vaccines., Competing Interests: Declaration of competing interest The authors declare that they do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted., (Copyright © 2021. Published by Elsevier B.V.)

Mink enteritis virus (MEV) is a parvovirus that causes acute enteritis in mink. The capsid protein VP2 of MEV is a major immunogenicity that is important for disease prevention. In this study, this protein was expressed in Spodoptera frugiperda 9 cells using a recombinant baculovirus system and was observed to self-assemble into virus-like particles (VLPs) with a high hemagglutination (HA) titer (1:2 16 ). A single-dose injection of VLPs (HA titer, 1:256) resulted in complete protection of mink against virulent MEV challenge for at least 180 days. These data suggest that these MEV VLPs could be used as a vaccine for the prevention of viral enteritis in mink.

The porcine circovirus type 2 (PCV2) capsid (Cap) protein and porcine parvovirus (PPV) VP2 protein have been studied in vaccines to control postweaning multisystemic wasting syndrome (PMWS). Virus-like particle (VLP) vaccines are nonreplicative vectors that deliver epitopes and induce immune responses. However, most VLP vaccines are recombinant proteins expressed in eukaryotic systems and are expensive and complex. In this study, the full-length PCV2-Cap and PPV-VP2 proteins were expressed in Escherichia coli , which self-assembled into VLPs. The highly soluble proteins were purified using Ni-chelating affinity chromatography. The proteins self-assembled into VLPs of ∼20 nm (Cap VLP) and 25 nm (VP2 VLP) in diameter. The immunogenicities of Cap VLP and VP2 VLP were determined in piglets coinfected with PPV and PCV2 postimmunization. The results suggested that Cap VLP and VP2 VLP did not antagonize each other. The combined vaccine induced stronger humoral and cellular immune responses and provided the best protection against PPV and PCV2 coinfection. On a farm containing PMWS-infected pigs, the combined Cap VLP and VP2 VLP vaccine significantly improved piglet growth indices; the average daily weight gains were significantly higher than those of the Cap VLP vaccine and nonimmunized groups. Thus, Cap and VP2 protein expression in E. coli is feasible for large-scale VLP vaccine production. The combined vaccine may be a promising candidate vaccine for better preventing PMWS-associated diseases coinfected with PCV2 and PPV.

The capsid of human papillomavirus (HPV) spontaneously arranges into a T = 7 icosahedral particle with 72 L1 pentameric capsomeres associating via disulfide bonds between Cys175 and Cys428. Here, we design a capsomere-hybrid virus-like particle (chVLP) to accommodate multiple types of L1 pentamers by the reciprocal assembly of single C175A and C428A L1 mutants, either of which alone encumbers L1 pentamer particle self-assembly. We show that co-assembly between any pair of C175A and C428A mutants across at least nine HPV genotypes occurs at a preferred equal molar stoichiometry, irrespective of the type or number of L1 sequences. A nine-valent chVLP vaccine-formed through the structural clustering of HPV epitopes-confers neutralization titers that are comparable with that of Gardasil 9 and elicits minor cross-neutralizing antibodies against some heterologous HPV types. These findings may pave the way for a new vaccine design that targets multiple pathogenic variants or cancer cells bearing diverse neoantigens.

Nervous necrosis virus (NNV) reassortant strains RGNNV/SJNNV have emerged as a potent threat to the Mediterranean marine aquaculture industry, causing viral encephalopathy and retinopathy (VER) in Senegalese sole (Solea senegalensis). In this study, a cheap and practical vaccine strategy using bacterial inclusion bodies made of the coat protein of a virulent reassortant strain of this betanodavirus was devised. The nanostructured recombinant protein nanoparticles, VNNV-C NP , were administered without adjuvant to two groups of juvenile sole, one by intraperitoneal injection and the other by oral intubation. Specific antibodies were raised in vivo against the NNV coat protein via both routes, with a substantial specific antibody expansion in the injected group 30 days post homologous prime boost. Expression levels of five adaptive immune-related genes, cd8a, cd4, igm, igt and arg2, were also quantified in intestine, spleen and head kidney. Results showed cd4 and igm were upregulated in the head kidney of injected fish, indicating activation of an adaptive systemic response, while intubated fish exhibited a mucosal response in the intestine. Neither route showed significant differential expression of cd8a. The specific antibody response elicited in vivo and the lack of any signs of toxicity over the 6-week study period in young fish (n = 100), evidences the potential of the nanoparticle as a vaccine candidate., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: Porcine circovirus type 2 (PCV2) is the causative agent of postweaning multisystemic wasting syndrome, and is associated with a number of other diseases. PCV2 is widely distributed in most developed swine industries, and is a severe economic burden. With an eye to developing an effective, safe, and convenient vaccine against PCV2-associated diseases, we have constructed a recombinant Bacillus subtilis strain (B. subtilis-Cap) that expresses the PCV2 capsid protein (Cap)., Methods: Electroporation of a plasmid shuttle vector encoding the PCV2 Cap sequence was use to transform Bacillus subtilis. Flow cytometry was used to evaluate in vitro bone marrow derived dendritic cell (BM-DC) maturation and T cell proliferation induced by B. subtilis-Cap. Orally inoculated piglets were used for in vivo experiments; ELISA and western blotting were used to evaluate B. subtilis-Cap induced PCV2-specific IgA and IgG levels, as well as the secretion of cytokines and the expression of Toll-like receptor 2 (TLR2) and Toll-like receptor 9 (TLR9)., Results: We evaluated the immune response to B. subtilis-Cap in vitro using mouse BM-DCs and in vivo using neonatal piglets orally inoculated with B. subtilis-Cap. Our results showed that the recombinant B. subtilis-Cap activated BM-DCs, significantly increased co-stimulatory molecules (CD40 and CD80) and major histocompatibility complex II, and induced allogenic T cells proliferation. Piglets immunized with B. subtilis-Cap had elevated levels of PCV2-specific IgA in the mucosal tissues of the digestive and respiratory tract, and PCV2-specific IgG in serum (P < 0.05 or P < 0.01). Ileal immunocompetent cells, such as the IgA-secreting cells (P < 0.01), intestinal intraepithelial lymphocytes (IELs) (P < 0.01), CD3 + T lymphocytes (P < 0.01) and CD4 + T lymphocytes (P < 0.01) increased significantly in the B. subtilis-Cap immunized piglets. Additionally, B. subtilis-Cap inoculation resulted in increased the expression of TLR2 and TLR9 (P < 0.01), and induced the secretion of cytokines IL-1β, IL-6, interferon-γ, and β-defensin 2 (P < 0.01)., Conclusions: We constructed a prototype PCV2 vaccine that can be administered orally and elicits a more robust humoral and cellular immunity than inactivated PCV2. B. subtilis-Cap is a promising vaccine candidate that is safe, convenient, and inexpensive. Further in vivo research is needed to determine its full range of efficacy in pigs.

Pigeon circovirus (PiCV) is able to infect racing and meat pigeons of all ages and is a key factor that triggers young pigeon disease syndrome (YPDS). PiCV vaccine research has been impeded because PiCV cannot be grown or propagated in cell cultures. Virus-like particles (VLPs), which can be generated by a wide range of expression systems, have been shown to have outstanding immunogenicity and constitute promising vaccines against a wide range of pathogens. Cap protein, which contains neutralizing antibody epitopes, is the only capsid protein of PiCV. In this study, the baculovirus expression system was utilized to express the PiCV Cap protein, which was self-assembled into VLPs with a spherical morphology and diameters of 15-18 nm. Specific antibodies against the Cap protein were induced after BALB/c mice immunized intramuscularly (i.m.) with VLPs combined with adjuvant. Based on these findings, PiCV VLPs may be a promising candidate vaccine against PiCV., Competing Interests: Declaration of competing interest The authors report no conflicts of interest in this work., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

We have previously shown that rotavirus (RV) inner capsid protein VP6 has an adjuvant effect on norovirus (NoV) virus-like particle- (VLP-) induced immune responses and studied the adjuvant mechanism in immortalized cell lines used as antigen-presenting cells (APCs). Here, we investigated the uptake and presentation of RV VP6 and NoV GII.4 VLPs by primary bone marrow-derived dendritic cells (BMDCs). The adjuvant effect of VP6 on GII.4 VLP presentation and NoV-specific immune response induction by BMDC in vivo was also studied. Intracellular staining demonstrated that BMDCs internalized both antigens, but VP6 more efficiently than NoV VLPs. Both antigens were processed and presented to antigen-primed T cells, which responded by robust interferon γ secretion. When GII.4 VLPs and VP6 were mixed in the same pulsing reaction, a subpopulation of the cells had uptaken both antigens. Furthermore, VP6 copulsing increased GII.4 VLP uptake by 37% and activated BMDCs to secrete 2-5-fold increased levels of interleukin 6 and tumor necrosis factor α compared to VLP pulsing alone. When in vitro -pulsed BMDCs were transferred to syngeneic BALB/c mice, VP6 improved NoV-specific antibody responses. The results of this study support the earlier findings of VP6 adjuvant effect in vitro and in vivo ., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this paper., (Copyright © 2020 Kirsi Tamminen et al.)

Virus-like particles (VLPs), i.e. molecular assemblies that resemble the geometry and organization of viruses, are promising platforms for therapeutics and imaging. Understanding the assembly and cellular uptake pathways of VLPs can contribute to the development of new antiviral drugs and new virus-based materials for the delivery of drugs or nucleic acid-based therapies. Here we report the assembly of capsid proteins of the cowpea chlorotic mottle virus (CCMV) around DNA into defined structures at neutral pH. Depending on the type of DNA used, we are able to create spherical structures of various diameters and rods of various lengths. In order to determine the shape dependency, the cellular uptake routes and intracellular positioning of these formed polymorphic VLPs in RAW264.7, HeLa and HEK 293 cells are evaluated using flow cytometry analysis with specific chemical inhibitors for different uptake routes. We observed particular uptake routes for the various CCMV-based nanostructures, but the experiments point to clathrin-mediated endocytosis as the major route for cell entry for the studied VLPs. Confocal microscopy reveals that the formed VLPs enter the cells, with clear colocalization in the endosomes. The obtained results provide insight in the cargo dependent VLP morphology and increase the understanding of shape dependent uptake into cells, which is relevant in the design of new virus-based structures with applications in drug and gene delivery., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Infections with immunosuppressive pigeon circovirus (PiCV) pose the most severe health problem to the global pigeon breeding. The vaccination with immunogenic PiCV recombinant capsid protein (PiCV rCP) is a potential tool for disease control. Because of the high prevalence of PiCV asymptomatic infections, the subclinically infected pigeons will be vaccinated in practice. The aim of this study was to answer a question if vaccination of asymptomatic, infected with PiCV pigeons induces a similar immune response to PiCV rCP as in uninfected birds. One hundred and twenty 6-week-old carrier pigeons were divided into 4 groups (2 groups of naturally infected and uninfected with PiCV individuals). Birds from groups V and V1 were vaccinated twice with PiCV rCP mixed with an adjuvant, whereas pigeons from groups C and C1 were immunized with an adjuvant only. The expression of genes encoding IFN-γ, CD4, and CD8 T lymphocyte receptors; the number of anti-PiCV rCP IgY-secreting B cells (SBC) and anti-PiCV rCP IgY were evaluated 2, 21, 39 and 46 days post vaccination (dpv). Study results showed that the expression of CD8 and IFN-γ genes was higher in both groups of infected pigeons than in the uninfected birds, irrespective of vaccination. In the uninfected birds, the expression of these genes was insignificantly higher in the vaccinated pigeons. The anti-PiCV rCP IgY-SBC were detected on 2 and 23 dpv and seroconversion was noted on 23 and 39 dpv in V and V1 groups, respectively. In the light of the results obtained, it could be concluded that pigeon circovirus recombinant capsid protein elicits the immune response in both naturally infected and uninfected pigeons, but its rate varies depending on PiCV infectious status. The infection with PiCV masks the potential cellular immune response to the vaccination with PiCV rCP and leads to the suppression of humoral immunity., Competing Interests: The authors have declared that no competing interests exist.

Objectives: To assess the safety and immunogenicity of a nonadjuvant human papillomavirus (HPV) type 6 L1 virus-like particle (VLP) vaccine in recurrent respiratory papillomatosis (RRP) in local Chinese patients., Methods: Patients with RRP who had undergone surgical treatment before intramuscular administration of an escalating dose of HPV type 6 L1 VLPs (1, 5, and 25 µg at 4 weekly intervals) as part of their treatment were followed up for more than 10 years. Efficacy was assessed by detecting the vaccine-induced type-specific antibody titer, calculating the intersurgical interval, and observing recurrence or remission of papillomas after receiving the vaccine., Results: Nonadjuvant HPV vaccine was generally well tolerated, with no serious vaccine-related adverse episodes. It induced seroconversion for each vaccine-related HPV type. At week 12 (4 weeks after injecting 25 µg), the vaccine-induced type-specific antibody titer was significantly high. Analysis of all patients found a significant increase in the intersurgical interval and decrease in the scores. One patient (16.7%; female) experienced complete remission. Five patients (83.3%) (two males and three females) experienced partial remission. In total, complete or partial remission was achieved in six (100%) patients., Conclusions: Administration of nonadjuvant HPV type 6 L1 VLPs vaccine to RRP was generally well tolerated and highly immunogenic., (Copyright © 2019 The Voice Foundation. Published by Elsevier Inc. All rights reserved.)

Background: Oral vaccine is highly desired for infectious disease which is caused by pathogens infection through the mucosal surface. The design of suitable vaccine delivery system is ongoing for the antigen protection from the harsh gastric environment and target to the Peyer's patches to induce sufficient mucosal immune responses. Among various potential delivery systems, bacterial inclusion bodies have been widely used as delivery systems in the field of nanobiomedicine. However, a large number of heterologous complex proteins could be difficult to propagate in E. coli and fusion partners are often used to enhance target protein expression. As a safety concern the fusion protein need to be removed from the target protein to get tag-free protein, especially for the production of protein antigen in vaccinology. Until now, there is no report on how to remove fusion tag from inclusion body particles in vitro and in vivo. Coxsackievirus B3 (CVB3) is a leading causative agent of viral myocarditis and orally protein vaccine is high desired for CVB3-induced myocarditis. In this context, we explored a tag-free VP1 inclusion body nanoparticles production protocol though a truncated Ssp DnaX mini-intein spontaneous C-cleavage in vivo and also exploited the VP1 inclusion bodies as an oral protein nanoparticle vaccine to protect mice against CVB3-induced myocarditis., Results: We successfully produced the tag-free VP1 inclusion body nanoparticle antigen of CVB3 and orally administrated to mice. The results showed that the tag-free VP1 inclusion body nanoparticles as an effective antigen delivery system targeting to the Peyer's patches had the capacity to induce mucosal immunity as well as to efficiently protect mice from CVB3 induce myocarditis without any adjuvant. Then, we proposed the use of VP1 inclusion body nanoparticles as good candidate for oral vaccine to against CVB3-induced myocarditis., Conclusions: Our tag-free inclusion body nanoparticles production procedure is easy and low cost and may have universal applicability to produce a variety of tag-free inclusion body nanoparticles for oral vaccine.

Chicken infectious anemia virus is an important pathogen that causes severe anemia and immunosuppression in chickens, leading to serious economic losses worldwide in the poultry industry. However, no commercialized inactivated vaccine, subunit vaccine, or genetically engineered vaccine that is effective for controlling this virus is available. In this study, 3 recombinant plasmids were constructed to produce corresponding viral proteins in an Escherichia coli system. The immune effects of the subunit proteins accompanied by CpG-ODN or Freund's immune adjuvants were evaluated and analyzed in systemic animal experiments. The results showed that VP1 induced the highest antibody titers with the participation of VP2 protein, indicating better protection under combined treatment, and the CpG-ODN adjuvant induced higher antibody titers and smaller dispersion of antibody titers than Freund's adjuvants. This is the first study to demonstrate that VP1 protein formulated with VP2 and CpG-ODN adjuvant can induce highest antibody titers and markedly enhance the immune response, indicating its promise as a vaccine candidate., (© 2018 Poultry Science Association Inc.)

In some serotypes of adenovirus (Ad), the penton base and attached trimeric fiber assemble into dodecameric virus-like particles called penton-dodecahedron (Pt-Dd), which can be internalized and used to deliver the vaccine antigens and drugs. Fowl adenovirus serotype 4 (FAdV-4) is an important pathogen, causing seriously economic loss to poultry industry in China and other counties. Pt-Dd particles from FAdV-4 infected cells, as well as in those infected with recombinant human Ad expressing fiber-1, fiber-2, and penton base of FAdV-4, were visualized by transmission electron microscopy. For the first time, we proved that FAdV-4 produced Pt-Dd in infected cells. Pt-Dd can also be assembled by the overexpressed recombinant proteins fiber-1, fiber-2, and penton base. Pt-Dd, as well as the recombinant proteins fiber-1, fiber-2, and penton base, were then used to immunize chickens. The humoral immune response, expression of selected immune molecules and challenge results were used to evaluate the immune efficacy of the vaccine candidates. Pt-Dd induced the highest level of enzyme-linked immunosorbent assay antibodies and significant high levels (p < 0.05) of interferonγ, interleukin-4, and major histocompatibility complex II expression in peripheral blood mononuclear cells at 48 h post-infection. The challenge results showed that Pt-Dd, inactivated FAdV-4 vaccine, and fiber-1 induced the best protection (100%), followed by fiber-2 (80%) and penton base (67%). The present study showed that FAdV-4 -Pt-Dd and recombinant fiber-1 are promising FAdV-4 vaccine candidates and could be used to replace the tissue-sourced inactivated FAdV-4 vaccine., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Porcine rotavirus (PoRV) and porcine epidemic diarrhea virus (PEDV) usually co-infect pigs in modern large-scale piggery, which both can cause severe diarrhea in newborn piglets and lead to significant economic losses to the pig industry. The VP7 protein is the main coat protein of PoRV, and the S protein is the main structural protein of PEDV, which are capable of inducing neutralizing antibodies in vivo. In this study, a DNA vaccine pPI-2.EGFP.VP7.S co-expressing VP7 protein of PoRV and S protein of PEDV was constructed. Six 8-week-old mice were immunized with the recombinant plasmid pPI-2.EGFP.VP7.S. The high humoral immune responses (virus specific antibody) and cellular immune responses (IFN-γ, IL-4, and spleen lymphocyte proliferation) were evaluated. The immune effect through intramuscular injection increased with plasmid dose when compared with subcutaneous injection. The immune-enhancing effect of IFN-α adjuvant was excellent compared with pig spleen transfer factor and IL-12 adjuvant. These results demonstrated that pPI-2.EGFP.VP7.S possess the immunological functions of the VP7 proteins of PoRV and S proteins of PEDV, indicating that pPI-2.EGFP.VP7.S is a candidate vaccine for porcine rotaviral infection (PoR) and porcine epidemic diarrhea (PED).

Duck circovirus (DuCV) is an immunosuppressive pathogen that causes a huge economic loss in the avian industry. Efficient vaccination has become a necessary strategy for preventing DuCV infection in the breeding industry. Three DNA vaccines encoding the Capsid (Cap) protein of DuCV were developed in this study, which were based on the eukaryotic vector pcDNA3.1 containing (i) the full length of Cap gene, pcDNA3.1-Cap, (ii) the Cap gene with a deletion of its nuclear localization signal (NLS) peptide encoding sequence, pcDNA3.1-CapΔNLS, and (iii) the Cap gene without NLS but harboring a fragment encoding the secretory signal peptide of tissue plasminogen activator (tPA), pcDNA3.1-tPA-CapΔNLS. Production of Cap protein-derived antigens from these three DNA vaccines was confirmed in vitro. The deletion of the NLS coding sequence of the Cap gene changed the subcellular location of the Capsid protein from the nucleus to the cytoplasm. Secretion of the Cap protein was observed in pcDNA3.1-tPA-CapΔNLS-transfected cells. The immunogenicity of these three DNA vaccines was assessed in vivo by measuring Cap-specific antibody and related cytokine levels. The results demonstrated that all these vaccines could induce a significant, specific immune response to protect ducks from DuCV challenge. Notably, higher titers of Cap-specific antibody were produced in ducks vaccinated with pcDNA3.1-tPA-CapΔNLS, which provided the highest protective efficacy at a rate of 90% in the challenge experiment. Taken together, DNA vaccines expressing the DuCV Cap protein show promising immunogenicity, which can be enhanced by replacing the NLS of the Cap protein with a secretory signal peptide of tPA., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The present study was designed to characterize transduction of non-human primate brain and spinal cord with a modified adeno-associated virus serotype 2, incapable of binding to the heparan sulfate proteoglycan receptor, referred to as AAV2-HBKO. AAV2-HBKO was infused into the thalamus, intracerebroventricularly or via a combination of both intracerebroventricular and thalamic delivery. Thalamic injection of this modified vector encoding GFP resulted in widespread CNS transduction that included neurons in deep cortical layers, deep cerebellar nuclei, several subcortical regions, and motor neuron transduction in the spinal cord indicative of robust bidirectional axonal transport. Intracerebroventricular delivery similarly resulted in widespread cortical transduction, with one striking distinction that oligodendrocytes within superficial layers of the cortex were the primary cell type transduced. Robust motor neuron transduction was also observed in all levels of the spinal cord. The combination of thalamic and intracerebroventricular delivery resulted in transduction of oligodendrocytes in superficial cortical layers and neurons in deeper cortical layers. Several subcortical regions were also transduced. Our data demonstrate that AAV2-HBKO is a powerful vector for the potential treatment of a wide number of neurological disorders, and highlight that delivery route can significantly impact cellular tropism and pattern of CNS transduction., (Published by Elsevier Inc.)

Human papillomavirus (HPV) is the causative agent in genital warts and nearly all cervical, anogenital, and oropharyngeal cancers. Nine HPV types (6, 11, 16, 18, 31, 33, 45, 52, and 58) are associated with about 90% of cervical cancers and 90% of genital warts. HPV neutralization by vaccine-elicited neutralizing antibodies can block viral infection and prevent HPV-associated diseases. However, there is only one commercially available HPV vaccine, Gardasil 9, produced from Saccharomyces cerevisiae that covers all nine types, raising the need for microbial production of broad-spectrum HPV vaccines. Here, we investigated whether N-terminal truncations of the major HPV capsid proteins L1, improve their soluble expression in Escherichia coli. We found that N-terminal truncations promoted the soluble expression of HPV 33 (truncated by 10 amino acids [aa]), 52 (15 aa), and 58 (10 aa). The resultant HPV L1 proteins were purified in pentamer form and extensively characterized with biochemical, biophysical, and immunochemical methods. The pentamers self-assembled into virus-like particles (VLPs) in vitro, and 3D cryo-EM reconstructions revealed that all formed T = 7 icosahedral particles having 50-60-nm diameters. Moreover, we formulated a nine-valent HPV vaccine candidate with aluminum adjuvant and L1 VLPs from four genotypes used in this study and five from previous work. Immunogenicity assays in mice and non-human primates indicated that this HPV nine-valent vaccine candidate elicits neutralizing antibody titers comparable to those induced by Gardasil 9. Our study provides a method for producing a nine-valent HPV vaccine in E. coli and may inform strategies for the soluble expression of other vaccine candidates.

Rotavirus (RV) inner capsid VP6 protein is a potential non-live vaccine candidate due to high degree of conservation and immunogenicity, and ability to self-assemble into oligomeric structures, including nanotubes. These VP6 structures induce strong humoral and T cell immunity and protect mice against RV challenge. It has been suggested that intracellular neutralization by IgA antibody and VP6-specific CD4 + T cells mediate protection. We investigated generation of diverse CD4 + T cell subsets by intradermal and intranasal delivery of recombinant VP6 (rVP6) nanotubes in BALB/c mice. Production of antiviral cytokine interferon-γ (IFN-γ), interleukin-4 (IL-4) and pro-inflammatory cytokine IL-17 was analyzed following in vitro stimulation of immune cells. Cell surface CD107a expression was measured to determine VP6-specific cytotoxic T cells. Both parenteral and mucosal immunization with oligomeric rVP6 induced VP6-specific Th1, Th2 and Th17 cells. For the first time, cytotoxicity-related degranulation (CD107a surface expression) indicated that RV VP6-specific CD4 + T cells had cytotoxic T lymphocyte (CTL) phenotype. These findings demonstrate an ability of rVP6 nanostructures to induce heterogeneous CD4 + T cells with different effector functions, including CTLs with potential to lyse RV-infected cells, suggesting an additional mechanism of RV VP6-induced protection., (Copyright © 2018 Elsevier B.V. All rights reserved.)

One of the major factors limiting the effectiveness of cancer chemotherapy is inefficient drug delivery. Systems enabling efficient delivery and enhanced intracellular uptake appear particularly promising in this respect. Virus-like particle, adenoviral dodecahedron (Dd), employs receptor-mediated endocytosis for cell penetration and is able to deliver intracellularly dozens of cargo molecules attached to one particle. We focused on studying Dd properties in the context of cancer treatment, showing that intratumoral injection of Dd, assessed in mouse xenograft model, results in vector accumulation in tumor without spreading in off-target organs. Moreover, we demonstrated that Dd is a promising vector targeting leukocytes and drug-resistant cancer cells. Dd uptake by human blood cells analyzed in vitro indicated the preference for leukocytes in comparison to red blood cells and platelets. Furthermore, internalization of Dd-doxorubicin conjugate by drug-resistant cells leads to increased nuclear accumulation of doxorubicin and significant enhancement of cytotoxicity against target cancer cells., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Spherical particles (SPs) generated by thermally denatured tobacco mosaic virus (TMV) coat protein can act as an adjuvant, as they are able to enhance the magnitude and longevity of immune responses to different antigens. Here, the toxicity of TMV SPs was assessed prior to it being offered as a universal safe adjuvant for the development of vaccine candidates. The evaluation included nonclinical studies of a local tolerance following the single administration of TMV SPs, and of the local and systemic effects following repeated administrations of TMV SPs. These were conducted in mice, rats and rabbits. General health status, haematology and blood chemistry parameters were monitored on a regular basis. Also, reproductive and development toxicity were studied. No significant signs of toxicity were detected following single or repeated administrations of the adjuvant (TMV SPs). The absence of toxicological effects following the injection of TMV SPs is promising for the further development of recombinant vaccine candidates with TMV SPs as an adjuvant., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Porcine circovirus type 2 (PCV2) is a primary causative agent of postweaningmultisystemic wasting syndrome (PMWS), which has a significant economic impact on the swine industry. The capsid protein (Cap) encoded by ORF2 of the viral genome has been used effectively as a vaccine against PCV2 infection. The Cap protein can spontaneously assemble into virus-like particles (VLPs) that are safe and highly immunogenic for vaccine applications. Several expression systems, including bacteria, yeast and insect cells, have been utilized to produce PCV2 VLPs. However, in some cases, the recombinant Cap (rCap) proteins produced in bacteria and yeast do not assemble spontaneously. In this study, we expressed rCap protein using a silkworm-baculovirus expression vector system (silkworm-BEVS) for mass production of PCV2 VLPs and established a simple three-step protocol for its purification from pupae: extraction by detergent, ammonium sulfate precipitation and anion exchange column chromatography. Size-exclusion chromatography (SEC) analysis and transmission electron microscope (TEM) observation showed that purified rCap proteins formed VLPs with a similar morphology to that of the original virus. Furthermore, the VLPs produced in silkworms were capable of inducing neutralizing antibodies against PCV2 in mice. Our results demonstrated that the silkworm system is a powerful tool for the production of PCV2 VLPs and will be useful for the development of a reliable and cost-effective PCV2 vaccine.

Bluetongue virus (BTV), a vector-borne pathogen, is the causative agent of bluetongue disease in ruminants. In view of the recent emergence of BTV in regions previously known to be free from the disease and/or specific serotypes or strains, optimization of the currently available vaccination strategies to control the spread of vector-borne bluetongue is crucial. The main objective of the current study was to develop a subunit vaccine candidate targeting BTV-16, a strain previously isolated in China from sheep with obvious clinical signs. To this end, five polyhistidine-tagged recombinant proteins (BTV-16 VP2, VP3, VP7, NS2 and a truncated version of VP5 (VP5-41amino acids) were expressed using the baculovirus or Escherichia coli expression system for characterization of protective activity. To determine ovine and murine immune responses to the five proteins, sheep and mice were immunized twice at 4- and 2-week intervals, respectively, with one of two different protein combinations in Montanide TM ISA201 VG adjuvant or placebo. Data from the competitive enzyme linked immunosorbent assay revealed significantly higher antibody titers in immunized than control animals. Expressed VP5 and NS2 induced a protein-specific humoral response. Interestingly, a serum neutralization test against the BTV-1 serotype showed promising cross-serotype immune response by the vaccine. Based on the collective data, we suggest that these recombinant purified proteins present promising candidates for the design of effective novel vaccines against BTV., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Nanoparticulate vaccines can potentiate immune responses by site-specific drainage to lymph nodes (LNs). This approach may benefit from a nanoparticle engineering method with fine control over size and codelivery of antigen and adjuvant. Here, we applied the flash nanocomplexation (FNC) method to prepare nanovaccines via polyelectrolyte complexation of chitosan and heparin to coencapsulate the VP1 protein antigen from enterovirus 71, which causes hand-foot-mouth disease (HFMD), with tumor necrosis factor α (TNF) or CpG as adjuvants. FNC allows for reduction of the nanovaccine size to range from 90 to 130 nm with relatively narrower size distribution and a high payload capacity. These nanovaccines reached both proximal and distal LNs via subcutaneous injection and subsequently exhibited prolonged retention in the LNs. The codelivery induced strong immune activation toward a Th1 response in addition to a potent Th2 response, and conferred effective protection against lethal virus challenge comparable to that of an approved inactivated viral vaccine in mouse models of both passive and active immunization setting. In addition, these nanovaccines also elicited strong IgA titers, which may offer unique advantages for mucosal protection. This study addresses the issues of size control, antigen bioactivity retention, and biomanufacturing to demonstrate the translational potential of a subunit nanovaccine design.

Due to the limitations and safety issues of the two currently approved live attenuated rotavirus (RV) vaccines "RotaTeq and Rotarix," studies on nonreplicating sources of RV vaccines and search for proper RV antigens are actively carried out. The adjuvant activity of NSP4 and highly immunogenic properties of RV VP6 protein prompted us to consider the construction of a NSP4 112-175 -VP6 fusion protein and to assess the anti-VP6 IgG, IgA, and IgG subclass responses induced by Escherichia coli-derived NSP4-VP6 fusion protein compared to that of VP6 protein with/without formulation in Montanide ISA 50V2 (M50) in BALB/c mice. Results indicated to the proper expression of the fused NSP4-VP6 and VP6 proteins in E. coli. Intraperitoneal immunization by M50 formulated NSP4-VP6 fusion protein (M5+NSP4-VP6) induced the highest titration of VP6-specific IgG and IgA responses compared to the other groups. Indeed, the presence of NSP4 resulted to the induction of stronger humoral immune responses against the fused protein compared to that elicited by administration of VP6 protein alone (with/without M50 formulation), implying the adjuvant properties of NSP4 for the fused protein. Moreover, the "M50+NSP4-VP6" formulation induced higher serum IgG2a titers than IgG1 and increased Interferon-γ levels, despite unchanged interleukin-4 amounts compared to other groups, indicating Th1-oriented responses with a possible role of NSP4. In conclusion, this study further highlights the potentiality of NSP4-VP6 fusion protein as an efficient and cost-effective immunogen in the field of RV vaccine development.

Porcine circovirus type 2 (PCV2) is an economically important pathogen in domestic pigs and wild boars all around the world. To understand the molecular epidemiology of PCV2 strains circulating in central China and to provide a potential vaccine candidate strain, we analyzed the genetic variations of 46 PCV2 isolates circulating from 2009 to 2016 in Henan Province (24 detected in the field from 2009-2013 and 22 from 2013-2016) and evaluated the efficacy of an isolate as a vaccine candidate strain in a mouse model. We found that PCV2b was the predominant genotype and PCV2b-1C was the main subtype. The PCV2 isolate DF-1, which had a virus titer of 10 6.5 TCID 50 /mL and a stable genome, was selected and used to immunize Kunming mice. Enzyme-linked immunosorbent assay (ELISA), immunoperoxidase monolayer assay (IPMA), and virus neutralization test (VNT) results indicated that the DF-1 vaccine candidate strain could elicit a level of specific antibodies and neutralizing antibodies similar to those induced by a commercial vaccine. Polymerase chain reaction (PCR) detection of virus in vaccinated mice after challenge revealed that DF-1 vaccination was effective in clearing the virus in different tissues. Hence, the PCV2 isolate DF-1, a circulating subtype of PCV2b-1C, might be used as a potential vaccine candidate strain.

Coxsackievirus A10 (CVA10) has emerged worldwide as one of the main pathogens of hand, foot, and mouth disease (HFMD) in recent years. However, there is currently no commercial vaccine available to prevent CVA10 infection. Here we report the development of a recombinant virus-like particle (VLP) based candidate vaccine for CVA10. Co-expression of the capsid protein precursor P1 and the protease 3CD of CVA10 in Pichia pastoris resulted in cleavage of P1 into three capsid subunit proteins VP0, VP1, and VP3. These three subunit proteins co-assembled into CVA10 VLPs, which were visualized as spherical particles with a diameter of ∼30 nm under electron microscope. Immunization studies showed that CVA10 VLP could efficiently induce antigen-specific serum antibodies in mice. The anti-VLP sera were able to potently neutralize homologous and heterologous CVA10 strains. Importantly, passively transferred anti-VLP sera fully protected recipient neonatal mice from lethal CVA10 infection. In addition, neonatal mice born to the VLP-immunized dams were also completely protected from CVA10 lethal challenge. Collectively, these data show that CVA10 VLP represents a promising CVA10 vaccine candidate., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Nervous necrosis virus (NNV) infection causes viral nervous necrosis, inflicting serious economic losses in marine fish cultivation. Vaccination is the most effective choice for controlling and preventing viral infection. Virus-like particles (VLPs) are considered a novel vaccine platform because they are not infectious and they induce neutralizing antibodies efficiently. In the present study, we investigated the effect of the recombinant orange-spotted grouper NNV (OSGNNV) capsid proteins produced in Escherichia coli and cell-free self-assembled into VLPs on protective immune responses in orange-spotted grouper following immersion, intramuscular injection and oral immunization. We found the OSGNNV VLPs elicited neutralizing antibody with high efficacy, and provided the fish with full protection against OSGNNV challenge. In addition, the cell-free self-assembled OSGNNV VLPs did not contain residual host cell components and was safer compared with the intracellular assembled VLPs. Thus, oral vaccination is a more convenient and preferred route for fish vaccination. Our results show that the fish fed four times with a diet supplemented with 50-200 μg/g OSGNNV VLPs at 7-day intervals have sufficient protection. These findings demonstrate that cell-free self-assembled OSGNNV VLPs have potential as oral vaccines in grouper., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Nervous necrosis virus (NNV), also known as betanodavirus, has been recently implicated in mass mortalities of cultured marine fish. An effective vaccine is urgently needed to protect fish against this virus. However, parenteral immunization methods are very stressful. Individual immunization for thousands of fish is very labor intensive and expensive. Therefore, we expressed NNV coat protein in tobacco chloroplasts and used it as an oral vaccine to induce immunities in fish followed by challenges with NNV. Our results revealed that mice (IgG and IgA) and fish (IgM) immunized with the oral vaccine developed significantly higher antibody titers against the NNV coat protein. Fish were partially protected against viral challenge. Taken together, our results demonstrated that a plant-based vaccine could effectively induce immune response and protect groupers against NNV. The present method could be used to develop oral fish vaccine in the future.

The spread of hydropericardium syndrome has recently become serious in China since 2015. There is, therefore, an urgent need for new, safe and effective vaccines that prevent the disease. Here, the immune protection induced by Escherichia coli-expressed capsid proteins of fowl adenovirus serotype 4, including fiber-1, fiber-2, penton base and hexon (loop-1 region) were compared in chickens at different inoculation amounts. According to challenge mortalities and tissue gross/micro lesion results, fiber-2 induced the best protection, followed by fiber-1 and hexon. Fiber-1 and fiber-2 provided complete protection against 10 5.5 TCID 50 viral load challenge with 100 or 50μg doses per chicken, respectively. Penton could induce effective protection only at the high dosage of 200μg per chicken. The immunoprotective characteristics of these FAdV-4 capsid proteins may prove useful for developing subunit vaccines to control hydropericardium syndrome., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Enterovirus D68 (EV-D68) has been increasingly associated with severe respiratory illness and neurological complications in children worldwide. However, no vaccine is currently available to prevent EV-D68 infection. In the present study, we investigated the possibility of developing a virus-like particle (VLP)-based EV-D68 vaccine. We found that co-expression of the P1 precursor and 3CD protease of EV-D68 in Pichia pastoris yeast resulted in the generation of EV-D68 VLPs, which were composed of processed VP0, VP1, and VP3 capsid proteins and were visualized as ~30 nm spherical particles. Mice immunized with these VLPs produced serum antibodies capable of specifically neutralizing EV-D68 infections in vitro. The in vivo protective efficacy of the EV-D68 VLP candidate vaccine was assessed in two challenge experiments. The first challenge experiment showed that neonatal mice born to the VLP-immunized dams were fully protected from lethal EV-D68 infection, whereas in the second experiment, passive transfer of anti-VLP sera was found to confer complete protection in the recipient mice. Collectively, these results demonstrate the proof-of-concept for VLP-based broadly effective EV-D68 vaccines.

HSV-1 is a mucosal and nerve pathogen, whose morbidity shows an increasing tendency. Although several antiviral drugs exist, there is no cure for viral latency for virtually all carriers. There is an urgent need for an HSV-1 vaccine to control infection and limit its spread and recurrence. The UL18 gene, encoding a vital component of capsids, is one of the essential genes of HSV-1. Deletion of UL18 from HSV-1 may be exploited as a new approach to develop an attenuated vaccine. The purpose of this study was to construct a DNA vaccine with a full-length UL18 gene deletion of the HSV-1 genome that can induce an effective immune response. A UL18-knockdown plasmid (BAC-HSV-1ΔUL18) was constructed using the bacterial markerless gene knockout system, consisting of the functional pREDI plasmid and BAC-HSV-1 plasmid. Mice were immunized weekly for 3 weeks, and at 1 week post immunization, blood and splenocyte samples of vaccinated and control groups of mice were prepared for immunogenicity assessment. The level of immune response was evaluated using a DTH assay, cytokine determination, and splenocyte proliferation assay. Combination of the pREDI plasmid and BAC-HSV-1 plasmid provides an effective bacterial markerless gene knockout system. Using two-step homologous recombination with the UL18 homologous recombination fragment constructed by multistep PCR amplification, BAC-HSV-1ΔUL18 plasmid vaccine was successfully constructed and was found to significantly enhance cellular immune responses.

The oncolytic effect of Canine Parvovirus ns1 gene and Chicken Anemia vp3 gene in naturally occurring cases of Canine Transmissible Venereal Tumor (CTVT) is being reported. Dogs suffering from CTVT (N = 18) were systematically randomized into three groups viz. A, B, and C (n = 6). Animals of the groups A, B, and C received 100 µg of the ns1 gene, vp3 gene, and ns1  +  vp3 gene combination, respectively, for three weeks intratumorally at weekly intervals; results were normalized against base values before commencement of therapy and after complete remission that were taken as negative and positive controls, respectively. Initiation of oncolytic gene therapy arrested the further progression of the tumor but most of the animals in the study underwent incomplete remission, indicating incomplete activity of ns1 and vp3 genes. The oncolytic effect of the treatments was in the order ns1 > vp3 > ns1 + vp3. Oncolysis was accompanied by decreased mitotic index and AgNOR count, and increased TUNEL positive cells and CD4 + lymphocyte counts. Our findings show that Canine Parvovirus ns1 may eventually find an important role as an oncolytic agent.

A subunit protein vaccine candidate based on norovirus (NoV) virus-like particles (VLPs) and rotavirus (RV) VP6 protein against acute childhood gastroenteritis has been proposed recently. RV VP6 forms different oligomeric nanostructures, including tubes and spheres when expressed in vitro, which are highly immunogenic in different animal models. We have shown recently that recombinant VP6 nanotubes have an adjuvant effect on immunogenicity of NoV VLPs in mice. In this study, we investigated if the adjuvant effect is dependent upon a VP6 dose or different VP6 structural assemblies. In addition, local and systemic adjuvant effects as well as requirements for antigen co-delivery and co-localization were studied. The magnitude and functionality of NoV GII.4-specific antibodies and T cell responses were tested in mice immunized with GII.4 VLPs alone or different combinations of VLPs and VP6. A VP6 dose-dependent adjuvant effect on GII.4-specific antibody responses was observed. The adjuvant effect was found to be strictly dependent upon co-administration of NoV GII.4 VLPs and VP6 at the same anatomic site and at the same time. However, the adjuvant effect was not dependent on the types of oligomers used, as both nanotubes and nanospheres exerted adjuvant effect on GII.4-specific antibody generation and, for the first time, T cell immunity. These findings elucidate the mechanisms of VP6 adjuvant effect in vivo and support its use as an adjuvant in a combination NoV and RV vaccine., (© 2017 The Authors. Clinical & Experimental Immunology published by John Wiley & Sons Ltd on behalf of British Society for Immunology.)

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

We report on the design of a phase I, non-randomized, open-label study of idiotypic DNA vaccination in patients with B-cell non-Hodgkin's lymphoma (ISRCTN31090206). The study uses DNA fusion gene vaccination encoding patient-specific single chain variable fragment, or idiotype, linked to an immunostimulatory sequence. Two types of immunostimulatory sequence are being explored: potato virus X coat protein and human chemokine MIP3α. Linear polyethylenimine with low molecular weight (8 kDa) is used as a synthetic vehicle for vaccine delivery. Humoral and T-cellular immune responses to vaccination will be measured by ELISA and ELISPOT, respectively. The primary study endpoints are safety, tolerability and immunogenicity of DNA-PEI vaccination.

Porcine parvovirus (PPV) is a causative agent of reproductive failure in pregnant sows. Classical inactivated vaccine is extensively used to control PPV infection, but problems concerning safety, such as incomplete inactivation may occur. In this study, a novel subunit vaccine against PPV based on virus-like particles (VLPs) formed from the complete PPV VP2 protein expressed in a prokaryotic system with co-expressed chaperones is reported. The VLPs have a similar size, shape, and hemagglutination property to the PPV. Immunization with these VLPs stimulated the neutralization antibody and hemagglutination inhibition (HI) antibody responses in mice and guinea pigs. The lymphocyte proliferation response and cytokine secretion was also induced in immunized guinea pigs comparable to those immunized with PPV inactivated vaccine. In addition, immunization with VLPs also significantly reduced the PPV content in the spleen of guinea pigs 14 days after the challenge with intact virus. These studies suggest that PPV VLPs created as described here could be a potential candidate for vaccine development., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The integration of a targeted delivery with a tumour-selective agent has been considered an ideal platform for achieving high therapeutic efficacy and negligible side effects in cancer therapy. Here, we present engineered protein nanoparticles comprising a tumour-selective oncolytic protein and a targeting moiety as a new format for the targeted cancer therapy. Apoptin from chicken anaemia virus (CAV) was used as a tumour-selective apoptotic protein. An EGFR-specific repebody, which is composed of LRR (Leucine-rich repeat) modules, was employed to play a dual role as a tumour-targeting moiety and a fusion partner for producing apoptin nanoparticles in E. coli, respectively. The repebody was genetically fused to apoptin, and the resulting fusion protein was shown to self-assemble into supramolecular repebody-apoptin nanoparticles with high homogeneity and stability as a soluble form when expressed in E. coli. The repebody-apoptin nanoparticles showed a remarkable anti-tumour activity with negligible side effects in xenograft mice through a cooperative action of the two protein components with distinct functional roles. The repebody-apoptin nanoparticles can be developed as a systemic injectable and tumour-selective therapeutic protein for targeted cancer treatment., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The researches on chlamydia in recent years show that chlamydia bacteriophage may be a potential and effective means to solve the clinical infection of chlamydia trachomatis (Ct). We investigated the biological effect of chlamydiaphage phiCPG1 capsid protein Vp1 on Ct both in McCoy cells and genital tract of mice. Different concentrations of Vp1 were co-incubated with Ct E serotype strain in McCoy cells. Female BALB/c mice were used to establish Ct E strain-induced urogenital infection model. They were randomly divided into five groups and given different treatments on the fifth day after Ct inoculation. Animals in groups 1 and 2 were given 30 μL different concentrations of Vp1 in the genital tract respectively, those in group 3 were intramuscularly injected with 30 μL Vp1, those in the infected group did not receive any intervention, and those in the control group received 30 μL PBS in the genital tract. The vaginal discharge was collected to identify the live chlamydia by cell culture and gene fragment by real time PCR different days after infection. Inhibition rate of 100 μg/mL and 50 μg/mL Vp1 proteins against Ct E strain in the McCoy cell cultures was 91% and 79% respectively. The number of intracellular Ct inclusion in the McCoy cells co-cultured with vaginal discharge of group 1 and group 2 was less than in the infected group, and that in group 1 was less than in group 2, on the 7th day after Ct inoculation. Real-time PCR showed that chlamydia concentration of the vaginal discharge in group 2 was lower than in the infected group, and that in group 1 was lower than in group 2 on the 10th day. It was suggested that Vp1 capsid proteins had inhibitory effect on the proliferation of Ct serovar E strain in cell culture and mouse genital tract.

A human adenovirus (Ad5) vectored foot-and-mouth disease virus (FMDV) O1-Manisa subunit vaccine (Ad5-O1Man) was engineered to deliver FMDV O1-Manisa capsid and capsid-processing proteins. Swine inoculated with Ad5-O1Man developed an FMDV-specific humoral response as compared to animals inoculated with an empty Ad5-vector. Vaccinated animals were completely protected against homologous challenge at 7 or 21 days post-vaccination. Potency studies exhibited a PD50 of about 10 7 pfu/animal while a dose of 4×10 7 pfu/animal fully protected swine against FMDV intradermal challenge. In-vitro cross-neutralization analysis distinctly predicted that swine vaccinated with Ad5-O1Man would be protected against challenge with homologous FMDV O1Man Middle East-South Asia (ME-SA) topotype and also against recent outbreak strains of Mya-98 South East Asia (SEA) lineage including O1-UK-2001 and O1-South Korea-2010. These results indicate that recombinant Ad5-O1Man is an effective, safe and cross-reacting vaccine that could potentially be used preventively and in outbreak situations, to control FMDV O Mya-98 lineage in swine., (Published by Elsevier Inc.)

Recent studies have shown that a cytoplasmic virus called Leishmaniavirus (LRV) is present in some Leishmania species and acts as a potent innate immunogen, aggravating lesional inflammation and development in mice. In humans, the presence of LRV in Leishmania guyanensis and in L. braziliensis was significantly correlated with poor treatment response and symptomatic relapse. So far, no clinical effort has used LRV for prophylactic purposes. In this context, we designed an original vaccine strategy that targeted LRV nested in Leishmania parasites to prevent virus-related complications. To this end, C57BL/6 mice were immunized with a recombinant LRV1 Leishmania guyanensis viral capsid polypeptide formulated with a T helper 1-polarizing adjuvant. LRV1-vaccinated mice had significant reduction in lesion size and parasite load when subsequently challenged with LRV1+ Leishmania guyanensis parasites. The protection conferred by this immunization could be reproduced in naïve mice via T-cell transfer from vaccinated mice but not by serum transfer. The induction of LRV1 specific T cells secreting IFN-γ was confirmed in vaccinated mice and provided strong evidence that LRV1-specific protection arose via a cell mediated immune response against the LRV1 capsid. Our studies suggest that immunization with LRV1 capsid could be of a preventive benefit in mitigating the elevated pathology associated with LRV1 bearing Leishmania infections and possibly avoiding symptomatic relapses after an initial treatment. This novel anti-endosymbiotic vaccine strategy could be exploited to control other infectious diseases, as similar viral infections are largely prevalent across pathogenic pathogens and could consequently open new vaccine opportunities., Competing Interests: The authors have declared that no competing interests exist.

Glioblastoma (GBM) is the most common malignant brain tumor in adults. We designed an adeno-associated virus (AAV) vector for intracranial delivery of the secreted HSP70-targeted peptide APOPTIN derived from Apoptin to GBM tumors. We applied this therapy to GBM models using human U87MG glioma cells and GBM xenograft models in mice. In U87MG and U251MG cells, conditioned medium from AAV2-apoptin-derived peptide (ADP)-expressing cells induced 83% and 78% cell death. In mice bearing intracranial U87MG tumors treated with AAV2-ADP, treatment resulted in a significant decrease in tumor growth and longer survival in mice bearing orthotopic invasive GBM brain tumors. These data indicate that ssAAV2-ADP injection in the left hemisphere effectively prevented ipsilateral tumor growth but was insufficient to prevent distal tumor growth in the contralateral hemisphere. However, the systemic route is the most effective approach for treating widely dispersed tumors. In summary, systemic delivery of AAV2-ADP is an attractive approach for invasive GBM treatment., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: Assembly of recombinant capsid proteins into virus-like particles (VLPs) still represents an interesting challenge in virus-based nanotechnologies. The structure of VLPs has gained importance for the development and design of new adjuvants and antigen carriers. The potential of Tobacco etch virus capsid protein (TEV CP) as adjuvant has not been evaluated to date., Findings: Two constructs for TEV CP expression in Escherichia coli were generated: a wild-type version (TEV-CP) and a C-terminal hexahistidine (His)-tagged version (His-TEV-CP). Although both versions were expressed in the soluble fraction of E. coli lysates, only His-TEV-CP self-assembled into micrometric flexuous filamentous VLPs. In addition, the His-tag enabled high yields and facilitated purification of TEV VLPs. These TEV VLPs elicited broader IgG2-specific antibody response against a novel porcine reproductive and respiratory syndrome virus (PRRSV) protein when compared to the potent IgG1 response induced by the protein alone., Conclusions: His-TEV CP was purified by immobilized metal affinity chromatography and assembled into VLPs, some of them reaching 2-μm length. TEV VLPs administered along with PRRSV chimeric protein changed the IgG2/IgG1 ratio against the chimeric protein, suggesting that TEV CP can modulate the immune response against a soluble antigen.