Immunogenicity and immunodominant linear B-cell epitopes of a new DNA-based tetravalent vaccine against four major enteroviruses causing hand, foot, and mouth disease.

Hand, foot, and mouth disease (HFMD) poses a significant public health threat primarily caused by four major enteroviruses: enterovirus 71 (EV71), coxsackieviruses A16, A10, and A6. Broadly protective immune responses are essential for complete protection against these major enteroviruses. In this study, we designed a new tetravalent immunogen for HFMD, validated it in silico, in vivo evaluated the immunogenicity of the DNA-based tetravalent vaccine in mice, and identified immunogenic B-cell and T-cell epitopes. A new tetravalent immunogen, VP1me, was designed based on the chimeric protein and epitope-based vaccine principles. It contains a complete EV71 VP1 protein and six reported neutralizing B-cell epitopes derived from the four major enteroviruses causing HFMD. In silico validation using multiple immunoinformatic tools indicated good attributes of the VP1me immunogen suitable for vaccine development. The VP1me-based DNA vaccine efficiently induced both humoral and cellular immune responses in BALB/cAJcl mice. A combination of in silico prediction and immunoassays enabled the identification of immunogenic linear B-cell and CD8 T-cell epitopes within the VP1me immunogen. Immunodominant linear B-cell epitopes were identified in six regions of VP1me, with one epitope located at the N-terminus of the VP1 protein (aa 9-23) regarded as a novel epitope. Interestingly, some B-cell epitopes could also induce the CD8 T-cell response, suggesting their dual functions in immune stimulation. These results lay the groundwork for further development of VP1me as a new vaccine candidate.

Humoral and Cellular Immune Responses Induced by Bivalent DNA Vaccines Expressing Fusion Capsid Proteins of Porcine Circovirus Genotypes 2a and 2b.

Porcine circovirus type 2 (PCV2) is the main causative agent of porcine circovirus-associated disease (PCVAD) that profoundly impacts the swine industry worldwide. While most of the commercial PCV vaccines are developed based on PCV genotype 2a (PCV2a), PCV genotype 2b (PCV2b) has become predominant since 2003. In this study, we developed and evaluated DNA-based bivalent vaccines covering both PCV2a and PCV2b. We generated a new immunogen, PCV2b-2a, by combining consensus sequences of the PCV2a and PCV2b capsid proteins (Cap2a and Cap2b) in a form of fusion protein. We also examined whether modifications of the PCV2b-2a fusion protein with a signal sequence (SS) and granulocyte macrophage-colony stimulating factor (GM-CSF) fusing with interleukine-4 (IL-4) (GI) could further improve the vaccine immunogenicity. An immunogenicity study of BALB/cAJcl mice revealed that the DNA vector pVAX1 co-expressing PCV2b-2a and GI (pVAX1.PCV2b-2a-GI) was most potent at inducing both antibody and cellular immune responses against Cap2a and Cap2b. Interestingly, the vaccines skewed the immune response towards Th1 phenotype (IgG2a > IgG1). By performing ELISA and ELISpot with predicted epitope peptides, the three most immunogenic B cell epitopes and five putative T cell epitopes were identified on Cap2a and Cap2b. Importantly, our DNA vaccines elicited broad immune responses recognizing both genotype-specific and PCV2-conserved epitopes. Sera from mice immunized with the DNAs expressing PCV2b-2a and PCV2b-2a-GI significantly inhibited PCV2a cell entry at serum dilution 1:8. All these results suggest a great potential of our PCV2b-2a-based vaccines, which can be further developed for use in other vaccine platforms to achieve both vaccine efficacy and economical production cost.

Recent progress and advances towards developing enterovirus 71 vaccines for effective protection against human hand, foot and mouth disease (HFMD).

The main pathogen causing severe and neurotrophic hand, foot and mouth disease (HFMD) is enterovirus A71 (EV71). EV71 infection is among the major cause of serious public health burden and economic loss especially in the Asia-pacific region. Yet, no specific anti-viral treatment against this life-threatening infection is currently available. Thus, the best way to control EV71 infection is by vaccination with an effective and safe vaccine. Several strategies are being employed to develop vaccines against EV71. These include conventional and modern recombinant vaccine strategies. Conventional vaccines such as inactivated EV71 vaccines are the most studied and advanced vaccines against HFMD. Recombinant HFMD vaccines developed based on the recombinant DNA technology have been employed but are mostly at early or late preclinical development stage. In this article, we discuss the recent progress and advances in modern recombinant strategies of EV71 vaccine development including subunit, VLP, epitope-based, DNA, and vector-based vaccines, as well as conventional approaches, focusing on their various prospects, advantages and disadvantages.

Amphiphilic Chitosan Bearing Double Palmitoyl Chains and Quaternary Ammonium Moieties as a Nanocarrier for Plasmid DNA.

Amphiphilic chitosan, bPalm-CS-HTAP, having N-(2-((2,3-bis(palmitoyloxy)propyl)amino)-2-oxoethyl) (bPalm) groups as double hydrophobic tails and O-[(2-hydroxyl-3-trimethylammonium)] propyl (HTAP) groups as hydrophilic heads was synthesized and evaluated for its self-assembly properties and potential as a gene carrier. The degree of bis-palmitoyl group substitution (DS bPalm) and the degree of quaternization (DQ) were approximately 2 and 56%, respectively. bPalm-CS-HTAP was found to assemble into nanosized spherical particles with a hydrodynamic diameter (D H) of 265.5 ± 7.40 nm (PDI = 0.5) and a surface charge potential of 40.1 ± 0.04 mV. bPalm-CS-HTAP condensed the plasmid pVAX1.CoV2RBDme completely at a bPalm-CS-HTAP:pDNA ratio of 2:1. The self-assembled bPalm-CS-HTAP/pDNA complexes could enter HEK 293A and CHO cells and enabled gene expression at negligible cytotoxicity compared to commercial PEI (20 kDa). These results suggested that bPalm-CS-HTAP can be used as a promising nonviral gene carrier.

Immunodominant linear B cell epitopes in the spike and membrane proteins of SARS-CoV-2 identified by immunoinformatics prediction and immunoassay.

SARS-CoV-2 continues to infect an ever-expanding number of people, resulting in an increase in the number of deaths globally. With the emergence of new variants, there is a corresponding decrease in the currently available vaccine efficacy, highlighting the need for greater insights into the viral epitope profile for both vaccine design and assessment. In this study, three immunodominant linear B cell epitopes in the SARS-CoV-2 spike receptor-binding domain (RBD) were identified by immunoinformatics prediction, and confirmed by ELISA with sera from Macaca fascicularis vaccinated with a SARS-CoV-2 RBD subunit vaccine. Further immunoinformatics analyses of these three epitopes gave rise to a method of linear B cell epitope prediction and selection. B cell epitopes in the spike (S), membrane (M), and envelope (E) proteins were subsequently predicted and confirmed using convalescent sera from COVID-19 infected patients. Immunodominant epitopes were identified in three regions of the S2 domain, one region at the S1/S2 cleavage site and one region at the C-terminus of the M protein. Epitope mapping revealed that most of the amino acid changes found in variants of concern are located within B cell epitopes in the NTD, RBD, and S1/S2 cleavage site. This work provides insights into B cell epitopes of SARS-CoV-2 as well as immunoinformatics methods for B cell epitope prediction, which will improve and enhance SARS-CoV-2 vaccine development against emergent variants.

Immunodominant and Neutralizing Linear B-Cell Epitopes Spanning the Spike and Membrane Proteins of Porcine Epidemic Diarrhea Virus.

Porcine epidemic diarrhea virus (PEDV) is the causative agent of PED, an enteric disease that causes high mortality rates in piglets. PEDV is an alphacoronavirus that has high genetic diversity. Insights into neutralizing B-cell epitopes of all genetically diverse PEDV strains are of importance, particularly for designing a vaccine that can provide broad protection against PEDV. In this work, we aimed to explore the landscape of linear B-cell epitopes on the spike (S) and membrane (M) proteins of global PEDV strains. All amino acid sequences of the PEDV S and M proteins were retrieved from the NCBI database and grouped. Immunoinformatics-based methods were next developed and used to identify putative linear B-cell epitopes from 14 and 5 consensus sequences generated from distinct groups of the S and M proteins, respectively. ELISA testing predicted peptides with PEDV-positive sera revealed nine novel immunodominant epitopes on the S protein. Importantly, seven of these novel immunodominant epitopes and other subdominant epitopes were demonstrated to be neutralizing epitopes by neutralization-inhibition assay. Our findings unveil important roles of the PEDV S2 subunit in both immune stimulation and virus neutralization. Additionally, our study shows the first time that the M protein is also the target of PEDV neutralization with seven neutralizing epitopes identified. Conservancy profiles of the epitopes are also provided. In this study, we offer immunoinformatics-based methods for linear B-cell epitope identification and a more complete profile of linear B-cell epitopes across the PEDV S and M proteins, which may contribute to the development of a greater next-generation PEDV vaccine as well as peptide-based immunoassays.

Co-expression of self-cleaved multiple proteins derived from Porcine Reproductive and Respiratory Syndrome Virus by bi-cistronic and tri-cistronic DNA vaccines.

Porcine Reproductive and Respiratory Syndrome caused by Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) remains one of the important diseases in swine industry. A vaccine that is safe, effective and also elicit broad immune response against multiple antigens is desirable. In this study, we developed multi-cistronic DNA vaccines capable of co-expressing multiple structural proteins derived from PRRSV. To preserve the structure and function of each antigen protein, we employed self-cleaving 2A peptides to mediate separation of multiple proteins expressed by multi-cistronic genes. Six bi-cistronic genes encoding PRRSV GP5 and M proteins were generated, by which each construct contains different 2A sequences derived from Foot-and-mouth disease virus (F2A), porcine teschovirus-1 (P2A) and Thosea asigna virus (T2A) either with or without furin cleavage site (Fu). Vectored by the mammalian expression plasmid pTH, all six bi-cistronic genes co-expressed the proteins GP5 and M at comparable level. Importantly, all six types of 2A sequences could mediate a complete self-cleavage of the GP5 and M. We next generated tri-cistronic DNA vaccines co-expressing the PRRSV proteins GP5, M and N. All homologous and heterologous combinations of P2A and F2A in tri-cistronic genes yielded a complete self-cleavage of the GP5, M and N proteins. Our study reports a success in co-expression of multiple PRRSV structural proteins in discrete form from a single vaccine and confirms feasibility of developing one single vaccine that provides broad immune responses against PRRSV.

Direct suppression of human islet dedifferentiation, progenitor genes, but not epithelial to mesenchymal transition by liraglutide.

ß-cell dedifferentiation has been accounted as one of the major mechanisms for ß-cell failure; thus, is a cause to diabetes. We study direct impacts of liraglutide treatment on ex vivo human dedifferentiated islets, and its effects on genes important in endocrine function, progenitor states, and epithelial mesenchymal transition (EMT). Human islets from non-diabetic donors, were purified and incubated until day 1 and day 4, and were determined insulin contents, numbers of insulin (INS+) and glucagon (GCG+) cells. The islets from day 3 to day 7 were treated with diabetic drugs, the long acting GLP-1 receptor agonist, liraglutide. As observed in pancreatic islets of type 2 diabetic patients, ex vivo dedifferentiated islets showed more than 50% reduced insulin contents while number of glucagon increased from 10% to about 20%. ß-cell specific genes: PDX1, MAFA, as well as ß-cell functional markers: GLUT1 and SUR1, were significantly depleted more than 40%. Notably, we found increased levels of glucagon regulator, ARX and pre-glucagon transcripts, and remarkably upregulated progenitor expressions: NEUROG3 and ALDH1A identified as ß-cell dysfunction markers in diabetic models. Hyperglucagonemia was often observed in type 2 patients that could lead to over production of gluconeogenesis by the liver. Liraglutide treatments resulted in decreased number of GCG+ cells, increased numbers of GLP-1 positive cells but did not alter elevated levels of EMT marker genes: ACTA2, CDH-2, SNAIL2, and VIM. These effects of liraglutide were blunted when FOXO1 transcripts were depleted. This work illustrates that ex vivo human isolated islets can be used as a tool to study different aspects of ß-cell dedifferentiation. Our novel finding suggests a role of GLP-1 pathway in beta-cell maintenance in FOXO1-dependent manner. Importantly, dedifferentiated islets ex vivo is a useful model that can be utilized to verify the actions of potential drugs to diabetic ß-cell failure.

Dendritic cells enter lymph vessels by hyaluronan-mediated docking to the endothelial receptor LYVE-1.

Trafficking of tissue dendritic cells (DCs) via lymph is critical for the generation of cellular immune responses in draining lymph nodes (LNs). In the current study we found that DCs docked to the basolateral surface of lymphatic vessels and transited to the lumen through hyaluronan-mediated interactions with the lymph-specific endothelial receptor LYVE-1, in dynamic transmigratory-cup-like structures. Furthermore, we show that targeted deletion of the gene Lyve1, antibody blockade or depletion of the DC hyaluronan coat not only delayed lymphatic trafficking of dermal DCs but also blunted their capacity to prime CD8+ T cell responses in skin-draining LNs. Our findings uncovered a previously unknown function for LYVE-1 and show that transit through the lymphatic network is initiated by the recognition of leukocyte-derived hyaluronan.

Antiviral Compounds Against Nucleocapsid Protein of Porcine Epidemic Diarrhea Virus.

Porcine epidemic diarrhea (PED) is a severe diarrhea disease in swine that is caused by porcine epidemic diarrhea virus (PEDV). Nucleocapsid (N) protein is the RNA-binding protein of PEDV, which plays an important role for virus life cycle. The aim of this research was to screen and characterize the compounds that could inhibit the activity of PEDV N protein. The gene encoding PEDV N protein obtained from PEDV Thai isolate was cloned and expressed in E. coli. Its amino acid sequence was employed to generate the three dimensional structure by homology modeling. There were 1,286 compounds of FDA-approved drug database that could virtually bind to the RNA-binding region of N protein. Three compounds, trichlormethiazide, D-(+) biotin, and glutathione successfully bound to the N protein, in vitro, with the IC50 at 8.754 mg/mL, 0.925 mg/mL, and 2.722 mg/mL. Antiviral activity in PEDV-infected Vero cells demonstrated that the effective concentration of trichlormethiazide, D-(+) biotin, and glutathione in inhibiting PEDV replication were 0.094, 0.094 and 1.5 mg/mL. This study demonstrated a strategy applied for discovery of antiviral agents capable of inhibiting PEDV N protein and PEDV replication. The compounds identified here exhibited a potential use as therapeutic agents for controlling PEDV infection.

T cells induced by recombinant chimpanzee adenovirus alone and in prime-boost regimens decrease chimeric EcoHIV/NDK challenge virus load.

The popularity of nonreplicating adenoviruses of chimpanzee origin (ChAdVs) as vectors for subunit vaccines is on the rise. This is mainly for their excellent safety and impressive immunogenicity observed in human studies to date. Here, we recloned the chimpanzee adenovirus sero type 68 (ChAdV-68), also designated SAdV-25 and AdC68, genome and demonstrated its straightforward genetic manipulation facilitated by the use of bacterial artificial chromosome recombineering. To generate the ChAdV68.GagB vaccine, the HIV-1 consensus clade B Gag-derived Tg was inserted into the E1 region. In part confirming previous observations, the ChAdV68.GagB vaccine alone and in heterologous prime-boost regimens with plasmid DNA- and modified vaccinia virus Ankara (MVA)-vectored vaccines induced robust polyfunctional HIV-1-specific CD8(+) and CD4(+) T-cell responses with a gut-homing phenotype. Importantly, we showed that when a single epitope is expressed as an immunodominant CD8(+) T-cell determinant, responses elicited by ChAdV68.GagB alone and in combination lowered surrogate challenge EcoHIV/NDK (where EcoHIV is chimeric ecotropic HIV) virus load in mice both at the peak T-cell frequencies 2 weeks after vaccination and 16 weeks later indicating development of protective effector memory. These results parallel the immunogenicity of similar vaccine regimens in macaques and an ongoing phase I/IIa trial in humans, and support further development of vaccines vectored by ChAdVs.

Protective efficacy of serially up-ranked subdominant CD8+ T cell epitopes against virus challenges.

Immunodominance in T cell responses to complex antigens like viruses is still incompletely understood. Some data indicate that the dominant responses to viruses are not necessarily the most protective, while other data imply that dominant responses are the most important. The issue is of considerable importance to the rational design of vaccines, particularly against variable escaping viruses like human immunodeficiency virus type 1 and hepatitis C virus. Here, we showed that sequential inactivation of dominant epitopes up-ranks the remaining subdominant determinants. Importantly, we demonstrated that subdominant epitopes can induce robust responses and protect against whole viruses if they are allowed at least once in the vaccination regimen to locally or temporally dominate T cell induction. Therefore, refocusing T cell immune responses away from highly variable determinants recognized during natural virus infection towards subdominant, but conserved regions is possible and merits evaluation in humans.

Ovine atadenovirus, a novel and highly immunogenic vector in prime-boost studies of a candidate HIV-1 vaccine.

Ovine adenovirus type 7 (OAdV) is the prototype member of the genus Atadenovirus. No immunity to the virus has so far been detected in human sera. We describe the construction and evaluation of a candidate HIV-1 vaccine based on OAdV and its utilisation alone and in combination with plasmid-, human adenovirus type 5 (HAdV5; a Mastadenovirus)-, and modified vaccinia Ankara (MVA)-vectored vaccines. All vectors expressed HIVA, an immunogen consisting of HIV-1 clade A consensus Gag-derived protein coupled to a T cell polyepitope. OAdV.HIVA was genetically stable, grew well and expressed high levels of protein from the Rous sarcoma virus promoter. OAdV.HIVA was highly immunogenic in mice and efficiently primed and boosted HIV-1-specific T cell responses together with heterologous HIVA-expressing vectors. There were significant differences between OAdV and HAdV5 vectors in priming of naïve CD8(+) T cell responses to HIVA and in the persistence of MHC class I-restricted epitope presentation in the local draining lymph nodes. OAdV.HIVA primed T cells more rapidly but was less persistent than AdV5.HIVA and thus induced a qualitatively distinct T cell response. Nevertheless, both vectors primed a response in mice that reduced viral titres in a surrogate challenge model by three to four orders of magnitude. Thus, OAdV is a novel, underexplored vaccine vector with potential for further development for HIV-1 and other vaccines. The data are discussed in the context of the latest HIV-1 vaccine developments.

Novel HIV-1 clade B candidate vaccines designed for HLA-B*5101(+) patients protected mice against chimaeric ecotropic HIV-1 challenge.

Novel candidate HIV-1 vaccines have been constructed, which are tailor-designed for HLA-B*5101(+) patients infected with HIV-1 clade B. These vaccines employ novel immunogen HIVB-B*5101 derived from consensus HIV-1 clade B Gag p17 and p24 regions coupled to two Pol-derived B*5101-restricted epitopes, which are together with a third B*5101 epitope in Gag dominant in HIV-1-infected long-term non-progressing patients. Both plasmid DNA and modified vaccinia virus Ankara (MVA) vectors supported high expression levels of the HIVB-B*5101 immunogen in cultured cells. Heterologous DNA prime-recombinant MVA boost regimen induced efficiently HIV-1-specific CD8(+) T-cell responses in BALB/c mice. These vaccine-elicited T cells were multifunctional, killed efficiently target cells in vivo, and protected mice against challenge with ecotropic HIV-1/NL4-3 and ecotropic HIV-1/NDK chimaeric viruses with HIV-1 clade B or D backbones, respectively, and ecotropic murine leukemia virus gp80 envelope, and therefore did so in the absence of anti-HIV-1 gp120 antibodies. These results support further development of HIVB-B*5101 vaccines in combined heterologous-modality regimens. The use of allele-specific vaccines in humans is discussed in the context of other developments in the HIV-1 field.

Molecular cloning and functional expression of the Penaeus monodon 5-HT receptor.

Serotonin (5-HT) mediates a number of diverse physiological functions in crustaceans by interacting with various 5-HT receptor subtypes. A putative 5-HT receptor cloned from the ovary of the black tiger prawn (Penaeus monodon) consisted of 2291 nucleotides, encoding a putative 5-HT(1Pem) receptor protein of 591 amino acids. Transient expression of 5-HT(1Pem) in HEK293 cells demonstrated a saturable [3H]-5-HT binding with a Kd of 10.43+/-1.13 nM and Bmax of 1.53+/-0.06 pmol/mg. The putative 5-HT(1Pem) receptor is expressed in all tissues examined and is constitutively expressed in the ovary during ovarian maturation and spent phase. Polyclonal antibodies against the third intracellular loop (i3 loop) of the 5-HT receptor showed that the 5-HT(1Pem) receptor protein was expressed in the trabeculae of ovarian stages 1 and 2 but on the cortical rod and surrounding the oocyte membrane of stages 3 and 4, suggesting that receptor localization plays a critical role in regulating ovarian maturation and spawning in penaeus shrimp.

YHV-protease dsRNA inhibits YHV replication in Penaeus monodon and prevents mortality.

Yellow head virus infects cultured shrimps and causes severe mortality resulting in a great economic loss. Haemolymph injection of dsRNA(pro) corresponding to the protease motif of YHV genome resulted in a complete inhibition of YHV replication. The effect of dsRNA lasted for at least 5 days. Injecting sequence-unrelated dsRNA(gfp) or dsRNA(TSV-pol) also resulted in an inhibition of YHV replication but at a comparatively much less extent. Shrimp mortality was monitored for 10 days when more than 90% shrimps receiving no dsRNA died within 8 dpi. However, those receiving dsRNA(pro) showed no mortality. A partial mortality was observed among the shrimps receiving dsRNA(gfp) or dsRNA(TSV-pol). Thus, Penaeus monodon possesses the sequence-specific protection to YHV infection, most likely through the RNAi pathway, in addition to sequence-independent protection. It gives a new notion that dsRNA induction of antiviral immunity in shrimp goes through two pathways, sequence-independent and sequence-dependent.

Silencing of yellow head virus replication in penaeid shrimp cells by dsRNA.

RNA interference (RNAi) has been shown to inhibit viral replication in some animals and plants. Whether the RNAi is functional in shrimp remains to be demonstrated. In vitro transcribed dsRNAs of YHV helicase, polymerase, protease, gp116, and gp64 were transfected into shrimp primary cell culture and found to inhibit YHV replication. dsRNA targeted to nonstructural genes (protease, polymerase, and helicase) effectively inhibited YHV replication. Those targeted structural genes (gp116 and gp64) were the least effective. These findings are the first evidence that RNAi-mediated gene silencing is operative in shrimp cells. This could be a powerful tool for studying gene function and to develop effective control of viral infection in shrimp.