a poultry-specific intestinal protozoan parasite, is histomonosis's etiological agent. Since treatment or prophylaxis options are no longer available in various countries, histomonosis can lead to significant production losses in chickens and mortality in turkeys. The surfaceome of microbial pathogens is a crucial component of host-pathogen interactions.
View Article and Find Full Text PDFHepatitis B virus (HBV) core (HBV-C) antigens with homologous or heterologous HIV-tat48-57-like (HBV-C149tat) cationic domains non-specifically bind cellular RNA in vector-transfected cells. Here, we investigated whether RNA-binding to cationic domains influences the immunogenicity of endogenously expressed antigens delivered by DNA vaccination. We initially evaluated induction of HBV-C (K/C93)-specific CD8 T cell responses in C57BL/6J (B6) and 1.
View Article and Find Full Text PDFThe HBV core protein self-assembles into particles and encapsidates immune-stimulatory bacterial RNA through a cationic COOH-terminal (C150-183) domain. To investigate if different cationic domains have an impact on the endogenous RNA-binding of HBV-C antigens in mammalian cells, we developed a strep-tag (st) based expression/purification system for HBV-C/RNA antigens in vector-transfected HEK-293 cells. We showed that HBV-stC but not HBV-stC149 particles (lacking the cationic domain) capture low amounts of mammalian RNA.
View Article and Find Full Text PDFUnlabelled: Chronic hepatitis B virus (HBV) infection remains the most common risk factor for hepatocellular carcinoma (HCC). Efficient suppression of HBV viremia and necroinflammation as a result of nucleos(t)ide analogue treatment is able to reduce HCC incidence; nevertheless, hepatocarcinogenesis can occur in the absence of active hepatitis, correlating with high HBV surface antigen (HBsAg) levels. Nuclear factor κB (NF-κB) is a central player in chronic inflammation and HCC development.
View Article and Find Full Text PDFDevelopment of active targeted immunotherapeutics is a rapid developing field in the arena of chronic infectious diseases. The question of repeated, closely spaced administration of immunotherapeutics to achieve a rapid impact on the replicating agent is an important one. We analyzed here, using a prototype adenovirus-based immunotherapeutic encoding Core and Polymerase from the hepatitis B virus (Ad-HBV), the influence of closely spaced repeated immunizations on the level and quality of induced HBV-specific and vector-specific immune responses in various mouse models.
View Article and Find Full Text PDFLittle is known about whether presentation of endogenous and exogenous hepatitis B virus (HBV) surface antigens on APCs targeted by vaccination and/or virus-harboring hepatocytes influences de novo priming of CD8(+) T cells. We showed that surface antigen-expressing transfectants exclusively display a K(b) /S190 epitope, whereas cells pulsed with recombinant surface particles (rSPs) exclusively present a K(b) /S208 epitope to CD8(+) T cells. The differential presentation of these epitopes largely reflects the selective, but not exclusive, priming of K(b) /S190- and K(b) /S208-specific T cells in C57BL/6 mice by endogenous/DNA- or exogenous/protein-based vaccines, respectively.
View Article and Find Full Text PDFImmunodominance hierarchies operating in immune responses to viral antigens limit the diversity of the elicited T cell responses. The L(d)/S(28-39)-restricted CD8 T cell response to the hepatitis B surface antigen (HBsAg or S) prevents copriming of D(d)- and K(b)-restricted CD8 T cell responses. We exchanged L to V at position S(39) of HBsAg to construct mutant S(L39V).
View Article and Find Full Text PDFBased on their role for the induction of T-cell responses, dendritic cells (DCs) are popular candidates in cancer vaccine development. We established a novel single-step intracellular delivery of peptide/poly(I:C) complexes for antigen loading and Toll-like receptor-3 (TLR3)-mediated maturation of human DCs using a cell-penetrating peptide (tat(49-57): RKKRRQRRR) as delivery vector. Towards this end, a cationic tat-sequence was fused with an antigenic, major histocompatibility complex (MHC) class I-binding melanoma epitope (Melan-A/Mart-1 sequence: ELAGIGILTV) and then mixed with negatively charged poly(I:C) dsRNA to form peptide/nucleic acid complexes.
View Article and Find Full Text PDFImmunodominance limits the TCR diversity of specific antiviral CD8 T cell responses elicited by vaccination or infection. To prime multispecific T cell responses, we constructed DNA vaccines that coexpress chimeric, multidomain Ags (with CD8 T cell-defined epitopes of the hepatitis B virus (HBV) surface (S), core (C), and polymerase (Pol) proteins and/or the OVA Ag as stress protein-capturing fusion proteins. Priming of mono- or multispecific, HLA-A*0201- or K(b)-restricted CD8 T cell responses by these DNA vaccines differed.
View Article and Find Full Text PDFUnlabelled: Only small populations of nonactivated, nonproliferating Foxp3(+) CD4 regulatory T cell (T(R)) cells are found in the nonparenchymal cell compartment of the mouse liver while liver-draining celiac nodes contain expanded, activated T(R) cell populations (similar to other lymph nodes). Liver Foxp3(+) CD4 T(R) cells suppress activation of T cell responses. Polyclonal, systemic T cell activation in vivo (via anti-CD3 antibody injection) is accompanied by intrahepatic accumulation of T blasts and a rapid but transient intrahepatic increase of activated, proliferating Foxp3(+) CD4 T(R) cells.
View Article and Find Full Text PDFA priority in current vaccine research is the development of multivalent vaccines that support the efficient priming of long-lasting CD8(+) T-cell immunity. We developed a novel vaccination strategy that used synthetic, cationic (positively charged), and antigenic peptides complexed to negatively charged nucleic acids: antigenic, major histocompatibility complex-class I-binding epitopes fused with a cationic sequence derived from the HIV tat protein (tat50-57: KKRRQRRR) were mixed with nucleic acids (e.g.
View Article and Find Full Text PDFIn DNA vaccination, an exciting new immunization technique with potential applications in clinical medicine, expression plasmid DNA containing antigen-encoding sequences cloned under heterologous promoter control are delivered by techniques that lead in vivo to antigen expression in transfected cells. DNA vaccination efficiently primes both humoral and cellular immune responses. We developed a novel expression system for DNA vaccines in which a fusion protein with a small, N-terminal, viral DnaJ-like sequence (J domain) is translated in frame with C-terminal antigen-encoding sequences.
View Article and Find Full Text PDFDNA vaccines encoding heat shock protein (hsp)-capturing, chimeric peptides containing antigenic determinants of the tumor-associated Ag (TAA) gp70 (an envelope protein of endogenous retrovirus) primed stable, specific, and tumor-protective CD8 T cell immunity. Expression of gp70 transcripts was detectable in most normal tissues but was particularly striking in some (but not all) tumor cell lines tested (including the adenocarcinoma cell line CT26). An approximately 200 residue gp70 fragment or its L(d)-binding antigenic AH1 peptide cloned in-frame behind an hsp-capturing (cT(272)) or noncapturing (T(60)) N-terminal large SV40 tumor Ag sequence was expressed as either hsp-binding or -nonbinding chimeric Ags.
View Article and Find Full Text PDFWe investigated the specific and cross-reactive CD8 T cell immunity to three natural variants (of different geno/serotype) of the small hepatitis B surface Ag (or S protein). The D(d)-binding variants of the S(201-209) epitope showed different immunogenicity. The loss of the consensus C-terminal (P9) anchor abrogated its immunogenicity.
View Article and Find Full Text PDFNeoplastic transformation of mature B cells can be triggered by class-switch recombination of the immunoglobulin gene, which aberrantly targets a protooncogene and promotes translocation. Class-switch recombination is initiated by the B-cell-specific protein activation-induced cytidine deaminase (AID). Using immunohistochemistry with a newly generated monoclonal antibody and quantitative reverse-transcription-polymerase chain reaction (RT-PCR) on microdissected tissue from lymph node, tonsil, and thymus, we demonstrate that AID expression is found in secondary lymphoid organs outside germinal centers and in the thymic medulla at substantial levels.
View Article and Find Full Text PDFTo test whether simple expression units used in DNA vaccines can generate immunogenic, MHC class I-binding epitopes by translating other than the primary open reading frame (ORF), we constructed a vector (pCI/SX) that encodes the small hepatitis B surface Ag in the primary ORF, and a C-terminal fragment (residue 344-832) of the polymerase (Pol) in an alternative (out-of-frame) reading frame. pCI/SX efficiently primed multispecific, HLA-A2-restricted CD8+ T cell responses to epitopes of hepatitis B surface Ag and of Pol (Pol3, Pol(803-811)). Pol3-containing products generated from pCI/SX were detected only by T cell assays, but not by biochemical assays.
View Article and Find Full Text PDFRepeated injections of hepatitis D antigen (HDAg) delivered either as a recombinant protein, or expressed from a DNA vaccine elicited no (or only very low) antibody responses in inbred mouse strains. Codelivery of oligonucleotides (ODN) with immune-stimulating sequences (ISS) with the protein antigen, or ISS in DNA vaccines (encoding HDAg) did not overcome the low intrinsic immunogenicity of this small viral antigen for B cells. In contrast, codelivery of immunogenic, heterologous proteins (either mixed to recombinant HDAg as recombinant proteins, or fused to HDAg sequences as chimeric antigens expressed from DNA vaccines) provided specific, CD4+ T cell-dependent "help" that supported efficient priming of antibody responses to HDAg.
View Article and Find Full Text PDFA hepatitis B virus (HBV)-derived sequence that encodes the 832-residue polymerase (Pol) protein of HBV in the primary open reading frame (ORF), and the three (large, middle and small) hepatitis B surface antigen (HBsAg) variants in an alternative ORF was used. This sequence was cloned into expression vectors in which Pol was expressed under heterologous (HCMV, SV40 or metallothionin) promoter control. Some Pol-encoding vectors coexpressed Pol as well as readily detectable amounts of HBsAg.
View Article and Find Full Text PDFVaccines for the prophylactic and/or therapeutic immunization against hepatotropic pathogens (e.g., hepatitis B and hepatitis C virus) should establish long-lasting, specific antiviral effector/memory CD8+ T cell immunity in the liver.
View Article and Find Full Text PDFWe explored strategies to codeliver DNA- and peptide-based vaccines in a way that enhances the immunogenicity of both components of the combination vaccine for T cells. Specific CD8(+) T cell responses to an antigenic peptide are primed when the peptide is fused to a cationic peptide domain that is bound to plasmid DNA or oligonucleotides (ODN; with or without CpG motifs). Plasmid DNA mixed with antigenic/cationic peptides or histones forms large complexes with different biological properties depending on the molar ratios of peptide/protein and polynucleotide.
View Article and Find Full Text PDFA priority in current vaccine research is the development of adjuvants that support the efficient priming of long-lasting, CD4(+) T cell help-independent CD8(+) T cell immunity. Oligodeoxynucleotides (ODN) with immune-stimulating sequences (ISS) containing CpG motifs facilitate the priming of MHC class I-restricted CD8(+) T cell responses to proteins or peptides. We show that the adjuvant effect of ISS(+) ODN on CD8(+) T cell priming to large, recombinant Ag is enhanced by binding them to short, cationic (arginine-rich) peptides that themselves have no adjuvant activity in CD8(+) T cell priming.
View Article and Find Full Text PDFThe incorporation of linear and conformational antibody-binding epitopes into polyepitope, chimeric antigens with satisfactory immunogenicity is a challenge. We selectively expressed antigen fragments encoding the linear e2 epitope (C(79-149)) of hepatitis B virus (pre)core antigen (HBc/eAg) and the conformational 'a' epitope (S(80-180)) of hepatitis B surface antigen (HBsAg) in a novel system. The domains were expressed as chimeric antigens containing either heat shock protein (hsp)73-binding simian virus 40 large tumor antigen (e.
View Article and Find Full Text PDFThe immunodominant, conformational "a" determinant of hepatitis B surface Ag (HBsAg) elicits Ab responses. We selectively expressed the Ab-binding, glycosylated, native a determinant (residue 120-147) of HBsAg in a fusion protein containing C-terminally the HBsAg fragment SII (residue 80-180) fused to a SV40 T-Ag-derived hsp73-binding 77 aa (T(77)) or non-hsp-binding 60 aa (T(60)) N terminus. A DNA vaccine encoding non-hsp-binding secreted T(60)-SII fusion protein-stimulated murine Ab responses with a similar efficacy as a DNA vaccine encoding the secreted, native, small HBsAg.
View Article and Find Full Text PDFPriming specific Th1 immunity by recombinant hepatitis B core antigen (HBcAg) depends on its arginine (Arg)-rich, 34-36-residue-long C terminus, and nucleotides bound to it. This adjuvant activity intrinsic to HBcAg facilitates priming of Th1 immunity to co-delivered, unrelated antigens, such as hepatitis B surface antigen (HBsAg), or ovalbumin (OVA) that prime specific Th2 immunity when injected without adjuvants. We loaded immune-stimulating, CpG-containing oligodeoxynucleotides (ODN) to the HBcAg-derived Arg-rich peptides C-1 (HBcAg(150-157), RRRDRGRS) or C-2 (HBcAg(164-179), SPRRRRSQSPRRRRSQ).
View Article and Find Full Text PDFMHC-I-restricted CTL responses of H-2(d) (L(d+) or L(d-)) and F(1) H-2(dxb) mice to hepatitis B surface Ag (HBsAg) are primed by either DNA vaccines or HBsAg particles. The D(d)/S(201-209) and K(d)/S(199-208) epitopes are generated by processing endogenous HBsAg; the K(b)/S(208-215) epitope is generated by processing exogenous HBsAg; and the L(d)/S(28-39) epitope is generated by exogenous as well as endogenous processing of HBsAg. DNA vaccination primed high numbers of CTL specific for the L(d)/S(28-39) HBsAg epitope, low numbers of CTL specific for the D(d)/S(201-209) or K(d)/S(199-208) HBsAg epitopes in BALB/c mice, and high numbers of D(d)/S(201-209)- and K(d)/S(199-208)-specific CTL in congenic H-2(d)/L(d-) dm2 mice.
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