Additional Epitopes Research Articles

Hepatitis B virus-like particles access major histocompatibility class I and II antigen presentation pathways in primary dendritic cells

Virus-like particles (VLPs) represent high density displays of viral proteins that efficiently trigger immunity. VLPs composed of the small hepatitis B virus envelope protein (HBsAgS) are useful vaccine platforms that induce humoral and cellular immune responses. Notably, however, some studies suggest HBsAgS VLPs impair dendritic cell (DC) function. Here we investigated HBsAgS VLP interaction with DC subsets and antigen access to major histocompatibility complex (MHC) class I and II antigen presentation pathways in primary DCs. HBsAgS VLPs impaired plasmacytoid DC (pDC) interferon alpha (IFNα) production in response to CpG in vitro, but did not alter conventional DC (cDC) or pDC phenotype when administered in vivo. To assess cellular immune responses, HBsAgS VLPs were generated containing the ovalbumin (OVA) model epitopes OVA257–264 and OVA323–339 to access MHCI and MHCII antigen presentation pathways, respectively; both in vitro and following immunisation in vivo. HBsAgS VLP-OVA257–264 elicited CTL responses in vivo that were not enhanced by inclusion of an additional MHCII helper epitope. HBsAgS VLP-OVA257–264 administered in vivo was cross-presented by CD8+ DCs, but not CD8− DCs. Therefore, HBsAgS VLPs can deliver antigen to both MHCI and MHCII antigen presentation pathways in primary DCs and promote cytotoxic and helper T cell priming despite their suppressive effect on pDCs.

Combination peptide immunotherapy based on T‐cell epitope mapping reduces allergen‐specific IgE and eosinophilia in allergic airway inflammation

Peptide immunotherapy using soluble peptides containing allergen-derived immunodominant T-cell epitopes holds therapeutic promise for allergic asthma. Previous studies in BALB/c mice using the immunodominant peptide epitope of chicken ovalbumin (p323-339) have been unable to demonstrate therapeutic effects in ovalbumin-induced allergic airway inflammation. We have previously shown that intravenous application of p323-339 can effectively tolerise p323-339-reactive T cells in a non-allergic model in C57BL/6 mice. This study aimed to assess the effects of using p323-339 immunotherapy in a C57BL/6 model of ovalbumin-induced allergic airway inflammation, identify any additional epitopes recognized by the ovalbumin-responsive T-cell repertoire in C57BL/6 mice and assess the effects of combination peptide immunotherapy in this model. Ovalbumin-reactive T-cell lines were generated from ovalbumin-immunized C57BL/6 mice and proliferative responses to a panel of overlapping peptides covering the ovalbumin sequence were assessed. Soluble peptides (singly or combined) were administered intravenously to C57BL/6 mice before the induction of ovalbumin-induced allergic airway inflammation. Peptide immunotherapy using the 323-339 peptide alone did not reduce the severity of allergic airway inflammation. An additional immunodominant T-cell epitope in ovalbumin was identified within the 263-278 sequence. Combination peptide immunotherapy, using the 323-339 and 263-278 peptides together, reduced eosinophilia in the bronchoalveolar lavage and ovalbumin-specific IgE, with apparent reductions in interleukin-5 and interleukin-13. Characterization of the T-cell response to a model allergen has allowed the development of combination peptide immunotherapy with improved efficacy in allergic airway inflammation. This model holds important potential for future mechanistic studies using peptide immunotherapy in allergy.

Targeting of Conserved Gag-Epitopes in Early HIV Infection Is Associated with Lower Plasma Viral Load and Slower CD4+T Cell Depletion

We aimed to investigate whether the character of the immunodominant HIV-Gag peptide (variable or conserved) targeted by CD8(+) T cells in early HIV infection would influence the quality and quantity of T cell responses, and whether this would affect the rate of disease progression. Treatment-naive HIV-infected study subjects within the OPTIONS cohort at the University of California, San Francisco, were monitored from an estimated 44 days postinfection for up to 6 years. CD8(+) T cells responses targeting HLA-matched HIV-Gag-epitopes were identified and characterized by multicolor flow cytometry. The autologous HIV gag sequences were obtained. We demonstrate that patients targeting a conserved HIV-Gag-epitope in early infection maintained their epitope-specific CD8(+) T cell response throughout the study period. Patients targeting a variable epitope showed decreased immune responses over time, although there was no limitation of the functional profile, and they were likely to target additional variable epitopes. Maintained immune responses to conserved epitopes were associated with no or limited sequence evolution within the targeted epitope. Patients with immune responses targeting conserved epitopes had a significantly lower median viral load over time compared to patients with responses targeting a variable epitope (0.63 log(10) difference). Furthermore, the rate of CD4(+) T cell decline was slower for subjects targeting a conserved epitope (0.85% per month) compared to subjects targeting a variable epitope (1.85% per month). Previous studies have shown that targeting of antigens based on specific HLA types is associated with a better disease course. In this study we show that categorizing epitopes based on their variability is associated with clinical outcome.

Low GILT Expression is Associated with Poor Patient Survival in Diffuse Large B-Cell Lymphoma

The major histocompatibility complex (MHC) class II-restricted antigen processing pathway presents antigenic peptides acquired in the endocytic route for the activation of CD4+ T cells. Multiple cancers express MHC class II, which may influence the anti-tumor immune response and patient outcome. Low MHC class II expression is associated with poor survival in diffuse large B-cell lymphoma (DLBCL), the most common form of aggressive non-Hodgkin lymphoma. Therefore, we investigated whether gamma-interferon-inducible lysosomal thiol reductase (GILT), an upstream component of the MHC class II-restricted antigen processing pathway that is not regulated by the transcription factor class II transactivator, may be important in DLBCL biology. GILT reduces protein disulfide bonds in the endocytic compartment, exposing additional epitopes for binding to MHC class II and facilitating antigen presentation. In each of four independent gene expression profiling cohorts with a total of 585 DLBCL patients, low GILT expression was significantly associated with poor overall survival. In contrast, low expression of a classical MHC class II gene, HLA-DRA, was associated with poor survival in one of four cohorts. The association of low GILT expression with poor survival was independent of established clinical and molecular prognostic factors, the International Prognostic Index and the cell of origin classification, respectively. Immunohistochemical analysis of GILT expression in 96 DLBCL cases demonstrated variation in GILT protein expression within tumor cells which correlated strongly with GILT mRNA expression. These studies identify a novel association between GILT expression and clinical outcome in lymphoma. Our findings underscore the role of antigen processing in DLBCL and suggest that molecules targeting this pathway warrant investigation as potential therapeutics.

An efficient T-cell epitope discovery strategy using in silico prediction and the iTopia assay platform

Functional T-cell epitope discovery is a key process for the development of novel immunotherapies, particularly for cancer immunology. In silico epitope prediction is a common strategy to try to achieve this objective. However, this approach suffers from a significant rate of false-negative results and epitope ranking lists that often are not validated by practical experience. A high-throughput platform for the identification and prioritization of potential T-cell epitopes is the iTopiaTM Epitope Discovery SystemTM, which allows measuring binding and stability of selected peptides to MHC Class I molecules. So far, the value of iTopia combined with in silico epitope prediction has not been investigated systematically. In this study, we have developed a novel in silico selection strategy based on three criteria: (1) predicted binding to one out of five common MHC Class I alleles; (2) uniqueness to the antigen of interest; and (3) increased likelihood of natural processing. We predicted in silico and characterized by iTopia 225 candidate T-cell epitopes and fixed-anchor analogs from three human tumor-associated antigens: CEA, HER2 and TERT. HLA-A2-restricted fragments were further screened for their ability to induce cell-mediated responses in HLA-A2 transgenic mice. The iTopia binding assay was only marginally informative while the stability assay proved to be a valuable experimental screening method complementary to in silico prediction. Thirteen novel T-cell epitopes and analogs were characterized and additional potential epitopes identified, providing the basis for novel anticancer immunotherapies. In conclusion, we show that combination of in silico prediction and an iTopia-based assay may be an accurate and efficient method for MHC Class I epitope discovery among tumor-associated antigens.

Isotype modulates epitope specificity, affinity, and antiviral activities of anti–HIV-1 human broadly neutralizing 2F5 antibody

The constant heavy chain (CH1) domain affects antibody affinity and fine specificity, challenging the paradigm that only variable regions contribute to antigen binding. To investigate the role of the CH1 domain, we constructed IgA2 from the broadly neutralizing anti-HIV-1 2F5 IgG1, and compared 2F5 IgA2 and IgG binding affinity and functional activities. We found that 2F5 IgA2 bound to the gp41 membrane proximal external region with higher affinity than IgG1. Functionally, compared with IgG1, 2F5 IgA2 more efficiently blocked HIV-1 transcytosis across epithelial cells and CD4(+) cell infection by R5 HIV-1. The 2F5 IgG1 and IgA2 acted synergistically to fully block HIV-1 transfer from Langerhans to autologous CD4(+) T cells and to inhibit CD4(+) T-cell infection. Epitope mapping performed by screening a random peptide library and in silico docking modeling suggested that along with the 2F5 IgG canonical ELDKWA epitope on gp41, the IgG1 recognized an additional 3D-conformational epitope on the gp41 C-helix. In contrast, the IgA2 epitope included a unique conformational motif on the gp41 N-helix. Overall, the CH1 region of 2F5 contributes to shape its epitope specificity, antibody affinity, and functional activities. In the context of sexually transmitted infections such as HIV-1/AIDS, raising a mucosal IgA-based vaccine response should complement an IgG-based vaccine response in blocking HIV-1 transmission.

Dendritic Cells Loaded With mRNA Encoding Full-length Tumor Antigens Prime CD4+ and CD8+ T Cells in Melanoma Patients

It is generally thought that dendritic cells (DCs) loaded with full-length tumor antigen could improve immunotherapy by stimulating broad T-cell responses and by allowing treatment irrespective of the patient's human leukocyte antigen (HLA) type. To investigate this, we determined the specificity of T cells from melanoma patients treated with DCs loaded with mRNA encoding a full-length tumor antigen fused to a signal peptide and an HLA class II sorting signal, allowing presentation in HLA class I and II. In delayed-type hypersensitive (DTH)-biopsies and blood, we found functional CD8(+) and CD4(+) T cells recognizing novel treatment-antigen-derived epitopes, presented by several HLA types. Additionally, we identified a CD8(+) response specific for the signal peptide incorporated to elicit presentation by HLA class II and a CD4(+) response specific for the fusion region of the signal peptide and one of the antigens. This demonstrates that the fusion proteins contain newly created immunogenic sequences and provides evidence that ex vivo-generated mRNA-modified DCs can induce effector CD8(+) and CD4(+) T cells from the naive T-cell repertoire of melanoma patients. Thus, this work provides definitive proof that DCs presenting the full antigenic spectrum of tumor antigens can induce T cells specific for novel epitopes and can be administered to patients irrespective of their HLA type.

Nodal proteins are target antigens in Guillain‐Barré syndrome

Neurofascin-186 (NF186), neuronal cell adhesion molecule (NrCAM), and gliomedin are adhesion molecules playing a central role in the formation of nodes of Ranvier. In Guillain-Barré syndrome (GBS), immune attack toward the nodes may participate in the disabilities. Autoantibodies to NF186 and gliomedin have been detected in a rat model of GBS. Here, we investigated the prevalence of antibodies against nodal adhesion molecules in patients with GBS or chronic inflammatory demyelinating polyneuropathy (CIDP). Sera from 100 GBS patients, 50 CIDP patients, 80 disease controls, and 50 healthy controls were tested for their ability to bind the nodes of Ranvier. To characterize the antigens, we performed cell binding assays against NF186, gliomedin, contactin, and NrCAM. We found that 43% of patients with GBS and 30% of patients with CIDP showed IgG fixation at nodes or paranodes. In eight patients with GBS or CIDP, we identified that IgG antibodies recognized the native extracellular domain of NF186, gliomedin, or contactin. Also, 29 patients showed IgM against nodal adhesion molecules. However, we did not detect IgM fixation at nodes or paranodes. Antibodies to gliomedin or NF186 were mostly detected in demyelinating and axonal GBS, respectively. The adsorption of the antibodies to their soluble antigens abolished IgG deposition at nodes and paranodes in nerves, indicating these were specific to NF186, gliomedin, and contactin. In conclusion, gliomedin, NF186, and contactin are novel target antigens in GBS. At nodes, additional epitopes are also the targets of IgG. These results suggest that antibody attack against nodal antigens participates in the etiology of GBS.

Cofilin Nuclear-Cytoplasmic Shuttling Affects Cofilin-Actin Rod Formation During Stress

Cofilin protein is involved in regulating the actin cytoskeleton during typical steady state conditions, as well as during cell stress conditions where cofilin saturates F-actin, forming cofilin-actin rods. Cofilin can enter the nucleus through an active nuclear localization signal (NLS), accumulating in nuclear actin rods during stress. Here, we characterize the active nuclear export of cofilin through a leptomycin-B-sensitive, CRM1-dependent, nuclear export signal (NES). We also redefine the NLS of cofilin as a bipartite NLS, with an additional basic epitope required for nuclear localization. Using fluorescence lifetime imaging microscopy (FLIM) and Förster resonant energy transfer (FRET) between cofilin moieties and actin, as well as automated image analysis in live cells, we have defined subtle mutations in the cofilin NLS that allow cofilin to bind actin in vivo and affect cofilin dynamics during stress. We further define the requirement of cofilin-actin rod formation in a system of cell stress by temporal live-cell imaging. We propose that cofilin nuclear shuttling is critical for the cofilin-actin rod stress response with cofilin dynamically communicating between the nucleus and cytoplasm during cell stress.

Myasthenogenicity of the main immunogenic region and endogenous muscle nicotinic acetylcholine receptors

In myasthenia gravis (MG) and experimental autoimmune MG (EAMG), many pathologically significant autoantibodies are directed at the main immunogenic region (MIR), a conformation-dependent region at the extracellular tip of α1 subunits of muscle nicotinic acetylcholine receptors (AChRs). Human muscle AChR α1 MIR sequences were integrated into Aplysia ACh-binding protein (AChBP). The chimera was potent in inducing both acute and chronic EAMG, though less potent than Torpedo electric organ AChR. Wild-type AChBP also induced EAMG but was less potent, and weakness developed slowly without an acute phase. AChBP is more closely related in sequence to neuronal α7 AChRs that are also homomeric; however, autoimmune responses were induced to muscle AChR, but not to neuronal AChR subtypes. The greater accessibility of muscle AChRs to antibodies, compared to neuronal AChRs, may allow muscle AChRs to induce self-sustaining autoimmune responses. The human α1 subunit MIR is a potent immunogen for producing pathologically significant autoantibodies. Additional epitopes in this region or other parts of the AChR extracellular domain contribute significantly to myasthenogenicity. We show that an AChR-related protein can induce EAMG. Thus, in principle, an AChR-related protein could induce MG. AChBP is a water-soluble protein resembling the extracellular domain of AChRs, yet rats that developed EAMG had autoantibodies to AChR cytoplasmic domains. We propose that an initial autoimmune response, directed at the MIR on the extracellular surface of muscle AChRs, leads to an autoimmune response sustained by muscle AChRs. Autoimmune stimulation sustained by endogenous muscle AChR may be a target for specific immunosuppression.

Characterization of immunologic properties of a second HLA-A2 epitope from a granule protease in CML patients and HLA-A2 transgenic mice

AbstractThe serine proteases, neutrophil elastase (HNE) and proteinase 3 (PR3), are aberrantly expressed in human myeloid leukemias. T-cell responses to these proteins have been correlated with remission in patients with chronic myeloid leukemia (CML). Human PR3/HNE-specific CD8+ T cells predominantly recognize a nonameric HLA-A2–restricted T-cell epitope called PR1 which is conserved in both Ags. However, CML patients have CD8+ T cells in peripheral blood recognizing an additional HLA-A2 epitope termed PR2. To assess immunologic properties of these Ags, novel recombinant vaccinia viruses (rVV) expressing PR3 and HNE were evaluated in HLA-A2 transgenic (Tg) mice (HHDII). Immunization of HHDII mice with rVV-PR3 elicited a robust PR3-specific CD8+ T-cell response dominated by recognition of PR2, with minimal recognition of the PR1 epitope. This result was unexpected, because the PR2 peptide has been reported to bind poorly to HLA. To account for these findings, we proposed that HHDII mice negatively selected PR1-specific T cells because of the presence of this epitope within murine PR3 and HNE, leading to immunodominance of PR2-specific responses. PR2-specific splenocytes are cytotoxic to targets expressing naturally processed PR3, though PR1-specific splenocytes are not. We conclude that PR2 represents a functional T-cell epitope recognized in mice and human leukemia patients. These studies are registered at www.clinicaltrials.gov as NCT00716911.

Molecular Mechanisms of Induction of Antigen-Specific Allograft Tolerance by Intranasal Peptide Administration

We have previously shown that intranasal (i.n.) administration of a single MHC class II-restricted HY peptide to female mice induces tolerance to up to five additional epitopes expressed on test male grafts, a phenomenon known as linked suppression. In this study, we investigated the molecular mechanisms involved both in the induction phase following peptide administration and during linked suppression after grafting. We report that following initial i.n. administration, peptide is widely disseminated and is presented by functionally immature dendritic cells. These fail to cause optimal stimulation of the responding HY-specific CD4(+) T cells that express genes characteristic of regulatory T cells. Following i.n. peptide plus LPS administration, causing immunization, HY-specific CD4(+) T cells express genes characteristic of activated T cells. We further find that following male skin grafting, HY-specific CD8(+) T cells from peptide-treated tolerant mice display both quantitative and qualitative differences compared with similar cells from untreated mice that reject their grafts. In tolerant mice there are fewer HY-specific CD8(+) cells and they express several genes characteristic of exhausted T cells. Furthermore, associated with specific chemokine receptor and integrin expression, HY-specific CD8(+) T cells show more limited migration from the graft draining lymph node into other tissues.

Specificity of Mimotope-Induced Anti-High Molecular Weight-Melanoma Associated Antigen (HMW-MAA) Antibodies Does Not Ensure Biological Activity

Vaccines based on peptide mimics (mimotopes) of conformational tumor antigen epitopes have been investigated for a variety of human tumors including breast cancer, tumors expressing the carcinoembryonic antigen, B cell lymphoma, neuroblastoma, and melanoma. In our previous work, we designed a vaccine based on a mimotope of the high molecular weight-melanoma associated antigen (HMW-MAA) that elicited HMW-MAA-specific antibodies (Abs) with anti-tumor activity in vitro and in vivo. In this study, we aimed to identify mimotopes of additional distinct HMW-MAA epitopes, since they could be used to construct a polymimotope melanoma vaccine. For this purpose, random peptide phage libraries were screened with the anti-HMW-MAA monoclonal antibodies (mAbs) VT80.12 and VF1-TP43 yielding one peptide ligand for each mAb. Both peptides inhibited the binding of the corresponding mAb to the HMW-MAA. Furthermore, when coupled to the carrier protein keyhole limpet hemocyanin (KLH), both HMW-MAA mimotopes elicited peptide-specific Abs in rabbits or BALB/c mice, but only the mimotope isolated with the mAb VT80.12 elicited HMW-MAA-specific Abs and only in mice. However, the latter Abs had no detectable effect on HMW-MAA expressing human melanoma cells in vitro. These results describe limitations related to the phage display technique and emphasize the need to characterize the functional properties of the mAb utilized to isolate mimotopes of the corresponding epitopes.

A simple quantitative affinity capturing assay of poliovirus antigens and subviral particles by single-domain antibodies using magnetic beads

Recently, single-domain recombinant antibody fragments (VHHs or nanobodies) against poliovirus type 1 were isolated. To examine the antigenicity of poliovirus using these recombinant VHHs, an alternative technique mimicking protein A immunoprecipitation had to be developed that was designed specifically for VHHs. The current study validated an affinity capturing assay that is based on the magnetic separation of unbound antigen and antigen–VHH complexes. The technique is simple, fast, reliable, quantitative and inexpensive and was employed to assess the reactivity of 15 VHHs for native infectious poliovirus (N antigen), heat-denatured virus (H antigen) and 14S subviral particles. Three distinct subsets of VHHs were tentatively distinguished based on their specificity for the antigens: one that binds only to 14S precursors, another that binds to the H antigen and a third that binds to the N antigen. Some VHHs of the latter two subsets bound 14S subviral particles with equal affinity but others had at least 100-fold less affinity for the precursors. All neutralizing VHHs were demonstrated to recognize N antigen and all N-specific VHHs were shown to be neutralizing. This study corroborates the findings that VHHs mainly target conformational epitopes and that they target additional epitopes as compared to classical antibodies. The described technique may be useful for epitope mapping and tracking conformational changes of proteins.

Autoimmune hepatitis and antigen-specific T regulatory cells: When can we send in the regulators?

Autoimmune hepatitis and antigen-specific T regulatory cells: When can we send in the regulators?

Impact of the conjugation method on the immunogenicity of Streptococcus pneumoniae serotype 19F polysaccharide in conjugate vaccines.

7vCRM (Pfizer, Inc.) and PHiD-CV (GlaxoSmithKline Biologicals) are two pneumococcal conjugate vaccines licensed for the prevention of invasive pneumococcal disease and acute otitis media caused by the vaccine serotypes of Streptococcus pneumoniae. Neither vaccine contains serotype 19A, but both contain the closely related serotype 19F. No decrease in the incidence of serotype 19A disease has been observed following the introduction of 7vCRM, suggesting that this serotype 19F-containing vaccine provides limited cross-protection against serotype 19A. To investigate the impact that conjugation methods may have on antipolysaccharide immune responses and to determine whether this limited cross-protection is characteristic of the serotype 19F polysaccharide or rather of the 19F-CRM (cross-reacting material) conjugate, we compared naturally induced antibodies against serotypes 19F and 19A with antibodies induced after vaccination with different pneumococcal conjugate vaccines. We found that conjugation of the serotype 19F polysaccharide using reductive amination (as in 7vCRM) resulted in the formation of at least one additional epitope that is not present in the native form of the 19F polysaccharide or following 19F conjugation using a bifunctional spacer (as in the prototype vaccine 7vOMPC) or cyanylation (as in PHiD-CV). We also found that pneumococcal vaccines conjugated using cyanylation induce more opsonophagocytic antibodies against serotype 19F and a considerably higher level of cross-opsonophagocytic antibodies against serotype 19A than vaccines conjugated using reductive amination. In conclusion, these results suggest that the conjugation method can influence the functionality of the antibodies induced against the homologous serotype 19F and the cross-reactive serotype 19A of S. pneumoniae.

Alternative Translational Reading Frames as a Novel Source of Epitopes for an Expanded CD8 T-Cell Repertoire: Use of a Retroviral System to Assess the Translational Requirements for CTL Recognition and Lysis

CD8 T-cell responses constitute a key host defense mechanism against tumor cells and a variety of viral infections, including retroviral infections that lead to acquired immunodeficiency. However, both for tumor cells and for many viral infections, there can be a relative paucity of immunodominant protective CD8 T-cell responses. For retroviruses, their rapid and error-prone replication, coupled with initial CD8 T-cell immunoselection of epitope-variant, retroviral quasi-species, are major impediments to sustaining a protective CD8 T-cell response. To approach this limitation of functional CD8 T-cell epitopes, here we further characterize an underappreciated source of additional T-cell epitopes: cryptic determinants, in particular those encoded in unconventional, alternative reading frames (ARFs). By use of the CD8 T-cell epitope, SYNTGRFPPL, which we have defined as encoded by the +1NT ARF of the gag gene of the LP-BM5 retrovirus that causes murine AIDS, we further characterize the regulation of ARF-epitope expression. Specifically, we examine the translation initiation requirements for production of sufficient epitope for effector CD8 T-cell recognition. Such translation must arise from rare frame-shifting events, making it crucial to understand any other constraints on epitope production, and therefore on the ability of the anti-Kd/SYNTGRFPPL CD8 T cells to protect from LP-BM5 pathogenesis and retroviral load, as we have previously shown. The data herein demonstrate that ARF epitope production depends entirely on conventional AUG-initiated translation, and that the more proximal in-frame ARF AUG is most important. However, maximal epitope production for protective CD8 T-cell lytic function also requires synergy of this initiation codon with a counterpart conventional AUG codon upstream in the same ARF (ORF 2), and with the classic ORF 1 AUG that initiates conventional gag polyprotein translation. These results have implications for the design of ARF-epitope-based vaccines, both to counter retroviral pathogenesis, as well as potentially more broadly, including in tumor systems.

Immunogenic comparison for two different recombinant chimeric peptides (CP12 and CP22) containing one or two copies of three linear B cell epitopes from β-hCG subunit

To develop a superior chimeric peptide (CP) vaccine of human chorionic gonadotropin (hCG), two CP antigens (named CP12 and CP22) encoding one or two copies of three linear B cell epitopes from the β-hCG subunit and six foreign T cell epitopes, including two promiscuous TCEs from hepatitis B surface antigen and tetanus toxoid, were constructed and biosynthesized. The hCG CP12 and CP22 of 21 or 23 kDa, respectively, were expressed in Escherichia coli at the level of ∼1% of total cell proteins when inserted into thermo-inducible pBV221 expression vector. The purified CP12 and CP22 proteins with >95% relative homogeneity are immunogenic, and elicited antibodies against the β5, β9 and β8 BCEs of β-hCG in both rabbits and three different inbred strains of mice. A mouse uterine weight study in Balb/c mice demonstrated that the CP12 and CP22 antigens with an additional β5 neutralizing epitope enhanced the in vivo bio-neutralization capacity of the induced antibodies compared to the C-terminal immunogen of β-hCG. We propose that the biosynthesized CP22, possessing with two copies of three BCEs, represents a novel candidate antigen for an hCG contraceptive or tumor therapeutic vaccine.

Characterization of CD8+ T-cell response in acute and resolved hepatitis A virus infection

In contrast to the infection with other hepatotropic viruses, hepatitis A virus (HAV) always causes acute self-limited hepatitis, although the role for virus-specific CD8 T cells in viral containment is unclear. Herein, we analyzed the T cell response in patients with acute hepatitis by utilizing a set of overlapping peptides and predicted HLA-A2 binders from the polyprotein. A set of 11 predicted peptides from the HAV polyprotein, identified as potential binders, were synthesized. Peripheral blood mononuclear cells (PBMCs) from patients were tested for IFNγ secretion after stimulation with these peptides and ex vivo with HLA-A2 tetramers. Phenotyping was carried out by staining with the activation marker CD38 and the memory marker CD127. Eight out of 11 predicted HLA-A2 binders showed a high binding affinity and five of them were recognized by CD8+ T cells from patients with hepatitis A. There were significant differences in the magnitude of the responses to these five peptides. One was reproducibly immunodominant and the only one detectable ex vivo by tetramer staining of CD8+ T cells. These cells have an activated phenotype (CD38hi CD127lo) during acute infection. Three additional epitopes were identified in HLA-A2 negative patients, most likely representing epitopes restricted by other HLA-class I-alleles (HLA-A11, B35, B40). Patients with acute hepatitis A have a strong multi-specific T cell response detected by ICS. With the tetramer carrying the dominant HLA-A2 epitope, HAV-specific and activated CD8+ T cells could be detected ex vivo. This first description of the HAV specific CTL-epitopes will allow future studies on strength, breadth, and kinetics of the T-cell response in hepatitis A.

A phase I clinical trial of dendritic cell immunotherapy in HCV-infected individuals

HCV patients who fail conventional interferon-based therapy have limited treatment options. Dendritic cells are central to the priming and development of antigen-specific CD4(+) and CD8(+) T cell immunity, necessary to elicit effective viral clearance. The aim of the study was to investigate the safety and efficacy of vaccination with autologous dendritic cells loaded with HCV-specific cytotoxic T cell epitopes. We examined the potential of autologous monocyte-derived dendritic cells (MoDC), presenting HCV-specific HLA A2.1-restricted cytotoxic T cell epitopes, to influence the course of infection in six patients who failed conventional therapy. Dendritic cells were loaded and activated ex vivo with lipopeptides. In this phase 1 dose escalation study, all patients received a standard dose of cells by the intradermal route while sequential patients received an increased dose by the intravenous route. No patient showed a severe adverse reaction although all experienced transient minor side effects. HCV-specific CD8(+) T cell responses were enumerated in PBMC by ELIspot for interferon-gamma. Patients generated de novo responses, not only to peptides presented by the cellular vaccine but also to additional viral epitopes not represented in the lipopeptides, suggestive of epitope spreading. Despite this, no increases in ALT levels were observed. However, the responses were not sustained and failed to influence the viral load, the anti-HCV core antibody response and the level of circulating cytokines. Immunotherapy using autologous MoDC pulsed with lipopeptides was safe, but was unable to generate sustained responses or alter the outcome of the infection. Alternative dosing regimens or vaccination routes may need to be considered to achieve therapeutic benefit.