Design of TLR2-ligand-synthetic long peptide conjugates for therapeutic vaccination of chronic HBV patients

Synthetic long peptide (SLP) vaccination is a promising new treatment strategy for patients with a chronic hepatitis B virus (HBV) infection. We have previously shown that a prototype HBV-core protein derived SLP was capable of boosting CD4+ and CD8+ T cell responses in the presence of a TLR2-ligand in chronic HBV patients ex vivo. For optimal efficacy of a therapeutic vaccine in vivo, adjuvants can be conjugated to the SLP to ensure delivery of both the antigen and the co-stimulatory signal to the same antigen-presenting cell (APC). Dendritic cells (DCs) express the receptor for the adjuvant and are optimally equipped to efficiently process and present the SLP-contained epitopes to T cells. Here, we investigated TLR2-ligand conjugation of the prototype HBV-core SLP. Results indicated that TLR2-ligand conjugation reduced cross-presentation efficiency of the SLP-contained epitope by both monocyte-derived and naturally occurring DC subsets. Importantly, cross-presentation was improved after optimization of the conjugate by either shortening the SLP or by placing a valine-citrulline linker between the TLR2-ligand and the long SLP, to facilitate endosomal dissociation of SLP and TLR2-ligand after uptake. HBV-core SLP conjugates also triggered functional patient T cell responses ex vivo. These results provide an import step forward in the design of a therapeutic SLP-based vaccine to cure chronic HBV

CD4+ T Cells in Chronic Hepatitis B and T Cell-Directed Immunotherapy

The impaired T cell responses observed in chronic hepatitis B (HBV) patients are considered to contribute to the chronicity of the infection. Research on this impairment has been focused on CD8+ T cells because of their cytotoxic effector function; however, CD4+ T cells are crucial in the proper development of these long-lasting effector CD8+ T cells. In this review, we summarize what is known about CD4+ T cells in chronic HBV infection and discuss the importance and opportunities of including CD4+ T cells in T cell-directed immunotherapeutic strategies to cure chronic HBV

Flow Cytometric Clinical Immunomonitoring Using Peptide–MHC Class II Tetramers: Optimization of Methods and Protocol Development

With the advent of novel strategies to induce tolerance in autoimmune and autoimmune-like conditions, clinical trials of antigen-specific tolerizing immunotherapy have become a reality. Besides safety, it will be essential to gather mechanistic data on responding CD4+ T cells to assess the effects of various immunomodulatory approaches in early-phase trials. Peptide–MHC class II (pMHCII) multimers are an ideal tool for monitoring antigen-specific CD4+ T cell responses in unmanipulated cells directly ex vivo. Various protocols have been published but there are reagent and assay limitations across laboratories that could hinder their global application to immune monitoring. In this methodological analysis, we compare protocols and test available reagents to identify sources of variability and to determine the limitations of the tetramer binding assay. We describe a robust pMHCII flow cytometry-based assay to quantify and phenotype antigen-specific CD4+ T cells directly ex vivo from frozen peripheral blood mononuclear cell samples, which we suggest should be tested across various laboratories to standardize immune-monitoring results

Antibodies specific for carbamylated proteins precede the onset of clinical symptoms in mice with collagen induced arthritis.

OBJECTIVE: The immune response to post-translationally modified antigens is a key characteristic of rheumatoid arthritis. Carbamylation is such a posttranslational modification. Recently, we demonstrated that autoantibodies recognizing carbamylated proteins are present in sera of rheumatoid arthritis. The molecular mechanisms underlying the break of tolerance and hence the induction of anti-CarP antibody responses are unknown as well as their appearance in mouse models for systemic arthritis. Therefore we analyzed their appearance in the mouse collagen-induced arthritis model. METHODS: collagen induced arthritis was induced by immunization with type II collagen in complete Freund's adjuvant. Arthritis severity was monitored by clinical scoring and anti-CarP antibody levels were determined by ELISA. RESULTS: Anti-CarP antibodies were detectable in mice with collagen induced arthritis. We did not detect ACPA in mice with collagen induced arthritis. The specificity of the antibodies for carbamylated proteins was confirmed by inhibition assays and immunoblotting. Injection with complete Freund's adjuvant without type II collagen could also induce anti-CarP antibodies, however, in mice with arthritis, the anti-CarP antibody response was stronger and developed more rapidly. The onset of collagen induced arthritis was preceded by an increase of anti-CarP IgG2a levels in the serum. CONCLUSION: In mice with collagen induced arthritis we did not observe an immune response against citrullinated antigens, but we did observe an immune response against carbamylated antigens. This anti-CarP response already appeared before disease onset, indicating that collagen induced arthritis can be used as an in vivo model to study anti-CarP antibodies. Our data also indicate that the tolerance to carbamylated proteins, in contrast to the response to citrullinated proteins, is easily broken and that arthritis boosts the immune response against these proteins. The anti-CarP response in mice with CIA can be used as a model for immune responses to post-translationally modified proteins

Anti-CarP antibodies can be detected in mice.

<p>(<b>A</b>) Schematic picture of the carbamylation process. (<b>B</b>) DBA/1J mice were immunized with CII in CFA. The antibody binding to FCS and Carbamylated-FCS (Ca-FCS) was determined by ELISA. The OD value for Ca-FCS binding and FCS binding of each sample are connected with a line. Statistical analysis was performed using a Wilcoxon paired test (n = 29). (<b>C</b>) Sera from anti-CarP positive mice were pre-incubated with different concentrations of Ca-FCS and FCS. The Ig binding to FCS and Ca-FCS was determined by ELISA (n = 4). (<b>D</b>) C57Bl/6 mice were immunized with CII in CFA. Equal amounts of Ca-FCS and FCS were blotted on a membrane. The presence of antibodies reactive to the Ca-FCS or FCS on the blots was analyzed by incubating the blots with sera from the immunized mice. A representative example of 2 independent experiments is depicted.</p

Anti-citrullinated protein antibodies (ACPA) could not be detected in mice with CIA.

<p>CIA was induced in DBA/1J mice. After 70–90 days, serum was harvested and anti-CarP and ACPA levels were determined by ELISA. Every symbol represents an individual mice and the line indicates the median. (<b>A</b>) IgG1 levels. (<b>B</b>) IgG2a levels. Statistical analysis was performed using a kruskall-wallis test followed by a Dunn's Multiple comparison test (* p<0.05).</p

Kinetics of the anti-CarP response during CIA.

<p>(<b>A</b>) CIA was induced in DBA/1J mice (n = 19). The mice were immunized at day 0 and boosted at day 21. Blood was harvested from the mice at the indicated time points and serum was stored at −80. The anti-CarP antibody levels were determined in all serum samples simultaneously by ELISA at the end of the experiment. The data shown are the pooled data from 2 independent experiments that showed a similar trend. (<b>B</b>) Anti-CarP IgG2a levels were determined by ELISA. Arbitrary units were calculated using a standard curve of pooled serum from mice with CIA. Every symbol represents one individual mouse and the line indicates the median. (<b>C</b>) Anti-CarP IgG2a antibody levels are plotted on the left Y axes and indicated by the dots with the solid line. The clinical score is plotted on the right Y-axis and indicated by the squares and the dashed line. The error bars indicate the SEM.</p

Mice with CIA harbor anti-CarP antibodies.

<p>CIA was induced in DBA/1J mice. After 70–90 days, serum of naïve non-immunized mice (HC; squares) and mice with CIA (dots) was harvested and anti-CarP levels were determined by ELISA. Arbitrary units were calculated using a standard curve of pooled serum from mice with CIA. The number indicates the number of mice per group. (<b>A</b>) Total Ig levels (<b>B</b>) Total Ig levels plotted against the clinical score (<b>C</b>) IgG1 levels (<b>D</b>) IgG1 levels plotted against the clinical score (<b>E</b>) IgG2a levels (<b>F</b>) IgG2a levels plotted against the clinical score. The data is pooled data from 4 independent experiments. Every symbol represents one mouse and the bar indicates the median. Statistical analysis was performed using a Mann-Whitney test (** p<0.01, *** p<0.001).</p

Anti-CarP can be detected in CFA immunized mice.

<p>DBA/1J were immunized with CII (CIA; (n = 19)) or PBS in CFA followed by a booster with CII or PBS in IFA (CFA; n = 9). Anti-CarP IgG2a levels were determined by ELISA. The data shown are the pooled data from 2 independent experiments that showed a similar trend. CFA +PBS immunized mice are depicted as circles and CIA mice are depicted as squares. The error bars indicate the SEM. In the right panel the area under curve is depicted. Every symbol represents one individual mouse and the line indicates the median. Statistical analysis was performed by comparing the area under the curve followed by a Mann-Whitney test (** p<0.01).</p

Crossreactivity to vinculin and microbes provides a molecular basis for HLA-based protection against rheumatoid arthritis

The HLA locus is the strongest risk factor for anti-citrullinated protein antibody (ACPA)(+) rheumatoid arthritis (RA). Despite considerable efforts in the last 35 years, this association is poorly understood. Here we identify (citrullinated) vinculin, present in the joints of ACPA(+) RA patients, as an autoantigen targeted by ACPA and CD4(+) T cells. These T cells recognize an epitope with the core sequence DERAA, which is also found in many microbes and in protective HLA-DRB1*13 molecules, presented by predisposing HLA-DQ molecules. Moreover, these T cells crossreact with vinculin-derived and microbial-derived DERAA epitopes. Intriguingly, DERAA-directed T cells are not detected in HLA-DRB1*13(+) donors, indicating that the DERAA epitope from HLA-DRB1*13 mediates (thymic) tolerance in these donors and explaining the protective effects associated with HLA-DRB1*13. Together our data indicate the involvement of pathogen-induced DERAA-directed T cells in the HLA-RA association and provide a molecular basis for the contribution of protective/predisposing HLA allele