Vitamin C supramolecular hydrogel for enhanced cancer immunotherapy

Vitamin C (VitC) has shown great promise to promote cancer immunotherapy, however, its high hydrophilicity makes it quickly excreted, leading to limited therapeutic efficiency even with frequent high-dose administration. Herein, we provide a pioneering report about the employment of VitC amphiphile self-assembled nanofiber hydrogels for enhanced cancer immunotherapy. Specifically, driven by hydrogen bonding and hydrophobic interactions, the synthesized VitC amphiphile, consisting of a hydrophilic VitC headgroup and a hydrophobic alkyl chain, could self-assemble into an injectable nanofiber hydrogel with self-healing properties. The formed VitC hydrogel not only serves as a reservoir for VitC but also acts as an effective delivery platform for stimulator of interferon genes (STING) agonist-4 (SA). Interestingly, the VitC hydrogel itself exhibits antitumor effects by upregulating genes related to interferon (IFN) signaling, apoptotic signaling and viral recognition and defense. Moreover, the SA-encapsulated VitC hydrogel (SA@VitC hydrogel) synergistically activated the immune system to inhibit the progression of both local and abscopal tumors

Genome-wide identification of Chlamydia trachomatis antigens associated with trachomatous trichiasis.

PURPOSE: Chlamydia trachomatis is the leading infectious cause of blindness. The goal of the current study was to search for biomarkers associated with C. trachomatis-induced ocular pathologies. METHODS: We used a whole genome scale proteome array to systematically profile antigen specificities of antibody responses to C. trachomatis infection in individuals from trachoma-endemic communities with or without end-stage trachoma (trichiasis) in The Gambia. RESULTS: When 61 trichiasis patients were compared with their control counterparts for overall antibody reactivity with organisms of different chlamydial species, no statistically significant difference was found. Both groups developed significantly higher titers of antibodies against C. trachomatis ocular serovars A and B than ocular serovar C, genital serovar D, or Chlamydia psittaci, whereas the titers of anti-Chlamydia pneumoniae antibodies were the highest. When antisera from 33 trichiasis and 26 control patients (with relatively high titers of antibodies to C. trachomatis ocular serovars) were reacted with 908 C. trachomatis proteins, 447 antigens were recognized by at least 1 of the 59 antisera, and 10 antigens by 50% or more antisera, the latter being designated as immunodominant antigens. More importantly, four antigens were preferentially recognized by the trichiasis group, with antigens CT414, CT667, and CT706 collectively reacting with 30% of trichiasis antisera but none from the normal group, and antigen CT695 reacting with 61% of trichiasis but only 31% of normal antisera. On the other hand, eight antigens were preferentially recognized by the control group, with antigens CT019, CT117, CT301, CT553, CT556, CT571, and CT709 together reacting with 46% of normal antisera and none from the trichiasis group, whereas antigen CT442 reacted with 35% of normal and 19% of trichiasis antisera respectively. CONCLUSIONS: The current study, by mapping immunodominant C. trachomatis antigens and identifying antigens associated with both ocular pathology and protection, has provided important information for further understanding chlamydial pathogenesis and the development of subunit vaccines

Chlamydia trachomatis GlgA is secreted into host cell cytoplasm.

Glycogen has been localized both inside and outside Chlamydia trachomatis organisms. We now report that C. trachomatis glycogen synthase (GlgA) was detected in both chlamydial organism-associated and -free forms. The organism-free GlgA molecules were localized both in the lumen of chlamydial inclusions and in the cytosol of host cells. The cytosolic GlgA displayed a distribution pattern similar to that of a known C. trachomatis-secreted protease, CPAF. The detection of GlgA was specific since the anti-GlgA antibody labeling was only removed by preabsorption with GlgA but not CPAF fusion proteins. GlgA was detectable at 12h and its localization into host cell cytosol only became apparent at 24h after infection. The cytosolic localization of GlgA was conserved among all C. trachomatis serovars. However, the significance of the GlgA secretion into host cell cytoplasm remains unclear since, while expression of chlamydial GlgA in HeLa cells increased glycogen stores, it did not affect a subsequent infection with C. trachomatis. Similar to several other C. trachomatis-secreted proteins, GlgA is immunogenic in women urogenitally infected with C. trachomatis, suggesting that GlgA is expressed and may be secreted into host cell cytosol during C. trachomatis infection in humans. These findings have provided important information for further understanding C. trachomatis pathogenic mechanisms

Reactivity of GlgA with antisera from women infected and rabbits immunized with <i>Chlamydia trachomatis</i> organisms.

<p>(<b>A</b>) Chlamydial GST fusion proteins or GST alone were resolved in a SDS polyacrylamide gel and stained with a Coomassie blue dye. All GST fusion proteins along with their gene names/ORF numbers and fusion protein molecular weights were listed on top of the gel image. Note that besides the GST-containing degradation fragments, the major bands are full length fusion proteins (marked with circles) for all GST fusion proteins. (B) The above fusion proteins listed along the X-axis were reacted with antisera from women diagnosed with acute <i>Chlamydia trachomatis</i> infection (STI patients, n=20, panel a) or tubal factor infertility (TFI patients, n=24, b) or from normal women without <i>C. trachomatis</i> infection (n=10, c) or from rabbits intramuscularly immunized with dead <i>Chlamydia trachomatis</i> organisms (Rabbits, n=7, c) in ELISA assays. The OD values obtained at the wavelength of 405nm in the format of mean plus/minus standard deviation were displayed along the Y-axis. Any reactivity with an OD value equal to or above the mean plus 2 standard deviations is defined as positive (+ve), which is noted on top of each bar. Note that 12 of the 20 STI, 7 of the 24 TFI and 0 of the 10 normal women or 7 rabbit antisera positively reacted with GlgA.</p

Localization of GlgA in <i>C. trachomatis</i>-infected cells.

<p>HeLa cells infected with <i>C. trachomatis</i> serovar L2 at an MOI 0.5 were processed either 24h or 40h post infection for co-staining with mouse antibodies recognizing individual chlamydial proteins visualized with a goat anti-mouse IgG conjugated with Cy3 (red), a rabbit antibody to chlamydial organisms visualized with a Cy2-conjugated goat anti-rabbit IgG (green) and DNA dye Hoechst (blue). (A) The mouse antisera raised with 6 GST fusion proteins as indicated on top of each panel were used to localize the corresponding endogenous chlamydial proteins after 1:1000 dilution. Note that only the anti-GST-CT798 (GlgA; panels f & l) detected signals that were free of chlamydial organisms. (B) The anti-GlgA antiserum labeling was repeated at multiple dilutions: 1:100 (panel a), 1:1000 (b) and 1:5000 (c). Note that the antibody detected both intra-inclusion (yellow & red arrowheads) and extra-inclusion (red arrow) signals under a conventional fluorescence microscope at all dilutions. (C) The mouse anti-GlgA antiserum labeling at 1:1000 dilution was further observed under a confocal microscope. Note that the intra-inclusion labeling with anti-GlgA polyclonal antibody displayed two distinct patterns [overlapping with chlamydial organisms (yellow arrowheads) and free of the organisms (red arrowheads)] while all anti-HSP60 antibody labelings overlapped with the organisms (panels d-f). The images were taken under an objective lens of either 100x (conventional fluorescence microscopy) or 60x (confocal microscopy).</p

Western blot detection of GlgA in fractions of <i>C. trachomatis</i>-infected cells.

<p>Normal HeLa lysates (lane 1) or HeLa cells with Chlamydia trachomatis (Ct) serovar L2 infection (lane 2) were fractionated into pellets (lane 3) and supernatants (S100, lane 4) along with purified chlamydial reticulate bodies (RB, lane 5) or elementary body (EB, lane 6) were resolved in SDS polyacrylamide gel and the resolved protein bands were transferred onto nitrocellulose membrane for Western blot detection with anti-GlgA (panel a), anti-CPAF (b), anti-CT813 (c), anti-MOMP (d) and anti-human HSP70 (e) antibodies. Note that CPAF was detected only in either Ct-HeLa lysates (lane 2) or Ct-HeLa S100 (lane 4) while GlgA was detected in all samples containing chlamydial organisms or derived from <i>Chlamydia trachomatis</i>-infected cells.</p

Time course expression and secretion of GlgA during <i>C. trachomatis</i> infection.

<p>HeLa cell infected with <i>C. trachomatis</i> serovar L2 at MOI of 0.5 were processed at various times after infection (as indicated on the top) for immunofluorescence staining as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068764#pone-0068764-g001" target="_blank">Figure 1</a>. The labelings of anti-GlgA (polyclonal antiserum, panels a to f) and anti-CPAF (mAb 100a; g to l) were visualized with a goat anti-mouse IgG conjugated with Cy3 (red) while the rabbit anti-chlamydial organism labelings were visualized with a goat anti-rabbit IgG-Cy2 conjugate (green). Images amplified in separate panels were marked with white squares and the corresponding amplified images were labeled the same letters followed by the number 1. Note that both GlgA (c & c₁) and CPAF (i & i1) proteins were first detected associated with the chlamydial inclusions at 12h while secretion out of the inclusions was first detected at 24h (panel d for GlgA; j for CPAF) post infection (red arrows). The secreted GlgA and CPAF remained in the infected cells throughout the infection cycle. The % of cells positive for GlgA (CT798) secretion into host cell cytoplasm was compared to those positive for CPAF secretion (B & C). HeLa cells infected with L2 for 40h were processed and triple labeled with mouse antibodies (red) against GlgA (CT798), CPAF or GlgC (CT489) in combination with a rabbit antibody against chlamydial organisms (green) and a DNA dye (blue). The images were taken under a 20X objective lens. The areas marked with dashed squares in images a, e & i were magnified in the corresponding panels on the right. White arrows point to inclusions without secretion of GlgA (images a-d) or CPAF (e–h) into host cell cytosol. GlgC was used as a secretion-negative control (panels i to l). The number of cells with positive secretion was counted and calculated into % of secretion-positive cells (C). The results were expressed as mean plus standard deviation and data came from 4 independent experiments. Please note that the % of cells secreting either GlgA or CPAF were similar (p>0.05, Fisher Exact).</p

Effect of chlamydial GlgA expression on glycogen synthesis and subsequent chlamydial infection.

<p>HeLa cells were transfected with pFlag plasmid alone or pFlag plasmid encoding chlamydial GlgA gene. Twenty-four hours after transfection, the cell samples were processed for immunofluorescence labelings with mouse anti-FLAG or anti-GlgA (A), harvested for glycogen detection (B) or further infected with <i>C. trachomatis</i> L2 (C). (A) The images were taken 24h after transfection under a 60X objective lens using a conventional fluorescence microscope. (B) HeLa cells with or without transfection with pFLAG or pFLAG-GlgA or infection with L2 were harvested 24h post-transfection or 48h post-infection for glycogen measurements. The results coming from 5 independent experiments were expressed as µg per ml of samples as shown along the Y-axis. Note that both exogenous GlgA overexpression and chlamydial infection significantly enhanced the levels of glycogen synthesis in HeLa cells (Student t-test). (C) Some of the transfected cells were further infected with chlamydial organisms and 40h after chlamydial infection, the cultures were processed for immunofluorescence labelings with mouse anti-FLAG to identify positive transfection cells and rabbit anti-chlamydial EBs to identify chlamydial inclusions. Number of total and <i>C. trachomatis</i>-infected cells was counted from 5 random views of each coverslip. The infection rate was calculated for anti-Flag positive (solid bar) and negative (open bar) cells respectively and expressed as mean plus/minus standard deviation along the Y-axis. Note that the infection rates displayed no statistically significant differences between cells with or without transfection from either pFLAG vector alone or pFLAG/GlgA recombinant plasmid-transfected cultures (p>0.05, Fisher Exact).</p

Secretion of GlgA into host cytosol by different biovars/serovars of <i>Chlamydia trachomatis</i>.

<p>HeLa cells were infected with different serovars of chlamydial organisms representing 3 major biovars of <i>Chlamydia trachomatis</i> as indicated on top of each panel. The infected cultures were subjected to immunofluorescence labelings as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068764#pone-0068764-g004" target="_blank">Figure 4</a> legend. Note that GlgA secretion into host cell cytosol was detected in all <i>Chlamydia trachomatis</i> serovars assayed.</p

Antibody specificity validation.

<p>The mouse anti-GlgA (polycolonal antibody, pAb at 1:1000), anti-CPAF (monoclonal antibody, clone# 100a, IgG1) and anti-Pgp3 (clone# 2H4, IgG2a) antibodies were preabsorbed without (panels a, e & i) or with GST-GlgA (b, f & j), GST-CPAF (c, g & k) and GST-Pgp3 (d, h & l) fusion proteins prior to reacting with <i>C. trachomatis</i>-infected cultures as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068764#pone-0068764-g001" target="_blank">Figure 1</a> legend. Note that the antibody labelings were removed by the corresponding (f, g & k) but not irrelevant fusion proteins. (B) HeLa cell lysates, Chlamydia trachomatis (Ct)-infected HeLa cell lysates, GST-GlgA or GST-CPAF fusion proteins were resolved in SDS-polyacrylamide gel and transferred onto nitrocellulose membrane for Western blot detection with mouse anti-GlgA (αGlgA at 1:4000 dilution, panel a), αCPAF (mAb 100a, b), αMOMP (mAb MC22, c) and αhuman HSP70 (mAb W27, d). Note that these antibodies only detected their corresponding antigens without any significant cross-reactivity.</p