Solubility Controlling Peptide Tags of Opposite Charges Generate a Bivalent Immune Response Against Dengue ED3 Serotypes 3 and 4.

We previously demonstrated that a protein's immunogenicity could be substantially increased by attaching a hydrophobic solubility controlling peptide tag (SCP-tag) producing small sub-visible aggregates. Here, we report the oligomerization of Dengue envelop protein domain 3 (ED3), and consequently, its immunogenicity increase by mixing ED3s attached with SCP-tags of opposite charges at equimolar concentration. We used ED3 of serotype 3 (D3ED3) and serotype 4 (D4ED3), which are, respectively, moderately and poorly immunogenic, and their SCP tagged variants constructed by attaching either a C-termini 5-Aspartic acid (C5D) or a 5-Lysine (C5K) tag. Light scattering indicated that the isolated tagged ED3s remained monomeric, but mixing the C5D and C5K tagged ED3s at equimolar concentration generated sub-visible aggregates or oligomers of ~500 nm through electrostatic interaction. In addition, the oligomerized ED3s remained in a native-like state, as assessed by fluorescence spectroscopy and circular dichroism. The in vivo immunogenicity of the D3ED3 and D4ED3 oligomers generated by the charged tags increased by 5 and 16 fold, respectively. Furthermore, injection of heterotypic ED3 oligomers (D3C5D+D4C5K) induced an immune response against both D3ED3 and D4ED3 in 3 of 4 responsive mice, and the IgG titer of the bivalent anti-D3C5D-D4C5K sera was over 100 times higher than that generated by co-injecting the untagged D3ED3 and D4ED3 (D3+D4). Altogether, these observations suggest that SCP-tags could be used as a platform for producing a long-sought tetravalent dengue vaccine.

Nanometer-Sized Aggregates Generated Using Short Solubility Controlling Peptide Tags Do Increase the In Vivo Immunogenicity of a Nonimmunogenic Protein.

Subvisible aggregates of proteins are suspected to cause adverse immune response, and a recent FDA guideline has recommended the monitoring of micrometer-sized aggregates (2-10 µm) though recognizing that the underlying mechanism behind aggregation and immunogenicity remains unclear. Here, we report a correlation between the immunogenicity and the size of nanometer-scaled aggregates of a small 6.5 kDa model protein, bovine pancreatic trypsin inhibitor (BPTI) variant. BPTI-19A, a monomeric and nonimmunogenic protein, was oligomerized into subvisible aggregates with hydrodynamic radii (Rh) of 3-4 nm by attaching hydrophobic solubility controlling peptide (SCP) tags to its C-terminus. The results showed that the association of nonimmunogenic BPTI into nanometer-sized subvisible aggregates made it highly immunogenic, as assessed by the IgG antibody titers of the mice's sera. Overall, the study emphasizes that subvisible aggregates, as small as a few nanometers, which are presently ignored, are worth monitoring for deciphering the origin of undesired immunogenicity of therapeutic proteins.

Anti-Dengue ED3 Long-Term Immune Response With T-Cell Memory Generated Using Solubility Controlling Peptide Tags.

Recombinant proteins are an attractive choice as a safe alternative to traditional live attenuated vaccines. However, most small-size proteins are poorly immunogenic, and adjuvants, whose mode of action remain to be fully clarified, are needed for increasing their immunogenicity. Here, we report the effects of short solubility controlling peptide tags (SCP-tags) on the immunogenicity of DENV3 envelope protein domain 3 (3ED3; 103 residues, 11.46 kDa) in ICR and Swiss albino model mice. The attachment of a 4-Ile SCP-tag (C4I-tag) increased the hydrodynamic radius of 3ED3 from 2.2 ± 0.09 to 111 ± 146 nm as assessed by dynamic light scattering in phosphate buffered saline at 37°C, indicating that the C4I-tag oligomerized 3ED3. Immunization at 30 µg/dose showed that the untagged 3ED3 was not or poorly immunogenic, whereas the C4I-tag increased its immunogenicity by up to 39-fold as assessed by the IgG level measured using ELISA. Moreover, the increased antibody level was sustained for over 6 months after immunization and a high number of effector and central memory T cells were generated. These observations provide solid and quantitative evidence for the hypothesis that subvisible aggregates with hydrodynamic radii of 100 nm can increase immunogenicity and that SCP-tag can establish a long-term, target-specific immune response in a way adequate for the development of a peptide/protein-based DENV vaccine.

Radical hysterectomy with or without para-aortic lymphadenectomy for patients with stage IB2, IIA2, and IIB cervical cancer: outcomes for a series of 308 patients.

BACKGROUND: Although many studies have already shown that lymph node metastasis is one of the major prognostic factors for cervical cancer, the therapeutic significance of para-aortic lymphadenectomy for the surgical treatment of cervical cancer remains controversial. METHODS: A total of 308 patients diagnosed with stage IB2, IIA2, or IIB cervical cancer and treated with radical hysterectomy were retrospectively investigated to assess the incidence of para-aortic lymph node metastasis and the clinicopathological factors linked to cervical cancer prognosis. RESULTS: Para-aortic lymph node metastases were pathologically confirmed in 13 of the 136 patients (9.6 %) who underwent para-aortic lymphadenectomy. The incidence of para-aortic lymph node metastasis was significantly higher in the patients who had common iliac lymph node metastases (odds ratio 31.5, p < 0.001) according to logistic regression analysis. Common iliac lymph node metastasis was related to risk of recurrence (hazard ratio 2.43, p = 0.003) and death (hazard ratio 2.62, p = 0.007) in Cox regression analysis. Kaplan-Meier analysis and Cox regression analysis showed that para-aortic lymphadenectomy did not have a positive impact on survival in 308 patients or 140 pN1 patients, but para-aortic lymphadenectomy was related to better overall survival with a marginal trend toward significance (p = 0.053) in 30 patients with common iliac lymph node metastasis. CONCLUSIONS: Indication for para-aortic lymphadenectomy in the surgical treatment of stage IB2, IIA2, or IIB cervical cancer needs to be individualized. Patients with common iliac lymph node metastasis are possible candidates, and a prospective study is needed to clarify this issue.

Positive peritoneal cytology at interval surgery is a poor prognostic factor in patients with stage T3c advanced ovarian carcinoma: A retrospective study.

AIM: The purpose of our study is to investigate clinically significant prognostic factors at the time of interval surgery (IS), comprising interval look surgery and interval debulking surgery, for T3c (International Federation of Gynecology and Obstetrics stage IIIc to IV) advanced ovarian cancer (AOC) patients during primary treatment. METHODS: We reviewed records of patients with T3c AOC who underwent IS following neoadjuvant chemotherapy or up-front primary debulking surgery with adjuvant chemotherapy at our institution between January 1996 and December 2010. For analysis of prognostic factors, cytology of peritoneal exfoliative cells at IS was added to clinicopathological variables. RESULTS: A retrospective analysis was performed on 50 cases. The median age was 61.1 years (range, 38-78), with median follow-up of 45.9 months (range, 12-122). Macroscopic tumors were completely resected in 32 cases (64%) at IS. Univariate analyses of clinicopathological factors for IS identified preoperative serum cancer antigen-125 levels (≥20 IU/mL; P = 0.0539), number of residual lesions at IS (≥20; P = 0.0554), incomplete surgery at IS (P = 0.0171) and positive peritoneal cytology at IS (P = 0.0015) as significant factors for prognosis regarding progression-free survival (PFS). Multivariate analysis identified positive peritoneal cytology (P = 0.0303) as a unique independent predictor of poor prognosis in PFS. CONCLUSION: Positive peritoneal cytology at IS appears to be a significant factor for poor prognosis in PFS, which may provide useful information for post-IS chemotherapy planning. IS in the treatment of AOC may be useful for not only complete resection, but also for identification of patients with poor prognosis.

High-risk human papillomavirus correlates with recurrence after laser ablation for treatment of patients with cervical intraepithelial neoplasia 3: a long-term follow-up retrospective study.

AIM: The purpose of our study was to evaluate the efficacy of laser ablation as a conservative treatment for cervical intraepithelial neoplasia 3 (CIN3) and assess whether the human papillomavirus (HPV) test is useful to predict recurrence after treatment. MATERIALS AND METHODS: A total of 134 patients who received laser ablation for treatment of CIN3 were enrolled in this study. During the follow-up period, patients were followed with cytological and colposcopic evaluations. Recurrence of CIN3 was regarded as the primary end-point. HPV genotype was tested before and after treatment. Post-treatment cumulative recurrence rates were estimated and comparisons by both patient age and HPV genotype were performed. RESULTS: Overall cumulative recurrence rate of CIN3 in the first year after treatment was 22.6% for all patients. No significant correlation was shown between patient age and recurrence. Patients infected by specific genotypes (16, 18, 31, 33, 52, and 58) frequently failed to clear the infection after treatment. The 1-year recurrence-free survival in those positive after treatment for eight high-risk genotypes (16, 18, 31, 33, 35, 45, 52, and 58) was significantly lower (66.7%), compared to that in those positive for other high-risk types (78.6%). The recurrence-free survival of those who remained HPV-positive after treatment was significantly lower than those who turned negative. CONCLUSION: Laser ablation should be performed prudently with appropriate patient counseling about recurrence rate. Considering its minimal invasiveness, laser ablation is effective, especially for young patients who are negative for eight high-risk genotypes. With regard to HPV testing, although genotyping has significant value for predicting recurrence, screening for all genotypes warrants further evaluation.

Three-dimensional structural diversity-oriented peptidomimetics based on the cyclopropylic strain.

Conformationally restricted peptidomimetics comprising eight stereoisomeric scaffolds with three-dimensional structural diversity were designed based on the structural features of cyclopropane, that is, cyclopropylic strain, which mimic wide-ranging tetrapeptide conformations covering ß-turns through ß-strands. Stereoselective synthesis of the designed peptidomimetics led to the identification of nonpeptidic melanocortin-4 receptor ligands.

The prevalence of cervical regulatory T cells in HPV-related cervical intraepithelial neoplasia (CIN) correlates inversely with spontaneous regression of CIN.

PROBLEM: Local adaptive cervical regulatory T cells (Tregs) are the most likely direct suppressors of the immune eradication of cervical intraepithelial lesion (CIN). PD-1 expression on T cells induces Tregs. No studies have quantitatively analyzed the Tregs and PD-1+ cells residing in CIN lesions. METHOD OF STUDY: Cervical lymphocytes were collected using cytobrushes from CIN patients and analyzed by FACS analysis. Comparisons were made between populations of cervical Tregs and PD-1+ CD4+ T cells in CIN regressors and non-regressors. RESULTS: A median of 11% of cervical CD4+ T cells were Tregs, while a median of 30% were PD-1+ cells. The proportions of cervical CD4+ T cells that were Tregs and/or PD-1+ cells were significantly lower in CIN regressors when compared with non-regressors. CONCLUSIONS: The prevalence of cervical tolerogenic T cells correlates inversely with spontaneous regression of CIN. Cervical Tregs may play an important role in HPV-related neoplastic immunoevasion.

A possible coagulation-independent mechanism for pregnancy loss involving ß(2) glycoprotein 1-dependent antiphospholipid antibodies and CD1d.

PROBLEM ß(2) glycoprotein1 (ß(2) GP1)-dependent antiphospholipid antibodies (aPL) increase the risk for recurrent pregnancy loss. We address whether anti-ß(2) GP1 antibodies can interact with phosphatidylserine (PS)-bearing CD1d on trophoblast cells and induce local inflammation. METHODS CD1d-bearing choriocarcinoma cells were used in flow cytometry and immunoprecipitation experiments. CD1d-mediated cytokine induction was assessed using antibody cross-linking. Cytokine production during co-culture of decidual lymphocytes with CD1d-bearing cells was also examined. RESULTS Trophoblast surface-expressed CD1d forms a complex with PS-bound ß(2) GP1. Anti-ß(2) GP1 mAb cross-linking causes IL12p70 release from CD1d-bearing cells. IL12p70 release from CD1d-bearing trophoblast cells was also induced during co-culture with human decidual lymphocytes. The addition of anti-ß2GP1 mAb to co-cultures resulted in a three-fold increase in IL12p70 secretion. IFNγ secretion from decidual lymphocytes was also induced during co-culture with anti-ß2GP1 mAbs. CONCLUSIONS ß(2) GP1-dependent IL12 release from CD1d-bearing trophoblast in the presence of aPL may link the antiphospholipid syndrome to pregnancy loss via an inflammatory mechanism.

Characterization of gut-derived intraepithelial lymphocyte (IEL) residing in human papillomavirus (HPV)-infected intraepithelial neoplastic lesions.

PROBLEM: Mucosal T cells are the most likely direct effectors in host anti-human papillomavirus adaptive immunity and regression of cervical intraepithelial neoplasia (CIN) lesions. There are no studies addressing intraepithelial lymphocytes (IELs) in CIN lesions. METHOD OF STUDY: Cervical lymphocytes were collected using cytobrushes from patients with CIN and analyzed by FACS analysis. Comparisons were made between populations of cervical T cells in CIN regressors and non-regressors. RESULTS: A median of 74% of cervical lymphocytes were CD3(+) T cells. Populations of integrin αEß7(+) IEL in CIN lesions varied markedly among patients (6-57%). Approximately half of integrin ß7(+) T cells were CD45RA-negative memory T cells. The number of integrin αEß7(+) cells among cervical T cells was significantly higher in CIN regressors when compared to non-regressors. CONCLUSION: Higher cervical IEL numbers are associated with spontaneous regression of CIN. Accumulation of cervical integrin αEß7(+) IEL may be necessary for local adaptive effector functions.

CD1d, a sentinel molecule bridging innate and adaptive immunity, is downregulated by the human papillomavirus (HPV) E5 protein: a possible mechanism for immune evasion by HPV.

CD1d and CD1d-restricted natural killer T (NKT) cells serve as a natural bridge between innate and adaptive immune responses to microbes. CD1d downregulation is utilized by a variety of microbes to evade immune detection. We demonstrate here that CD1d is downregulated in human papillomavirus (HPV)-positive cells in vivo and in vitro. CD1d immunoreactivity was strong in HPV-negative normal cervical epithelium but absent in HPV16-positive CIN1 and HPV6-positive condyloma lesions. We used two cell lines for in vitro assay; one was stably CD1d-transfected cells established from an HPV-negative cervical cancer cell line, C33A (C33A/CD1d), and the other was normal human vaginal keratinocyte bearing endogenous CD1d (Vag). Flow cytometry revealed that cell surface CD1d was downregulated in both C33A/CD1d and Vag cells stably transfected with HPV6 E5 and HPV16 E5. Although the steady-state levels of CD1d protein decreased in both E5-expressing cell lines compared to empty retrovirus-infected cells, CD1d mRNA levels were not affected. Confocal microscopy demonstrated that residual CD1d was not trafficked to the E5-expressing cell surface but colocalized with E5 near the endoplasmic reticulum (ER). In the ER, E5 interacted with calnexin, an ER chaperone known to mediate folding of CD1d. CD1d protein levels were rescued by the proteasome inhibitor, MG132, indicating a role for proteasome-mediated degradation in HPV-associated CD1d downregulation. Taken together, our data suggest that E5 targets CD1d to the cytosolic proteolytic pathway by inhibiting calnexin-related CD1d trafficking. Finally, CD1d-mediated production of interleukin-12 from the C33A/CD1d cells was abrogated in both E5-expressing cell lines. Decreased CD1d expression in the presence of HPV E5 may help HPV-infected cells evade protective immunological surveillance.

Oral immunization with a Lactobacillus casei vaccine expressing human papillomavirus (HPV) type 16 E7 is an effective strategy to induce mucosal cytotoxic lymphocytes against HPV16 E7.

Although many clinical trials on human papillomavirus (HPV) therapeutic vaccines have been performed, clinical responses have not been consistent. We have addressed mucosal cytotoxic cellular immune responses to HPV16 E7 after oral immunization of mice with recombinant Lactobacillus casei expressing HPV16 E7 (LacE7). C57BL/6 mice were orally exposed to 0.1-100mg/head of attenuated LacE7 or vehicle (Lac) vaccines at weeks 1, 2, 4, and 8. Responses to subcutaneous or intramuscular injection of an HPV16 E7 fusion protein using the same timing protocol were used for comparison. Oral immunization with LacE7 elicited E7-specific IFN gamma-producing cells (T cells with E7-type 1 immune responses) among integrin alpha 4 beta 7(+) mucosal lymphocytes collected from gut mucosa. An induction of E7-specific granzyme B-producing cells (E7-CTL) exhibiting killer responses toward HPV16 E7-positive cells was also observed. The induction of T cells with specific mucosal E7-type 1 immune responses was greater after oral immunization with LacE7 when compared to subcutaneous or intramuscular antigen delivery. Oral immunization with Lactobacillus-based vaccines was also able to induce mucosal cytotoxic cellular immune responses. This novel approach at a therapeutic HPV vaccine may achieve more effective clinical responses through its induction of mucosal E7-specific CTL.

Expression of autotaxin, an ectoenzyme that produces lysophosphatidic acid, in human placenta.

PROBLEM: Lysophosphatidic acid (LPA) is a bioactive lipid mediator and thought to play an important role in pregnancy. Plasma LPA is produced by autotaxin (ATX), and ATX activity in plasma increases during pregnancy paralleled with gestational weeks and decreases to near the non-pregnant level soon after delivery. However, the source of increased ATX during pregnancy is still uncertain. We hypothesized that the source of increased ATX might be placenta. METHOD OF STUDY: We investigated the protein and mRNA expression of ATX in human placenta using immunohistochemistry and RT-PCR, respectively. RESULTS: At all 3 gestational trimesters, immunohistochemical staining for placenta tissues revealed the most marked positive staining of ATX protein in trophoblasts. Real-time PCR revealed that mRNA amounts of ATX in placenta tissues paralleled with gestational weeks, i.e. ATX level in plasma. CONCLUSION: These findings suggest that trophoblasts might produce ATX and its bioactive resultant substance, LPA, paralleled with gestational weeks.

Expression of CD1d and ligand-induced cytokine production are tissue specific in mucosal epithelia of the human lower reproductive tract.

Mucosal epithelia of the human lower reproductive tract (vagina, cervix, and penile urethra) are exposed to sexually transmitted microbes, including Chlamydia trachomatis. The in vivo susceptibility of each tissue type to infection with C. trachomatis is quite distinct. CD1d is expressed on the surface of antigen-presenting cells, including mucosal epithelial cells, and interacts specifically with invariant NKT cells. Invariant NKT cells play a role in both innate and adaptive immune responses to microbes. Here we assessed CD1d expression in normal reproductive tissues by using immunohistochemistry. Immortalized epithelial cell lines from the human lower reproductive tract (vagina, endocervix, and penile urethra) were examined for CD1d expression and for ligand-induced cytokine production induced by CD1d cross-linking. CD1d expression in normal tissue was strong in the vagina but weak in the endocervix and penile urethra. Gamma interferon exposure induced CD1d transcription in all of the cell types studied, with the strongest induction in vaginal cells. Flow cytometry revealed cell surface expression of CD1d in vaginal and penile urethral epithelial cells but not in endocervical cells. Ligation of surface-expressed CD1d by monoclonal antibody cross-linking promoted interleukin-12 (IL-12) and IL-15, but not IL-10, production in vaginal and penile urethral cells. No induction was demonstrated in endocervical cells. CD1d-mediated cytokine production in penile urethral cells was abrogated by C. trachomatis infection. Basal deficiency in CD1d-mediated immune responsiveness may result in susceptibility to sexually transmitted agents. Decreased CD1d-mediated signaling may help C. trachomatis evade detection by innate immune cells.