Oral vaccination against HPV E7 for treatment of cervical intraepithelial neoplasia grade 3 (CIN3) elicits E7-specific mucosal immunity in the cervix of CIN3 patients.

BACKGROUND: Cervical intraepithelial neoplasia grade 3 (CIN3) is a mucosal precancerous lesion caused by high-risk human papillomavirus (HPV). Induction of immunological clearance of CIN3 by targeting HPV antigens is a promising strategy for CIN3 therapy. No successful HPV therapeutic vaccine has been developed. METHODS: We evaluated the safety and clinical efficacy of an attenuated Lactobacillus casei expressing modified full-length HPV16 E7 protein in patients with HPV16-associated CIN3. Ten patients were vaccinated orally during dose optimization studies (1, 2, 4, or 6 capsules/day) at weeks 1, 2, 4, and 8 (Step 1). Seven additional participants were only tested using the optimized vaccine formulation (Step 2), giving a total of 10 patients who received optimized vaccination. Cervical lymphocytes (CxLs) and peripheral blood mononuclear cells (PBMCs) were collected and E7 specific interferon-γ-producing cells were counted (E7 cell-mediated immune responses: E7-CMI) by ELISPOT assay. All patients were re-evaluated 9 weeks after initial vaccine exposure using cytology and biopsy to assess pathological efficacy. RESULTS: No patient experienced an adverse event. E7-CMI in both CxLs and PBMCs was negligible at baseline. All patients using 4-6 capsules/day showed increased E7-CMI in CxLs, whereas patients using 1-2 capsules/day did not. No patient demonstrated an increase in E7-CMI in their PBMCs. In comparison between patients of cohorts, E7-CMI at week 9 (9 wk) in patients on 4 capsules/day was significantly higher than those in patients on 1, 2, or 6 capsules/day. Most patients (70%) taking the optimized dose experienced a pathological down-grade to CIN2 at week 9 of treatment. E7-CMI in CxLs correlated directly with the pathological down-grade. CONCLUSIONS: Oral administration of an E7-expressing Lactobacillus-based vaccine can elicit E7-specific mucosal immunity in the uterine cervical lesions. We are the first to report a correlation between mucosal E7-CMI in the cervix and clinical response after immunotherapy in human mucosal neoplasia.

Evaluation of Mdh1 protein as an antigenic candidate for a vaccine against candidiasis.

Candida albicans malate dehydrogenase (Mdh1p) has been screened by previous proteome studies as a candidate for a vaccine against candidiasis. In this study, recombinant Mdh1 protein with a His-tag was produced in Escherichia coli and evaluated as an immunogenic protein against candidiasis. Mdh1p was administrated to mice by two methods subcutaneous injection and intranasal administration before challenging them with a lethal dose of C. albicans. After vaccination of Mdh1p, antibody responses were observed. To evaluate the vaccination effect of Mdh1p, survival tests were performed after 35 d. Although all control mice died within 24 d or 25 d, 100% and 80% of mice survived with subcutaneous and intranasal administration, respectively. Therefore, our results indicate that, among C. albicans antigens examined thus far, Mdh1p is currently the most effective antigen for use as a vaccine for C. albicans.

Oral Immunization Against Candidiasis Using Lactobacillus casei Displaying Enolase 1 from Candida albicans.

Candidiasis is a common fungal infection that is prevalent in immunocompromised individuals. In this study, an oral vaccine against Candida albicans was developed by using the molecular display approach. Enolase 1 protein (Eno1p) of C. albicans was expressed on the Lactobacillus casei cell surface by using poly-gamma-glutamic acid synthetase complex A from Bacillus subtilis as an anchoring protein. The Eno1p-displaying L. casei cells were used to immunize mice, which were later challenged with a lethal dose of C. albicans. The data indicated that the vaccine elicited a strong IgG response and increased the survival rate of the vaccinated mice. Furthermore, L. casei acted as a potent adjuvant and induced high antibody titers that were comparable to those induced by strong adjuvants such as the cholera toxin. Overall, the molecular display method can be used to rapidly develop vaccines that can be conveniently administered and require minimal processing.

An oral vaccine against candidiasis generated by a yeast molecular display system.

Enolase 1 (Eno1p) of Candida albicans is an immunodominant antigen. However, conventional technologies for preparing an injectable vaccine require purification of the antigenic protein and preparation of an adjuvant. To develop a novel type of oral vaccine against candidiasis, we generated Saccharomyces cerevisiae cells that display the Eno1p antigen on their surfaces. Oral delivery of the engineered S. cerevisiae cells prolonged survival rate of mice that were subsequently challenged with C. albicans. Given that a vaccine produced using molecular display technology avoids the need for protein purification, this oral vaccine offers a promising alternative to the use of conventional and injectable vaccines for preventing a range of infectious diseases.

Engineered hepatitis B virus surface antigen L protein particles for in vivo active targeting of splenic dendritic cells.

Dendritic cells (DCs) are key regulators of adaptive T-cell responses. By capturing exogenous antigens and presenting antigen-derived peptides via major histocompatibility complex molecules to naïve T cells, DCs induce antigen-specific immune responses in vivo. In order to induce effective host immune responses, active delivery of exogenous antigens to DCs is considered important for future vaccine development. We recently generated bionanocapsules (BNCs) consisting of hepatitis B virus surface antigens that mediate stringent in vivo cell targeting and efficient endosomal escape, and after the fusion with liposomes (LP) containing therapeutic materials, the BNC-LP complexes deliver them to human liver-derived tissues in vivo. BNCs were further modified to present the immunoglobulin G (IgG) Fc-interacting domain (Z domain) derived from Staphylococcus aureus protein A in tandem. When mixed with IgGs, modified BNCs (ZZ-BNCs) displayed the IgG Fv regions outwardly for efficient binding to antigens in an oriented-immobilization manner. Due to the affinity of the displayed IgGs, the IgG-ZZ-BNC complexes accumulated in specific cells and tissues in vitro and in vivo. After mixing ZZ-BNCs with antibodies against DCs, we used immunocytochemistry to examine which antibodies delivered ZZ-BNCs to mouse splenic DCs following intravenous injection of the ZZ-BNCs. ZZ-BNCs displaying anti-CD11c monoclonal antibodies (α-CD11c-ZZ-BNCs) were found to accumulate with approximately 62% of splenic DCs, and reside within some of them. After the fusion with liposomes containing antigens, the α-CD11c-ZZ-BNCs could elicit the respective antibodies more efficiently than other nontargeting control vaccines, suggesting that this DC-specific nanocarrier is promising for future vaccines.

Japanese encephalitis virus structural and nonstructural proteins expressed in Escherichia coli induce protective immunity in mice.

Ectodomain of Japanese encephalitis virus (JEV) E protein [domains I through III (D1-3), domains I and II (D1-2) and domain III (D3)] and the nonstructural protein 1 (NS1) were expressed in Escherichia coli, and administered to BALB/c mice via the intranasal (i.n.) route. The E protein, but not the NS1, induced JEV-specific serum IgG with virus-neutralization capacity in vitro. When mice were lethally challenged with JEV, i.n. immunization with D1-3, D1-2, D3, or a mouse brain-derived formalin-inactivated JE vaccine conferred complete protection, while an 80% protection rate was observed in the NS1 immunized mice. Cytokine analysis of the cervical lymph nodes of mice i.n. immunized with D1-3 or NS1 revealed antigen-specific IL-2 and IL-17 responses, but no IFN-γ T cell response, were observed. This study demonstrates for the first time the i.n. vaccine efficacy of the E. coli-expressed recombinant JEV proteins.

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.

[Generation of mucosal vaccine utilizing lactobacillus display system].

Lactic acid bacteria (LAB) are a group of Gram-positive, and generally recognized as safe bacteria. LAB have been used as the starter for the fermentation food (i.e., cheese, yoghurt and kimuchi etc.). On the other hand, several studies of LAB as delivery vehicles have focused on the generation of mucosal vaccine. We have developed novel surface display system based on PgsA gene, which isolated from Bacillus subtilis chungkookjang. We introduce the Lactobacillus surface display system by using the PgsA anchor protein and its application. HPV oncogene, E7, is a reliable target protein since E7 is expressed in the CIN lesion. Although many studies have demonstrated these vaccines elicit systemic immune responses to HPV E6/E7, few studies have shown mucosal immune responses. There is no therapeutic vaccine utilizing oral administration and there is no clinical trial which addresses cervical mucosal cellular immune responses to the vaccine. Our recent progress is production of a mucosal vaccine to treat cervical intraepithelial neoplasia (CIN) that has potential of cervical cancer. The vaccine is expected to help the vast number of women suffering from high grade CIN. Lac-E7 is a candidate for new therapeutic vaccine for cervical intraepithelial neoplasia.

Display of alpha-amylase on the surface of Lactobacillus casei cells by use of the PgsA anchor protein, and production of lactic acid from starch.

We developed a new cell surface engineering system based on the PgsA anchor protein from Bacillus subtilis. In this system, the N terminus of the target protein was fused to the PgsA protein and the resulting fusion protein was expressed on the cell surface. Using this new system, we constructed a novel starch-degrading strain of Lactobacillus casei by genetically displaying alpha-amylase from the Streptococcus bovis strain 148 with a FLAG peptide tag (AmyAF). Localization of the PgsA-AmyA-FLAG fusion protein on the cell surface was confirmed by immunofluorescence microscopy and flow cytometric analysis. The lactic acid bacteria which displayed AmyAF showed significantly elevated hydrolytic activity toward soluble starch. By fermentation using AmyAF-displaying L. casei cells, 50 g/liter of soluble starch was reduced to 13.7 g/liter, and 21.8 g/liter of lactic acid was produced within about 24 h. The yield in terms of grams of lactic acid produced per gram of carbohydrate utilized was 0.60 g per g of carbohydrate consumed at 24 h. Since AmyA was immobilized on the cells, cells were recovered after fermentation and used repeatedly. During repeated utilization of cells, the lactic acid yield was improved to 0.81 g per g of carbohydrate consumed at 72 h. These results indicate that efficient simultaneous saccharification and fermentation from soluble starch to lactic acid were carried out by recombinant L. casei cells with cell surface display of AmyA.

Display of active enzymes on the cell surface of Escherichia coli using PgsA anchor protein and their application to bioconversion.

We have developed a novel Escherichia coli cell surface display system by employing PgsA as an anchoring motif. In our display system, C-terminal fusion to PgsA anchor protein from Bacillus subtilis was used. The enzymes selected for display were alpha-amylase (AmyA) from Streptococcus bovis 148 and lipase B (CALB) from Candida antarctica. The molecular mass values of AmyA and CALB are approximately 77 and 34 kDa, respectively. The enzymes were displayed on the surface as a fusion protein with a FLAG peptide tag at the C terminus. Both the PgsA-AmyA-FLAG and PgsA-CALB-FLAG fusion proteins were shown to be displayed by immunofluorescence labeling using anti-FLAG antibody. The displayed enzymes were active forms, and AmyA and CALB activities reached 990 U/g (dry cell weight) and 4.6 U/g (dry cell weight), respectively. AmyA-displaying E. coli cells grew utilizing cornstarch as the sole carbon source, while CALB-displaying E. coli cells catalyzed enantioselective transesterification, indicating that they are effective whole-cell biocatalysts. Since a target enzyme with a size of 77 kDa and an industrially useful lipase have been successfully displayed on the cell surface of E. coli for the first time, PgsA protein is probably a useful anchoring motif to display various enzymes.