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 0.1–100 mg/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 IFNγ-producing cells (T cells with E7-type1 immune responses) among integrin α4β7⁺ 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-type1 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.     

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.     

Immunization with the conjugate vaccine Vi-CRM(197) against Salmonella Typhi induces Vi-specific mucosal and systemic immune responses in mice

Typhoid fever is a public health problem, especially among young children in developing countries. To address this need, a glycoconjugate vaccine Vi-CRM(197), composed of the polysaccharide antigen Vi covalently conjugated to the non-toxic mutant of diphtheria toxin CRM(197), is under development. Here, we assessed the antibody and cellular responses, both local and systemic, following subcutaneous injection of Vi-CRM(197). The glycoconjugate elicited Vi-specific serum IgG titers significantly higher than unconjugated Vi, with prevalence of IgG1 that persisted for at least 60 days after immunization. Vi-specific IgG, but not IgA, were present in intestinal washes. Lymphocytes proliferation after restimulation with Vi-CRM(197) was observed in spleen and mesenteric lymph nodes. These data confirm the immunogenicity of Vi-CRM(197) and demonstrate that the vaccine-specific antibody and cellular immune responses are present also in the intestinal tract, thus strengthening the suitability of Vi-CRM(197) as a promising candidate vaccine against Salmonella Typhi.     

Comparative evaluation of the antibody in lymphocyte supernatant (ALS) and enzyme-linked immunospot (ELISPOT) assays for measuring mucosal immune responses to Shigella antigens

Accurately assessing mucosal immune responses to candidate vaccines remains a technical challenge. ELISPOT is widely used as a surrogate of mucosal immune response by directly enumerating circulating antibody secreting cells (ASCs), while antibody in lymphocyte supernatant (ALS) titers the total amount of antibody secreted by ASC ex vivo using ELISA. ALS is more practical than ELISPOT because the ASC supernatant is frozen for ELISA that can be conducted at any time, with any antigen, and in any laboratory. We compared IgA and IgG responses to serotype-specific Shigella LPS using ELISPOT and ALS in subjects following vaccination or infection with Shigella. ALS results correlated well with ELISPOT results, and the ALS method was both sensitive and specific for the detection of antibody responses against Shigella LPS. Based on these observations, the ALS assay is a practical and flexible alternative to ELISPOT for measuring mucosal IgA responses to Shigella LPS antigen.     

表达人乳头瘤病毒16型E6E7重组腺病毒疫苗对小鼠的免疫和抗肿瘤效应

目的 评价HPV16 E6E7的复制缺陷型重组5型腺病毒(PK-HPV-ad5)治疗性疫苗对实验小鼠免疫应答和抗肿瘤的生物学效应.方法 使用基因重组技术构建PK-HPV-ad5疫苗,并通过小鼠免疫试验,检测小鼠总抗体和特异性IFNγ,同时将造模小鼠分成疫苗组和对照组,分别对其进行抑瘤试验、TC-1肿瘤细胞挑战试验和肿瘤切除后防复发试验.结果 HPV16 E6E7诱导的总抗体第12天的水平相对较高(1:400～1:600);3批次疫苗特异性IFNγ在第14天与对照组比较分别升高8.6、5.9和8.9倍,差异有统计学意义(t=15.721、6.967和14.342,P均＜0.01).抑瘤试验表明疫苗剂量为107IU/只时小鼠肿瘤生长率为0,与对照组比较差异有统计学意义(确切概率法,P＜0.01),3批次疫苗验证有效剂量为107IU/只时肿瘤抑制率可达80％(8/10)以上.TC-1肿瘤细胞挑战试验结果显示:小鼠先接种疫苗能引起特异性的免疫应答,并能保护90％(9/10)的小鼠免受TC-1肿瘤细胞的攻击;肿瘤切除后防止复发试验提示在注射相同剂量疫苗时,对104个/只和105个/只肿瘤细胞造模小鼠,第0、5天免疫组肿瘤复发数少于第5,8天免疫组(1/10,4/10 vs 8/10,7/10).结论 PK-HPV-ad5疫苗能诱导小鼠产生特异性的免疫应答,对抗肿瘤复发有治疗潜力.     

Incorporation of immunostimulatory motifs in the transcribed region of a plasmid DNA vaccine enhances Th1 immune responses and therapeutic effect against Mycobacterium tuberculosis in mice

T-helper type 1 (Th1) immune response is involved in the development of protective immunity against Mycobacterium tuberculosis. Thus, an increase in Th1 and cellular immune responses should lead to enhanced anti-mycobacterial activity. In this study, we aimed to improve Th1 immune responses to a DNA vaccine by adding potentially immunostimulatory nucleotide sequences into the transcribed region downstream of the antigen. The Mycobacterium leprae gene for hsp65, codon-optimized for expression in mammalian cells, was inserted into pVAX1 with and without 3′-sequences containing CpG and dsRNA motifs. When the plasmid contained both motifs, transfected murine macrophage-like RAW264.7 cells showed markedly increased levels of mRNA for immune molecules of Th1 (IFN-α, IL-12) and Th17 (IL-17, IL-23 and IL-6) responses and for T cell co-stimulatory molecules (CD80 and CD86) but not for a Th2 response (IL-4 and IL-10). Immunized mice showed substantially increased serum anti-Hsp65 IgG2a antibody levels and IFN-γ production by spleen cells, confirming enhancement of the Th1 response in vivo. Furthermore, when non-vaccinated mice were infected with H37Rv by low-dose aerosol challenge, and then 4 weeks later were treated with plasmids by intramuscular injection, the mice that had been treated with plasmids containing immunostimulatory motifs showed an enhanced reduction in mycobacterial loads in lung and spleen. We conclude that DNA vaccines may be made more highly immunogenic and more effective for treatment by including transcribed stimulatory sequences.     

A novel fusion protein-based vaccine comprising a cell penetrating and immunostimulatory peptide linked to human papillomavirus (HPV) type 16 E7 antigen generates potent immunologic and anti-tumor responses in mice

The ultimate success of cancer vaccination is dependent upon the generation of tumor-specific CTLs. In this study, we designed and evaluated a novel fusion protein comprising a cell penetrating and immunostimulatory peptide corresponding to residues 32-51 of the Limulus polyphemus protein (LALF_32-51) linked to human papillomavirus (HPV) 16 E7 antigen (LALF_32-51-E7). We demonstrated that LALF_32-51 penetrates the cell membrane and delivers E7 into cells. In a preclinical model of HPV16-induced cervical carcinoma we showed that vaccination with adjuvant-free LALF_32-51-E7 fusion protein significantly improves the presentation of E7-derived peptides to T-cells in vitro and induces suppression of tumor growth.     

A novel therapeutic vaccine composed of a rearranged human papillomavirus type 16 E6/E7 fusion protein and Fms-like tyrosine kinase-3 ligand induces CD8+ T cell responses and antitumor effect

The development of cervical cancer is mainly caused by infection with high risk genotypes of human papillomavirus, particularly type 16 (HPV16), which accounts for more than 50% of cervical cancer. The two early viral oncogenes, E6 and E7, are continuously expressed in cervical cancer cells and are necessary to maintain the malignant cellular phenotype, thus providing ideal targets for immunotherapy of cervical cancer. In this study, a novel vaccine strategy was developed based on a rationally shuffled HPV16 E6/E7 fusion protein, the addition of Fms-like tyrosine kinase-3 ligand (Flt3L) or the N domain of calreticulin (NCRT), and the usage of a CpG adjuvant. Four recombinant proteins were constructed: m16E6E7 (mutant E6/E7 fusion protein), rm16E6E7 (rearranged mutant HPV16 E6/E7 fusion protein), Flt3L-RM16 (Flt3L fused to rm16E6E7), and NCRT-RM16 (NCRT fused to rm16E6E7). Our results suggest that Flt3L-RM16 was the most potent of these proteins in terms of inducing E6- and E7-specific CD8⁺ T cell responses. Additionally, Flt3L-RM16 significantly induced regression of established E6/E7-expressing TC-1 tumors. Higher doses of Flt3L-RM16 trended toward higher levels of antitumor activity, but these differences did not reach statistical significance. In summary, this study found that Flt3L-RM16 fusion protein is a promising therapeutic vaccine for immunotherapy of HPV16-associated cervical cancer.