Elucidation of immunological pathophysiology of various diseases in obstetrics and gynecology and the research for the development of new prevention and therapeutics

Kei Kawana, MD., PhD.

Associate Professor
Department of Obstetrics and Gynecology, Graduate School of Medicine,
The University of Tokyo

 Etiology of various diseases in the field of obstetrics and gynecology is closely related to immunological disorders, including lack of immune responses, immunological hyper-stimulation and autoimmunity. In turn, immunological approach can be one of the strategy for the treatment and one of the predictive biomarker for the diagnosis. Therfore, immunity and inflammation are critical factors for knowledge of gynecologic and obstetrical diseases.
 In this study, we focused on “immunology” to address clearance of gynecologic neoplasm, prevention of infectious diseases at reproductive tract, and resolution of inflammatory gynecologic and obstetrical disorders. From the viewpoint of reproductive health, preventive medicine should be an ultimate strategy which prevents functional disorder of gynecologic organs, including vagina, uterus, ovary, fallopian tube, and pelvic peritoneum. The goal of our studies is preventive medicine to keep reproductive health in female.

(1) Development of novel HPV vaccines: prophylactic and thepeutics

<A novel broad-spectrum prophylactic HPV vaccine>

 Lack of permissive and productive cell cultures for the human papillomaviruses (HPVs) has hindered the study of virus-neutralizing antibodies and infection. We developed a cell-free system generating infectious HPV16 pseudovirions. HPV16 L1/L2 capsids were disassembled with 2-mercaptoethanol (2-ME), a reducing agent, and reassembled by removal of 2-ME in the presence of a b-galactosidase expression plasmid. Plasmid DNA purified together with the reassembled capsids was resistant to DNase I digestion. The reassembled pseudovirions mediated DNA transfer to COS-1 cells, as monitored by induced b-galactosidase activity. Transfer was inhibited by anti-HPV16 L1 antiserum but not by antisera against L1s of HPV6 and HPV18. Furthermore, we demonstrated that HPV-16 L2 region of aa 69?81 contains a type-common immunodeterminant exposed on the surface of HPV virions using monoclonal antibody analysis against HPV16L1/L2 capsid.
 Then, we examined the neutralizing activity of mouse monoclonal antibodies (MAbs) that recognize surface epitopes in HPV16 minor capsid protein L2. Two MAbs binding to a synthetic peptide with the HPV16 L2 sequence of amino acids (aa) 108 to 120 were found to inhibit pseudoinfections with HPV16 as well as HPV6. Antisera raised by immunizing BALB/c mice with the synthetic peptide had a cross-neutralizing activity similar to that of the MAb. The data indicate that HPV16 and -6 have a common cross-neutralization epitope (located within aa 108 to 120 of L2 in HPV16), suggesting that this epitope may be shared by other genital HPVs.

<Therapeutic HPV vaccine for treatment of HPV-related neoplasm using mucosal immunity>

 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 attenuated LacE7 vaccines at weeks 1, 2, 4, and 8. Oral immunization with LacE7 elicited E7-specific IFN?-producing cells (T cells with E7-type1 immune responses) among integrin ?4?7+ mucosal lymphocytes collected from gut mucosa. 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.

 On the other hand, 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. Cervical lymphocytes were collected using cytobrushes from patients with CIN and analyzed by FACS analysis. A median of 74% of cervical lymphocytes were CD3+ T cells. Populations of integrin aEb7+ IEL in CIN lesions varied markedly among patients (6?57%). Approximately half of integrin b7+ T cells were CD45RA-negative memory T cells. The number of integrin aEb7+ cells among cervical T cells was significantly higher in CIN regressors when compared to non-regressors. Higher cervical IEL numbers are associated with spontaneous regression of CIN.
 Taken together these our rationales, we conducted Ph. I/IIa clinical trial to evaluate the safety and clinical efficacy of an attenuated Lactobacillus casei expressing mod-ified full-length HPV16 E7 protein in patients with HPV16-associated CIN3. Ten patients were vaccinatedorally during dose optimization studies (1, 2, 4, or 6 capsules/day) at weeks 1, 2, 4, and 8 (Step 1). Sevenadditional participants were only tested using the optimized vaccine formulation (Step 2), giving a totalof 10 patients who received optimized vaccination. Cervical lymphocytes (CxLs) and peripheral bloodmononuclear cells (PBMCs) were collected and E7 specific IFN?-producing cells were counted (E7 cell-mediated immune responses: E7-CMI). All patients were re-evaluated 9 weeks after initial vaccine exposure using cytology and biopsy to assess pathological efficacy. No patient experienced an adverse event. All patients using 4?6 capsules/day showed increased E7-CMI in CxLs, whereas patients using1?2 capsules/day did not. No patient demonstrated an increase in E7-CMI in their PBMCs. In comparison between patients of cohorts, E7-CMI 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. Oral administration of an E7-expressing Lactobacillus-based vaccine can elicit E7-specificmucosal immunity in the uterine cervical lesions. We are the first to report a correlation between mucosalE7-CMI in the cervix and clinical response after immunotherapy in human mucosal neoplasia.

(2) Omega-3 polyunsaturated fatty acids (omega-3 PUFAs) may be a novel therapeutic and prophylactic agents against acute and chronic inflammatory diseases in obstetrics and gynecology

 Omega-3 polyunsaturated fatty acids such as eicosapentaenoic acid (EPA) have anti-inflammatory effects. Preterm birth is an important problem in modern obstetrics and one of the main causes is an inflammation. We showed that abundance of omega-3 fatty acids reduced the incidence of preterm birth induced by LPS with fat-1 mice, capable of converting omega-6 to omega-3 fatty acids. We also indicated that the gene expression of IL-6 and IL-1? in uteruses and the number of cervical infiltrating macrophages were reduced in fat-1 mice. The analyses of lipid metabolomics showed the high level of 18-hydroxyeicosapentaenoate in fat-1 mice, which was derived from EPA and was metabolized to anti-inflammatory product named resolving E3 (RvE3). We finally showed that the administration of RvE3 to LPS-exposed pregnant wild type mice lowered the incidence of preterm birth. Our data suggest that RvE3 could be a potential new therapeutic for the prevention of preterm birth.
 Endometriosis is characterized by the presence of endometrial tissue on the peritoneum and an exaggerated inflammatory environment around ectopic tissues. Peritoneal endometriosis was reproduced using a mouse model in which murine endometrial fragments were inoculated into the peritoneal cavity of mice. Fat-1 mice, in which omega-6 can be converted to omega-3 PUFAs, or wild type mice, in which it cannot, were used for the endometriosis model to address the actions of omega-3 PUFAs on the development of endometriotic lesions. The number and weight of cystic endometriotic lesions in fat-1 mice two weeks after inoculation were significantly less than half to those of controls. Mediator lipidomics revealed that cystic endometriotic lesions and peritoneal fluids were abundant in 12/15-hydroxyeicosapentaenoic acid (12/15-HEPE), derived from eicosapentaenoic acid (EPA), and their amount in fat-1 mice was significantly larger than that in controls. 12/15-Lipoxygenase (12/15-LOX)-knockout (KO) and control mice with or without EPA administration were assessed for the endometriosis model. EPA administration decreased the number of lesions in controls but not in 12/15-LOX-KO mice. The peritoneal fluids in EPA-fed 12/15-LOX-KO mice contained reduced levels of EPA metabolites such as 12/15-HEPE and EPA derived RvE3 even after EPA administration. cDNA microarrays of endometriotic lesions revealed that IL-6 expression in fat-1 mice was significantly lower than that in controls. These results suggest that both endogenous and exogenous EPA-derived PUFAs protect against the development of endometriosis through their anti-inflammatory effects and, in particular, the 12/15-LOX-pathway products of EPA may be key mediators to suppress endometriosis.

 Our study provided attractive insights into etiology of various gynecologic and obstetrical diseases which are involved in immunity and inflammation. Furthermore, we conducted and will conduct clinical trials to develop novel therapeutics and strategy for treatment. Finally, our goal is to prevent these women’s diseases to keep reproductive health.