SMFM Papers www.AJOG.org Efficacy of polymeric encapsulated C5a peptidase–based group B streptococcus vaccines in a murine model Donna A. Santillan, PhD; Karishma K. Rai, MD; Mark K. Santillan, MD; Yogita Krishnamachari, PhD; Aliasger K. Salem, PhD; Stephen K. Hunter, MD, PhD OBJECTIVE: The purpose was to examine in mice the efficacy of vari- RESULTS: Thirty microgram doses of the 75:25 and 50:50 PLGA for- ous polymeric-encapsulated C5a peptidase vaccine formulations in mulations generate the highest and most sustained C5a peptidase– eliciting a long-term immune response and preventing group B strepto- specific immune responses. Mice that received encapsulated C5a pep- coccus (GBS) infection. tidase were significantly protected from vaginal colonization compared STUDY DESIGN: C5a peptidase was encapsulated in semiperme- with mice that received empty microspheres. able microspheres of poly(lactide-coglycolide) (PLGA). Female ICR CONCLUSION: Encapsulated C5a peptidase elicited significant immune mice were immunized with 0, 10, or 30 ␮g of encapsulated C5a responses and protection against a GBS challenge. C5a peptidase mi- peptidase within 2 different formulations of PLGA polymers. Booster crosphere encapsulation has potential as a GBS vaccine. doses were given at weeks 4 and 8. Antibody responses were mea- sured by enzyme-linked immunosorbent assay at weeks 4, 8, 11, Key words: antigen encapsulation, C5a peptidase, group B and 40. Vaginal challenges with GBS types 1a, III, and V were per- streptococcus, microparticle, microsphere, mouse, poly(lactide- formed at week 12. coglycolide), vaccine Cite this article as: Santillan DA, Rai KK, Santillan MK, et al. Efficacy of polymeric encapsulated C5a peptidase–based group B streptococcus vaccines in a murine model. Am J Obstet Gynecol 2011;205:249.e1-8. roup B streptococci are Gram-posi- colonize the intestinal and urogenital tract adults and pregnant women in the top Gtive diplococci that produce polysac- of healthy adults without causing any com- tier for vaccine development.3 They sug- charide capsules.1 There are 10 different plications. However, GBS infections can gested the vaccine would have the great- Group B Streptococcus (GBS) serotypes, cause serious problems in pregnancy. est effect if given as part of routine pre- based on the antigenic variation of the cap- In 1985, the Institute of Medicine natal care for all women in their first sular polysaccharides. These serotypes are from the National Academies of Sciences pregnancy. Maternal complications of Ia, Ib, and II through IX. A recent interna- indicated that a vaccine for GBS should GBS infections in pregnancy include uri- tional study most commonly isolated sero- be a high priority. In a 1999 report, they nary tract infections, chorioamnionitis, 2 types III and V from patients. GBS can again listed a GBS vaccine for high-risk postpartum wound infection, pyelone- phritis, postpartum endometritis, and sepsis. For the neonate, preterm prema- From the Divisions of Reproductive Health Research (Drs D. A. Santillan, M. K. Santillan, ture rupture of membranes (PPROM), and Hunter) and Maternal-Fetal Medicine (Drs Rai, M. K. Santillan, and Hunter), neonatal pneumonia, sepsis, meningitis, Department of Obstetrics and Gynecology, University of Iowa Hospitals and Clinics, and the or death can occur as a consequence of Division of Pharmaceutics, University of Iowa College of Pharmacy (Drs Krishnamachari and 4-8 Salem), Iowa City, IA. GBS. Because GBS infection continues to be Presented, in part, at the 31st Annual Meeting of the Society for Maternal-Fetal Medicine, San Francisco, CA, Feb. 7-12, 2011. a serious concern during pregnancy, the Received March 10, 2011; revised April 26, 2011; accepted June 7, 2011. updated guidelines from the Centers for Reprints: Stephen K. Hunter, MD, PhD, University of Iowa Hospitals and Clinics, Department of Disease Control and Prevention con- Obstetrics and Gynecology, 200 Hawkins Dr., Iowa City, IA 52242. [email protected]. tinue to recommend universal culture- This work was supported in part by the Eunice Kennedy Shriver National Institute of Child Health based screening of all pregnant women at and Human Development, National Institutes of Health (NIH), Grant no. 1R03HD056006 (S.K.H.); 35-37 weeks’ gestation.9,10 Currently, if a Children’s Miracle Network and the University of Iowa Children’s Hospital (D.A.S.); NIH pregnant woman screens positive for Interdisciplinary Immunology Postdoctoral Training Program, Grant no. T32 AI 007260 (D.A.S.); NIH Interdisciplinary Training Program in Pain Research, Grant no. T32 NS045549 (K.K.R.); NIH GBS, she will receive intravenous antibi- University of Iowa Institute for Clinical and Translational Science Grant no. KL2 RR024980-2 otics during labor to prevent early-onset (M.K.S.); Department of Obstetrics and Gynecology, American Cancer Society Grant no. GBS disease in the child, which can in- RSG-09-015-01-CDD (A.K.S.); National Cancer Institute/NIH Grant nos. 1R21CA13345-01/ clude sepsis and death.9 Recent studies 1R21CA128414-01A2/UI, Mayo Clinic Lymphoma SPORE (A.K.S.); Pharmaceutical Research demonstrate that up to 24% of all preg- and Manufacturers of America (PhRMA) Foundation (A.K.S.); and support from a Guillory Fellowship (Y.K.). nant women receive antibiotic prophy- 0002-9378/$36.00 • © 2011 Mosby, Inc. All rights reserved. • doi: 10.1016/j.ajog.2011.06.024 laxis for GBS. Between 50% and 75% of neonates exposed through the birth ca- SEPTEMBER 2011 American Journal of Obstetrics & Gynecology 249.e1 SMFM Papers www.AJOG.org nal of GBS-infected mothers will be highly conserved multifunctional surface and 50:50) would affect these immune colonized. protein that is expressed on the surface of responses and the ability of vaccinated Many shortcomings exist in the cur- all serotypes of both group A streptococcus mice to prevent GBS colonization and to rent therapy of antibiotic prophylaxis. (GAS) and group B streptococcus tes- pass GBS protection to pups of vacci- These shortcomings are especially evi- ted.17-20 C5a peptidase (ScpB) expressed nated dams. dent in cases in which a woman has a lack by GBS is 98% identical in sequence to that of prenatal care, delivers before being expressed by GAS.21,22 Structurally, C5a screened, delivers before the culture re- peptidase contains 5 domains including a MATERIALS AND METHODS sults return, has a rapid labor and does subtisilin-like protease domain, a pro- C5a peptidase encapsulation not finish receiving all of the antibiotic tease-associated domain, and 3 fibronectin C5a peptidase, guanosine monophosphate dose(s), or is allergic to antibiotics. In type III domains.23 The enzymatic activity prepared and greater than 98-99% pure, addition, the development of antibiotic of the peptidase is highly specific for C5a, was generously provided by Pfizer (Gro- resistance is an increasing problem. cleaving the chemotaxin at its polymor- ton, CT). The C5a peptidase was microen- Penicillin-tolerant strains of GBS have phonuclear leukocyte binding site.24 capsulated in PLGA microspheres. PLGA been identified, and resistance to other Recent evidence also suggests that C5a (50:50, inherent viscosity, 0.4 dL/g) and antibiotics has been documented.8,11 A peptidase may bind fibronectin to pro- PLGA (75:25, inherent viscosity, 0.51 recent study found 91% of strains iso- mote cellular invasion.25,26 Cheng et al27 dL/g) were purchased from Lactel Absorb- lated were resistant to erythromycin.12 demonstrated that ScpB increased the able Polymers (Cupertino, CA). Polyvinyl The consequences of a GBS infection in immunogenicity of a GBS type III poly- alcohol (PVA; 87-89% hydrolyzed, molec- pregnancy require that treatment be saccharide vaccine in mice when the 2 ular weight 30-67,000 Da) was purchased given. However, this large-scale use of were coupled. In addition, the investiga- from Sigma-Aldrich (St Louis, MO). antibiotics is contributing to the devel- tors found that ScpB was also found to Encapsulation was done using a water- opment of antibiotic resistance. Without induce the production of GBS serotype- in-oil-in-water (w/o/w) double-emulsion prevention strategies, such as vaccina- independent antibodies.27 technique as described previously.16,32,33 tion, our first-line antibiotic therapies We have previously shown that encap- Briefly, the internal aqueous phase con- are going to become useless against GBS. sulating C5a peptidase within micro- sisted of 3.6 mg peptidase equivalent to 6.6 Most importantly, this traditional ap- spheres composed of a copolymer of mg lyophilized powder C5a peptidase sol- proach does not prevent preterm deliv- lactic and glycolic acids, poly(lactide-co- ubilized in 500 ␮L of 1% (weight/volume) ery or PPROM or protect against late- glycolide) (PLGA), was able to induce aqueous solution of PVA as a surfactant. onset disease caused by GBS infection.13 systemic and mucosal immune response This was emulsified into an oil phase con- A GBS vaccine could overcome these in mice. Furthermore, this vaccine pro- taining 200 mg of PLGA 50:50 or PLGA pitfalls. vided protection in mice against GBS se- 75:25 dissolved in 5 mL dichloromethane Development of a vaccine for GBS has rotype III in vaginal and pup challenge (DCM) using a microtip probe sonicator. been hindered by several factors. First, studies.28 The PLGA polymer–based mi- This primary water/oil emulsion was then there are 10 serotypes of GBS that are based crospheres are able to act as an adjuvant poured into 50 mL of external aqueous on antigenic variation of the capsular poly- to the vaccine and are safe for use in hu- phase containing 1% (weight/volume) saccharides. Purified capsular polysaccha- mans and has been used for many years PVA as a surfactant and rapidly homoge- rides, without adjuvants, have elicited in resorbable sutures, bone plates, and nized using a high-speed homogenizer at weak immune responses in vaccines, and a commercial depot drug delivery formu- 9500 rpm for 30 seconds to form the sec- multivalent vaccine would be necessary to lations.29 The antigen release profile by ondary w/o/w emulsion.