Efficacy of Polymeric Encapsulated C5a Peptidase–Based Group B

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 measured 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. Stirring was then provide protection against the multiple PLGA microsphere–based vaccines is continued using a magnetic stirrer until GBS serotypes because each polysaccha- largely dependent on the lactide/gly- complete evaporation of DCM. The mi- ride can target only the serotype from colide ratio. Copolymers with a higher crospheres were collected by centrifuga- which it was derived. Furthermore, poly- lactide/glycolide ratio have a longer deg- tion at 5000 ϫ g for 10 minutes, washed 3 saccharide-based vaccines were unable to radation profile because lactic acid is hy- times with deionized water, and lyophi- elicit significant mucosal immune re- drophobic.30,31 PLGA microspheres have lized overnight. sponses.14-16 A mucosal immune response been studied for use in numerous vaccines. To quantitate encapsulation efficiency would be critical in completely eliminating We hypothesized that encapsulation of protein for dosing purposes, 30 mg of maternal GBS colonization. Eliminating of C5a peptidase within PLGA micro- lyophilized PLGA microspheres con- colonization by all GBS serotypes would spheres would induce specific systemic taining C5a peptidase were dissolved in give the best chance of preventing the in- and mucosal immune responses that 3.0 mL of 1 M NaOH containing 5.0% fection from being vertically passed to the would afford protection against multiple (weight/volume) sodium dodecyl sulfate child during the birthing process. serotypes of GBS. We further hypothe- and incubated for 24 hours at room tem- In the current study, we further evalu- sized that differences in antigen doses (0, perature. After centrifugation (4000 ϫ g ated the use of streptococcal C5a peptidase 10, and 30 ␮g) and PLGA microsphere for 10 minutes at room temperature), as the vaccine antigen. C5a peptidase is a lactide-glycolide formulations (75:25 the supernatant was assayed for protein

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concentration using the bicinchonic acid FIGURE 1 assay (Thermo Scientific, Swedesboro, Vaccine microspheres NJ) following the manufacturer’s proto- col. All the measurements were done in PLGA 50:50 microspheres PLGA 75:25 microspheres triplicate. A B In vitro release profile Thirty to 40 mg of C5a-peptidase– Day 0 loaded PLGA microspheres were incubated in 2-3 mL phosphate buffered sa- line (PBS; pH 7.4). Two hundred microliters of samples were withdrawn at predetermined time intervals. The sample was centrifuged at 10,000 rpm for 5 minutes, and the supernatant was analyzed using a bicinchoninic assay to de- C D termine C5a content. The sedimented microspheres were dispersed in 200 ␮L Day 30 of PBS and replaced back instantly into the release samples. Scanning electron microscopy was performed as described previously at days 0 and 30 of the release profile assay.28,32,33

Administration of vaccine Scanning electron microscopy of vaccines at A and B, days 0 and C and D, 30 during the in vitro Female ICR mice (Charles River Breed- release profile. Microspheres from both formulations were round and of uniform size. The average size ␮ ing Laboratories, Portage, MI) 5-7 weeks sphere at day 0 was 3-4 m. Microspheres were completely degraded by day 30 with debris of the old were vaccinated either through an low-molecular-weight fractions of lactic acid and glycolic acid left behind. intramuscular or intranasal route. For all Santillan. C5a peptidase-based group B streptococci vaccines. Am J Obstet Gynecol 2011. doses of the intramuscular vaccine, the vaccine was administered in 100 ␮L into responses in serum and vaginal washes as into the vagina of 5 mice of each vacci- the right upper leg. For all doses of the described previously.28 nation group. After 48 hours, vaginal intranasal administration, 50 ␮L of vac- Samples producing a significant dif- washings were obtained and 2 dilutions cine was administered into each nostril ference when further diluted are consid- were plated on blood agar plates. Plates (100 ␮L total volume). Booster doses ered to have a larger immune response were incubated for 24 hours at 37oC with were administered in the same manner than samples in which a significant dif- 5% CO2. After 24 hours, plates were as- as the initial vaccination and were given ference was observed in only smaller di- sessed for growth of GBS. If no colonies at weeks 4 and 8. For the pup challenge lutions. The optical density (OD)405 were evident, plates were incubated for experiment, the vaccine was adminis- reading for each dilution was compared another 24 hours and growth of GBS col- tered intranasally and boosters were between each vaccine formulation group onies was again assessed. Cyclic adeno- given at weeks 2 and 4. and the empty microsphere control. The sine monophosphate tests and Gram largest dilution that remained statisti- staining were used to determine whether Determination of immune response cally significant in the OD405 compari- questionable beta-hemolytic colonies Mice were bled via the submandibular sons was considered the titer. The largest were GBS.34 The presence of at least 1 route at weeks 4, 8, 11, and 40. Serum dilution tested was 1:100,000. Animals GBS colony on a plate was counted as a was isolated using serum separator tubes were housed at the University of Iowa positive plate. Results of each vaccine (Becton Dickinson, Lincoln Park, NJ) and all experiments were performed ac- group were compared against the group per the manufacturer’s recommenda- cording to Institutional Animal Care and of mice receiving empty microspheres tions, frozen, and stored at –80°C. Con- Use Committee–approved protocols. (75:25, 0 ␮g). currently, vaginal washes were obtained by pipetting 100 ␮L of PBS 40-50 times. Vaginal colonization studies Pup protection studies Washes were frozen and stored at –80°C. At 12 weeks, 1 ϫ 106 colony-forming At 48 hours of age, pups were injected Colormetric enzyme linked immu- units of GBS serotypes Ia, III, and V with a 70% lethal dose of GBS serotype V nosorbant assay (ELISA) was used to (ATCC 12400, 12403, and 700046, re- (1.8 ϫ 107 colony-forming units) intra- measure the C5a peptidase–specific im- spectively, American Type Culture Col- peritoneally. These pups were born to munoglobulin (Ig) G and IgA antibody lection, Manassas, VA) were pipetted dams who received the 50:50 30 ␮g for-

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used. Statistical signiﬁcance was desig- FIGURE 2 ␣ ϭ Release proﬁle of antigen nated at 0.05. All statistical analyses were performed with SigmaStat 11 software (Systat Software, Inc, Point Rich- mond, CA).

RESULTS Microsphere formulation Both the C5a-loaded PLGA (50:50, 0.41 dL/g) microspheres and the C5a-loaded PLGA (75:25, 0.51 dL/g) microspheres exhibited a mean particle size of 3-4 ␮m (Figure 1, A and B). There was no significant difference in the particle size of both the formulations. In terms of entrapment efficiency, the amount of C5a peptidase loaded per mil- The release profiles demonstrate an initial burst phase followed by a longer release of antigen. ligram of PLGA (50:50, 0.41 dL/g) was 12 ␮ Santillan. C5a peptidase-based group B streptococci vaccines. Am J Obstet Gynecol 2011. g, whereas that of PLGA (75:25, 0.51 dL/g) was 16 ␮g. The overall entrapment efficiency was about 65%. mulation of the vaccine. Pup survival groups. For continuous variables, a Stu- C5a peptidase release profile was assessed at 48 hours after injection. dent t test or Mann Whitney U test was from PLGA microspheres Statistical analysis utilized for comparisons. For differences Both the 50:50 and 75:25 PLGA formu- Descriptive statistics described and com- in proportions of dichotomous vari- lations demonstrated a similar biphasic 2 pared the characteristics of our study ables, a ␹ or a Fisher’s exact test was burst rate in vitro. There was an initial burst in the first 2 days that resulted in the release of approximately 35-40% of the FIGURE 3 C5a peptidase. The 50:50 formulation is Average C5a peptidase–specific titers 95% degraded by day 25, whereas the 75:25 C5a peptidase IgG C5a peptidase IgA formulation is approximately 60% at that point (Figure 2). By day 30, the micro- 100000 100000 spheres were completely degraded (Figure 80000 80000 1, C and D). 60000 60000 40000 serum 40000 Immune response 20000 50:50 30ug 20000 To compare the strength and duration of 50:50 30ug 0 75:25 30ug 0 75:25 30ug C5a peptidase–specific IgG and IgA im- 4 weeks 75:25 10ug 8 weeks 4 weeks 75:25 10ug 11 8 weeks 40 11 mune responses of mice vaccinated with weeks 40 weeks weeks weeks various microsphere formulations and

Titers 1:Y doses of encapsulated C5a peptidase, an 100000 100000 ELISA was performed on serum and vag- 80000 80000 inal mucosal samples. When average ti- 60000 60000 ters were calculated regardless of route of Vaginal 40000 40000 administration, the 30 ␮g doses of the washes 20000 20000 75:25 and 50:50 formulations elicited the 50:50 30ug 50:50 30ug 0 75:25 30ug 0 75:25 30ug highest titers at 40 weeks for C5a pepti- 75:25 10ug 4 weeks 4 weeks 75:25 10ug 8 weeks 8 weeks 11 11 dase–specific IgG responses in serum 40 40 weeks weeks weeks weeks and in vaginal washes (Figure 3). The Serum and vaginal wash samples were obtained at weeks 4, 8, 11, and 40. The blue bars represent titers 75:25 30 ␮g dose led to the highest C5a from all mice immunized with 75:25 10 ␮g. Purple bars represent all mice immunized with 75:25 30 ␮g peptidase–specific IgA titer at 40 weeks doses. Green bars represent the titers of mice receiving the 50:50 30 ␮g vaccine. These results are the in serum and vaginal washes. average titers for the vaccine doses and are irrespective of route of administration of the vaccine. When we analyzed the C5a peptidase– Santillan. C5a peptidase-based group B streptococci vaccines. Am J Obstet Gynecol 2011. specific antibody titers with respect to route of vaccine administration, we

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found that same results were achieved for mice vaccinated via the intramuscu- TABLE 1 lar route (Table 1). Titers of 1:100,000 Intramuscular vaccine administration titers were achieved by both 75:25 30 ␮g and Variable 4 weeks, n 8 weeks, n 11 weeks, n 40 weeks, n ␮ 50:50 30 g PLGA microspheres by 4 Serum C5a-IgG weeks and were sustained through 40 ...... 75:25 10 ␮g 1000 (27) 100,000 (27) 100,000 (27) 1000 (11) weeks for serum C5 peptidase–specific ...... ␮ 75:25 30 ␮g 100,000 (28) 100,000 (26) 100,000 (25) 100,000 (4) IgG, whereas the 75:25 10 g titer ...... dropped to 1000 at 40 weeks. 50:50 30 ␮g 100,000 (28) 100,000 (27) 100,000 (27) 100,000 (6) ...... In serum, the C5a-IgA response was Serum C5a-IgA not detectable for the 75:25 30 ␮g dose ...... 75:25 10 ␮g 100,000 (27) 100,000 (27) 100,000 (27) 0 (11) until 8 weeks; by 11 weeks both the 75:25 ...... ␮ ␮ 75:25 30 ␮g 0 (28) 10,000 (26) 100,000 (25) 10,000 (4) 30 g and 50:50 30 g doses were ...... 1:100,000. By 40 weeks these serum titers 50:50 30 ␮g 0 (28) 0 (27) 100,000 (27) 10,000 (6) ␮ ...... were reduced to 1:10,000 for the 30 g Vaginal C5-IgG doses and were not detectable for the 10 ...... 75:25 10 ␮g 100,000 (27) 100,000 (27) 100,000 (27) 1000 (11) ␮g dose. The vaginal C5a-specific titers ...... 75:25 30 ␮g 100,000 (28) 10,000 (26) 100 (25) 100,000 (4) were inconsistent...... The vaginal washes of mice inoculated 50:50 30 ␮g 100,000 (28) 10,000 (27) 10,000 (27) 100,000 (6) ␮ ␮ ...... with the 50:50 30 g and 75:25 30 g vac- Vaginal C5a-IgA cines had C5a-IgG titers of 1:100,000 at ...... 75:25 10 ␮g 100,000 (27) 100,000 (27) 0 (27) 0 (11) 40 weeks despite titers of 1:10,000 and ...... 75:25 30 ␮g 10,000 (28) 100,000 (26) 100 (25) 100,000 (4) 1:100 at 11 weeks, respectively. C5a pep- ...... tidase–specific IgA antibodies were not 50:50 30 ␮g 1000 (28) 0 (27) 10,000 (27) 10,000 (6) ...... detectable after 8 weeks in mice vacci- Titers are shown as 1:Y (Y represents the dilution). A Student t test was used to determine the significant titer. The largest nated with 75:25 10 ␮g. However, at 40 dilution tested that was significantly different from mice receiving 75:25 0 ␮g is the reported titer. weeks, the vaginal washes had titers of Santillan. C5a peptidase-based group B streptococci vaccines. Am J Obstet Gynecol 2011. 1:100,000 and 1:10,000 with 75:25 30 ␮g and 50:50 30 ␮g, respectively. and inserted 1 ϫ 106 colony-forming were colonized demonstrated colonies Intranasal administration resulted in units of serotypes Ia, III, and V (n ϭ 5 per that were too numerous to count, often more variable titers (Table 2). Each vac- serotype per vaccine group). even in the smallest dilution plated, cine was able to generate a 1:100,000 The results were compared against whereas in mice that were protected, the C5a-IgG titer in serum by 4 weeks and those from mice vaccinated with empty plates showed no evidence of GBS sustain this titer through 11 weeks. How- microspheres. Not all mice receiving growth. Without being able to count col- ever, all of the titers dropped by 40 empty microspheres were colonized onies, it is impossible to perform statis- weeks. Also in serum, the C5a-IgA titer (Table 3). Without regard to which en- tical analysis based on the number of reached the maximum dilution tested at capsulated vaccine the mice received, the GBS colonies. 1:100,000 for all vaccines at week 8 and mice that received a vaccine were signif- Because we did not observe any signifi- then dropped to 1:10,000 by week 11. In icantly protected against colonization cant protection against serotype V in the the vaginal washes, C5a-IgG titers of (27 of 90 positive) in comparison with vaginal colonization studies, we were in- 1:100,000 were measured for each of the mice that received the empty micro- terested in whether the IgG antibodies that vaccine formulations at weeks 8 and 11, spheres (18 of 30 positive) (P ϭ .005). cross the placenta in combination with any whereas the maximal 1:100,000 C5a-IgA The intramuscular 30 ␮g doses of the IgA antibodies in the dam’s milk were able titer was found only at week 8 in the vag- 75:25 and 50:50 formulations were able to afford protection to pups. Because we inal wash samples. to prevent colonization by serotypes 1a achieved high C5a-IgG titers in serum, we and III (0 of 5 positive) (Figure 4). When hypothesized that the C5a peptidase IgG Protection by immunization all serotypes were considered together, the antibodies that cross the placenta would be In previous work, we demonstrated that intranasal 30 ␮g doses of the 75:25 and able to better protect pups than the weaker the 50:50 30 ␮g dose administered intra- 50:50 PLGA microsphere formulations mucosal immune response that would be nasally was able to prevent GBS coloni- trended toward significantly inhibited vag- necessary to prevent vaginal colonization. zation of the vaginal vault by serotype inal colonization in mice (P ϭ .06). In pup protection studies, pups were in- III.28 We hypothesized that the 30 ␮g We also tested mice that were vacci- jected intraperitoneally with a 70% lethal doses of the 75:25 and 50:50 PLGA mi- nated with unencapsulated antigen, and dose of serotype V. crosphere formulations would be able to this vaccine did not significantly impede In comparing survival of pups between protect against multiple serotypes of vaginal colonization (Table 3). Of note, nonvaccinated (3 of 10) and vaccinated GBS. We used 15 mice from each group most blood agar plates from mice that mice (4 of 5), we observed a notable higher

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COMMENT TABLE 2 Intranasal vaccine administration titers In our previous work, we demonstrated that by encapsulating C5a peptidase Variable 4 weeks, n 8 weeks, n 11 weeks, n 40 weeks, n within microspheres composed of PLGA, we Serum C5a-IgG were able to elicit antibody responses in ...... 75:25 10 ␮g 100,000 (29) 100,000 (29) 100,000 (26) 100 (10) serum and in the vagina of mice against ...... GBS and that these responses were suffi- 75:25 30 ␮g 100,000 (28) 100,000 (27) 100,000 (25) 100 (4) ...... cient to protect against vaginal coloniza- 50:50 30 ␮g 100,000 (29) 100,000 (28) 100,000 (27) 1000 (4) 28 ...... tion by serotype III. This protection Serum C5a-IgA was also conferred to pups of vaccinated ...... 75:25 10 ␮g 10,000 (29) 100,000 (29) 10,000 (26) 100,000 (10) dams. In this study, our primary objec- ...... tive was to determine the duration of the 75:25 30 ␮g 100,000 (28) 100,000 (27) 10,000 (25) 100,000 (4) ...... immune response to various vaccine for- 50:50 30 ␮g 0 (29) 100,000 (28) 10,000 (27) 0 (4) ...... mulations and doses. In addition, our Vaginal C5a-IgG ...... secondary goal was to determine 75:25 10 ␮g 10,000 (29) 100,000 (29) 100,000 (26) 10,000 (10) whether this univalent vaccine was able ...... 75:25 30 ␮g 100,000 (28) 100,000 (27) 100,000 (25) 0 (4) to protect against vaginal colonization ...... by multiple serotypes of GBS. Further- 50:50 30 ␮g 10,000 (29) 100,000 (28) 100,000 (27) 10,000 (4) ...... more, we wanted to compare whether Vaginal C5a-IgA ...... different formulations of the PLGA mi- 75:25 10 ␮g 0 (29) 100,000 (29) 10,000 (26) 10,000 (10) crospheres vaccine (75:25 and 50:50) ...... ␮ 75:25 30 ␮g 100,000 (28) 100,000 (27) 10,000 (25) 0 (4) and different doses (0, 10, and 30 g) ...... were more effective in protecting from 50:50 30 ␮g 0 (29) 100,000 (28) 10,000 (27) 100 (4) ...... vaginal colonization by GBS. Titers are shown as 1:Y (Y represents the dilution). A Student t test was used to determine the significant titer. The largest dilution tested that was significantly different from mice receiving 75:25 0 ␮g is the reported titer. We achieved our main objective by de- Santillan. C5a peptidase-based group B streptococci vaccines. Am J Obstet Gynecol 2011. termining whether there are differences in titers between vaccine formulations and doses. We have demonstrated that, survival for pups born to vaccinated dams bred within a similar time frame and by in general, the 30 ␮g doses of the 75:25 (30% vs 80%). Although this was not a sta- small litter sizes. However, these results and 50:50 PLGA microsphere formula- ϭ tistically significant difference (P .11) in were similar to the significant improve- tions generate the highest and most sus- a 2-tailed Fisher’s exact test, we were lim- ment in survival that we previously re- 28 tained C5a peptidase–specific IgG and ited by the number of female mice that ported with serotype III. IgA antibody responses. At weeks 4, 8, and 11, we did not detect any significant TABLE 3 differences in the IgG or IgA titers be- Vaginal colonization tween the PLGA 75:25 and 50:50 micro- ␮ Mice colonized with GBS, n sphere formulations at the 30 g dose. At week 40, there were also no differ- Vaccine formulation Dose Route Ia (P value) III (P value) V (P value) Total (P value) ences in the C5a peptidase–specific IgG re- 75:25 0 ␮g IM 3/5 (n/a) 2/5 (n/a) 3/5 (n/a) 8/15 (n/a) ...... sponses. The titers were higher at week 40 75:25 10 ␮g IM 3/5 (1) 2/5 (1) 2/5 (.58) 7/15 (1) for the 75:25 30 ␮g vaccine compared with ...... ␮ 75:25 30 ␮g IM 0/5 (.16) 0/5 (.16) 3/5 (1) 3/15 (.07) the 50:50 30 g vaccine. Because we mea- ...... sured similar particle sizes and we adjusted 50:50 30 ␮g IM 0/5 (.16) 0/5 (.16) 4/5 (.54) 4/15 (.27) ...... our dosing based on the actual amount of Free C5a peptidase 30 ␮g IM 3/5 (1) 5/5 (.08) 3/5 (1) 11/15 (.45) ...... protein encapsulated, these factors cannot 75:25 0 ␮g IN 4/5 (n/a) 2/5 (n/a) 4/5 (n/a) 10/15 (n/a) account for this difference. For additional ...... 75:25 10 ␮g IN 1/5 (.21) 2/5 (1) 2/5 (.29) 5/15 (.08) consistency, the same volume was used to ...... administer each vaccine. The slower deg- 75:25 30 ␮g IN 1/5 (.21) 1/5 (1) 2/5 (.29) 4/15 (.06) ...... radation rate of PLGA 75:25 in compari- 50:50 30 ␮g IN 2/5 (.29) 1/5 (1) 1/5 (.21) 4/15 (.06) ...... son with PLGA 50:50 may affect the IgA Free C5a peptidase 30 ␮g IN 3/5 (.58) 3/5 (1) 4/5 (1) 10/15 (1) titers over a longer time period...... P values represent 2-tailed Fisher exact comparing the 75:25 0 ␮g dose of each route of administration against each of the Furthermore, we found that mice re- other vaccination groups. ceiving the encapsulated C5a peptidase GBS, group B streptococcus; IM, intramuscular; IN, intranasal; n/a, not available. ␮ ␮ Santillan. C5a peptidase-based group B streptococci vaccines. Am J Obstet Gynecol 2011. (including 75:25 10 g, 75:25 30 g, and 50:50 30 ␮g) were significantly protected

249.e6 American Journal of Obstetrics & Gynecology SEPTEMBER 2011 www.AJOG.org SMFM Papers from vaginal colonization compared FIGURE 4 with mice that received empty micro- Vaginal GBS serotype Ia challenge spheres (75:25 0 ␮g). It is a strength of this study that we used very stringent guidelines for this experiment by identi- fying any number of GBS colonies as a positive plate. We could not statistically compare the number of colonies per plate because the number of colonies was innumerable on many of the plates from mice receiving the empty microspheres. Although the vaginal challenge studies were able to demonstrate trends toward significance and promise for this vaccine to protect against serotypes 1a and III, the number of mice used per group was too small to detect any significant differences between vaccination subgroups. These results did indicate that this en- Prior to challenge at 12 weeks, vaginas of mice were washed and samples were assessed for GBS capsulation approach and antigen have growth (not detected). Forty-eight hours after infection with serotype Ia, the vagina of each mouse was merit for use as a GBS vaccine and warrant washed and samples were grown to assess growth of beta-hemolytic GBS colonies. future studies to further expand on their GBS, group B streptococcus. ability to protect against the 10 GBS sero- Santillan. C5a peptidase-based group B streptococci vaccines. Am J Obstet Gynecol 2011. types. Because we have now identified the optimal vaccine formulation and dose, we after the last booster dose.36-39 In con- against serotype V. We may also try alter- will be able to utilize larger groups of mice trast, we administered our last booster nate routes of administration such as vag- for subgroup analysis in future studies. dose at week 8, and our last titer was inal vaccination. Intravaginal immuniza- We found more fluctuations in the anti- measured at week 40, more than 220 tion has recently been shown to elicit body responses in the intranasal group days later. Knowledge of the duration of higher IgG antibody responses than other than in the intramuscular group. These the immune response will be especially vaccination strategies.43 observations in the titers of vaginal washes important in knowing how often women The results presented here contribute likely are due to difficulties in equally dis- greatly to the field of GBS vaccine devel- tributing the mucus between dilutions in will need to be vaccinated to at least be opment as well as the use of PLGA mi- the ELISA as well to variations in mucosal protected throughout pregnancy. crosphere–based vaccines for mucosal IgG and IgA production with the mouse We and others have had difficulty pro- 39-41 immune responses. We are continuing estrous cycle.35 We may have experienced tecting mice from serotype V. Sero- to pursue the development of a GBS vac- less variable results if we had isolated the type V has also been shown to induce low cine and to better understand the muco- vagina and extracted the antibodies from levels of antibodies in older human pa- 42 sal immune response to our vaccine in the tissue.36 Because we used the mice to tients. Our future studies will work to addition to methods of evasion by differ- measure titers at several time points, it was determine the mechanisms of serotype V ent GBS serotypes. f not feasible for us to have a large enough to evade an immune response. Although sample group to kill mice at each time we did not observe significant protection from vaginal colonization, the systemic point for vaginal tissue. These variations REFERENCES IgG response passed from the dam to the may have been minimized by one of the 1. Kasper DL, Baker CJ. 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