Objective
The purpose was to examine in mice the efficacy of various polymeric-encapsulated C5a peptidase vaccine formulations in eliciting a long-term immune response and preventing group B streptococcus (GBS) infection.
Study Design
C5a peptidase was encapsulated in semipermeable microspheres of poly(lactide-coglycolide) (PLGA). Female ICR mice were immunized with 0, 10, or 30 μg of encapsulated C5a peptidase within 2 different formulations of PLGA polymers. Booster doses were given at weeks 4 and 8. Antibody responses were measured by enzyme-linked immunosorbent assay at weeks 4, 8, 11, and 40. Vaginal challenges with GBS types 1a, III, and V were performed at week 12.
Results
Thirty microgram doses of the 75:25 and 50:50 PLGA formulations generate the highest and most sustained C5a peptidase–specific immune responses. Mice that received encapsulated C5a peptidase were significantly protected from vaginal colonization compared with mice that received empty microspheres.
Conclusion
Encapsulated C5a peptidase elicited significant immune responses and protection against a GBS challenge. C5a peptidase microsphere encapsulation has potential as a GBS vaccine.
Group B streptococci are Gram-positive diplococci that produce polysaccharide capsules. There are 10 different Group B Streptococcus (GBS) serotypes, based on the antigenic variation of the capsular polysaccharides. These serotypes are Ia, Ib, and II through IX. A recent international study most commonly isolated serotypes III and V from patients. GBS can colonize the intestinal and urogenital tract of healthy adults without causing any complications. However, GBS infections can cause serious problems in pregnancy.
In 1985, the Institute of Medicine from the National Academies of Sciences indicated that a vaccine for GBS should be a high priority. In a 1999 report, they again listed a GBS vaccine for high-risk adults and pregnant women in the top tier for vaccine development. They suggested the vaccine would have the greatest effect if given as part of routine prenatal care for all women in their first pregnancy. Maternal complications of GBS infections in pregnancy include urinary tract infections, chorioamnionitis, postpartum wound infection, pyelonephritis, postpartum endometritis, and sepsis. For the neonate, preterm premature rupture of membranes (PPROM), neonatal pneumonia, sepsis, meningitis, or death can occur as a consequence of GBS.
Because GBS infection continues to be a serious concern during pregnancy, the updated guidelines from the Centers for Disease Control and Prevention continue to recommend universal culture-based screening of all pregnant women at 35-37 weeks’ gestation. Currently, if a pregnant woman screens positive for GBS, she will receive intravenous antibiotics during labor to prevent early-onset GBS disease in the child, which can include sepsis and death. Recent studies demonstrate that up to 24% of all pregnant women receive antibiotic prophylaxis for GBS. Between 50% and 75% of neonates exposed through the birth canal of GBS-infected mothers will be colonized.
Many shortcomings exist in the current therapy of antibiotic prophylaxis. These shortcomings are especially evident in cases in which a woman has a lack of prenatal care, delivers before being screened, delivers before the culture results return, has a rapid labor and does not finish receiving all of the antibiotic dose(s), or is allergic to antibiotics. In addition, the development of antibiotic resistance is an increasing problem.
Penicillin-tolerant strains of GBS have been identified, and resistance to other antibiotics has been documented. A recent study found 91% of strains isolated were resistant to erythromycin. The consequences of a GBS infection in pregnancy require that treatment be given. However, this large-scale use of antibiotics is contributing to the development of antibiotic resistance. Without prevention strategies, such as vaccination, our first-line antibiotic therapies are going to become useless against GBS. Most importantly, this traditional approach does not prevent preterm delivery or PPROM or protect against late-onset disease caused by GBS infection. A GBS vaccine could overcome these pitfalls.
Development of a vaccine for GBS has been hindered by several factors. First, there are 10 serotypes of GBS that are based on antigenic variation of the capsular polysaccharides. Purified capsular polysaccharides, without adjuvants, have elicited weak immune responses in vaccines, and a multivalent vaccine would be necessary to provide protection against the multiple GBS serotypes because each polysaccharide can target only the serotype from which it was derived. Furthermore, polysaccharide-based vaccines were unable to elicit significant mucosal immune responses. A mucosal immune response would be critical in completely eliminating maternal GBS colonization. Eliminating colonization by all GBS serotypes would give the best chance of preventing the infection from being vertically passed to the child during the birthing process.
In the current study, we further evaluated the use of streptococcal C5a peptidase as the vaccine antigen. C5a peptidase is a highly conserved multifunctional surface protein that is expressed on the surface of all serotypes of both group A streptococcus (GAS) and group B streptococcus tested. C5a peptidase (ScpB) expressed by GBS is 98% identical in sequence to that expressed by GAS. Structurally, C5a peptidase contains 5 domains including a subtisilin-like protease domain, a protease-associated domain, and 3 fibronectin type III domains. The enzymatic activity of the peptidase is highly specific for C5a, cleaving the chemotaxin at its polymorphonuclear leukocyte binding site.
Recent evidence also suggests that C5a peptidase may bind fibronectin to promote cellular invasion. Cheng et al demonstrated that ScpB increased the immunogenicity of a GBS type III polysaccharide vaccine in mice when the 2 were coupled. In addition, the investigators found that ScpB was also found to induce the production of GBS serotype-independent antibodies.
We have previously shown that encapsulating C5a peptidase within microspheres composed of a copolymer of lactic and glycolic acids, poly(lactide-co-glycolide) (PLGA), was able to induce systemic and mucosal immune response in mice. Furthermore, this vaccine provided protection in mice against GBS serotype III in vaginal and pup challenge studies. The PLGA polymer–based microspheres are able to act as an adjuvant to the vaccine and are safe for use in humans and has been used for many years in resorbable sutures, bone plates, and commercial depot drug delivery formulations. The antigen release profile by PLGA microsphere–based vaccines is largely dependent on the lactide/glycolide ratio. Copolymers with a higher lactide/glycolide ratio have a longer degradation profile because lactic acid is hydrophobic. PLGA microspheres have been studied for use in numerous vaccines.
We hypothesized that encapsulation of C5a peptidase within PLGA microspheres would induce specific systemic and mucosal immune responses that would afford protection against multiple serotypes of GBS. We further hypothesized that differences in antigen doses (0, 10, and 30 μg) and PLGA microsphere lactide-glycolide formulations (75:25 and 50:50) would affect these immune responses and the ability of vaccinated mice to prevent GBS colonization and to pass GBS protection to pups of vaccinated dams.
Materials and Methods
C5a peptidase encapsulation
C5a peptidase, guanosine monophosphate prepared and greater than 98-99% pure, was generously provided by Pfizer (Groton, CT). The C5a peptidase was microencapsulated in PLGA microspheres. PLGA (50:50, inherent viscosity, 0.4 dL/g) and PLGA (75:25, inherent viscosity, 0.51 dL/g) were purchased from Lactel Absorbable Polymers (Cupertino, CA). Polyvinyl alcohol (PVA; 87-89% hydrolyzed, molecular weight 30-67,000 Da) was purchased from Sigma-Aldrich (St Louis, MO).
Encapsulation was done using a water-in-oil-in-water (w/o/w) double-emulsion technique as described previously. Briefly, the internal aqueous phase consisted of 3.6 mg peptidase equivalent to 6.6 mg lyophilized powder C5a peptidase solubilized in 500 μL of 1% (weight/volume) aqueous solution of PVA as a surfactant. This was emulsified into an oil phase containing 200 mg of PLGA 50:50 or PLGA 75:25 dissolved in 5 mL dichloromethane (DCM) using a microtip probe sonicator. This primary water/oil emulsion was then poured into 50 mL of external aqueous phase containing 1% (weight/volume) PVA as a surfactant and rapidly homogenized using a high-speed homogenizer at 9500 rpm for 30 seconds to form the secondary w/o/w emulsion. Stirring was then continued using a magnetic stirrer until complete evaporation of DCM. The microspheres were collected by centrifugation at 5000 × g for 10 minutes, washed 3 times with deionized water, and lyophilized overnight.
To quantitate encapsulation efficiency of protein for dosing purposes, 30 mg of lyophilized PLGA microspheres containing C5a peptidase were dissolved in 3.0 mL of 1 M NaOH containing 5.0% (weight/volume) sodium dodecyl sulfate and incubated for 24 hours at room temperature. After centrifugation (4000 × g for 10 minutes at room temperature), the supernatant was assayed for protein concentration using the bicinchonic acid assay (Thermo Scientific, Swedesboro, NJ) following the manufacturer’s protocol. All the measurements were done in triplicate.
In vitro release profile
Thirty to 40 mg of C5a-peptidase–loaded PLGA microspheres were incubated in 2-3 mL phosphate buffered saline (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 determine C5a content. The sedimented microspheres were dispersed in 200 μL 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.
Administration of vaccine
Female ICR mice (Charles River Breeding Laboratories, Portage, MI) 5-7 weeks old were vaccinated either through an intramuscular or intranasal route. For all doses of the intramuscular vaccine, the vaccine was administered in 100 μL into the right upper leg. For all doses of the intranasal administration, 50 μL of vaccine was administered into each nostril (100 μL total volume). Booster doses were administered in the same manner as the initial vaccination and were given at weeks 4 and 8. For the pup challenge experiment, the vaccine was administered intranasally and boosters were given at weeks 2 and 4.
Determination of immune response
Mice were bled via the submandibular route at weeks 4, 8, 11, and 40. Serum was isolated using serum separator tubes (Becton Dickinson, Lincoln Park, NJ) per the manufacturer’s recommendations, frozen, and stored at –80°C. Concurrently, vaginal washes were obtained by pipetting 100 μL of PBS 40-50 times. Washes were frozen and stored at –80°C. Colormetric enzyme linked immunosorbant assay (ELISA) was used to measure the C5a peptidase–specific immunoglobulin (Ig) G and IgA antibody responses in serum and vaginal washes as described previously.
Samples producing a significant difference when further diluted are considered to have a larger immune response than samples in which a significant difference was observed in only smaller dilutions. The optical density (OD) 405 reading for each dilution was compared between each vaccine formulation group and the empty microsphere control. The largest dilution that remained statistically significant in the OD 405 comparisons was considered the titer. The largest dilution tested was 1:100,000. Animals were housed at the University of Iowa and all experiments were performed according to Institutional Animal Care and Use Committee–approved protocols.
Vaginal colonization studies
At 12 weeks, 1 × 10 6 colony-forming units of GBS serotypes Ia, III, and V (ATCC 12400, 12403, and 700046, respectively, American Type Culture Collection, Manassas, VA) were pipetted into the vagina of 5 mice of each vaccination group. After 48 hours, vaginal washings were obtained and 2 dilutions were plated on blood agar plates. Plates were incubated for 24 hours at 37°C with 5% CO 2 . After 24 hours, plates were assessed for growth of GBS. If no colonies were evident, plates were incubated for another 24 hours and growth of GBS colonies was again assessed. Cyclic adenosine monophosphate tests and Gram staining were used to determine whether questionable beta-hemolytic colonies were GBS. The presence of at least 1 GBS colony on a plate was counted as a positive plate. Results of each vaccine group were compared against the group of mice receiving empty microspheres (75:25, 0 μg).
Pup protection studies
At 48 hours of age, pups were injected with a 70% lethal dose of GBS serotype V (1.8 × 10 7 colony-forming units) intraperitoneally. These pups were born to dams who received the 50:50 30 μg formulation of the vaccine. Pup survival was assessed at 48 hours after injection.
Statistical analysis
Descriptive statistics described and compared the characteristics of our study groups. For continuous variables, a Student t test or Mann Whitney U test was utilized for comparisons. For differences in proportions of dichotomous variables, a χ 2 or a Fisher’s exact test was used. Statistical significance was designated at α = 0.05. All statistical analyses were performed with SigmaStat 11 software (Systat Software, Inc, Point Richmond, CA).
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