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Journal of Infectious Diseases and Therapeutics, 2015, 3, 8-20

Assessment of the Bordetella pertussis BpCNIC0311 Strain as a Producing Strain of Genetically Detoxified Toxoid (PTg), Filamentous Hemagglutinin (FHA) and Type 2 Pertactin (Prn2) Diógenes Quintana-Vázquez*, Maité Delgado, Gilda Lemos, Edelgis Coizeau, Yassel Ramos, Yanet Támbara, Luis Javier Gonzales, Gerardo E. Guillén and Anabel Álvarez Center for Genetic Engineering and Biotechnology, CIGB Ave. 31 e/ 158 y 190, Cubanacán, Playa, P.O. Box 11600, Havana, Cuba Abstract: Pertussis is a vaccine preventable disease caused by the Gram-negative bacterium Bordetella pertussis (B. pertussis). Acellular vaccines as regard to whole cell vaccines are less reactogenic and very similar in efficacy. An increased incidence of B. pertussis infections in countries where acellular vaccines are widely used has questioned their effectiveness. In fact, the circulating strains differ from vaccine strains due to variations in protein sequences associated to the evolutionary process of B. pertussis. Furthermore, vaccine antigens change throughout the manufacturing process caused by chemical treatments for toxin inactivation. Both aspects should be considered when looking into the loss of efficacy of acellular vaccines. In this paper we study the performance of a new producing strain, BPCNIC0311. The strain expresses the genetically inactivated pertussis toxoid (PTg) and type 2 pertactin (Prn2), the most frequently occurring pertactin variant in clinical isolates. As a result, the new strain is able to grow stably at high cell density in a chemically defined culture medium. Similarly, the strain stably expresses high levels of PTg, FHA and Prn2 antigens which allow for suitable yields after purification. High purity and adequate physicochemical features of the antigens were obtained. The three components formulated in alum, as a stand-alone pertussis preparation, were highly immunogenic and induced a protective immune response in mice against the reference strain BP18323. It is concluded that the new strain can be used to obtain sufficient amounts of PTg, FHA and Prn2 for preclinical studies of an improved-updated acellular vaccine against whooping cough.

Keywords: Bordetella pertussis, pertussis strains, Whooping Cough/prevention & control, Immunoglobulin IgG/ IgG2a / IgG2b, Protein purification, Pertussis Vaccine, Acellular vaccine, Vaccination, Mice, Inbred BALB C/ outbred OF1. INTRODUCTION Pertussis or whooping cough is a highly infectious human disease caused by the Gram-negative bacterium Bordetella pertussis (B. pertussis). This often deadly disease was rapidly tackled in the United States (1940s) with stand-alone pertussis inactivated whole cell (wP) vaccines. Soon afterwards (1950’s), it was combined with diphtheria and tetanus toxoid to produce DTP. The combined DTP vaccines were decisive in reducing pertussis mortality and morbidity [1]. Because of safety concerns, acellular pertussis (aP) vaccines were developed in the 1980’s in Japan and also further marketed as combined DTaP vaccines. This generation of aP vaccines contained two pertussis components: the purified pertussis toxoid (PT) and filamentous hemagglutinin (FHA). Later, vaccine efficacy studies suggested that vaccines that additionally contained pertactin (Prn) and/or fimbrial antigens were more effective [2]. Consequently, another generation of vaccines containing three or five pertussis components ( three) was available for vaccination programs. *Address correspondence to this author at the Center for Genetic Engineering and Biotechnology, CIGB Ave. 31 e/ 158 y 190, Cubanacán, Playa, PO box 11600, Havana, Cuba; Tel: 5372504547; Fax: 5372504494; E-mail: [email protected] E-ISSN: 2310-9386/15

The aP vaccines have fewer adverse effects than wP vaccines whether in the primary series or in booster doses. In terms of efficacy, multi-component ( three) aP vaccines are 84-85% effective in preventing whooping cough in children, although their efficacy may be less than the wP vaccines of maximum efficacy [3]. Safety issues on wP have led to adverse public perceptions that limit its use. Severe injury and fatality are sometimes recorded in vulnerable infants after wP vaccination; these events cannot, however, be attributed to the non compliance of quality standards of the vaccine [4]. Moreover, since Pertussis is not a lifethreatening illness in adolescents and adults, the use of wP vaccines is avoided in these cases. Therefore, it is important for new developments to have vaccine safety as the benchmark. Whereas acellular vaccines promised to be effective and less reactogenic, the increased incidence of Pertussis in countries with high vaccination coverage has questioned its effectiveness [5]. This is related to the fact that B. pertussis is a pathogen in an ongoing process of evolution at the expense of genome reduction and antigen variation [6, 7]. On the other hand, regulatory and proprietary issues have made © 2015 Pharma Publisher

Assessment of the Bordetella pertussis BpCNIC0311 Strain

vaccine manufacturing a quiescent process. Consequently, vaccines are currently made from strains that were isolated several decades ago. In the Netherlands, as well as in other countries, at the time the aP vaccines were introduced, the antigens found in the producer strains were already different from the circulating isolates [8]. The significance of this antigenic discrepancy has been evaluated in the mouse model by different laboratories with results suggesting a relationship between sequence variation and the loss of vaccine functionality [9-11]. This antigenic gap increases when the structural surfaces of antigens are modified by chemical agents such as formaldehyde, glutaraldehyde or hydrogen peroxide during toxin inactivation in production [12]. Hence, both viewpoints should be considered in order to obtain improved pertussis vaccine candidates. In this sense, the pertussis toxin was rendered inactive through recombinant DNA techniques [13]. The genetically detoxified/inactive pertussis toxin (PTg) retained its native structure and consequently induced a superior immune response (in quality and quantity) than the toxin inactivated by other procedures [12]. PTg was safe in the combined DTaP vaccine; it was effective in humans (84%) at a PTg dosage five times lower than that used for chemically detoxified PT and had a longer lasting protection pattern [14]. However, this promising new generation of acellular vaccines was hampered for reasons of intellectual property and withdrawn from the market decades ago. At present, no vaccines can benefit from PTg and similarly, no vaccines contain the Prn variant present in strains isolated from patients. In this paper we studied the performance of a new strain, BPCNIC0311, as an antigen producer. The strain expresses the genetically inactivated pertussis toxoid (PTg) and type 2 pertactin (Prn2), which is the Prn variant most frequently occurring in clinical isolates. The strain was studied in regard to: growth in a chemically defined medium, expression levels, yields and physicochemical quality of antigens after purification using the reported methods. The biological activity and ability of these molecules to confer protective immunity were also evaluated. MATERIALS AND METHODS Bacterial Strain The BpCNIC0311 B. pertussis strain was used to obtain PTg, FHA and Prn2 antigens. The strain was

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obtained in the Laboratory of Molecular Biology and Biotechnology of the National Center for Scientific Research as part of a collaboration service agreement and delivered to the Center for Genetic Engineering and Biotechnology both in Havana, Cuba. The strain is a Bp165 derivative that was genetically modified to produce the pertussis toxoid PTg (ptxA2, 9K/129G) under the regulation of the ptxP1 promoter and the pertactin 2 protein (prn2). The fhaB gene encoding filamentous hemagglutinin (FHA) remained as a wild type. The genetic and microbiological characterization was included in the technical dossier sent to the CIGB. The strain was received in a Stainer Scholte medium (SS) [15] with 30% glycerol and stored at -70°C. Culture Medium The THIJS medium described by Thalen et al [16] was used to grow the BpCNIC0311. One liter of the medium contained 1.87 g of monosodium glutamate, 1.83 g of 90% L-Lactate, 0.24 g of L-proline, 3.3 g of NaCl, 0.1 g of NH4Cl, 0.5 g of KH2PO4, 0.5 g of KCl, 0.1 g of MgCl2 6H2O, 1.525 g of Tris base, 0.01 g of FeSO4 H2O, 0.02 g of CaCl2 2H2O, 0.04 g of Lcysteine hydrochloride, 0.1 g of glutathione (reduced), 0.02 g of L-ascorbic acid, and 0.004 g of nicotinic acid. The monosodium glutamate, L-Lactate, NaCl, KH2PO4, KCl, MgCl2 6H2O Tris base, and CaCl2 2H2O were prepared in a basal formulation and filter sterilized. The remaining medium (supplement) was prepared at a one-hundredfold concentration and filtersterilized. The final pH of the complete medium was adjusted to 7.3. All chemicals were reagents purchased from Applichem GmbH, Germany. Frozen Seed To obtain single colonies, a volume of 50 uL containing the strain in 30% glycerol was used for -4 decuple dilutions up to 10 . The strain was then sown in Charcoal Agar (Difco) plates without blood. The plates were incubated for 96 h at 35°C with a relative humidity > 70%. The isolated colonies were used to prepare a bacterial suspension with an Abs 530 nm = 0.1 that was used to smear on fresh Charcoal Agar plates. After 72 h of growth the homogeneous layer was used to inoculate a two-liter flask containing 200 mL of the THIJS medium at a final Abs 530 nm = 0.1. The culture was grown for 24 h at 35°C while stirring at 180 rpm. After this time, anhydrous glycerol was added to a final concentration of 30%. This seed culture was distributed in aliquots and stored at -80°C.

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Quintana-Vázquez et al.

Growth Conditions

Protein Identification

Two-liter flasks containing 200 mL of the THIJS medium were inoculated at Abs 530 nm = 0.1-0.3 from the frozen seed and grown 24 h at 35°C and 180 rpm. The cultures were then transferred to a 7.5 L roundbottomed bioreactor (Marubishi Co LTD, Japan). The bacterium was grown in batch mode with a working volume of 3 liters. Fermentation was carried out at 35°C. The level of dissolved oxygen remained above 20% while stirring at 100 rpm and aeration of 1 vvm. pH was controlled with 10% phosphoric acid and foaming was controlled by adding Glanapon DG (Bussetti & Co GmbH), as needed. Fermentation was stopped when the culture reached Abs 530 nm  4. Samples were taken from a sterile port every 3 to 6 h. The identity and concentration of the PTg was estimated by ELISA. Culture purity was verified by Gram staining and plating on tryptone soy agar plates.

Monoisotopic masses of tryptic peptides from PTg, FHA and Prn2 were used to identify the proteins in a non-redundant database by peptide mass fingerprinting (PMF) using MASCOT (http://www.matrixscience.com). Additionally, the most intense signals of every spectrum were fragmented by collision induced dissociation. The MS/MS ion search option of the MASCOT software was used to confirm protein identity. The parameter settings considered were: enzyme treatment (up to one missed cleavage), peptide mass error (0.1 Da), and the modification of cysteine (propionamide cysteine). Other (variable) modifications such as methionine sulfoxide and the deamidation of glutamine and asparagine were also taken into account. Molecular weight of the protein and taxonomy were not restricted. Purification of PTg, FHA and Prn2

Electrophoresis Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) on 12% (w/v) acrylamide was performed for separate proteins as described by Laemmli [17]. The molecular size standard for protein, RPN5800 (GE Healthcare), was used as a reference. Samples were mixed with Tris-glycine SDS reducing sample buffer (2) and boiled for 3 min. Before loading into polyacrylamide gels, samples were incubated with 5% acrylamide (w/v) for 1 hour at 25 °C. Bands were visualized by silver staining [18]. The purity of obtained proteins was estimated by densitometry. Mass Spectrometric Analysis The identity of the PTg, FHA and Prn2 proteins was confirmed by mass spectrometric analyses using a hybrid quadrupole-orthogonal time-of-flight mass spectrometer with a nanospray ion source (QTOF-2TM, Micromass, UK). The SDS-PAGE protein bands (Imidazol Zinc staining, [19] were digested in-gel with trypsin as described above [20]. The mixture of tryptic peptides was desalted by ZipTips (Millipore, USA) and loaded into the borosilicate nanoflow tips and submitted to 900 and 35 V of capillary and cone voltage, respectively. To acquire the ESI-MS/MS spectra, the first quadrupole was used to select the precursor ion within a window of 3 Th. Argon gas was used in the -2 collision chamber at ~3 10 Pa pressure, and collision energy ranging from 20 to 30 eV was set to fragment precursor ions. Data acquisition and processing were performed using MassLynx v3.5 (Micromass, UK).

The purification of PTg and FHA was achieved using a modified protocol of the Özcengiz procedure [21]. Briefly, the fermentations were stopped at 18 h, then separation/clarification was performed by centrifugation of the broth at 10 000 g for 30 min at 4°C. The supernatant was used to obtain PTg and FHA while the cell precipitate was used for Prn2 purification. The supernatant was then filter-sterilized by a double pore 0.45 + 0.2 m membrane cartridge. The filtered supernatant was evaluated in Bordet Gengou sheep blood-agar plates to monitor the presence of viable B. pertussis. At room temperature, 0.7 liter of 10 M Urea was gradually added to 2.85 liter of the B. pertussis free cell supernatant under constant shaking at 150 rpm. The sample was then adjusted to pH = 6.0 with 10% orthophosphoric acid. The sample was applied to a COO EMD Fractogel column (XK 50 mm /5 cm) equilibrated with 20 mM phosphate, 20 mM NaCL, 2 M Urea, pH = 6.0 pH = 6.0 (Buffer A). Endotoxin impurities were sequentially washed with 20 column volumes of equilibrium buffer containing 0.1% Triton X100, pH = 6 in buffer A. The PTg was eluted by increasing the pH of buffer A to 7.4. The FHA protein was eluted washing the column with 20 mM phosphate, 200 mM NaCL, 2 M Urea, pH = 8.0. The PTg and FHA eluates were dialyzed against the 20 mM Naphosphate buffer containing 0.3 M NaCl, pH =8.0 and stored at 2-8 °C. Glycerol was added up to 50% v/v and samples were kept at -70 °C for long-term storage. The Prn2 protein was detached from the surface of B. pertussis as previously reported [22]. For this, approximately 24 g (wet weight) of the cell biomass

Assessment of the Bordetella pertussis BpCNIC0311 Strain

was resuspended in a volume of 240 mL with 10 mM Tris, 150 mM NaCL pH = 8.0 and incubated for 60 min at 60°C. The cell extract was assessed in Bordet Gengou sheep blood-agar plates to detect the presence of viable B. pertussis. After centrifugation at 10 000 g for 30 min the supernatant was treated for 4 h at 4°C with 0.15 M calcium phosphate pH = 8 to precipitate nucleic acid contaminants. After centrifugation at 10 000 g for 30 min the supernatant was filtered by a double pore 0.45 + 0.2 m membrane cartridge. The sample was then dialyzed using Amicon with a cut-off value of 50 kDa and 10 mM Tris pH = 8.8. The sample was applied to a DEAE equilibrated with diafiltration buffer. Then the column was washed with 10% elution buffer (10 mM Tris, 100 mM NaCl, pH = 8.8) before eluting pertactin with 30% elution buffer. The Prn2 eluate was dialyzed against the 50mM Naphosphate buffer containing 0.3 M NaCl, pH =8.0 and stored at -20 °C. The purification (column) procedures for the three proteins was carried out at T=22 °C. Protein Reference for ELISA The following NIBSC reference proteins were used as standards for ELISA: PTx (JNIH 90/518), Prn (NIBSC 90/520) and FHA (NIBSC 90/654). ELISA for Identity The identity of B. pertussis antigens were estimated by domestically produced ELISA tests (manuscript in preparation). Briefly, high binding polystyrene 96-well ® ELISA microplates (Costar Corning Incorporated, USA) were sensitized with 100 μl/well anti-FHA or Prn mAbs (1-10 g/mL) diluted in 0.1 M carbonate/ bicarbonate pH 9.6 (except PTg ELISA, in which 40 g/mL fetuin was used as a capture reagent) and incubated for 1h at 37 °C. After coating, the plates were washed three times with 200 μl/well PBST (PBS containing 0.05 % Tween 20) and then blocked with 200 μl/well blocking buffer (with 1% BSA, w/v, and 0.1% Tween 20 in PBS) for 1-2 hours at 37°C. The blocking reagent was discarded by tapping the plates. After blocking, 100 μL of the tests samples, standard curve and controls were added in appropriate dilutions in the sample buffer (0.1% BSA diluted in PBS containing 1% Tween 20) and incubated for 1-2 hours at 37 °C. After the washing step, 100 μl/well of the detector antibody (HRP conjugated mouse antiS1subunit of PT, Prn or FHA monoclonal antibody) diluted in the sample buffer were added and incubated for 30 minutes at 37°C. Following washing, 100 μl of the TMB chromogen preparation (Sigma) diluted in

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0.05 M Phosphate-Citrate Buffer was added. After incubating for 10 min at room temperature in the dark, the reaction was stopped by adding 50 μl/well of 2 M H2SO4. Absorbance was measured at 450 nm using an ELISA plate reader. The protein concentration of PTg was calculated using the GraphPad Prism program. Immunogenicity Assay in Mice The PTg, FHA and Prn2 were evaluated in Balb/c and OF1 mice strains. Groups of female mice (n = 10) were immunized with a formulation containing 5 μg of PTg, 2.5 μg of FHA, 2.5 μg of Prn2 adsorbed on 80 μg of the aluminum hydroxide gel adjuvant (HCL Biosector). The placebo group was inoculated with buffer phosphate plus alum instead of protein. The Adacel vaccine (Sanofi Pasteur) was used as the positive control (n=2) and diluted 1:2.5 with phosphate buffer. All formulations were administered subcutaneously with a total volume of 500 μL. Mice received two doses at days 0 and 21, and were bled at days -2, 21 and 42. The IgG titers against PTg, FHA, Prn1 and Prn2 in the sera were estimated by the ELISA described below. ELISA for Immunogenicity Three independent enzyme-linked immunosorbent assays were performed to analyze the specific antibodies to PTg, FHA and Prn2. For this, polystyrene ® high binding 96-well ELISA plates (Costar Corning Incorporated, USA) were coated with 2 g/mL of PTg, 2 g/mL of Prn (Prn1 or Prn2) or 5 g/mL of FHA in 0.05 M carbonate buffer, pH 9.6, for 3 h at 37 °C (100 L/well). After washing once with PBS supplemented with 0.05% Tween 20 (PBST) the plates were blocked with 4% skim milk (Oxoid) in PBST for 1 h at 37° C. Then, diluted test sera was added to the wells and incubated for 2 h. An anti-serum was included as the reference curve with 100 arbitrary titer units. After washing four times with PBST, the plates were incubated for 2 h with 2000 diluted horseradish peroxidase-conjugated goat anti-mouse IgG (Sigma, St. Louis, MI). Following extensive washing ophenylenediamine (0.5 mg/mL), dissolved in 0.15 M sodium citrate buffer pH=5.0, was added to each well. Immunoglobulin classes and subclasses in serum were determined against PTg, FHA and Prn2 using a mouse isotyping kit (Sigma). The sera from the immunized groups were also tested after the second dose against the following linear peptides (coating, 5 μg of peptide/mL): P1, RGDAPAGGAVPGGAVPGGAV PGGFGPGGFGPVLDGW, located at the R1 variable

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region of Prn1; and P2, RGDAPAGGAVPGGAVPG GFGPGGFGPGGFGPGGFGPVLDGW, located at the R1 variable region of Prn2. The differences observed were analyzed using t comparison tests. The plates were read by a Micro ELISA auto reader at 450 nm. Intracerebral Challenge The intracerebral challenge was performed by the Biological assays Department of the National Center for Biologicals (Biocen, Mayabeque, Cuba) as part of a collaboration service agreement with the Center for Genetic Engineering and Biotechnology in Havana, Cuba. Groups of female OF1 mice (n = 17) with 11-13 g of weight were immunized intraperitoneally with three dilutions of immunogens: PTg/FHA/Prn2, PTg/FHA/ Prn2-1, PTg/FHA and the reference vaccine VPR (99)11. For the immunogen PTg/FHA/Prn2, formulation A consisted of 5 μg of PTg, 2.5 μg of FHA and 2.5 μg of Prn2 adsorbed to 80 μg of an aluminum hydroxide gel adjuvant (HCL Biosector). Formulations B and C were 5-fold and 25-fold dilutions of formulation A, respectively. Similar antigen proportions and dilutions were used for PTg/FHA/Prn2-1 and PTg/FHA formulations. The B. pertussis (cellular) reference vaccine, VPR (99)11, was used as follows: Dose A, 0.5 IU; Dose B, 0.1 IU and Dose C, 0.02 IU. VPR (99)11 is used as the national reference standard in the intracerebral challenge assay in Cuba with an assigned activity of 4 IU/ampoule. The placebo group was inoculated with alum-buffered phosphate. After 21 days, the lethal B. pertussis strain 18323 was administrated intracerebrally. For this, a bacterial 5 suspension (25 μL) with approximately 2.3  10 CFU suspended in PBS containing 1% casamino acids was injected into mice brains using a 0.25-mL syringe. The challenged animals were observed for 14 days and survival rate was recorded. RESULTS AND DISCUSSION The incidence of Pertussis has been increasing in infants [23], adolescents and adults of highly vaccinated populations [24]. This epidemiological status could be explained by the fact that commercial vaccines have limited efficacy against the strains in circulation. Actually, the microorganism has gradually changed the relevant virulence factors, the same present in acellular vaccines [8]. Nonetheless, the industry continues to manufacture the vaccines with B. pertussis strains that circulated decades ago. Additionally, certain technological factors burden the gap between the vaccines and circulating strains.

Quintana-Vázquez et al.

Vaccine production requires the use of chemicals to treat the pertussis toxin (PT), and detoxify it (PTd). The FHA and Prn proteins, depending on the production process, are not exempted from chemical treatments, either when they are purified together/mixed (PT+FHA), or due to the presence of PT as a minor contaminant in pure samples of FHA or Prn. The insights of this have been studied and it was demonstrated that chemicals change the structure of the purified components and consequently affect antipertussis immune response [25, 26]. However, acellular vaccines have been clinically effective for decades against the circulating strains of B. pertussis [27]. The debate about the change of incidence should be properly interpreted having into account multiple interrelated factors such as: type of vaccine, manufacturing, vaccination programs, population coverage and density, waning of vaccine immunity, adaptation of the microorganism, and improvement in diagnosis, among others. From a vaccine centered approach, the introduction of certain changes could improve the protection status conferred by commercially available aP vaccines. Some authors have advised the replacement of PTd (chemically detoxified) by PTg (genetically inactivated) [28]. Other studies suggest the inclusion of emerging variants of protective antigens present in vaccines such as pertactin [8]. This study evaluates the suitability of the BpCNIC0311 strain as an antigen producer for PTg, FHA and Prn2. BpCNIC0311 has been genetically modified to express pertussis toxoid PTg and pertactin protein type 2, Prn2. The PTg mutant has the advantage that chemical inactivation is avoided, thus the native structure of epitopes involved in vaccine efficacy should be largely preserved [26]. On the other hand, the replacement of Prn1 by Prn2 ensures that the most prevalent Prn variant in the strains circulating worldwide be present in future vaccines. The following features were examined to ensure a feasible performance of the strain: 1) its growth on a solid medium prepared without using defribinated blood as the supplement; 2) its growth when transferring it from a solid to a liquid medium (chemically defined) with Abs530nm > 1 after 24 hours; 3) its growth in stirred cultures (bioreactor) reaching appropriate cell densities: Abs530nm > 3 and > 4 g wet weight-cell pellet / Liter of broth; 4) the PTg levels should behave stably at the end of fermentation; 5) the expression levels of PTg, FHA and Prn2 proteins must allow to obtain

Assessment of the Bordetella pertussis BpCNIC0311 Strain

enough quantities of each pure antigen > 1 mg / Liter of broth; 6) the antigens should be obtained with appropriate physical integrity at the end of the purification process; 7) once formulated, the antigens should be immunogenic in mice at a reported human dose; and 8) the antigens must be able to confer dose dependent protection in the mouse intracerebral challenge assay (MICA) against the reference strain, BP18323. Criteria 1 and 2 were fulfilled during the preparation of the frozen seed. Then, the BpCNIC0311 strain was grown in stirred cultures in the THIJS medium. This chemically defined culture medium has shown to be advantageous in respect to the conventional SS medium [16]. As shown in Figure 1, the BpCNIC0311 strain experienced a rapid growth in the first 18 hours of culture, reaching an average cell density of Abs530nm = 4.5 and values of wet weight > 8 g/Liter of broth. At

Figure 1: Growth of BpCNIC 0311 and PTg production. The bacteria were grown in 3 L of the THIJS medium in a Marubishi bioreactor in batch mode. The controlled parameters were: temperature, 35.5 °C; stirring at 100 rpm; pH = 7.3 and pO2 > 20%. The graph represents three independent batch fermentations. Each time point is an average of two independent samples (coefficient of variance < 5%).

the end of fermentation PTg production was stable at about 10 g/mL (2-2.5 mg/mL/Abs). Preliminary attempts showed that after 18 h the culture reaches the stationary phase and PTg production stops and drops gradually (data not shown). Similar results have been obtained when BPCNIC0311 has been grown at the 30 L scale in the THIJS medium (manuscript in

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preparation). Results are consistent with a study that showed that B. pertussis strains cultured in the THIJS medium led to shorter cultivation periods and higher expression levels of PT [29]. Unpublished studies in our laboratory, showed that the BpCNIC0311 strain grown in the SS medium reached lower average cell densities, Abs530nm = 2.4; with longer cultivation periods, 28 - 30 hours; as well as lower PTg production, 1-1.3 μg PTg / mL / Abs. So far, the results suggest that the BPCNIC0311 strain has a suitable performance during fermentation in a chemically defined medium. It is known that the quality of the fermentation process has a crucial impact on downstream processing. The complex mixture of unidentified compounds, particularly proteases, contained in the fermentation broth of the new strain could hamper the final goal. Thus, an important aspect is to explore the feasibility of obtaining pure proteins from previously reported successful methods. For this, the culture was clarified by centrifugation and the supernatant was filtered. The cell biomass was resuspended and then heated at 60 °C for Prn2 extraction. Notably, no viable colonies of B. pertussis were observed in samples of the filtered culture supernatant containing PTg and FHA, or after heating the resuspended biomass during Prn2 extraction. Both inactivation steps make it possible for the following purification stages to be carried out in a laboratory under Biosafety Level 1. Then, the PTg and FHA were purified from the filtered culture supernatant using cation exchange chromatography COO . On the other hand, the soluble Prn2 protein from the extraction step was treated with calcium phosphate gel before performing further purification by anion exchange chromatography, + DEAE . Proteins were obtained with a high level of purity (> 95%) evidenced by the absence of contaminant protein bands (Figure 2A and 2B) and low levels of endotoxins (Table 1) according to the values reported for acellular pertussis vaccines (< 100 EU / Dose) [30]. Pure antigens were specifically recognized by monoclonal antibodies generated against native proteins (Figure 2C). Similarly, the three antigen proteins reacted positively in an identity assay using a sandwich ELISA (data not shown). An important evaluation for the selection of B. pertussis as a producing host strain is that related to the physical integrity of the protein produced. As in previous results, the pure proteins migrated according

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Quintana-Vázquez et al.

Figure 2: SDS–polyacrylamide gel electrophoresis and Western blot of purified B. pertussis proteins on 12.5% gel. Approximately 1 μg of Prn and FHA, and 3.5 μg of PTg were loaded in each line. The gels were developed with silver staining. A) PTg and FHA purified proteins from the culture supernatants of batches 1 to 3. B) Prn purified from cell pellet extracts from batches 1 to 3, C) Specific recognition of the proteins (batch 1) by Western blot using the mAbs anti-PT S1 subunit, anti-FHA and anti-Prn respectively. Table 1: Yield and Endotoxin Level for the Purified Proteins PTg, FHA and Prn2 mg/L

EU/Dose

PTg*

FHA

Prn

PTg (5 g)

FHA (2.5 g)

Prn (2.5 g)

batch 1

1.89

2.8

2.05

1.03

0.34

0.2

batch 2

1.94

2.23

1.58

2.31

0.5

0.02

batch 3

1.87

1.77

1.66

0.95

0.14

0.02

Mean

1.9

2.26

1.76

1.43

0.33

0.08

Average Total

5.32

6.34

4.93

-

-

-

Protein concentration was estimated by Lowry. mg/L: amount of purified proteins per liter of broth. EU/Dose: amount of residual endotoxin (EU, endotoxin unit) present in 5 g, 2.5 g and 2.5 g of PTg, FHA and Prn respectively. Total Average: the average of three batches of the total amount of protein for approximately three liters.* Protein concentration for PTg does not differ significantly when estimated by ELISA-PTg.

to their predicted sizes. The PTg was partitioned into four bands that migrated at between 28 and 17 kDa corresponding to subunits S1, S2, S3 and S4, S5 [31]. The presence of subunit S1 in purified PTg from the BPCNIC0311 strain is of utmost importance. It has been reported that serum antibody response to PT, following either infection or vaccination, reacts almost exclusively against the 28-kDa enzymatic S1 subunit [32]. On the other hand, the Prn2 protein showed a

distinctive aberrant migration pattern at about 72 kDa, which is due to the relative abundance of proline in the primary structure [33]. Similarly, the FHA protein migrated at approximately 220 kDa with the characteristic degradation bands of sizes over 100 kDa. The faint bands observed are chain scission of not cell-associated FHA, eventually formed due to shear forces related to stirring and aeration during fermentation [34]. The FHA protein obtained is

Assessment of the Bordetella pertussis BpCNIC0311 Strain

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Figure 3: Mass spectrometry analysis of the S1 subunit of PTg from the BpCNIC0311 strain. A: SDS-PAGE/Imidazol-zinc staining, PTg indicates the lane corresponding to the purified PTg toxoid (sample); the blue line indicates the band at the height of approximately 27-28 kDa corresponding to the S1 subunit. B: Mass spectrum of the tryptic digests of the S1 band. K9 and G129 indicate the signals corresponding to mutated peptides. These signals were fragmented to obtain sequence information. 9 134 The MS / MS spectra made it possible to confirm both mutations: DDPPATVYK and LAGALATYQSGYLAHR containing the 9 129 K and G mutations respectively.

comparable in quality to previous results [21], suggesting that the molecule could be immunogenic and functional, since it has been demonstrated that during natural infection the protective response is generated against those FHA components with sizes greater than 92 kDa [35]. The purified products were further analyzed by mass spectrometry. The highest intensity signals were selected for the MS/MS analysis and submitted to an automatic search with the MASCOT software. The program identified Bordetella pertussis sequences associated to PT, FHA and Prn2 with a high confidence level. The scores were above the levels set for the three proteins. Importantly, as shown in Figure 3, the 9 129 K and G amino acid mutations of PTg (S1 subunit) were confirmed by the MS/MS sequence analysis. As shown in Table 1, the yields of the purified proteins were similar for PTg, FHA and Prn. However, the human dose reported for the genetically detoxified pertussis toxin during a controlled trial was PTg, 5 g, FHA, 2.5 g; Prn, 2.5 g [14]. The twofold quantities used for PTg make it the limiting active ingredient when estimating the maximum number of doses of a given production process. In this sense, an average of 380 human doses per liter of broth, more than 1100 doses /process at 3 L laboratory scale, can be estimated.

Results suggest that the BPCNIC0311 host stably expresses PTg, FHA and Prn2 with a high quality and at satisfactory amounts. In contrast with our results, a study using similar purification methods obtained yields 2.6 fold higher for the PT (toxin) [21]. However, in that study the purified proteins are intermediate products that must still be chemically treated, which implies subsequent additional steps that influence the final yields. Additionally, the growth and antigen expression were at the expense of using static cultures and longer cultivation periods (5 days). These growth conditions are not cost-effective at an industrial scale, where stirring and aeration, among others, are crucial variables in sustaining batch to batch yields. Furthermore, in our study the conditions for culture are devoid of blood and casein hydrolysates. Particularly, the latter is a poorly defined component responsible for the large differences observed in growth curves in stirred cultures of B. pertussis [36]. No less important, because of its animal origin, is that both products are not suitable reagents for an industry that must produce parenteral molecules for human use, mainly infants. The biological activity of the purified PTg, FHA and Prn2 proteins was studied in inbred Balb/c and outbred OF1 mouse strains. The PTg, 5 g, FHA, 2.5 g and

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Prn, 2.5 g proteins were mixed and adsorbed to aluminum hydroxide. Additionally, a control formulation was assayed containing a similar immunogen proportion but including PRN2-1 instead of Prn2. The PRN2-1 is a recombinant hybrid protein that covers the complete sequences/regions of natural Prn1 and Prn2 pertactins. In a previous study, the PRN2-1 hybrid protein proved to be more immunogenic in Balb/c mice (stand-alone) than the recombinant Prn1 or Prn2 proteins [37]. After the first dose (Figures 4A and 4B), significant IgG antibody titers against the three proteins (-PTg, -

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FHA and -Prn2) were detected in the sera of immunized mice of both strains. The percentages of seroconversion were equal or higher than 60%. No specific activity was detected in the sera of mice receiving placebo (data not shown). In Balb/c mice, regardless of the formulation assayed, a significant pattern of immunogenicity was found with the following order: -PTg > -FHA > -Prn (Prn1 or Prn2) (Figure 4A). However, this pattern was not observed in OF1 mice where differences between proteins were only obtained for -FHA > -Prn2 (Figure 4B). The formulation containing PRN2-1 instead of Prn2 was also highly immunogenic in OF1 mice. The sera of

Figure 4: Immunogenicity of a formulation containing PTg, FHA and Prn2 in Balb/c (inbred) and OF1 (outbred) mice strains (n=10). Bars represent the IgG antibody titers that are specific to PTg, FHA or Prn (Prn1 or Prn2) proteins at 21 and 42 days after the first and second dose respectively. Symbol - is an abbreviation of antibody titer against a given antigen protein. The error bars represent the standard deviation. AU stands for arbitrary units. The symbol % above the bars means percentage of seropositivity in groups with partial seroconversion. Control vaccine: it is a dilution (1:2.5) of the commercial ADACEL vaccine; therefore every mouse received 1 g of the detoxified pertussis toxin (PT), 2 g of filamentous hemagglutinin (FHA) and 1.2 g of pertactin (Prn). In all cases twice the titer value of placebo was subtracted from the corresponding samples. The MannWhitney test was used for statistical analyses; the p values are shown in inset tables.

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Figure 5: Immunoglobulin types after the second dose in groups immunized with PTg/FHA/ Prn2 and PTg/FHA/ Prn2-1. A, B and C correspond respectively to the Anti-PTg, Anti-Prn2 and Anti-FHA reactions of immunoglobulin IgG subtypes in pooled sera pairs of OF1 mice diluted 1/250. Bars represent means ± standard deviation. D: The same sera samples were tested for the recognition of the variable region R1 of Prn1 and Prn2. The % stands for the percentage of seropositivity. The calculated average was for the positive sera.

mice immunized with PTg/ FHA/PRN2-1 showed differences in -PTg> -Prn (Prn1 or Prn2) and FHA> -Prn (Prn1 or Prn2). At the end of the study (after two doses), regardless of the mouse strain used or formulation tested, the pattern: -PTg > -FHA > Prn, significantly determined the specific activity of the immune responses. Similarly, the antibodies induced by the natural Prn2 or the recombinant PRN2-1equally recognized both Prn1 and Prn2 proteins. The sera of both formulations were pooled by pairs and further evaluated against linear peptides from the

variable region R1 of Prn1 and R1 of Prn2 (Figure 5D). The sera from Balb/c mice immunized with both formulations recognized the variable region 1 of Prn1 and Prn2 with a percentage of seropositivity > 80%. The linear peptides of both variable R1 regions were recognized by 40% of OF1 mice sera. The sera of OF1 mice (outbred) were additionally tested against each one of the three antigens to determine the profile of IgG isotypes. As shown in Figure 5, the assayed formulations PTg/FHA/ Prn2 and PTg/FHA/ PRN2-1 activated an immune response containing IgG1, IgG2a and IgG2b immunoglobulin subclasses. As previously

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reported in mice, IgG2a and IgG2b have been involved in cellular host responses to bacterial infections [38, 39]. The mice immunized with the PTg/FHA/ Prn2 immunogen showed significantly higher levels of antiPrn2 IgG2a respect to the mice immunized with PTg/FHA/ PRN2-1. Importantly, the use of Prn2 or PRN2-1 did not change the profile of immunoglobulin subclasses specific to the protection antigen leader PTg. The experimental formulations were further evaluated in their ability to confer dose-dependent protection. Hence, groups of mice were immunized once with the immunogens adsorbed to alum using three dosage levels: PTg/FHA/Prn2, PTg/FHA/Prn2-1, the controls PTg/FHA and the reference vaccine VPR (99)11. Dose A represents an infant’s dose (PTg, 5 g, FHA, 2.5 g; Prn, 2.5 g) [14], while Dose B and Dose C are fivefold and twenty-fivefold dilutions of Dose A. As shown in Figure 6, all the mice receiving the placebo died. However, mice immunized either with Dose A or Dose B significantly resisted the lethal dose with the BP18323 reference strain, showing survival rates of > 94% and > 70 % respectively. On the other hand, mice receiving the 25-fold dilution (Dose C, survival < 15 %) showed no statistical differences (p > 0.05) from the placebo group (data not shown). Results are consistent with a recent study showing that a modified intra-cerebral challenge assay (modified by Kendrick, MICA) distinguished mice vaccinated with acellular vaccines from unvaccinated mice and gave a significant dose-response relationship [40]. Interestingly, mice immunized with the control formulation devoid of pertactin PTg/FHA (Dose B), showed lower survival rates, although there was no statistical significance. This observation could denote the contribution of pertactin in protection. Further approaches should be made, such as obtaining other new molecules as for example fimbrial antigens, among others. This is an important aspect since recent reports show emerging strains that do not express pertactin [5, 41], which could reduce the efficacy of an acellular preparation based solely on three pertussis components. Although these are preliminary results at a laboratory scale, they show the potential of the BPCNIC0311 strain as an antigen producer. The results met the different evaluation criteria proposed. The new strain is able to grow stably in different culture media, either solid or liquid, which are devoid of components derived from animal milk and blood.

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Similarly, the strain is able to stably express the three antigens at adequate levels for purification. The antigens can be obtained with high purity and physicochemical quality by the methods previously reported. The antigens formulated in alum induced a protective immune response in mice against the reference strain BP18323. It is concluded that the new strain can be used to obtain sufficient amounts of PTg, FHA and Prn2 for preclinical studies aimed to develop an improved-updated acellular vaccine against Whooping Cough.

Figure 6: Protection against intracerebral challenge with a lethal dose of B. pertussis 18323. Mice groups were immunized with each immunogen at a human dose (Dose A) and a fivefold dilution (Dose B) of the human dose (see also material and methods). Mice were challenged on day 21 after immunization. The survival of the challenged mice was recorded for 15 days. Bars represent the survival rate at day 15. The statistical analysis was performed comparing each of the survival curves with respect to the placebo, by means the Log-rank (Mantel-Cox) test. The result is representative of two independent assays.

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Received on 08-01-2015

Accepted on 17-01-2015

Published on 20-03-2015

DOI: http://dx.doi.org/10.14205/2310-9386.2015.03.01.2

© 2015 Quintana-Vázquez et al.; Licensee Pharma Publisher. This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited.