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Contents lists available at ScienceDirect
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Development of a recombinant fusion protein vaccine formulation to protect against Streptococcus pyogenes
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Garry Morefield a,∗ , Graham Touhey a , Fangjia Lu b , Anisa Dunham b , Harm HogenEsch b
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VaxForm, LLC, Bethlehem, PA 18015 United States Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West Lafayette, IN 47907 United States
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Article history: Received 6 February 2014 Received in revised form 22 April 2014 Accepted 25 April 2014 Available online xxx
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Keywords: Streptococcus pyogenes Group A strep Aluminum adjuvant Vaccine formulation
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1. Introduction
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Diseases resulting from infection by group A streptococcus (GAS) are an increasing burden on global health. A novel vaccine was developed targeting infection by Streptococcus pyogenes. The vaccine incorporates a recombinant fusion protein antigen (SpeAB) which was engineered by combining inactive mutant forms of streptococcal pyrogenic exotoxin A (SpeA) and streptococcal pyrogenic exotoxin B (SpeB) from S. pyogenes. A rational, scientific approach to vaccine development was utilized to determine optimal formulation conditions with aluminum adjuvants. Investigations of the pH stability profile of SpeAB concluded the antigen was most stable near pH 8. Incorporation of the stabilizers sucrose and mannitol significantly enhanced the stability of the antigen. Vaccines were formulated in which most of the SpeAB was adsorbed to the adjuvant or remained in solution. A SpeAB vaccine formulation, stabilized with sucrose, in which the antigen remains adsorbed to the aluminum adjuvant retained the greatest potency as determined by evaluation of neutralizing antibody responses in mice. This vaccine has great potential to provide a safe and effective method for prevention of GAS disease. © 2014 Published by Elsevier Ltd.
Streptococcus pyogenes is a Gram-positive bacterium that causes a wide variety of diseases such as strep throat, scarlet fever, necrotizing fasciitis, streptococcal toxic shock syndrome, and impetigo. These disease states are generally referred to as Group A Streptococcal (GAS) Diseases. Mortality from GAS disease is a global issue with an estimated 500,000 deaths attributed to S. pyogenes infection annually [1]. In the United States there are an estimated 10 million cases of non-invasive disease each year and between 9000 and 11,500 cases of invasive disease [2]. Disease resulting from invasive infection can have mortality rates as high as 35%. Additionally, there is growing evidence for the role of GAS infection in the causation of pediatric autoimmune neuropsychiatric disorders [3,4]. In general GAS infection is treated with a course of antibiotics, however antibiotic treatment does not guarantee prevention of complications associated with infection such as glomerulonephritis. Combined with the low availability of antibiotics in developing
Abbreviations: GAS, group A streptococcal; SpeA, streptococcal pyrogenic exotoxin A; SpeB, streptococcal pyrogenic exotoxin B; SpeAB, recombinant fusion protein of SpeA and SpeB. ∗ Corresponding author at: 116, Research Drive, Bethlehem, PA 18015, United States. Tel.: +1 610 573 9620. E-mail address: garry.morefi[email protected] (G. Morefield).
countries and the emergence of antibiotic-resistant strains the need is great for a safe and effective vaccine for the prevention of GAS disease [5–8]. A recombinant fusion protein antigen (SpeAB) was engineered combining Streptococcal pyrogenic exotoxin A (SpeA) with Streptococcal pyrogenic exotoxin B (SpeB) from S. pyogenes [9]. SpeA is a superantigen which enables bacteria to evade immune responses by disabling recognition by adaptive immunity [10]. SpeA crosslinks major histocompatibility complex class II molecules with T-cell receptors resulting in polyclonal activation of T cells. The systemically increased levels of inflammatory cytokines can induce toxic shock [11,12]. The SpeA utilized in the fusion protein antigen was genetically mutated at the leucine located at position 42 disabling superantigen activity. SpeB is an extracellular cysteine protease that is expressed by most M protein serotypes of S. pyogenes. SpeB is not homologous in structure to SpeA and has no superantigen functionality [13]. SpeB is not only secreted but also retained at the bacterial cell surface where it actively binds glycoproteins [14]. Analysis of sera obtained from patients with invasive GAS infection has demonstrated SpeB specific antibody production [15]. SpeB utilized in the fusion protein antigen was genetically mutated at the cysteine located at position 47 inactivating the enzyme [16]. There is high risk in development of novel vaccine candidates as evidenced by the failure of the majority of novel vaccines at the
http://dx.doi.org/10.1016/j.vaccine.2014.04.092 0264-410X/© 2014 Published by Elsevier Ltd.
Please cite this article in press as: Morefield G, et al. Development of a recombinant fusion protein vaccine formulation to protect against Streptococcus pyogenes. Vaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.04.092
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preclinical and early clinical stages of development [17]. It is critical to initiate formulation investigations early in development to understand the critical quality attributes of the vaccine and how to optimize those parameters for production of a robust product. In order to gain knowledge of the formulation factors that may impact potency and stability of the SpeAB antigen in the final formulation we employed a rational, scientific approach to development [18]. Through this approach the biophysical characteristics of the antigen are determined, potential excipient stabilizers are evaluated, the interactions with adjuvants are optimized, and stability of the final product is investigated. Biophysical characterization studies employ a variety of analytical techniques for determining how formulation attributes such as pH, buffer species, and stabilizers impact the physical state of the antigen [19,20]. The formulation components and attributes selected from the biophysical characterization studies affect the interaction of antigen with adjuvants that may be incorporated into the vaccine. For the SpeAB vaccine aluminum adjuvants were investigated for enhancement of the immune response. Primary mechanisms of adsorption of antigens to aluminum adjuvants are electrostatic interactions, hydrophobic interactions, and ligand exchange [21–23]. Formulation characteristics impact the balance of these forces influencing the adsorption state of the antigen and the strength those interactions. Both the adsorption state and strength of binding may affect potency of vaccines [24–26]. Therefore, it is important to understand what the adsorption state is and how that influences the potency of the vaccine. Finally, it is important not just to understand these parameters at a single point in time immediately following production of the vaccine, but to understand if any changes occur in the formulation over time which may adversely impact the potency of the SpeAB antigen. Therefore, the stability of SpeAB was investigated under typical refrigerated storage conditions as well as stressed conditions of elevated temperature. Stability data confirms the appropriate quality attributes were optimized during development of the vaccine. By following this approach to vaccine development the traditional risks associated with early phase development are minimized. This allows for rapid development of a safe and effective vaccine able to reduce the burden of GAS disease in the general population.
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Solutions of 100 g/ml SpeAB were prepared at pHs 7.0–8.0 with 20 mM Tris (Sigma, St. Louis, MO) or phosphate buffer (Sigma, St. Louis, MO). Ultraviolet spectra from 240 to 380 nm were obtained using a BioSpec 1601 spectrophotometer (Shimadzu, Japan) between 25 and 80 ◦ C in 2.5 ◦ C increments. The samples were incubated at each temperature for 5 min prior to obtaining the spectra. The second derivative with a triangular smooth was applied to the data and peak minima associated with the phenylalanine, tyrosine, and tryptophan residues were recorded. The Tm for each buffer condition was determined by calculating the inflection point of the curve on a plot of peak minima versus temperature. For stabilizer evaluation, 100 g/ml SpeAB was prepared in 20 mM Tris with or without 10% added sucrose or mannitol. Analysis by ELISA was performed on samples diluted 25 fold in 20 mM Tris, 150 mM NaCl, pH 9. 100 l was placed in duplicate in the appropriate well of a 96 well plate. Standards were prepared by performing six, 2-fold serial dilutions starting at 4 g/ml. The plate was incubated for 1 h at 37 ◦ C and then washed three times with 100 l/well of PBS (20 mM PO4 , 150 mM NaCl, pH 7.4). The plate was blocked with 100 g/ml BSA in PBS. Following washing, 100 l/well
of a 1:25,000 dilution of rat anti-SpeAB antisera was added to the plate. After incubation and washing 100 l/well of a 1:70,000 dilution of goat anti-rat IgG-HRP (Rockland, Gilbertsville, PA) was added to the plate and the plate was incubated as before. The plate was washed and 100 l/well of TMB (KPL, Gaithersburg, MD) was added and the plate was incubated for 20 min at room temperature. The reaction was stopped by addition of 100 l/well 3 M H2 SO4 and the absorbance read at 450 nm using a UVmax plate reader (Molecular Devices, Sunnyvale, CA). 2.2. Interactions with aluminum adjuvants Aluminum hydroxide adjuvant (Alhydrogel® , Brenntag, Denmark) was treated with phosphate at P:Al molar ratios of 0–2. After combination, the adjuvant and phosphate were mixed for 1 h at room temperature. Formulations were prepared containing 50 g/ml SpeAB and 1.7 mg/ml Al and mixed for 30 min at room temperature. The formulations were then centrifuged at 10,000 × g RCF for 5 min and the supernatant was collected. Finally, the protein concentration of the supernatant was determined by BCA assay (Pierce, Rockford, IL) and the percent adsorbed calculated by mass balance. In association with the in vivo potency study, a short term stability study at 5 ◦ C was performed by monitoring aspect, pH, and percent adsorption of the adjuvanted formulations on days 0 and 14. The pH of each formulation was measured using a Symphony pH meter (VWR, Radnor, PA). The percent adsorption was determined as previously described. The aspect of each formulation was evaluated by visual inspection of the vials in front of both a white and black background. 2.3. In vivo potency The potency of SpeAB formulations were determined in 7–8 week old female BALB/c mice (Harlan, Indianapolis, IN). There were 10 mice per group in those receiving SpeAB and 5 mice per group in those receiving adjuvant only. Mice were administered 0.1 ml of vaccine IM on days 0 and 14 of the study. Sera were collected from mice on days 14 and 28. To determine IgG titer 96 well plates were coated overnight at 4 ◦ C with 100 l of 10 g/ml SpeAB in 0.1 M carbonate buffer at pH 9.6. Plates were washed three times with 0.05% Tween in PBS (PBS-T). The plates were blocked with 200 l of 1% (w/v) BSA/PBS-T per well for one hour at 37 ◦ C. After washing, serial dilutions of serum samples in BSA/PBS-T was added in triplicate and incubated for 2 h at 37 ◦ C. Following washing 100 l of 1:5000 diluted peroxidase-conjugated goat anti-mouse IgG (Sigma, St. Louis, MO) was added. The plates were washed and 100 l TMB (Sigma) was added. After incubation at room temperature for 20 min, 50 l of 2 M sulfuric acid was added to stop the reaction. The absorbance was read at 450 nm in a BioTEK synergy HT microplate reader (BioTEK, Winooski, VT). Titers were calculated as the dilution at which the OD reading reached 0.2. To determine if the differences between groups were statistically different, logtransformed titers were analyzed by one-way ANOVA with post hoc analysis by Tukey’s multiple comparison test. Differences were considered significant at p < 0.05. To determine the neutralizing antibody response serum samples were heat-inactivated for 30 min at 56 ◦ C. They were diluted 1:25 and incubated for 1 h at 37 ◦ C with SpeA (Toxin Technology, Sarasota, FL) at 80 ng/ml. Human peripheral blood mononuclear cells (PBMC; Zen-bio, Research Triangle Park, NC) were diluted to 2 × 106 cells/ml in RPMI1640 supplemented with 10% fetal calf serum 100 U/ml penicillin, 100 g/ml streptomycin, and 0.25 g/ml amphotericin B and added to the wells of a 96 well plate at 100 l/well. Triplicate wells 20 ng/ml SpeA (positive control), medium only (negative control), or 20 ng/ml mixed with
Please cite this article in press as: Morefield G, et al. Development of a recombinant fusion protein vaccine formulation to protect against Streptococcus pyogenes. Vaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.04.092
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mouse serum. After 48 h incubation at CO2 , the supernatants were collected and analyzed by ELISA for the concentration of IFN␥. The neutralizing activity was calculated as 100 − (IFN␥ sample/IFN␥ control) × 100. The statistical significance of differences of the group means was determined by one-way ANOVA with post hoc analysis by Tukey’s multiple comparison test. Differences were considered significant at p < 0.05.
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The pH/stability profile of SpeAB was investigated at accelerated temperature conditions in both phosphate and Tris buffered saline systems. Prior to initiating the investigation the melting temperature of the antigen in both buffers was determined by derivative spectroscopy. The lowest melting temperature observed for all formulations was 55 ◦ C (data not shown). Therefore, a temperature of 45 ◦ C was selected to determine the pH/stability profile to minimize the impact of physical transitions of the antigen that occur above the melting point. The stability of SpeAB solutions was evaluated following 2 days of storage at 45 ◦ C and pHs 7.0, 7.5, and 8.0 (Fig. 1). The temperature sensitivity of the pH of Tris buffered solutions was accounted for in preparation of the study samples. There were no significant differences in the concentration of SpeAB remaining between the two buffer systems at any of the pHs investigated. However, there was significantly more SpeAB remaining at pH 7.5 and 8 than at pH 7. While the concentration of SpeAB at pH 8 appears to be higher than at 7.5, this difference was not found to be statistically significant. For subsequent investigations the Tris buffered system at pH 8 was selected to maximize stability of the antigen in solution as well as to minimize sources of phosphate, which has the potential to interact with aluminum adjuvants in the final formulations. To investigate further enhancement of stability formulations containing added sucrose or mannitol were monitored at accelerated temperature. (Fig. 2) The data demonstrated that the addition of either sucrose or mannitol significantly enhanced the stability of SpeAB in solution over buffer alone. By day 9 nearly all of the SpeAB in the formulation without stabilizer had degraded, while the formulations containing stabilizer had nearly all of the SpeAB intact. This suggests either sucrose or mannitol would be appropriate stabilizers to include in the final vaccine formulation.
Fig. 2. Addition of 10% sucrose or mannitol to SpeAB in 20 mM Tris buffer pH 8 significantly enhances antigen stability at 45 ◦ C.
3.2. Interactions with aluminum adjuvants
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To investigate the interactions of SpeAB with aluminum adjuvants a series of aluminum hydroxide adjuvants with increasing levels of phosphate treatment were prepared to modify the adjuvant surface charge. The SpeAB was then formulated with each of these adjuvants and the level of adsorption was determined (Fig. 3). The level of SpeAB adsorbed to the adjuvant was inversely proportional to the amount of phosphate treatment applied to the aluminum hydroxide adjuvant. This suggests that electrostatic interactions are one of the primary adsorption forces in the formulation. However, since the adsorption level did not approach 0% until the phosphate treatment ratio approached 2:1, this suggests that other adsorption forces such as hydrophobic interactions are also present in the system. Once the mechanism of SpeAB interaction with aluminum adjuvants was better understood, investigations were performed to determine conditions for adsorbed and non-adsorbed formulations in the sucrose stabilized buffer system. As before, formulations were prepared utilizing aluminum hydroxide adjuvants that had been modified with phosphate (Fig. 3). The interaction of SpeAB with the aluminum adjuvants in the sucrose stabilized formulations followed the same trend as in the non-stabilized formulations. Target adsorption levels for adsorbed (>80%) and non-adsorbed (20% adsorbed Homogeneous suspension of white particles 7.5–8.5 >20% adsorbed Homogeneous suspension of white particles 7.5–8.5 >20% adsorbed
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0.4, 2, or 5 g SpeAB on days 0 and 14. A short term stability study bracketing the vaccine administration dates was also performed to ensure attributes of the various vaccine formulations, such as aspect, pH, and percent adsorption, did not change significantly over the course of the potency evaluation (Table 1). Results from the stability study demonstrate all formulations remained within their target values for the duration of the study. Sera was obtained on days 14 and 28 and analyzed for antigen specific immune responses. Serum titers of anti-SpeAB IgG were dose dependent (Fig. 4). Both adsorbed and non-adsorbed
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Experimental groups Fig. 5. The functional activity of the mouse anti-sera to neutralize wild type SpeA toxin was evaluated by monitoring the inhibition of INF␥ production by human peripheral blood mononuclear cells following incubation with anti-sera/toxin mixtures.
Please cite this article in press as: Morefield G, et al. Development of a recombinant fusion protein vaccine formulation to protect against Streptococcus pyogenes. Vaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.04.092
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Acknowledgments
Using a rational, scientific approach we were able to develop a potent and stable vaccine targeting GAS disease. Through biophysical characterization and accelerated stability studies it was determined that SpeAB was most stable near pH 8 in both Tris and phosphate buffers. Tris was selected as the formulation buffer since the pKa of Tris is closer to 8 than that of phosphate, resulting in increased buffer capacity. Additionally, Tris has a lower potential to impact adsorption of SpeAB with aluminum adjuvants so the adsorption state of the antigen can be more easily controlled in the final product. When investigating further stabilization of SpeAB sucrose and mannitol were selected based on their abilities to act as stabilizing osmolytes. The principle mechanism of stabilization of proteins by these excipients is through a process called preferential exclusion [27]. In this process the solute, such as sucrose, is excluded from the immediate domain of the protein, thereby maintain greater hydration of the antigen. When accelerated stability studies were performed it was determined that both sucrose and mannitol were able to significantly enhance the stability of SpeAB to an equivalent magnitude. Therefore, either excipient would be suitable for use in the final formulation. To determine if the adsorption state of SpeAB impacted potency formulations in which the antigen was adsorbed or remained in solution were produced. Based on the amino acid sequence of SpeAB it was anticipated that at the formulation pH of 8 there should be electrostatic attractive forces present between the antigen and aluminum hydroxide adjuvant. The data demonstrated that this was the case as the majority of SpeAB was adsorbed in the formulation with aluminum hydroxide adjuvant. Adsorption decreased as phosphate treatment level increased inducing electrostatic repulsive forces in the system. However, the low slope of the curve indicates that other forces such as hydrophobic interactions or hydrogen bonding are acting in the formulation as well. Another possibility since SpeAB is a fusion protein and SpeA has an acidic pI while SpeB has a basic pI is that there is directional electrostatic adsorption occurring in the formulation. The directional adsorption may allow SpeAB to remain adsorbed to the surface of the aluminum adjuvant in the present of some electrostatic repulsive forces. The potency of the adsorbed and non-adsorbed formulations was evaluated in mice at three different doses. The results of the potency study indicate that the presence of an aluminum adjuvant significantly enhances SpeAB IgG titer as well as functional antibody responses. The immune response was further enhanced when SpeAB was adsorbed to the aluminum adjuvant in the formulation and of the three doses tested 5 g induced the greatest potency. The SpeAB vaccine developed here has great potential to protect against GAS disease. The cysteine protease SpeB has been found to be broadly expressed in many clinical isolates of GAS [28,29]. There have been reports that point mutations in csrRS result in reduced production of speB [30]. However, while there may be a decreased expression of SpeB in these mutants there is not a lack of expression. Therefore, the SpeB portion of the antigen maintains the potential to provide protection against a broad range of GAS serotypes. While the super antigen, SpeA, is typically found in a smaller percentage of circulating strains of S.pyogenes it is associated with serotypes causing invasive disease [31–33]. The SpeAB antigen is able to induce a protective response in mice against the superantigen demonstrating its potential to reduce the prevalence of invasive disease. By combining these two antigens into a single vaccine there is the potential to significantly decrease the burden of both invasive and non-invasive GAS disease globally.
This work is supported by the US Army Medical Research and Q3 Materiel Command under Contract No.W81XWH-12-C-0183. The views, opinions and/or findings contained in this report are those of the author(s) and should not be construed as an official Department of the Army position, policy or decision unless so designated by other documentation. In conducting research using animals, the investigator(s) adhered to the Animal Welfare Act Regulations and other Federal statutes relating to animals and experiments involving animals and the principles set forth in the current version of the Guide for Care and Use of Laboratory Animals, National Research Council. The authors thank Dr. Robert Ulrich for the gift of SpeAB fusion protein. References [1] W.H.O. http://www.who.int/child adolescent health/documents/ivb 05 14/ en/ [2] Centers for Disease Control and Prevention. Online, Available at: http://www. cdc.gov/ncidod/dbmd/diseaseinfo/groupastreptococcal t.htm. [3] Mell LK, Davis RL, Owens D. Association between streptococcal infection and obsessive-compulsive disorder, Tourette’s syndrome, and tic disorder. Pediatrics 2005;116(July (1)):56–60. [4] Leckman JF, King RA, Gilbert DL, Coffey BJ, Singer HS, Dure 4th LS, et al. Streptococcal upper respiratory tract infections and exacerbations of tic and obsessive-compulsive symptoms: a prospective longitudinal study. J Am Acad Child Adolesc Psychiatr 2011;50(February (2)):108–18. [5] Reinert RR, Lutticken R, Al-Lahham A. High-level fluoroquinolone resistance in a clinical Streptoccoccus pyogenes isolate in Germany. Clin Microbiol Infect 2004;10:659–62. [6] Buxbaum A, Forsthuber S, Sauermann R, Gattringer R, Graninger W, Georgopoulos A. Development of macrolide-resistance and comparative activity of telithromycin in streptococci in Austria, 1996–2002. Int J Antimicrob Agents 2004;24:397–400. [7] Stevens DL. Streptococcal toxic shock syndrome associated with necrotizing fasciitis. Annu Rev Med 2000;51:271–88. [8] Musser JM, Krause RM. The revival of group A streptococcal diseases with a commentary on staphylococcal toxic shock syndrome. In: Krause RM, Fauci A, editors. Emerging infections New York: Academic Press; 1998. p. 185–218. [9] Ulrich R. Vaccine based on a ubiquitous cysteinyl protease and streptococcal pyrogenic exotoxin A protects against Streptococcus pyogenes sepsis and toxic shock. J Immune Based Ther Vaccine 2008;6:1. [10] Ulrich RG, Bavari S, Olson M. Bacterial superantigens in human diseases: structure, function and diversity. Trends Microbiol 1995;3:463–8. [11] Norrby-Teglund A, Pauksens K, Norgren M, Holm SE. Correlation between serum TNF alpha and IL-6 levels and severity of group A streptococcal infections. Scand J Infect Dis 1995;27:125–30. [12] Stevens DL, Bryant AE, Hackett SP, Chang A, Peer G, Kosanke S, et al. Group A streptococcal bacteremia: the role of tumor necrosis factor in shock and organ failure. J Infect Dis 1996;173:619–26. [13] Kapur V, Topouzis S, Majesky MW, Li LL, Hamrick MR, Hamill RJ, et al. A conserved Streptococcal pyogenes extracellular cysteine protease cleaves human fibronectin and degrades vitronectin. Microb Patho 1993;15:327–46. [14] Hytonen J, Haataja S, Gerlach D, Podbielski A, Finne J. The SpeB virulence factor of Streptococcus pyogenes, a multifunctional secreted and cell surface molecule with strepadhesin, laminin-binding and cysteine protease activity. Mol Microbiol 2001;39:512–9. [15] Akesson P, Rasmussen M, Mascini E, von Pawel-Rammingen U, Janulczyk R, Collin M, et al. Low antibody levels against cell wall-attached proteins of Streptococcus pyogenes predispose for severe invasive disease. J Infect Dis 2004;189:797–804. [16] Gubba S, Low DE, Musser JM. Expression and characterization of group A Streptococcus extracellular cysteine protease recombinant mutant proteins and documentation of seroconversion during human invasive disease episodes. Infect Immun 1998;66:765–70. [17] Douglas RG, Sadoff J, Samant V. The vaccine industry. In: Plotkin S, Orenstien W, Offit P, editors. Vaccines UK: Elsevier; 2008. p. 37–44. [18] Morefield G. A rational systematic approach for the development of vaccine formulations. AAPS J 2011;13(2):191–200. [19] Peek LJ, Brey RN, Middaugh CR. A rapid, three-step process for the preformulation of a recombinant ricin toxin A-chain vaccine. J Pharm Sci 2007;96: 44–60. [20] Peek LJ, Brandau DT, Jones LS, Joshi SB, Middaugh CR. A systematic approach to stabilizing EBA-175 RII-NG for use as a malaria vaccine. Vaccine 2006;24:5839–51. [21] Seeber SJ, White JL, Hem SL. Predicting the adsorption of proteins by aluminium-containing adjuvants. Vaccine 1991;9:201–3. [22] Al-Shakhshir RH, Regnier FE, White JL, Hem SL. Contribution of electrostatic and hydrophobic interactions to the adsorption of proteins by aluminumcontaining adjuvants. Vaccine 1995;13:41–4.
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[29] Mathur P, Bhardwaj N, Gupta G, Punia P, Tak V, John NV, et al. Outbreak of Streptococcus pyogenes emm type 58 in a high dependency unit of a level1 trauma center of India. Indian J Crit Care Med 2014 Feb;18(February (2)): 77–82. [30] Tatsuno I, Okada R, Zhang Y, Isaka M, Hasegawa T. Partial loss of CovS function in Streptococcus pyogenes causes severe invasive disease. BMC Res Notes 2013;28(March (6)):126. [31] Silva-Costa C, Carric¸o JA, Ramirez M, Melo-Cristino J. Scarlet fever is caused by a limited number of Streptococcus pyogenes lineages and is associated with the exotoxin genes ssa, speA and speC. Pediatr Infect Dis J 2014;33(March (3)):306–10. [32] Kittang BR, Skrede S, Langeland N, Haanshuus CG, Mylvaganam H. emm gene diversity, superantigen gene profiles and presence of SlaA among clinical isolates of group A, C and G streptococci from western Norway. Eur J Clin Microbiol Infect Dis 2011;30(March (3)):423–33. [33] Plainvert C1, Doloy A, Loubinoux J, Lepoutre A, Collobert G, Touak G, et al. Invasive group A streptococcal infections in adults, France (2006–2010). Clin Microbiol Infect 2012;18(July (7)):702–10.
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Contents lists available at ScienceDirect
Vaccine journal homepage: www.elsevier.com/locate/vaccine
Development of a recombinant fusion protein vaccine formulation to protect against Streptococcus pyogenes
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Garry Morefield a,∗ , Graham Touhey a , Fangjia Lu b , Anisa Dunham b , Harm HogenEsch b
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VaxForm, LLC, Bethlehem, PA 18015 United States Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, West Lafayette, IN 47907 United States
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Article history: Received 6 February 2014 Received in revised form 22 April 2014 Accepted 25 April 2014 Available online xxx
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Keywords: Streptococcus pyogenes Group A strep Aluminum adjuvant Vaccine formulation
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1. Introduction
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Diseases resulting from infection by group A streptococcus (GAS) are an increasing burden on global health. A novel vaccine was developed targeting infection by Streptococcus pyogenes. The vaccine incorporates a recombinant fusion protein antigen (SpeAB) which was engineered by combining inactive mutant forms of streptococcal pyrogenic exotoxin A (SpeA) and streptococcal pyrogenic exotoxin B (SpeB) from S. pyogenes. A rational, scientific approach to vaccine development was utilized to determine optimal formulation conditions with aluminum adjuvants. Investigations of the pH stability profile of SpeAB concluded the antigen was most stable near pH 8. Incorporation of the stabilizers sucrose and mannitol significantly enhanced the stability of the antigen. Vaccines were formulated in which most of the SpeAB was adsorbed to the adjuvant or remained in solution. A SpeAB vaccine formulation, stabilized with sucrose, in which the antigen remains adsorbed to the aluminum adjuvant retained the greatest potency as determined by evaluation of neutralizing antibody responses in mice. This vaccine has great potential to provide a safe and effective method for prevention of GAS disease. © 2014 Published by Elsevier Ltd.
Streptococcus pyogenes is a Gram-positive bacterium that causes a wide variety of diseases such as strep throat, scarlet fever, necrotizing fasciitis, streptococcal toxic shock syndrome, and impetigo. These disease states are generally referred to as Group A Streptococcal (GAS) Diseases. Mortality from GAS disease is a global issue with an estimated 500,000 deaths attributed to S. pyogenes infection annually [1]. In the United States there are an estimated 10 million cases of non-invasive disease each year and between 9000 and 11,500 cases of invasive disease [2]. Disease resulting from invasive infection can have mortality rates as high as 35%. Additionally, there is growing evidence for the role of GAS infection in the causation of pediatric autoimmune neuropsychiatric disorders [3,4]. In general GAS infection is treated with a course of antibiotics, however antibiotic treatment does not guarantee prevention of complications associated with infection such as glomerulonephritis. Combined with the low availability of antibiotics in developing
Abbreviations: GAS, group A streptococcal; SpeA, streptococcal pyrogenic exotoxin A; SpeB, streptococcal pyrogenic exotoxin B; SpeAB, recombinant fusion protein of SpeA and SpeB. ∗ Corresponding author at: 116, Research Drive, Bethlehem, PA 18015, United States. Tel.: +1 610 573 9620. E-mail address: garry.morefi[email protected] (G. Morefield).
countries and the emergence of antibiotic-resistant strains the need is great for a safe and effective vaccine for the prevention of GAS disease [5–8]. A recombinant fusion protein antigen (SpeAB) was engineered combining Streptococcal pyrogenic exotoxin A (SpeA) with Streptococcal pyrogenic exotoxin B (SpeB) from S. pyogenes [9]. SpeA is a superantigen which enables bacteria to evade immune responses by disabling recognition by adaptive immunity [10]. SpeA crosslinks major histocompatibility complex class II molecules with T-cell receptors resulting in polyclonal activation of T cells. The systemically increased levels of inflammatory cytokines can induce toxic shock [11,12]. The SpeA utilized in the fusion protein antigen was genetically mutated at the leucine located at position 42 disabling superantigen activity. SpeB is an extracellular cysteine protease that is expressed by most M protein serotypes of S. pyogenes. SpeB is not homologous in structure to SpeA and has no superantigen functionality [13]. SpeB is not only secreted but also retained at the bacterial cell surface where it actively binds glycoproteins [14]. Analysis of sera obtained from patients with invasive GAS infection has demonstrated SpeB specific antibody production [15]. SpeB utilized in the fusion protein antigen was genetically mutated at the cysteine located at position 47 inactivating the enzyme [16]. There is high risk in development of novel vaccine candidates as evidenced by the failure of the majority of novel vaccines at the
http://dx.doi.org/10.1016/j.vaccine.2014.04.092 0264-410X/© 2014 Published by Elsevier Ltd.
Please cite this article in press as: Morefield G, et al. Development of a recombinant fusion protein vaccine formulation to protect against Streptococcus pyogenes. Vaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.04.092
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preclinical and early clinical stages of development [17]. It is critical to initiate formulation investigations early in development to understand the critical quality attributes of the vaccine and how to optimize those parameters for production of a robust product. In order to gain knowledge of the formulation factors that may impact potency and stability of the SpeAB antigen in the final formulation we employed a rational, scientific approach to development [18]. Through this approach the biophysical characteristics of the antigen are determined, potential excipient stabilizers are evaluated, the interactions with adjuvants are optimized, and stability of the final product is investigated. Biophysical characterization studies employ a variety of analytical techniques for determining how formulation attributes such as pH, buffer species, and stabilizers impact the physical state of the antigen [19,20]. The formulation components and attributes selected from the biophysical characterization studies affect the interaction of antigen with adjuvants that may be incorporated into the vaccine. For the SpeAB vaccine aluminum adjuvants were investigated for enhancement of the immune response. Primary mechanisms of adsorption of antigens to aluminum adjuvants are electrostatic interactions, hydrophobic interactions, and ligand exchange [21–23]. Formulation characteristics impact the balance of these forces influencing the adsorption state of the antigen and the strength those interactions. Both the adsorption state and strength of binding may affect potency of vaccines [24–26]. Therefore, it is important to understand what the adsorption state is and how that influences the potency of the vaccine. Finally, it is important not just to understand these parameters at a single point in time immediately following production of the vaccine, but to understand if any changes occur in the formulation over time which may adversely impact the potency of the SpeAB antigen. Therefore, the stability of SpeAB was investigated under typical refrigerated storage conditions as well as stressed conditions of elevated temperature. Stability data confirms the appropriate quality attributes were optimized during development of the vaccine. By following this approach to vaccine development the traditional risks associated with early phase development are minimized. This allows for rapid development of a safe and effective vaccine able to reduce the burden of GAS disease in the general population.
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Solutions of 100 g/ml SpeAB were prepared at pHs 7.0–8.0 with 20 mM Tris (Sigma, St. Louis, MO) or phosphate buffer (Sigma, St. Louis, MO). Ultraviolet spectra from 240 to 380 nm were obtained using a BioSpec 1601 spectrophotometer (Shimadzu, Japan) between 25 and 80 ◦ C in 2.5 ◦ C increments. The samples were incubated at each temperature for 5 min prior to obtaining the spectra. The second derivative with a triangular smooth was applied to the data and peak minima associated with the phenylalanine, tyrosine, and tryptophan residues were recorded. The Tm for each buffer condition was determined by calculating the inflection point of the curve on a plot of peak minima versus temperature. For stabilizer evaluation, 100 g/ml SpeAB was prepared in 20 mM Tris with or without 10% added sucrose or mannitol. Analysis by ELISA was performed on samples diluted 25 fold in 20 mM Tris, 150 mM NaCl, pH 9. 100 l was placed in duplicate in the appropriate well of a 96 well plate. Standards were prepared by performing six, 2-fold serial dilutions starting at 4 g/ml. The plate was incubated for 1 h at 37 ◦ C and then washed three times with 100 l/well of PBS (20 mM PO4 , 150 mM NaCl, pH 7.4). The plate was blocked with 100 g/ml BSA in PBS. Following washing, 100 l/well
of a 1:25,000 dilution of rat anti-SpeAB antisera was added to the plate. After incubation and washing 100 l/well of a 1:70,000 dilution of goat anti-rat IgG-HRP (Rockland, Gilbertsville, PA) was added to the plate and the plate was incubated as before. The plate was washed and 100 l/well of TMB (KPL, Gaithersburg, MD) was added and the plate was incubated for 20 min at room temperature. The reaction was stopped by addition of 100 l/well 3 M H2 SO4 and the absorbance read at 450 nm using a UVmax plate reader (Molecular Devices, Sunnyvale, CA). 2.2. Interactions with aluminum adjuvants Aluminum hydroxide adjuvant (Alhydrogel® , Brenntag, Denmark) was treated with phosphate at P:Al molar ratios of 0–2. After combination, the adjuvant and phosphate were mixed for 1 h at room temperature. Formulations were prepared containing 50 g/ml SpeAB and 1.7 mg/ml Al and mixed for 30 min at room temperature. The formulations were then centrifuged at 10,000 × g RCF for 5 min and the supernatant was collected. Finally, the protein concentration of the supernatant was determined by BCA assay (Pierce, Rockford, IL) and the percent adsorbed calculated by mass balance. In association with the in vivo potency study, a short term stability study at 5 ◦ C was performed by monitoring aspect, pH, and percent adsorption of the adjuvanted formulations on days 0 and 14. The pH of each formulation was measured using a Symphony pH meter (VWR, Radnor, PA). The percent adsorption was determined as previously described. The aspect of each formulation was evaluated by visual inspection of the vials in front of both a white and black background. 2.3. In vivo potency The potency of SpeAB formulations were determined in 7–8 week old female BALB/c mice (Harlan, Indianapolis, IN). There were 10 mice per group in those receiving SpeAB and 5 mice per group in those receiving adjuvant only. Mice were administered 0.1 ml of vaccine IM on days 0 and 14 of the study. Sera were collected from mice on days 14 and 28. To determine IgG titer 96 well plates were coated overnight at 4 ◦ C with 100 l of 10 g/ml SpeAB in 0.1 M carbonate buffer at pH 9.6. Plates were washed three times with 0.05% Tween in PBS (PBS-T). The plates were blocked with 200 l of 1% (w/v) BSA/PBS-T per well for one hour at 37 ◦ C. After washing, serial dilutions of serum samples in BSA/PBS-T was added in triplicate and incubated for 2 h at 37 ◦ C. Following washing 100 l of 1:5000 diluted peroxidase-conjugated goat anti-mouse IgG (Sigma, St. Louis, MO) was added. The plates were washed and 100 l TMB (Sigma) was added. After incubation at room temperature for 20 min, 50 l of 2 M sulfuric acid was added to stop the reaction. The absorbance was read at 450 nm in a BioTEK synergy HT microplate reader (BioTEK, Winooski, VT). Titers were calculated as the dilution at which the OD reading reached 0.2. To determine if the differences between groups were statistically different, logtransformed titers were analyzed by one-way ANOVA with post hoc analysis by Tukey’s multiple comparison test. Differences were considered significant at p < 0.05. To determine the neutralizing antibody response serum samples were heat-inactivated for 30 min at 56 ◦ C. They were diluted 1:25 and incubated for 1 h at 37 ◦ C with SpeA (Toxin Technology, Sarasota, FL) at 80 ng/ml. Human peripheral blood mononuclear cells (PBMC; Zen-bio, Research Triangle Park, NC) were diluted to 2 × 106 cells/ml in RPMI1640 supplemented with 10% fetal calf serum 100 U/ml penicillin, 100 g/ml streptomycin, and 0.25 g/ml amphotericin B and added to the wells of a 96 well plate at 100 l/well. Triplicate wells 20 ng/ml SpeA (positive control), medium only (negative control), or 20 ng/ml mixed with
Please cite this article in press as: Morefield G, et al. Development of a recombinant fusion protein vaccine formulation to protect against Streptococcus pyogenes. Vaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.04.092
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mouse serum. After 48 h incubation at CO2 , the supernatants were collected and analyzed by ELISA for the concentration of IFN␥. The neutralizing activity was calculated as 100 − (IFN␥ sample/IFN␥ control) × 100. The statistical significance of differences of the group means was determined by one-way ANOVA with post hoc analysis by Tukey’s multiple comparison test. Differences were considered significant at p < 0.05.
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Fig. 1. The stability of SpeAB in 20 mM Tris or phosphate buffers between pH 7 and 8 was determined following storage for 2 days at 45 ◦ C.
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The pH/stability profile of SpeAB was investigated at accelerated temperature conditions in both phosphate and Tris buffered saline systems. Prior to initiating the investigation the melting temperature of the antigen in both buffers was determined by derivative spectroscopy. The lowest melting temperature observed for all formulations was 55 ◦ C (data not shown). Therefore, a temperature of 45 ◦ C was selected to determine the pH/stability profile to minimize the impact of physical transitions of the antigen that occur above the melting point. The stability of SpeAB solutions was evaluated following 2 days of storage at 45 ◦ C and pHs 7.0, 7.5, and 8.0 (Fig. 1). The temperature sensitivity of the pH of Tris buffered solutions was accounted for in preparation of the study samples. There were no significant differences in the concentration of SpeAB remaining between the two buffer systems at any of the pHs investigated. However, there was significantly more SpeAB remaining at pH 7.5 and 8 than at pH 7. While the concentration of SpeAB at pH 8 appears to be higher than at 7.5, this difference was not found to be statistically significant. For subsequent investigations the Tris buffered system at pH 8 was selected to maximize stability of the antigen in solution as well as to minimize sources of phosphate, which has the potential to interact with aluminum adjuvants in the final formulations. To investigate further enhancement of stability formulations containing added sucrose or mannitol were monitored at accelerated temperature. (Fig. 2) The data demonstrated that the addition of either sucrose or mannitol significantly enhanced the stability of SpeAB in solution over buffer alone. By day 9 nearly all of the SpeAB in the formulation without stabilizer had degraded, while the formulations containing stabilizer had nearly all of the SpeAB intact. This suggests either sucrose or mannitol would be appropriate stabilizers to include in the final vaccine formulation.
Fig. 2. Addition of 10% sucrose or mannitol to SpeAB in 20 mM Tris buffer pH 8 significantly enhances antigen stability at 45 ◦ C.
3.2. Interactions with aluminum adjuvants
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To investigate the interactions of SpeAB with aluminum adjuvants a series of aluminum hydroxide adjuvants with increasing levels of phosphate treatment were prepared to modify the adjuvant surface charge. The SpeAB was then formulated with each of these adjuvants and the level of adsorption was determined (Fig. 3). The level of SpeAB adsorbed to the adjuvant was inversely proportional to the amount of phosphate treatment applied to the aluminum hydroxide adjuvant. This suggests that electrostatic interactions are one of the primary adsorption forces in the formulation. However, since the adsorption level did not approach 0% until the phosphate treatment ratio approached 2:1, this suggests that other adsorption forces such as hydrophobic interactions are also present in the system. Once the mechanism of SpeAB interaction with aluminum adjuvants was better understood, investigations were performed to determine conditions for adsorbed and non-adsorbed formulations in the sucrose stabilized buffer system. As before, formulations were prepared utilizing aluminum hydroxide adjuvants that had been modified with phosphate (Fig. 3). The interaction of SpeAB with the aluminum adjuvants in the sucrose stabilized formulations followed the same trend as in the non-stabilized formulations. Target adsorption levels for adsorbed (>80%) and non-adsorbed (20% adsorbed Homogeneous suspension of white particles 7.5–8.5 >20% adsorbed Homogeneous suspension of white particles 7.5–8.5 >20% adsorbed
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0.4, 2, or 5 g SpeAB on days 0 and 14. A short term stability study bracketing the vaccine administration dates was also performed to ensure attributes of the various vaccine formulations, such as aspect, pH, and percent adsorption, did not change significantly over the course of the potency evaluation (Table 1). Results from the stability study demonstrate all formulations remained within their target values for the duration of the study. Sera was obtained on days 14 and 28 and analyzed for antigen specific immune responses. Serum titers of anti-SpeAB IgG were dose dependent (Fig. 4). Both adsorbed and non-adsorbed
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Experimental groups Fig. 5. The functional activity of the mouse anti-sera to neutralize wild type SpeA toxin was evaluated by monitoring the inhibition of INF␥ production by human peripheral blood mononuclear cells following incubation with anti-sera/toxin mixtures.
Please cite this article in press as: Morefield G, et al. Development of a recombinant fusion protein vaccine formulation to protect against Streptococcus pyogenes. Vaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.04.092
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4. Discussion
Acknowledgments
Using a rational, scientific approach we were able to develop a potent and stable vaccine targeting GAS disease. Through biophysical characterization and accelerated stability studies it was determined that SpeAB was most stable near pH 8 in both Tris and phosphate buffers. Tris was selected as the formulation buffer since the pKa of Tris is closer to 8 than that of phosphate, resulting in increased buffer capacity. Additionally, Tris has a lower potential to impact adsorption of SpeAB with aluminum adjuvants so the adsorption state of the antigen can be more easily controlled in the final product. When investigating further stabilization of SpeAB sucrose and mannitol were selected based on their abilities to act as stabilizing osmolytes. The principle mechanism of stabilization of proteins by these excipients is through a process called preferential exclusion [27]. In this process the solute, such as sucrose, is excluded from the immediate domain of the protein, thereby maintain greater hydration of the antigen. When accelerated stability studies were performed it was determined that both sucrose and mannitol were able to significantly enhance the stability of SpeAB to an equivalent magnitude. Therefore, either excipient would be suitable for use in the final formulation. To determine if the adsorption state of SpeAB impacted potency formulations in which the antigen was adsorbed or remained in solution were produced. Based on the amino acid sequence of SpeAB it was anticipated that at the formulation pH of 8 there should be electrostatic attractive forces present between the antigen and aluminum hydroxide adjuvant. The data demonstrated that this was the case as the majority of SpeAB was adsorbed in the formulation with aluminum hydroxide adjuvant. Adsorption decreased as phosphate treatment level increased inducing electrostatic repulsive forces in the system. However, the low slope of the curve indicates that other forces such as hydrophobic interactions or hydrogen bonding are acting in the formulation as well. Another possibility since SpeAB is a fusion protein and SpeA has an acidic pI while SpeB has a basic pI is that there is directional electrostatic adsorption occurring in the formulation. The directional adsorption may allow SpeAB to remain adsorbed to the surface of the aluminum adjuvant in the present of some electrostatic repulsive forces. The potency of the adsorbed and non-adsorbed formulations was evaluated in mice at three different doses. The results of the potency study indicate that the presence of an aluminum adjuvant significantly enhances SpeAB IgG titer as well as functional antibody responses. The immune response was further enhanced when SpeAB was adsorbed to the aluminum adjuvant in the formulation and of the three doses tested 5 g induced the greatest potency. The SpeAB vaccine developed here has great potential to protect against GAS disease. The cysteine protease SpeB has been found to be broadly expressed in many clinical isolates of GAS [28,29]. There have been reports that point mutations in csrRS result in reduced production of speB [30]. However, while there may be a decreased expression of SpeB in these mutants there is not a lack of expression. Therefore, the SpeB portion of the antigen maintains the potential to provide protection against a broad range of GAS serotypes. While the super antigen, SpeA, is typically found in a smaller percentage of circulating strains of S.pyogenes it is associated with serotypes causing invasive disease [31–33]. The SpeAB antigen is able to induce a protective response in mice against the superantigen demonstrating its potential to reduce the prevalence of invasive disease. By combining these two antigens into a single vaccine there is the potential to significantly decrease the burden of both invasive and non-invasive GAS disease globally.
This work is supported by the US Army Medical Research and Q3 Materiel Command under Contract No.W81XWH-12-C-0183. The views, opinions and/or findings contained in this report are those of the author(s) and should not be construed as an official Department of the Army position, policy or decision unless so designated by other documentation. In conducting research using animals, the investigator(s) adhered to the Animal Welfare Act Regulations and other Federal statutes relating to animals and experiments involving animals and the principles set forth in the current version of the Guide for Care and Use of Laboratory Animals, National Research Council. The authors thank Dr. Robert Ulrich for the gift of SpeAB fusion protein. References [1] W.H.O. http://www.who.int/child adolescent health/documents/ivb 05 14/ en/ [2] Centers for Disease Control and Prevention. Online, Available at: http://www. cdc.gov/ncidod/dbmd/diseaseinfo/groupastreptococcal t.htm. [3] Mell LK, Davis RL, Owens D. Association between streptococcal infection and obsessive-compulsive disorder, Tourette’s syndrome, and tic disorder. Pediatrics 2005;116(July (1)):56–60. [4] Leckman JF, King RA, Gilbert DL, Coffey BJ, Singer HS, Dure 4th LS, et al. Streptococcal upper respiratory tract infections and exacerbations of tic and obsessive-compulsive symptoms: a prospective longitudinal study. J Am Acad Child Adolesc Psychiatr 2011;50(February (2)):108–18. [5] Reinert RR, Lutticken R, Al-Lahham A. High-level fluoroquinolone resistance in a clinical Streptoccoccus pyogenes isolate in Germany. Clin Microbiol Infect 2004;10:659–62. [6] Buxbaum A, Forsthuber S, Sauermann R, Gattringer R, Graninger W, Georgopoulos A. Development of macrolide-resistance and comparative activity of telithromycin in streptococci in Austria, 1996–2002. Int J Antimicrob Agents 2004;24:397–400. [7] Stevens DL. Streptococcal toxic shock syndrome associated with necrotizing fasciitis. Annu Rev Med 2000;51:271–88. [8] Musser JM, Krause RM. The revival of group A streptococcal diseases with a commentary on staphylococcal toxic shock syndrome. In: Krause RM, Fauci A, editors. Emerging infections New York: Academic Press; 1998. p. 185–218. [9] Ulrich R. Vaccine based on a ubiquitous cysteinyl protease and streptococcal pyrogenic exotoxin A protects against Streptococcus pyogenes sepsis and toxic shock. J Immune Based Ther Vaccine 2008;6:1. [10] Ulrich RG, Bavari S, Olson M. Bacterial superantigens in human diseases: structure, function and diversity. Trends Microbiol 1995;3:463–8. [11] Norrby-Teglund A, Pauksens K, Norgren M, Holm SE. Correlation between serum TNF alpha and IL-6 levels and severity of group A streptococcal infections. Scand J Infect Dis 1995;27:125–30. [12] Stevens DL, Bryant AE, Hackett SP, Chang A, Peer G, Kosanke S, et al. Group A streptococcal bacteremia: the role of tumor necrosis factor in shock and organ failure. J Infect Dis 1996;173:619–26. [13] Kapur V, Topouzis S, Majesky MW, Li LL, Hamrick MR, Hamill RJ, et al. A conserved Streptococcal pyogenes extracellular cysteine protease cleaves human fibronectin and degrades vitronectin. Microb Patho 1993;15:327–46. [14] Hytonen J, Haataja S, Gerlach D, Podbielski A, Finne J. The SpeB virulence factor of Streptococcus pyogenes, a multifunctional secreted and cell surface molecule with strepadhesin, laminin-binding and cysteine protease activity. Mol Microbiol 2001;39:512–9. [15] Akesson P, Rasmussen M, Mascini E, von Pawel-Rammingen U, Janulczyk R, Collin M, et al. Low antibody levels against cell wall-attached proteins of Streptococcus pyogenes predispose for severe invasive disease. J Infect Dis 2004;189:797–804. [16] Gubba S, Low DE, Musser JM. Expression and characterization of group A Streptococcus extracellular cysteine protease recombinant mutant proteins and documentation of seroconversion during human invasive disease episodes. Infect Immun 1998;66:765–70. [17] Douglas RG, Sadoff J, Samant V. The vaccine industry. In: Plotkin S, Orenstien W, Offit P, editors. Vaccines UK: Elsevier; 2008. p. 37–44. [18] Morefield G. A rational systematic approach for the development of vaccine formulations. AAPS J 2011;13(2):191–200. [19] Peek LJ, Brey RN, Middaugh CR. A rapid, three-step process for the preformulation of a recombinant ricin toxin A-chain vaccine. J Pharm Sci 2007;96: 44–60. [20] Peek LJ, Brandau DT, Jones LS, Joshi SB, Middaugh CR. A systematic approach to stabilizing EBA-175 RII-NG for use as a malaria vaccine. Vaccine 2006;24:5839–51. [21] Seeber SJ, White JL, Hem SL. Predicting the adsorption of proteins by aluminium-containing adjuvants. Vaccine 1991;9:201–3. [22] Al-Shakhshir RH, Regnier FE, White JL, Hem SL. Contribution of electrostatic and hydrophobic interactions to the adsorption of proteins by aluminumcontaining adjuvants. Vaccine 1995;13:41–4.
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