Received date: November 01, 2015; Accepted date: December 18, 2015; Published date: December 21, 2015
Citation: Suvorov A, Dukhovlinov I, Leontieva G, Kramskaya T, Koroleva I, et al. (2015) Chimeric Protein PSPF, a Potential Vaccine for Prevention Streptococcus pneumoniae Infection. J Vaccines Vaccin 6:304. doi: 10.4172/2157-7560.1000304
Copyright: © 2015 Suvorov A, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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A chimeric protein, designated PSPF (Pneumococcus Surface Proteins and Flagellin) was constructed from conservative and immunogenic fragments of S. pneumoniae surface proteins PspA, Spr1875, PsaA and the Salmonella typhiurium flagellin terminal domains FliC1, FliC2 functioning as adjuvant. PSPF was highly immunogenic in mice and induced protection from virulent S. pneumonia strains. All the subtypes of S. pneumoniae under study were recognized by anti-PSPF sera. Thus, the chimeric recombinant PSPF protein is a promising vaccine candidate for immunization of humans against S. pneumoniae.
According to WHO data, Streptococcus pneumoniae is among the most common bacterial infections responsible for mortality among children in developing countries [1,2]. S. pneumoniae is also the most often cause of bacterial pneumonia, meningitis, bacteremia, bronchitis, sinusitis and otitis media. In Europe and the US, the annual incidence of invasive pneumococcal disease ranges from 10 to 100 cases per 100000 of the population . S. pneumoniae isolates are covered by a thick polysaccharide capsule, which provides protection against opsonization and phagocytosis. Pneumococcal capsule polysaccharides (PSs) are the first surface elements recognized by an immune system and characterized by high antigenic variability. At present 91 S. pneumoniae serotypes have been discovered and the number is constantly growing . The increasing frequency of the pneumococcal isolates resistant to commonly used antibiotics complicates control of pneumococcal infections [4-6]. Current pneumococcal vaccines are all based on PSs antigens. Therefore, the effect of a polysaccharide-based vaccine is restricted to the PS serotypes included in the vaccine, and the coverage varies due to different geographical serotype distributions . Another disadvantage of PS vaccines are based on the weak immunological memory . In this respect, surface proteins of S. pneumoniae which are also exposed and may be reached by the opsonising antibodies are advantageous vaccine candidates due to the smaller degree of variability and higher immunogenicity.
The goal of the work was to study immunogenicity and preventive effect of chimeric protein, composed of the fragments of three surface proteins of S. pneumoniae. The choice of conservative and immunogenic fragments of surface proteins PsaA, PspA and Spr1875 to include in the chimeric vaccine was based on previous studies, which reported their ability to induce protective antibodies [9-13]. PsaA is a highly conservative lipoprotein expressed by several serotypes and essential for virulence of S. pneumoniae . PspA (Pneumococcal surface protein A) is a surface protein known to contribute to S. pneumonia virulence by binding human lactoferrin and by impeding complement activation in vivo and in vitro [10,11]. The surface protein Spr1875 (also known as LysM domain-containing protein) exhibits immunogenic properties and contributes to S. pneumoniae virulence due to its role in the development of pneumococcal meningitis [12,13].
Salmonella typhiurium flagelin FliC N- and C-terminal fragments known to interact with Toll-like receptor-5 (TLR-5) were included into the vaccine molecule as an activator of innate and adaptive immune responses [14,15]. All these components assembled in one molecule were expressed in E.coli system, isolated and tested for immunogenicity and the ability to provide the appearance of protective IgG.
Modeling the recombinant chimeric protein, artificial gene and expression plasmid
Amino acid sequence of chimeric protein based on PspA, Spr1875, PsaA and FliC fragments, separated with appropriate flexible bridges (called PSPF) was designed in silico according to the previously accumulated knowledge of the immunogenic domains and putative three dimensional structures. Nucleotide sequence of the chimeric protein was optimized for expression in E. coli (Figures 1, 2). Synthesis of nucleotide sequence of PSPF was performed by elongation of the overlapping oligonucleotides . The synthetic chimeric gene was cloned in pET24b+ vector (Invitrogen), producing the expression plasmid pPSPF, which was transformed into E. coli BL21 (Invitrogen). The gene encoding for chimeric protein devoid of S.typhimurium flagellin fragments (PSP) was inserted into pET25b+ giving expression plasmid pPSP, which was also transformed into E. coli BL21(DE3).
Figure 1: The sequence and structure of the chimeric protein PSPF. A) Schematic representation of the immunogenic domains in the protein molecule1-16 T7-tag (from pET24b+), 17-185 N-terminal fragment 34 of flagellin FliC (green), 186-197 GC-flexible bridge, 198-292 C-terminal fragment of flagellin FliC (green), 293-296 GC-flexible bridge, 297-402 PspA (blue), 403-406 GC-flexible bridge, 407-474 Spr1875 (brown), 475-479 GC-flexible bridge, 480-552 PsaA (red), 553-560 His-tag (from pET24b+), Flexible bridges and tags are underlined, In PSP protein the amino acids 17-296 were deleted, The predicted 3D- structure of PSPF shown in α-helixes and β-layers.
Artificial protein structure prediction
The sequence of the designed artificial protein PSPF was virtually cut into domains and the 3D structures of these domains were found in public databases or ab initio. The final refinement of the overall 3D structure was performed employing I-Trasser algorithm . The reliability of the structure prediction by I-Trasser is 70%. Prediction of intrinsic disorder and disorder-based binding sites of the designed artificial protein PSPF and its parent proteins were evaluated by several predictors of the PONDR family, such as PONDR® VLXT , PONDR® VSL2 , PONDR® VL3 , and PONDR® FIT . Disorder-based binding sites in the PSPF protein were identified by the ANCHOR algorithm .
Chimeric proteins expression and purification
Recombinant E.coli with plasmids encoding for PSPF and PSP were grown in PYP5052 medium containing 0.2% lactose according to Studier’s method . The proteins PSPF and PSP were extracted from the E. coli cells by cell lysis in 20 mM Tris-HCl pH 7.5, 5 mM EDTA, 1 mM PMSF and sonication (7 rounds of 30 sec pulses with 30 sec pauses, ultrasound frequency 22 kHz). Bacterial lysates were centrifuged and the pellets were suspended in 1M urea. After the next centrifugation the pellets were suspended in 2M urea and centrifuged again. The pellets were dissolved in 20 mM Na-phosphate buffer, with 0.5M NaCl, 6M guanidine-HCl, pH 8.0 and subjected to Ni-NTA (Invitrogen) purification chromatography according to producer's protocol. After final dialysis against 20 mM tris-HCl pH 7.5, 5 mM EDTA and 1 mM PMSF the obtained products contained the chimeric proteins PSPF and PSP with purity of 97% and concentration 1 mg/ml with LPS concentration not exceeding 0.016 EU/mg evaluated by Endosafe LAL assay (Charles River).
S. pneumoniae strains
S. pneumoniae clinical isolates were obtained from the collection of the Research Institute of Pediatric Infections (St. Petersburg, Russia) and two strains were purchased from ATCC (ATCC® 6319™ type 19, ATCC BAA-1667-type 6) (Table 1). Pneumococci were cultured in anaerobic conditions at 37°Ð¡ for 18 hours in THB medium with 20% horse serum (Difco). Schaedler agar with sheep red blood cells was used as a solid medium.
Table 1: Streptococcus pneumonia strains used in the study.
The study was performed using BALB/c inbred mice (female, 8 weeks old, 18-20 g) obtained from «Pushino» nursery. All animals were housed in illuminated and temperature controlled conditions. Mice were offered standard commercial feed and water ad libidum and treated according to the Guide for the Care and Use of laboratory animals of the Institute of Experimental Medicine.
The experimental design
The animal study of vaccine described in Tables 2-3. In short, three groups of animals were injected with three different dosages of vaccine. Two groups used as controls: the reference vaccine group and a group of mice received placebo.
|Immunogen||Groups||The first immuni-zation dose||The second immuni-zation dose||The mode of
S. pneumonia? infection
|Number of animals|
|PSPF + adjuvant||1||5 µg/mouse||2.5 µg/mouse||intraperitoneally||10|
|2||10 µg/mouse||5 µg/mouse||intraperitoneally||10|
|3||20 µg/mouse||10 µg/mouse||intraperitoneally||10|
|Adjuvant only||4||No PSPF(control group)||intraperitoneally||10|
|z||5||1 vial/8 mice||1vial /16 mice||intraperitoneally||10|
Table 2: Scheme of immunization.
|Day 0||The first vaccination: groups 1-3, 5
Adjuvant injection in control group: group 4
|Day 21||Taking the blood from mice to identify specific antibodies (IgG) titer|
|Day 22||The second vaccination (½of the first vaccination dose): groups 1-3, 5 Adjuvant injection in control group: group 4|
|Day 35||Taking the blood from mice to identify specific antibodies (IgG) titer|
|Day 42||Taking the blood from mice to identify specific antibodies (IgG) titer|
|Day 49||Taking the blood from mice to identify specific antibodies (IgG) titer|
|Day 50||Intranasal infection with S.pneumoniae strain 73 serotype 3|
|Day 53||Intraperitoneal infection with S.pneumoniae strain 442 serotype 19F|
|Days 54-63||Monitoring of the animals survival|
Table 3: Schedule of immunization.
Human pneumococcal conjugate vaccine (Prevnar 7) containing 2 µg of each polysaccharide of 4, 6B, 9V, 14,18C, 19F, 23F serotypes and 20 micrograms of carrier protein CRM197 per dose was used as reference vaccine.
The route of administration
Chimeric proteins PSPF and PSP and reference vaccine were administered subcutaneously. Each injection of chimeric proteins contained 0.1 ml of the adjuvant Imject® Alum (Thermo Scientific) and 0.1 ml of the protein solution in PBS buffer. A placebo group of mice were injected with 0.1 ml of PBS and 0.1 ml of adjuvant. A reference vaccine group immunized with Prevnar 7 in proportion 1 human dose per 8 mice.
Serum IgG titer detection
Blood was collected from submaxillary vein. Specific IgG levels were determined by ELISA in 96-well ELISA plates (Nunc) coated overnight at 4°C with the PSP or PSPF proteins. The diluted sample (100 µl) was added to duplicate wells and incubated 1 h at 37°Ð¡. HRP-labeled goat anti-mouse IgG antibodies (Sigma) diluted in blocking buffer were added (100 µl/well). After incubation at room temperature for 1 hr, plates were developed with TMB substrate (BD Bioscience) according to the manufacturer’s instructions. ELISA endpoint titers were defined as inverse value of the highest dilution that yielded an OD value above 2 times the mean of negative control wells.
Interaction of PSPF- specific sera with different S. pneumoniae serotypes
The study was performed using dot -blot assay on nitrocellulose membranes. Daily cultures of S. pneumoniae were washed in PBS and three microliters of each bacterial suspension were applied to nitrocellulose membranes and dried. The membrane was incubated in blocking buffer (5% dry milk dissolved in PBS pH 7.4). After the incubation, membrane was treated with mice sera containing PSPF-specific antibodies with titer 1:400000 diluted 1000 times in blocking buffer. Membrane was placed in a conjugate solution (anti-mouse IgG (Fc-specific)-peroxidase). Color was developed in ready to use TMB Liquid Substrate System for Membranes (Sigma) for 3-5 minutes.
Estimation of S. pneumoniae lethal doses
Bacterial aliquots of S. pneumoniae were inoculated into mice by intraperitoneal or intranasal administration. Survival level was monitored for 10 days. The minimum bacterial doses which caused the death of the 50 or 100% of the animals were evaluated as LD50 or LD100.
Protective efficacy of recombinant vaccine
Ten S. pneumoniae strains belonging to ten different serotypes were analyzed (Table 1). Only two of these, strain 442 (serotype 19F) and strain 73 (serotype 3) were highly virulent for the mice and were used to investigate the vaccination effectiveness in lethal models of pneumococcal infection. All bacterial strains were tested for recognitions by PSPF-specific IgG antibodies in dot-blot ELISA.
Vaccinated and controlled mice were infected with virulent strains by intraperitoneal or intranasal administration on the 50th day after the first immunization. Intraperitoneal infection was induced with LD100 of the strain 442 (serotype 19F). One infective dose contained 3 × 102 CFU of the pathogen. Intranasal infection was induced with LD50 of the strain 73 (serotype 3). One infectious dose contained 1 × 107CFU of the pathogen. Each experimental group included 8-10 mice.
Data analysis included the estimation of the medium values and the standard deviation (M ± σ). Statistical relevance was estimated employing t-test by Student or U-test by Mann- Whitney. Differences were considered significant at p<0.05.
The survival curves of mice were compared for significance using the log-rank test. Differences were considered significant at p<0.05
Molecular design of artificial chimeric protein PSPF
Developed chimeric recombinant protein PSPF contains the fragments of S. pneumonia proteins PspA, PsaA, Spr1875 and S. typhimurium flagellin FliC. PspA is a surface protein showing high heterogeneity between pneumococcal strains . In S. pneumonia PspA varies in size from 439 to 649 amino acids and contains three major domains: the N-terminal 288 amino acid functional helical domain, 82 amino acid flexible proline-rich segments, and the C-terminal choline-binding domain responsible for cell wall binding. Protective Mabs against PspA predominately recognize the fragment between 192-260 amino acids of PspA [25,26]. Based on these data the conservative fragment of PspA corresponding to this region was included into chimeric protein (Figure 1).
Fragment containing amino acids from of the A-chain of the surface protein PsaA (480-552 in the protein as shown in Figure 1) was included into chimeric protein because this fragment contains epitopes that are immunogenic and protective in mice .
Spr1875 fragment of amino acids from corresponding to the amino acids from 94 to 162 in the original protein contains the epitopes immunogenic in mice and devoid of presumptive immunosuppressive regions located in the C-terminal part of the molecule was also included into chimeric protein . All these fragments of S, pneumoniae proteins and two immunostimulatory S.typhimurium flagellin FliC N- and C-terminal fragments, responsible for TLR5 signaling [28,29] were connected with appropriate GS-flexible bridges. The total length of PSPF is 560 amino acids (Figure 1A).
Analysis of the amino acid sequence of this protein performed with ProtParam program  has shown that the molecular weight of the chimeric protein is 59.3 kDa, pI is 4.71. The predicted 3D-structure of the obtained protein vaccine (Figure 1B) shows that it contains two domains; one of them is composed of S. pneumoniae protein fragments and the second-of flagellin N- and C-termini. The structure obtained predicts that flagellin termini will have no constrains in their interaction with TLR-5.
According to NCBI Reference Sequence Database  the fragments of conservative surface proteins included into the PSPF were present in the majority of S. pneumonia strains and when applied for immunization could be protective against the most part of pneumococcal strains.
Intrinsic disorder in PSPF and parent proteins
Computational analysis revealed that all parent proteins included in PSPF contain significant amounts of predicted intrinsic disorder. A multi-parametric approach for the prediction of the overall predisposition revealed that chimeric PSPF protein also possesses high degree of intrinsic disorder. Figure 2 illustrates the disorder-based “assembly” of the designed artificial protein by representing the PONDR® VSL2-based disorder profiles of the parent FliC, PspA, Spr1875, and PsaA proteins together with the disorder propensity plot of the resulting chimeric PSPF protein. Figure 2 clearly shows that similar to the parent proteins, the hybrid PSPF protein is expected to be mostly disordered. Curiously, distributions of the disorder propensities in the FliC, PspA, Spr1875, and PsaA regions incorporated in this protein differ significantly from distribution of their disorder predispositions within the corresponding parent proteins, indicating that disorder propensity is content-dependent.
The majority of the PONDR curves (Figure 3A) are located above the 0.5 threshold, thereby indicating that the PSPF protein is expected to be highly disordered, since scores above 0.5 are considered to correspond to the disordered residues/regions. Often, intrinsically disordered proteins and regions are involved in protein-protein interactions and molecular recognitions there are numerous reports emphasizing that these proteins are able to undergo at least partial disorder-to-order transitions upon binding, which is crucial for recognition, regulation, and signaling [18,32]. The results of the analysis employing ANCHOR algorithm shown in Figure 3B indicate that this protein is expected to have at least 9 disorder-based binding sites (residues 20-29, 65-73, 211-216, 260-306, 339-382, 388-459, 473-483, 514-523, and 536-547). These regions have significant potential to be binding sites for an appropriate but typically unidentified partner protein.
Effect of FliC portion of the vaccine on immunogenicity
To compare the immunogenicity of PSPF and PSP, the mice were immunized with both proteins in the doses of 20 µg/mouse for prime and 10 µg/mouse for boost immunizations three weeks later. Figure 4A shows, that the presence of flagellin domain in PSPF increased specific antibody titers by 1.0-1.5 orders of magnitude at all the time points tested. Thus we could suppose that this effect was achieved by the immune-stimulatory activity of the flagellin domain included in PSPF.
Figure 4: Immunogenicity of chimeric proteins in BALB/c mice. A) Stimulation of immune response by PSPF or PSP with or without Salmonella flagellin fragments Mice (n=10) were immunized subcutaneously with the chimeric proteins PSPF and PSP in doses 20 μg and three weeks later with 10 μg. Specific anti-PSP IgG titers on says 35, 42, 49 and 70 were assessed. Results are shown as mean ± SD. *Differencies between the groups immunized by PSPF or PSP were statistically significant on 35, 42 and 49 days after immunization. B) Immunogenicity 95 of PSPF protein in different dosage. Mice (n=10) were immunized subcutaneously with the chimeric protein in doses of 5, 10 and 20 μg and three weeks later with one-half of each dose (boost). Specific IgG titers on days 21, 35, 42, 49 and 70 were assessed. Results are shown as mean ± SD.
Immunogenic properties of the PSPF applied in different doses
After the immunization, the titers of anti-PSPF antibodies in mice sera were determined by regimen indicated in Tables 2 and 3. Stimulation of the immune response in all the experimental groups were detected on the 14th day after the booster vaccination (35th day of the experiment), but the mean dose (10 μg/mouse) provided a statistically significant increase of the humoral immune response level as compared with the other experimental groups. However, starting from day 42 of the vaccination, this group showed the most pronounced downward trend in the quantity of circulating antibodies and after 28 days from the second vaccination (day 49 of the experiment), the level of specific circulating antibodies in all experimental groups was virtually identical. Specific IgG continued to circulate at the same level in all the groups for the following 20 days (Figure 4B).
After the intraperitoneal infection, all the mice from control group (placebo) died on the second day of experiment. Death rates of immunized mice differed among the groups. The best protection was observed in the group of mice immunized with the lowest dose of the vaccine (Figure 5A). Mice vaccinated twice with recombinant vaccine stimulated the development of a specific immune response, which provided 25-40% protection against lethal intraperitoneal infection. In the group of mice immunized with the reference vaccine, the mortality rate was close to the control value-88%. Thus, the level of protection provided by the recombinant pneumococcal vaccine was higher compared to the reference vaccine.
Figure 5: Surviving of animals in different groups after infection. A. Surviving of animals in different groups after peritoneal infection. The X axis shows the time after infection (days), on an axis Y - survival rate of animals (%). Infection was induced with 3x102 CFU/mouse S. pneumoniae strain 442 (type 19F), B) Surviving of animals in different groups after intranasal infection. The X axis shows the time after infection (days), on an axis Y - survival rate of individuals (%). Infection was induced with 1 × 107 CFU/mouse S. pneumoniae strain 73 (type 3). Each experimental group included 10 mice.
Death of the animals after intranasal infection in control group started on the second day and on the third day in the groups immunized with PSPF. The mortality of animals immunized with PSPF was 40% after 10 days of monitoring. Death rate of animals in control group was 63% (Figure 5B). This experiment was repeated three times with different concentrations of vaccine protein. In all the cases the difference was statistically significant. The protective level did not depend on the dose of the vaccine.
Thus, the PSPF immunization showed protective effect against S.pneumoniae intranasal infection.
Interaction of PSPF-specific sera with S. pneumoniae strains listed in Table 1 was studied employing the dot-blot method. The sera recognized all S. pneumoniae strains under study belonging to different serotypes in contrast to control sera taken from non-immunized animals (Figure 6).
At present several vaccines against S. pneumoniae infections were introduced in the public health systems. FDA approved two different types of vaccines: pneumococcal conjugate vaccines and pneumococcal 23-valent polysaccharide vaccine . Polysaccharide vaccines implemented on the market have been proved both efficient and safe but they have several very important disadvantages. As there are over 90 PS serotypes and serotypes distribution differs in geographical areas, it is difficult to create one universal vaccine based only on polysaccharides. Polysaccharides are T-cell independent antigens, inducing short-term immunity without immune memory. Pneumococcal conjugated vaccines due to the carrier protein CRM197 are better recognized by T-cells and induce long-lasting immunity . However, though the CRM197 is over 100 times less toxic than diphtheria toxin, it still retains some toxicity, especially in high doses .
The above considerations advocate for development of the protein vaccines against S. pneumonia. Several proteins have been determined on the cell surface including PsaA, PspA, PcsB, StkP, Spr1875 and many others . Many of them have been proved immunogenic and were able to induce protection against S.pneumoniae in the animal models. It is also determined that vaccines based on the epitopes from several proteins provide superior protection relatively to monovalent protein vaccines. The approach based on several epitopes in one chimeric protein allows avoiding the problem of the strains antigenic variability like in case of using PspA based vaccines, which are impeded by the high degree of genetic mozaicism and epitope variations [24,25]. On the other hand, implementation of the number of separate proteins and adjuvants in vaccine combination makes the vaccine Ð° lot more difficult for standardization and it increases the cost. Major advantage of chimeric protein vaccine beside the cost effectiveness is their simplicity of production and its control, relatively high degree of conservation and T – dependent type of immune memory response [37-39]. These considerations resulted in construction and experimental testing of a new recombinant chimeric immunogenic protein PSPF comprising fragments of pneumococcal conservative proteins PspA, Spr1875, PsaA and flagellin domains (FliC). The molecule was analyzed in silico by several computer engines and several constructs have been generated employing DNA synthesis techniques with the following cloning and expression in the E.coli vectors. Salmonella typhimurium flagellin FliC N- and C-terminal fragments were included as "internal adjuvant" due to their ability to interact with Toll-like receptor-5 (TLR-5). Conserved N- and C- termini of flagellin cooperatively interact to generate a TLR-5 binding site and the middle highly immunogenic and pyrogenic part of the molecule can be deleted without the loss of TLR-5 stimulating activity [28,29].
Including the FliC-domains in PSPF significantly increased the level of the humeral immune response relatively to PSP, which does not possess this region.
Double subcutaneous immunization of mice with PSPF induce development of immune response typical for T-dependent antigens characterized with significant booster effects after the second immunization. We were not able to see the difference after immunizing with three doses of PSPF (20, 10 or 5 µg/dose) and it suggests that PSPF can be applied with less doses and provide little immunologic burden on the immunized individuals.
The ability of the strains under study to bind PSPF-specific antibodies has been tested in vitro employing dot-blot assay. All the strains belonging to the major serotypes were able to precipitate chimeric molecule – specific IgG equally. The ability of the anti-PSPF IgG to react with all S. pneumonia strains under study reflects the broad specificity of the post vaccination immunity.
Protective features of antibodies against PSPF were studied on two S. pneumonia strains, expressing significant virulence in mice. It was discovered that the immunized animals were significantly better protected relatively the control group of animals and group of mice vaccinated by reference vaccine Prevnar. Protection was determined both after nasal or intraperitoneal immunization.
This study has shown the efficiency of new recombinant chimeric protein PSPF which included the PsaA, PspA and Spr1875 epitopes assembled in one molecule as vaccine against S. pneumoniae infection in a mouse model. The PSPF immunization induced specific immune response against the range of S. pneumoniae serotypes and dramatically decreased the mortality after intraperitoneal and intranasal infection. The PSPF immunization showed stronger immune response than chimeric protein PSP without FliC portion and better protective effect in comparison with the reference conjugated pneumococcal vaccine. Results of protection did not depend on the dosage of vaccine, which correlated with similar dynamics of IgG production. Most likely lot smaller doses of PSPF could provide the protection. Thus, the new chimeric protein PSPF can be considered a promising vaccine candidate against S. pneumoniae.
We thank Professor P.P. Cleary and Aiden Griffin for useful remarks and language editing. This work was supported by State contract No 14.613.21.0023.