Pneumococcal surface protein A and new approaches for pneumococcal vaccine development

The problem of pneumococcal infections is pressing for the whole world. Existing vaccines based only on pneumococci polysaccharide antigens or polysaccharide antigens and diphtherial anatoxin are not capable of protecting from all serotypes of the microorganism. Reasonability of creation of pneumococcal vaccine based on surface proteins of Streptococcus pneumoniae is discussed in the literature. One of such key pneumococcal proteins is pneumococcal surface protein A (PSPA), because it is detected in all the S. pneumoniae strains, has cross activity and switches B-cell immune response to T-cell. Currently the development of conjugated vaccine based on surface proteins and capsule polysaccharides of pneumococcus seems promising.     

Pneumococcal Virulence Factors: Structure and Function

The overall goal for this review is to summarize the current body of knowledge about the structure and function of major known antigens of Streptococcus pneumoniae, a major gram-positive bacterial pathogen of humans. This information is then related to the role of these proteins in pneumococcal pathogenesis and in the development of new vaccines and/or other antimicrobial agents. S. pneumoniae is the most common cause of fatal community-acquired pneumonia in the elderly and is also one of the most common causes of middle ear infections and meningitis in children. The present vaccine for the pneumococcus consists of a mixture of 23 different capsular polysaccharides. While this vaccine is very effective in young adults, who are normally at low risk of serious disease, it is only about 60% effective in the elderly. In children younger than 2 years the vaccine is ineffective and is not recommended due to the inability of this age group to mount an antibody response to the pneumococcal polysaccharides. Antimicrobial drugs such as penicillin have diminished the risk from pneumococcal disease. Several pneumococcal proteins including pneumococcal surface proteins A and C, hyaluronate lyase, pneumolysin, autolysin, pneumococcal surface antigen A, choline binding protein A, and two neuraminidase enzymes are being investigated as potential vaccine or drug targets. Essentially all of these antigens have been or are being investigated on a structural level in addition to being characterized biochemically. Recently, three-dimensional structures for hyaluronate lyase and pneumococcal surface antigen A became available from X-ray crystallography determinations. Also, modeling studies based on biophysical measurements provided more information about the structures of pneumolysin and pneumococcal surface protein A. Structural and biochemical studies of these pneumococcal virulence factors have facilitated the development of novel antibiotics or protein antigen-based vaccines as an alternative to polysaccharide-based vaccines for the treatment of pneumococcal disease.     

Discovery of novel Streptococcus pneumoniae antigens by screening a whole-genome lambda-display library

Streptococcus pneumoniae is a causative agent of otitis media, pneumonia, meningitis and sepsis in humans. For the development of effective vaccines able to prevent pneumococcal infection, characterization of bacterial antigens involved in host immune response is crucial. In order to identify pneumococcal proteins recognized by host antibody response, we created an S. pneumoniae D39 genome library, displayed on lambda bacteriophage. The screening of such a library, with sera either from infected individuals or mice immunized with the S. pneumoniae D39 strain, allowed identification of phage clones carrying S. pneumoniae B-cell epitopes. Epitope-containing fragments within the families of the histidine-triad proteins (PhtE, PhtD), the choline-binding proteins (PspA, CbpD) and zinc metalloproteinase B (ZmpB) were identified. Moreover, library screening also allowed the isolation of phage clones carrying three distinct antigenic regions of a hypothetical pneumococcal protein, encoded by the ORF spr0075 in the R6 strain genome sequence. In this work, Spr0075 is first identified as an expressed S. pneumoniae gene product, having an antigenic function during infection.     

Pneumococcal virulence factors: structure and function.

The overall goal for this review is to summarize the current body of knowledge about the structure and function of major known antigens of Streptococcus pneumoniae, a major gram-positive bacterial pathogen of humans. This information is then related to the role of these proteins in pneumococcal pathogenesis and in the development of new vaccines and/or other antimicrobial agents. S. pneumoniae is the most common cause of fatal community-acquired pneumonia in the elderly and is also one of the most common causes of middle ear infections and meningitis in children. The present vaccine for the pneumococcus consists of a mixture of 23 different capsular polysaccharides. While this vaccine is very effective in young adults, who are normally at low risk of serious disease, it is only about 60% effective in the elderly. In children younger than 2 years the vaccine is ineffective and is not recommended due to the inability of this age group to mount an antibody response to the pneumococcal polysaccharides. Antimicrobial drugs such as penicillin have diminished the risk from pneumococcal disease. Several pneumococcal proteins including pneumococcal surface proteins A and C, hyaluronate lyase, pneumolysin, autolysin, pneumococcal surface antigen A, choline binding protein A, and two neuraminidase enzymes are being investigated as potential vaccine or drug targets. Essentially all of these antigens have been or are being investigated on a structural level in addition to being characterized biochemically. Recently, three-dimensional structures for hyaluronate lyase and pneumococcal surface antigen A became available from X-ray crystallography determinations. Also, modeling studies based on biophysical measurements provided more information about the structures of pneumolysin and pneumococcal surface protein A. Structural and biochemical studies of these pneumococcal virulence factors have facilitated the development of novel antibiotics or protein antigen-based vaccines as an alternative to polysaccharide-based vaccines for the treatment of pneumococcal disease.     

Novel vaccine strategies with protein antigens of Streptococcus pneumoniae

Infections caused by Streptococcus pneumoniae (pneumococcus) are a major cause of mortality throughout the world. This organism is primarily a commensal in the upper respiratory tract of humans, but can cause pneumonia in high-risk persons and disseminate from the lungs by invasion of the bloodstream. Currently, prevention of pneumococcal infections is by immunization with vaccines which contain capsular polysaccharides from the most common serotypes causing invasive disease. However, there are more than 90 antigenically distinct serotypes and there is concern that serotypes not included in the vaccines may become more prevalent in the face of continued use of polysaccharide vaccines. Also, certain high-risk groups have poor immunological responses to some of the polysaccharides in the vaccine formulations. Protein antigens that are conserved across all capsular serotypes would induce more effective and durable humoral immune responses and could potentially protect against all clinically relevant pneumococcal capsular types. This review provides a summary of work on pneumococcal proteins that are being investigated as components for future generations of improved pneumococcal vaccines.     

Acquisition of pneumococci specific effector and regulatory Cd4+ T cells localising within human upper respiratory-tract mucosal lymphoid tissue

The upper respiratory tract mucosa is the location for commensal Streptococcus (S.) pneumoniae colonization and therefore represents a major site of contact between host and bacteria. The CD4(+) T cell response to pneumococcus is increasingly recognised as an important mediator of immunity that protects against invasive disease, with data suggesting a critical role for Th17 cells in mucosal clearance. By assessing CD4 T cell proliferative responses we demonstrate age-related sequestration of Th1 and Th17 CD4(+) T cells reactive to pneumococcal protein antigens within mucosal lymphoid tissue. CD25(hi) T cell depletion and utilisation of pneumococcal specific MHCII tetramers revealed the presence of antigen specific Tregs that utilised CTLA-4 and PDL-1 surface molecules to suppress these responses. The balance between mucosal effector and regulatory CD4(+) T cell immunity is likely to be critical to pneumococcal commensalism and the prevention of unwanted pathology associated with carriage. However, if dysregulated, such responses may render the host more susceptible to invasive pneumococcal infection and adversely affect the successful implementation of both polysaccharide-conjugate and novel protein-based pneumococcal vaccines.     

Immune response and protective efficacy of S9 ribosomal protein of Streptococcus pneumoniae in a model of sepsis

Vaccination is the most promising strategy to reduce the incidence of pneumococcal infection. Although there are vaccines available, all of them are based on polysaccharide antigens (conjugated or not). In addition to their high cost, those vaccines do not cover all serotypes. To overcome these hindrances, we evaluated the immunogenicity and the protective efficacy of the S9 ribosomal protein of Streptococcus pneumoniae with the aim of developing a protein-based vaccine in the future. The gene encoding the S9 ribosomal protein was cloned in pET21-a expression vector, and the recombinant S9 protein was used to immunize mice. Significantly higher levels of anti-S9 immunoglobulin G were achieved (with predominance of immunoglobulin G1) in comparison with the control. Antibodies elicited against S.?pneumoniae protein extract in rabbit recognized the recombinant S9 protein by Western blot, thus demonstrating its immunogenicity. Moreover, mice immunized with recombinant S9 protein and challenged with a virulent strain of S.?pneumoniae presented a significant reduction of bacteremia after 24?h of infection as compared with the control. However, in the S9-immunized mice the onset of death was insignificantly delayed, but all of them died by the fourth day postinfection.     

Quantitative analysis of Streptococcus pneumoniae TIGR4 response to in vitro iron restriction by 2-D LC ESI MS/MS

Understanding the growth of bacterial pathogens in a micronutrient restricted host environment can identify potential virulence proteins that help overcome this nutritional barrier to productive infection. In this study, we investigated the pneumococcal protein expression response to iron limitation using an in vitro model. We identified S. pneumoniae TIGR4 proteins by 2-D LC ESI MS/MS and determined significant changes in protein expression in response to iron restriction using computer-intensive random resampling methods. Differential protein expression was studied in the context of a S. pneumoniae TIGR4 protein interaction network using Pathway Studio. Our analysis showed that pneumococcal iron restriction response was marked by increased expression of known virulence factors like PsaA. It involved changes in the expression of stress response, and phase variation and biofilm formation proteins. The net effect of changes in all these biological processes could increase the virulence of S. pneumoniae TIGR4 during in vivo infection.     

T(H)17-based vaccine design for prevention of Streptococcus pneumoniae colonization

Streptococcus pneumoniae is a leading cause of mortality in young children. While successful conjugate polysaccharide vaccines exist, a less expensive serotype-independent protein-based pneumococcal vaccine offers a major advancement for preventing life-threatening pneumococcal infections, particularly in developing nations. IL-17A-secreting CD4+ T cells (T(H)17) mediate resistance to mucosal colonization by multiple pathogens including S. pneumoniae. Screening an expression library containing >96% of predicted pneumococcal proteins, we identified antigens recognized by T(H)17 cells from mice immune to pneumococcal colonization. The identified antigens also elicited IL-17A secretion from colonized mouse splenocytes and human PBMCs suggesting that similar responses are primed during natural exposure. Immunization of two mouse strains with identified antigens provided protection from pneumococcal colonization that was significantly diminished in animals treated with blocking CD4 or IL-17A antibodies. This work demonstrates the potential of proteomic screening approaches to identify specific antigens for the design of subunit vaccines against mucosal pathogens via harnessing T(H)17-mediated immunity.     

Nasal immunization of mice with Lactobacillus casei expressing the Pneumococcal Surface Protein A: induction of antibodies, complement deposition and partial protection against Streptococcus pneumoniae challenge

Strategies for the development of new vaccines against Streptococcus pneumoniae infections try to overcome problems such as serotype coverage and high costs, present in currently available vaccines. Formulations based on protein candidates that can induce protection in animal models have been pointed as good alternatives. Among them, the Pneumococcal Surface Protein A (PspA) plays an important role during systemic infection at least in part through the inhibition of complement deposition on the pneumococcal surface, a mechanism of evasion from the immune system. Antigen delivery systems based on live recombinant lactic acid bacteria (LAB) represents a promising strategy for mucosal vaccination, since they are generally regarded as safe bacteria able to elicit both systemic and mucosal immune responses. In this work, the N-terminal region of clade 1 PspA was constitutively expressed in Lactobacillus casei and the recombinant bacteria was tested as a mucosal vaccine in mice. Nasal immunization with L. casei-PspA 1 induced anti-PspA antibodies that were able to bind to pneumococcal strains carrying both clade 1 and clade 2 PspAs and to induce complement deposition on the surface of the bacteria. In addition, an increase in survival of immunized mice after a systemic challenge with a virulent pneumococcal strain was observed.     

Lactococcus lactis as an adjuvant and delivery vehicle of antigens against pneumococcal respiratory infections

Most studies of Lactococcus lactis as delivery vehicles of pneumococcal antigens are focused on the effectiveness of mucosal recombinant vaccines against Streptococcus pneumoniae in animal models. At present, there are three types of pneumococcal vaccines: capsular polysaccharide pneumococcal vaccines (PPV), protein-polysaccharide conjugate pneumococcal vaccines (PCV) and protein-based pneumococcal vaccines (PBPV). Only PPV and PCV have been licensed. These vaccines, however, do not represent a definitive solution. Novel, safe and inexpensive vaccines are necessary, especially in developing countries. Probiotic microorganisms such as lactic acid bacteria (LAB) are an interesting alternative for their use as vehicles in pneumococcal vaccines due to their GRAS (Generally Recognized As Safe) status. Thus, the adjuvanticity of Lactococcus lactis by itself represents added value over the use of other bacteria, a question dealt with in this review. In addition, the expression of different pneumococcal antigens as well as the use of oral and nasal mucosal routes of administration of lactococcal vaccines is considered. The advantages of nasal live vaccines are evident; nonetheless, oral vaccines can be a good alternative when the adequate dose is used. Another point addressed here is the use of live versus inactivated vaccines. In this sense, few researchers have focused on inactivated strains to be used as vaccines against pneumoccoccus. The immunogenicity of live vaccines is better than the one afforded by inactivated ones; however, the probiotic-inactivated vaccine combination has improved this matter considerably. The progress made so far in the protective immune response induced by recombinant vaccines, the successful trials in animal models and the safety considerations of their application in humans suggest that the use of recombinant vaccines represents a good short-term option in the control of pneumococcal diseases.     

Characterization of Streptococcus pneumoniae isolated from children with otitis media

Streptococcus pneumoniae is the main causative agent of acute otitis media in children. Serotype-based vaccines have provided some protection against otitis media, but not as much as anticipated, demonstrating the need for alternative vaccine options. Pneumococcal otitis media isolates were obtained from children 5 years old or younger from hospitals around Mississippi in the prevaccine era (1999-2000). These isolates were compared by capsular typing, pneumococcal surface protein A (PspA) family typing, antibiotic susceptibility, and DNA fingerprinting. Our study shows that there is great genetic variability among pneumococcal clinical isolates of otitis media, except with regard to PspA. Therefore, efforts focused on the development of a PspA-based pneumococcal vaccine would be well placed.     

Analysis of recombinant acylated pneumococcal surface adhesin A of Streptococcus pneumoniae by mass spectrometry

Streptococcus pneumoniae pneumococcal surface adhesin A (PsaA) is a species-common, immunogenic surface lipoprotein. In this study, the psaA gene was expressed as a nonfusion acylated protein in an Escherichia coli expression system. Yields of pure recombinant PsaA (rPsaA) were 8-10 mg/liter of fermentation culture. Analysis of rPsaA tryptic digests by HPLC-electrospray mass spectrometry (MS) confirmed 98% of the expected protein sequence. GC/MS data demonstrated very similar acylation of native and rPsaA by C12:0-C22:0 fatty acids, with C16 and C18 predominating. Negative ion electrospray MS/MS analysis of the rPsaA lipid anchor released by Pronase-E confirmed that the structure was based on an N-terminal palmitoylcysteine (Pam(3)Cys). Electrospray MS heterogeneity analysis of intact rPsaA indicated that all of the observed heterogeneity could be accounted for by the fatty acid distributions. The availability of well-characterized rPsaA will facilitate the continued research and development of protein-based vaccines for the prevention of pneumococcal disease.     

Mucosal vaccination with a multicomponent adenovirus-vectored vaccine protects against Streptococcus pneumoniae infection in the lung

Streptococcus pneumoniae is a major bacterial respiratory pathogen. Current licensed pneumococcal polysaccharide and polysaccharide–protein conjugate vaccines are administered by an intramuscular injection. In order to develop a new-generation vaccine that can be administered in a needle-free mucosal manner, we have constructed early 1 and 3 gene regions (E1/E3) deleted, replication-defective adenoviral vectors encoding pneumococcal surface antigen A (PsaA), the N-fragment of pneumococcal surface protein A (N-PspA), and the detoxified mutant pneumolysin (PdB) from S. pneumoniae strain D39. Intranasal vaccination with the three adenoviral vectors (Ad/PsaA, Ad/N-PspA, and Ad/PdB) in mice resulted in robust antigen-specific serum immunoglobulin G responses, as demonstrated by an enzyme-linked immunosorbent assay. In addition, nasal mucosal vaccination with the combination of the three adenoviral vectors conferred protection against S. pneumoniae strain D39 colonization in mouse lungs. Taken together, these data demonstrate the feasibility of developing a mucosal vaccine against S. pneumoniae using recombinant adenoviruses for antigen delivery.     

Streptococcus pneumoniae heat shock protein 70 does not induce human antibody responses during infection

Mouse monoclonal antibodies (mAbs) were developed against Streptococcus pneumoniae in search for potential common pneumococcal proteins as vaccine antigens. mAb 230,B-9 (IgG1) reacted by immunoblotting with a 70-kDa protein which was isolated by immunoaffinity chromatography and subsequent preparative electrophoresis. N-terminal amino acid sequencing showed homology to that of heat shock protein 70 (hsp70). The hsp70 epitope reactive with mAb 230,B-9 was found in all the pneumococci examined as well as in other streptococci and enterococci. The epitope was not expressed in several other examined Gram-positive or -negative bacteria. Pneumococcal hsp70 has by other investigators been proposed to be a vaccine candidate. Binding experiments using flow cytometry showed that the epitope was not surface-exposed on live exponential phase grown S. pneumoniae. Human patient sera did not react with affinity-purified pneumococcal hsp70. Therefore the pneumococcal hsp70 does not seem to be of special interest in a vaccine formulation. The human sera contained antibodies to high molecular proteins co-purified with hsp70. Some of these proteins could be the pneumococcal surface protein A.     

SP0454, a putative threonine dehydratase, is required for pneumococcal virulence in mice

Increasing pressure in antibiotic resistance and the requirement for the design of new vaccines are the objectives of clarifying the putative virulence factors in pneumococcal infection. In this study, the putative threonine dehydratase sp0454 was inactivated by erythromycin-resistance cassette replacement in Streptococcus pneumoniae CMCC 31203 strain. The sp0454 mutant was tested for cell growth, adherence, colonization, and virulence in a murine model. The Δsp0454 mutant showed decreased ability for colonization and impaired ability to adhere to A549 cells. However, the SP0454 polypeptide or its antiserum did not affect pneumococcal CMCC 31203 adhesion to A549 cells. The sp0454 deletion mutant was less virulent in a murine intranasal infection model. Real-time RT-PCR analysis revealed significant decrease of the pneumococcal surface antigen A expression in the sp0454 mutant. These results suggest that SP0454 contributes to virulence and colonization, which could be explained in part by modulating the expression of other virulence factors, such as psaA in pneumococcal infection.     

The epidemiology of pneumococcal infection in children in the developing world.

Pneumonia causes about three million deaths a year in young children, nearly all of which are in developing countries. Streptococcus pneumoniae (the pneumococcus) is the most important bacterial cause of pneumonia in young children and so is likely to be responsible for a high proportion of these deaths. The pneumococcus is also responsible for a substantial proportion of the 100,000-500,000 deaths that occur from meningitis in children each year. The incidence of invasive pneumococcal disease in children in the developing world is several times higher than in industrialized countries. This discrepancy may, in part, be due to socio-economic differences but genetic factors may also play a role. Children with sickle cell disease have a substantially increased risk of invasive pneumococcal infection and a search is being made for other possible genetic risk factors. Infection with human immunodeficiency virus (HIV) also predisposes to invasive pneumococcal disease and so the incidence of this disease in young children is expected to rise as increasing numbers of African and Asian children are born with a perinatally acquired HIV infection. Until recently, pneumococcal infections could be treated effectively with penicillin, a cheap and safe antibiotic. However, pneumococci that are resistant to penicillin are becoming prevalent in many countries, necessitating a change to more costly antibiotics which may be beyond the reach of the health services of poor, developing countries. The spread of antibiotic resistance has provided an added stimulus to the development of vaccines that might be able to prevent pneumococcal disease in infants. Recently developed polysaccharide-protein conjugate vaccines show promise and are now undergoing field trials. How deployment of these vaccines will influence the balance between invasive pneumococcal infections and asymptomatic nasopharyngeal carriage of pneumococci is uncertain.     

Dendritic cell‐targeting DNA‐based nasal adjuvants for protective mucosal immunity to Streptococcus pneumoniae

To develop safe vaccines for inducing mucosal immunity to major pulmonary bacterial infections, appropriate vaccine antigens (Ags), delivery systems and nontoxic molecular adjuvants must be considered. Such vaccine constructs can induce Ag‐specific immune responses that protect against mucosal infections. In particular, it has been shown that simply mixing the adjuvant with the bacterial Ag is a relatively easy means of constructing adjuvant‐based mucosal vaccine preparations; the resulting vaccines can elicit protective immunity. DNA‐based nasal adjuvants targeting mucosal DCs have been studied in order to induce Ag‐specific mucosal and systemic immune responses that provide essential protection against microbial pathogens that invade mucosal surfaces. In this review, initially a plasmid encoding the cDNA of Flt3 ligand (pFL), a molecule that is a growth factor for DCs, as an effective adjuvant for mucosal immunity to pneumococcal infections, is introduced. Next, the potential of adding unmethylated CpG oligodeoxynucleotide and pFL together with a pneumococcal Ag to induce protection from pneumococcal infections is discussed. Pneumococcal surface protein A has been used as vaccine for restoring mucosal immunity in older persons. Further, our nasal pFL adjuvant system with phosphorylcholine‐keyhole limpet hemocyanin (PC‐KLH) has also been used in pneumococcal vaccine development to induce complete protection from nasal carriage by Streptococcus pneumoniae . Finally, the possibility that anti‐PC antibodies induced by nasal delivery of pFL plus PC‐KLH may play a protective role in prevention of atherogenesis and thus block subsequent development of cardiovascular disease is discussed.

     

Mucosal immunisation with novel Streptococcus pneumoniae protein antigens enhances bacterial clearance in an acute mouse lung infection model

Streptococcus pneumoniae contains many proteins that have not been evaluated as potential protective vaccine antigens. In this study we isolated proteins from a serotype 3 strain of S. pneumoniae for use in mouse immunisation studies. Separation of the protein mix was achieved by SDS-PAGE electrophoresis followed by electro-elution to isolate individual proteins. This procedure successfully separated 21 fractions from which six proteins were selected based on purity and quantity and were initially denoted by their molecular masses: 14-, 34-, 38-, 48-, 57- and 75-kDa. The immunogenicity of these proteins was investigated in a mucosal immunisation model in mice involving a primary inoculation to the intestinal Peyer's patches followed by an intra-tracheal boost two weeks later. The immune response was assessed by enhancement of pulmonary clearance of infection, recruitment of phagocytes to the lungs and induction of an antibody response. Two of the proteins, the 14-kDa identified as a L7/L12 ribosomal protein, and the 34-kDa identified as glyceraldehyde-3-phosphate dehydrogenase resulted in up to 99% and 94%, respectively, enhanced clearance of infection within 5 h following pulmonary challenge with S. pneumoniae. This study has shown that novel pneumococcal proteins have the potential to be vaccine candidates to enhance clearance of an acute mucosal S. pneumoniae infection.     

Experimental infection of rhesus macaques with Streptococcus pneumoniae: a possible model for vaccine assessment

BACKGROUND: We explored the possibility of using normal adult rhesus macaques for the preclinical assessment of safety, immunogenicity, and efficacy of newly developed vaccines against Streptococcus pneumoniae infection of the lung. METHODS: Our primary objective was to determine whether an intra-bronchial inoculum of at least 10(6)S. pneumoniae colony-forming units, or one as high as 10(8)-10(9) organisms, could detectably survive in rhesus macaques for a period longer than 1-2 weeks. If so, we hypothesized, it would be possible to observe signs of pneumonia commonly observed in humans, and discriminate between vaccinated/protected animals and controls. Infection was detectable in bronchoalveolar lavage fluids 3-5 weeks post-inoculation. RESULTS: The clinical course of disease mimicked aspects of that of human pneumococcal pneumonia. Signs of inflammation typical of the disease in humans, such as elevated concentrations of neutrophils and of pro-inflammatory cytokines in bronchoalveolar lavage fluids were also observed. CONCLUSIONS: These findings underscore the utility of this model to assess the safety, immunogenicity, and efficacy of newly developed S. pneumoniae vaccines.