A Live Recombinant Avirulent Oral Salmonella Vaccine Expressing Pneumococcal Surface Protein A Induces Protective Responses against Streptococcus pneumoniae

A live oral recombinant Salmonella vaccine strain expressing pneumococcal surface protein A (PspA) was developed. The strain was attenuated with Δcya Δcrp mutations. Stable expression of PspA was achieved by the use of the balanced-lethal vector-host system, which employs an asd deletion in the host chromosome to impose an obligate requirement for diaminopimelic acid. The chromosomal Δasd mutation was complemented by a plasmid vector possessing the asd⁺ gene. A portion of the pspA gene from Streptococcus pneumoniae Rx1 was cloned onto a multicopy Asd⁺ vector. After oral immunization, the recombinant Salmonella-PspA vaccine strain colonized the Peyer’s patches, spleens, and livers of BALB/cByJ and CBA/N mice and stimulated humoral and mucosal antibody responses. Oral immunization of outbred New Zealand White rabbits with the recombinant Salmonella strain induced significant anti-PspA immunoglobulin G titers in serum and vaginal secretions. Polyclonal sera from orally immunized mice detected PspA on the S. pneumoniae cell surface as revealed by immunofluorescence. Oral immunization of BALB/cJ mice with the PspA-producing Salmonella strain elicited antibody to PspA and resistance to challenge by the mouse-virulent human clinical isolate S. pneumoniae WU2. Immune sera from orally immunized mice conferred passive protection against otherwise lethal intraperitoneal or intravascular challenge with strain WU2.     

Comparison of the PspA Sequence from Streptococcus pneumoniae EF5668 to the Previously Identified PspA Sequence from Strain Rx1 and Ability of PspA from EF5668 To Elicit Protection against Pneumococci of Different Capsular Types

PspA (pneumococcal surface protein A) is a serologically varied virulence factor of Streptococcus pneumoniae. In mice, PspA has been shown to elicit an antibody response that protects against fatal challenge with encapsulated S. pneumoniae, and the protection-eliciting residues have been mapped to the α-helical N-terminal half of the protein. To date, a published DNA sequence for pspA is available only for S. pneumoniae Rx1, a laboratory strain. PspA/EF5668 (EF5668 indicates the strain of origin of the PspA) is serologically distinct from PspA/Rx1. Sequencing of the gene encoding PspA/EF5668 revealed 71% identity with that of PspA/Rx1. The greatest amount of divergence between the two proteins was seen in their α-helical portions, which are surface exposed and probably under selective pressure to diversify serologically. In spite of the diversity within the α-helical regions of PspAs, we have observed that recombinant PspA (rPspA)/EF5668, like rPspA/Rx1, can elicit cross-protection against pneumococci of different capsular and PspA serological types.     

Genetic immunization with the region encoding the alpha-helical domain of PspA elicits protective immunity against Streptococcus pneumoniae

Pneumococcal surface protein A (PspA) is a pneumococcal virulence factor capable of eliciting protection against pneumococcal infection in mice. Previous studies have demonstrated that the protection is antibody mediated. Here we examined the ability of pspA to elicit a protective immune response following genetic immunization of mice. Mice were immunized by intramuscular injections with a eukaryotic expression vector encoding the alpha-helical domain of PspA/Rx1. Immunization induced a PspA-specific serum antibody response, and immunized mice survived pneumococcal challenge. Survival and antibody responses occurred in a dose-dependent manner, the highest survival rates being seen with doses of 10 microg or greater. The ability of genetic immunization to elicit cross-protection was demonstrated by the survival of immunized mice challenged with pneumococcal strains differing in capsule and PspA types. Also, immunized mice were protected from intravenous and intratracheal challenges with pneumococci. Similar to the results seen with immunization with PspA, the survival of mice genetically immunized with pspA was antibody mediated. There was no decline in the level of protection 7 months after immunization. These results support the use of genetic immunization to elicit protective immune responses against extracellular pathogens.     

Molecular localization of variable and conserved regions of pspA and identification of additional pspA homologous sequences in Streptococcus pneumoniae.

PspA is anchored to the surface of all pneumococci by the C-terminal end of the molecule. The N-terminal half of PspA is known to be serologically variable and to be able to elicit protective immune responses. Molecular analysis with DNA probes spanning different regions of pspA was carried out to identify homologous sequences among pneumococcal isolates. At high stringency, DNA probes derived from the 3'-half of pspA (encoding the C-terminal half of PspA) hybridized to all of 37 pneumococcal isolates tested, representing 20 capsular serotypes and 12 PspA serotypes. Most strains had two sequences highly homologous to this region of pspA. Using derivatives of strain Rx1, with insertion mutations in pspA, it was possible to identify the functional pspA sequence. At 50% stringency, the 3' pspA probes also detected lytA and additional sequences. lytA encodes autolysin and shares homology with the 3' portion of pspA. A probe derived from the 5'-half of pspA (encoding the N-terminal half of PspA) hybridized with only 75% of strains and generally detected only one of the two sequences recognized by the 3' probes. Thus, the 3'-half of pspA appears to contain more highly conserved sequences than the 5'-half of pspA and shares homology with several additional sequences, suggesting that the pneumococcus might make several proteins that interact with the surface by the same mechanism as PspA.     

Correlation between In Vitro Complement Deposition and Passive Mouse Protection of Anti-Pneumococcal Surface Protein A Monoclonal Antibodies

The shortcomings of the licensed polysaccharide-based pneumococcal vaccine are driving efforts toward development of a protein-based vaccine that is serotype independent and effective in all age groups. An opsonophagocytic killing assay (OPKA) is used to evaluate the antibody response against polysaccharide-based pneumococcal vaccines. However, the OPKA is not reliable for noncapsular antigens. Thus, there is a need to develop an in vitro surrogate for protection for protein vaccine candidates like pneumococcal surface antigen A (PspA). PspA is a serologically variable cell surface virulence factor. Based on its sequence, PspA has been classified into families 1 (clade 1 and 2), 2 (clades 3, 4 and 5), and 3 (clade 6). Here, we report the characterization of 18 IgG anti-PspA monoclonal antibodies (anti-PspA^hkR36A MAbs) generated from mice immunized with heat-killed strain R36A (clade 2). An enzyme-linked immunosorbent assay (ELISA)-based analysis of the reactivity of the MAbs with recombinant PspAs from the 6 clades indicated that they were family 1 specific. This was confirmed by flow cytometry using a hyperimmune serum generated against PspA from R36A. Eight MAbs that bind at least one clade 1- and clade 2-expressing strain were evaluated for complement deposition, bactericidal activity, and passive protection. The anti-PspA^hkR36A MAb-dependent deposition of complement on pneumococci showed a positive correlation with passive protection against strain WU2 (r = 0.8783, P = 0.0041). All of our protective MAbs showed bactericidal activity; however, not all MAbs that exhibited bactericidal activity conferred protection in vivo. The protective MAbs described here can be used to identify conserved protection eliciting B cell epitopes for engineering a superior PspA-based vaccine.     

PspA Family Fusion Proteins Delivered by Attenuated Salmonella enterica Serovar Typhimurium Extend and Enhance Protection against Streptococcus pneumoniae

Pneumococcal surface protein A (PspA) is highly immunogenic and can induce a protective immune response against pneumococcal infection. PspA is divided into two major families based on serological variability: family 1 and family 2. To provide broad protection, PspA proteins from pneumococcal strains Rx1 (family 1) and EF5668 (family 2) were combined to form two PspA fusion proteins, PspA/Rx1-EF5668 and PspA/EF5668-Rx1. Each protein was fused to a type II secretion signal and delivered by a recombinant attenuated Salmonella vaccine (RASV). Both PspA/Rx1-EF5668 and PspA/EF5668-Rx1 were synthesized in the RASV and secreted into the periplasm and supernatant. The fusion proteins reacted strongly with both anti-PspA/Rx1 and anti-PspA/EF5668 antisera. Oral immunization of BALB/c mice with RASV synthesizing either PspA fusion protein elicited serum immunoglobulin G (IgG) and mucosal IgA responses against both families of PspA. Analysis of IgG isotypes (IgG2a and IgG1) indicated a strong Th1 bias to the immune responses to both proteins. Sera from mice immunized with RASV synthesizing PspA/Rx1-EF5668 bound to the surface and directed C3 complement deposition on representative strains from all five PspA clades. Immunization with RASV synthesizing either protein protected mice against intraperitoneal challenge with Streptococcus pneumoniae WU2 strain (family 1), intravenous challenge with S. pneumoniae 3JYP2670 strain (family 2), and intranasal challenge with S. pneumoniae A66.1 (family 1). The PspA/Rx1-EF5668 protein elicited significantly greater protection than PspA/EF5668-Rx1, PspA/Rx1, or PspA/EF5668. These results indicate an RASV synthesizing a PspA fusion protein representing both PspA families constitutes an effective antipneumococcal vaccine, extending and enhancing protection against multiple strains of S. pneumoniae.Streptococcus pneumoniae is a human pathogen causing significant morbidity and mortality worldwide, especially in developing countries. It causes respiratory infections, otitis media, sinusitis, and invasive diseases such as pneumonia, meningitis, and bacteremia. S. pneumoniae causes more than 1 million deaths worldwide every year among children under 5 years of age (8, 11, 20). The current 23-valent capsular polysaccharide vaccine elicits immunity in individuals greater than 2 years of age, and the current conjugate polysaccharide-protein pneumococcal vaccine provides protection for those under the age of 2 years (23, 26, 33). However, protection is restricted to only the limited number of serotypes included in the vaccine formulation (26), and the expensive production costs limit its use in developing countries. Moreover, serotype replacement has been observed in vaccinated populations and an increase in infections by pneumococcal serotypes not included in the 7-valent conjugated polysaccharide vaccine has been described recently (29, 56). In some countries, as many as 66% of childhood strains would not be covered (26, 45). Treatment of pneumococcal diseases has become more challenging due to the increase in multiple-drug-resistant pneumococcal strains (58). These issues reinforce the need for more affordable, broadly protective strategies for immunization against pneumococcal infection.Several pneumococcal proteins have been under investigation as potential vaccine candidates, including pneumococcal surface protein A (PspA) (7, 10, 14), pneumococcal surface protein C (PspC) (12), and pneumolysin (1, 50). PspA is a virulence factor expressed by all clinical S. pneumoniae isolates. It consists of five domains: (i) a signal peptide, (ii) an α-helical and charged domain that bears a strong 7-residue periodicity typical of coiled-coil proteins (amino acids [aa] 1 to 288), (iii) a proline-rich region (aa 289 to 370) which spans the cell wall and is highly conserved in all S. pneumoniae strains, (iv) a choline-binding domain consisting of 10 20-aa repeats (aa 371 to 571) that anchors the protein to the cell surface, and (v) a C-terminal 17-aa tail (aa 572 to 589) (Fig. ​(Fig.1).1). The α-helical region is variable in length and amino acid sequence, but the antibodies against this region are protective and cross-reactive. PspA proteins have been grouped into three families encompassing six different clades based on the C-terminal 100 aa of the α-helical region (28). Family 1 is comprised of clades 1 and 2, family 2 is comprised of clades 3, 4, and 5, and family 3 consists of clade 6. S. pneumoniae strains expressing family 1 or 2 PspA proteins constitute 98% of clinical isolates (27, 28, 53). To accommodate this variability, it was proposed that a combination of two PspA antigens, one from PspA family 1 and one from PspA family 2, would elicit protection against the vast majority of S. pneumoniae strains (27, 28, 47). In addition to the α-helical region, the proline-rich domain has been shown to encode protective epitopes (S. Hollingshead, unpublished observation). This region of the protein is highly conserved compared to the α-helical region, making inclusion of the proline-rich domain important to achieve broad protection (4, 9, 28).Open in a separate windowFIG. 1.Schematic diagram of PspA and PspA fusion proteins. At the top is the entire PspA molecule containing the N-terminal α-helical domain (region A), the proline-rich region (region B), the choline-binding domain (region C), and the C-terminal tail (region D). Each recombinant fusion protein is shown with its distinct domains.Complement-mediated opsonin-dependent phagocytosis is an important defense mechanism against pneumococcal infections. It activates both the classical and alternative complement pathways, depositing C3b on the pneumococcal surface (13, 34, 35). PspA inhibits complement activation (60), and anti-PspA antibodies can overcome this effect (53), leading to increased C3 deposition on the bacterial surface and enhanced clearance. Anti-PspA-directed C3 complement deposition has been correlated with protection against S. pneumoniae challenge in mice (19). Therefore, measurement of C3 complement deposition on the pneumococcal surface directed by sera from vaccinated individuals could be an important correlate of protection.Previous work in our laboratory demonstrated that recombinant avirulent Salmonella enterica serovar Typhimurium vaccines (RASVs) can be used to deliver PspA cloned from S. pneumoniae strain Rx1 (family 1) and induce protection in mice against challenge with homologous family 1 S. pneumoniae strain WU2 (38-40, 48, 64). Using RASV to deliver antigens has many advantages, including low-cost vaccine production, needle-free delivery, and induction of strong mucosal immunity (16, 18). In this article, gene fragments encoding the α-helix domain of PspA from family 1 strain Rx1 and the α-helix domain and proline-rich region of family 2 strain EF5668 were used to construct gene fusions encoding two PspA fusion proteins, PspA/Rx1-EF5668 and PspA/EF5668-Rx1. These gene fusions were expressed and delivered by an RASV strain designed to regulate antigen expression by the availability of arabinose, resulting in regulated delayed antigen synthesis, to enhance and extend protection against S. pneumoniae clinical strains.     

Evidence for the simultaneous expression of two PspAs by a clone of capsular serotype 6BStreptococcus pneumoniae

Pneumococcal surface protein A (PspA) has been shown to be a serologically variable virulence factor ofStreptococcus pneumoniae.In mice, PspA can elicit antibodies capable of protecting them against otherwise fatal infections with encapsulated pneumococci. In previous studies it has been reported that almost all isolates have two apparently unlinked genomic sequences that are highly homologous to the 5′ and 3′ halves of Rx1pspA, although out MAbs to PspA have not detected more than one PspA in any given isolate ofS. pneumoniae.Recently, we have identified four isolates from a clone of capsular serotype 6B pneumococci (MC25-28) that simultaneously express two distinct PspAs. Each of the isolates (MC25-28) exhibited the same twoKpnI fragments (each containing aHind III site) that hybridized with Rx1pspA. MAbs specific for PspA detected two PspAs characterized by different molecular weights and different serologic patterns of reactivity (PspA type 6 detected by MAbs XiR278 and 2A4, and PspA type 34 detected only by MAb 7D2) in each of the four isolates. In previous studies XiR278 and 2A4 frequently have been observed to react with PspA epitopes of the same strain. Based on molecular weight data both epitopes were always present on the same molecule. Our present findings raise the possibility that pneumococci make a second serologically variable PspA which is generally not detected by currently available MAbs to PspA.     

Optimized immune response elicited by a DNA vaccine expressing pneumococcal surface protein a is characterized by a balanced immunoglobulin G1 (IgG1)/IgG2a ratio and proinflammatory cytokine production

We have previously shown that DNA immunization with PspA (pneumococcal surface protein A) DNA is able to elicit protection comparable to that elicited by immunization with PspA protein (with alum as adjuvant), even though the antibody levels elicited by DNA immunization are lower than those elicited by immunization with the protein. This work aims at characterizing the ability of sera to bind to the pneumococcal surface and to mediate complement deposition, using BALB/c wild-type and interleukin-4 knockout mice. We observed that higher anti-PspA levels correlated with intense antibody binding to the pneumococcal surface, while elevated complement deposition was observed with sera that presented balanced immunoglobulin G1 (IgG1)/IgG2a ratios, such as those from DNA-immunized mice. Furthermore, we demonstrated that gamma interferon and tumor necrosis factor alpha were strongly induced after intraperitoneal pneumococcal challenge only in mice immunized with the DNA vaccine. We therefore postulate that although both DNA and recombinant protein immunizations are able to protect mice against intraperitoneal pneumococcal challenge, an optimized response would be achieved by using a DNA vaccine and other strategies capable of inducing balanced Th1/Th2 responses.     

Mapping of Epitopes Recognized by Antibodies Induced by Immunization of Mice with PspA and PspC

Pneumococcal surface protein A (PspA) and pneumococcal surface protein C (PspC) are important candidates for an alternative vaccine against pneumococcal infections. Since these antigens show variability, the use of variants that do not afford broad protection may lead to the selection of vaccine escape bacteria. Epitopes capable of inducing antibodies with broad cross-reactivities should thus be the preferred antigens. In this work, experiments using peptide arrays show that most linear epitopes recognized by antibodies induced in mice against different PspAs were located at the initial 44 amino acids of the mature protein and that antibodies against these linear epitopes did not confer protection against a lethal challenge. Conversely, linear epitopes recognized by antibodies to PspC included the consensus sequences involved in the interaction with human factor H and secretory immunoglobulin A (sIgA). Since linear epitopes of PspA were not protective, larger overlapping fragments containing 100 amino acids of PspA of strain Rx1 were constructed (fragments 1 to 7, numbered from the N terminus) to permit the mapping of antibodies with conformational epitopes not represented in the peptide arrays. Antibodies from mice immunized with fragments 1, 2, 4, and 5 were capable of binding onto the surface of pneumococci and mediating protection against a lethal challenge. The fact that immunization of mice with 100-amino-acid fragments located at the more conserved N-terminal region of PspA (fragments 1 and 2) induced protection against a pneumococcal challenge indicates that the induction of antibodies against conformational epitopes present at this region may be important in strategies for inducing broad protection against pneumococci.     

Protective Immune Responses Elicited by Fusion Protein Containing PsaA and PspA Fragments

Streptococcus pneumoniae is an important pathogen accounting for a large number of deaths worldwide. Due to drawbacks of the current polysaccharide-based vaccine, the most promising way to generate an improved vaccine may be to utilize protection-eliciting pneumococcal proteins. Pneumococcal surface adhesin A (PsaA) and pneumococcal surface protein A (PspA) are two vaccine candidates which have been evaluated against S. pneumoniae infection in animal models or human clinical trials with encouraging results. In this study, the efficacy of the fusion protein PsaA–PspA, which includes PsaA part and PspA part, in inducing immunoprotective effects against fatal pneumococcal challenge was evaluated in an animal model. PspA part of PsaA–PspA fusion protein contains both family1 N-terminal region and family 2 N-terminal clade-defining region of PspA. Immunization with the PsaA–PspA fusion protein induced high levels of antibodies against both PsaA and PspA, which could bind to intact S. pneumoniae strains bearing different PspAs. Ex vivo stimulation of splenocytes from mice immunized with PsaA–PspA induced IL-17A secretion. Mice immunized with PsaA–PspA showed reduced S. pneumoniae levels in the blood and lungs compared with the PBS group after intranasal infection. Finally, mice immunized with PsaA–PspA fusion proteins were protected against fatal challenge with pneumococcal strains expressing different PspAs regardless of the challenge route. These results support the PsaA–PspA fusion protein as a promising vaccine strategy, as demonstrated by its ability to enhance the immune response and stimulate production of high titer antibodies against S. pneumoniae strains bearing heterologous PspAs, as well as confer protection against fatal challenge with PspA family 1 and family 2 strains.     

肺炎链球菌外膜蛋白PspA和PsaA的免疫原性比较

目的 比较肺炎链球菌(Streptococcus pneumoniae)表面黏附素A(PsaA)和表面蛋白A(PspA)的免疫原性.方法 电泳分析Sp6B亚型菌株的两种外膜蛋白基因及所编码蛋白相对分子质量的可变性,采用Western blot方法比较两种重组外膜蛋白对5种亚型菌株相应蛋白抗血清的交叉反应,采用酶联免疫吸附法(ELISA)分析两种外膜蛋白的抗体类型和抗体水平以及抗血清对5种亚型菌株的亲和性,以小鼠保护实验比较两种蛋白对5种亚型菌株的交叉保护作用.结果 两种蛋白产生的抗体亚型水平相当;PspA蛋白与其他亚型蛋白的交叉反应广泛性不如PsaA蛋白,只限于同一支系的蛋白之间;PspA抗体与病原菌具有可接近性,并具备交叉免疫保护作用.可作为一种更有效的抗原成分.结论 PspA对同家族同支系菌株攻击具有交叉免疫保护作用,可作为一种更有效的抗原成分.     

A pneumococcal vaccine combination with two proteins containing PspA families 1 and 2 can potentially protect against a wide range of <Emphasis Type="Italic">Streptococcus pneumoniae</Emphasis> strains

Streptococcus pneumoniae is an important pathogen accounting for a large number of deaths worldwide. Despite the multitude of capsular polysaccharide vaccines used to guard against pneumococcal disease, fatal pneumococcal disease remains epidemic due to the narrow range of protection afforded by the capsular polysaccharide vaccines and rate of change in serotypes. The most promising solution is to develop an improved protein-based vaccine with broad protection. In this study, we tested a bivalent vaccine containing antigens mixed with the fusion protein PsaA-pneumococcal surface protein A (PspA)23 and single protein PspA4, including conserved PsaA and PspA from clades 2, 3, and 4 with coverage for families 1 and 2. The vaccine induced a significant increase of anti-PspA IgG, which demonstrated cross-reactivity with the 22 different S. pneumoniae strains from serotypes contained in PPV23 by Western blot. The wide ranging protection was determined by challenging mice with S. pneumoniae from PspA clades 1 to 5. Bacterial loads in the blood and lung and survival rate after challenge were measured. After immunization, the number of bacteria in mice was significantly reduced. The clearance rates with all strains were greater than 90% in the lung, and bacterial loads in the blood were decreased to lower than 10 CFU/ml. The survival rates in immunized animals also were greatly increased (all over 50%) compared with controls. Therefore, this bivalent PspA vaccine may be a good substitute for capsular polysaccharide vaccines.     

A Novel PspA Protein Vaccine Intranasal Delivered by Bacterium-Like Particles Provides Broad Protection Against Pneumococcal Pneumonia in Mice

Background: Streptococcus pneumoniae is a major pathogen accounting for a large number of pneumococcal disease in worldwide. Due to the mucosal immune pathway induces both systemic and mucosal immune responses, the potential strategy to prevent pneumococcal disease may be to develop a mucosal vaccine.

Method: In this study, we developed an intranasal pneumococcal protein vaccine based on a bacterium-like particle (BLP) delivery system. PspA is expressed and exposed on the surface of all pneumococcal strains, which confers the potential to induce immune responses to protect against pneumococcal infection. We fused one of the pneumococcal surface proteins (PspA, family2 clade4) with the protein anchor (PA) protein in order to display PspA on the surface of BLPs.

Result: The current results showed that intranasal immunization with BLPs/PspA-PA efficiently induced both PspA-specific IgG in the serum and PspA-specific IgA in mucosal washes. And intranasal immunization of BLPs/PspA-PA could provide complete protection in a mouse challenge model with pneumococci of different two clades of both homologous and heterologous PspA families.

Discussion and conclusion: Thus, targeted delivery of multiple bacterial antigens via BLPs may prevent pneumococcal disease by inducing both systemic and mucosal immune responses.     

Development of 5-valent conjugate pneumococcal protein A – Capsular polysaccharide pneumococcal vaccine against invasive pneumococcal disease

In this study, we synthesized a 5-valent pneumococcal conjugate vaccine, which was prepared with the pneumococcal capsular polysaccharides (PCPs) (from Streptococcus pneumoniae 1, 5, 6B, 19F, 23F) and pneumococcal surface protein A (PspA) mediated by 1,4-butanediol diglycidyl ether. The PspA cloned from serotype 19 strain showed good cross-immune response to 1, 5, 6B, and 23F serotypes of Streptococcus pneumonia (S. pneumoniae). Analysis of the maturation process of conjugate polyclonal antibody showed that conjugation with the protein carrier converted the polysaccharide from a weak T cell-independent (TI) antigen to a T cell-dependent (TD) antigen, although antibodies affinity to polysaccharide was not as strong as it to PspA in conjugate. We used an invasive disease mouse model to evaluate the protective efficacy of this conjugate vaccine. Active and passive protection against intraperitoneal challenge with virulent type 6B strain showed that the median survival times for mice immunized with conjugate were significantly longer than that of mice treated with capsular polysaccharides or PspA alone. Our study's results showed that immunization of the 5-valent PspA-capsular polysaccharides conjugate vaccine could afford strong protection to mice against the invasion of 1, 5, 6B, 19F, 23F serotypes S. pneumoniae.     

Immunization of Mice with Single PspA Fragments Induces Antibodies Capable of Mediating Complement Deposition on Different Pneumococcal Strains and Cross-Protection

PspA is an important candidate for a vaccine with serotype-independent immunity against pneumococcal infections. Based on sequence relatedness, PspA has been classified into three families comprising six clades. We have previously addressed the cross-reactivity of antibodies against PspA fragments containing the N-terminal and proline-rich regions of PspA from clades 1 to 5 (PspA1, PspA2, PspA3, PspA4, and PspA5) by Western blot analysis and reported that anti-PspA4 and anti-PspA5 were able to recognize pneumococci expressing PspA proteins from all of the clades analyzed. We have now analyzed the functional capacity of these antibodies to bind and to mediate complement deposition on intact bacteria in vitro. Our results show that both PspA4 and PspA5 elicit antibodies that are able to bind and to mediate complement deposition efficiently on pneumococcal strains bearing PspA proteins from clades 1 to 5. Moreover, mice immunized with PspA4 and PspA5 were protected against an intranasal lethal challenge with strains expressing PspA proteins from the two major families. PspA4 and PspA5 are thus able to induce antibodies with a high degree of cross-reactivity in vitro, which is reflected in cross-protection of mice. We have also analyzed the contribution of the nonproline (NonPro) block within the conserved proline-rich region to the reactivity of anti-PspA antibodies, and the results indicate that N-terminal α-helical region, the blocks of proline repeats, and the NonPro region can influence the degree of cross-reactivity of antibodies to PspA.Streptococcus pneumoniae is an important human pathogen, being responsible for millions of deaths worldwide every year. The pneumococcal disease burden could be greatly reduced by the use of the current seven-valent conjugate vaccine, but the high cost and restricted serotype coverage limit its widespread use, especially in developing countries. New-generation vaccines containing up to 13 serotypes are expected to increase vaccine coverage, but the serotype replacement in colonization and disease by nonvaccine serotypes observed with the use of the seven-valent conjugate vaccine (8-9, 11) further emphasizes the importance of the development of alternative vaccines. Protein antigens such as PspA (pneumococcal surface protein A) could be used to induce serotype-independent immunity at a low cost (24).PspA is present in all isolated pneumococcal strains and was shown to be an important virulence factor, interfering with complement deposition (19, 21, 25), killing by apolactoferrin (23), and immune adherence to erythrocytes (12). It has been shown to induce protection in mice in carriage, pneumonia, and fatal systemic models (2, 4, 16). Mature PspA is composed of a mosaic structure with four domains: an α-helical N-terminal domain, a proline-rich region, a choline-binding domain, and a short hydrophobic tail (10, 27-28). PspA shows variability in the surface-exposed N-terminal region, and a classification was proposed based on sequence relatedness of the C-terminal portion of the α-helix, the clade-defining region. It has been classified into three families encompassing six clades. Family 1 (Fam1) is composed of clades 1 and 2, Fam2 includes clades 3, 4, and 5, and Fam3, which is rarely isolated, comprises clade 6 (10). Since the degree of similarity seems to be reflected in cross-reactivity, it has been proposed that a broad-coverage vaccine should contain at least one fragment from each of the two major families.Immunization of healthy adults with a single recombinant fragment of PspA in a phase I clinical trial showed the induction of cross-reactive antibodies (14) that were able to induce passive protection in mice challenged intravenously (3). The natural exposure of adults to several pneumococcal strains might be responsible for the cross-reactivity detected, with the immunization with PspA acting as a booster dose.Because of the diversity observed in PspA, it is extremely important to analyze whether each fragment selected to compose a vaccine is indeed able to induce cross-protection. We have previously addressed the degree of cross-reactivity of antibodies to recombinant fragments including the N-terminal and proline-rich regions of PspA proteins from clades 1 to 5 (PspA1, PspA2, PspA3, PspA4, and PspA5) by Western blot analysis of 35 strains isolated in Brazil. As expected, we have observed higher cross-reactivity within the same clade. Within Fam1, anti-PspA1 serum also showed cross-reaction with PspA2-expressing strains, while anti-PspA2 showed reaction restricted to the same clade. Within Fam2, anti-PspA3 serum also showed reactivity restricted to PspA3-expressing strains, while anti-PspA5 and, more strikingly, anti-PspA4 sera showed a broad recognition capacity, being able to react with strains expressing PspA proteins from clades 1 to 5 (7). The ability of sera to recognize a pneumococcal strain by Western blot analysis does not necessarily correlate with their capacity to induce protection in vivo though. In fact, the levels of antibodies to PspA detected by enzyme-linked immunosorbent assay (ELISA) or through surface staining of the bacteria failed to provide a useful correlate of protection (22). Based on the strong evidence supporting the importance of complement in protection against pneumococcal disease, it was proposed that in vitro complement deposition mediated by antibody may be used as a surrogate assay for the prediction of protection induced by surface antigens of pneumococci (15). This work aimed at further characterizing antibodies against the PspA1, PspA2, PspA3, PspA4, and PspA5 N-terminal fragments in terms of their capacity to mediate C3 deposition on the surface of pneumococci expressing PspA proteins from different clades. Moreover, protection of mice against a lethal intranasal challenge with strains expressing PspA from Fam1 or Fam2 was also analyzed. The basis for the broad reactivity observed in the anti-PspA4 serum by Western blot analysis was also further investigated. Of the five PspA fragments analyzed, PspA4 was the only one containing a nonproline (NonPro) block within the proline-rich region. Not all native PspA proteins include this region: of 24 PspA sequences analyzed by Hollingshead and collaborators (10), 14 were shown to have this NonPro block. We have thus examined whether this region would be responsible for increased cross-reactivity.     

Combined prime-boost immunization with systemic and mucosal pneumococcal vaccines based on Pneumococcal surface protein A to enhance protection against lethal pneumococcal infections

Limited protective effects of commercially available vaccines necessitate the development of novel pneumococcal vaccines. We recently reported a pneumococcal systemic vaccine containing two proteins, Pneumococcal surface protein A (PspA of family 1 and 2) and a bacterium-like particle-based pneumococcal mucosal vaccine containing PspA2 and PspA4 fragments, both eliciting broad protective immune responses. We had previously reported that subcutaneous (s.c.+s.c.+s.c.) immunization with the systemic vaccine induced more pronounced humoral serum IgG responses, while intranasal (i.n.+i.n.+i.n.) immunization with the mucosal vaccine elicited a more pronounced mucosal secretory IgA (sIgA) response. We hypothesized that a combinatorial administration of the two vaccines might elicit more pronounced and broader protective immune responses. Therefore, this study aimed to determine the efficacy of combinatorial prime-boost immunization using both systemic and mucosal vaccines for a pneumococcal infection. Combinatorial prime-boost immunization (s.c.+i.n. and i.n.+s.c.) induced not only IgG, but also mucosal sIgA production at high levels. Systemic priming and mucosal boosting immunization (s.c.+i.n.) provided markedly better protection than homologous prime-boost immunization (s.c.+s.c.+s.c. and i.n.+i.n.+i.n.). Moreover, it induced more robust Th1 and Th17 cell-mediated immune responses than mucosal priming and systemic boosting immunization (i.n.+s.c.). These results indicate that combinatorial prime-boost immunization potentially induces a robust systemic and mucosal immune response, making it an optimal alternative for maximum protection against lethal pneumococcal infections.

     

Invasive and Noninvasive Streptococcus pneumoniae Capsule and Surface Protein Diversity following the Use of a Conjugate Vaccine

The 13-valent pneumococcal conjugate vaccine (PCV13) was introduced in the United States in 2010 for the prevention of invasive pneumococcal disease (IPD) and otitis media. While many studies have reported its potential efficacy for IPD, not much is known about the epidemiology of noninvasive disease following its introduction. We characterized the capsular types and surface protein genes of noninvasive pediatric pneumococcal isolates collected between 2002 and 2010 (n = 1,058) at Children''s of Alabama following the introduction of PCV7 and tested a subset of noninvasive and previously characterized IPD isolates for the presence of the pspA, pspC, and rrgC genes, which encode protection-eliciting proteins. PCV7 serotypes had dramatically decreased by 2010 (P < 0.0001), and only 50% of all noninvasive infections were caused by the PCV13 capsular serotypes. Serotype 19A accounted for 32% of the noninvasive isolates, followed by serotypes 35B (9%), 19F (7%), and 6C (6%). After 7 years of PCV7 usage, there were no changes in the frequencies of the pspA or pspC genes; 96% of the strains were positive for family 1 or 2 pspA genes, and 81% were also positive for pspC. Unexpectedly, more noninvasive than invasive strains were positive for rrgC (P < 0.0001), and the proportion of rrgC-positive strains in 2008 to 2010 was greater than that in 2002 to 2008 (IPD, P < 0.02; noninvasive, P < 0.001). Serotypes 19F, 19A, and 35B were more frequently rrgC positive (P < 0.005) than other serotypes. A vaccine containing antigens, such as PspA, PspC, and/or RrgC, can provide coverage against most non-PCV13-type pneumococci. Continued surveillance is critical for optimal future vaccine development.     

Enhanced Protective Antibody Responses to PspA after Intranasal or Subcutaneous Injections of PspA Genetically Fused to Granulocyte-Macrophage Colony-Stimulating Factor or Interleukin-2

Antibody to pneumococcal surface protein A (PspA) has been shown to be protective for Streptococcus pneumoniae infections in mice. In an attempt to define a model for inducing protective antibody to PspA in the absence of adjuvant, we designed two genetic fusions, PspA–interleukin-2 [IL-2]) and PspA–granulocyte-macrophage colony-stimulating factor (GM-CSF). These constructs maintained high cytokine function in vitro, as tested by their activity on IL-2 or GM-CSF-dependent cell lines. While intranasal immunization with PspA induced no detectable anti-PspA response, both PspA–IL-2 and PspA–GM-CSF stimulated high immunoglobulin G1 (IgG1) antibody responses. Interestingly, only the PspA–IL-2, not the PspA–GM-CSF, construct stimulated IgG2a antibody responses, suggesting that this construct directed the response along a TH1-dependent pathway. Comparable enhancement of the anti-PspA response with similar isotype profiles was observed after subcutaneous immunization as well. The enhancement observed with PspA–IL-2 was dependent on IL-2 activity in that it was not seen in IL-2 receptor knockout mice, while PspA in alum induced high-titer antibody in these mice. The antibody was tested for its protective activity in a mouse lethality model using S. pneumoniae WU-R2. Passive transfer of 1:90 dilutions of sera from mice immunized with PspA–IL-2 and PspA–GM-CSF elicited protection of CBA/N mice against intravenous challenge with over 170 50% lethal doses of capsular type 3 strain WU2. Only 0.17 μg or less of IgG antibody to PspA was able to provide passive protection against otherwise fatal challenge with S. pneumoniae. The data demonstrate that designing protein-cytokine fusions may be a useful approach for mucosal immunization and can induce high-titer systemic protective antibody responses.     

Live attenuated Streptococcus pneumoniae strains induce serotype-independent mucosal and systemic protection in mice

Streptococcus pneumoniae is an important human pathogen causing both mucosal (otitis media and pneumonia) and systemic (sepsis and meningitis) diseases. Due to increasing rates of antibiotic resistance, there is an urgent need to improve prevention of pneumococcal disease. Two currently licensed vaccines have been successful in reducing pneumococcal disease, but there are limitations with their use and effectiveness. Another approach for prevention is the use of live attenuated vaccines. Here we investigate the safety and protection induced by live attenuated strains of S. pneumoniae containing combinations of deletions in genes encoding three of its major virulence determinants: capsular polysaccharide (cps), pneumolysin (ply), and pneumococcal surface protein A (pspA). Both the cps and ply/pspA mutants of a virulent type 6A isolate were significantly attenuated in a mouse model of sepsis. These attenuated strains retained the ability to colonize the upper respiratory tract. A single intranasal administration of live attenuated vaccine without adjuvant was sufficient to induce both systemic and mucosal protection from challenge with a high dose of the parent strain. Immunization with cps mutants demonstrated cross-protective immunity following challenge with a distantly related isolate. Serum and mucosal antibody titers were significantly increased in mice immunized with the vaccine strains, and this antibody is required for full protection, as microMT mice, which do not make functional, specific antibody, were not protected by immunization with vaccine strains. Thus, colonization by live attenuated S. pneumoniae is a potentially safe and less complex vaccine strategy that may offer broad protection.