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.     

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.     

PspA, a surface protein of Streptococcus pneumoniae, is capable of eliciting protection against pneumococci of more than one capsular type.

Monoclonal antibodies against pneumococcal surface protein A (PspA) have been shown to protect mice from fatal pneumococcal infection. PspA is highly variable serologically, raising the possibility that PspA from one strain might not be able to elicit protective responses against strains which possess serologically different PspA. We have prepared a lambda gt11 library of pneumococcal genomic DNA and identified a clone expressing PspA. The recombinant PspA in this phage lysate elicited protection against pneumococcal infections with three strains of two different capsular serotypes. This finding demonstrated that PspA could elicit a protective response in the absence of other pneumococcal antigens. The observed protection was probably antibody mediated because it could be passively transferred with immune sera. Lambda lysates producing pneumococcal proteins other than PspA failed to elicit protection against fatal pneumococcal infection.     

Serum and mucosal antibody responses to pneumococcal protein antigens in children: relationships with carriage status

Streptococcus pneumoniae causes significant morbidity and mortality especially in children. Some pneumococcal protein antigens can protect mice against infection. Little information is available concerning the nature of naturally acquired protective immunity to pneumococci in humans induced by these antigens. This study investigates the relationships between systemic and local antibody production and carriage in children. Children undergoing adenoidectomy (n=112, ages 2-12 years) were studied. Nasopharyngeal swabs were collected for pneumococcal culture. Serum and saliva were assayed for antibodies to several pneumococcal proteins: choline binding protein A (CbpA), pneumolysin (Ply), pneumococcal surface adhesin A (PsaA) and pneumococcal surface protein A (PspA). Adenoidal mononuclear cells (MNC) were cultured with pneumococcal culture supernatants or recombinant proteins. Cell culture supernatants were analyzed for antigen-specific antibodies. Carriage rates fell with age and serum levels of anti-CbpA, Ply and PspA rose. Anti-CbpA and -Ply serum and salivary IgG antibody levels were higher in children who were culture negative than those who were colonized. Antigen stimulation increased respective antigen-specific IgG production by adenoidal MNC and these responses were greater in those who were colonized than in culture-negative children. Antibodies to CbpA and Ply may protect children aged 2 years and older against pneumococcal colonization. Adenoids may be important local induction and effector sites for both mucosal and systemic antibody production to pneumococcal proteins in children.     

Capsule does not block antibody binding to PspA, a surface virulence protein of Streptococcus pneumoniae

Of the proteins on the surface of Streptococcus pneumoniae, one of those best able to elicit protection against pneumococcal infection is pneumococcal surface protein A (PspA). Although this protein is attached to the membrane molecule, lipoteichoic acid, which is well beneath the capsule, PspA's ability to inhibit complement deposition and killing by apolactoferrin, suggests that it must have surface exposure. This study provides quantitative data showing that the capsular polysaccharide on types 2 and 3 pneumococci provides little or no masking ability of antibodies to bind PspA. Capsule was even observed to enhance, rather than inhibit the binding of two protective monoclonal antibodies to their epitopes on cell surface PspA. These results with antibodies to PspA are in contrast to binding by antibodies to the phosphocholine (PC) epitope of the lipoteichoic and teichoic acids. The binding of antibody to PC was largely, but not completely, blocked by capsular polysaccharide.     

Pneumococcal surface protein A is expressed in vivo, and antibodies to PspA are effective for therapy in a murine model of pneumococcal sepsis

Pneumococcal surface protein A (PspA) is an immunogenic protein expressed on the surface of all strains of Streptococcus pneumoniae (pneumococcus) and induces antibodies which protect against invasive infection in mice. Pneumococci used for infectious challenge in protection studies are typically collected from cultures grown in semisynthetic medium in vitro. The purpose of these studies is to confirm that PspA is expressed by pneumococci during growth in vivo at a level sufficient for antibodies to PspA to be protective. Mice were actively immunized with purified PspA or by passive transfer of monoclonal antibody (MAb) and challenged with a capsular type 3 strain in diluted whole blood from bacteremic mice. All were protected against challenge with 10 times the 50% lethal dose (LD(50)), and mice challenged with 1,000 times the LD(50) had increased survival compared with controls. Additionally, nonimmune mice treated with MAbs to PspA or PspA immune serum at 6 and 12 h after infection with 10 times the LD(50) also showed increased survival. Northern blot analysis of RNA from pneumococci grown either in vitro or in vivo showed similar levels of PspA mRNA. These results demonstrate that PspA is expressed in vivo in a mouse model and that immunization with PspA induces antibodies to an antigen which is expressed during the course of invasive infection. Immunotherapy with antibodies to PspA may have some utility in treating pneumococcal infections in humans.     

A 43-kilodalton pneumococcal surface protein, PspA: isolation, protective abilities, and structural analysis of the amino-terminal sequence.

PspA is an antigenically variable surface protein of Streptococcus pneumoniae that appears to be essential for full pneumococcal virulence. In addition, monoclonal antibodies to PspA protect mice against infection with specific strains of pneumococci virulent for mice. In this study, we have isolated the 43-kDa N-terminal half of the native 84-kDa PspA and determined the sequence of the first 45 amino acids. This sequence, the first obtained for a pneumococcal surface protein, is consistent with that of an amphiphatic coiled-coil alpha helix with a 7-residue periodicity common to fibrous proteins such as tropomyosin and streptococcal M protein. The 7-residue periodicity begins with residue 8 and extends throughout the remaining sequence for nearly 11 turns of the helix. Mice immunized with this purified PspA segment were protected from fatal pneumococcal challenge, thus demonstrating that those PspA epitopes eliciting protection were present in the N-terminal half of the molecule.     

Natural materno-fetal transfer of antibodies to PspA and to PsaA

PspA and PsaA are Streptococcus pneumoniae surface proteins and potential pneumococcal vaccine antigens. The aim of this study was to characterize the transplacental transfer of antibodies to PspA and to PsaA. Paired mother and cord blood sera were obtained at delivery from 28 women. Concentrations of antibodies against PspA, PsaA, tetanus toxoid (vaccine-induced antibodies) and P6-outer membrane protein (OMP) of nontypeable Haemophilus influenzae were determined by ELISA. Antibodies to PspA of the IgG, IgG1 and IgG2 antibodies were also determined. The geometric mean percentage (GM%) of the paired infant:mother antibody were calculated. Results: The GM% of the infant:mother antibody concentrations against PspA, PsaA and P6-OMP antibodies were 64.7% (3.3 micro g/ml in infants vs. 5.1 micro g/ml in mothers), 50.4% (6.8 micro g/ml vs. 13.5 micro g/ml) and 66.7% (5.6 micro g/ml vs. 8.4 micro g/ml), respectively; the GM% of antibodies against tetanus toxoid was 104.5% (4.6 micro g/ml vs. 4.4 micro g/ml). Transplacental transfer of IgG1 was more efficient than that of IgG2 (approximately 120%vs. 65%). A transplacental transfer of antibodies to PspA and to PsaA exist. Moreover, these data suggest an active placental transfer of IgG1 antibodies to PspA since the concentration of these antibodies were consistently higher in cord sera than in the mother's sera.     

Pneumococcal Diversity: Considerations for New Vaccine Strategies with Emphasis on Pneumococcal Surface Protein A (PspA)

Streptococcus pneumoniae is a problematic infectious agent, whose seriousness to human health has been underscored by the recent rise in the frequency of isolation of multidrug-resistant strains. Pneumococcal pneumonia in the elderly is common and often fatal. Young children in the developing world are at significant risk for fatal pneumococcal respiratory disease, while in the developed world otitis media in children results in substantial economic costs. Immunocompromised patients are extremely susceptible to pneumococcal infection. With 90 different capsular types thus far described, the diversity of pneumococci contributes to the challenges of preventing and treating S. pneumoniae infections. The current capsular polysaccharide vaccine is not recommended for use in children younger than 2 years and is not fully effective in the elderly. Therefore, innovative vaccine strategies to protect against this agent are needed. Given the immunogenic nature of S. pneumoniae proteins, these molecules are being investigated as potential vaccine candidates. Pneumococcal surface protein A (PspA) has been evaluated for its ability to elicit protection against S. pneumoniae infection in mouse models of systemic and local disease. This review focuses on immune system responsiveness to PspA and the ability of PspA to elicit cross-protection against heterologous strains. These parameters will be critical to the design of broadly protective pneumococcal vaccines.     

肺炎链球菌毒力蛋白在侵入性感染中的免疫原性评价

目的观察肺炎链球菌表面蛋白A(PspA)、肺炎链球菌表面黏附素A(PsaA)及肺炎链球菌溶菌素(Ply)激发自然途径感染肺炎链球菌机体保护性体液免疫应答的情况,评价这三种毒力蛋白作为治疗性肺炎链球菌疫苗候选抗原的免疫原性。方法采集侵入性肺炎链球菌感染患者(实验组)及同期确诊为侵入性其它细菌感染患者(对照第一组)和同期确诊为非感染性疾病患者(对照第二组)各36例急性期(第0+3天)及恢复期(第21±3天)血清样本,酶联免疫吸附法(ELISA)检测患者血清中肺炎链球菌3种毒力蛋白抗原特异性抗体IgG水平变化。结果实验组与两组对照组间第(0+3)天血清的3种肺炎链球菌毒力蛋白特异性抗体水平无显著性差异(P>0.05)。第一组对照组恢复期较急性期血清中3种毒力蛋白特异性抗体水平无显著变化(P>0.05),而实验组恢复期血清3种肺炎链球菌毒力蛋白特异性抗体水平明显高于急性期血清抗体水平8～30倍(P<0.01),其中PspA和Ply蛋白抗原特异性抗体水平升高更明显,血清中针对PspA家族1(PspA-Faml)特异性抗体水平高于针对PspA家族2(PspA-Fam2)的特异性抗体水平(P<0.01)。结论 PsaA,P...     

Protection of the elderly from pneumococcal pneumonia with a protein-based vaccine?

Vaccines exist to protect children and adults from pneumococcal infection. The adult vaccine contains capsular polysaccharides from those pneumococci causing the vast majority of pneumococcal infection around the world. This vaccine is, however, poorly immunogenic and not as protective as would be desired. The vaccine for children is a seven-valent conjugate vaccine, which is highly protective against invasive infection and offers some protection against otitis media and pneumococcal carriage. The capsular types in the vaccine are not all appropriate for the developing world and the vaccine is too expensive for use in the developing world. As a result of these problems there have been extensive efforts to develop pneumococcal vaccines for adults and children based on cross-reactive protein antigens. The molecules used are in general virulence factors and the antibodies to them neutralize their function, thus reducing the virulence of the infecting bacteria. Studies in humans have revealed that the proteins studied are invariably immunogenic in humans, as at least low levels of antibody are seen following colonization or infection. Studies in mice have demonstrated that vaccines containing more than one of these virulence proteins are generally more protective than those involving just one. Proteins that have been studied the most in mice are pneumococcal surface protein A (PspA), PspC, PsaA, and pneumolysin. PspA has been used in human safety trials and was shown to elicit antibodies that can protect mice from otherwise fatal pneumococcal infections.     

Enhanced immunogenicity of pneumococcal surface adhesin A by genetic fusion to cytokines and evaluation of protective immunity in mice

Immunization of mice with pneumococcal surface adhesin A (PsaA) emulsified in complete Freund's adjuvant (CFA) provides protection against systemic infection with Streptococcus pneumoniae. Because the use of CFA is not acceptable in humans, we sought to develop alternative means of enhancing the immunogenicity of protein antigens of potential use in pneumococcal vaccines. We designed a series of genetic constructs in which coding sequences for PsaA were linked to sequences encoding either murine interleukin-2 (mIL-2), mIL-4, or two copies of an immunostimulatory nonapeptide derived from mIL-1beta. The PsaA-cytokine constructs were cloned and expressed in Escherichia coli. Mice immunized twice with PsaA-IL-2, or PsaA-IL-4 responded with PsaA-specific antibody production comparable in magnitude to that of mice primed with PsaA in CFA and boosted with PsaA in incomplete Freund's adjuvant (PsaA-Adj). Antibodies elicited by PsaA-Adj were predominantly of the immunoglobulin G1 (IgG1) subclass, while PsaA-IL-2 and PsaA-IL-4 elicited substantial amounts of IgG2a in addition to IgG1. Mice immunized with PsaA-Adj or PsaA-IL-4 were partially protected against intraperitoneal challenge with virulent S. pneumoniae (30% overall survival beyond 15 days postchallenge). Mice immunized with PsaA and no adjuvant or PsaA-IL-2 exhibited 0 or 5% survival rates, respectively, following challenge. In contrast, mice immunized twice with capsular polysaccharide were 100% protected. The modest levels of protection seen in mice immunized with PsaA and its more immunogenic derivatives may be explained in part by the relative inaccessibility of antibody to PsaA on the surface of encapsulated S. pneumoniae.     

The putative proteinase maturation protein A of Streptococcus pneumoniae is a conserved surface protein with potential to elicit protective immune responses

Surface-exposed proteins often play an important role in the interaction between pathogenic bacteria and their host. We isolated a pool of hydrophobic, surface-associated proteins of Streptococcus pneumoniae. The opsonophagocytic activity of hyperimmune serum raised against this protein fraction was high and species specific. Moreover, the opsonophagocytic activity was independent of the capsular type and chromosomal genotype of the pneumococcus. Since the opsonophagocytic activity is presumed to correlate with in vivo protection, these data indicate that the protein fraction has the potential to elicit species-specific immune protection with cross-protection against various pneumococcal strains. Individual proteins in the extract were purified by two-dimensional gel electrophoresis. Antibodies raised against three distinct proteins contributed to the opsonophagocytic activity of the serum. The proteins were identified by mass spectrometry and N-terminal amino acid sequencing. Two proteins were the previously characterized pneumococcal surface protein A and oligopeptide-binding lipoprotein AmiA. The third protein was the recently identified putative proteinase maturation protein A (PpmA), which showed homology to members of the family of peptidyl-prolyl cis/trans isomerases. Immunoelectron microscopy demonstrated that PpmA was associated with the pneumococcal surface. In addition, PpmA was shown to elicit species-specific opsonophagocytic antibodies that were cross-reactive with various pneumococcal strains. This antibody cross-reactivity was in line with the limited sequence variation of ppmA. The importance of PpmA in pneumococcal pathogenesis was demonstrated in a mouse pneumonia model. Pneumococcal ppmA-deficient mutants showed reduced virulence. The properties of PpmA reported here indicate its potential for inclusion in multicomponent protein vaccines.     

Immune responses to novel pneumococcal proteins pneumolysin,PspA, PsaA,and CbpA in adenoidal B cells from children

Studies of mice suggest that pneumococcal proteins, including PspA, pneumolysin, PsaA, and CbpA, are promising vaccine candidates. To determine whether these proteins are good mucosal immunogens in humans, adenoidal lymphocytes from 20 children who had adenoidectomies were isolated and tested by ELISpot for antigen-specific antibody-secreting cells (ASCs). Cells were also cultured for 7 days in the presence of a concentrated culture supernatant (CCS) from a type 14 strain of pneumococcus which contained secreted pneumococcal proteins, including PspA, pneumolysin, PsaA, and CbpA, and then tested by ELISpot. ELISpot assays done on freshly isolated cells detected ASCs to all four antigens in most children studied. However, there were differences both between antigens and between isotypes. The densities of immunoglobulin G (IgG) ASCs against both PsaA and CbpA were significantly higher than those of ASCs for PspA and PdB (pneumolysin toxoid B) (P < 0.001). For all antigens, the numbers of IgA ASCs tended to be lower than those of both IgG and IgM ASCs. The numbers of anti-CbpA and -PsaA IgA ASCs were higher than those of anti-PdB IgA ASCs (P < 0.01). Concentrations of IgA antibodies to PspA and PsaA in saliva correlated with the numbers of IgA ASCs to PspA and PsaA in freshly isolated adenoidal cells, but no such correlation was found between salivary IgG antibody concentrations and IgG ASCs to the four antigens in adenoidal cells. In cultured cells, anti-PspA, -PsaA, and -CbpA IgG ASCs proliferated significantly, but only two of eight samples showed >2-fold increases in anti-CbpA and -PspA IgA ASCs after CCS stimulation. The results suggest that CbpA, PsaA, and PspA may be good upper respiratory mucosal antigens in children. Adenoids may be important inductive sites for memory IgG responses and important sources of salivary IgA. Some protein antigens may also prime for mucosal IgA memory. These data support the effort to explore mucosal immunization against pneumococcal infection.     

Pneumococcal surface protein A (PspA) is serologically highly variable and is expressed by all clinically important capsular serotypes of Streptococcus pneumoniae.

Pneumococcal surface protein A (PspA) has been shown previously to elicit antibodies protective against pneumococcal infection and to be necessary for full pneumococcal virulence in mice. The protein was originally defined by the two mouse monoclonal antibodies Xi64 and Xi126, which together recognized PspA on 14% of pneumococcal isolates. Some PspA molecules reacted with both antibodies, but most reacted with only one or the other. In the present study we demonstrated that PspA is produced by all pneumococci, confirming our hypothesis that there are variants of PspA which are not detected by Xi64 and Xi126. We produced a rabbit antiserum and five additional monoclonal antibodies specific for PspA for these studies. The rabbit antiserum reacted with each of 95 pneumococcal isolated tested, comprising 16 capsular serotypes. One or more of the seven monoclonal anti-PspA antibodies reacted with 95% (53 of 57) of pneumococcal isolates tested. The specificity of the monoclonal and polyclonal antibodies to PspA was confirmed in two ways: (i) by detection of molecules on wild-type pneumococci that are identical in molecular weight to those detected in Western blots (immunoblots) with Xi64 and Xi126 and (ii) by the use of mutants of Streptococcus pneumoniae that fail to produce PspA or that produce a truncated form of PspA. By using the seven monoclonal antibodies, we observed 31 PspA types among the 57 isolates. When the 53 strains reactive with the monoclonal antibodies were analyzed by capsular type as well as by serologic type and molecular weight of PspA, we observed 50 different clonotypes of pneumococci.     

Novel protein-based pneumococcal vaccines administered with the Th1-promoting adjuvant IC31 induce protective immunity against pneumococcal disease in neonatal mice

Streptococcus pneumoniae is responsible for many vaccine-preventable deaths, annually causing around 1 million deaths in children younger than 5 years of age. A new generation of pneumococcal vaccines based on conserved proteins is being developed. We evaluated the immunogenicities and protective efficacies of four pneumococcal protein vaccine candidates, PcsB, StkP, PsaA, and PspA, in a neonatal mouse model. Mice were immunized three times and challenged intranasally with virulent pneumococci. All four proteins were immunogenic in neonatal mice, and antibody (Ab) responses were significantly enhanced by the novel adjuvant IC31, which consists of an antibacterial peptide (KLKL5KLK) and a synthetic oligodeoxynucleotide, ODN1a, that signals through Toll-like receptor 9 (TLR9). Two single proteins, StkP and PspA, combined with IC31 significantly reduced pneumococcal bacteremia but had no effects on lung infection. Three proteins, PcsB, StkP, and PsaA, were evaluated with alum or IC31. IC31 enhanced Ab responses and avidity to all three proteins, whereas alum enhanced Ab responses and avidity to StkP and PsaA only. Mice receiving the trivalent protein formulation with IC31 had significantly reduced bacteremia and lung infection compared to unvaccinated mice, but the level of protection was dependent on the dose of IC31. When PspA was added to the trivalent protein formulation, the dose of IC31 needed to obtain protective immunity could be reduced. These results demonstrate that a novel pneumococcal protein-based vaccine is immunogenic at an early age of mice and emphasize the benefits of using a combination of conserved proteins and an effective adjuvant to elicit potent protective immunity against invasive pneumococcal disease.     

Linkage Units in Cell Walls of Gram-Positive Bacteria

Abstract

Pneumococcal infection persists as a major cause of pneumonia, otitis media, and meningitis in infants. Children less than 2 years of age show the highest incidence of pneumococcal diseases.

Production of monoclonal antibody (MAb) to polysaccharide (PS) and binding characteristics to PS epitopes were studied. Removal of the O-acetyl group from 9V PS by alkali hydrolysis resulted in a decreased binding with rabbit 9V antiserum (AS). However, the binding reaction with 9V MAb was less affected by the loss of O-acetyl content. Type 9V IgG MAb provided passive protection and enhanced the opsonophagocytic activity of polymorphonuclear (PMN) leukocytes to kill type 9V pneumococci.

The pathogenecity of pneumococci is attributed to various virulence factors distributed on the cell surface, including capsular polysaccharide and protein antigens, for example, pneumolysin, autolysin, pneumococcal surface protein A (PspA), pneumococcal surface adhesion (PsaA), and hemin binding protein. Some of these protein antigens may be used as a component to combine with pneumococcal PS vaccine or as a carrier of conjugate vaccine.

Clinical trials of pneumococcal conjugate vaccines showed that covalent linkage of capsular PS to protein carriers improved the immunogenicity of the PS. Development of glycoconjugate vaccine for selected pneumococcal types will help solve the problem of poor immunogenecity of PS vaccine in young children used for prevention of pneumococcal infection.     

Immunization of mice with combinations of pneumococcal virulence proteins elicits enhanced protection against challenge with Streptococcus pneumoniae

The vaccine potential of a combination of three pneumococcal virulence proteins was evaluated in an active-immunization-intraperitoneal-challenge model in BALB/c mice, using very high challenge doses of Streptococcus pneumoniae. The proteins evaluated were a genetic toxoid derivative of pneumolysin (PdB), pneumococcal surface protein A (PspA), and a 37-kDa metal-binding lipoprotein referred to as PsaA. Mice immunized with individual proteins or combinations thereof were challenged with high doses of virulent type 2 or type 4 pneumococci. The median survival times for mice immunized with combinations of proteins, particularly PdB and PspA, were significantly longer than those for mice immunized with any of the antigens alone. A similar effect was seen in a passive protection model. Thus, combinations of pneumococcal proteins may provide the best non-serotype-dependent protection against S. pneumoniae.     

Preclinical evaluation of the Pht proteins as potential cross-protective pneumococcal vaccine antigens

Current pneumococcal vaccines are composed of capsular polysaccharides (PS) of various serotypes, either as free PS or as protein-PS conjugates. The use of pneumococcus protein antigens that are able to afford protection across the majority of serotypes is envisaged as a relevant alternative and/or complement to the polysaccharides. In this context, based on several studies, the Pht protein family emerged as relevant vaccine candidates. The purpose of the present study was to evaluate the Pht protein family in several preclinical mouse models. Immunization with these antigens was compared with immunization with other pneumococcal antigens, such as CbpA, PspA, and PsaA. In a nasopharyngeal colonization model and in a lung colonization model, the Phts were found to be superior to the other candidates in terms of efficacy of protection and serotype coverage. Likewise, vaccination with PhtD allowed higher animal survival rates after lethal intranasal challenge. Finally, a passive transfer model in which natural anti-PhtD human antibodies were transferred into mice demonstrated significant protection against lethal intranasal challenge. This indicates that natural anti-PhtD human antibodies are able to protect against pneumococcal infection. Our findings, together with the serotype-independent occurrence of the Phts, designate this protein family as valid candidate antigens to be incorporated in protein-based pneumococcal vaccines.     

Differential PsaA-, PspA-, PspC-, and PdB-specific immune responses in a mouse model of pneumococcal carriage

Larger numbers of pneumococci were detected in the nasal tract compared to the lung, cervical lymph nodes, and spleen 1, 2, 4, 7, 14, and 21 days after nasal challenge with Streptococcus pneumoniae strain EF3030. In this mouse model of pneumococcal carriage, peripheral S. pneumoniae pneumococcal surface adhesin A (PsaA)-specific humoral responses (immunoglobulin G2a [IgG2a] > IgG1 = IgG2b > IgG3) were significantly higher than pneumococcal surface protein A (PspA)-specific, genetic toxoid derivative of pneumolysin (PdB)-specific, or pneumococcal surface protein C (PspC)-specific serum antibody levels. However, PspA-specific mucosal IgA antibody levels were significantly higher than those against PsaA, PdB, and PspC. In general, both PsaA- and PspA-specific lung-, cervical lymph node-, nasal tract-, and spleen-derived CD4(+) T-cell cytokine (interleukin-4, interleukin-6, granulocyte-macrophage colony-stimulating factor, gamma interferon, and tumor necrosis factor alpha) and proliferative responses were higher than those for either PspC or PdB. Taken together, these findings suggest that PsaA- and PspA-specific mucosal responses as well as systemic humoral and T helper cell cytokine responses are predominantly yet differentially induced during pneumococcal carriage.