Protective role of PhtD and its amino and carboxyl fragments against pneumococcal sepsis

The implementation of polysaccharide-based vaccines has massively reduced the incidence of invasive pneumococcal diseases. However, there is great concern regarding serotype replacement and the increase in antibiotic resistant strains expressing non-vaccine capsular types. In addition, conjugate vaccines have high production costs, a limiting factor for their implementation in mass immunization programs in developing countries. These limitations have prompted the development of novel vaccine strategies for prevention of Streptococcus pneumoniae infections. The use of conserved pneumococcal antigens such as recombinant proteins or protein fragments presents an interesting serotype-independent alternative. Pht is a family of surface-exposed proteins which have been evaluated as potential vaccine candidates with encouraging results. The present work investigated the immune responses elicited by subcutaneous immunization of mice with the polyhistidine triad protein D (PhtD) and its amino and carboxyl terminal fragments. The proteins were immunogenic and protective against pneumococcal sepsis in mice. Antibodies raised against PhtD increased complement C3b deposition on the pneumococcal surface, mainly mediated by the alternative pathway. Sera from mice immunized with PhtD and PhtD_Cter promoted an increase in bacterial uptake by mouse phagocytes. The interaction of PhtD with the complement system regulator factor H was investigated in silico and in vitro by ELISA and western blot, confirming PhtD as a factor-H binding protein. Our results support the inclusion of PhtD and more specifically, its C-terminal fragment in a multicomponent serotype independent vaccine and suggests a role for the complement system in PhtD-mediated protection.     

The global distribution and diversity of protein vaccine candidate antigens in the highly virulent Streptococcus pnuemoniae serotype 1

Serotype 1 is one of the most common causes of pneumococcal disease worldwide. Pneumococcal protein vaccines are currently being developed as an alternate intervention strategy to pneumococcal conjugate vaccines. Pre-requisites for an efficacious pneumococcal protein vaccine are universal presence and minimal variation of the target antigen in the pneumococcal population, and the capability to induce a robust human immune response. We used in silico analysis to assess the prevalence of seven protein vaccine candidates (CbpA, PcpA, PhtD, PspA, SP0148, SP1912, SP2108) among 445 serotype 1 pneumococci from 26 different countries, across four continents. CbpA (76%), PspA (68%), PhtD (28%), PcpA (11%) were not universally encoded in the study population, and would not provide full coverage against serotype 1. PcpA was widely present in the European (82%), but not in the African (2%) population. A multi-valent vaccine incorporating CbpA, PcpA, PhtD and PspA was predicted to provide coverage against 86% of the global population. SP0148, SP1912 and SP2108 were universally encoded and we further assessed their predicted amino acid, antigenic and structural variation. Multiple allelic variants of these proteins were identified, different allelic variants dominated in different continents; the observed variation was predicted to impact the antigenicity and structure of two SP0148 variants, one SP1912 variant and four SP2108 variants, however these variants were each only present in a small fraction of the global population (<2%). The vast majority of the observed variation was predicted to have no impact on the efficaciousness of a protein vaccine incorporating a single variant of SP0148, SP1912 and/or SP2108 from S. pneumoniae TIGR4. Our findings emphasise the importance of taking geographic differences into account when designing global vaccine interventions and support the continued development of SP0148, SP1912 and SP2108 as protein vaccine candidates against this important pneumococcal serotype.     

Pneumococcal carriage and acute otitis media induce serum antibodies to pneumococcal surface proteins CbpA and PhtD in children

We assessed the development and role of serum anti-CbpA and -PhtD in early childhood in relation to pneumococcal exposure. Serum IgG concentrations to CbpA and PhtD were measured with enzyme immunoassay in serum samples collected at the ages of 6, 12, 18, and 24 months from 50 healthy children and from 50 adults. Furthermore, antibodies to CbpA, PhtD and the C-terminal fragment of PhtD (PhtD C) were measured in serum samples collected at 12 (N = 286) and 18 months (N = 259) to evaluate the risk of subsequent pneumococcal acute otitis media (AOM) in relation to antibody concentrations. The increase in anti-CbpA and -PhtD concentrations was related to prior pneumococcal exposure. At 12 and 18 months, in the risk model of pneumococcal AOM adjusted for prior pneumococcal AOM, higher concentrations of anti-CbpA, but not anti-PhtD, were associated with a lowered risk of subsequent pneumococcal AOM. In conclusion, pneumococcal exposure induces the development of serum anti-CbpA and -PhtD in early childhood. Anti-CbpA antibodies may play a role in the prevention of subsequent pneumococcal AOM during the second year of life.     

In vivo screen of genetically conserved Streptococcus pneumoniae proteins for protective immunogenicity

We evaluated 52 different E. coli expressed pneumococcal proteins as immunogens in a BALB/c mouse model of S. pneumoniae lung infection. Proteins were selected based on genetic conservation across disease-causing serotypes and bioinformatic prediction of antibody binding to the target antigen. Seven proteins induced protective responses, in terms of reduced lung burdens of the serotype 3 pneumococci. Three of the protective proteins were histidine triad protein family members (PhtB, PhtD and PhtE). Four other proteins, all bearing LPXTG linkage domains, also had activity in this model (PrtA, NanA, PavB and Eng). PrtA, NanA and Eng were also protective in a CBA/N mouse model of lethal pneumococcal infection. Despite data inferring widespread genomic conservation, flow-cytometer based antisera binding studies confirmed variable levels of antigen expression across a panel of pneumococcal serotypes. Finally, BALB/c mice were immunized and intranasally challenged with a viulent serotype 8 strain, to help understand the breadth of protection. Those mouse studies reaffirmed the effectiveness of the histidine triad protein grouping and a single LPXTG protein, PrtA.     

Vaccination with a Streptococcus pneumoniae trivalent recombinant PcpA,PhtD and PlyD1 protein vaccine candidate protects against lethal pneumonia in an infant murine model

Streptococcus pneumoniae infections continue to cause significant worldwide morbidity and mortality despite the availability of efficacious serotype-dependent vaccines. The need to incorporate emergent strains expressing additional serotypes into pneumococcal polysaccharide conjugate vaccines has led to an identified need for a pneumococcal protein-based vaccine effective against a broad scope of serotypes. A vaccine consisting of several conserved proteins with different functions during pathogenesis would be preferred. Here, we investigated the efficacy of a trivalent recombinant protein vaccine containing pneumococcal choline-binding protein A (PcpA), pneumococcal histidine triad D (PhtD), and genetically detoxified pneumolysin (PlyD1) in an infant mouse model. We found the trivalent vaccine conferred protection from lethal pneumonia challenges using serotypes 6A and 3. The observed protection with trivalent PcpA, PhtD, and PlyD1 vaccine in infant mice supports the ongoing study of this candidate vaccine in human infant clinical trials.     

A protein-based pneumococcal vaccine protects rhesus macaques from pneumonia after experimental infection with Streptococcus pneumoniae

Infections caused by Streptococcus pneumoniae are a major cause of mortality throughout the world. Protein-based pneumococcal vaccines are envisaged to replace or complement the current polysaccharide-based vaccines. In this context, detoxified pneumolysin (dPly) and pneumococcal histidine triad protein D (PhtD) are two potential candidates for incorporation into pneumococcal vaccines. In this study, the protective efficacy of a PhtD-dPly vaccine was evaluated in a rhesus macaque (Macaca mulatta) model of pneumonia. The animals were immunized twice with 10 μg of PhtD and 10 μg of dPly formulated in the Adjuvant System AS02 or with AS02 alone, before they were challenged with a 19F pneumococcal strain. The survival was significantly higher in the protein-vaccinated group and seemed to be linked to the capacity to greatly reduce bacterial load within the first week post-challenge. Vaccination elicited high concentrations of anti-PhtD and anti-Ply antibodies and a link was found between survival and antibody levels. In conclusion, AS02-adjuvanted PhtD-dPly vaccine protects against S. pneumoniae-induced pneumonia. It is probable that the protection is at least partially mediated by PhtD- and Ply-specific antibodies.     

Immunization with pneumococcal elongation factor Tu enhances serotype-independent protection against Streptococcus pneumoniae infection

Vaccination is an effective strategy to prevent pneumococcal diseases. Currently, licensed vaccines include the pneumococcal polysaccharide vaccine (PPSV) and the pneumococcal conjugate vaccine (PCV), which target some of the most common of the 94 serotypes of S. pneumoniae based on their capsular composition. However, it has been reported that PPSV is not effective in children aged less than 2?years old and PCV induces serotype replacement, which means that the pneumococcal population has changed following widespread introduction of these vaccines, and the non-vaccine serotypes have increased in being the cause of invasive pneumococcal disease. Therefore, it is important that there is development of novel pneumococcal vaccines to either replace or complement current polysaccharide-based vaccines. Our previous study suggested that S. pneumoniae releases elongation factor Tu (EF-Tu) through autolysis followed by the induction of proinflammatory cytokines in macrophages via toll-like receptor 4, that may contribute to the development of pneumococcal diseases. In this study, we investigated the expression of EF-Tu in various S. pneumoniae strains and whether EF-Tu could be an antigen candidate for serotype-independent vaccine against pneumococcal infection. Western blotting and flow cytometry analysis revealed that EF-Tu is a common factor expressed on the surface of all pneumococcal strains tested, as well as intracellularly. In addition, we demonstrate that immunization with recombinant (r) EF-Tu induced the production of inflammatory cytokines and the IgG1 and IgG2a antibodies in mice, and increased the CD4⁺ T-cells proportion in splenocytes. We also reveal that anti-EF-Tu serum increased the phagocytic activity of mouse peritoneal macrophages against S. pneumoniae infection, independent of their serotypes. Finally, our results indicate that mice immunized with rEF-Tu were significantly and non-specifically protected against lethal challenges with S. pneumoniae serotypes (2 and 15A). Therefore, pneumococcal EF-Tu could be an antigen candidate for the serotype-independent vaccine against pneumococcal infection.     

Safety,reactogenicity and immunogenicity of a novel pneumococcal protein-based vaccine in adults: A phase I/II randomized clinical study

Background

New vaccines containing highly conserved Streptococcus pneumoniae proteins such as pneumolysin toxoid (dPly) and histidine-triad protein D (PhtD) are being developed to provide broader protection against pneumococcal disease. This study evaluated the safety, reactogenicity and immunogenicity of different pneumococcal protein-containing formulations in adults.

Methods

In a phase I double-blind study (www.clinicaltrials.gov: NCT00707798), healthy adults (18–40 years) were randomized (1:2:2:2:2:2:2) to receive two doses of one of six investigational vaccine formulations 2 months apart, or a single dose of the control 23-valent pneumococcal polysaccharide vaccine (23PPV; Pneumovax23™, Sanofi Pasteur MSD) followed by placebo. The investigational formulations contained dPly alone (10 or 30 μg), or both dPly and PhtD (10 or 30 μg each) alone or combined with the polysaccharide conjugates of the 10-valent pneumococcal non-typeable Haemophilus influenzae protein D conjugate vaccine (PHiD-CV; Synflorix™, GlaxoSmithKline Vaccines). Two groups primed with a formulation containing dPly and PhtD (10 or 30 μg each) continued to the follow-up phase II study (NCT00896064), in which they received a booster dose at 5–9 months after primary vaccination.

Results

Of 156 enrolled and vaccinated adults, 146 completed the primary immunization and 43 adults received a booster dose. During primary and booster vaccination, for any formulation, ≤8.9% of doses were followed by grade 3 solicited local or general adverse events. No fever >39.5 °C (oral temperature) was reported. Unsolicited adverse events considered causally related to vaccination were reported following ≤33.3% of investigational vaccine doses. No serious adverse events were reported for adults receiving investigational vaccine formulations. Formulations containing dPly with or without PhtD were immunogenic for these antigens; polysaccharide conjugate-containing formulations were also immunogenic for those 10 polysaccharides.

Conclusion

Investigational vaccine formulations containing dPly and PhtD were well tolerated and immunogenic when administered to healthy adults as standalone protein vaccine or combined with PHiD-CV conjugates.     

Safety and immunogenicity of an investigational vaccine containing two common pneumococcal proteins in toddlers: A phase II randomized clinical trial

Background

To provide broader protection against pneumococcal disease, new vaccines containing conserved Streptococcus pneumoniae proteins are being developed. This study assessed the safety, reactogenicity and immunogenicity of four formulations containing pneumococcal proteins pneumolysin toxoid (dPly) and histidine triad protein (PhtD) in toddlers.

Methods

In this phase II, multicenter, observer-blind study (www.clinicaltrials.gov: NCT00985751) conducted in the Czech Republic, toddlers (12–23 months) were randomized (1:1:1:1:1) to receive one of four investigational vaccine formulations (10 or 30 μg each of dPly and PhtD, alone or in combination with polysaccharide conjugates from the pneumococcal non-typeable Haemophilus influenzae protein-D conjugate vaccine [PHiD-CV]), or the licensed PHiD-CV, in a 2-dose primary series plus booster at study months 0, 2 and 6. Solicited local and general symptoms were recorded within seven days post-vaccination, unsolicited symptoms within 31 days post-vaccination, and serious adverse events (SAEs) during the entire study period. Antibody concentrations against the vaccine components were measured pre-vaccination, one month post-dose 2, pre- and one month post-booster.

Results

257 toddlers were enrolled and vaccinated. Percentages of solicited local and general symptoms following the different investigational formulations were generally within the same ranges as for PHiD-CV. After each dose, grade 3 fever (>40.0 °C, rectal measurement) was reported for maximum one toddler in each group with no differences between investigational formulations and PHiD-CV during primary vaccination. 23 SAEs were reported for 17 toddlers, with distribution balanced between all groups except the group receiving 30 μg dPly/PhtD with PHiD-CV-conjugates (no SAEs reported). None of the SAEs were considered to be vaccine-related.For all pneumococcal protein-containing formulations, anti-PhtD and anti-Ply antibody geometric mean concentrations increased from pre-vaccination to post-dose 2 and from pre- to post-booster vaccination.

Conclusion

All investigational vaccine formulations were well-tolerated and immunogenic when administered to toddlers as a 2-dose primary vaccination followed by a booster dose.     

Protection against pneumococcal infection elicited by immunization with multiple pneumococcal heat shock proteins

Heat shock proteins (HSPs) play important roles in the pathogenesis of pneumococcal infection, and they are considered as potential protein vaccine antigens. In this study, we investigated the efficacy of immunization with pneumococcal HSPs, including ClpP (hsp100/Clp peptidase subunit), DnaJ (hsp40) and GroEL (hsp60), to protect against pneumococcal carriage, lung colonization and sepsis in mouse models using different serotypes of Streptococcus pneumoniae. In a nasopharyngeal colonization model by serotype 6B or 14 and in a lung colonization model by serotype 19F, immunization with pneumococcal HSPs could elicit effective protection. Likewise, vaccination with ClpP, DnaJ or GroEL allowed significantly longer mouse survival times after lethal intranasal challenge with serotype pneumococcal 2, 3 or 4. Interestingly, combinations of these HSPs could consistently enhance the protection against nasopharynx carriage, lung colonization as well as invasive infection caused by different pneumococcal serotypes. In an in vitro killing assay, anti-sera against ClpP, DnaJ or GroEL could kill S. pneumoniae by polymorphonuclear leukocytes in a complement-dependent way, and combinations of multiple anti-sera against these HSPs could increase the killing ability compared with single anti-sera. Finally, passive immunization studies with anti-sera against pneumococcal HSPs also demonstrated that an additive effect could be achieved by using multiple anti-sera when compared with single anti-sera. Thus, inclusion of multiple pneumococcal HSPs is important for the development of protein-based pneumococcal vaccines.     

Immunogenicity of pneumococcal conjugate vaccine formulations containing pneumococcal proteins,and immunogenicity and reactogenicity of co-administered routine vaccines – A phase II,randomised, observer-blind study in Gambian infants

Background

Two conserved pneumococcal proteins, pneumolysin toxoid (dPly) and pneumococcal histidine triad protein D (PhtD), combined with 10 polysaccharide conjugates from the pneumococcal non-typeable Haemophilus influenzae protein D-conjugate vaccine (PHiD-CV) in two investigational pneumococcal vaccine (PHiD-CV/dPly/PhtD) formulations were immunogenic and well-tolerated when administered to Gambian children. Here, we report immunogenicity of the polysaccharide conjugates, and immunogenicity and reactogenicity of co-administered routine vaccines.

Efficacy,safety and immunogenicity of a pneumococcal protein-based vaccine co-administered with 13-valent pneumococcal conjugate vaccine against acute otitis media in young children: A phase IIb randomized study

BackgroundNative American populations experience a substantial burden of pneumococcal disease despite use of highly effective pneumococcal conjugate vaccines (PCVs). Protein-based pneumococcal vaccines may extend protection beyond the serotype-specific protection elicited by PCVs.MethodsIn this phase IIb, double-blind, controlled trial, 6–12 weeks-old Native American infants randomized 1:1, received either a protein-based pneumococcal vaccine (dPly/PhtD) containing pneumolysin toxoid (dPly, 10 µg) and pneumococcal histidine triad protein D (PhtD, 10 µg) or placebo, administered along with 13-valent PCV (PCV13) at ages 2, 4, 6 and 12–15 months. Other pediatric vaccines were given per the routine immunization schedule. We assessed vaccine efficacy (VE) against acute otitis media (AOM) and acute lower respiratory tract infection (ALRI) endpoints. Immunogenicity, reactogenicity and unsolicited adverse events were assessed in a sub-cohort and serious adverse events were assessed in all children.Results1803 infants were randomized (900 dPly/PhtD; 903 Control). VE against all episodes of American Academy of Pediatrics (AAP)-defined AOM was 3.8% (95% confidence interval: −11.4, 16.9). Point estimates of VE against other AOM outcomes ranged between 2.9% (−9.5, 14.0) and 5.2% (−8.0, 16.8). Point estimates of VE against ALRI outcomes ranged between −4.4% (−39.2, 21.8) and 2.0% (−18.3, 18.8). Point estimates of VE tended to be higher against first than all episodes but the confidence intervals included zero. dPly/PhtD vaccine was immunogenic and had an acceptable reactogenicity and safety profile after primary and booster vaccination in Native American infants.ConclusionsThe dPly/PhtD vaccine was immunogenic and well tolerated, however, incremental efficacy in preventing AAP-AOM over PCV13 was not demonstrated.Clinical trials registrationNCT01545375 (www.clinicaltrials.gov)     

High pneumococcal serotype specific IgG,IgG1 and IgG2 levels in serum and the middle ear of children with recurrent acute otitis media receiving ventilation tubes

Recurrent acute otitis media (AOM), frequently caused by Streptococcus pneumoniae, is a major paediatric health problem. A reduced antibody response against pneumococcal polysaccharides may contribute to an increased susceptibility to AOM. Using a multiplex bead-based assay we measured IgG, IgG1 and IgG2 levels against 11 pneumococcal polysaccharides in serum samples from 166 children below 3 years of age with a history of at least 3 episodes of acute otitis media receiving ventilation tubes, and 61 healthy controls. Pneumococcal serotype specific IgG was also determined in 144 middle ear effusion samples. Pneumococcal serotype specific IgG, IgG1 and IgG2 levels were similar in children with or without AOM, except for IgG and IgG1 levels against serotype 5, which were significantly higher in children with a history of frequent AOM (IgG: 137.5 μg/ml vs. 84.0 μg/ml; p = 0.02; IgG1: 24.5 μg/ml vs. 18.2 μg/ml; p = 0.05). The age-related development of pneumococcal serotype-specific IgG, IgG1 and IgG2 levels was similar in children with or without a history of AOM. Pneumococcal serotype specific IgG was present in middle ear effusion and these levels correlated significantly with serum titres. Children with a history of frequent AOM receiving ventilation tubes do not have a deficient IgG, IgG1 or IgG2 response against pneumococcal polysaccharides, either induced by vaccination or due to natural exposure. The strong correlation between IgG levels in serum and the middle ear suggests parenteral pneumococcal conjugate vaccination induces antibodies in the middle ear which may therefore contribute to reducing the burden of AOM.     

Adjuvant system AS02V enhances humoral and cellular immune responses to pneumococcal protein PhtD vaccine in healthy young and older adults: Randomised,controlled trials

BackgroundThe protection elicited by polysaccharide pneumococcal vaccines against community-acquired pneumonia in older adults remains debatable. Alternative vaccine targets include well-conserved pneumococcal protein antigens, such as pneumococcal histidine triad protein D (PhtD).ObjectiveTo evaluate humoral and cellular immune responses and safety/reactogenicity following immunisation with PhtD vaccine with or without adjuvant (alum or AS02_V) in older (≥65 years) and young (18–45 years) healthy adults.MethodsTwo phase I/II, single-blind, parallel-group studies were conducted in 150 older and 147 young adults. Participants were randomised to receive 2 doses (months 0 and 2) of PhtD 30 μg, PhtD 10 μg plus alum, PhtD 30 μg plus alum, PhtD 10 μg plus AS02_V or PhtD 30 μg plus AS02_V, or the 23-valent polysaccharide pneumococcal vaccine (23PPV) at month 0 with placebo (saline solution) at month 2. Safety/reactogenicity was assessed. PhtD-specific antibody, T cell and memory B cell responses were evaluated.ResultsSolicited adverse events were more common in young participants and with adjuvanted vaccines. No vaccine-related serious adverse events were reported. Although anti-PhtD geometric mean antibody concentrations (GMCs) were consistently lower in the older adult cohort than in young adults, GMCs in the older cohort following PhtD 30 μg plus AS02_V were comparable to those induced by plain PhtD or PhtD 30 μg plus alum in the young cohort. Compared with alum adjuvant, AS02_V adjuvant system was associated with an increased frequency of PhtD-specific CD4 cells in both cohorts and a significantly higher specific memory B cell response in the older cohort, similar to responses obtained in the young cohort.ConclusionThe improved immune response to PhtD vaccine containing the AS02_V adjuvant system in comparison to alum suggests that the reduced immune response to vaccines in older adults can be partially restored to the response level observed in young adults. ClinicalTrials.gov identifiers: NCT00307528/NCT01767402.     

Immune responses and protection against Streptococcus pneumoniae elicited by recombinant Bordetella pertussis adenylate cyclase (CyaA) carrying fragments of pneumococcal surface protein A,PspA

Streptococcus pneumoniae is a common agent of important human diseases such as otitis media, pneumonia, meningitis and sepsis. Current available vaccines that target capsular polysaccharides induce protection against invasive disease and nasopharyngeal colonization in children, yet their efficacy is limited to the serotypes included in the formulations. The virulence factor Pneumococcal Surface Protein A (PspA) interacts with host immune system and helps the bacteria to evade phagocytosis. Due to its essential role in virulence, PspA is an important vaccine candidate. Here we have tested a delivery system based on the adenylate cyclase toxin of Bordetella pertussis (CyaA) to induce immune responses against PspA in mice. CyaA was engineered to express fragments of the N-terminal region of PspAs from clades 2 and 4 (A2 and A4) and the resulting proteins were used in immunization experiments in mice. The recombinant CyaA-A2 and CyaA-A4 proteins were able to induce high levels of anti-PspA antibodies that reacted with pneumococcal strains expressing either PspA2 or PspA4. Moreover, reactivity of the antibodies against pneumococcal strains that express PspAs from clades 3 and 5 (PspA3 and PspA5) was also observed. A formulation containing CyaA-A2 and CyaA-A4 was able to protect mice against invasive pneumococcal challenges with isolates that express PspA2, PspA4 or PspA5. Moreover, a CyaA-A2-A4 fusion protein induced antibodies at similar levels and with similar reactivity as the formulation containing both proteins, and protected mice against the invasive challenge. Our results indicate that CyaA-PspA proteins are good candidates to induce broad protection against pneumococcal isolates.     

Trivalent pneumococcal protein recombinant vaccine protects against lethal Streptococcus pneumoniae pneumonia and correlates with phagocytosis by neutrophils during early pathogenesis

ObjectiveDue to the fact that current polysaccharide-based pneumococcal vaccines have limited serotype coverage, protein-based vaccine candidates have been sought for over a decade to replace or complement current vaccines. We previously reported that a trivalent Pneumococcal Protein recombinant Vaccine (PPrV), showed protection against pneumonia and sepsis in an infant murine model. Here we investigated immunological correlates of protection of PPrV in the same model.MethodsC57BL/6J infant mice were intramuscularly vaccinated at age 1–3 weeks with 3 doses of PPrV, containing pneumococcal histidine triad protein D (PhtD), pneumococcal choline binding protein A (PcpA), and detoxified pneumolysin mutant PlyD1. 3–4 weeks after last vaccination, serum and lung antibody levels to PPrV components were measured, and mice were intranasally challenged with a lethal dose of Streptococcus pneumoniae (Spn) serotype 6A. Lung Spn bacterial burden, number of neutrophils and alveolar macrophages, phagocytosed Spn by granulocytes, and levels of cytokines and chemokines were determined at 6, 12, 24, and 48 h after challenge.ResultsPPrV vaccination conferred 83% protection against Spn challenge. Vaccinated mice had significantly elevated serum and lung antibody levels to three PPrV components. In the first stage of pathogenesis of Spn induced pneumonia (6–24 h after challenge), vaccinated mice had lower Spn bacterial lung burdens and more phagocytosed Spn in the granulocytes. PPrV vaccination led to lower levels of pro-inflammatory cytokines IL-6, IL-1β, and TFN-α, and other cytokines and chemokines (IL-12, IL-17, IFN-γ, MIP-1b, MIP-2 and KC, and G-CSF), presumably due to a lower lung bacterial burden.ConclusionTrivalent PPrV vaccination results in increased serum and lung antibody levels to the vaccine components, a reduction in Spn induced lethality, enhanced early clearance of Spn in lungs due to more rapid and thorough phagocytosis of Spn by neutrophils, and correspondingly a reduction in lung inflammation and tissue damage.     

Mucosal and systemic immunization with a novel attenuated pneumococcal vaccine candidate confer serotype independent protection against Streptococcus pneumoniae in mice

Despite the availability of effective vaccines, Streptococcus pneumoniae is still one of the major infectious diseases causing substantial morbidity and mortality in children under 5 years old. In this study, we demonstrate the protective efficacy of S. pneumoniae SPY1, a novel live attenuated vaccine strain against pneumococcal infection in murine models. This strain was characterized by defects in three important pneumococcal virulence factors including capsule, teichoic acids and pneumolysin. The lactate dehydrogenase assays and in vivo animal experiments demonstrated a significantly attenuated virulence and a reduced nasopharyngeal colonization for the SPY1 strain. We also show that mucosal and systemic immunization with the live SPY1 strain induced protective immune responses against pneumococci. Mucosal immunization with SPY1 offered better protection against colonization challenge with strains TIGR4 and serotype 19F than systemic SPY1 immunization. In invasive infection models, mucosal vaccination with the SPY1 strain conferred complete protection against D39 and clinical serotype 6B and 3 strains. Notably, intranasal vaccination with the SPY1 strain conferred superior protection against pneumococcal invasive disease compared with the commercial available vaccines. SPY1 strain was shown to elicit high levels of serotype-independent antibodies and a mixed cellular immune response. Besides, the SPY1 serum was able to passively protect mice against invasive challenge with D39 strain, indicating the protective effect of the antibody-mediated responses. Together, the SPY1 strain may be a promising live vaccine strain to protect pneumococcal infection.     

Evaluation of serotype-specific pneumococcal IgG measurement using two different polysaccharides from ATCC and SSI Diagnostica

Streptococcus pneumoniae (pneumococcus) is a major cause of morbidity and mortality especially in infants and elderly people. Pneumococcus capsular polysaccharide has been characterised and more than 90 different serotypes have been identified. Serotype-specific antibodies against the capsular polysaccharide are produced during infection.At present, many countries follow the WHO pneumococcal ELISA IgG measurement protocol, in which polysaccharides from ATCC are used as antigens. In recent years, serotype specific polysaccharides from different producers have been tested in pneumococcal antibody assay's. In this project, purified serotype specific pneumococcal antigens from SSI Diagnostica and from ATCC were compared. In general, the data showed that both types of polysaccharide could be used as antigens. Furthermore, the effect of adsorption using different combinations of adsorption procedures was tested, showing similar results using CWPSmulti or CWPS + 22F.     

A modified surface killing assay (MSKA) as a functional in vitro assay for identifying protective antibodies against pneumococcal surface protein A (PspA)

Streptococcus pneumoniae causes otitis media, meningitis and pneumonia in patients worldwide; predominantly affecting young children, the elderly, and the immune compromised. Current vaccines against invasive pneumococcal disease are based on the polysaccharide capsules of the most clinically relevant serotypes. Due to serotype replacement, non-vaccine serotypes of S. pneumoniae have become more clinically relevant and as a result pneumococcal vaccines are becoming increasingly complex. These events emphasize the need to evaluate the potential for pneumococcal cross-reactive proteins to contribute to future vaccines. Antibody elicited by the immunization of humans with pneumococcal surface protein A (PspA) can passively protect mice from infection. However, robust in vitro functional assays for antibody to PspA are not available to predict the protective capacity of immune serum. For polysaccharide based vaccines, a standardized opsonophagocytosis killing assay (OPKA) is used. Antibody to PspA, however, does not work well in the standard OPKA. The present studies take advantage of past observations that phagocytosis is more efficient on tissue surfaces than in solution. In a modified surface killing assay (MSKA), monoclonal antibody to PspA, in the presence of complement, opsonized pneumococci for killing by phagocytes on an agar surface. Five monoclonal antibodies to PspA were tested; three demonstrated increased amounts of killing compared to the diluent control and protected mice by passive protection against type 3 pneumococci. The two antibodies that were not functional in the MSKA also failed to protect mice. Thus, an MSKA might be useful as a functional assay for immunity to PspA.     

Trivalent pneumococcal protein vaccine protects against experimental acute otitis media caused by Streptococcus pneumoniae in an infant murine model

BackgroundCurrently licensed serotype-based pneumococcal vaccines are effective in preventing invasive pneumococcal diseases, but less effective in preventing non-bacteremic pneumonia and acute otitis media (AOM). We previously reported that a trivalent pneumococcal protein recombinant vaccine (PPrV) protected against pneumonia in a murine model. Here we evaluated PPrV protection against AOM in an infant murine model.MethodsC57BL/6J mice were intramuscularly vaccinated at 1–3 weeks of age with monovalent pneumococcal histidine triad protein D (PhtD), or pneumococcal choline binding protein A (PcpA), or detoxified pneumolysin (PlyD1), or trivalent vaccine, and transtympanically challenged at 7–8 weeks of age with 1 × 10² CFU of pneumococcal strain BG7322 (6A) or 1 × 10⁴ CFU of pneumococcal nontypeable strain 0702064 MEF. Serum IgG titers were determined by ELISA. At 24 and 48 h post infection (hpi), animals were sacrificed and middle ear fluid (MEF) samples were collected to determine pneumococcal CFUs.ResultsWe found that vaccination of infant mice with monovalent and trivalent pneumococcal proteins elicited significant serum IgG antibody responses to corresponding component proteins. Vaccination with PhtD reduced BG7322 bacterial burdens in MEF at both 24 (p = 0.05) and 48 hpi (p = 0.16). Vaccination with PcpA significantly reduced the bacterial burdens in MEF at both 24 (p = 0.02) and 48 hpi (p = 0.004), and PlyD1 significantly reduced bacterial burden in MEF at 48 hpi (p = 0.02). Vaccination with trivalent PPrV (PhtD, PcpA and PlyD1) significantly reduced Spn burdens in MEF at both 24 (p = 0.001) and 48 hpi (p < 0.0001). Similar reductions of bacterial burdens were found when the vaccinated animals were challenged with a non-typeable Spn strain. Vaccinated mice had significantly milder inflammatory cytokine levels (IL-1β, IL-6, TNF-α, MIP-2 and KC) in middle ears at 24 hpi (all p values < 0.05).ConclusionTrivalent PPrV confers protection against pneumococcal AOM in an infant murine model.