Vaccine 33 (2015) 993–1000
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Trivalent pneumococcal protein recombinant vaccine protects against lethal Streptococcus pneumoniae pneumonia and correlates with phagocytosis by neutrophils during early pathogenesis Qingfu Xu a , Naveen Surendran a , David Verhoeven a , Jessica Klapa a , Martina Ochs b , Michael E. Pichichero a,∗ a b
Rochester General Hospital Research Institute, Rochester General Hospital, Rochester, NY 14621, USA Sanofi Pasteur, 1541 Ave Marcel Merieux, 69280 Marcy L’Etoile, France
a r t i c l e
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Article history: Received 29 September 2014 Received in revised form 4 December 2014 Accepted 6 January 2015 Available online 15 January 2015 Keywords: Streptococcus pneumoniae Infant murine model Pneumococcal protein vaccine Phagocytosis Neutrophils Alveolar macrophages
a b s t r a c t Objective: Due 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. Methods: C57BL/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. Results: PPrV 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-1, and TFN-␣, and other cytokines and chemokines (IL-12, IL-17, IFN-␥, MIP-1b, MIP-2 and KC, and G-CSF), presumably due to a lower lung bacterial burden. Conclusion: Trivalent 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. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Invasive infectious diseases caused by Streptococcus pneumoniae (Spn), such as pneumonia, sepsis and meningitis continue to be a major cause of morbidity and mortality throughout the world [1–3]. To date, 94 distinct serotypes have been documented based on capsular composition [4]. Current licensed pneumococcal
∗ Corresponding author at: Rochester General Research Institute, Rochester General Hospital, 1425 Portland Avenue, Rochester, NY 14621, USA. Tel.: +1 585 922 2411. E-mail address: [email protected] (M.E. Pichichero). http://dx.doi.org/10.1016/j.vaccine.2015.01.014 0264-410X/© 2015 Elsevier Ltd. All rights reserved.
vaccines include 23-valent polysaccharide vaccine (PPV-23), and 7-, 10-, 13-valent pneumococcal polysaccharide conjugate vaccines (PCV-7, -10, -13). PPV-23 is currently recommended in the elderly and high-risk adults as it is poorly immunogenic in children, especially under 2 years of age [5]. The current polysaccharide-based vaccines have significantly reduced invasive pneumococcal diseases (IPD) [6–8]. However, since these vaccines have limited serotype coverage, after introduction of PCV-7, and more recently PCV-13, pneumococcal infections caused by non-vaccine replacement serotypes increased significantly [9,10]. Pneumococcal protein vaccine candidates therefore have been sought to complement or replace current capsule-based vaccines. [1,2,11,12]
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Multiple pneumococcal proteins have been studied as vaccine candidates [2,11,12]. Pneumococcal histidine triad protein D (PhtD) [13–18], pneumococcal choline binding protein A (PcpA) [15,17,19,20], and pneumolysin (Ply) [17,21–25] are highly conserved, immunogenic and elicit protection against pneumococcal disease in animal models. PhtD is an especially attractive vaccine candidate due to its strong immunogenicity, efficacy in protection against nasopharyngeal colonization, and among the other pneumococcal histidine triad proteins in Spn, PhtD is the most highly conserved across pneumococcal serotypes [26,27]. PhtD and PcpA play a role in adherence of Spn to human NP epithelial cells [16,20]. Pneumolysin (Ply) is a cholesterol-dependent cytolysin that is a key virulence factor contributing to bacterial pathogenesis at both early and late stages of infection [21]. Monovalent vaccine candidates of PhtD (clinical trial# NCT01444001) [13], PcpA (NCT01444339) [15] and a genetically detoxified pneumolysin mutant (PlyD1, NCT01444352) [21], a bivalent vaccine candidate composed of PhtD and PcpA (NCT01444339) [15], and a trivalent vaccine candidate composed of PhtD, PcpA and PlyD1 (NCT01764126) [12] hereafter referred to as Pneumococcal Protein recombinant Vaccine (PPrV), have all shown sufficient promise to enter human clinical trials. However, the mechanism of protection elicited by these pneumococcal protein vaccines remains unclear. Children less than 2 years of age have the greatest risk of pneumococcal infections [28]. An ideal animal model for vaccine study should more closely represent the maturing immunological state of corresponding vaccination age in young children. We recently developed an infant murine vaccination model [29] in which animals were immunized at 1–3 week of age, immunologically similar to 6 months to 2 year of age in children, and found that intramuscular vaccination with PPrV provided protection against pneumonia and sepsis caused by lethal challenge of Spn serotypes 6A and 3 [29]. The protective mechanism against pneumonia by pneumococcal protein vaccine candidates has not been completely understood [30]. Phagocytosis of Spn by neutrophils and alveolar macrophages may play an important role in protection against infections [31]. In this study we show an association between PPrV vaccination and enhanced neutrophil-mediated phagocytosis in the lungs of infant mice during early pathogenesis, resulting in protection against potentially lethal intranasal challenge. 2. Materials and methods 2.1. Animals and experimental design Paired male and female C57BL/6J mice, 8–10 weeks old, were purchased from Jackson Laboratory (JAX@ Mice) to breed. Newborn mice were weaned at 4 weeks of age, and thereafter the female and male mice were housed separately, 4–5 mice per cage. Infant mice were intramuscularly vaccinated in the hind leg with trivalent PPrV vaccine at age 1, 2 and 3 weeks and intranasally challenged with a lethal dose of Spn at age 7–8 weeks. The lungs were collected at 6, 12, 24, 48 h post inoculation (hpi) to determine bacterial burdens, granulocyte phagocytosis by antibiotic protection assay, innate immune responses by flow cytometry (FACS), and lung cytokines and chemokine levels by Luminex beads-based multiplex assay. Animals were housed in a SPF BSLII murine facility at the Rochester General Hospital Research Institute (RGHRI), and all experiments were approved by Institutional Animal Care and Use Committee at RGHRI. 2.2. Vaccination PPrV components included 18 g/ml of PhtD, 4 g/ml of PcpA and 100 g/ml of PlyD1 and aluminum salt adjuvant. Infant mice of
a same litter were randomly allocated to vaccinated and unvaccinated groups. Vaccinated groups were given PPrV intramuscularly at age 6–8 days, and boosted twice at age 13–15, 19–21 days with 0.9 g of PhtD, 0.2 g of PcpA and 5 g of PlyD1 in 50 l, and control groups were given the adjuvant in parallel as described previously [29]. The mice were anesthetized with isofluorane (1%) in 100% oxygen with a delivery rate of 5 L/min. 2.3. Spn challenge Spn strain BG3722, serotype 6A [29] was freshly cultured to mid-logarithmic growth phase in Todd Hewitt Broth containing 1% yeast extract (THBY, Difco) and 17% fetal bovine serum. The mice were intranasally inoculated with 1.1–1.3 × 106 CFU in 40 l PBS under anesthesia as described above. Challenge dosages first were calculated based on OD600 values and an optimized convert ratio, and actual challenge CFU were determined on 5% sheep blood agar plate. 2.4. Lung bacterial burden Left lung lobe was homogenized into 250 l of pre-cooled PBS using a tissue grinder. 10 l of 10-fold serial diluted PBShomogenate was plated onto 5% sheep blood agar plates overnight at 37 ◦ C to quantify the number of CFUs of Spn per left lung lobes. 2.5. Enzyme-linked immunosorbent assay (ELISA) Antibody titers in serum and lung homogenate were determined by ELISA as described previously [29]. 2.6. Spn phagocytosis assay of lung granulocyte cells The four right lobes were minced, enzymatically digested in 10% DMEM containing 1 mg/ml of Collagenase Type IV (Worthington biochemical company, MA) and 0.02 mg/ml of DNaseI Type II (Sigma) for 1 h at 37◦ C, and then filtrated with a 70 m-filter. The cells were subsequently washed in DPBS containing 1% fetal bovine serum (FBS) and treated with 2 ml Ammonium-Chloride-Potassium lysis buffer (ACK, Gibco) for 2 min to lyse red blood cells. After the wash steps some of the lung cells were used for further isolation of granulocytes for the phagocytosis assay, and some for FACS analysis as described below. Granulocytes were isolated by Ficoll density gradient centrifugation and then treated with 20 g/ml ciprofloxacin and 0.5% penicillin/streptomycin in 10% Dulbecco’s Modified Eagle’s Medium (DMEM Gibco) for 1 h at 4 ◦ C to eliminate extracellular bacteria. After two washes with wash buffer the granulocytes were lysed with 1× saponin buffer (permeabilization buffer from BD Biosciences) for 15 min at room temperature, and then 100 l lysate as well as 10 l of 10-fold serial dilutions of lysate were cultured on TSA II blood plates overnight at 37 ◦ C with 5% CO2 to quantify the intracellular CFU of Spn in granulocytes per right lung lobes. 3. FACS analysis For flow cytometry, cells were surface stained in two sets with a combination of fluorochrome labeled anti-mouse innate or adaptive immune cell markers: CD45-Pacific blue, Ly6C/6G-APC/Cy7, Ly6G-APC/Cy7, F4/80-APC, CD11C-FITC, I-A/I-E-Pe/Cy7, CD11b-PE, CD86-Alexafluor 700, CD103-Percp/Cy5.5, CD3-APC, ␥␦-PE (GL3), CD4-FITC, CD8-Percp/Cy5.5, CD44-Alexafluor 700, CD62L-Pe/Cy7, and CD69-APC-Cy7 antibodies (all from Biolegend, San Diego, CA). The cell viability was determined using LIVE/DEAD® Fixable Aqua Dead Cell Stain Kit (Life Technologies). After washing with DPBS, fluorescence was measured by an LSRII flow cytometer (BD
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Biosciences) and analyzed using FlowJo software (Tree Star, Portland OR). Cells were gated based on FSC, SSC characteristics, and after exclusion of debris, dead cells and doublets, lung leukocytes were identified by CD45 staining. A sequential gating strategy was employed to identify populations expressing specific markers: alveolar macrophages (CD11c+ F4/80+ ) and neutrophils (CD11b+ Ly6G+ ). Percentages of neutrophils and alveolar macrophages to lung total live cells were determined. 3.1. Cytokines and chemokines measurement Left lung lobe was homogenized into 250 l of pre-cooled DPBS using a tissue grinder, and was spun at 14,000 × g for 20 min, and the clarified supernatant stored at −80 ◦ C. Cytokines and chemokines in lung homogenate samples were quantified using Milliplex Map Mouse Cytokine/Chemokine Magnetic Bead Panel including G-CSF, IFN␥, IL-1 IL-4, IL-6, IL-10, IL-12 (p70), IL-17, KC, LIX, MIP-1, MIP-2, and TNF-␣ on a Bio-Plex 200 instrument (Bio-Rad) according to the manufacturer’s instructions. 3.2. Statistics The statistical tests were performed using Prism software (Graph Pad, La Jolla, CA). Results of lung bacterial loads, intracellular bacterial CFU in granulocytes, and cytokine/chemokine concentrations of lung homogenate were expressed as means ± standard error (SE). Differences between vaccinated and non-vaccinated groups were analyzed by non-parametric two-tailed Mann–Whitney U test for antibody levels, lung bacterial loads, and intracellular bacterial CFU, and two-way ANOVA Fisher’s LSD for cytokine/chemokine levels. For the purpose of statistical analysis, undetectable samples were arbitrarily assigned a value equivalent to half the lower limit of detection as described previously [32]. Survival curves were estimated by the Kaplan–Meier method, and compared by the log-rank (Mantel–Cox) test. P values < 0.05 were considered statistically significant. 4. Results 4.1. Trivalent PPrV vaccination elicits serum and lung antibody response to three pneumococcal proteins We evaluated antibody responses to the three component proteins of PPrV in serum and lung of mice after three vaccinations with a PPrV. We found that PPrV elicited both serum and lung homogenate IgG, but not IgA responses against all three components. IgG titers in serum (geometric mean, GM ± SE, ng/ml, n = 14) of PPrV vaccinated mice were 3738 ± 339 for PhtD, 3167 ± 706 for PcpA, and 90.2 ± 27 ng/ml for PlyD1 compared to non-detectable in control mice (p < 0.001 for all 3 antigen). IgG antibody titers in lung (GM ± SE, endpoint titer, n = 9) of PPrV vaccinated mice were 53.8 ± 41 and undetectable for controls (p = 0.01) for PhtD, 403 ± 343 (p = 0.03) for PcpA and 11.9 ± 7.9 (p = 0.03) for PlyD1, respectively, compared to non-detectable in control mice. IgA antibody titers to PPrV components were undetectable in either serum or lung of vaccinated and control mice (data not shown). The results agree with our previous reports [29]. 4.2. Trivalent PPrV vaccination protects against lethal Spn challenge We evaluated protection of mice vaccinated with the PPrV against lethal Spn challenge. After challenge with 1 × 106 CFU of Spn BG7322, 100% (4/4) of the non-vaccinated mice died by 96 h, whereas 83% (5/6) of vaccinated mice survived out to 2 weeks and
Fig. 1. Protection against lethal Spn challenge in infant mouse vaccination model. C57BL/6J infant mice were vaccinated intramuscularly with trivalent vaccine at 1, 2 and 3 weeks of age, and then challenged intranasally at 7–8 weeks of age with 1.2 × 106 CFU of Spn BG7322. (n = 6 in vaccinated and n = 4 in non-vaccinated group).
were then sacrificed (Fig. 1). The results are consistent with our previous report although the survival rate was slightly lower [29]. 4.3. Vaccination with the trivalent vaccine enhanced bacterial clearance at early times after challenge We compared bacterial burdens in the lungs of vaccinated and non-vaccinated mice at different times after challenge (Fig. 2). The vaccinated mice had a 7.8-fold decrease in GM bacterial load in lung at 12 h post infection (hpi) (4314 vs 33940, p = 0.11), a 17.2fold decrease at 24 h after challenge (804 vs 13805, p = 0.064) and a 54-fold decrease at 48 h (16123 vs 870751, p = 0.01), compared to control mice. 4.4. Phagocytosis by granulocytes correlates with protection in trivalent vaccinated mice against Spn challenge To evaluate the effects of PPrV vaccination on phagocytosis, we quantitated the number of intracellular Spn in lung granulocytes in vaccinated and non-vaccinated mice at different time points after intranasal challenge (Fig. 3). We found that detection of more intracellular Spn was associated with protection of vaccinated mice. Compared with control mice, the GM CFU of phagocytosed Spn in granulocytes of vaccinated mice was 8.2-fold higher at 6 hpi (301.9 vs. 36.6, p = 0.002), 7.2-fold higher at 12 hpi (51.0 vs. 7.1, p = 0.006), 3.3-fold higher at 24 hpi (34.9 vs. 10.6, p = 0.026) and 2.5-fold higher at 48 hpi (82.4 vs. 33.0, p = 0.53) 4.5. Effect of vaccination on the level of neutrophil and alveolar macrophages recruitment in early stage of infection We evaluated the influence of PPrV vaccination on recruitment of neutrophils/total live lung cells at 6, 12, 24 and 48 hpi. The results are summarized in Fig. 4. We found that neutrophil levels were comparable at 6 hpi for both vaccinated and control mice. However, at 12 hpi (p = 0.002) and 24 hpi (p = 0.03) neutrophil numbers were significantly higher in the non-vaccinated mice which remained elevated at 48 hpi, when the experiment ended. In contrast, the vaccinated mice did not experience further rises in neutrophil numbers between 6 and 24 hpi, but more gradually attained a similar number of neutrophils as the control at 48 hpi. We also evaluated the influence of PPrV vaccination on recruitment of alveolar macrophages/total live lung cells at 6, 12, 24, and 48 h after intranasal challenge. The macrophage levels in both the vaccinated and non-vaccinated mice showed a gradual increase from 6 to 48 hpi. Interestingly, the control mice had higher alveolar
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Fig. 2. Comparison of bacterial lung loads after lethal Spn challenge between vaccinated and non-vaccinated. Infant mice were i.m. vaccinated with trivalent at 1, 2, 3 weeks of age, and i.n. challenged at 7–8 weeks of age with 1 × 106 CFU of Spn BG7322. The CFU of Spn in lung left lobe homogenate was determined at 6 (A), 12 (B), 24 (C) and 48 (D) h after challenge.
macrophage numbers at 6 hpi (p = 0.02) compared to vaccinated mice. 4.6. Vaccination leads to lower levels of lung cytokines and chemokines after challenge We investigated an associated or a relationship between lung inflammatory cell infiltrates and pro-inflammatory cytokine and chemokine levels in lung in vaccinated versus control mice. At 6 hpi, vaccinated mice had similar levels of pro-inflammatory cytokines
IL-6, IL-1 and TFN-␣ (Fig. 5A–C; p = 0.2–0.3). At 12, 24, and 48 hpi, control mice had overall higher levels of pro-inflammatory cytokines than vaccinated mice; 7–24 fold higher IL-6, 3–4 fold higher IL-1, and 3–5 fold higher TNF-␣ (Fig. 5A, 5B, 5C; all p values 0.05). 5. Discussion Limited pneumococcal serotype coverage and replacement of vaccine serotypes with non-vaccine serotype strains has continued to emerge with increased rapidity during the current PCV era [33–35] thereby stimulating high interest in non-serotype specific protein-based vaccines [2,12,36]. We recently reported that trivalent PPrV provides protection against pneumonia and sepsis in an infant murine model [29]. In this study, we investigated several immunological correlates of protection and found that trivalent PPrV vaccination resulted in increased serum and lung antibody levels to the vaccine components, reduction in pneumococcal infection lethality, enhanced lung Spn clearance early in pathogenesis due to more rapid and thorough ingestion of Spn by granulocytes, presumably mainly neutrophils, and correspondingly a reduction in lung inflammation and tissue damage. The results are fully consistent with the known role of antibody as an opsonin of Grampositive bacteria to promote neutrophil ingestion in the early phase of disease pathogenesis and strongly encourage further study of this new vaccine candidate. Neutrophils play an important role in the innate immune response [31,37]. Recruitment of neutrophils is one of the most important components of the initial immune response in the lung against bacterial infection [31]. Human and mouse neutrophils have shown opsonophagocytotic activity to kill Spn [38,39].
Neutrophils can infiltrate into lung at an early stage of lung Spn infection [40,41]. In this study, we found that lung bacterial clearance correlated with higher level of phagocytosis by neutrophils at an early stage in pathogenesis after a potential lethal challenge of Spn by the physiologically relevant intranasal route. The vaccinated mice showed lower neutrophil recruitment and higher phagocytosis than control mice, a result suggestive that vaccination led to a more efficient antibody-mediated neutrophil activation against Spn infection at an early stage in pathogenesis. We observed that phagocytosis of Spn by granulocytes started within 6 h after challenge. Vaccinated mice had significantly higher levels of phagocytosis by granulocytes through the first 24 h after challenge. There was a decreasing trend of lung bacterial burden among vaccinated mice from 6 (p = 0.67), to 12 (p = 0.11), to 24 (p = 0.06), to 48 (p = 0.01) h after challenge. Although the trend was clear, the difference did not reach statistical significant at an individual time point until 48 h. The significant difference in lung bacterial burden at 48 h in vaccinated mice we interpret to indicate an accumulated efficiency in killing bacteria during early pathogenesis. Moreover, the data suggest that there might be a critical bacterial load whereby above a certain level, the host immune system can no longer control the bacteria. We interpret the data to suggest that very early time, 6 h, is important in survival in the vaccinated mice. Although the number of lung alveolar macrophages increased during the 12–48 hpi of observation in both groups, we did not identify significant differences in the levels of macrophages or dendritic cells (data not shown) between vaccinated and non-vaccinated mice. Antibody-mediated opsonization and phagocytosis is an important defense mechanism against Spn [42–44]. Antibodies to Spn capsular polysaccharides [43,44] and to pneumococcal surface protein A (PspA) [42] mediate opsonophagocytic killing of Spn. This
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study and our previous study [29] have shown that vaccination with trivalent PPrV elicits high sera and lung mucosal IgG responses to PhtD, PcpA and PlyD1 in mice. Anti-PhtD and anti-PcpA antibodies inhibit Spn NP colonization and attachment to lung epithelia by reducing adherence to those respiratory epithelial cells [16,20], and enhance lung bacterial clearance [29], while anti-PlyD1 inhibits [29] pneumolysin mediated lung damage and inflammation [22]. The results of this current work strongly suggest that during the first 48 h after challenge, PPrV specific antibodies mediate phagocytosis of Spn by neutrophils, resulting in protection from lethal sepsis and pneumonia. Others have previously shown that anti-sera from mice immunized with PspA or fusion protein of PspA–PlyD elicit serotype-independent opsonophagocytic activity against Spn using murine peritoneal cells (neutrophils and macrophages) [42,45]. We found that PPrV vaccinated infant mice had significantly lower levels of IL-17 in lung homogenates at 24 but not 48 hpi compared to non-vaccinated mice. Prior work by other groups has demonstrated an IL-17-mediated (Th17) protective mechanism against Spn colonization and infection [46–48]. Design of vaccine candidates to protect against Spn colonization by stimulating Th17 cells is being pursued [49]. IL-17A promotes pneumococcal clearance by recruiting neutrophils [46]. Spn colonization and infection lead to increased IL-17A in sera and lung in mice [48] and man. Mucosal immunization with recombinant fusion protein of DnaJ protein (a heat shock protein) and Ply [3], and a trivalent vaccine containing cell wall polysaccharide (CWPS), Ply, and surface adhesin A (PsaA) [50], has been shown to confer protective immunity against Spn colonization and infection via IL-17A in mice. Spleenocytes [3] and whole blood cells [50] from vaccinated mice produce significantly more IL-17A in response to stimulation with DnaJ protein [3], and stimulation with killed whole-cell pneumococcal antigen (WCA) [50] compared to non-vaccinated mice. Low levels of IL-17 in our experiments can be explained by the lower lung bacterial loads resulting from antibody-mediated phagocytosis. We also observed lower levels of Th1 polarizing cytokines such as IL-12 and IFN-␥ as well as the pro-inflammatory cytokines in PPrV vaccinated compared to unvaccinated mice. This observation also can be explained by lower lung bacterial loads resulting from antibody-mediated phagocytosis. This study has limitations. Intracellular live Spn and not actual total phagocytized (live and killed) bacteria in the granulocytes were used as the measure of phagocytic activity. We did eliminate counting of any attached bacteria to the granulocytes by exposure of the cells to antibiotic that would have killed exposed Spn but the total phagocytized bacteria were almost certainly more than the reported intracellular live bacteria detected. In addition, alveolar macrophages are regarded as a first line of defense in the lung. We did not determine phagocytosis by macrophages in the lung and therefore cannot exclude the possibility that alveolar macrophages also mediated protection by the trivalent vaccine in lung during early pathogenesis after challenge. Furthermore, our study reports an association between PPrV protection against pneumococcal pneumonia and phagocytosis of Spn by granulocytes, not a direct experimental mechanism. Further studies therefore are needed to directly define the role of neutrophils and macrophages in antibody-mediated phagocyte-mediated protection induced by PPrV.
Acknowledgments This work was supported by an Investigator initiated grant awarded to MEP by Sanofi Pasteur in Canada. We thank Robert Zagursky for advising on experiments, and reviewing and editing the manuscript. We also thank Ted Nicolosi for assistance in
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sample processing and FACS and Emily Newman for assistance with sample collection.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.vaccine.2015. 01.014.
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[39] Lysenko ES, Ratner AJ, Nelson AL, Weiser JN. The role of innate immune responses in the outcome of interspecies competition for colonization of mucosal surfaces. PLoS Pathog 2005;1:e1. [40] Dominis-Kramaric M, Bosnar M, Kelneric Z, Glojnaric I, Cuzic S, Parnham MJ, et al. Comparison of pulmonary inflammatory and antioxidant responses to intranasal live and heat-killed Streptococcus pneumoniae in mice. Inflammation 2011;34:471–86. [41] Wilson R, Cohen JM, Jose RJ, de Vogel C, Baxendale H, Brown JS. Protection against Streptococcus pneumoniae lung infection after nasopharyngeal colonization requires both humoral and cellular immune responses. Mucosal Immunol 2014, http://dx.doi.org/10.1038/mi.2014.95, advance online publication 29 October 2014. [42] Arulanandam BP, Lynch JM, Briles DE, Hollingshead S, Metzger DW. Intranasal vaccination with pneumococcal surface protein A and interleukin-12 augments antibody-mediated opsonization and protective immunity against Streptococcus pneumoniae infection. Infect Immun 2001;69:6718–24. [43] Parkkali T, Vakevainen M, Kayhty H, Ruutu T, Ruutu P. Opsonophagocytic activity against Streptococcus pneumoniae type 19F in allogeneic BMT recipients before and after vaccination with pneumococcal polysaccharide vaccine. Bone Marrow Transplant 2001;27:207–11. [44] Melin M, Jarva H, Siira L, Meri S, Kayhty H, Vakevainen M. Streptococcus pneumoniae capsular serotype 19F is more resistant to C3 deposition and less sensitive to opsonophagocytosis than serotype 6B. Infect Immun 2009;77:676–84. [45] Ferreira DM, Darrieux M, Silva DA, Leite LC, Ferreira Jr JM, Ho PL, et al. Characterization of protective mucosal and systemic immune responses elicited by pneumococcal surface protein PspA and PspC nasal vaccines against a respiratory pneumococcal challenge in mice. Clin Vaccine Immunol 2009;16: 636–45. [46] Wang W, Zhou A, Zhang X, Xiang Y, Huang Y, Wang L, et al. Interleukin 17A promotes pneumococcal clearance by recruiting neutrophils and inducing apoptosis through a p38 mitogen-activated protein kinase-dependent mechanism in acute otitis media. Infect Immun 2014;82:2368–77. [47] Marques JM, Rial A, Munoz N, Pellay FX, Van Maele L, Leger H, et al. Protection against Streptococcus pneumoniae serotype 1 acute infection shows a signature of Th17- and IFN-gamma-mediated immunity. Immunobiology 2012;217:420–9. [48] Wright AK, Bangert M, Gritzfeld JF, Ferreira DM, Jambo KC, Wright AD, et al. Experimental human pneumococcal carriage augments IL-17A-dependent Tcell defence of the lung. PLoS Pathog 2013;9:e1003274. [49] Moffitt K, Skoberne M, Howard A, Gavrilescu LC, Gierahn T, Munzer S, et al. Toll-like receptor 2-dependent protection against pneumococcal carriage by immunization with lipidated pneumococcal proteins. Infect Immun 2014;82:2079–86. [50] Lu YJ, Forte S, Thompson CM, Anderson PW, Malley R. Protection against Pneumococcal colonization and fatal pneumonia by a trivalent conjugate of a fusion protein with the cell wall polysaccharide. Infect Immun 2009;77:2076–83.
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Trivalent pneumococcal protein recombinant vaccine protects against lethal Streptococcus pneumoniae pneumonia and correlates with phagocytosis by neutrophils during early pathogenesis Qingfu Xu a , Naveen Surendran a , David Verhoeven a , Jessica Klapa a , Martina Ochs b , Michael E. Pichichero a,∗ a b
Rochester General Hospital Research Institute, Rochester General Hospital, Rochester, NY 14621, USA Sanofi Pasteur, 1541 Ave Marcel Merieux, 69280 Marcy L’Etoile, France
a r t i c l e
i n f o
Article history: Received 29 September 2014 Received in revised form 4 December 2014 Accepted 6 January 2015 Available online 15 January 2015 Keywords: Streptococcus pneumoniae Infant murine model Pneumococcal protein vaccine Phagocytosis Neutrophils Alveolar macrophages
a b s t r a c t Objective: Due 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. Methods: C57BL/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. Results: PPrV 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-1, and TFN-␣, and other cytokines and chemokines (IL-12, IL-17, IFN-␥, MIP-1b, MIP-2 and KC, and G-CSF), presumably due to a lower lung bacterial burden. Conclusion: Trivalent 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. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Invasive infectious diseases caused by Streptococcus pneumoniae (Spn), such as pneumonia, sepsis and meningitis continue to be a major cause of morbidity and mortality throughout the world [1–3]. To date, 94 distinct serotypes have been documented based on capsular composition [4]. Current licensed pneumococcal
∗ Corresponding author at: Rochester General Research Institute, Rochester General Hospital, 1425 Portland Avenue, Rochester, NY 14621, USA. Tel.: +1 585 922 2411. E-mail address: [email protected] (M.E. Pichichero). http://dx.doi.org/10.1016/j.vaccine.2015.01.014 0264-410X/© 2015 Elsevier Ltd. All rights reserved.
vaccines include 23-valent polysaccharide vaccine (PPV-23), and 7-, 10-, 13-valent pneumococcal polysaccharide conjugate vaccines (PCV-7, -10, -13). PPV-23 is currently recommended in the elderly and high-risk adults as it is poorly immunogenic in children, especially under 2 years of age [5]. The current polysaccharide-based vaccines have significantly reduced invasive pneumococcal diseases (IPD) [6–8]. However, since these vaccines have limited serotype coverage, after introduction of PCV-7, and more recently PCV-13, pneumococcal infections caused by non-vaccine replacement serotypes increased significantly [9,10]. Pneumococcal protein vaccine candidates therefore have been sought to complement or replace current capsule-based vaccines. [1,2,11,12]
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Multiple pneumococcal proteins have been studied as vaccine candidates [2,11,12]. Pneumococcal histidine triad protein D (PhtD) [13–18], pneumococcal choline binding protein A (PcpA) [15,17,19,20], and pneumolysin (Ply) [17,21–25] are highly conserved, immunogenic and elicit protection against pneumococcal disease in animal models. PhtD is an especially attractive vaccine candidate due to its strong immunogenicity, efficacy in protection against nasopharyngeal colonization, and among the other pneumococcal histidine triad proteins in Spn, PhtD is the most highly conserved across pneumococcal serotypes [26,27]. PhtD and PcpA play a role in adherence of Spn to human NP epithelial cells [16,20]. Pneumolysin (Ply) is a cholesterol-dependent cytolysin that is a key virulence factor contributing to bacterial pathogenesis at both early and late stages of infection [21]. Monovalent vaccine candidates of PhtD (clinical trial# NCT01444001) [13], PcpA (NCT01444339) [15] and a genetically detoxified pneumolysin mutant (PlyD1, NCT01444352) [21], a bivalent vaccine candidate composed of PhtD and PcpA (NCT01444339) [15], and a trivalent vaccine candidate composed of PhtD, PcpA and PlyD1 (NCT01764126) [12] hereafter referred to as Pneumococcal Protein recombinant Vaccine (PPrV), have all shown sufficient promise to enter human clinical trials. However, the mechanism of protection elicited by these pneumococcal protein vaccines remains unclear. Children less than 2 years of age have the greatest risk of pneumococcal infections [28]. An ideal animal model for vaccine study should more closely represent the maturing immunological state of corresponding vaccination age in young children. We recently developed an infant murine vaccination model [29] in which animals were immunized at 1–3 week of age, immunologically similar to 6 months to 2 year of age in children, and found that intramuscular vaccination with PPrV provided protection against pneumonia and sepsis caused by lethal challenge of Spn serotypes 6A and 3 [29]. The protective mechanism against pneumonia by pneumococcal protein vaccine candidates has not been completely understood [30]. Phagocytosis of Spn by neutrophils and alveolar macrophages may play an important role in protection against infections [31]. In this study we show an association between PPrV vaccination and enhanced neutrophil-mediated phagocytosis in the lungs of infant mice during early pathogenesis, resulting in protection against potentially lethal intranasal challenge. 2. Materials and methods 2.1. Animals and experimental design Paired male and female C57BL/6J mice, 8–10 weeks old, were purchased from Jackson Laboratory (JAX@ Mice) to breed. Newborn mice were weaned at 4 weeks of age, and thereafter the female and male mice were housed separately, 4–5 mice per cage. Infant mice were intramuscularly vaccinated in the hind leg with trivalent PPrV vaccine at age 1, 2 and 3 weeks and intranasally challenged with a lethal dose of Spn at age 7–8 weeks. The lungs were collected at 6, 12, 24, 48 h post inoculation (hpi) to determine bacterial burdens, granulocyte phagocytosis by antibiotic protection assay, innate immune responses by flow cytometry (FACS), and lung cytokines and chemokine levels by Luminex beads-based multiplex assay. Animals were housed in a SPF BSLII murine facility at the Rochester General Hospital Research Institute (RGHRI), and all experiments were approved by Institutional Animal Care and Use Committee at RGHRI. 2.2. Vaccination PPrV components included 18 g/ml of PhtD, 4 g/ml of PcpA and 100 g/ml of PlyD1 and aluminum salt adjuvant. Infant mice of
a same litter were randomly allocated to vaccinated and unvaccinated groups. Vaccinated groups were given PPrV intramuscularly at age 6–8 days, and boosted twice at age 13–15, 19–21 days with 0.9 g of PhtD, 0.2 g of PcpA and 5 g of PlyD1 in 50 l, and control groups were given the adjuvant in parallel as described previously [29]. The mice were anesthetized with isofluorane (1%) in 100% oxygen with a delivery rate of 5 L/min. 2.3. Spn challenge Spn strain BG3722, serotype 6A [29] was freshly cultured to mid-logarithmic growth phase in Todd Hewitt Broth containing 1% yeast extract (THBY, Difco) and 17% fetal bovine serum. The mice were intranasally inoculated with 1.1–1.3 × 106 CFU in 40 l PBS under anesthesia as described above. Challenge dosages first were calculated based on OD600 values and an optimized convert ratio, and actual challenge CFU were determined on 5% sheep blood agar plate. 2.4. Lung bacterial burden Left lung lobe was homogenized into 250 l of pre-cooled PBS using a tissue grinder. 10 l of 10-fold serial diluted PBShomogenate was plated onto 5% sheep blood agar plates overnight at 37 ◦ C to quantify the number of CFUs of Spn per left lung lobes. 2.5. Enzyme-linked immunosorbent assay (ELISA) Antibody titers in serum and lung homogenate were determined by ELISA as described previously [29]. 2.6. Spn phagocytosis assay of lung granulocyte cells The four right lobes were minced, enzymatically digested in 10% DMEM containing 1 mg/ml of Collagenase Type IV (Worthington biochemical company, MA) and 0.02 mg/ml of DNaseI Type II (Sigma) for 1 h at 37◦ C, and then filtrated with a 70 m-filter. The cells were subsequently washed in DPBS containing 1% fetal bovine serum (FBS) and treated with 2 ml Ammonium-Chloride-Potassium lysis buffer (ACK, Gibco) for 2 min to lyse red blood cells. After the wash steps some of the lung cells were used for further isolation of granulocytes for the phagocytosis assay, and some for FACS analysis as described below. Granulocytes were isolated by Ficoll density gradient centrifugation and then treated with 20 g/ml ciprofloxacin and 0.5% penicillin/streptomycin in 10% Dulbecco’s Modified Eagle’s Medium (DMEM Gibco) for 1 h at 4 ◦ C to eliminate extracellular bacteria. After two washes with wash buffer the granulocytes were lysed with 1× saponin buffer (permeabilization buffer from BD Biosciences) for 15 min at room temperature, and then 100 l lysate as well as 10 l of 10-fold serial dilutions of lysate were cultured on TSA II blood plates overnight at 37 ◦ C with 5% CO2 to quantify the intracellular CFU of Spn in granulocytes per right lung lobes. 3. FACS analysis For flow cytometry, cells were surface stained in two sets with a combination of fluorochrome labeled anti-mouse innate or adaptive immune cell markers: CD45-Pacific blue, Ly6C/6G-APC/Cy7, Ly6G-APC/Cy7, F4/80-APC, CD11C-FITC, I-A/I-E-Pe/Cy7, CD11b-PE, CD86-Alexafluor 700, CD103-Percp/Cy5.5, CD3-APC, ␥␦-PE (GL3), CD4-FITC, CD8-Percp/Cy5.5, CD44-Alexafluor 700, CD62L-Pe/Cy7, and CD69-APC-Cy7 antibodies (all from Biolegend, San Diego, CA). The cell viability was determined using LIVE/DEAD® Fixable Aqua Dead Cell Stain Kit (Life Technologies). After washing with DPBS, fluorescence was measured by an LSRII flow cytometer (BD
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Biosciences) and analyzed using FlowJo software (Tree Star, Portland OR). Cells were gated based on FSC, SSC characteristics, and after exclusion of debris, dead cells and doublets, lung leukocytes were identified by CD45 staining. A sequential gating strategy was employed to identify populations expressing specific markers: alveolar macrophages (CD11c+ F4/80+ ) and neutrophils (CD11b+ Ly6G+ ). Percentages of neutrophils and alveolar macrophages to lung total live cells were determined. 3.1. Cytokines and chemokines measurement Left lung lobe was homogenized into 250 l of pre-cooled DPBS using a tissue grinder, and was spun at 14,000 × g for 20 min, and the clarified supernatant stored at −80 ◦ C. Cytokines and chemokines in lung homogenate samples were quantified using Milliplex Map Mouse Cytokine/Chemokine Magnetic Bead Panel including G-CSF, IFN␥, IL-1 IL-4, IL-6, IL-10, IL-12 (p70), IL-17, KC, LIX, MIP-1, MIP-2, and TNF-␣ on a Bio-Plex 200 instrument (Bio-Rad) according to the manufacturer’s instructions. 3.2. Statistics The statistical tests were performed using Prism software (Graph Pad, La Jolla, CA). Results of lung bacterial loads, intracellular bacterial CFU in granulocytes, and cytokine/chemokine concentrations of lung homogenate were expressed as means ± standard error (SE). Differences between vaccinated and non-vaccinated groups were analyzed by non-parametric two-tailed Mann–Whitney U test for antibody levels, lung bacterial loads, and intracellular bacterial CFU, and two-way ANOVA Fisher’s LSD for cytokine/chemokine levels. For the purpose of statistical analysis, undetectable samples were arbitrarily assigned a value equivalent to half the lower limit of detection as described previously [32]. Survival curves were estimated by the Kaplan–Meier method, and compared by the log-rank (Mantel–Cox) test. P values < 0.05 were considered statistically significant. 4. Results 4.1. Trivalent PPrV vaccination elicits serum and lung antibody response to three pneumococcal proteins We evaluated antibody responses to the three component proteins of PPrV in serum and lung of mice after three vaccinations with a PPrV. We found that PPrV elicited both serum and lung homogenate IgG, but not IgA responses against all three components. IgG titers in serum (geometric mean, GM ± SE, ng/ml, n = 14) of PPrV vaccinated mice were 3738 ± 339 for PhtD, 3167 ± 706 for PcpA, and 90.2 ± 27 ng/ml for PlyD1 compared to non-detectable in control mice (p < 0.001 for all 3 antigen). IgG antibody titers in lung (GM ± SE, endpoint titer, n = 9) of PPrV vaccinated mice were 53.8 ± 41 and undetectable for controls (p = 0.01) for PhtD, 403 ± 343 (p = 0.03) for PcpA and 11.9 ± 7.9 (p = 0.03) for PlyD1, respectively, compared to non-detectable in control mice. IgA antibody titers to PPrV components were undetectable in either serum or lung of vaccinated and control mice (data not shown). The results agree with our previous reports [29]. 4.2. Trivalent PPrV vaccination protects against lethal Spn challenge We evaluated protection of mice vaccinated with the PPrV against lethal Spn challenge. After challenge with 1 × 106 CFU of Spn BG7322, 100% (4/4) of the non-vaccinated mice died by 96 h, whereas 83% (5/6) of vaccinated mice survived out to 2 weeks and
Fig. 1. Protection against lethal Spn challenge in infant mouse vaccination model. C57BL/6J infant mice were vaccinated intramuscularly with trivalent vaccine at 1, 2 and 3 weeks of age, and then challenged intranasally at 7–8 weeks of age with 1.2 × 106 CFU of Spn BG7322. (n = 6 in vaccinated and n = 4 in non-vaccinated group).
were then sacrificed (Fig. 1). The results are consistent with our previous report although the survival rate was slightly lower [29]. 4.3. Vaccination with the trivalent vaccine enhanced bacterial clearance at early times after challenge We compared bacterial burdens in the lungs of vaccinated and non-vaccinated mice at different times after challenge (Fig. 2). The vaccinated mice had a 7.8-fold decrease in GM bacterial load in lung at 12 h post infection (hpi) (4314 vs 33940, p = 0.11), a 17.2fold decrease at 24 h after challenge (804 vs 13805, p = 0.064) and a 54-fold decrease at 48 h (16123 vs 870751, p = 0.01), compared to control mice. 4.4. Phagocytosis by granulocytes correlates with protection in trivalent vaccinated mice against Spn challenge To evaluate the effects of PPrV vaccination on phagocytosis, we quantitated the number of intracellular Spn in lung granulocytes in vaccinated and non-vaccinated mice at different time points after intranasal challenge (Fig. 3). We found that detection of more intracellular Spn was associated with protection of vaccinated mice. Compared with control mice, the GM CFU of phagocytosed Spn in granulocytes of vaccinated mice was 8.2-fold higher at 6 hpi (301.9 vs. 36.6, p = 0.002), 7.2-fold higher at 12 hpi (51.0 vs. 7.1, p = 0.006), 3.3-fold higher at 24 hpi (34.9 vs. 10.6, p = 0.026) and 2.5-fold higher at 48 hpi (82.4 vs. 33.0, p = 0.53) 4.5. Effect of vaccination on the level of neutrophil and alveolar macrophages recruitment in early stage of infection We evaluated the influence of PPrV vaccination on recruitment of neutrophils/total live lung cells at 6, 12, 24 and 48 hpi. The results are summarized in Fig. 4. We found that neutrophil levels were comparable at 6 hpi for both vaccinated and control mice. However, at 12 hpi (p = 0.002) and 24 hpi (p = 0.03) neutrophil numbers were significantly higher in the non-vaccinated mice which remained elevated at 48 hpi, when the experiment ended. In contrast, the vaccinated mice did not experience further rises in neutrophil numbers between 6 and 24 hpi, but more gradually attained a similar number of neutrophils as the control at 48 hpi. We also evaluated the influence of PPrV vaccination on recruitment of alveolar macrophages/total live lung cells at 6, 12, 24, and 48 h after intranasal challenge. The macrophage levels in both the vaccinated and non-vaccinated mice showed a gradual increase from 6 to 48 hpi. Interestingly, the control mice had higher alveolar
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Fig. 2. Comparison of bacterial lung loads after lethal Spn challenge between vaccinated and non-vaccinated. Infant mice were i.m. vaccinated with trivalent at 1, 2, 3 weeks of age, and i.n. challenged at 7–8 weeks of age with 1 × 106 CFU of Spn BG7322. The CFU of Spn in lung left lobe homogenate was determined at 6 (A), 12 (B), 24 (C) and 48 (D) h after challenge.
macrophage numbers at 6 hpi (p = 0.02) compared to vaccinated mice. 4.6. Vaccination leads to lower levels of lung cytokines and chemokines after challenge We investigated an associated or a relationship between lung inflammatory cell infiltrates and pro-inflammatory cytokine and chemokine levels in lung in vaccinated versus control mice. At 6 hpi, vaccinated mice had similar levels of pro-inflammatory cytokines
IL-6, IL-1 and TFN-␣ (Fig. 5A–C; p = 0.2–0.3). At 12, 24, and 48 hpi, control mice had overall higher levels of pro-inflammatory cytokines than vaccinated mice; 7–24 fold higher IL-6, 3–4 fold higher IL-1, and 3–5 fold higher TNF-␣ (Fig. 5A, 5B, 5C; all p values 0.05). 5. Discussion Limited pneumococcal serotype coverage and replacement of vaccine serotypes with non-vaccine serotype strains has continued to emerge with increased rapidity during the current PCV era [33–35] thereby stimulating high interest in non-serotype specific protein-based vaccines [2,12,36]. We recently reported that trivalent PPrV provides protection against pneumonia and sepsis in an infant murine model [29]. In this study, we investigated several immunological correlates of protection and found that trivalent PPrV vaccination resulted in increased serum and lung antibody levels to the vaccine components, reduction in pneumococcal infection lethality, enhanced lung Spn clearance early in pathogenesis due to more rapid and thorough ingestion of Spn by granulocytes, presumably mainly neutrophils, and correspondingly a reduction in lung inflammation and tissue damage. The results are fully consistent with the known role of antibody as an opsonin of Grampositive bacteria to promote neutrophil ingestion in the early phase of disease pathogenesis and strongly encourage further study of this new vaccine candidate. Neutrophils play an important role in the innate immune response [31,37]. Recruitment of neutrophils is one of the most important components of the initial immune response in the lung against bacterial infection [31]. Human and mouse neutrophils have shown opsonophagocytotic activity to kill Spn [38,39].
Neutrophils can infiltrate into lung at an early stage of lung Spn infection [40,41]. In this study, we found that lung bacterial clearance correlated with higher level of phagocytosis by neutrophils at an early stage in pathogenesis after a potential lethal challenge of Spn by the physiologically relevant intranasal route. The vaccinated mice showed lower neutrophil recruitment and higher phagocytosis than control mice, a result suggestive that vaccination led to a more efficient antibody-mediated neutrophil activation against Spn infection at an early stage in pathogenesis. We observed that phagocytosis of Spn by granulocytes started within 6 h after challenge. Vaccinated mice had significantly higher levels of phagocytosis by granulocytes through the first 24 h after challenge. There was a decreasing trend of lung bacterial burden among vaccinated mice from 6 (p = 0.67), to 12 (p = 0.11), to 24 (p = 0.06), to 48 (p = 0.01) h after challenge. Although the trend was clear, the difference did not reach statistical significant at an individual time point until 48 h. The significant difference in lung bacterial burden at 48 h in vaccinated mice we interpret to indicate an accumulated efficiency in killing bacteria during early pathogenesis. Moreover, the data suggest that there might be a critical bacterial load whereby above a certain level, the host immune system can no longer control the bacteria. We interpret the data to suggest that very early time, 6 h, is important in survival in the vaccinated mice. Although the number of lung alveolar macrophages increased during the 12–48 hpi of observation in both groups, we did not identify significant differences in the levels of macrophages or dendritic cells (data not shown) between vaccinated and non-vaccinated mice. Antibody-mediated opsonization and phagocytosis is an important defense mechanism against Spn [42–44]. Antibodies to Spn capsular polysaccharides [43,44] and to pneumococcal surface protein A (PspA) [42] mediate opsonophagocytic killing of Spn. This
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study and our previous study [29] have shown that vaccination with trivalent PPrV elicits high sera and lung mucosal IgG responses to PhtD, PcpA and PlyD1 in mice. Anti-PhtD and anti-PcpA antibodies inhibit Spn NP colonization and attachment to lung epithelia by reducing adherence to those respiratory epithelial cells [16,20], and enhance lung bacterial clearance [29], while anti-PlyD1 inhibits [29] pneumolysin mediated lung damage and inflammation [22]. The results of this current work strongly suggest that during the first 48 h after challenge, PPrV specific antibodies mediate phagocytosis of Spn by neutrophils, resulting in protection from lethal sepsis and pneumonia. Others have previously shown that anti-sera from mice immunized with PspA or fusion protein of PspA–PlyD elicit serotype-independent opsonophagocytic activity against Spn using murine peritoneal cells (neutrophils and macrophages) [42,45]. We found that PPrV vaccinated infant mice had significantly lower levels of IL-17 in lung homogenates at 24 but not 48 hpi compared to non-vaccinated mice. Prior work by other groups has demonstrated an IL-17-mediated (Th17) protective mechanism against Spn colonization and infection [46–48]. Design of vaccine candidates to protect against Spn colonization by stimulating Th17 cells is being pursued [49]. IL-17A promotes pneumococcal clearance by recruiting neutrophils [46]. Spn colonization and infection lead to increased IL-17A in sera and lung in mice [48] and man. Mucosal immunization with recombinant fusion protein of DnaJ protein (a heat shock protein) and Ply [3], and a trivalent vaccine containing cell wall polysaccharide (CWPS), Ply, and surface adhesin A (PsaA) [50], has been shown to confer protective immunity against Spn colonization and infection via IL-17A in mice. Spleenocytes [3] and whole blood cells [50] from vaccinated mice produce significantly more IL-17A in response to stimulation with DnaJ protein [3], and stimulation with killed whole-cell pneumococcal antigen (WCA) [50] compared to non-vaccinated mice. Low levels of IL-17 in our experiments can be explained by the lower lung bacterial loads resulting from antibody-mediated phagocytosis. We also observed lower levels of Th1 polarizing cytokines such as IL-12 and IFN-␥ as well as the pro-inflammatory cytokines in PPrV vaccinated compared to unvaccinated mice. This observation also can be explained by lower lung bacterial loads resulting from antibody-mediated phagocytosis. This study has limitations. Intracellular live Spn and not actual total phagocytized (live and killed) bacteria in the granulocytes were used as the measure of phagocytic activity. We did eliminate counting of any attached bacteria to the granulocytes by exposure of the cells to antibiotic that would have killed exposed Spn but the total phagocytized bacteria were almost certainly more than the reported intracellular live bacteria detected. In addition, alveolar macrophages are regarded as a first line of defense in the lung. We did not determine phagocytosis by macrophages in the lung and therefore cannot exclude the possibility that alveolar macrophages also mediated protection by the trivalent vaccine in lung during early pathogenesis after challenge. Furthermore, our study reports an association between PPrV protection against pneumococcal pneumonia and phagocytosis of Spn by granulocytes, not a direct experimental mechanism. Further studies therefore are needed to directly define the role of neutrophils and macrophages in antibody-mediated phagocyte-mediated protection induced by PPrV.
Acknowledgments This work was supported by an Investigator initiated grant awarded to MEP by Sanofi Pasteur in Canada. We thank Robert Zagursky for advising on experiments, and reviewing and editing the manuscript. We also thank Ted Nicolosi for assistance in
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sample processing and FACS and Emily Newman for assistance with sample collection.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.vaccine.2015. 01.014.
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