Vaccine 32 (2014) 3205–3210
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Vaccine journal homepage: www.elsevier.com/locate/vaccine
Vaccination with a Streptococcus pneumoniae trivalent recombinant PcpA, PhtD and PlyD1 protein vaccine candidate protects against lethal pneumonia in an infant murine model David Verhoeven, Qingfu Xu, Michael E. Pichichero ∗ Rochester General Hospital Research Institute, Rochester General Hospital, 1425 Portland Avenue, Rochester, NY 14621, United States
a r t i c l e
i n f o
Article history: Received 11 December 2013 Received in revised form 24 March 2014 Accepted 1 April 2014 Available online 13 April 2014 Keywords: Streptococcus pneumoniae Pneumolysin Pneumococcal histidine triad D protein Pneumococcal choline binding protein A Infant vaccination
a b s t r a c t 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. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Despite the availability of efficacious capsular serotype-based vaccines, significant morbidity and mortality from infections caused by Streptococcus pneumoniae (Spn) persist, due in part to vaccine serotype replacement and limited availability of pneumococcal conjugate vaccines [1,2]. Development of efficacious pneumococcal protein vaccines represent an alternative strategy to expanding pneumococcal conjugate vaccines by targeting conserved proteins between serotypes [3,4] and the reduced costs of these vaccines could allow for greater use worldwide [5]. Human antibodies to pneumococcal choline-binding protein A (PcpA) have been detected in children with pneumococcal bacteremia and pneumonia [6]. In monovalent vaccines containing PcpA, protection from pneumonia and a delay in morbidity after sepsis challenge has been shown [7] with antibodies affording the protection in vitro [8]. The contribution of PcpA to protection in a trivalent vaccine for both children and adults needs further evaluation to better understand its contribution to vaccine efficacy.
∗ Corresponding author. Tel.: +1 585 922 2411. E-mail addresses: [email protected], [email protected] (M.E. Pichichero). http://dx.doi.org/10.1016/j.vaccine.2014.04.004 0264-410X/© 2014 Elsevier Ltd. All rights reserved.
In animal models, vaccine candidates containing PhtD have been studied against sepsis, pneumonia and nasopharyngeal colonization [9–13]. A two component, PhtD–dPly (detoxified pneumolysin), vaccine protected rhesus macaques from pneumonia [14]. Human antibodies to PhtD have been shown to be functional in a mouse passive protection sepsis model and in a Phase I study PhtD vaccine was shown to be safe and immunogenic in adults [15]. Natural colonization or infection by Spn stimulates antibodies directed against PhtD but antibody levels have not been shown to correlate with disease prevention [11,16–18]. Human anti-PhtD antibodies have been shown to reduce adhesion of Spn to human nasopharyngeal epithelial cells in vitro [19] but it is not known whether a PhtD protein vaccination would offer similar protective antibodies in the lungs. Moreover, the level of protection afforded by PhtD vaccination in the context of a trivalent vaccine is unknown. Vaccines using chemically detoxified pneumolysin (dPly) provide protection in animal studies [20–24]. Recently developed genetically detoxified Ply (PlyD1) vaccine has been shown to provide protection in adult mice against nasopharyngeal challenge [22]. Spn colonization leads to lower Ply-specific plasma IgG levels in young children compared to other Spn proteins in children [16] and therefore anti-PlyD1 IgG responses would be important to study to better understand the efficacy of a potential trivalent
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Vaccination and Challenge Schedule 7 days old Infant Mice
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Difco), to 108 cfu/ml. Bacteria were washed twice with PBS and resuspended at 25 × 106 cfu/ml in PBS (dilution plating confirmed CFUs). Mice were anesthetized with Isoflurane and 40 l of bacteria (106 cfu/ml) were instilled into the nose of each mouse as an intranasal challenge. 2.4. Antibody assays
Adult Mice
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Fig. 1. Infant mice were injected at times shown with trivalent vaccines containing PcpA, PhtD, and PlyD1. Injections were into both hind leg caudal muscles (25 l/muscle). Four weeks after the third vaccination, mice were challenged by intranasal instillation with Spn.
vaccine containing this component. Phase I studies of PlyD1 have shown it to be safe and immunogenic in adults [25]. Here, we studied the efficacy and potential mechanisms of protection against lethal pneumonia induced by a trivalent vaccine containing PcpA, PhtD and PlyD1 (derived from a serotype 6B strain). Intranasal challenges with heterologous serotypes 6A and 3 were used to demonstrate cross serotype protection. We used an infant mouse challenge model that mirrors immune responses to human infants age about 1 year old. An adult mouse challenge model was included as a control. Differences in lung responses in young mice and adult mice would affect the level of protection in unvaccinated mice after challenge [26]. The foundation for this study was the premise that any new vaccine against Spn that will target primarily children for vaccination should be studied in an infant animal model that more closely parallels the immunobiology of the intended age of the human vaccinees. This is the first study using a trivalent vaccine containing PcpA, PhtD and PlyD1 in infant mice to determine protection against lethal pneumonia and the correlates of protection critical for future efficacy clinical trials in children.
2.4.1. Serum antibody titers After vaccinations serum was obtained by tail bleed and by cardiac puncture after Spn challenge. Recombinant proteins were plated on Immunlon II ELISA plates (ThermoFisher, Hampton NH) at 0.5 g per well overnight at 4 ◦ C, then blocked with nonfat milk. Antibody levels were detected after addition of secondary rabbit anti-mouse AP (Jackson Immuno, West Grove PA) at 1:10,000 dilution. An in house reference serum with known antibody concentrations was included on all plates. Calculation of total IgG was calculated from a standardized curve using a reference serum. Endpoint titers for subclasses of IgG were performed with specific donkey secondary antibodies AP (Jackson Immuno) at 1:10,000 dilution. 2.4.2. Lung antibody titers After the tertiary vaccine, lung tissue (10 mg) was ground with a pestle in 500 l of cold PBS containing 1% Triton X-100 and clarified by centrifugation. ELISAs for IgG and IgA were performed as previously stated for PcpA, PhtD, and PlyD1 with an endpoint cutoff of 3× the standard deviation of the mean titer for unvaccinated mice. 2.5. Lung bacterial counts CFUs were determined by dilution plating from clarified lung extracts (48 h post-challenge), processed by mortar and pestle and resuspended in 1 ml of PBS onto TSA II plates containing gentamicin (BD Biosciences, San Jose CA). Plates were incubated overnight at 37 ◦ C.
2. Methods and materials
2.6. Histology
2.1. Animals Six-week old male and female C57BL/6 mice were purchased from NCI and housed in a SPF BSLII murine facility at Rochester General Hospital Research Institute (RGHRI). C57BL/6 infant mice were obtained by breeding at RGHRI. All procedures were IACUC approved.
Lungs were obtained 48 h post infection after vascular perfusion with PBS. The lobes were clamped at the bronchioles and perfused with 4% Buffered Formalin. 5 m sections were cut and H&E stained (AML labs). Image files were processed with Adobe Photoshop (San Jose, CA) with auto levels selected. We assumed equal distribution of Spn into the large left lobes. Histopathological scores were based on published methodology [22].
2.2. Vaccinations
2.7. Bacterial adhesion assay
Recombinant PcpA [27], PhtD [15] and PlyD1 [28] proteins obtained from Sanofi Pasteur [27] were derived from a serotype 6B Spn strain. Dose ranging studies were performed using aluminum hydroxide (Alum) as an adjuvant. Unvaccinated controls received Alum alone. Three vaccinations were given in an accelerated weekly schedule since infant mice rapidly age (Fig. 1). All vaccinations were administered as two intramuscular injections into the hind caudal muscles for both infant and adult mice.
Spn was grown in THY pre-chelated with Chelax beads (Sigma) overnight [29]. In both studies, 108 bacteria were stained in 1 mg/ml of FITC (Sigma) at 4 ◦ C for 1 h and then washed with PBS. Approximately, 106 Spn cells were incubated with antibodies from vaccinated mice that was diluted 1:10 in DMEM 10% media, with or without prior chelation and supplementation (as described above), for 1 h at 37 ◦ C in the presence of 1 g/ml guinea pig complement (Fisher). Bacteria were transferred onto primary lung epithelial (Type II) (Cell Biosystems), grown to 95% confluency in 8-well chamber slides, and then incubated for 5 h. Cells were washed 3 times with PBS and fixed for 5 min in cold acetone then air-dried. Cells were rehydrated in PBS before mounting in Vectashield. Bacterial adherence was imaged with an Axioshop (Zeiss) with a FITC filter.
2.3. Spn challenge BG3722, a serotype 6A strain, was obtained from Sanofi Pasteur. WU2, serotype 3, was a gift from Dr. David Briles. Bacteria were grown in Todd Hewitt Broth with 1% yeast extract (THY,
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Fig. 2. Trivalent vaccination leads to higher IgG responses to PcpA, PhtD, and Ply in adult than infant mice. Serum IgG responses were determined for infant and adult mice using (A) lower antigen concentrations in a trivalent vaccine (0.2 g PcpA, 0.9 g PhtD, 5 g PlyD1) or (B) higher antigen concentrations (5 g PcpA, 2.5 g PhtD, 15.2 g PlyD1). (n = 5 each group for 2 independent experiments).
2.8. PlyD toxicity assay BG7322 was grown in THY. FITC labeled or unlabeled Spn was neutralized as described above with antibodies generated in infant and adult mice vaccinated with PlyD1 or the trivalent vaccine. Spn was then added to primary epithelial and endothelial lung cells (Cell Biosystems) and incubated at 37 ◦ C for 5 h. Cells were washed 3 times with PBS and Sytox Orange (Invitrogen) was added at 0.1 l/ml in PBS and incubated for 5 min followed by washing 3 times with PBS and then fixation, mounting, and imaging as described above. 2.9. Statistics Data was analyzed by two tailed Student’s T-tests or ANOVA and results with a p < 0.05 considered significant. Data was analyzed by Prism software (Graph Pad, La Jolla CA). 3. Results 3.1. Serum antibody generation in infant and adult mice with trivalent vaccine We evaluated two different dosages for each antigen and found that a higher dose of antigens led to significantly higher antigenspecific IgG serum concentrations in adult mice for only PcpA (p = 0.008) while adult IgG specific titers for PhtD were lower with higher dosage antigen (p = 0.02) (Fig. 2). In infant mice, the higher doses led to higher IgG responses to PcpA and Ply but lower responses to PhtD although the differences were not statistically significant. Based on these results, we proceeded to test the lower
dose. Importantly, at the lower dosage levels, vaccination increased antibody levels to all three components of the trivalent vaccine in both infant and adult mice as compared to sham vaccinated controls.
3.2. Vaccination using trivalent PcpA, PhtD and PlyD1 We assessed survival of vaccinated infant and adult mice after intranasal challenge that caused lethal bacteremic pneumonia in control mice. We found that 90% of infant and 100% of adult trivalent vaccinated mice were protected from lethal pneumonia caused by a serotype 6A strain (Fig. 3A). In addition, 70% of trivalent vaccinated infant mice were protected from lethal pneumonia caused by WU2 strain; a serotype 3 strain (Fig. 3B). Thus trivalent vaccination conferred cross serotype protection from lethal pneumonia.
3.3. Antibody levels in lung We examined the levels of IgG in the lungs of trivalent vaccinated animals after the third vaccination. Significantly higher IgG titers were measured for all three antigens in vaccinated compared to control mice (Fig. 4A–C). Parenteral vaccination did not increase IgA in the lungs, similar to the results in serum (data not shown).
3.4. Bacterial lung burden and histopathology Trivalent vaccinated infant and adult mice had a significant 0.5–0.7 log reduction in the amount of lung bacterial burden 48 h post-challenge (Fig. 4D). Trivalent vaccinated infant and adult mice
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Fig. 3. Vaccination with trivalent vaccine confers protection from lethal pneumonia. (A) Survival curves for trivalent (0.2 g PcpA, 0.9 g PhtD, 5 g PlyD1) vaccinated infant and adult mice were assessed after challenge. (n = 5 each group for 2 independent experiments). (B) Survival curves for vaccinated infants after challenge with WU2 strain. (n = 5 each group for 2 independent experiments).
had reduced histopathological scores as compared to unvaccinated controls (Fig. 4E–F). 3.5. Antibodies reduce Spn adherence and neutralize epithelial damage We found abundant adherence of Spn to lung epithelial cells in the absence of Spn specific serum antibodies (Fig. 5A and B). Serum antibodies derived from trivalent vaccinated mice led to significant reductions in the ability of Spn to bind to primary lung epithelial cells from both vaccinated infant and adult mice (Fig. 5C and D). To determine whether serum antibodies generated to PlyD1 could protect lung epithelial damage from the toxicity of native Ply
and thus prevent potential translocation of Spn into the lung vasculature, we performed an in vitro neutralization assay using primary lung cells (Fig. 5E–J). We found that primary lung cells incubated with serum antibodies derived from PlyD1 vaccinated infant and adult mice had significantly less cell damage after 5 h of incubation in the presence of Spn. 4. Discussion A multi-component protein-based vaccine that protects from all Spn strains would be a significant advance [30]. Here we investigated the protective effect of a trivalent vaccine consisting of Spn proteins, PcpA, PhtD and PlyD1 in an infant murine challenge
Fig. 4. Bacterial lung load and histopathology of trivalent vaccinated mice. IgG titers were determined from 10 mg of lung extract for (A) PcpA, (B) PhtD, and (C) Ply. (n = 10 mice each group). (D) Lung bacterial burdens at 48 h post-infection in unvaccinated (control) or trivalent vaccinated infant and adult mice. (n = 5 each group for 2 independent experiments). (E) Lung tissue histology in 48 h post-infected trivalent vaccinated or unvaccinated mice. Representative histology from infant mice is shown at 20× magnification. (F) Histopathologic scores were determined in vaccinated or unvaccinated mice 48 h post-challenge. (n = 5 each group for 2 independent experiments). * p-value < 0.05
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Fig. 5. Trivalent vaccination leads to a reduction in Spn induced epithelial damage to primary lung epithelial cells. FITC labeled Spn was incubated with serum from vaccinated or unvaccinated mice and with guinea pig complement for 1 h prior to adding to C57BL/6 derived primary lung epithelial cells. Spn/No antibody control contained non-specific mouse serum. White = Spn. (A–D) Spn adherence was determined on primary lung epithelial cells either in the absence or presence of antibodies obtained from trivalent vaccinated infant and adult mice. Representative microscopy is shown at magnification of 20×. (E–I) Spn was incubated with serum from vaccinated or unvaccinated mice for 5 h to allow for Ply mediated membrane damage. White = Sytox orange that stains membrane damaged cells. (n = 4 each from randomly chosen frozen serum for 2 independent experiments). Representative microscopy with magnification of 20× is shown.
model, with adult comparators. Importantly each protein was designed from serotype 6B and we tested the protection afforded by the trivalent vaccine against strains expressing heterologous serotypes 6A and 3 challenge strains to demonstrate cross serotype protection. Using monovalent vaccines, infant and adult mice showed similar increases in IgG antibody specific titer to the vaccinated antigens except for PlyD1 where infant mice elicited minimal titers. After monovalent PhtD, PcpA and PlyD1 vaccination, subtyping of IgG specific antibody responses demonstrated a dominance of IgG1 antibodies implying a strong Th2 or TFH CD4 T-cell memory response. Lung bacterial loads were reduced with the trivalent vaccine in both infant and adult mice. Our results are in agreement with previous studies showing protection from Spn challenge after vaccination with PcpA and PhtD in adult mice [7,10,31]. It has been reported that PlyD1 vaccination prevents lung histopathology after lethal challenge by limiting inflammation in adult mice [22,32,33]. Of interest, even with lower antibody titers directed to PlyD1 in infant mice compared to adult mice, tissue histopathology was limited suggesting that the titers of antibody in the infants were adequate to neutralize the damage of lung epithelial cells caused by Ply. PlyD1 may offer better protection of lung epithelium from the toxic effects of Ply and subsequent reduction of bacteria from dissemination into the capillaries that line the epithelial barrier in the alveolar space. However, we observed high levels of protection only in mice vaccinated with the trivalent vaccine suggesting that reduction of bacteremia and reduction in lung bacterial burdens are both necessary for optimal protection. Investigation of the role of antibody from the lung would be of interest in a future study. Adherence of Spn to primary lung epithelial cells was reduced using antisera from mice vaccinated with the trivalent vaccine and mixtures of antibodies against PhtD and PcpA. In prior studies of natural colonization by Spn in young children, antibodies to either PcpA [19] or PhtD [8] blocked adherence of Spn to human lung cells. IgG specific antibody responses increased with higher dose vaccinations in adult mice. In comparison a higher dose trivalent vaccines elicited an increased (PcpA), decreased (PhtD) and similar (PlyD1) antibody response in infant mice. Although this may imply some mechanism of antigen presentation interference for
PhtD with the trivalent vaccine in infant mice, vaccination with the trivalent Spn vaccine provided protection from pneumonia, lung tissue damage, and subsequent sepsis. Moreover, in our challenge studies the trivalent vaccine resulted in 100% protection in adult mice and 70–90% protection in infant mice upon lethal challenge. In summary, we have shown that vaccination with a trivalent protein-based vaccine containing PcpA, PhtD and PlyD1 can confer protection against lethal pneumonia in infant mice. The mechanism of protection appears to be by reduction of Spn binding to airway epithelium by antibodies directed to PcpA and PhtD while antibodies to Ply significantly reduce epithelial damage and probably subsequent inflammation. This study highlights the interplay necessary between vaccine-induced antibodies for prevention of adherence to respiratory epithelial cells and direct neutralization of toxic virulence factors to prevent epithelial damage. Future studies in infant mice to further define the contribution of phagocytosis and the requirement for complement activation in antibody-mediated protection are underway.
Acknowledgments This work was supported by an Investigator initiated grant awarded to MEP by Sanofi Pasteur. We thank Dr. Robert Zagursky for assistance in manuscript preparation and Sheldon Perry, Jessica Klapa, and Karin Pryharski for technical assistance.
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Contents lists available at ScienceDirect
Vaccine journal homepage: www.elsevier.com/locate/vaccine
Vaccination with a Streptococcus pneumoniae trivalent recombinant PcpA, PhtD and PlyD1 protein vaccine candidate protects against lethal pneumonia in an infant murine model David Verhoeven, Qingfu Xu, Michael E. Pichichero ∗ Rochester General Hospital Research Institute, Rochester General Hospital, 1425 Portland Avenue, Rochester, NY 14621, United States
a r t i c l e
i n f o
Article history: Received 11 December 2013 Received in revised form 24 March 2014 Accepted 1 April 2014 Available online 13 April 2014 Keywords: Streptococcus pneumoniae Pneumolysin Pneumococcal histidine triad D protein Pneumococcal choline binding protein A Infant vaccination
a b s t r a c t 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. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Despite the availability of efficacious capsular serotype-based vaccines, significant morbidity and mortality from infections caused by Streptococcus pneumoniae (Spn) persist, due in part to vaccine serotype replacement and limited availability of pneumococcal conjugate vaccines [1,2]. Development of efficacious pneumococcal protein vaccines represent an alternative strategy to expanding pneumococcal conjugate vaccines by targeting conserved proteins between serotypes [3,4] and the reduced costs of these vaccines could allow for greater use worldwide [5]. Human antibodies to pneumococcal choline-binding protein A (PcpA) have been detected in children with pneumococcal bacteremia and pneumonia [6]. In monovalent vaccines containing PcpA, protection from pneumonia and a delay in morbidity after sepsis challenge has been shown [7] with antibodies affording the protection in vitro [8]. The contribution of PcpA to protection in a trivalent vaccine for both children and adults needs further evaluation to better understand its contribution to vaccine efficacy.
∗ Corresponding author. Tel.: +1 585 922 2411. E-mail addresses: [email protected], [email protected] (M.E. Pichichero). http://dx.doi.org/10.1016/j.vaccine.2014.04.004 0264-410X/© 2014 Elsevier Ltd. All rights reserved.
In animal models, vaccine candidates containing PhtD have been studied against sepsis, pneumonia and nasopharyngeal colonization [9–13]. A two component, PhtD–dPly (detoxified pneumolysin), vaccine protected rhesus macaques from pneumonia [14]. Human antibodies to PhtD have been shown to be functional in a mouse passive protection sepsis model and in a Phase I study PhtD vaccine was shown to be safe and immunogenic in adults [15]. Natural colonization or infection by Spn stimulates antibodies directed against PhtD but antibody levels have not been shown to correlate with disease prevention [11,16–18]. Human anti-PhtD antibodies have been shown to reduce adhesion of Spn to human nasopharyngeal epithelial cells in vitro [19] but it is not known whether a PhtD protein vaccination would offer similar protective antibodies in the lungs. Moreover, the level of protection afforded by PhtD vaccination in the context of a trivalent vaccine is unknown. Vaccines using chemically detoxified pneumolysin (dPly) provide protection in animal studies [20–24]. Recently developed genetically detoxified Ply (PlyD1) vaccine has been shown to provide protection in adult mice against nasopharyngeal challenge [22]. Spn colonization leads to lower Ply-specific plasma IgG levels in young children compared to other Spn proteins in children [16] and therefore anti-PlyD1 IgG responses would be important to study to better understand the efficacy of a potential trivalent
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Vaccination and Challenge Schedule 7 days old Infant Mice
Inject
14 days Inject Bleed
21 days
7 wks
Inject Bleed
Bleed Infect
Difco), to 108 cfu/ml. Bacteria were washed twice with PBS and resuspended at 25 × 106 cfu/ml in PBS (dilution plating confirmed CFUs). Mice were anesthetized with Isoflurane and 40 l of bacteria (106 cfu/ml) were instilled into the nose of each mouse as an intranasal challenge. 2.4. Antibody assays
Adult Mice
6wks
7 wks
8 wks
12 wks
Inject
Inject Bleed
Inject Bleed
Bleed Infect
Intramuscular injections into both hind caudal muscles (25 l each)
Fig. 1. Infant mice were injected at times shown with trivalent vaccines containing PcpA, PhtD, and PlyD1. Injections were into both hind leg caudal muscles (25 l/muscle). Four weeks after the third vaccination, mice were challenged by intranasal instillation with Spn.
vaccine containing this component. Phase I studies of PlyD1 have shown it to be safe and immunogenic in adults [25]. Here, we studied the efficacy and potential mechanisms of protection against lethal pneumonia induced by a trivalent vaccine containing PcpA, PhtD and PlyD1 (derived from a serotype 6B strain). Intranasal challenges with heterologous serotypes 6A and 3 were used to demonstrate cross serotype protection. We used an infant mouse challenge model that mirrors immune responses to human infants age about 1 year old. An adult mouse challenge model was included as a control. Differences in lung responses in young mice and adult mice would affect the level of protection in unvaccinated mice after challenge [26]. The foundation for this study was the premise that any new vaccine against Spn that will target primarily children for vaccination should be studied in an infant animal model that more closely parallels the immunobiology of the intended age of the human vaccinees. This is the first study using a trivalent vaccine containing PcpA, PhtD and PlyD1 in infant mice to determine protection against lethal pneumonia and the correlates of protection critical for future efficacy clinical trials in children.
2.4.1. Serum antibody titers After vaccinations serum was obtained by tail bleed and by cardiac puncture after Spn challenge. Recombinant proteins were plated on Immunlon II ELISA plates (ThermoFisher, Hampton NH) at 0.5 g per well overnight at 4 ◦ C, then blocked with nonfat milk. Antibody levels were detected after addition of secondary rabbit anti-mouse AP (Jackson Immuno, West Grove PA) at 1:10,000 dilution. An in house reference serum with known antibody concentrations was included on all plates. Calculation of total IgG was calculated from a standardized curve using a reference serum. Endpoint titers for subclasses of IgG were performed with specific donkey secondary antibodies AP (Jackson Immuno) at 1:10,000 dilution. 2.4.2. Lung antibody titers After the tertiary vaccine, lung tissue (10 mg) was ground with a pestle in 500 l of cold PBS containing 1% Triton X-100 and clarified by centrifugation. ELISAs for IgG and IgA were performed as previously stated for PcpA, PhtD, and PlyD1 with an endpoint cutoff of 3× the standard deviation of the mean titer for unvaccinated mice. 2.5. Lung bacterial counts CFUs were determined by dilution plating from clarified lung extracts (48 h post-challenge), processed by mortar and pestle and resuspended in 1 ml of PBS onto TSA II plates containing gentamicin (BD Biosciences, San Jose CA). Plates were incubated overnight at 37 ◦ C.
2. Methods and materials
2.6. Histology
2.1. Animals Six-week old male and female C57BL/6 mice were purchased from NCI and housed in a SPF BSLII murine facility at Rochester General Hospital Research Institute (RGHRI). C57BL/6 infant mice were obtained by breeding at RGHRI. All procedures were IACUC approved.
Lungs were obtained 48 h post infection after vascular perfusion with PBS. The lobes were clamped at the bronchioles and perfused with 4% Buffered Formalin. 5 m sections were cut and H&E stained (AML labs). Image files were processed with Adobe Photoshop (San Jose, CA) with auto levels selected. We assumed equal distribution of Spn into the large left lobes. Histopathological scores were based on published methodology [22].
2.2. Vaccinations
2.7. Bacterial adhesion assay
Recombinant PcpA [27], PhtD [15] and PlyD1 [28] proteins obtained from Sanofi Pasteur [27] were derived from a serotype 6B Spn strain. Dose ranging studies were performed using aluminum hydroxide (Alum) as an adjuvant. Unvaccinated controls received Alum alone. Three vaccinations were given in an accelerated weekly schedule since infant mice rapidly age (Fig. 1). All vaccinations were administered as two intramuscular injections into the hind caudal muscles for both infant and adult mice.
Spn was grown in THY pre-chelated with Chelax beads (Sigma) overnight [29]. In both studies, 108 bacteria were stained in 1 mg/ml of FITC (Sigma) at 4 ◦ C for 1 h and then washed with PBS. Approximately, 106 Spn cells were incubated with antibodies from vaccinated mice that was diluted 1:10 in DMEM 10% media, with or without prior chelation and supplementation (as described above), for 1 h at 37 ◦ C in the presence of 1 g/ml guinea pig complement (Fisher). Bacteria were transferred onto primary lung epithelial (Type II) (Cell Biosystems), grown to 95% confluency in 8-well chamber slides, and then incubated for 5 h. Cells were washed 3 times with PBS and fixed for 5 min in cold acetone then air-dried. Cells were rehydrated in PBS before mounting in Vectashield. Bacterial adherence was imaged with an Axioshop (Zeiss) with a FITC filter.
2.3. Spn challenge BG3722, a serotype 6A strain, was obtained from Sanofi Pasteur. WU2, serotype 3, was a gift from Dr. David Briles. Bacteria were grown in Todd Hewitt Broth with 1% yeast extract (THY,
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Fig. 2. Trivalent vaccination leads to higher IgG responses to PcpA, PhtD, and Ply in adult than infant mice. Serum IgG responses were determined for infant and adult mice using (A) lower antigen concentrations in a trivalent vaccine (0.2 g PcpA, 0.9 g PhtD, 5 g PlyD1) or (B) higher antigen concentrations (5 g PcpA, 2.5 g PhtD, 15.2 g PlyD1). (n = 5 each group for 2 independent experiments).
2.8. PlyD toxicity assay BG7322 was grown in THY. FITC labeled or unlabeled Spn was neutralized as described above with antibodies generated in infant and adult mice vaccinated with PlyD1 or the trivalent vaccine. Spn was then added to primary epithelial and endothelial lung cells (Cell Biosystems) and incubated at 37 ◦ C for 5 h. Cells were washed 3 times with PBS and Sytox Orange (Invitrogen) was added at 0.1 l/ml in PBS and incubated for 5 min followed by washing 3 times with PBS and then fixation, mounting, and imaging as described above. 2.9. Statistics Data was analyzed by two tailed Student’s T-tests or ANOVA and results with a p < 0.05 considered significant. Data was analyzed by Prism software (Graph Pad, La Jolla CA). 3. Results 3.1. Serum antibody generation in infant and adult mice with trivalent vaccine We evaluated two different dosages for each antigen and found that a higher dose of antigens led to significantly higher antigenspecific IgG serum concentrations in adult mice for only PcpA (p = 0.008) while adult IgG specific titers for PhtD were lower with higher dosage antigen (p = 0.02) (Fig. 2). In infant mice, the higher doses led to higher IgG responses to PcpA and Ply but lower responses to PhtD although the differences were not statistically significant. Based on these results, we proceeded to test the lower
dose. Importantly, at the lower dosage levels, vaccination increased antibody levels to all three components of the trivalent vaccine in both infant and adult mice as compared to sham vaccinated controls.
3.2. Vaccination using trivalent PcpA, PhtD and PlyD1 We assessed survival of vaccinated infant and adult mice after intranasal challenge that caused lethal bacteremic pneumonia in control mice. We found that 90% of infant and 100% of adult trivalent vaccinated mice were protected from lethal pneumonia caused by a serotype 6A strain (Fig. 3A). In addition, 70% of trivalent vaccinated infant mice were protected from lethal pneumonia caused by WU2 strain; a serotype 3 strain (Fig. 3B). Thus trivalent vaccination conferred cross serotype protection from lethal pneumonia.
3.3. Antibody levels in lung We examined the levels of IgG in the lungs of trivalent vaccinated animals after the third vaccination. Significantly higher IgG titers were measured for all three antigens in vaccinated compared to control mice (Fig. 4A–C). Parenteral vaccination did not increase IgA in the lungs, similar to the results in serum (data not shown).
3.4. Bacterial lung burden and histopathology Trivalent vaccinated infant and adult mice had a significant 0.5–0.7 log reduction in the amount of lung bacterial burden 48 h post-challenge (Fig. 4D). Trivalent vaccinated infant and adult mice
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Fig. 3. Vaccination with trivalent vaccine confers protection from lethal pneumonia. (A) Survival curves for trivalent (0.2 g PcpA, 0.9 g PhtD, 5 g PlyD1) vaccinated infant and adult mice were assessed after challenge. (n = 5 each group for 2 independent experiments). (B) Survival curves for vaccinated infants after challenge with WU2 strain. (n = 5 each group for 2 independent experiments).
had reduced histopathological scores as compared to unvaccinated controls (Fig. 4E–F). 3.5. Antibodies reduce Spn adherence and neutralize epithelial damage We found abundant adherence of Spn to lung epithelial cells in the absence of Spn specific serum antibodies (Fig. 5A and B). Serum antibodies derived from trivalent vaccinated mice led to significant reductions in the ability of Spn to bind to primary lung epithelial cells from both vaccinated infant and adult mice (Fig. 5C and D). To determine whether serum antibodies generated to PlyD1 could protect lung epithelial damage from the toxicity of native Ply
and thus prevent potential translocation of Spn into the lung vasculature, we performed an in vitro neutralization assay using primary lung cells (Fig. 5E–J). We found that primary lung cells incubated with serum antibodies derived from PlyD1 vaccinated infant and adult mice had significantly less cell damage after 5 h of incubation in the presence of Spn. 4. Discussion A multi-component protein-based vaccine that protects from all Spn strains would be a significant advance [30]. Here we investigated the protective effect of a trivalent vaccine consisting of Spn proteins, PcpA, PhtD and PlyD1 in an infant murine challenge
Fig. 4. Bacterial lung load and histopathology of trivalent vaccinated mice. IgG titers were determined from 10 mg of lung extract for (A) PcpA, (B) PhtD, and (C) Ply. (n = 10 mice each group). (D) Lung bacterial burdens at 48 h post-infection in unvaccinated (control) or trivalent vaccinated infant and adult mice. (n = 5 each group for 2 independent experiments). (E) Lung tissue histology in 48 h post-infected trivalent vaccinated or unvaccinated mice. Representative histology from infant mice is shown at 20× magnification. (F) Histopathologic scores were determined in vaccinated or unvaccinated mice 48 h post-challenge. (n = 5 each group for 2 independent experiments). * p-value < 0.05
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Fig. 5. Trivalent vaccination leads to a reduction in Spn induced epithelial damage to primary lung epithelial cells. FITC labeled Spn was incubated with serum from vaccinated or unvaccinated mice and with guinea pig complement for 1 h prior to adding to C57BL/6 derived primary lung epithelial cells. Spn/No antibody control contained non-specific mouse serum. White = Spn. (A–D) Spn adherence was determined on primary lung epithelial cells either in the absence or presence of antibodies obtained from trivalent vaccinated infant and adult mice. Representative microscopy is shown at magnification of 20×. (E–I) Spn was incubated with serum from vaccinated or unvaccinated mice for 5 h to allow for Ply mediated membrane damage. White = Sytox orange that stains membrane damaged cells. (n = 4 each from randomly chosen frozen serum for 2 independent experiments). Representative microscopy with magnification of 20× is shown.
model, with adult comparators. Importantly each protein was designed from serotype 6B and we tested the protection afforded by the trivalent vaccine against strains expressing heterologous serotypes 6A and 3 challenge strains to demonstrate cross serotype protection. Using monovalent vaccines, infant and adult mice showed similar increases in IgG antibody specific titer to the vaccinated antigens except for PlyD1 where infant mice elicited minimal titers. After monovalent PhtD, PcpA and PlyD1 vaccination, subtyping of IgG specific antibody responses demonstrated a dominance of IgG1 antibodies implying a strong Th2 or TFH CD4 T-cell memory response. Lung bacterial loads were reduced with the trivalent vaccine in both infant and adult mice. Our results are in agreement with previous studies showing protection from Spn challenge after vaccination with PcpA and PhtD in adult mice [7,10,31]. It has been reported that PlyD1 vaccination prevents lung histopathology after lethal challenge by limiting inflammation in adult mice [22,32,33]. Of interest, even with lower antibody titers directed to PlyD1 in infant mice compared to adult mice, tissue histopathology was limited suggesting that the titers of antibody in the infants were adequate to neutralize the damage of lung epithelial cells caused by Ply. PlyD1 may offer better protection of lung epithelium from the toxic effects of Ply and subsequent reduction of bacteria from dissemination into the capillaries that line the epithelial barrier in the alveolar space. However, we observed high levels of protection only in mice vaccinated with the trivalent vaccine suggesting that reduction of bacteremia and reduction in lung bacterial burdens are both necessary for optimal protection. Investigation of the role of antibody from the lung would be of interest in a future study. Adherence of Spn to primary lung epithelial cells was reduced using antisera from mice vaccinated with the trivalent vaccine and mixtures of antibodies against PhtD and PcpA. In prior studies of natural colonization by Spn in young children, antibodies to either PcpA [19] or PhtD [8] blocked adherence of Spn to human lung cells. IgG specific antibody responses increased with higher dose vaccinations in adult mice. In comparison a higher dose trivalent vaccines elicited an increased (PcpA), decreased (PhtD) and similar (PlyD1) antibody response in infant mice. Although this may imply some mechanism of antigen presentation interference for
PhtD with the trivalent vaccine in infant mice, vaccination with the trivalent Spn vaccine provided protection from pneumonia, lung tissue damage, and subsequent sepsis. Moreover, in our challenge studies the trivalent vaccine resulted in 100% protection in adult mice and 70–90% protection in infant mice upon lethal challenge. In summary, we have shown that vaccination with a trivalent protein-based vaccine containing PcpA, PhtD and PlyD1 can confer protection against lethal pneumonia in infant mice. The mechanism of protection appears to be by reduction of Spn binding to airway epithelium by antibodies directed to PcpA and PhtD while antibodies to Ply significantly reduce epithelial damage and probably subsequent inflammation. This study highlights the interplay necessary between vaccine-induced antibodies for prevention of adherence to respiratory epithelial cells and direct neutralization of toxic virulence factors to prevent epithelial damage. Future studies in infant mice to further define the contribution of phagocytosis and the requirement for complement activation in antibody-mediated protection are underway.
Acknowledgments This work was supported by an Investigator initiated grant awarded to MEP by Sanofi Pasteur. We thank Dr. Robert Zagursky for assistance in manuscript preparation and Sheldon Perry, Jessica Klapa, and Karin Pryharski for technical assistance.
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