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  • Good afternoon. Today I am presenting my dissertation presentation entitled “Polyreactive and antigen-specific B-cell antibody response to Streptococcus pneumoniae”.
  • The bacteria we study in the lab is Streptococcus pneumoniae also referred to as pneumococcus. This bacteria readily colonizes the human nasopharynx. It is a gram-positive, bullet-shaped diplococci that grow in pairs or chains as shown here in a sputum smear. Pneumococcus is alpha-hemolytic meaning it partially lyses red blood cells. When grown on blood agar, the red hemoglobulin is oxidized and turns green giving the bacteria a dark green color. Over 90 different serotypes of pneumococcus have been identified. Serotypes are distinguished by the structure of the capsular polysaccharide. Vaccination elicits protective antibodies against pneumococcal polysaccharide resulting in bacterial clearance.
  • Streptococcus pneumoniaeis responsible for considerable morbidity and mortality worldwide. Incidence and mortality in children under the age of five is highest in underdeveloped countries, shown on the map in red and orange, due to lack of vaccination and treatment. In the U.S, pneumococcus is responsible for 3,000 cases of meningitis, 50,000 cases of bacteremia, 500,000 cases of pneumonia and 7 million cases of otitis media each year. Invasive pneumococcal disease presents a characteristic age distribution with the majority of cases occurring in children under the age of 2 and adults older than 65. Other populations at high risk for invasive pneumococcal disease include HIV-positive, functionally or surgically asplenic, common variable immunodeficiency, IgG deficiency, IgA deficiency and impaired polysaccharide responsiveness. The immune system in these populations are defective in response to pneumococcal infection and are unable to efficiently clear S. pneumoniae allowing for increased colonization and pathogenesis of disease.
  • A common mode of colonization is inhalation of Streptococcus pneumoniae in aerosolized particles. Many individuals are natural carriers of pneumococcus and do not become ill. There are two types of pneumococcal disease: mucosal and invasive. When pneumococcimove from its natural reservoir in the nasopharynx, invasive pneumococcal diseasemay develop.In children, ascension of pneumococci tothe Eustachian tubes and colonization of the middle ear results in the mucosal disease otitis media. Pneumococcal infection of the lungs causes pneumonia. Prolonged infection leads invasive pneumococcal disease by allowing pneumococci to reach the blood stream causingbacteremia. If pneumococciinfect cerebral spinal fluid or the central nervous system, it may result in encephalitis or meningitis.
  • Streptococcus pneumoniaehas several virulence factors that help it evade the host immune system. Pneumococcal capsular polysaccharide prevents complement mediated opsonophagocytosis. The cell wall aids in attachment to host epithelial cells and induces inflammation. Pneumococcal surface protein C also facilitates in host cell attachment and pneumococcal surface protein A prevents binding of complement and bactericidal enzymes. Pneumococcal suface antigen A helps maintain the cell’s biological functions. Pneumolysin is cytoxic to host cells.
  • During the initial stages of infection, innate immunity can remove pneumococci through non-specific complement mediated pathways. However if innate immunity is not sufficient, specific antibodies against pneumococcal surface molecules are necessary for clearance.
  • In 1891 anti-serum obtained from an individual infected with pneumococcus was successful in protecting a naïve individual from pneumococcal disease. By the mid-20th century, penicillin was successful in treating infections caused by gram-positive organisms however mutations soon led to penicillin resistant pneumococci. This set in motion the development of pneumococcal vaccines. In the 1977, a 14 valent purified pneumococcal polysaccharide vaccine was licensed. This vaccine was later updated in 1983 to 23 valent to include additional clinically relevant pneumococcal serotypes. Pneumococcal polysaccharide is poorly immunogenic in children under the age of 2 due to their underdeveloped immune systems. Therefore in 2000, a 7 valent pneumococcal conjugate vaccine (PCV) was licensedcontaining pneumococcal polysaccharides conjugated to a protein.
  • The best way to prevent invasive pneumococcal disease is to vaccinate.The 23-valent pneumococcal polysaccharide vaccine has an 80% protective efficacy in healthy young adults and is recommended for all elderly adults over the age of 65.Administration of the pneumococcal conjugate vaccine decreased the incidence of invasive pneumococcal disease in children under the age of 5 by 80%. After introduction of pneumococcal conjugate vaccine the incidence of invasive pneumococcal disease in the elderly also dropped. This phenomenon, referred to as herd immunity, is the result of decreased spread of disease to adults because a large percentage of children are vaccinated.
  • In our lab we study the immune response to pneumococcal polysaccharide vaccine. After establishing the immune response to this vaccine in healthy young adults as a positive control, we can compare the immune response to pneumococcal polysaccharide vaccine in the HIV-positive and elderly populations. In our lab we focus on two pneumococcal polysaccharides, 14 and 23F. These two serotypes were chosen because they are commonly isolated from all three populations, healthy, elderly and HIV+ and they both have different net charges. PPS14 is neutral while PPS23F is negative. For these studies we analyzed the B-cell response to pneumococcal polysaccharide vaccine in healthy young adultvolunteers two ways. First we developed a method to directly label anti-pneumococcal polysaccharide cells enabling us to identify the phenotype of B cells that respond to the pneumococcal polysaccharide vaccine. Second we characterized antibodies that bound multiple pneumococcal polysaccharides by sequence and kinetic analysis.
  • We hypothesized that pneumococcal polysaccharide vaccine responding B cells are IgM memory cells.Previous investigators have suggested CD27+IgM+ memory cells play an important role in protection from pneumococcal infection mainly because populations at an increased risk for infection are deficient in IgM memory cells. For the second project we hypothesized that human polyreactive anti-pneumococcal polysaccharideantibodies are of low avidity but elicit protection from pneumococcal challenge. Murine studies have identified B1 cells that spontaneously secrete polyreactive antibodies but also had functional activity.
  • This is a diagram of our unique methodology for isolating pneumococcal polysaccharide binding B cells. First we vaccinate our volunteer with Pneumovax® which is the 23 valent pneumococcal polysaccharide vaccine licensed in the US. Blood is drawn on day 0 pre-vaccination and day 7 and 28 post-vaccination. B cells are analyzed using flow cytometry and fluorescently labeled pneumococcal polysaccharide 14 and 23F on day 0 and 7 to compare peripheral B cells pre and post-vaccination. Pneumococcal polysaccharide binding B cells from day 7 were single cell sorted into 96 well plates and expanded for 21 days. After 21 days of growth, cell supernatants were tested by ELISA for immunoglobulin secretion and binding to pneumococcal polysaccharide. Serum from day 0 and 28 were evaluated for antibody concentration, antibody isotype and opsonophagocytic activity.
  • Serum samples were obtained pre- and 28 days post-vaccination. Using ELISA, 18 serum sampleswere tested for pneumococcal polysaccharide 14 and 23F-specific IgG, IgM and IgA antibody concentrations. Pre-vaccination concentrations are represented by open red circles and post-vaccination concentrations are represented by blue triangles. Serum antibody titers are displayed in micrograms per milliliter. Post-vaccination all individuals had a significant increase in concentration of anti-pneumococcal polysaccharide IgG and IgM antibody levels but not necessarily IgA. This data confirms that the healthy young adult volunteers used in this study respond to vaccination with Pneumovax.
  • Serum samples were also tested for opsonophagocytic activity. Pre-vaccination values are again represented by open red circles and open blue triangles represent post-vaccination values. The opsonophagocytic index is the reciprocal dilution of antibody needed to kill 50% of pneumococci. Pre-vaccination there were virtually no functional antibodies against pneumococcus in the periphery however post-vaccination all donors had a significant increase in antibody induced phagocytosis. Therefore this population displayed a functional immune response post-vaccination.
  • Fluorescently labeled pneumococcal polysaccharide was tested for preservation of native epitopes. Positive control serum, 89SF was pre-incubated with labeled or unlabeled pneumococcal polysaccharide. Percent inhibition of binding was measured by ELISA with unlabeled pneumococcal polysaccharide coated plates. Incremental addition of inhibitory pneumococcal polysaccharide, increased inhibition in a dose dependent manner with homologous pneumococcal polysaccharide but not with heterologous pneumococcal polysaccharide indicating specificity of binding and preservation of pneumococcal polysaccharide structure after labeling with fluorescent markers, cascade blue or DTAF. 30% of cells are non-specifically binding to PPS because we only achieved 70% inhibition. The only way to separate this is to stain cells with two PPS and eliminate the double-stained non specific B cells. This remaining percentage (20-32%) of non specific staining likely expresses the unselected B cell phenotype. The unselected B cell phenotype is only 14-16% C27+IgM+ which is much lower than PPS-specific B cells. Therefore since the majority of non-specific B cells are not IgM memory this would have actually diminished the IgM memory phenotype in our PPS-specific B cell population. Since the majority of unselected B cells for both PPS are CD27+IgM- B cells is unlikely that contaminating non-specific B cells changed the percentage of switched memory B cells in the PPS-specific population.
  • To further confirm functionality and specificity of the labeled polysaccharides, hybridoma cells with a specificity for pneumococcal polysaccharide 14 and 23F were incubated with labeled polysaccharide. Hybridoma cells expressing antibody specific for PPS23F were stained with both fluorescently labeled pneumococcal polysaccharide 14 (y-axis) and pneumococcal polysaccharide 23F (x-axis). These Pneumococcal polysaccharide 23F-specific hybridoma cells stained only with pneumococcal polysaccharide 23F in a dose dependent manner while no pneumococcal polysaccharide 23F-specific hybridoma cells stained with fluorescently labeled pneumococcal polysaccharide 14. The same results were observed with Pneumococcal polysaccharide 14-specific hybridoma cells. These experiments confirm specificity and function of labeled pneumococcal polysaccharide.
  • CD19+ B cells were stained with labeled pneumococcal polysaccharide pre and post-vaccination. Pre-vaccination approximately 0.5% of B cells in the periphery were specific for pneumococcal polysaccharide 14 or pneumococcal polysaccharide 23F. Seven days post-vaccination there was a significant increase in the percentage of pneumococcal polysaccharide-specific B cells in peripheral blood.
  • To determine the phenotype of B cells that respond to vaccination with Pneumovax, pre- vaccination and 7 days post-vaccination circulating peripheral blood mononuclear cells were isolated and labeled for analysis by flow cytometry with fluorescently labeled anti-CD19, anti-CD27, anti-IgM and pneumococcal polysaccharide 14 or 23F. The following phenotypes were determined, CD27-IgM+ naïve B cells shown in green, CD27-IgM- naïve class-switched B cells in purple, CD27+IgM+ memory B cells in red and CD27+IgM- switched memory B cells in blue. The phenotype of unselected and selected B cells pre- and post-vaccination were compared. Pre-vaccination, unselected B cells were not different from pneumococcal polysaccharide-selected B cells with all three groups having a predominance of CD27-IgM+ naïve B cells. However post-vaccination there is a big difference between unselected and selected B cell phenotypes. In pneumococcal polysaccharide-selected B cells there is a dramatic shift from naïve B cells to IgM memory B cells.
  • In sharp contrast to both the unselected and pre-vaccination pneumococcal polysaccharide-selected B cells, the minority of post-vaccination pneumococcal polysaccharide-specific B cells consisted of naïve CD27- B cells. There is a significant decrease in CD27-IgM+ naïve and CD27-IgM-naïve, class-switched B cells with a significant increase in CD27+IgM+ memory cells for both pneumococcal polysaccharides post-vaccination.
  • Healthy donor peripheral blood mononuclear cells were stained with antibodies to CD19, CD27, IgM and fluorescently labeled Pneumococcal polysaccharide 14. CD19+ B cells were gated on Pneumococcal polysaccharide 14. Pre- and post-vaccination the majority of unselected pneumococcal polysaccharide B cells were CD27-IgM+. Post vaccination there is a shift from naïve to IgM memory pneumococcal polysaccharide-specific B cells. CD27+IgM+ Pneumococcal polysaccharide 14 specific B cells increased from 28.3% pre-vaccination to 68.5% post-vaccination. Similar results were observed with Pneumococcal polysaccharide23F.
  • In this analysis itwas crucial to have the right tools. We can now look at the phenotype of these cells because we developed a unique reagent, fluorescently labeled pneumococcal polysaccharide and identified pneumococcal polysaccharide-responding B cells after vaccination using flow cytometry. CD27+IgM+ B cells increased in healthy young volunteers post-vaccination with the pneumococcal polysaccharide vaccine. Post-vaccination pneumococcal polysaccharide specific antibody titers and opsonophagocytic activity correlated with the increase in CD27+IgM+ memory B cells. CD27+IgM+ cells play a crucial role in the immune response to pneumococcal polysaccharide.
  • When specific pneumococcal polysaccharide antibodies are not present, natural antibodies serve as the first line of defense against pneumococcal infection. Natural antibodies are not antigen specific but can clear the pathogen through neutralization, complement and lysis.
  • By comparison,IgG1 is more flexible than IgG2. (Show difference in number of disulfide bonds, amino acids in constant region and flexibility). IgG1 is recognized by all FcYR on effector cells making IgG1 more effective in mediating phagocytosis. There are four different kinds of antibody movement. (Go over each one) Restriction of any of these four types of movement limits the antibody’s ability to interact with antigens.
  • After testing cell supernatant by ELISA for immunoglobulin secretion and binding to pneumococcal polysaccharide from single cell sort, which was the last part of the diagram I showed you, several monoclonal polyreactive pneumococcal polysaccharide antibodies were detected. These polyreactive pneumococcal antibodies bound pneumococcal polysaccharide 4 show in blue, pneumococcal polysaccharide 6B in red, pneumococcal polysaccharide 14 in green and pneumococcal polysaccharide 23F in purple. Both isotypes, IgG1 and IgG2, of all identified polyreactive antibodies bound multiple pneumococcal polysaccharides with varying optical densities.
  • Protective antibodies induce opsonophagocytosis of pneumococci. All polyreactive variable chains in both IgG1 and IgG2 possessed functional activity against pneumococcal serotypes 14 and 23F. The IgG1 isotype of each monoclonal is represented in green and the IgG2 isotype of each monoclonal antibody is shown in purple. Bars measure the killing efficiency of each monoclonal. There were some minor differences in killing efficiency between monoclonal antibody isotypes and pneumococcal polysaccharides but nothing statistically significant.
  • Sequence analysis of the polyreactive anti-pneumococcal polysaccharide variable regions revealed several characteristics. Variable light chain CDR3 lengths were conserved with all but one monoclonal having a length of 9 amino acids. Variable light chain CDR3s also had varying mutations with varying V and J gene usage. In contrast, variable heavy chain CDR3 lengths differed greatly. There was also a large range of mutations, different J gene usage and mainly VH3 gene usage.
  • To further analyze the trends seen in polyreactive antibodies, % homology to germline and CDR3 length was compared to pneumococcal polysaccharide-specific CDR3 sequences. Polyreactive antibodies are shown in red and specific anti-pneumococcal polysaccharide antibodies are show in blue. Additional polyreactive and pneumococcal polysaccharide specific antibodies against pneumococcal polysaccharide 3, 6B, 14 and 23F are from previously published sequences reported by several investigators. While there was no difference in percent homology to germline in either the variable heavy chain or variable light chain CDR3 sequences there was an increase in the polyreactive variable heavy CDR3 length when compared to pneumococcal polysaccharide-specific sequences.
  • Binding of an antigen to an antibody’s binding groove is influenced by the nature of its amino acid sequence. Several factors such as charge and flexibility alter the antigen-antibody interaction. Three groups of amino acids: flexible, positive and negative were counted. The flexible group of amino acids include arginine (R), tryptophan (W) and tyrosine (Y). Positive amino acids are arginine (R), histidine (H) and lysine (K). The negative amino acid group contains aspartic acid (D) and glutamic acid (E). The number of flexible, positive and negative amino acids in the variable heavy and variable light chain CDR3s from polyreactive in red and pneumococcal polysaccharide-specific in blue were compared. While there was no trend in amino acid expression between polyreactive and pneumococcal polysaccharide-specific variable light CDR3s, there was a significant increase in the number of flexible amino acids expressed in polyreactive variable heavy CDR3s.
  • To compare the avidity of IgG1 and IgG2 recombinant antibodies with identical variable regions, surface plasmon resonance was used. Surface plasmon resonance technology measures molecular interactions in real-time. Our experimental model detected interactions between polyreactive pneumococcal polysaccharide antibodies and different concentrations of pneumococcal polysaccharide to determine a binding constant. The gold chip was first coated with an anti-human antibody to ensure that all of the polyreactive antibodies will be in the correct orientation for interaction with pneumococcal polysaccharide. A single wavelength of light from a laser is shown through a prism and the prism rests underneath the gold chip. When there are changes in molecular mass on the gold chip due to molecular interactions, the angle in which light refracts through the prism changes. These changes are recorded by a computer and displayed as refractive index units.
  • The binding constants for the polyreactive antibodies are listed in following tables. To make these values easier to compare they are also presented in bar graphs with IgG1 in green and IgG2 in purple. The y-axis is reversed because smaller numbers mean better binding. All polyreactive antibodies were weakly avid with all binding constants in the micromolar range. As a point of reference, a pneumococcal polysaccharide-specific control antibody measured in the nanomolar range which is 10-9, a much stronger avidity. Overall IgG1 bound both pneumococcal polysaccharide 14 and 23F more avidly. Since OPSA doesn't show a difference, the importance in the difference in binding is not clear at this time. This observation may be restricted to polyreactive antibodies and completely different for specific antibodies.
  • To further investigate the role of the constant region in polyreactive antibody avidity, antibodies were cleaved into F(ab)’2 fragments by enzymatic digestion with pepsin. Surface plasmonresponance analysis of F(ab)’2 fragments revealed no difference in avidity between IgG1 and IgG2. These results suggests that constant heavy domains 2 and 3 are necessary for antibody stability and restriction of movement.
  • In summary polyreactive pneumococcal polysaccharide antibodies possess the following characteristics- all but one monoclonal had a variable light CDR3 length of 9 AA. The variable heavyCDR3 sequences were various lengths, had a large range of mutations, expressed mainly the VH3 gene family and had a significantly higher number of flexible amino acids when compared to pneumococcal polysaccharide-specific variable heavy CDR3s. Flexible amino acids are thought to increase the plasticity of the antibody’s antigen binding groove allowing for recognition of multiple, large antigens such as bacterial polysaccharides.For both pneumococcal polysaccharides, polyreactive IgG1 antibodies had an overall higher avidity compared to IgG2 although this did not translate to improved functional activity.
  • Overall these studies have analyzed the normal immune response to pneumococcal polysacccharide vaccine in healthy young adults. The phenotype of human B cells that respond to pneumococcal polysaccharide vaccine had not been identified. Our lab developed a unique tool to specifically identify these cells. Flow cytometry using fluorescently labeled pneumococcal polysaccharide revealed that IgM memory B cells are crucial in the immune response to thepneumococcal polysaccharide vaccine. Knowing the phenotype of these cells increases the understanding of cell surface expression and will help us to identify individuals at a greater risk for invasive pneumococcal disease.Polyreactive pneumococcal polysaccharide-binding B cells were characterized and found to express a higher percentage of flexible amino acids when compared to pneumococcal polysaccharide-specific variable heavy CDR3s. Overall polyreactive IgG1 antibodies bound both pneumococcal polysaccharides more avidly when compared to IgG2 . Similar studies using pneumococcal polysaccharide-specific IgG1 and IgG2 antibodies need to be performed. If the results are similar this may suggest that IgG1 may be the preferable isotype in the immune response to pneumococcal polysaccharides which is important for future vaccine and adjuvant development.
  • In our future studies we will analyze the B cell response to pneumococcal polysaccharide vaccine in the elderly and HIV-positive. Recent studies have revealed similar characteristics in their immune response to pneumococcal polysaccharide. Both populations exhibit polyclonal activation, increased immunoglobulin secretion, activation of resting B cells, a decrease in naïve B cells and a decrease in CD27+IgM+ B cells.
  • Any questions?
  • FIGURE 6. Phenotype analyses of B cells in the human peripheral blood. Healthy donor PBMC sample was stained with Abs to CD19, CD27, IgM, and fluo- rescently labeled PPS14 (A, B) and 23F (C, D). CD19+ B cells (shown in histogram, dotted line = isotype control) were gated on PPS23F or PPS14. PPS14- or 23F-specific B cells (CD19+PPS14+, CD19+PPS23F+) and PPS14- or 23F-negative B cells (CD19+PPS142, CD19+ PPS23F2) were separated into CD27+IgM+, CD27+IgM2, CD272IgM+, and CD272IgM2. In each sample, 75,000 events were recorded. Representative data of FACS analyses: (A and C), prevaccination; (B and D), postvaccination of PPS14 (A, B) or PPS23F (C, D).
  • Theoretically every well in our 96 well plate has PPS specific B cells. However few if any B cells survived in each plate. After isolation of polyreactive B cells but not single PPS binding B cells this suggested that the cell supernatant cocktail used allowed for the survival of polyreactive but not specific B cells. We tried to optimize the mixture over two years but eventually had to abandon the project due to cost and time restraints. T cell replacement factor for , IL-2 for growth and function of T cells and to facilitate production of immunoglobulin by B cells, IL-4 to stimulate B cell proliferation, poke weed mitogen EL4-B5 Irradiated mouse thymoma cells
  • Dissertation presentation

    1. 1. Polyreactive and antigen-specific B-cell antibody response to Streptococcus pneumoniae Rebecca Thompson March 29, 2012
    2. 2. Streptococcus pneumoniae • Colonizes the nasopharynx • Over 90 identified serotypes • Serotypes are determined by capsular polysaccharide (PPS) • Vaccines induce protective antibodies against PPS Streptococcus pneumoniae in sputum smear. Gram stain (1200X) LeBeau 2009
    3. 3. Epidemiology • Streptococcus pneumoniae is responsible for considerable morbidity and mortality worldwide. • Invasive pneumococcal disease (IPD) exhibits a characteristic age distribution with the majority of cases occurring • Very young (<2 years old) • Elderly (>65 years old) • Other high risk populations • Immune systems in these populations are defective in response to pneumococcal infection Mortality rate* per 100,000 children under five years of age due to Incidence of Invasive Pneumococcal Disease in U.S. Streptococcus pneumoniae, 2000 200 160 2000 Rate per 100,000 persons 120 80 2010 40 dum <10 0 * HIV+ve deaths excluded 10- <100 100-<300 <1 1 2-4 3-17 18-34 35-49 50-64 ≥ 65 Total my Age (in years) 300-<500http://www.who.int/nuvi/pneumococcus/decision_implementation/en/index.html >500 Data adapted from www.cdc.gov/abcs
    4. 4. Pathogenesis1. Colonization • Inhalation of S. pneumoniae in aerosolized particles • Natural carriage2. Otitis Media3. Pneumonia4. Meningitis/Encephalitis
    5. 5. Virulence Factors PsaA Pneumolysin Cell membrane Capsule Cell wall PspC PspA
    6. 6. Immune Response• Innate • Complement mediated • Not antibody specific• Adaptive • Antibody mediated • Antibodies against surface molecules • Role of these in clearance of bacteria are yet to be determined • Antigen specific
    7. 7. Pneumococcal Therapies • Anti-serum • 1891 serum protected from pneumococcal disease • Antibiotics • Mid-20th century, penicillin was successful in treating infections with gram-positive organisms • Mutations soon lead to penicillin resistant pneumococcus • Purified pneumococcal polysaccharide vaccine • 1977 14 valent purified pneumococcal polysaccharide vaccine (PPV) was licensed • 1983 updated to 23 valent • Pneumococcal conjugate vaccine • In 2000, a 7 valent pneumococcal conjugate vaccine (PCV)
    8. 8. Prevention• The most effective form of prevention is vaccination• The 23-valent PPV has an 80% protective efficacy in healthy young adults• Administration of the PCV decreased the incidence of IPD in children under the age of 5 by 80%
    9. 9. Purpose• We analyzed the B-cell response to PPS in healthy young volunteers two ways • Developed a method to directly label anti- PPS cells • Characterization of polyreactive anti-PPS antibodies
    10. 10. Hypotheses• Anti-PPS B cells which respond to PPV are IgM memory cells• Human polyreactive anti-PPS antibodies are low avidity but elicit protection from pneumococcal challenge
    11. 11. Isolation of PPS B cells • Vaccinate volunteer Isolation of B cells with Pneumovax® 7 days post- vaccination • Draw blood at day 0, 7 and 28 Flow cytometry with • Analyze B cells at fluorescently labeled PPS to identify PPS- day 0 and 7 specific B cells • Single cell sort PPS binding B cells from day 7 • Analyze antibody titers at day 0 and 28 • Opsonophagocytic assay day 0 and 28 Testing of single B cell culture supernatant by ELISA
    12. 12. Healthy immune response after vaccination Pre-immunization PPS14 PPS23F Post-immunization p< 0.0001 p< 0.0001 IgG level (mg/ml) IgG level (mg/ml) IgG level (mg/ ml) p< 0.0001 p< 0.001 IgM level (mg/ml) IgM level (mg/ml) p< 0.03 NS IgA level (mg/ml) IgA level (mg/ml)
    13. 13. Healthy immune response after vaccination Pre-immunization PPS14 Post-immunization PPS23F p<0.0001 p<0.0001Opsonophagocytic Index Opsonophagocytic Index Pre-immunization Post-immunization Pre-immunization Post-immunization
    14. 14. Specific binding offluorescently labeled PPS
    15. 15. Specific staining of PPS hybridomas
    16. 16. Percentage of PPS specific CD19+ B cells pre- and post-vaccination
    17. 17. Phenotype of B lymphocytesthat respond to Pneumovax® Pre-vaccination Post-vaccination * * * = p<0.0001
    18. 18. Phenotype of B lymphocytes that respond to Pneumovax® PPS14 - Selected PPS23F - Selected ## ** * ## * #* = p<0.05, ** = p<0.0001 # = p<0.002, ## = p<0.0001
    19. 19. FIGURE.6.Phenotype of B lymphocytesthat respond to Pneumovax®A BC D
    20. 20. Summary• Developed fluorescently labeled PPS to identify PPS-specific B cells using flow cytometry• CD27+ IgM+ B cells increased after PPV in healthy young volunteers• Antibody titers and opsonophagocytic activity also correlated with the increase in CD27+ IgM+ memory B cells• These cells play a crucial role in the immune response to PPS
    21. 21. Natural Antibodies Pneumococcal polysaccharide IL-5 B1 cell • Natural Antibodies • Direct neutralization of pathogen • Activation of complement • Complement mediated lysisImmunology Today, Volume 21, Issue 12, 1 December 2000, Pages 624-630 Adrian F Ochsenbein, Rolf M Zinkernagel
    22. 22. Antibody structure• Antibodies have two functional regions Variable region • Variable • Capable of specific recognition and binding to epitope • Constant • Classically thought to contribute to effector functions • Complement activation C C H H Hinge region • Mediation of immune 2 2 phagocytosis Constant region C C • Antibody-dependent H H cytotoxicity 3 3
    23. 23. Recombinant human immunoglobulin expression vectors IgG1 IgG2
    24. 24. IgG1 vs. IgG2 • IgG1 is more flexible than IgG2 • 2 vs 4 disulfide bonds in hinge region 127 • More amino acids in constant region 117 • Recognized by all FcΥR on effector cells • IgG2 is a poor activator of 43 32 99 107 complement • Low capacity to bind C1q due to decreased flexibility IgG1 IgG2 • Recognized by FcΥRII which is a low affinity receptor Fab Fab elbow arm bending rotation Fab arm waving Fc tail waggingThe Journal of Immunology October 1, 1997 vol. 159 no. 7 3372-3382
    25. 25. Polyreactive antibodies 0.8 Polyreactive Antibodies binding to Multiple PPS 0.7 PPS4 0.6 0.5 PPS6BOD 490nm 0.4 PPS14 0.3 0.2 PPS23F 0.1 0 IgG1 IgG2 IgG1 IgG2 IgG1 IgG2 IgG1 IgG2 IgG1 IgG2 IgG1 IgG2 21B2 24F5 33G8 32E8 31E2 31B5
    26. 26. Opsonophagocytic activity of polyreactive antibodies 100 Opsonophagocytic Activity PPS14 100 Opsonophagocytic Activity PPS23F 90 90 80 80 70 70 IgG1Maximum percent killing Maximum percent killing 60 60 IgG1 50 50 40 IgG2 40 IgG2 30 30 20 20 10 10 0 0 33g8 32e8 31e2 31b5 21b2 24f5 33g8 32e8 31e2 31b5 21b2 24f5
    27. 27. Sequences of polyreactivepneumococcal antibodiesClone VH CDR3 # of Mutations % Homology VH Gene JH Gene34C10 AKDSRGSTPRAFDP 12 97 3-23 JH5b33G8 ARDGRQQWLVRPYYYGMDV 5 99 1-18 JH6b32E8 TKWRWQQSEFDY 29 93 3-7 JH4b31E2 AKDSRGSTPRAFDP 36 88 3-66 JH5b31B5 AKKAFSGYSPFDY 20 94 3-23 JH4b21B2 ARNGGVGATDPPYYYYGMDV 5 99 3-30 JH6b24F5 AKDRSLREYSSSWYYPFYYYGMDV 4 99 3-23 JH6b33E2 AREEYYGSGFDP 4 99 3-48 JH5bClone Vk CDR3 # of Mutations % Homology Vk Gene Jk Gene34C10 QKYNSAPFT 12 99 A20 JK333G8 QQANSFSLT 10 99 L5 JK432E8 QRSSGGPIS 35 90 O12 JK531E2 QKYNGAPFT 14 97 A20 JK331B5 QQYDRSPLT 15 98 A27 JK421B2 QRSSGGPIS 37 89 O12 JK524F5 QKYNGAPFT 15 93 A20 JK333E2 QQHYNTPT 14 98 B3 JK5
    28. 28. Polyreactive vs. specific PPS antibodies Homology to Germline Average CDR3 Length 25 100 98 20 Polyreactive 96 94 PPS3 Polyreactive # of amino acids 15% Homology 92 PPS6B 90 PPS-specific 88 10 PPS14 86 84 dumm 5 PPS23F 82 y Series6 80 0 VH CDR3 Vk CDR3 VH CDR3 Vk CDR3
    29. 29. Polyreactive vs. specific PPS antibodies Average Number of Amino Acid Groups in Variable Average Number of Amino Acid Groups in Variable 7 *** Heavy Chain CDR3 7 Light Chain CDR3 6 6 Polyreactive PolyreactiveAverage number of amino acids Average number of amino acids 5 5 PPS3 PPS3 4 PPS6B 4 PPS6B 3 PPS14 3 PPS14 PPS23F 2 2 PPS23F 1 Series12 1 Series6 0 0 Flexible AA - RWY Positive AA - RHK Negative AA - DE Flexible AA - RWY Positive AA - RHK Negative AA - DE
    30. 30. Surface Plasmon Resonance Pneumococcal polysaccharide Human polyreactive anti- pneumococcal antibodiesAnti-human antibodies Gold chip Prism mRIU Time Laser Plotted data
    31. 31. Polyreactive antibody avidity IgG1 vs. IgG2 0.0 0.0 Binding to PPS14 Binding to PPS23F 0.2 0.2 0.4 0.4 0.6 0.6 0.8 0.8KD uM KD uM 1.0 1.0 1.2 1.2 1.4 1.4 1.6 1.6 1.8 1.8 33G8 32E8 31B5 21B2 24F5 33G8 32E8 31B5 21B2 24F5 IgG1 1.02 x E-6 0.57 x E-6 0.67 x E-6 0.76 x E-6 0.28 x E-6 IgG1 1.7 x E-6 0.42 x E-6 1.2 x E-6 0.29 x E-6 0.51 x E-6 IgG2 0.12 x E-6 0.97 x E-6 0.99 x E-6 5.3 x E-6 1.4 x E-6 IgG2 0.73 x E-6 0.302 x E-6 1.4 x E-6 1.5 x E-6 1.005 x E-6
    32. 32. Polyreactive antibody avidity IgG1 vs. IgG2 F(ab)’2 fragment Pepsin digestion KD PPS14 PPS23F Clone IgG1 F(ab)’2 IgG2 F(ab)’2 IgG1 F(ab)’2 IgG2 F(ab)’2 33G8 0.49492 uM 0.5001 uM 0.500 uM 0.4989 uM 21B2 0.5359 uM 1.35362 uM 0.69445 uM 0.80091 uM 24F5 1.07 uM 1.16921 uM 0.98 uM 1.00 uM
    33. 33. Summary• All but one Mab VL CDR3 length – 9 AA• VH CDR3 • Various CDR3 lengths • Large range of mutations • Mostly VH3 • Significantly higher number of flexible amino• For both PPS14 and 23F, IgG1 had an overall higher avidity
    34. 34. Conclusion1. Developed unique tool – labeled PPS • Established IgM memory B cells crucial in PPV response • Helps to identify individuals at risk • Further understanding of cell surface expression2. Characterized polyreactive B cells • Express high % of flex AA3. IgG1 may be preferable isotype in immune response to PPS • Important for future vaccine and adjuvant development
    35. 35. Future Studies• Analyze PPS immune response in the elderly and HIV+• Studies have shown both populations have • Polyclonal activation • Hypergammaglobulinemia • Activation of resting B cells • Decrease naïve B cells • Decrease CD27+IgM+ B cells• By understanding the impaired immune response in these populations guidelines can be established to improve the immune response to vaccination
    36. 36. Clinical significance• Establish a healthy control response to PPS to aid in identifying immune dysfunction in HIV+ and elderly • Compare phenotype of PPS-specific B cells • Compare VH3 usage • Compare anti-PPS antibody titers and opsonophagocytic activity• When is it best to vaccinate?• Is there a benefit to vaccination every 5 years?
    37. 37. Thank you• Major advisor, Dr. M. A. Julie • Dr. Gary McLean for providing the Westerink M.D. recombinant human expression• Academic advisory committee vectors • J. David Dignam, Ph.D. • Dr. Sandra Romero-Steiner for • Dr. Deepak Malhotra, M.D., Ph.D. serotypes of S. pneumoniae used in • Randall Ruch, Ph.D. opsonophagocytic assays • R. Mark Wooten, Ph.D. • Pfizer, Inc. for donating hybridomas• Noor Khaskhely, M.D.,Ph.D. for all specific for PPS14 and PPS23F of this help with flow cytometry and • Dr. Sadik Khuder for statistical B cell acquisition analysis• The present and past members of the • The volunteers who have participated Westerink lab for their support and in our study help • These studies were supported by • Noor Khaskhely, M.D., Ph.D, Kristin National Institute of Health grants Malhotra, David Leggat, M.S., Anita AG081558 and AG05978. Iyer, M.S., Jason Mosakowski, Jieying Wang, Chris Selleck and S. Louise Smithson, Ph.D.
    38. 38. Questions? Strange Matter - Antibiotic Resistance Recruitment by Nick D. Kim.
    39. 39. Phenotype of B lymphocytesthat respond to Pneumovax® A B C D
    40. 40. B cell Culture Cocktail• TRF• IL-2• IL-4• PMA• EL4-B5