scholarly journals Modified Opsonization, Phagocytosis, and Killing Assays To Measure Potentially Protective Antibodies against Pneumococcal Surface Protein A

2013 ◽  
Vol 20 (10) ◽  
pp. 1549-1558 ◽  
Author(s):  
Calvin C. Daniels ◽  
Kyung-Hyo Kim ◽  
Robert L. Burton ◽  
Shaper Mirza ◽  
Melissa Walker ◽  
...  
Keyword(s):  
Capsular Polysaccharide ◽  
Protein A ◽  
Surface Protein ◽  
Pneumococcal Surface Protein A ◽  
Target Strain ◽  
Human Sera ◽  
Protective Antibodies ◽  
Normal Human ◽  
Type 3

ABSTRACTThe standard opsonophagocytosis killing assay (OPKA) for antibodies to pneumococcal capsular polysaccharide was modified to permit an evaluation of the protection-mediating antibodies to pneumococcal surface protein A (PspA). We found that by increasing the incubation time with the complement and phagocytes from 45 min to 75 min, the protective activity was readily detected. In another modification, we used a capsule type 2 target strain that expressed PspA but not pneumococcal surface protein C (PspC). With these modifications separately or in combination, rabbit antisera to the recombinant α-helical or proline-rich domains of PspA mediated >50% killing of the target strain. The ability of normal human sera to mediate the killing of pneumococci in this modified OPKA correlated with their levels of antibodies to PspA and their ability to protect mice against fatal infection with a type 3 strain. Passive protection of mice against pneumococci and killing in the modified OPKA were lost when normal human sera were adsorbed with recombinant PspA (rPspA) on Sepharose, thus supporting the potential utility of the modified OPKA to detect protective antibodies to PspA. In the standard OPKA, monoclonal antibodies to PspA were strongly protective in the presence of subprotective amounts of anti-capsule. Thus, the currently established high-throughput OPKA for antibodies to capsule could be modified in one of two ways to permit an evaluation of the opsonic efficacy of antibodies to PspA.

scholarly journals Impaired Function of Antibodies to Pneumococcal Surface Protein A but Not to Capsular Polysaccharide in Mexican American Adults with Type 2 Diabetes Mellitus

2012 ◽  
Vol 19 (9) ◽  
pp. 1360-1369 ◽  
Author(s):  
Christine E. Mathews ◽  
Eric L. Brown ◽  
Perla J. Martinez ◽  
Upasana Bagaria ◽  
Moon H. Nahm ◽  
...  
Keyword(s):  
Diabetes Mellitus ◽  
Type 2 Diabetes ◽  
Type 2 Diabetes Mellitus ◽  
Capsular Polysaccharide ◽  
Protein A ◽  
Surface Protein ◽  
Complement C3 ◽  
Enzyme Linked Immunosorbent Assay ◽  
Complement Deposition

ABSTRACTThe goal of the study was to determine baseline protective titers of antibodies toStreptococcus pneumoniaesurface protein A (PspA) and capsular polysaccharide in individuals with and individuals without type 2 diabetes mellitus. A total of 561 individuals (131 individuals with diabetes and 491 without) were screened for antibodies to PspA using a standard enzyme-linked immunosorbent assay (ELISA). A subset of participants with antibodies to PspA were retested using a WHO ELISA to determine titers of antibodies to capsular polysaccharide (CPS) (serotypes 4, 6B, 9V, 14, 18C, 19A, 19F, and 23F). Functional activity of antibodies was measured by assessing their ability to enhance complement (C3) deposition on pneumococci and promote killing of opsonized pneumococci. Titers of antibodies to protein antigens (PspA) were significantly lower in individuals with diabetes than controls without diabetes (P= 0.01), and antibodies showed a significantly reduced complement deposition ability (P= 0.02). Both antibody titers and complement deposition were negatively associated with hyperglycemia. Conversely, titers of antibodies to capsular polysaccharides were either comparable between the two groups or were significantly higher in individuals with diabetes, as was observed for CPS 14 (P= 0.05). The plasma specimens from individuals with diabetes also demonstrated a higher opsonophagocytic index against CPS serotype 14. Although we demonstrate comparable protective titers of antibodies to CPS in individuals with and individuals without diabetes, those with diabetes had lower PspA titers and poor opsonic activity strongly associated with hyperglycemia. These results suggest a link between diabetes and impairment of antibody response.

scholarly journals Genetic Alteration of Capsule Type but Not PspA Type Affects Accessibility of Surface-Bound Complement and Surface Antigens of Streptococcus pneumoniae

2003 ◽  
Vol 71 (1) ◽  
pp. 218-225 ◽  
Author(s):  
Melanie Abeyta ◽  
Gail G. Hardy ◽  
Janet Yother
Keyword(s):  
Streptococcus Pneumoniae ◽  
Immune Responses ◽  
Protein A ◽  
Surface Protein ◽  
Surface Antigens ◽  
Capsular Polysaccharides ◽  
Capsule Type ◽  
Type 3 ◽  
Determining Factor

ABSTRACT The Streptococcus pneumoniae capsular polysaccharides and pneumococcal surface protein A (PspA) are major determinants of virulence that are antigenically variable and capable of eliciting protective immune responses. By genetically switching the pspA genes of the capsule type 2 strain D39 and the capsule type 3 strain WU2, we showed that the different abilities of antibody to PspA to protect against these strains was not related to the PspA type expressed. Similarly, the level of specific antibody binding to PspA, other surface antigens, and surface-localized C3b did not depend on the PspA type but instead was correlated with the capsule type. The type 3 strain WU2 and an isogenic derivative of D39 that expresses the type 3 capsule bound nearly identical amounts of antibody to PspA and other surface antigens, and these amounts were less than one-half the amount observed with the type 2 parent strain D39. Expression of the type 3 capsule in D39 also reduced the amount of C3b deposited and its accessibility to antibody, resulting in a level intermediate between the levels observed with WU2 and D39. Despite these effects, the capsule type was not the determining factor in anti-PspA-mediated protection, as both D39 and its derivative expressing the type 3 capsule were more resistant to protection than WU2. The specific combination of PspA and capsule type also did not determine the level of protection. The capsule structure is thus a major determinant in accessibility of surface antigens to antibody, but certain strains appear to express other factors that can influence antibody-mediated protection.

scholarly journals Conjugation of Polysaccharide 6B from Streptococcus pneumoniae with Pneumococcal Surface Protein A: PspA Conformation and Its Effect on the Immune Response

2013 ◽  
Vol 20 (6) ◽  
pp. 858-866 ◽  
Author(s):  
Catia T. Perciani ◽  
Giovana C. Barazzone ◽  
Cibelly Goulart ◽  
Eneas Carvalho ◽  
Joaquin Cabrera-Crespo ◽  
...  
Keyword(s):  
Immune Response ◽  
Streptococcus Pneumoniae ◽  
Conjugate Vaccine ◽  
Reductive Amination ◽  
Capsular Polysaccharide ◽  
Protective Antigen ◽  
Protein A ◽  
Surface Protein ◽  
Promising Alternative ◽  
Pneumococcal Surface Protein A

ABSTRACTDespite the substantial beneficial effects of incorporating the 7-valent pneumococcal conjugate vaccine (PCV7) into immunization programs, serotype replacement has been observed after its widespread use. As there are many serotypes currently documented, the use of a conjugate vaccine relying on protective pneumococcal proteins as active carriers is a promising alternative to expand PCV coverage. In this study, capsular polysaccharide serotype 6B (PS6B) and recombinant pneumococcal surface protein A (rPspA), a well-known protective antigen fromStreptococcus pneumoniae, were covalently attached by two conjugation methods. The conjugation methodology developed by our laboratory, employing 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) as an activating agent through carboxamide formation, was compared with reductive amination, a classical methodology. DMT-MM-mediated conjugation was shown to be more efficient in coupling PS6B to rPspA clade 1 (rPspA1): 55.0% of PS6B was in the conjugate fraction, whereas 24% was observed in the conjugate fraction with reductive amination. The influence of the conjugation process on the rPspA1 structure was assessed by circular dichroism. According to our results, both conjugation processes reduced the alpha-helical content of rPspA; reduction was more pronounced when the reaction between the polysaccharide capsule and rPspA1 was promoted between the carboxyl groups than the amine groups (46% and 13%, respectively). Regarding the immune response, both conjugates induced functional anti-rPspA1 and anti-PS6B antibodies. These results suggest that the secondary structure of PspA1, as well as its reactive groups (amine or carboxyl) involved in the linkage to PS6B, may not play an important role in eliciting a protective immune response to the antigens.

Protection induced by pneumococcal surface protein A (PspA) is enhanced by conjugation to a Streptococcus pneumoniae capsular polysaccharide

Vaccine ◽  
2008 ◽  
Vol 26 (23) ◽  
pp. 2925-2929 ◽  
Author(s):  
Fátima C.L. Csordas ◽  
Cátia T. Perciani ◽  
Michelle Darrieux ◽  
Viviane M. Gonçalves ◽  
Joaquim Cabrera-Crespo ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Capsular Polysaccharide ◽  
Protein A ◽  
Surface Protein ◽  
Pneumococcal Surface Protein A

scholarly journals Relationship between Surface Accessibility for PpmA, PsaA, and PspA and Antibody-Mediated Immunity to Systemic Infection by Streptococcus pneumoniae

2005 ◽  
Vol 73 (3) ◽  
pp. 1304-1312 ◽  
Author(s):  
Dennis O. Gor ◽  
Xuedong Ding ◽  
David E. Briles ◽  
Michael R. Jacobs ◽  
Neil S. Greenspan
Keyword(s):  
Streptococcus Pneumoniae ◽  
Capsular Polysaccharide ◽  
Protein A ◽  
Age Groups ◽  
Pneumococcal Infection ◽  
Systemic Infection ◽  
Surface Protein ◽  
Target Antigen ◽  
Age Related ◽  
Type 3

ABSTRACT Antibodies to capsular polysaccharide (PS) are protective against systemic infection by Streptococcus pneumoniae, but the large number of pneumococcal serogroups and the age-related immunogenicity of pure PS limit the utility of PS-based vaccines. In contrast, cell wall-associated proteins from different capsular serotypes can be cross-reactive and immunogenic in all age groups. Therefore, we evaluated three pneumococcal proteins with respect to relative accessibility to antibody, in the context of intact pneumococci, and their ability to elicit protection against systemic infection by encapsulated S. pneumoniae. Sequences encoding pneumococcal surface adhesin A (PsaA), putative protease maturation protein A (PpmA), and the N-terminal region of pneumococcal surface protein A (PspA) from S. pneumoniae strain A66.1 were cloned and expressed in Escherichia coli. The presence of genes encoding PsaA, PpmA, and PspA in 11 clinical isolates was examined by PCR, and the expression of these proteins by each strain was examined by Western blotting with antisera raised to the respective recombinant proteins. We used flow cytometry to demonstrate that PspA was readily detectable on the surface of the pneumococcal strains analyzed, whereas PsaA and PpmA were not. Consistent with these observations, mice with passively or actively acquired antibodies to PspA or type 3 PS were equivalently protected from homologous systemic challenge with type 3 pneumococci, whereas mice with passively or actively acquired antibodies to PsaA or PpmA were not effectively protected. These experiments support the hypothesis that the extent of protection against systemic pneumococcal infection is influenced by target antigen accessibility to circulating host antibodies.

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

1998 ◽  
Vol 11 (4) ◽  
pp. 645-657 ◽  
Author(s):  
David E. Briles ◽  
Rebecca Creech Tart ◽  
Edwin Swiatlo ◽  
Joseph P. Dillard ◽  
Patricia Smith ◽  
...  
Keyword(s):  
Capsular Polysaccharide ◽  
Protein A ◽  
Pneumococcal Infection ◽  
Significant Risk ◽  
Surface Protein ◽  
Infectious Agent ◽  
The Elderly ◽  
Multidrug Resistant ◽  
Pneumococcal Surface Protein A ◽  
Potential Vaccine

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

scholarly journals Effects of PspA and Antibodies to PspA on Activation and Deposition of Complement on the Pneumococcal Surface

2004 ◽  
Vol 72 (1) ◽  
pp. 114-122 ◽  
Author(s):  
Bing Ren ◽  
Alexander J. Szalai ◽  
Susan K. Hollingshead ◽  
David E. Briles
Keyword(s):  
Complement Activation ◽  
Capsular Polysaccharide ◽  
Protein A ◽  
Surface Protein ◽  
Complement C3 ◽  
Mouse Serum ◽  
Classical Pathway ◽  
Important Virulence Factor ◽  
Almost All ◽  
Type 3

ABSTRACT Streptococcus pneumoniae infection is a frequent cause of pneumonia, otitis media, meningitis, and septicemia. Pneumococcal surface protein A (PspA) is an important virulence factor on the pathogen surface, and it is known to interfere with complement activation. In this study, flow cytometry was used to study the effects of PspA and antibodies to PspA on the deposition of complement C3 on the surface of a capsular type 3 strain, WU2, and its PspA− mutant, JY1119. Using naive mouse serum as a complement source, measurable deposition of C3 was observed within 4 min on PspA− pneumococci, and the amount of surface-bound C3 accumulated rapidly as the amount of serum was increased. In contrast, very little C3 was deposited on the PspA+ strain. In nonimmune mouse serum, the classical pathway was the dominant activation pathway triggered by PspA− pneumococci. Accordingly, EGTA blocked almost all of the complement activation. Moreover, a significant amount of C3 was still deposited on the PspA− strain when serum from factor B-deficient mice was used. This deposition was not observed on the PspA+ pneumococci, indicating that PspA may inhibit complement deposition via the classical pathway. Furthermore, under the conditions we tested, PspA also inhibited C3 deposition when the classical pathway was initiated by antibodies to capsular polysaccharide. Antibodies to PspA could overcome the anticomplementary effect of PspA, allowing for increased complement activation and C3 deposition onto PspA+ bacteria.

scholarly journals Protective humoral response against pneumococcal infection in mice elicited by recombinant bacille Calmette-Guérin vaccines expressing pneumococcal surface protein A.

1994 ◽  
Vol 180 (6) ◽  
pp. 2277-2286 ◽  
Author(s):  
S Langermann ◽  
S R Palaszynski ◽  
J E Burlein ◽  
S Koenig ◽  
M S Hanson ◽  
...  
Keyword(s):  
Capsular Polysaccharide ◽  
Gene Segment ◽  
Protein A ◽  
Pneumococcal Infection ◽  
Surface Protein ◽  
Cross Protection ◽  
Secreted Protein ◽  
Bacille Calmette Guerin ◽  
Immunodeficient Mice ◽  
Pneumococcal Surface Protein A

Pneumococcal surface protein A (PspA), a cell-surface protein present on all strains of pneumococci, has been shown to elicit protective antibody responses in mice in the absence of capsular polysaccharide. Whereas PspA is polymorphic, considerable cross-reactivity and cross-protection have been demonstrated among PspA proteins of pneumococci exhibiting different capsular and PspA serotypes. A gene segment encoding the nonrepetitive variable NH2-terminal portion of PspA has been cloned into three distinct recombinant Bacille Calmette-Guérin (rBCG) vectors, allowing for expression of PspA as a cytoplasmic or secreted protein, or a chimeric exported membrane-associated lipoprotein. All rBCG-PspA strains elicited comparable anti-PspA ELISA titers, ranging from 10(4) to 10(5) (reciprocal titers) in both BALB/c and C3H/HeJ mice. However, protective responses were observed only in animals immunized with the rBCG-PspA vaccines expressing PspA as a secreted protein or chimeric exported lipoprotein. In addition, anti-PspA immune sera elicited by the rBCG vaccines passively protected X-linked immunodeficient mice from lethal challenge with the highly virulent, encapsulated WU2 strain of Streptococcus pneumoniae and two additional virulent strains exhibiting heterologous PspA and capsular serotypes. These studies confirm previous PspA immunization studies showing cross-protection against heterologous serotypes of S. pneumoniae and demonstrate a potential for rBCG-based PspA vaccines to elicit protective humoral responses against pneumococcal disease in humans.

Sign in / Sign up

Export Citation Format

Share Document