Immunization of Mice with Single PspA Fragments Induces Antibodies Capable of Mediating Complement Deposition on Different Pneumococcal Strains and Cross-Protection

Abstract

PspA is an important candidate for a vaccine with serotype-independent immunity against pneumococcal infections. Based on sequence relatedness, PspA has been classified into three families comprising six clades. We have previously addressed the cross-reactivity of antibodies against PspA fragments containing the N-terminal and proline-rich regions of PspA from clades 1 to 5 (PspA1, PspA2, PspA3, PspA4, and PspA5) by Western blot analysis and reported that anti-PspA4 and anti-PspA5 were able to recognize pneumococci expressing PspA proteins from all of the clades analyzed. We have now analyzed the functional capacity of these antibodies to bind and to mediate complement deposition on intact bacteria in vitro. Our results show that both PspA4 and PspA5 elicit antibodies that are able to bind and to mediate complement deposition efficiently on pneumococcal strains bearing PspA proteins from clades 1 to 5. Moreover, mice immunized with PspA4 and PspA5 were protected against an intranasal lethal challenge with strains expressing PspA proteins from the two major families. PspA4 and PspA5 are thus able to induce antibodies with a high degree of cross-reactivity in vitro, which is reflected in cross-protection of mice. We have also analyzed the contribution of the nonproline (NonPro) block within the conserved proline-rich region to the reactivity of anti-PspA antibodies, and the results indicate that N-terminal ␣-helical region, the blocks of proline repeats, and the NonPro region can influence the degree of cross-reactivity of antibodies to PspA.

Pulmonary dry powder vaccine of pneumococcal antigen loaded nanoparticles

“…The N-terminal α-helix and the first block of the proline rich region of PspA from clade 4 (PspA4Pro), fragment with cross reactivity with PspA from other clades (Moreno et al, 2010), was cloned into pET37b + and expressed in E. coli BL21 (DE3). The protein was produced and purified using previously published methods with slight modifications Horta et al, 2012;Horta et al, 2014).…”

“…Among the different types of pneumococcal proteins such as pneumococcal surface protein A (PspA), pneumococcal surface protein C (PspC), pneumolysin; PspA is one of the most promising candidates and has been widely investigated by several groups (Briles et al, 2000a;Briles et al, 2000b;Haughney et al, 2013;Moreno et al, 2010;Ogunniyi et al, 2007;Ogunniyi et al, 2001;Vadesilho et al, 2012). The main function of PspA is preventing the deposition of complement on the surface of the bacterium thereby inhibiting opsonization and phagocytosis (Ren et al, 2004a;Ren et al, 2004b).…”

Evaluation of a Vaccine Formulation against Streptococcus pneumoniae Based on Choline-Binding Proteins

“…Cellular protein extracts from bacteria were analyzed for PspA production through Western blotting using anti-recombinant PspA4 (anti-rPspA4; a recombinant protein that includes the mature N-terminal ␣-helical region plus the proline-rich region of a PspA protein from clade 4) mouse serum (30). The membranes were incubated with anti-mouse IgG conjugated with peroxidase (Sigma-Aldrich, St. Louis, MO), and detection was performed with ECL Prime Western blotting reagent (GE Healthcare, United Kingdom).…”

“…Three weeks after the last immunization, mice were individually bled from the retroorbital plexus. Enzyme-linked immunosorbent assay (ELISA) was carried out as described previously in plates coated with 1 g/ml rPspA1 or rPspA4 (recombinant proteins that include the mature N-terminal ␣-helical region plus the proline-rich region of a PspA protein from clade 1 or 4, respectively) (30). Goat anti-mouse IgG conjugated with horseradish peroxidase was used as secondary antibody, and the titer was defined as the highest dilution with an A 492 of Ն0.1.…”

“…The purpose of these studies is to confirm some of the earlier work and to do the studies on a somewhat larger scale to determine whether it is possible and desirable to attempt to use one of these approaches to produce a pneumococcal vaccine. (30).…”