The modified surface killing assay distinguishes between protective and nonprotective antibodies to PspA

Pneumococcal surface protein A (PspA) elicits antibody protective against lethal challenge by Streptococcus pneumoniae and is a candidate noncapsular antigen for inclusion in vaccines. Evaluation of immunity to PspA in human trials would be greatly facilitated by an in vitro functional assay able to distinguish protective from nonprotective antibodies to PspA. Mouse monoclonal antibodies (MAbs) to PspA can mediate killing by human granulocytes in the modified surface killing assay (MSKA). To determine if the MSKA can distinguish between protective and nonprotective MAbs, we examined seven MAbs to PspA. All bound recombinant PspA, as detected by enzyme-linked immunosorbent assay and Western blotting; four gave strong passive protection against fatal challenge, two were nonprotective, and the seventh one only delayed death. The four that were able to provide strong passive protection were also most able to enhance killing in the MSKA, the two that were not protective in mice were not effective in the MSKA, and the MAb that was only weakly protective in mice was weakly effective in the MSKA (P < 0.001). One of the four most protective MAbs tested reacted to the proline-rich domain of PspA. Two of the other most protective MAbs and the weakly protective MAb reacted with a fragment from PspA’s a-helical domain (aHD), containing amino acids (aa) 148 to 247 from the N terminus of PspA. The fourth highly protective MAb recognized none of the overlapping 81- or 100-aa fragments of PspA. The two nonprotective MAbs recognized a more N-terminal aHD fragment (aa 48 to 147). IMPORTANCE The most important finding of this study is that the MSKA can be used as an in vitro functional assay. Such an assay will be critical for the development of PspA-containing vaccines. The other important findings relate to the locations and nature of the protection-eliciting epitopes of PspA. There are limited prior data on the locations of protection-eliciting PspA epitopes, but those data along with the data presented here make it clear that there is not a single epitope or domain of PspA that can elicit protective antibody and there exists at least one region of the aHD which seldom elicits protective antibody. Moreover, these data, in concert with prior data, strongly make the case that protective epitopes in the aHD are highly conformational (=100-amino-acid fragments of the aHD are required), whereas at least some protection-eliciting epitopes in the proline-rich domain are encoded by =15-amino-acid sequences.

Current status and perspectives on protein-based pneumococcal vaccines.

Despite the efforts to expand the availability of conjugate vaccines, pneumococcal diseases still pose an enormous burden worldwide. Therefore, several proteins have been investigated as alternative vaccines, alone or in combination with other antigens. With an increasing array of techniques, many of which arose from the publication of the bacterial genome, several proteins have been identified as potential vaccine candidates, and some have even progressed to clinical trials. Also, whole cell vaccines are being studied for the induction of broad ranging protective responses. Here, we briefly summarize the current knowledge on pneumococcal proteins that are being investigated as potential vaccine candidates against pneumococcal infections, and provide an insight on the future generation of protein-based vaccines against Streptococcus pneumoniae.

Evaluation of a vaccine formulation against Streptococcus pneumoniae based on choline-binding proteins.

Streptococcus pneumoniae has proteins that are attached to its surface by binding to phosphorylcholine of teichoic and lipoteichoic acids. These proteins are known as choline-binding proteins (CBPs). CBPs are an interesting alternative for the development of a cost-effective vaccine, and PspA (pneumococcal surface protein A) is believed to be the most important protective component among the different CBPs. We sought to use CBPs eluted from pneumococci as an experimental vaccine. Since PspA shows variability between isolates, we constructed strains producing different PspAs. We used the nonencapsulated Rx1 strain, which produces PspA from clade 2 (PspA2), to generate a pspA-knockout strain (Rx1 ΔpspA) and strains expressing PspA from clade 1 (Rx1 pspA1) and clade 4 (Rx1 pspA4). We grew Rx1, Rx1 ΔpspA, Rx1 pspA1, and Rx1 pspA4 in Todd-Hewitt medium containing 0.5% yeast extract and washed cells in 2% choline chloride (CC). SDS-PAGE analysis of the proteins recovered by a CC wash showed few bands, and the CBPs PspA and PspC (pneumococcal surface protein C) were identified by mass spectrometry analysis. Subcutaneous immunization of mice with these full-length native proteins without adjuvant led to significantly higher rates of survival than immunization with diluent after an intranasal lethal challenge with two pneumococcal strains and also after a colonization challenge with one strain. Importantly, immunization with recombinant PspA4 (rPspA4) without adjuvant did not elicit significant protection.

Mapping of epitopes recognized by antibodies induced by immunization of mice with PspA and PspC.

Pneumococcal surface protein A (PspA) and pneumococcal surface protein C (PspC) are important candidates for an alternative vaccine against pneumococcal infections. Since these antigens show variability, the use of variants that do not afford broad protection may lead to the selection of vaccine escape bacteria. Epitopes capable of inducing antibodies with broad cross-reactivities should thus be the preferred antigens. In this work, experiments using peptide arrays show that most linear epitopes recognized by antibodies induced in mice against different PspAs were located at the initial 44 amino acids of the mature protein and that antibodies against these linear epitopes did not confer protection against a lethal challenge. Conversely, linear epitopes recognized by antibodies to PspC included the consensus sequences involved in the interaction with human factor H and secretory immunoglobulin A (sIgA). Since linear epitopes of PspA were not protective, larger overlapping fragments containing 100 amino acids of PspA of strain Rx1 were constructed (fragments 1 to 7, numbered from the N terminus) to permit the mapping of antibodies with conformational epitopes not represented in the peptide arrays. Antibodies from mice immunized with fragments 1, 2, 4, and 5 were capable of binding onto the surface of pneumococci and mediating protection against a lethal challenge. The fact that immunization of mice with 100-amino-acid fragments located at the more conserved N-terminal region of PspA (fragments 1 and 2) induced protection against a pneumococcal challenge indicates that the induction of antibodies against conformational epitopes present at this region may be important in strategies for inducing broad protection against pneumococci.

Protection against nasal colonization with Streptococcus pneumoniae by parenteral immunization with a DNA vaccine encoding PspA (Pneumococcal surface protein A).

Pneumococcal surface protein A (PspA) is an important candidate for a cost-effective vaccine with broad coverage against Streptococcus pneumoniae. We have previously shown that intramuscular immunization with PspA as a DNA vaccine induces an immune response characterized by the induction of a balanced IgG1/IgG2a antibody response in BALB/c mice, which was able to efficiently mediate complement deposition onto intact bacteria and to induce protection against an intraperitoneal challenge. We now confirm the results in C57BL/6 mice and further show that the response induced by the DNA vaccine expressing PspA is able to mediate protection against colonization of the nasopharyngeal mucosa even though immunization was given parenterally. Moreover, a positive correlation was observed between IgG1 and the numbers of CFU recovered, whereas an inverse correlation was observed between nasal CFU levels and IgG2a. A positive correlation was also found for IgG1/IgG2a antibody ratios with CFU recovered from the nasopharynx. Therefore, reduction of nasal colonization was strongly associated with increased levels of serum IgG2a complement fixing antibody and low levels of IgG1 antibody which has much less complement fixing activity. Passive transfer of serum from animals immunized with the DNA vaccine expressing PspA was also able to reduce the fraction of mice with high density of colonization of the nasopharynx. Secretion of IFN-gamma, but not IL-17, was observed in splenocytes from mice immunized with the DNA vaccine.