Evaluation of a 74-kDa transferrin-binding protein from Moraxella (Branhamella) catarrhalis as a vaccine candidate.

An outer membrane protein from Moraxella catarrhalis with a mass of 74-kDa was isolated and evaluated as a vaccine candidate. The 74-kDa protein binds transferrin, and appears to be related to the other proteins from the organism that are reported to bind transferrin. The 74-kDa protein possessed conserved epitopes exposed on the bacterial surface. This is based on the reactivity with whole bacterial cells as well as complement dependent bactericidal activity of sera from mice immunized with the isolated proteins from the O35E and TTA24 isolates. However, there was divergence in the degree of antibody cross-reactivity with the protein from one strain to another. This serotypic divergence was reflected in both the complement-dependent bactericidal activities of the antibodies elicited in mice and the capacity of immune mice to clear the bacteria in a murine pulmonary model. Antibodies affinity purified from human plasma lacked bactericidal activity even though they were reactive with all the tested isolates. The 74-kDa protein appears to be a good vaccine candidate, but more studies are needed to understand its antigenic variability and whether antibodies toward it are protective.

Isolation and characterization of two proteins from Moraxella catarrhalis that bear a common epitope.

The UspA1 and UspA2 proteins of Moraxella catarrhalis are potential vaccine candidates for preventing disease caused by this organism. We have characterized both proteins and evaluated their vaccine potential using both in vitro and in vivo assays. Both proteins were purified from the O35E isolate by Triton X-100 extraction, followed by ion-exchange and hydroxyapatite chromatography. Analysis of the sequences of internal peptides, prepared by enzymatic and chemical cleavage of the proteins, revealed that UspA1 and UspA2 exhibited distinct structural differences but shared a common sequence including an epitope recognized by the monoclonal antibody 17C7. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), purified UspA1 exhibited a molecular weight of approximately 350, 000 when unheated and a molecular weight of 100,000 after being heated for 10 min at 100 degreesC. In contrast, purified UspA2 exhibited an apparent molecular weight of 240,000 by SDS-PAGE that did not change with the length of time of heating. Their sizes as determined by gel filtration were 1,150,000 and 830,000 for UspA1 and UspA2, respectively. Preliminary results indicate the proteins have separate functions in bacterial pathogenesis. Purified UspA1 was found to bind HEp-2 cells, and sera against UspA1, but not against UspA2, blocked binding of the O35E isolate to the HEp-2 cells. UspA1 also bound fibronectin and appears to have a role in bacterial attachment. Purified UspA2, however, did not bind fibronectin but had an affinity for vitronectin. Both proteins elicited bactericidal antibodies in mice to homologous and heterologous disease isolates. Finally, mice immunized with each of the proteins, followed by pulmonary challenge with either the homologous or a heterologous isolate, cleared the bacteria more rapidly than mock-immunized mice. These results suggest that UspA1 and UspA2 serve different virulence functions and that both are promising vaccine candidates.

Purification of an antigenic vaccine protein by selective displacement chromatography.

A recent advance in the state of the art of displacement chromatography has been the development of selective displacement chromatography. In this process, the bioproduct of interest is selectively displaced while impurities with lower retention are eluted in the induced salt gradient and higher retained impurities are desorbed after the breakthrough of the displacer front. In this manuscript, selective displacement chromatography is employed to purify an antigenic vaccine protein (AVP) from an industrial process stream. Displacers were screened and an operating regime plot was employed to establish appropriate conditions for selective displacement. The selective displacement process was successful and resulted in AVP that was equivalent in purity to product obtained at commercial production scale after conventional step gradient chromatography. Methods used to characterize the purified protein include size-exclusion chromatography, SDS-PAGE, isoelectric focusing, N-terminal amino acid sequence analysis, and amino acid composition analysis. This is the first report of the purification of a commercially and pharmaceutically significant protein using selective displacement chromatography and thereby sets the stage for the implementation of selective displacement chromatography for the downstream processing of biologicals.

Generation of atypical pulmonary inflammatory responses in BALB/c mice after immunization with the native attachment (G) glycoprotein of respiratory syncytial virus.

The feasibility of using the highly purified native attachment (G) protein in a subunit vaccine against respiratory syncytial virus (RSV) was examined in a murine model with or without the fusion (F) protein of RSV and the adjuvant QS-21. The studies established that QS-21 was more potent than AIOH as an adjuvant for both F and G glycoproteins. Augmented antigen-dependent killer cell activity and complement-assisted serum neutralizing and anti-F and G protein immunoglobulin G2a antibody titers were observed. Immunization with G/QS-21 generated immune responses that were characterized by low levels of antigen-dependent killer cell activity, elevated levels of interleukin-5 (IL-5) and percentages of eosinophils in the bronchoalveolar lavage fluids after challenge, and splenic immunocytes that secreted IL-5 but not gamma interferon (IFN-gamma) after in vitro stimulation with purified whole virus antigens. The pulmonary eosinophilia was similar to that induced by a facsimile of a formalin-inactivated vaccine used in previous clinical trials and was prevented by prior in vivo treatment with anti-IL-5 but not with control immunoglobulin G or anti-IFN-gamma neutralizing monoclonal antibodies. Thus the data implied that vaccination with G/QS-21 generated helper T-cell immune responses that were type 2 in nature. Alternatively, the data suggested that the helper T-cell immune responses elicited by F/QS-21 were more type 1 in character. Neither eosinophilia nor elevated levels of IL-5 were observed in the lungs of mice after challenge. Noteworthy levels of antigen-dependent killer cell activity was observed, and splenic immunocytes secreted copious quantities of IFN-gamma. Immunization with a combination vaccine composed of highly purified native F and G proteins plus QS-21 (F+G/QS-21) resulted in augmented complement-assisted serum neutralizing antibody titers compared with vaccination with either F/QS-21 or G/QS-21 alone. However, following vaccination with F+G/QS-21, the bronchoalveolar lavage fluids contained significant increases in IL-5 and percentages of eosinophils after challenge, the spleen cells appeared to secrete less IFN-gamma after in vitro stimulation, and there was no evidence of increased numbers of antigen-dependent killer cell precursors. Taken together, the data imply that native G protein influences the nature of the immune responses elicited by F/QS-21. The results therefore suggest that G, not F, protein has more potential to bias the host for atypical pulmonary inflammatory responses.