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ABSTRACTStreptococcus pneumoniaepneumolysin (PLY) is a virulence factor that causes toxic effects contributing to pneumococcal pneumonia. To date, deriving a PLY candidate vaccine with the appropriate detoxification and immune profile has been challenging. A pneumolysin protein that is appropriately detoxified and that retains its immunogenicity is a desirable vaccine candidate. In this study, we assessed the protective efficacy of our novel PlyD1 detoxified PLY variant and investigated its underlying mechanism of protection. Results have shown that PlyD1 immunization protected mice against lethal intranasal (i.n.) challenge with pneumococci and lung injury mediated by PLY challenge. Protection was associated with PlyD1-specific IgG titers andin vitroneutralization titers. Pretreatment of PLY with PlyD1-specific rat polyclonal antiserum prior to i.n. delivery of toxin reduced PLY-mediated lung lesions, interleukin-6 (IL-6) production, and neutrophil infiltration into lungs, indicating that protection from lung lesions induced by PLY is antibody mediated. Preincubation of PLY with a neutralizing monoclonal PLY antibody also specifically reduced the cytotoxic effects of PLY after i.n. inoculation in comparison to nonneutralizing monoclonal antibodies. These results indicate that the induction of neutralizing antibodies against PLY can contribute to protection against bacterial pneumonia by preventing the development of PLY-induced lung lesions and inflammation. Our detoxified PlyD1 antigen elicits such PLY neutralizing antibodies, thus serving as a candidate vaccine antigen for the prevention of pneumococcal pneumonia.

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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.

Currently marketed Streptococcus pneumoniae vaccines are based on polysaccharide capsular antigens from the most common strains. Pneumococcal histidine triad protein D (PhtD) is a conserved surface protein that is being evaluated as a candidate for a vaccine with improved serotype coverage. Here, we measured the functional activity of human anti-PhtD antibodies in a passive protection model wherein mice were challenged with a lethal dose of S. pneumoniae by intravenous injection. This functional activity was compared with anti-PhtD antibody concentrations measured by enzyme-linked immunosorbent assay (ELISA) to estimate the 50% protective dose (ED50). Anti-PhtD antibodies affinity purified from pooled normal human sera passively protected mice with an ED50 of 1679 ELISA units/ml (95% confidence interval, 1420–1946). Sera from subjects injected with aluminum-adjuvanted PhtD in a phase I trial had similar activity per unit of antibody (ED50 = 1331 ELISA units/ml [95% confidence interval, 762–2038]). Vaccine-induced activity in the passive protection model was blocked by pre-incubation with recombinant PhtD but not by a control S. pneumoniae antigen (LytB). These results show that human anti-PhtD antibodies, whether naturally acquired or induced by the PhtD candidate vaccine, are functional. This supports the development of the PhtD candidate as part of a broadly protective pneumococcal vaccine.

Vaccines based on conserved pneumococcal proteins are being investigated because serotype coverage by pneumococcal polysaccharide and polysaccharide conjugate vaccines is incomplete and may eventually decrease due to serotype replacement. Here, we examined the functionality of human antibodies induced by a candidate bivalent choline-binding protein A- pneumococcal histidine triad protein D (PcpA-PhtD) vaccine. Pre- and post-immune sera from subjects who had been vaccinated with the PcpA-PhtD candidate vaccine were tested in an established passive protection model in which mice were challenged by intravenous injection with Streptococcus pneumoniae serotype 3 strain A66.1. Serum antibody concentrations were determined by enzyme-linked immunosorbent assay (ELISA). Bacterial surface binding by serum antibodies was determined by a flow cytometry-based assay. Sera from 20 subjects were selected based on low activity of pre-immune samples in the passive protection model. Bacterial surface binding correlated more strongly with anti-PcpA (0.87; p < 0.0001) than with anti-PhtD (0.71; p < 0.0001). The odds ratio for predicting survival in the passive protection assay was higher for the anti-PcpA concentration (470 [95% confidence interval (CI), 46.8 to >999.9]) than for the anti-PhtD concentration (3.4 [95% CI, 1.9 to 5.6]) or bacterial surface binding (9.4 [95% CI, 3.6 to 24.3]). Pooled post-immune serum also protected mice against a challenge with S. pneumoniae serotype 3 strain WU2. Both anti-PcpA and anti-PhtD antibodies induced by the bivalent candidate vaccine mediate protection against S. pneumoniae. The results also showed that the ELISA titer might be useful as a surrogate for estimating the functional activity of antibodies induced by pneumococcal protein vaccines.

The pellet-associated portion of human brain alpha-L-fucosidase (which represents approx. 20% of the homogenate activity) was solubilized with 0.5% (w/v) Triton X-100, characterized with regard to several properties and compared with the corresponding properties of the soluble supernatant-fluid enzyme in an attempt to find a second alpha-L-fucosidase in human brain. The solubilized and soluble alpha-L-fucosidase activities exhibited complete stability after storage at 2-4 degrees C for up to 29 days, comparable thermostability after preincubation at 50 degrees C, comparable apparent Km values (0.07-0.08 mM) for 4-methylumbelliferyl alpha-L-fucopyranoside, comparable hydrophobicity, comparable isoelectric-focusing profiles (six major forms, with pI values between 4.5 and 5.8) and comparable immunoprecipitation curves (with the IgG fraction of antisera prepared against human liver alpha-L-fucosidase). Differences in three properties were found between solubilized and soluble alpha-L-fucosidase activities: the solubilized activity was less stable to storage at -20 degrees C, had a 0.5-pH-unit neutral shift in its pH optimum (6.0) and had smaller Mr forms after gel filtration on Sephadex G-200. The overall results indicate that the pellet-associated and soluble portions of human brain alpha-L-fucosidase are quite similar in most of their properties. Thus there is still no compelling evidence for the existence of a second mammalian alpha-L-fucosidase.