Broadly protective Shigella vaccine based on type III secretion apparatus proteins.

Shigella spp. are food- and waterborne pathogens that cause severe diarrheal and dysenteric disease associated with high morbidity and mortality. Individuals most often affected are children under 5 years of age in the developing world. The existence of multiple Shigella serotypes and the heterogenic distribution of pathogenic strains, as well as emerging antibiotic resistance, require the development of a broadly protective vaccine. All Shigella spp. utilize a type III secretion system (TTSS) to initiate infection. The type III secretion apparatus (TTSA) is the molecular needle and syringe that form the energized conduit between the bacterial cytoplasm and the host cell to transport effector proteins that manipulate cellular processes to benefit the pathogen. IpaB and IpaD form a tip complex atop the TTSA needle and are required for pathogenesis. Because they are common to all virulent Shigella spp., they are ideal candidate antigens for a subunit-based, broad-spectrum vaccine. We examined the immunogenicity and protective efficacy of IpaB and IpaD, alone or combined, coadministered with a double mutant heat-labile toxin (dmLT) from Escherichia coli, used as a mucosal adjuvant, in a mouse model of intranasal immunization and pulmonary challenge. Robust systemic and mucosal antibody- and T cell-mediated immunities were induced against both proteins, particularly IpaB. Mice immunized in the presence of dmLT with IpaB alone or IpaB combined with IpaD were fully protected against lethal pulmonary infection with Shigella flexneri and Shigella sonnei. We provide the first demonstration that the Shigella TTSAs IpaB and IpaD are promising antigens for the development of a cross-protective Shigella vaccine.

Formulation design and high-throughput excipient selection based on structural integrity and conformational stability of dilute and highly concentrated IgG1 monoclonal antibody solutions.

A systematic approach is presented to characterize and stabilize the higher order structural integrity of an immunoglobulin G (IgG1) monoclonal antibody (mAb) formulated at both low concentrations and as a highly concentrated solution. The conformational and colloidal stabilities of a recombinant humanized IgG1κ mAb at both 1 and 100 mg/mL were investigated as a function of solution temperature (10°C-87.5°C) and pH (3-8). Protein secondary structure was characterized using circular dichroism, whereas intrinsic (tryptophan) and extrinsic (8-anilino-1-naphthalenesulfonic acid) fluorescence spectroscopy measurements were used to evaluate the tertiary structure of the protein. Light scattering analysis was employed to monitor mAb aggregation behavior as a function of temperature and solution pH. These biophysical data sets were analyzed and summarized using a previously described empirical phase diagrams (EPDs) approach. The different phases observed in the EPD were correlated with the individual physical states of the IgG1 in solution (aggregated, native, unfolded, etc.). The temperature-dependent conformational stability profile of the mAb, at both 1 and 100 mg/mL, generally followed the order pH 6 ≥ pH 7 ≥ pH 8 > pH 5 > pH 4 ≥ pH 3. Analysis of the EPD apparent phase boundaries identified solution conditions of pH 4.5 near 60°C for the development of an excipient screening assay. A supplemented generally regarded as safe excipient library was screened using an aggregation assay (optical density at 350 nm) at low mAb concentrations (4 mg/mL) and potential stabilizers were identified. The ability of these excipients to prevent conformational alterations in high concentration mAb solutions (100 mg/mL) was determined by monitoring tertiary structure changes using an intrinsic fluorescence method. The results suggest that substantial increases in the onset temperature of thermal transitions (>5°C) are obtained in the presence of (a) 20% dextrose, (b) 20% sorbitol, and (c) 5% dextrose + 10% sorbitol. Similar stabilization effects were obtained at an intermediate (50 mg/mL) as well as low mAb concentrations (1 mg/mL).

H1N1 influenza virus-like particles: physical degradation pathways and identification of stabilizers.

A simple and rapid approach to vaccine stabilization has been applied to a novel virus-like particle (VLP) that contains the primary influenza antigens (hemagglutinin and neuraminidase surface proteins). A complement of spectroscopic and light scattering techniques was used to characterize the physical stability of influenza VLPs as a function of temperature and pH, two pharmaceutically relevant stress factors. The resulting data set was mathematically converted into a three-color empirical phase diagram (EPD) that illustrates changes in physical state as a function of these stress factors. Conditions of temperature and pH corresponding to apparent phase boundaries in the EPD were then used to screen for inhibitors of VLP aggregation from a library of generally recognized as safe compounds. Several potent inhibitors of VLP aggregation were identified; of these, trehalose, sorbitol, and glycine were all found to exert significant stabilizing effects on viral protein tertiary structure and/or membrane integrity.

Stabilization of measles virus for vaccine formulation.

An attenuated live measles virus (MV) was characterized by several biophysical methods as a function of temperature and pH. Following a method developed previously, the resultant light scattering and spectroscopic data were synthesized into an empirical phase diagram that visually and simultaneously represents the entire data set. Using this empirically-based phase diagram, screening assays were developed to identify potential vaccine stabilizers. Various compounds are shown by these assays to inhibit the temperature-induced aggregation of viral particles, and also to protect the integrity of the viral envelope. Accelerated stability assays show that, upon thermal challenge, MV formulated with these excipients retains its infectivity to a significant extent. Thus, the enhanced physical stability produced by this method is shown to protect the biological activity of this important but labile vaccine.

Physical stabilization of Norwalk virus-like particles.

Virus-like particles (VLPs) used as vaccine antigens often elicit strong immune responses due to their intrinsic repetitive, high-density display of epitopes, and the fact that the mammalian immune system is highly attuned to recognizing particles in the size range of viruses (20-150 nm). To retain these immunogenic qualities, vaccines that utilize virus-like particle (VLP) antigens should be formulated to stabilize both native conformational epitopes and the overall particulate nature of the VLP. This work describes a systematic approach for identifying potential stabilizers for formulation of Norwalk VLPs (NV-VLPs) in aqueous suspension. A number of excipients were screened for their ability to inhibit aggregation of NV-VLPs under conditions known to induce aggregation. Those compounds shown to inhibit aggregation were further evaluated under conditions of thermal stress and the NV-VLP structure was monitored using biophysical techniques such as CD, ANS fluorescence, and DSC to provide insight into the mechanisms by which stability was conferred. Increased thermal stability in the presence of chitosan glutamate, sucrose, and trehalose was correlated with stabilization of secondary and tertiary structural elements of NV-VLPs. These excipients may be useful for formulation of a stable NV-VLP vaccine.