Physical Characterization and Stabilization of a Lentiviral Vector Against Adsorption and Freeze-Thaw.

A replication-deficient lentiviral vector encoding the tumor antigen gene NY-ESO-1 was characterized in terms of vector morphology, particle size range, concentration, and zeta potential using a variety of physical methods. Environmentally stressed vector samples were then evaluated in terms of viral vector particle size and concentration by nanoparticle tracking analysis (NTA). These NTA stability results correlated reasonably well with a quantitative polymerase chain reaction assay for quantitation of viral genome copy number (r2 = 0.80). Approximately 40 pharmaceutical excipients were examined for their ability to stabilize the vector against exposure to an adsorptive container surface (glass) as well as freeze-thaw cycling using NTA as the screening method. Stabilizing additives that inhibited viral vector particle loss under these conditions included proline, lactose, and mannitol. Several candidate frozen liquid formulations that contained a combination of these lead excipients and various buffering agents were further evaluated for their ability to stabilize the viral vector. The additional benefit of lowering the Tris buffer concentration was observed. This study highlights the use of physical particle assays such as NTA for initial screening of stabilizing excipients to minimize vector loss due to container adsorption and freeze-thaw cycling to facilitate early formulation development of viral vector candidates in frozen liquid formulations.

Efficacy of broadly neutralizing monoclonal antibody PG16 in HIV-infected humanized mice.

Highly potent broadly neutralizing human monoclonal antibodies hold promise for HIV prophylaxis and treatment. We used the SCID-hu Thy/Liv and BLT humanized mouse models to study the efficacy of these antibodies, primarily PG16, against HIV-1 clades A, B, and C. PG16 targets a conserved epitope in the V1/V2 region of gp120 common to 70-80% of HIV-1 isolates from multiple clades and has extremely potent in vitro activity against HIVJR-CSF. PG16 was highly efficacious in SCID-hu mice as a single intraperitoneal administration the day before inoculation of R5-tropic HIV directly into their Thy/Liv implants and demonstrated even greater efficacy if PG16 administration was continued after Thy/Liv implant HIV inoculation. However, PG16 as monotherapy had no activity in humanized mice with established R5-tropic HIV infection. These results provide evidence of tissue penetration of the antibodies, which could aid in their ability to prevent infection if virus crosses the mucosal barrier.

Human antibodies reveal a protective epitope that is highly conserved among human and nonhuman influenza A viruses.

Influenza remains a serious public health threat throughout the world. Vaccines and antivirals are available that can provide protection from infection. However, new viral strains emerge continuously because of the plasticity of the influenza genome, which necessitates annual reformulation of vaccine antigens, and resistance to antivirals can appear rapidly and become entrenched in circulating virus populations. In addition, the spread of new pandemic strains is difficult to contain because of the time required to engineer and manufacture effective vaccines. Monoclonal antibodies that target highly conserved viral epitopes might offer an alternative protection paradigm. Herein we describe the isolation of a panel of monoclonal antibodies derived from the IgG(+) memory B cells of healthy, human subjects that recognize a previously unknown conformational epitope within the ectodomain of the influenza matrix 2 protein, M2e. This antibody binding region is highly conserved in influenza A viruses, being present in nearly all strains detected to date, including highly pathogenic viruses that infect primarily birds and swine, and the current 2009 swine-origin H1N1 pandemic strain (S-OIV). Furthermore, these human anti-M2e monoclonal antibodies protect mice from lethal challenges with either H5N1 or H1N1 influenza viruses. These results suggest that viral M2e can elicit broadly cross-reactive and protective antibodies in humans. Accordingly, recombinant forms of these human antibodies may provide useful therapeutic agents to protect against infection from a broad spectrum of influenza A strains.