Lessons Learned from the Development and Roll-Out of the rVSVΔG-ZEBOV-GP Zaire ebolavirus Vaccine to Inform Marburg Virus and Sudan ebolavirus Vaccines.

This review describes key aspects of the development of the rVSVΔG-ZEBOV-GP Ebola vaccine and key activities which are continuing to further expand our knowledge of the product. Extensive partnerships and innovative approaches were used to address the various challenges encountered during this process. The rVSVΔG-ZEBOV-GP Ebola vaccine was initially approved by the European Medicines Agency and prequalified by the World Health Organization in November 2019. It was approved by the United States Food and Drug Administration in December 2019 and approved in five African countries within 90 days of prequalification. The development resulted in the first stockpile of a registered Ebola vaccine that is available to support outbreak response. This also provides insights into how the example of rVSVΔG-ZEBOV-GP can inform the development of vaccines for Sudan ebolavirus, Marburg virus, and other emerging epidemic diseases in terms of the types of approaches and data needed to support product registration, availability, and the use of a filovirus vaccine.

Development of stable liquid formulations for adenovirus-based vaccines.

We have evaluated the stability profiles of adenovirus type-5 (Ad5)-based vaccine formulations to identify liquid formulations that are stable during long-term storage at 4 degrees C. By identifying the major physiochemical inactivation pathway(s) during storage, formulations of Ad5 were designed with specific pharmaceutical excipients leading to greatly enhanced stability. For example, results indicate that Ad5 is stabilized by non-ionic surfactants and cryoprotectants as well as excipients known to inhibit free-radical oxidation. A non-ionic surfactant is necessary to prevent adsorption of adenovirus to glass surfaces during storage, and a cryoprotectant is needed to prevent freeze-thaw-induced virus inactivation. In a base formulation (A105) containing sucrose as the cryoprotectant and polysorbate-80 as the non-ionic surfactant, metal-ion catalyzed free-radical oxidation is an important mechanism of Ad5 inactivation. The free-radical oxidation inhibitors ethanol and histidine, combined with the metal-ion chelator ethylenediaminetetraacetic acid (EDTA), were determined to be effective stabilizers of Ad5. Arrhenius plots of stability data are consistent with a first-order inactivation mechanism with apparent activation energies for virus inactivation of 26.5 +/- 0.9 and 28.7 +/- 0.6 kcal/mol in the absence and presence of free-radical oxidation inhibitors, respectively. Optimization of formulation pH, as well as the EDTA and ethanol concentrations, allowed for the identification of formulations that further enhanced long-term storage stability. For example, Ad5 in an optimized liquid formulation (A195) lost <0.1 logs of infectivity after 24 months of storage at 4 degrees C. The immunogenicity of a recombinant Ad5-based human immunodeficiency virus (HIV) vaccine candidate expressing HIV-1 gag (MRKAd5gag) formulated in A195, was shown to be equivalent to the same vaccine formulated in A105. Therefore, the use of EDTA, ethanol, and histidine did not significantly alter the immunogenicity of the vaccine in mice. The identification of 4 degrees C stable liquid formulations should significantly enhance the utility of Ad5 as a vector for vaccines and gene therapy.

Characterization and biological evaluation of a microparticle adjuvant formulation for plasmid DNA vaccines.

We describe the physiochemical characterization and immunological evaluation of plasmid DNA vaccine formulations containing a nonionic triblock copolymer adjuvant (CRL1005) in the presence and absence of a cationic surfactant, benzalkonium chloride (BAK). CRL1005 forms particles of 1-10 microns upon warming above its phase-transition temperature (approximately 6-8 degrees C) and the physical properties of the particles are altered by BAK. DNA/CRL1005 vaccines formulated with and without BAK were evaluated in rhesus macaques to determine the effect of CRL1005 and BAK on the ability of plasmid DNA to induce a cellular immune response. Immunogenicity results indicate that the addition of CRL1005 to human immunodeficiency virus-1 gag plasmid DNA formulated in phosphate-buffered saline leads to an enhancement in the gag-specific cellular immune response. Moreover, the addition of BAK to human immunodeficiency virus-1 gag plasmid DNA/CRL1005 formulations produces an additional enhancement in gag-specific cellular immunity. In vitro characterization studies of DNA/CRL1005 formulations indicate no detectable binding of DNA to CRL1005 particles in the absence of BAK, suggesting that the enhancement of cellular immunity induced by DNA/CRL1005 formulations is not due to enhanced DNA delivery. In the presence of BAK, however, results indicate that BAK binds to CRL1005 particles, producing cationic microparticles that bind DNA through electrostatic interactions. If BAK is present at the phase-transition temperature, it reduces the particle size from approximately 2 microns to approximately 300 nm, presumably by binding to hydrophobic surfaces during particle formation. Zeta potential measurements indicate that the surface charge of CRL1005-BAK particles changes from positive to negative upon DNA binding, and DNA bound to the surface of CRL1005-BAK particles was visualized by fluorescence microscopy. These results indicate that the addition of BAK to DNA/CRL1005 formulations leads to the formation of approximately 300 nm CRL1005-BAK-DNA particles that enhance the cellular immune response in rhesus monkeys.

Vaccine-induced immunity in baboons by using DNA and replication-incompetent adenovirus type 5 vectors expressing a human immunodeficiency virus type 1 gag gene.

The cellular immunogenicity of formulated plasmid DNA and replication-defective human adenovirus serotype 5 (Ad5) vaccine vectors expressing a codon-optimized human immunodeficiency virus type 1 gag gene was examined in baboons. The Ad5 vaccine was capable of inducing consistently strong, long-lived CD8(+)-biased T-cell responses and in vitro cytotoxic activities. The DNA vaccine-elicited immune responses were weaker than those elicited by the Ad5 vaccine and highly variable; formulation with chemical adjuvants led to moderate increases in the levels of Gag-specific T cells. Increasing the DNA-primed responses with booster doses of either Ad5 or modified vaccinia virus Ankara vaccines suggests a difference in the relative levels of cytotoxic and helper responses. The implications of these results are discussed.

Replication-incompetent adenoviral vaccine vector elicits effective anti-immunodeficiency-virus immunity.

Recent studies of human immunodeficiency virus type 1 (HIV-1) infection in humans and of simian immunodeficiency virus (SIV) in rhesus monkeys have shown that resolution of the acute viral infection and control of the subsequent persistent infection are mediated by the antiviral cellular immune response. We comparatively assessed several vaccine vector delivery systems-three formulations of a plasmid DNA vector, the modified vaccinia Ankara (MVA) virus, and a replication incompetent adenovirus type 5 (Ad5) vector-expressing the SIV gag protein for their ability to elicit such immune responses in monkeys. The vaccines were tested either as a single modality or in combined modality regimens. Here we show that the most effective responses were elicited by a replication-incompetent Ad5 vector, used either alone or as a booster inoculation after priming with a DNA vector. After challenge with a pathogenic HIV-SIV hybrid virus (SHIV), the animals immunized with Ad5 vector exhibited the most pronounced attenuation of the virus infection. The replication-defective adenovirus is a promising vaccine vector for development of an HIV-1 vaccine.