Project 2. cGMP manufacture of HIV-1 Env trimer sortase A-conjugated nanoparticles

点击翻译按钮获取中文摘要

ABSTRACT-PROJECT 2 A key goal of HIV-1 vaccine development is to induce long-lasting protective broadly neutralizing antibody (bnAb) responses that can inhibit HIV-1 infection. However, such protective immune responses have been difficult to induce in the setting of vaccination. One strategy to induce these protective responses is to present to the immune system the target for neutralizing antibodies, HIV-1 envelope (Env) trimers in their native form. Multimerizing Env trimers on nanoparticles (NPs) enhances their immunogenicity, thus a goal for immunogen design is to present multiple copies of well-folded Env trimers. Env is a metastable protein that adopts multiple conformations. The primary obstacle facing Env trimer NP vaccines is that Env fusion nanoparticles, recombinantly expressed as a single polypeptide chain, can adopt a mixture of well-folded and misfolded conformations. The misfolded Env trimers may induce non-neutralizing antibody responses that change Env conformation upon binding or compete with bnAb precursors for binding to HIV-1 Env. For HIV-1 Env trimer NPs to become a rapid and widely-used platform for HIV-1 vaccine design, a nanoparticle platform that easily incorporates well-folded Env trimers without the need for costly, year-long protein engineering experiments is needed. To overcome the hurdles of HIV-1 Env nanoparticle vaccines, we have innovated a rapid system for generating well-folded HIV-1 Env trimer nanoparticles using a sortase A conjugation method. The HIV-1 Env trimers and ferritin are produced separately and to a high purity, allowing for elimination of misfolded protein. By mixing HIV-1 Env trimer, ferritin and sortase A enzyme, the HIV-1 Env trimer is covalently linked to the ferritin nanoparticle. The resulting sortase A-conjugated nanoparticles (scNPs) display the Env trimer in the desired conformation. The overall goals of Project 2 are to translate sortase A-conjugated nanoparticles into a cGMP-capable process for generation of viral vaccines against numerous pathogens and to manufacture two HIV-1 bnAb-targeting immunogens for evaluation in a Phase I trial conducted by the HIV Vaccine Trials Network (HVTN). To accomplish this goal, we will first determine optimal expression system for ferritin and sortase A and establish target product profiles for platform production (Aim 1). Then, we will develop, manufacture, and quality release recombinant sortase A and ferritin platform molecules for the conjugation of Env stabilized trimer to ferritin nanoparticles (Aim 2). Next, we will cGMP produce and quality release >1 g of two different stabilized Env trimer scNP drug substances and drug products for toxicology studies and clinical testing in a Phase I clinical trial (Aim 3). The first Env trimer scNP, CH505 TF Env trimer scNP, will be used as the first boosting immunogen in a CH505 Env NP sequential vaccine targeting CD4 binding site bnAbs. Finally, we will produce an additional sequential Env trimer scNP to select for further maturation of the same CD4 binding site bnAb lineage (Aim 3). The impact of translating this platform to cGMP manufacturing is that it will enable rapid production and quicker testing in clinical trials of high-quality HIV-1 Env trimer NP vaccines.