A Method for Producing Protein Nanoparticles with Applications in Vaccines

Abstract

A practical method is described for synthesizing conjugated protein nanoparticles using thioether (thiol-maleimide) cross-linking chemistry. This method fills the need for a reliable and reproducible synthesis of protein conjugate vaccines for preclinical studies, which can be adapted to produce comparable material for clinical studies. The described method appears to be generally applicable to the production of nanoparticles from a variety of soluble proteins having different structural features. Examples presented include single-component particles of the malarial antigens AMA1, CSP and Pfs25, and two component particles comprised of those antigens covalently cross-linked with the immunogenic carrier protein EPA (a detoxified form of exotoxin A from Pseudomonas aeruginosa). The average molar masses (Mw) of particles in the different preparations ranged from 487 kDa to 3,420 kDa, with hydrodynamic radii (Rh) ranging from 12.1 nm to 38.3 nm. The antigenic properties and secondary structures of the proteins within the particles appear to be largely intact, with no significant changes seen in their far UV circular dichroism spectra, or in their ability to bind conformation-dependent monoclonal antibodies. Mice vaccinated with mixed particles of Pfs25 or CSP and EPA generated significantly greater antigen-specific antibody levels compared with mice vaccinated with the respective unmodified monomeric antigens, validating the potential of antigen-EPA nanoparticles as vaccines.

Nanoparticle Vaccines Adopting Virus-like Features for Enhanced Immune Potentiation

“…In a maleimide-thiol reaction, the nucleophilic thiolate anion attacks the π-bond of maleimide, forming the enolate intermediate and yielding the desired conjugate. This technique has been widely used to assemble nanoparticle vaccines [184][185][186]. In a study on refining a liposomal formulation of HIV vaccine, thiol chemistry was exploited to control the physiological conformation and density of the target antigen to modulate immune responses [186].…”

Decrease in Numbers of Naive and Resting B Cells in HIV-Infected Kenyan Adults Leads to a Proportional Increase in Total and Plasmodium falciparum–Specific Atypical Memory B Cells

“…The biochemical structure of AMA1 is shown in the supplemental data to demonstrate the potential biotinylation sites on the surface of domains 1 and 2 of the recombinant protein (Supplemental Fig. 1) (23). There is evidence that a significant number of B cells will bind PE, which limits many studies utilizing fluorescently labeled B cell Ags (20).…”

Protein-protein conjugate nanoparticles for malaria antigen delivery and enhanced immunogenicity

“…Our laboratory has pioneered conjugation of malaria antigens to protein carriers to generate effective immunogens and potential vaccines [ 45 – 49 ]. Chemical conjugation of malaria antigens to carrier proteins generates nanoparticles with enhanced immunogenicity [ 45 , 49 ], and some of these have progressed to clinical evaluation [ 50 ]. The lead antigens in our clinical trials are Pfs25 and Pfs230, TBV antigens expressed in zygotes/ookinetes and gametocytes, respectively.…”

“…Chemical conjugation of these antigens to the recombinant Exoprotein A (EPA) carrier increased functional immunogenicity in animal studies, and the conjugates have proven to be safe in human trials [ 50 ]. While these studies focused on EPA conjugates prepared by thioether chemistry [ 45 , 49 ], we are also exploring this platform technology with a variety of malaria antigens and carriers. For example, we find that both carriers and adjuvants can influence the level of immune response against conjugated malaria antigens [ 48 ].…”