Optimized cell-free synthesis of invasion plasmid antigen B and related compositions and methods of use

Inventors

Kapoor, Neeraj • FAIRMAN, Jeffery C.

Assignees

Vaxcyte Inc

Abstract

The present disclosure provides a cell-free method for synthesizing an Invasion Plasmid Antigen B (IpaB) antigen associated with a Shigella bacterium comprising exogenous addition of the purified chaperone protein IpgC to the cell-free synthesis mixture. The disclosure further provides IpaB antigen mutants comprising non-natural amino acids incorporated during cell-free synthesis, enabling covalent conjugation to a Shigella O-antigen polysaccharide. Further provided are Ipa B antigens and conjugates thereof, as well as immunogenic compositions prepared with the synthesized IpaB antigens and conjugates thereof and methods of use.

Core Innovation

The invention provides an Invasion Plasmid Antigen B (IpaB) polypeptide antigen in which 3 or 4 non-natural amino acids (nnAA) are incorporated at defined amino-acid positions of SEQ ID NO: 1, including nnAA incorporated at position K395 and at least one additional position selected from K299 and K368 and a position selected from K289, K436, and K470. The antigen includes IpaB mutants that bear nnAA with a click chemistry reactive group (an azide).

The invention further provides expression of the IpaB polypeptide antigen from a Shigella bacterium using cell-free protein synthesis (CFPS) in the presence of an exogenous IpgC chaperone protein. Purified exogenous IpgC is used as a chaperone to optimize soluble expression of IpaB and to enable production of the nnAA-bearing IpaB mutants for subsequent conjugation chemistry.

The invention also provides IpaB-Ops conjugates by incorporating azide-containing nnAA into IpaB and enabling click-conjugation to Shigella O-antigen polysaccharides (OPS), including OPS from Shigella serotypes 1a, 1b, 2a, 2b, 3b, 4a, 4b, 5a, 5b, 6, 7a, and 7b. Immunogenic compositions containing IpaB/OPS conjugates with excipients and adjuvants are described, and therapeutic and prophylactic vaccination methods are outlined for protection against Shigella dysentery.

From the background as reflected in the provided content, the problem is the need for improved Shigella dysentery immunogens that can present IpaB in a form that supports robust immunogenicity and effective protective immune responses. The described approach addresses this by generating nnAA-bearing IpaB using CFPS with an exogenous IpgC chaperone, and by forming IpaB-Ops conjugates to support immunogenic compositions intended for immunization against Shigella dysentery.

Claims Coverage

The provided independent claims are clm-00001 and clm-00011, which jointly cover (i) an nnAA-modified IpaB polypeptide antigen with defined residue positions and (ii) a CFPS method for expressing IpaB using an exogenous IpgC chaperone. Across these independent claims, the main inventive features include defined multi-site nnAA incorporation into IpaB (including K395) and CFPS expression with exogenous IpgC chaperone protein.

Together, the independent claims define an IpaB polypeptide antigen with 3 or 4 nnAA incorporated at specific residue positions (including K395) and define CFPS expression of the IpaB polypeptide antigen from a Shigella bacterium in the presence of an exogenous IpgC chaperone protein.

Stated Advantages

Documented Applications