Advances in Universal Influenza Virus Vaccine Design and Antibody Mediated Therapies Based on Conserved Regions of the Hemagglutinin

Abstract

The threat of novel influenza viruses emerging into the human population from animal reservoirs, as well as the short duration of protection conferred by licensed vaccines against human seasonal strains has spurred research efforts to improve upon current vaccines and develop novel therapeutics against influenza viruses. In recent years these efforts have resulted in the identification of novel, highly conserved epitopes for neutralizing antibodies on the influenza virus hemagglutinin protein, which are present in both the stalk and globular head domains of the molecule. The existence of such epitopes may allow for generation of novel therapeutic antibodies, in addition to serving as attractive targets of novel vaccine design. The aims of developing improved vaccines include eliciting broader protection from drifted strains, inducing long-lived immunity against seasonal strains, and allowing for the rational design of vaccines that can be stockpiled for use as pre-pandemic vaccines. In addition, an increased focus on influenza virus vaccine research has prompted an improved understanding of how the immune system responds to influenza virus infection.

Optimal activation of Fc-mediated effector functions by influenza virus hemagglutinin antibodies requires two points of contact

“…The digested F(ab′) 2 was then purified through a Protein A spin column [with the flow-through containing the F(ab′) 2 saved] and quantified using a NanoDrop 2000 spectrophotometer (extinction coefficient factor of 1.4). The bivalent F(ab′) 2 was assessed by SDS/PAGE in nonreducing conditions and detected with an anti-mouse F(ab′) 2 antibody conjugated to HRP (1:10,000) by Western blot analysis.…”

“…At 16 hpt, the cell monolayer was fixed with 100 μL of 3.7% paraformaldehyde for 10 min and then blocked with 100 μL of 5% nonfat milk/1× PBS for 30 min. PY102 F(ab′) 2 , control F(ab′) 2 , or no antibody was added at a starting concentration of 10 μg/mL and diluted fourfold, and a humanized [murine F(ab′) 2 with human Fc] mAb 6F12 was held constant at 10 μg/mL. The primary antibody mixtures were incubated onto the cells for 30 min at room temperature, after which the plates were washed four times with 1× PBS/0.1% Tween (TPBS).…”

“…In brief, a constant amount of a humanized [murine F(ab′) 2 and human Fc] 6F12 (10 μg/mL) and a variable amount of PY102 F(ab′) 2 (starting dilution of 10 μg/mL; diluted fourfold) were added onto WT HA-transfected HEK 293T cells, and an antihuman IgG-specific secondary antibody conjugated to horseradish peroxidase (HRP) was used to detect binding of the humanized 6F12. The humanized 6F12 alone bound equally well in the presence of either PY102 or control F(ab′) 2 (Fig. 3C), indicating that binding of PY102 F(ab′) 2 did not prevent 6F12 from binding to the stalk region.…”

“…By characterizing the interactions of several FLAG-tagged HAs with a FLAG-specific antibody, we show that in addition to Fc-FcγR engagement mediated by the FLAG-specific antibody, a second intermolecular bridge between the receptor-binding region of the HA and sialic acid on effector cells is required for optimal activation. Inhibition of this second molecular bridge, through the use of an F(ab′) 2 or the mutation of the sialic acid-binding site, renders the Fc-FcγR interaction unable to optimally activate effector cells. Our findings indicate that broadly reactive mAbs require two molecular contacts to possibly stabilize the immunologic synapse and potently induce antibody-dependent cell-mediated antiviral responses: (i) the interaction between the Fc of a mAb bound to HA with the FcγR of the effector cell and (ii) the interaction between the HA and its sialic acid receptor on the effector cell.…”

“…There is a vital need to improve influenza virus vaccination strategies. In the movement toward the development of a new generation of vaccines, one promising approach is to focus the immune response to target the conserved regions of the virus (2).…”