Neutralizing Antibodies Induced by Gene-Based Hydrodynamic Injection Have a Therapeutic Effect in Lethal Influenza Infection

Abstract

The influenza virus causes annual epidemics and occasional pandemics and is thus a major public health problem. Development of vaccines and antiviral drugs is essential for controlling influenza virus infection. We previously demonstrated the use of vectored immune-prophylaxis against influenza virus infection. We generated a plasmid encoding neutralizing IgG monoclonal antibodies (mAbs) against A/PR/8/34 influenza virus (IAV) hemagglutinin (HA). We then performed electroporation of the plasmid encoding neutralizing mAbs (EP) in mice muscles and succeeded in inducing the expression of neutralizing antibodies in mouse serum. This therapy has a prophylactic effect against lethal IAV infection in mice. In this study, we established a new method of passive immunotherapy after IAV infection. We performed hydrodynamic injection of the plasmid encoding neutralizing mAbs (HD) involving rapid injection of a large volume of plasmid-DNA solution into mice via the tail vein. HD could induce neutralizing antibodies in the serum and in several mucosal tissues more rapidly than in EP. We also showed that a single HD completely protected the mice even after infection with a lethal dose of IAV. We also established other isotypes of anti-HA antibody (IgA, IgM, IgD, and IgE) and showed that like anti-HA IgG, anti-HA IgA was also effective at combating upper respiratory tract IAV infection. Passive immunotherapy with HD could thus provide a new therapeutic strategy targeting influenza virus infection.

A Novel Gene Delivery Vector of Agonistic Anti-Radioprotective 105 Expressed on Cell Membranes Shows Adjuvant Effect for DNA Immunization Against Influenza

“…In our previous report (31) and others (29,30), HD can also induce gene expression in other organs, including the spleen. Clark and colleagues mainly focused on splenic B cells and demonstrated that in B cell-activating factor receptor (BAFFR)-deficient mice, which lack mature B cells but produce T1 B cells (57), anti-RP105 mAb bound to the antigen could also induce the production of antigen-specific antibodies (4,18).…”

“…Allophycocyanin (APC)-conjugated goat anti-mouse IgG(H+L) (Southern Biotech), FITC-conjugated rat anti-mouse IgG1 (RMG1-1; BioLegend, San Diego, CA, USA), APC-conjugated rat anti-mouse IgD (11-26c.2a), PerCP-conjugated or PE-conjugated rat anti-mouse B220 (RA3-6B2), biotinylated rat anti-mouse CD86 (GL-1), APC-conjugated streptavidin, PE-conjugated streptavidin, and FITC-conjugated rat anti-mouse IgM (eB121-15F9; eBioscience, San Diego, CA, USA) were purchased. We prepared biotinylated mouse anti-HA IgG1 ( 31 ), parental anti-RP105 mAb (rat), mouse anti-MD-1 (JR7G1) ( 35 ), and unconjugated anti-HA IgD ( 31 ). The parental anti-RP105 mAb (rat) was obtained from the supernatant of the hybridoma ( 14 ) ( Figures 1B, C ) or purified from ascites fluid, which was obtained from pristane (Funakoshi, Tokyo, Japan)-primed nude mice that were intraperitoneally inoculated with the hybridoma ( 36 , 37 ) using the caprylic acid (Wako, Tokyo, Japan)-ammonium sulfate (Katayama Kagaku, Osaka, Japan) precipitation method ( 38 ) ( Figures 2A, B ).…”

“…We performed HD ( 29 , 30 ) using a previously described method ( 31 ). Briefly, BALB/c mice and BALB/c RP105 -/- mice were injected with a PBS-containing plasmid (e.g., 5 µg/1.6 ml) into the tail vein, where the DNA volume was 8–12% of the body weight.…”

“…A single use of this method resulted in a long-term prophylactic efficacy before a lethal dose of influenza virus infection. In further studies, we performed hydrodynamic injections (HDs) that involved the rapid injection of a large volume of a plasmid-DNA solution into mice via the tail vein ( 29 , 30 ) and demonstrated that HD could induce rapid and potent neutralizing mAbs ( 31 ). A single use of this method resulted in therapeutic efficacy after a lethal dose of influenza virus infection.…”

DataSheet_2.pdf

“…After 14 days, their serum was obtained. The next day, the mice were intranasally infected with a lethal dose of A/PR8 virus (1,000 PFU/50 µl, 40 LD 50 ), as previously described (31,43). Three days post-infection, bronchoalveolar lavage specimens were obtained, and viral titers were determined by a plaque assay.…”