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, 1 (2), 127-31

C3d Enhancement of Antibodies to Hemagglutinin Accelerates Protection Against Influenza Virus Challenge

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C3d Enhancement of Antibodies to Hemagglutinin Accelerates Protection Against Influenza Virus Challenge

T M Ross et al. Nat Immunol.

Abstract

The ability of the C3d component of complement to enhance antibody responses and protective immunity to influenza virus challenges was evaluated using a DNA vaccine encoding a C3d fusion of the hemagglutinin (HA) from influenza virus. Plasmids were generated that encoded a transmembrane HA (tmHA), a secreted form of HA (sHA), or a sHA fused to three tandem copies of the murine homologue of the C3d (sHA-3C3d). Analysis of the titers, avidity maturation, and hemagglutinin-inhibition activity of raised antibody revealed that immunizations with sHA-3C3d DNA accelerated both the avidity maturation of antibody to HA and the appearance of hemagglutinin-inhibition activity. These accelerated antibody responses correlated to a more rapid appearance of protective immunity. They also correlated to complete protection from live virus challenge by a single vaccination at a dose ten times lower than the protective dose for non-C3d forms of HA.

Figures

Figure 1.
Figure 1.
Schematic representation of vector DNA vaccine constructs. (a) The pGA vector contains the cytomegalovirus immediate-early promoter (CMV-IE) plus intron A (IA) for initiating transcription of eukaryotic inserts and bovine growth hormone polyadenylation signal (BGH poly A) for termination of transcription. The vector also contains the Col E1 origin of replication for prokaryotic replication as well as the Kanamycin resistance gene (Kanr) for selection in antibiotic media. The λ T0 terminator has been placed 3′ to Kanr to increase the stability of eukaryotic inserts. Inserts were cloned into the vector using the Hind III and Bam HI restriction endonuclease sites. (b) Top, the wild-type transmembrane form of the HA protein used as a vaccine insert; middle, secreted form of the HA; bottom, the sHA-3C3d construct used as a vaccine insert. Linkers composed of two repeats of four glycines and a serine (G4S)2 were fused at the junctures of HA and C3d and between each C3d repeat. Rectangles indicate domains. 1, HA1; 2, HA2;TM, transmembrane.
Figure 2.
Figure 2.
Expression of vaccine constructs in vitro. Human embryonic kidney cells, 293T, were transfected with 2 μg of each vaccine plasmid. (a) Supernatant was collected and 1.5% of total volume was electrophoresis on a 10% polyacrylamide gel. Lane 1, sHA DNA; lane 2, sHA-3C3d DNA; lane 3, tmHA DNA; lane 4, pGA vector. (b) Cells were lysed and total protein was extracted in 300 μl volume of RIPA lysis buffer and 5% of the protein was electrophoresis on a 10% polyacrylamide gel. Lane 1, pGA vector; lane 2, sHA DNA; lane 3, sHA-3C3d DNA; lane 4, tmHA DNA.
Figure 3.
Figure 3.
Anti-HA IgG raised by gene gun inoculation of DNAs expressing HA proteins. Mice were immunized with different doses of vaccine plasmid. Half of the mice were primed at day 0 and boosted at week 4 (a,b) and half were given a single vaccination at day 0 (c,d). A ratio of the dose of DNA to specific IgG concentrations was obtained at week 14 (e,f). Sera were obtained from mice with vector (filled squares), sHA (open circles), tmHA (open squares) or sHA-3C3d (filled circles). Sera collected at the indicated times from each group were pooled for determination of specific IgG levels by ELISA. Data are represented as the average of three individual assays. Preimmune sera from mice had no detectable specific IgG.
Figure 4.
Figure 4.
Avidity of the anti-HA IgG raised by the three different HA DNA vaccines. Sera were analyzed from week 8 (a,b) and week 14 (c,d) in an A/PR/8/34 (H1N1)–specific NaSCN–displacement ELISA. Sera were obtained from mice inoculated. Sera were obtained from mice with sHA (open circles), tmHA (open squares) or sHA-3C3d (filled circles). Assays used pooled serum samples from each mouse group at a dilution of 1:300. Data are represented as the average of three independent experiments plus standard errors.
Figure 5.
Figure 5.
Hemagglutinin-inhibition antibodies and protection generated by HA DNA vaccines. The HI assay was read as the endpoint dilution of serum that still inhibited hemagglutination. Data are represented as the Log2 titer plus standard error for three to five individual serum samples collected at week 8 (a) and five individual serum samples collected at week 14 (b) from each mouse group. The data is represented as a Log2 titer of antibody to HA. (Open bars, vector; shaded bars, sHA; hatched bars, tmHA; filled bars, sHA-3C3d.)
Figure 6.
Figure 6.
Protection from weight loss after virus challenge. At week 8 (a,b) or week 14 (c–f) mice were challenged intranasally with a lethal dose of influenza virus, A/PR/8/34 (H1N1), and monitored daily for weight loss. The data are plotted as percentage of the average initial weight. (a,c) Mice were primed and boosted with a 1 μg dose of DNA vaccine. (b,d) Mice were primed and boosted with a 0.1 μg dose of DNA vaccine. (e) Mice were given a single 1 μg dose of DNA vaccine. (f) Mice were given a single 0.1 μg dose inoculum of DNA vaccine. Sera were obtained from mice with vector (filled squares), sHA (open circles), tmHA (open squares), sHA-3C3d (filled circles), naïve-mock (open triangles) or naïve-virus (filled triangles). The open cross indicates the time-point at which all five mice in a group succumbed to disease.

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