DNA vaccines encoding viral glycoproteins induce nonspecific immunity and Mx protein synthesis in fish

Abstract

Protective immunity by vaccination with plasmid DNA encoding a viral glycoprotein (G) has long been assumed to result from the induction of a specific immune response. We report here that the initial protection may be due to the induction of alpha/beta interferon, with long-term protection due to a specific response to the encoded viral G. DNA vaccines encoding the Gs of three serologically unrelated fish rhabdoviruses were used to vaccinate rainbow trout against a lethal challenge with infectious hematopoietic necrosis virus (IHNV). All three vaccines, each encoding the G gene of either IHNV (IHNV-G), snakehead rhabdovirus (SHRV) (SHRV-G), or spring viremia of carp virus (SVCV) (SVCV-G), elicited protective immunity against IHNV. Vaccinated fish were challenged at 30 or 70 days postvaccination with lethal doses of IHNV. At 30 days postvaccination, only 5% of fish that had received any of the G vaccines died, whereas more than 50% of the control fish succumbed to virus challenge. When fish were vaccinated and challenged at 70 days postvaccination, only 12% of the IHNV-G-vaccinated fish died compared to 68% for the SHRV-G- and 76% for the SVCV-G-vaccinated fish. Assays for trout Mx protein, an indicator of alpha/beta interferon induction, showed that only fish vaccinated with a G-containing plasmid produced high levels of Mx protein in the kidneys and liver. Interestingly, at day 7 after virus challenge, all of the fish vaccinated with the IHNV-G plasmid were negative for Mx, but the SHRV-G- and SVCV-G-vaccinated fish still showed detectable levels of Mx. These results suggest that DNA vaccines in fish induce an early, nonspecific antiviral protection mediated by an alpha/beta interferon and, later, a specific immune response.

FIG. 1

G amino acid sequence analysis. Alignment of the predicted amino acid sequences from SHRV, SVCV, and IHNV. ∗, amino acids conserved among all three genes.

FIG. 2

CPM for fish exposed to 10⁵ PFU (A) or 10³ PFU (B) of IHNV per ml at 30 dpv. The average mortality for the virus is shown on the ordinate and is plotted against the number of days postexposure. The range of mortality did not vary significantly between tanks and thus is not shown.

FIG. 3

CPM for fish exposed to 10⁵ PFU of IHNV per ml at 70 dpv. The average mortality for the virus is shown on the ordinate and is plotted against the number of days postexposure. The range of mortality did not vary significantly between tanks and thus is not shown.

FIG. 4

Mx expression at 0 and 7 dpc. Immunoblot analyses of kidney (K) and liver (L) tissues from fish that were vaccinated with IHNV, SHRV, and SVCV G vaccines or with a pcDNA3 or PBS control, and then subsequently challenged with 10⁵ PFU of IHNV per ml are shown. The immunoblots were probed with an Mx antiserum primary antibody and an alkaline phosphatase-conjugated donkey anti-rabbit secondary antibody as described in Materials and Methods.