Long-Term Protection Elicited by an Inactivated Vaccine Supplemented with a Water-Based Adjuvant against Infectious Hematopoietic Necrosis Virus in Rainbow Trout (Oncorhynchus mykiss)

Abstract

Previous inactivated vaccines against infectious hematopoietic necrosis (IHN) usually had a strong early immune protective effect but failed to provide long-term protection in rainbow trout (Oncorhynchus mykiss). To find a method for stabilizing the desired protective effect of IHN vaccines, we assessed the immune enhancement effect of four adjuvants on formaldehyde inactivated vaccine for IHN at 60 days postvaccination (dpv). The efficacy of a two-dose vaccination with the candidate adjuvant-formaldehyde inactivated vaccine for IHN was evaluated in terms of early protection and long-term protection (30 to 285 dpv). Neutralizing antibody titers were also measured at each time point. The Montanide GEL 02 PR (Gel 02) adjuvant significantly enhanced the immune protection provided by the IHN inactivated vaccine, whereas the immune-boosting effect of the other tested adjuvants lacked statistical significance. Both tested Gel 02-adjuvanted IHN inactivated vaccine dosages had a strong immune protection effect within 2 months postvaccination, with a relative percent of survival (RPS) of 89.01% to 100%, and the higher dosage provided complete protection at 204 dpv and a RPS of 60.79% on 285 dpv by reducing viral titers in rainbow trout. The neutralizing antibodies were observed only in vaccinated fish on 30 and 60 dpv. Through compatibility with an appropriate adjuvant, the highly immune protective effect of an IHN inactivated vaccine was prolonged from 60 dpv to at least 284 dpv; this novel adjuvant-IHN inactivated vaccine has promise as a commercial vaccine that provides the best available and longest duration of protection against IHN to rainbow trout. IMPORTANCE Infectious hematopoietic necrosis virus (IHNV) is one of the most serious pathogens threatening the global salmon and trout industry. However, there is currently only one commercialized infectious hematopoietic necrosis (IHN) vaccine, and it is inadequate for solving the global IHN problem. In this study, a promising adjuvanted inactivated vaccine with long-term protection was developed and comprehensively studied. We confirmed the presence of a late antiviral response stage in vaccinated rainbow trout that lacked detectable neutralizing antibodies, which are commonly recognized to be responsible for long-term specific protection in mammals. These findings further our understanding of unique features of fish immune systems and could lead to improved prevention and control of fish diseases.

Keywords: adjuvant; inactivated vaccine; infectious hematopoietic necrosis virus; long-term protection; rainbow trout.

FIG 1

The protective effect of the IHN inactivated vaccines prepared with formaldehyde or BPL in rainbow trout determined at 60 dpv. Two groups of rainbow trout, one with an average weight of 6 ± 2 g and the other with an average weight of 70 ± 8 g, were vaccinated with the IHN inactivated vaccines at dosages of 50 μL or 100 μL, respectively, and then were challenged with IHNV strain LN15 (100 TCID₅₀) at dosages of 50 μL or 100 μL, respectively. *, P < 0.05 versus the PBS control group. Different letters at the top of a bar indicate significant differences (P < 0.05).

FIG 2

Protective effect of the IHN inactivated vaccine alone or in combination with different adjuvants. Rainbow trout (weighing 7 ± 1.5 g) were vaccinated and challenged with IHNV strain LN15 (100 TCID₅₀) at a dosage of 50 μL at 60 dpv. The prepared Montanide ISA 763A VG-adjuvanted vaccine was difficult to inject, so it was not included in these experiments. *, P < 0.05 versus the PBS control group. **, P < 0.01 versus the PBS control group. Different letters at the top of a bar indicate significant differences (P < 0.05).

FIG 3

Protective effects elicited by different dosages of Montanide GEL 02 PR-adjuvanted IHN inactivated vaccine at 30 dpv. Fish mock-vaccinated with PBS were used as the negative control. No significant differences in mortality were observed between any replicate within each treatment group.

FIG 4

Protective effect of Montanide GEL 02 PR-adjuvanted IHN inactivated vaccine (containing 9 μL of inactivated IHNV) in rainbow trout at different postvaccination time points. The immunized fish were challenged with specific dosages of IHNV strain LN15 at different postvaccination time points (Table 5). No significant differences in mortality were observed between any replicates within each treatment. Different letters on the top of a bar indicate significant differences (P < 0.05).

FIG 5

Protective effect of Montanide GEL 02 PR-adjuvanted IHN inactivated vaccine (containing 45 μL of inactivated IHNV) in rainbow trout at different postvaccination time points. The immunized fish were challenged with specific dosages of IHNV strain at different postvaccination time points (Table 5). No significant differences in mortality were observed between any replicates within each treatment group. Different letters at the top of a bar indicate significant differences (P < 0.05).

FIG 6

Virus titers in the tissues and sera of vaccinated fish after IHNV challenge. Fish were challenged with IHNV stain LN15 at 285 dpv, and tissues (liver, spleen, and head kidney) were collected from the dead fish and from the surviving fish at 16 dpc. Serum samples were also collected from the surviving fish and titrated. Viral loads in the tissue and serum samples were statistically analyzed separately.