Montanide™ ISA 763A VG and ISA 761 VG induce different immune pathway responses in rainbow trout (Oncorhynchus mykiss) when used as adjuvant for an Aeromonas salmonicida bacterin.

Adjuvants are the helper substances that increase vaccine efficacy by enhancing the potency and longevity of specific immune responses to antigens. Most existing fish vaccines are presented in the form of oil-based emulsions delivered by intraperitoneal injection. The characterization of their mode of action is a valuable aid to future vaccine development, particularly for the potential identification and stimulation of specific immunological pathways related to the desired protective response. This study characterized the expression of selected immune-related genes in the peritoneal cavity, head kidney and spleen following the administration of two adjuvanted-bacterial vaccines thought to induce humoral (Montanide™ ISA 763A VG) or humoral and cell mediated (Montanide™ ISA 761 VG) immune responses, to determine if differences in responsiveness are readily apparent. The most informative site was the spleen, where Montanide™ ISA 763A VG + bacterin gave rise to upregulation of genes driving T-cell/lymphoid responses, namely IL-2, IL-15 and IL-21. This combined with upregulation of IFNγ1 and IFNγ2, IL-4/13B2, p35A1 and p40 (B1 and C) indicated that the induction of Th1 and possibly Th2 immunity was occurring in fish vaccinated with this adjuvant. Perhaps the most intriguing finding was the lack of a detectable Th1 response in fish given Montanide™ ISA 761 VG + bacterin, suggesting some other arm of the immune system is activated to give protection. Whatever the reason for the different responses detected, it is clear from the present study that the adjuvant used has a major impact on the responses elicited. Since these differences are readily detectable it allows, in principle, their use to help select the most appropriate adjuvants for inclusion into fish vaccines, where the type of response elicited may need to be tailored to a particular pathogen to confer protection.

Immunohistochemical examination of immune cells in adipose tissue of rainbow trout (Oncorhynchus mykiss) following intraperitoneal vaccination.

Mammalian perivisceral adipose has been shown to play an important role in the regulation of the peritoneal immune responses. Recently it has been demonstrated that peritoneal antigens are collected by leukocytes within the visceral adipose mass, and a broad range of immunomodulatory genes are differentially expressed in adipose tissue after intraperitoneal vaccination in rainbow trout. To assess the immune cell component in adipose, immunohistochemical analysis was used to examine B-cell, T-cell and antigen presenting cell (APC) numbers and distribution in rainbow trout adipose tissue 24 and 72 h post vaccination in comparison to control fish. The results of this study support previous work on mammals with omental milky spots in naïve fish found to contain APCs and T-cells which then increased in size, number and complexity following vaccination. It suggests that following peritoneal stimulation the visceral adipose mass in fish likely plays an important role in vaccine antigen uptake and presentation by APCs, as well as subsequent T-cell activation and differentiation.

Rainbow trout (Oncorhynchus mykiss) adipose tissue undergoes major changes in immune gene expression following bacterial infection or stimulation with pro-inflammatory molecules.

In mammals, visceral adipose is increasingly seen as playing an important role in immune function with numerous pro-inflammatory, anti-inflammatory and immune-modulating proteins and peptides being identified in adipocytes. Adipose is also now known as a tissue that has an important role in the regulation of peritoneal immune responses. Despite this, only lately has consideration been given to visceral adipose as an important immune tissue in fish, especially in the context of intraperitoneal vaccination. The present study demonstrates that fish visceral adipose is capable of expressing a large range of immune molecules in response to stimulation with a live bacterium (A. salmonicida), a bacterial PAMP (Y. ruckeri flagellin), and the pro-inflammatory cytokines IL-1ß, TNF-α3 and IFN-γ. Following infection and stimulation with flagellin and IL-1ß a large upregulation of pro-inflammatory and antimicrobial molecules was seen, with a high degree of overlap. TNF-α treatment affected relatively few genes and the effects were more modest. IFN-γ had the smallest impact on adipose but IFN-γ inducible genes showed some of the largest effects. Overall, it is clear that adipose tissue should be considered an active immune site in fish, capable of participating in and influencing immune responses.

Analysis of adipose tissue immune gene expression after vaccination of rainbow trout with adjuvanted bacterins reveals an association with side effects.

Most existing fish vaccines are presented in the form of oil-based emulsions delivered by intraperitoneal injection. Whilst very effective they are frequently associated with inflammatory responses that can result in clinically significant side-effects often involving the adipose tissue that is in direct contact with the vaccine. To explore the potential of immune gene expression changes in the adipose tissue of fish to be markers of vaccination efficacy or development of side-effects we have studied the response to a bacterial (Aeromonas salmonicida) vaccine administered with two different adjuvants. The first adjuvant was Montanide™ ISA 763A VG, thought to induce a mostly humoral response, and the second was Montanide™ ISA 761 VG that gives a more balanced humoral and cell mediated response. Following vaccination tissue samples were collected at days 3, 14 and 28 for RTqPCR analysis. Fifty immune genes were studied with a focus on a) pro-inflammatory associated molecules and b) adaptive immune response related molecules linked with possible Th1, Th2, Th17 and T-regulatory pathways, with the expression data analysed for associations with Speilberg post-vaccination side effect scores. The results showed that the adipose tissue is a particularly sensitive and discriminatory tissue for studying adjuvant effects. A clear upregulation of many immune genes occurred in response to both vaccine groups, which persisted over time and overlapped with the appearance of visible adhesions. Our analysis revealed a relationship between adipose tissue immune function and the development of vaccine-induced adhesions giving the potential to use immune gene expression profiling in this tissue to predict the side-effects seen.