Carbon nanotube-based DNA vaccine against koi herpesvirus given by intramuscular injection

Highlights

• Recombinant plasmid pcDNA-ORF149 was coupled to single walled carbon nanotubes (SWCNTs) to generate an anti-KHV vaccine.

• The vaccine SWCNTs-pcDNA-ORF149 conferred an RPS of 81.9% against challenge with KHV.

• The novel vaccine might provide an effective method of coping with KHV disease.

Abstract

Koi herpesvirus (KHV) also named Cyprinid Herpesvirus 3 (CyHV-3) is one of the most threatening pathogens affecting common carp production as well as the valued ornamental koi carp. The current commercial vaccines available are costly and potentially cause severe stress caused by live virus. KHV ORF149 gene has been proved encoding one of the main immunogenic proteins for KHV. In this study, we coupled a plasmid expression vector for ORF149 to single walled carbon nanotubes (SWCNTs) for an anti-KHV vaccine. The vaccine conferred an 81.9% protection against intraperitoneal challenge with KHV. Importantly, SWCNTs as a promising vehicle can enhanced the protective effects 33.9% over that of the naked DNA vaccine at the same dose. The protection was longer and serum antibody production, enzyme activities and immune-related gene expression were all induced in fish vaccinated with the nanotube-DNA vaccine compared with the DNA alone. Thereby, this study demonstrates that the ORF149 DNA vaccine loaded onto SWCNTs as a novel vaccine might provide an effective method of coping with KHV disease using intra-muscular vaccination.

Introduction

Aquaculture is a rapidly expanding economic sector worldwide and annual rate increases are estimated at 6.4% [1]. However, aquaculture is an economic activity with a high business risk. In fact, there are many factors that can cause adverse conditions that increase susceptibility to infections of the farmed species and that consequently pose a risk for the global production of aquaculture [2,3]. In particular, intensive rearing practices based on fish cultured in confined and controlled areas such as ponds or off-shore cages at high density all negatively affect the health status of cultured fish. This makes the control of disease outbreaks problematic and difficult [4,5].

Infection with Koi herpesvirus (KHV) [6] are a listed notifiable disease to the International Office of Epizootics (OIE, 2018). KHV has spread to most regions around the world due to the global fish trade and international ornamental koi shows [7]. First detected in the late 1990s, KHV has been found in Europe, Asia, North America and Africa and had caused serious worldwide losses in the carp and koi culture industries. As with all herpesviruses, KHV sets up a persistent infection in its host and can exist in a latent or state without obvious clinical signs [8]. Molecular analysis has demonstrated that the KHV isolates show little genomic variation as might be expected for a virus that is being rapidly disseminated by the global movement of infected fish [9].

KHVD is relatively host-specific, while only common carp and its ornamental subspecies, koi [10], are involved in the explosive losses reported globally [6]. In addition to its negative economical and societal impacts, KHV has also a negative environmental impact by affecting wild carp populations. In addition, hybrids of common carp and goldfish are partly susceptible to KHV infection although the mortality rate varies [11]. Cohabitation experiments indicated that some carp species such as goldfish, common bream (Abramis brama), silver carp and grass carp can carry KHV asymptomatically and transmit it to wild carp [12,13]. KHVD is highly contagious and extremely virulent with mortality rates of 80–100%. Carp of all ages can be infected with KHV but younger fish (1–3 months, 2.5–6 g) seem to be more susceptible to infection than mature fish (1 year old, 230 g) [14].

The koi subspecies is of economic importance as an ornamental fish in China. Because of its numerous colors and color combinations, the koi has grown into one of the most popular pet hobbies in the world [15]. Within the past few decades, the commercial production of koi has emerged as a major segment of the pet industry and the koi (especially high quality individuals) trade plays a major role in meeting a growing worldwide demand [16]. Intensive koi farming is associated with risk for the incidence and spread of KHVD that cause skin lesions, severe fin erosion, flaking scales, dark skin coloration, especially gill necrosis, one of the most distinct symptom of KHVD [17][18][19], and even increased mortality and resulting in lower prices.

Vaccine is widely accepted as an effective control against viral diseases [20]. Up to now, the commercialized KHV vaccine is attenuated vaccine, which has apparent stress response after immunization [21]. The efficacy of KHV inactivated virus vaccine is not satisfied. One of the most promising vaccine preparations against fish diseases is currently the DNA vaccine delivered intramuscularly that consists of plasmid DNA expression vectors that result in gene expression of pathogenic proteins in the muscle tissue of the vaccinated fish [22].

Compared with traditional vaccines, it is highly efficient, easy to prepare and has high stability. DNA vaccines prepared with KHV ORF25 and ORF81 as target genes have been shown to significantly reduce mortality to <20% indicating that DNA vaccines have favorable immune effects against KHV [23,24]. However, intramuscular injection of naked DNA vaccines generally induce only transient immune protection in fish [25]. The current challenge for DNA vaccines is the discovery of an effective carrier that can result in persistence of the expressed antigen.

Carbon nanotubes (CNTs) have been used as DNA carriers for vaccines and possess a large surface area in a small volume that translates to high surface reactivity and easy surface functionalization. Drugs and DNA have both been used as the cargo for CNTs for both diagnostic and therapeutic applications. These complexes can easily penetrate cell membrane and tissue barriers and achieve their biological effects by targeting the drug to the cell at a high dose [[26], [27], [28]]. Whether the CNT have correlation or negative impact to the fish immune-responses has been clarified by other researchers [[28], [29], [30]]. The previous research proved it does not take any negative effect on fish immune-responses and CNT itself could not induce immune protection.

Fish nanocarrier vaccines have also been developed such as chitosan nanomaterials loaded with a salmon anemia DNA vaccine that can be used orally. This vaccine significantly improved immune protection [31,32]. Single walled carbon nanotubes (SWCNTs) DNA vaccine against spring viremia of carp virus (SVCV) and grass carp reovirus (GCRV) have proved effective [[29], [30], [33]]. Mandarin fish immunized with SWCNT-MCP by immersion vaccination resulted in increased survival against challenge of infectious spleen and kidney necrosis virus (ISKNV) [34].

In the current study we used the KHV ORF 149 gene of 2100 bp with 699 predicted amino acids and theoretical molecular weight of 72 kDa, containing only one potential N-glycosylation and 93 O-glycosylation sites. The envelope glycoprotein encoded by ORF149 is a neutralizing epitope and most likely functions in virus attachment and cell penetration, and is therefore a promising target antigen for vaccine development and diagnostic testing [35]. In present work, on the basis of hydroxyl and amino condensation reactions, functionalized single-walled CNTs (o-SWCNTs) were used as carriers to load KHV ORF149 in an expression vector. We used this complex to immunize koi via intramuscular injection and evaluated the immune response elicited in vaccinated fish. This work provides a viable solution to KHVD and lays a foundation for future work on a wide range of CNTs-DNA vaccine delivery systems for fish.