Potency and efficacy of VP20-based vaccine against tilapia lake virus using different prime-boost vaccination regimens in tilapia

Highlights

• Tilapia inoculated with VP20 based vaccine induced strong humoral and cellular immune responses.

• Tilapias in the DNA prime-protein boost group developed a stronger immune response than those immunized with either the DNA or the protein alone.

• TiLV VP20 based vaccine significantly lower the viral loads and resulted in significant increases in survival.

• The DNA prime-protein boost immunization strategy showed the potential advantage over a solo vaccination for the prevention of TiLVD.

Abstract

Tilapia lake virus (TiLV) is a newly emerged pathogen responsible for high mortality and economic losses in the global tilapia industry. Vaccination is the most effective way to prevent and control viral diseases. There is currently no vaccine on the market to control TiLV infections. Recently, we identified that TiLV segment 8 encoded a protein (VP20) with immunogenicity sufficient for use as a vaccine antigen. In this study, the immune responses and protective efficacy elicited by VP20 with different prime-boost vaccination regimens (DNA only, protein only, and DNA plus protein) in tilapia was evaluated. The results indicated that both pV-optiVP20 plasmid and rVP20 protein induced humoral and cellular immune responses. Tilapia in the DNA prime-protein boost group developed significantly higher levels of antibody response compared with those immunized with either the DNA or the protein alone (P < 0.05). Furthermore, the highest mRNA levels of the genes TNF-α, IL-1β, IgM, CD4, MHC-Ia and MHC-II were induced following priming with the pV-optiVP20 and boosting with the rVP20. Additionally, tilapia inoculated with the pV-optiVP20 prime-rVP20 boost regimen had a 72.5% survival rate against challenge with the lethal TiLV 2017A compared with 50% or 52.5% survival using the pV-optiVP20 and the rVP20 vaccinations alone. These findings demonstrated that VP20-based vaccine could elicit both humoral and cellular immune responses and significantly protected the fish against infectious challenge by TiLV. The DNA prime-protein boost immunization strategy showed the potential advantage over a solo vaccination.

Introduction

Tilapia represents fish in the genus Oreochromis and are the second most farmed fish and these numbers combined with the wild caught populations in 2015 accounted for more than 6.4 M tons and production has been steadily increasing (FAO, 2016; Ng and Romano, 2013; FAO, 2017; Thammatorn et al., 2019; FAO, 2019). However, infections due to the emerging tilapia lake virus (TiLV) has resulted in mass mortality of wild and farmed Nile tilapia Oreochromis niloticus and caused a significant impact on tilapia aquaculture in many countries (Surachetpong et al., 2020). Currently, outbreaks have been reported in 16 countries in Asia, Africa, South and North America and involve 6 of the major tilapia producers and is a serious threat to the industry (Del-Pozo et al., 2017; Koesharyani et al., 2018; Mugimba et al., 2018; Surachetpong et al., 2017; Nicholson et al., 2017; FAO, 2018; Jansen et al., 2019; OIE, 2019; Surachetpong et al., 2020). These infections have resulted in high levels of morbidity and 20–90% mortality in field outbreaks and this had been reproduced in laboratory challenged fish (Eyngor et al., 2014; Tattiyapong et al., 2017; Jansen et al., 2019; Dong et al., 2017b; Surachetpong et al., 2017; Ferguson et al., 2014).

TiLV is a negative-sense single-stranded RNA virus containing 10 genomic seqments and is the only member of the genus Tilapinevirus in the family Amnoonviridae (ICTV, 2018; Del-Pozo et al., 2017; Surachetpong et al., 2017; Surachetpong et al., 2020). Its 10,323 kb total genome encodes 14 functional genes and 14 viral proteins although functions have not been assigned to all these proteins (Acharya et al., 2019; Bacharach et al., 2016; Surachetpong et al., 2017). Our previous research indicated that viral genome segment 8 encoded a 20 kDa protein (VP20) that was highly antigenic and induced high titers of specific anti-TiLV antibodies (Hu et al., 2012). Therefore, VP20 is a potential vaccine candidate against TiLV but this protein has not been assessed for its protective potential in tilapia.

Vaccination is the most effective way to prevent viral disease and many kinds of vaccines have proved successful in reducing the severity of fish viral diseases (Zeng et al., 2016; Hjeltnes et al., 2017; Jee et al., 2017; Matsuura et al., 2019). A recent study revealed that following tilapia exposure to TiLV, the surviving fish mount a robust and protective immune response which provide a complete protection against reinfection (Tattiyapong et al., 2020). It means that developing an efficacious vaccine against TiLV is very promising. There are four primary vaccine types: attenuated live vaccines, inactivated vaccines, subunit vaccines and DNA vaccines and all four types are already commercially available (Wang et al., 2020; Evensen and Leong, 2013). At present, both attenuated and inactivated TiLV vaccines are being developed and tested (Surachetpong et al., 2020; Zeng et al., 2021), but none of them are commercially available. In addition, inactivated vaccines are expensive to produce and susceptible to the loss of important immunogenicity during inactivation and live-attenuated vaccines carry a limited antigen mass and pose potential risks of virulence reversion and immunosuppression (Pitcovski et al., 2003; Celene et al., 2014). Furthermore, a major shortcoming of these two vaccine types is that they do not allow for the differentiation of vaccinated from naturally infected fish. In contrast, protein subunit and DNA vaccines solve the latter problem and the protein subunit vaccines are stabile and possess better safety profiles may not elicit complete T cell-mediated immunity (Ridpath, 2013; Pecora et al., 2012). Fortunately, plasmid DNA vaccines provide several advantages over classical vaccines and both humoral and cellular immunity can be induced with this vaccine type (Ferraro et al., 2011; Menon et al., 2017; Evensen and Leong, 2013; Chen et al., 2018; Hanne et al., 2018). Despite the great potential for plasmid-based vaccines, recent studies have concluded that plasmid vaccines used alone fail to induce significant protection against pathogens even when combined with molecular adjuvants (Lu, 2009; Liu, 2011). Interestingly, a DNA prime-protein boost regimen has proven more effective at eliciting an immune response than either the solo DNA or subunit vaccine immunizations (Pal et al., 2006; Li et al., 2006a, Li et al., 2006b; Liang et al., 2006; Kardani et al., 2016; Gupta and Garg, 2015; Costa et al., 2016).

In the present study, the S8 gene that encodes the VP20 protein of the TiLV isolate 2017A was codon-optimized for fish usage and cloned into plasmid vector pVAX1 (pVoptiVP20). Previous studies have shown that protein-based vaccines should be supplemented with adjuvants (Roozbehani et al., 2018; Kordbacheh et al., 2019). Aluminum is a safe and effective adjuvant that improved antigen uptake by antigen presenting cells and is widely used in vaccine formulations (He et al., 2015). Therefore, the VP20 subunit vaccine used in this study was prepared by purified recombinant VP20 protein combined with M402 enhanced aluminum adjuvant. Tilapia were vaccinated singly with pVoptiVP20 and VP20 subunit vaccine and together using VP20 subunit vaccine as a booster after DNA vaccination. We evaluated the immunogenicity and protective efficacy against TiLV infection that were elicited using these strategies to control TiLV infections. Our work may identify a novel, safe and effective vaccine and approach for controlling TiLV infections.