Immunogenic and antigenic analysis of recombinant NSP1 and NSP11 of PRRS virus

Abstract Background Porcine reproductive and respiratory syndrome virus (PRRSV) is an enveloped RNA virus in the order Nidovirales, family Arteriviridae, genus Betaarterivirus. Antibodies against nonstructural proteins (NSPs) from this virus can be found in pigs starting 4 days postinfection and they remain detectable for several months. Objective The goal of this study was to evaluate the immunogenicity and antigenic properties of recombinant proteins NSP1 and NSP11 expressed in Escherichia coli cells, as well as to assess the neutralization activity that they elicit. Methods We obtained the complete ORF‐1 genes coding for NSP1 and NSP11 from PRRSV using the VR‐2332 strain. Cloning was performed with the pET23a(+) vector with a histidine tag (His6), linearized by restriction enzyme digestion; the expression of the NSP1 and NSP11 clones was induced in OverExpress C41(DE3) chemically competent cells. Recombinant proteins were used to generate hyperimmune sera and we perform serological assays to confirm neutralizing antibodies. Results The expressed recombinant NSP1 and NSP11 were found to be immunogenic when injected in pigs, as well as demonstrated higher specificity in recognition of antigen in field sera from pigs positive infected with PRRSV. Furthermore, both NSP1 and NSP11 recombinant proteins elicited PRRSV neutralizing antibodies. Conclusions In this study, we demonstrated the immune humoral response to NSP 1 and NSP11, and neutralizing‐antibody response to PRRSV VR2332 strain in sera from hyperimmunized pigs.


INTRODUCTION
The causal agent of porcine reproductive and respiratory syndrome (PRRS) is an enveloped RNA virus from the order Nidovirales, family Arteriviridae, genus Betaarterivirus, subgenus Ampobarterivirus. The PRRS virus (PRRSV) has two known variants: Betaarterivirus suid 1, previously known as PRRSV-1, and B. suid 2, previously known as PRRSV-2 (King et al., 2018). Because PRRS increases fetal death rates by 20%-30% (Goyal, 1993), it has a strong negative economic impact on pork production. In the United States, economic losses due to PRSS have been estimated at 664 million dollars annually. On pig farms alone, losses are calculated at 302.06 million dollars, chiefly due to lower prices at time of weaning (Holtkamp et al., 2013;Neumann et al., 2005).
There are commercial vaccines available on the market worldwide, the most widely used contain the entire genome based on modified active virus (MLV) or inactive form. Most PRRS vaccines elicit specific humoral and cellular immune responses that confer protection against homologous parental strains and partial protection to heterologous strains, but there are safety concerns, such as a high mutation rate that causes reversion to virulence and recombination between vaccines and field strains ( Renukaradhya et al., 2015), in addition, the response they induce is late, which confers partial protection when the animals face the field virus. (Renukaradhya et al., 2015).
While previous studies have focused on the immune response against PRRSV structural proteins like N or GP5, their high variability and poor individual response to the virus have prevented us from finding a feasible strategy against this pathogen agent (Chand et al., 2012;Renukaradhya et al., 2015). Besides, we have demonstrated the neutralization capacity of NSP1 (Leng et al., 2017;Su et al., 2019), which have been used in this study.
PRRSV infects cells that participate in both the innate and the adaptive immune response, such as macrophages 'particularly with alveolar location and dendritic cells' (Lunney et al., 2010;Rossow, 1998;Welch & Calvert, 2010). NSP 11 has a function similar to NSP1 and antagonizes IFN type I, specifically IFNβ production (Montaner-Tarbes et al.,

2019
). An anti-NSP11 monoclonal antibody (mAb) created to test reactivity against genotype 1 and genotype 2 PRRSV was able to induce humoral immune response in PRRSV infected pigs. Furthermore, a Bcell epitope on the surface of NSP11 using a specific mAb was also able to induce humoral immune response in pigs infected with PRRSV (Jiang et al., 2017). The goal of this study was to evaluate immunogenicity, as well as the neutralization ability of recombinant NSP1 and NSP11 to contribute towards potential development of new generation PRRSV vaccines.
The cells were propagated in Eagle's minimal essential medium (MEM), supplemented with L-glutamine and 10% fetal bovine serum (FBS).

Producing recombinant proteins, cloning and expressing NSP1 and NSP11
We designed specific primers to amplify the entire ORF-1 genes that code for NSP1 and NSP11 from the PRRSV ATCC VR-2332 strain, via RT-PCR. These primers end sequences incorporated BamHI, XhoI and EcoR1 (Table 1), which correspond to enzymatic restriction sites.
We obtained viral RNA using the OneStep RT-PCR Kit® (Qiagen, Hilden, Germany), following the manufacturer's instructions. The samples were amplified for 35 cycles, preceded by an initial cDNA cycle at 50 • C for 30 min and a denaturing stage at 95 • C for 15 min. A final extension cycle was performed at 72 • C for 10 min. We used a mastercycler gradient thermocycler (Eppendorf, Hamburg, Germany) to carry out the RT-PCR, and both primers and test conditions are shown in Table 1.
We verified the presence of amplified fragments on 2% agarose gels with tris-acetate-EDTA buffer (TAE 1X) by staining with ethidium bromide and observing samples in a transilluminator. We used a

Purifying recombinant NSP1 and NSP11
Histidine-tagged, recombinant NSP1 and NSP11 were purified in a His-

Characterizing NSP1 and NSP11 by western blot
We transferred eluted proteins to a PVDF membrane and performed 1D/2D SDS-PAGE at 110 V for 1 h (4 • C). Following protein separation, the membranes were blocked with 3% BSA in PBS-T for 1 h. After this, membranes were washed with PBS-T and incubated for 1 h with a mouse monoclonal antibody (Roche Diagnostics, Basel, Switzerland) targeting the His6 label of the recombinant product, in a 1:4000 dilution. The membranes were then washed with PBS-T and incubated for 1 h with mouse anti-mouse polyclonal antibody, IgG isotype, linked to horseradish peroxidase (Sigma) in a 1:2000 dilution. We visualized the reaction using 3 mg/ml 3,3-diaminobenzidine (Sigma) in PBS-T and 30% hydrogen peroxide in a 1:1000 dilution.

Producing hyperimmune sera to NSPs
We immunized four York-Landrace pigs from a PRRS-free farm, two pigs each received either recombinant NSP1 or NSP11. The absence of anti-PRRSV antibodies was verified using the commercial HerdChek PRRS 2XR Ab ELISA test (IDEXX, Westbrook, MA). Each pig was vaccinated four times at 15 day intervals with 100 μg protein doses in a 1:1 protein-Montanide adjuvant emulsion, via deep intramuscular route in the neck. We established a baseline using a blood sample taken before the first immunization. Then, blood samples were taken 7 days following each immunization, to monitor serum antibody titres and determine when peak antibody titres occurred via ELISA tests.

Antigenicity of recombinant NSPs
To determine the antigenicity of the recombinant proteins, we developed an ELISA method (ELISA "in house") using purified recombinant NSPs as antigen sources and following the previous report (García-Plata, 2016). We used 50 negative and 50 positive sera (commercial kit) from pigs belonging to farms where PRRSV is present, and then compared the results with an ELISA "in house".
Before tests on field sera were run, we tested the purified recombinant proteins against known positive sera identified with high antibody titres and found recognition by these sera against both proteins.

Serum neutralization
A serum neutralization assay was performed to estimate the NSPs neutralizing capacity of hyperimmune sera. Sera were heat inactivated at 56 • C for 30 min, and then serum neutralization assay was performed following the method described by Leng et al., 2017

Replication and cloning of recombinant proteins
We confirmed replication of PRRSV in MA-104 cells via qRT-PCR (Table 2)

Purification and yield of recombinant proteins
Expressed proteins are located in the insoluble fraction of bacterial cells and were purified by inclusion antibodies under denaturation conditions. We obtained fractions in the concentration peaks in a polyacrylamide gel, and the presence of pure proteins was verified. Production yield was 597.5 μg/ml for NSP1 and 201.7 μg/ml for NSP11. Both recombinant proteins reacted with anti-His6 antibodies in immuno transfer. We found antigen and complete virus recognition for NSP1 or NSP11 in the different dilutions that were used in the western blot testing of hyperimmune sera (Figure 3).

Hyperimmune sera
Pigs immunized with recombinant NSPs seroconverted at different times postinoculation (Figure 4). Specific antibodies against NSP1 were detectable between days 30 and 45 postinfection and anti-NSP11 antibodies were detected on day 15 postinfection, the highest levels were observed on day 60, with an optical density value of 1.035.

Neutralization antibody
We detected antibodies for NSP1, NSP11 and PRRSV in hyperimmune sera. Negative serum samples showed no response to the test and the cytopathic effect to differentiate positive from negative responses was evident in cell cultures ( Figure 5). Hyperimmune sera showed a better response at lower infecting doses (100 and 300). For NSP1, we obtained protective titres of 1:32 at an infectious dose of 100, and of 1:16 titres at an infectious dose of 300. We found protective titres of 1:8 and 1:4 for NSP11 at infectious doses of 100 and 300, respectively.

DISCUSSION
No effective anti-PRRSV vaccine has been developed yet, and although DNA, subunit and attenuated vaccines have been tested, their potential as a substitute of the MLV anti-PRRS vaccine currently in use, is uncertain (Nan et al., 2017). An early and long-lasting immune response against PRRSV NSPs could be the key to this goal.
Some studies have reported the production of hyperimmune sera against PRRSV in mice (Bi et al., 2017), or by inoculating pigs with peptides (Díaz et al., 2009). In both these cases, the hyperimmune serum is targeted to B-cell epitopes, which have a higher probability of generating antibodies. On the other hand, PRRSV NSPs play a key role in the processing and maturation of the virion structural repertoire. Swine are known to mount antibody responses to these proteins with significant cross-reactivity, which seems to be equal to or higher than that induced by nucleocapsid protein (Johnson et al., 2007). and ORF2-7 + 3′UTR was exchanged between two infectious strains, that study also found that ORF1a is also a PRRSV neutralization region (Leng et al., 2017); several works have focused in detecting antibodies against viral structural proteins (Cancel-Tirado et al., 2004;Díaz et al., 2009;Wootton et al., 1998). These justifies the use of NSPs as components of a candidate vaccine that could elicit an early, protective immune response against PRRSV.
The time-course of antibody response showed that antibody response to NSP7 is comparable to the response to NSP1 and NSP2, as well as to the antigens used in the IDEXX commercial ELISA kit (Brown et al., 2009 (Gonin et al., 1999;Loemba et al., 1996;Yoon et al., 1994). However, few reports show that there is a neutralization region in PRRSV ORF1, mainly in NSP2, which has been better studied so far (Leng et al., 2017).
The main neutralizing activity against PRRSV has been reported to target the GP5 protein (Lopez & Osorio, 2004;Popescu et al., 2017;Vashisht et al., 2008). However, our results suggest that NSPs, in this case NSP1 and NSP11, can be recognized by Recombinant protein-based approaches allow us to rapidly and easily synthesize large amounts of a product whose characteristics are very similar to those of a natural protein, and as epitope-based vaccines have several benefits including safety, high specificity and ease of use (Nan et al., 2017). Herein, we demonstrated that NSP1 and NSP11 open a new approach to produce a protective recombinant vaccine to control PRRS.
These suggests that both proteins contain highly antigenic regions, however further studies are needed to confirm whether this humoral response provide cross-neutralization against heterologous PRRSV strains.

CONCLUSIONS
Our results indicate that recombinant PRRSV NSPs can be antigenic and immunogenic. This study is an initial exploration to propose the use of recombinant proteins as a novel immunogenic approach against PRRSV and/or to develop a more precise diagnostic tool.