Abstract
Porcine reproductive and respiratory syndrome (PRRS) is a devastating disease which impacts the pig industry worldwide. The disease is caused by PRRS viruses (PRRSV-1 and -2) which leads to abortions and other forms of reproductive failure in sows and severe respiratory disease in growing pigs. Current PRRSV vaccines provide limited protection; only providing complete protection against closely related strains. The development of improved PRRSV vaccines would benefit from an increased understanding of epitopes relevant to protection, including those recognized by antibodies which possess the ability to neutralize distantly related strains. In this work, a reverse vaccinology approach was taken; starting first with pigs known to have a broadly neutralizing antibody response and then investigating the responsible B cells/antibodies through the isolation of PRRSV neutralizing monoclonal antibodies (mAbs). PBMCs were harvested from pigs sequentially exposed to a modified-live PRRSV-2 vaccine as well as divergent PRRSV-2 field isolates. Memory B cells were immortalized and a total of 5 PRRSV-specific B-cell populations were isolated. All identified PRRSV-specific antibodies were found to be broadly binding to all PRRSV-2 isolates tested, but not PRRSV-1 isolates. Antibodies against GP5 protein, commonly thought to possess a dominant PRRSV neutralizing epitope, were found to be highly abundant, as four out of five B cells populations were GP5 specific. One of the GP5-specific mAbs was shown to be neutralizing but this was only observed against homologous and not heterologous PRRSV strains. Further investigation of these antibodies, and others, may lead to the elucidation of conserved neutralizing epitopes that can be exploited for improved vaccine design and lays the groundwork for the study of broadly neutralizing antibodies against other porcine pathogens.
Highlights
Porcine reproductive and respiratory syndrome virus (PRRSV) can spread rapidly among pigs through multiple routes of transmission, including aerosolization, causing severe respiratory disease that often leads to secondary infections and death in nursery age pigs [1]
Memory B cells from two pigs, BNW4 and BNW7, that had been exposed several times to both PRRSV ATP vaccine and field virus and whose sera neutralized a broad range of PRRSV strains (Supplementary Figure 1), were sorted and immortalized
A total of 480 multi-cell B cell populations were obtained from pig BNW7 through cell sorting based on binding of Alexa Fluor-647 (AF647) labeled PRRSV-2 VR2332, GFP presence, and surface expression of IgG
Summary
Porcine reproductive and respiratory syndrome virus (PRRSV) can spread rapidly among pigs through multiple routes of transmission, including aerosolization, causing severe respiratory disease that often leads to secondary infections and death in nursery age pigs [1]. Recent work has determined that the PRRSV GP2-GP3-GP4 minor glycoprotein complex is key to viral entry into the host macrophage, through its interactions with CD163 [21,22,23] Based on these findings, other studies have suggested sites of neutralization on GP4 and GP3 inducing renewed interest in the GP2-GP3-GP4 complex as a target for neutralization [24,25,26,27,28]. Passive immunization of sows with PRRSV-neutralizing antibodies have been shown to prevent viremia and viral transfer to the fetus, but an individual pig’s clearance of virus does not always correlate to a neutralizing antibody response [29, 30]. One antibody was determined to neutralize homologous but not heterologous PRRSV strains
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