The polarization status of porcine alveolar macrophages (PAMs) determines the infectivity of porcine reproductive and respiratory syndrome virus (PRRSV). PRRSV infection skews macrophage polarization toward an M2 phenotype, followed by T-cells inactivation. CD163, one of the scavenger receptors of M2 macrophages, has been described as a putative receptor for PRRSV. In this study, we examined two types of PRRSV-2-derived recombinant antigens, A1 (g6Ld10T) and A2 (lipo-M5Nt), for their ability to mediate PAM polarization and T helper (Th1) response. A1 and A2 were composed of different combination of ORF5, ORF6, and ORF7 in full or partial length. To enhance the adaptive immunity, they were conjugated with T cells epitopes or lipidated elements, respectively. Our results showed that CD163⁺ expression on PAMs significantly decreased after being challenged with A1 but not A2, followed by a significant increase in pro-inflammatory genes (TNF-α, IL-6, and IL-12). In addition, next generation sequencing (NGS) data show an increase in T-cell receptor signaling in PAMs challenged with A1. Using a co-culture system, PAMs challenged with A1 can induce Th1 activation by boosting IFN-γ and IL-12 secretion and TNF-α expression. In terms of innate and T-cell-mediated immunity, we conclude that A1 is regarded as a potential vaccine for immunization against PRRSV infection due to its ability to reverse the polarization status of PAMs toward pro-inflammatory phenotypes, which in turn reduces CD163 expression for viral entry and increases immunomodulation for Th1-type response.

PRRSV infects CD163-positive macrophages and skews their polarization toward an M2 phenotype, followed by T-cell inactivation. In our previous study, we found that recombinant protein A1 antigen derived from PRRSV-2 was a potential vaccine or adjuvant for immunization against PRRSV-2 infection due to its ability to repolarize macrophages into M1 subtype, thereby reducing CD163 expression for viral entry and promoting immunomodulation for Th1-type responses, except for stimulating Toll-like receptor (TLR) activation. The aim of our current study was to evaluate the effects of another two recombinant antigens, A3 (ORF6L5) and A4 (NLNsp10L11), for their ability to trigger innate immune responses including TLR activation. We isolated pulmonary alveolar macrophages (PAMs) from 8- to 12-week-old specific pathogen free (SPF) piglets and stimulated them with PRRSV (0.01 MOI and 0.05 MOI) or antigens. We also investigated the T-cell differentiation by immunological synapse activation of PAMs and CD4⁺ T-cells in the cocultured system. To confirm the infection of PRRSV in PAMs, we checked the expression of TLR3, 7, 8, and 9. Our results showed that the expression of TLR3, 7, and 9 were significantly upregulated in PAMs by A3 antigen induction, similar to the extent of PRRSV infection. Gene profile results showed that A3 repolarizes macrophages into the M1 subtype potently, in parallel with A1, as indicated by significant upregulation of proinflammatory genes (TNF-α, IL-6, IL-1β and IL-12). Upon immunological synapse activation, A3 potentially differentiated CD4 T cells into Th1 cells, determined by the expression of IL-12 and IFN-γ secretion. On the contrary, antigen A4 promoted regulatory T cell (T-reg) differentiation by significant upregulation of IL-10 expression. Finally, we concluded that the PRRSV-2 recombinant protein A3 provided better protection against PRRSV infection, suggested by its capability to reeducate immunosuppressive M2 macrophages into proinflammatory M1 cells. As M1 macrophages are prone to be functional antigen-presenting cells (APCs), they can call for TLR activation and Th1-type immune response within the immunological synapse.

Objective: The Baculovirus Expression Vector System (BEVS) is an established eukaryotic platform for high-yield recombinant protein production and is widely utilized for generating vaccine antigens, including recombinant classical swine fever virus E2 glycoprotein, African swine fever virus structural proteins (p72, p30, and p54), and porcine reproductive and respiratory syndrome virus envelope proteins GP5 and M. These antigens interact with macrophage-associated receptors, including CD163 and CD206, to modulate immune responses through macrophage polarization. However, whether baculovirus vector-derived components intrinsically influence macrophage polarization remains unclear, raising concerns regarding potential impacts on biological activity, vaccine efficacy, and downstream purification strategies. Methods: In this study, an M2-like tumor-associated macrophage (TAM) model capable of repolarizing toward an M1-like phenotype was established to distinguish the transcriptomic effects of the baculovirus vector control (VC) from those of the recombinant antigen (A1). Comparative RNA sequencing, Gene Set Enrichment Analysis (GSEA), functional enrichment analyses, and linear regression analysis were performed to evaluate vector- and antigen-associated transcriptional responses. Results: Comprehensive transcriptomic analysis showed that VC treatment induced only minimal transcriptomic alterations, with limited immune-related pathway enrichment and expression profiles that remained highly similar to those of untreated M2-like TAMs. Furthermore, linear regression analysis demonstrated negligible correlations between VC-induced and A1-induced transcriptional responses indicating that the limited changes associated with the vector backbone did not correspond to the receptor-mediated immune responses elicited by the recombinant antigen. In contrast, A1 stimulation induced distinct transcriptional reprogramming characterized by enrichment of pro-inflammatory and immune-activation pathways and a shift toward an M1-like macrophage phenotype. Conclusions: These findings suggest the negligible correspondence between VC- and A1-induced transcriptional profiles further supports the transcriptomic noises and signals of the baculovirus vector and inserted proteins, reinforcing its suitability as a platform for macrophage-targeted vaccine development while providing a basis for future optimization of downstream purification strategies.