Engineered antibodies show promise for blocking PRRSV entry in pigs

Porcine reproductive and respiratory syndrome virus (PRRSV) remains one of the most damaging pathogens of swine worldwide, causing reproductive failure in breeding herds and respiratory disease in pigs of all ages. A new review published in the journal Animal Diseases argues that the earliest step of infection, viral entry, is the most promising point at which antibody-based interventions can intervene, and it maps out which viral and host molecules are the best targets for engineered antibodies designed to block that step.

The review, authored by Siqi Zhao, Xudong Yin and colleagues with corresponding author Fang He, synthesizes current knowledge of PRRSV entry biology and antibody engineering. Its central argument is that not all entry-related targets are equally valuable. Some determinants are directly required for productive infection, while others merely facilitate binding or uptake in laboratory systems, and confusing the two categories risks directing antibody development toward targets that will not deliver broad protection in the field.

PRRSV entry is a multistep, receptor-mediated process. Viral envelope proteins first engage attachment and internalization factors on the host cell surface, then the virus is taken up through several endocytic routes, including clathrin-mediated endocytosis, macropinocytosis, and caveolae or lipid raft-related pathways. These uptake mechanisms operate within the broader receptor-mediated entry process rather than as alternatives to it, and their relative contributions vary with viral strain, cell type, and experimental conditions. Among all host molecules identified so far, the scavenger receptor CD163 stands apart: it is the essential receptor required for productive infection, whereas factors such as heparan sulfate, Siglec-1/CD169, HSPA8, MYH9, vimentin, and ATP1A1 function mainly as attachment or trafficking helpers in cell-based systems.

The genetic evidence for CD163’s central role is striking. Pigs lacking CD163, or lacking its SRCR5 domain specifically, show complete or near-complete resistance to PRRSV infection, while Siglec-1-deficient pigs remain fully susceptible. This distinction matters for antibody design because it identifies CD163 as the most biologically validated host target, while the auxiliary factors should be treated primarily as in vitro observations rather than evidence that the essential receptor can be bypassed in living animals.

On the viral side, the review highlights the minor glycoprotein complex GP2–GP3–GP4, which is closely associated with receptor binding, and the major envelope proteins GP5 and M, which participate in early virus–cell interactions as the M/GP5 complex. Among these, GP4 emerges as the most promising viral target. A recently described GP4-specific porcine monoclonal antibody recognizes a conserved GP4 epitope, interferes with the GP4–CD163 interaction, and shows broad neutralizing activity against both PRRSV-1 and PRRSV-2. Earlier epitope mapping studies had already identified neutralization-associated regions in GP4, but they also showed that antibody pressure on this protein can drive viral variation, meaning single-antibody strategies against GP4 remain vulnerable to antigenic drift.

GP3 is a second relevant viral target. The GP3-specific monoclonal antibody mAb-PN9cx3 showed broad-spectrum recognition and neutralizing activity against heterogeneous strains, reduced viral replication in porcine alveolar macrophages, and decreased tissue viral loads and lung lesions in challenged piglets. GP5, long regarded as the major envelope glycoprotein and a principal focus of neutralizing antibody research, presents a more difficult picture: many GP5-targeting antibodies show limited cross-strain neutralization, and recent work confirms that their activity can differ markedly between homologous and heterologous strains. The M protein, one of the most conserved PRRSV proteins, forms a disulfide-linked heterodimer with GP5 and has been identified as a ligand for sialoadhesin, but direct evidence supporting M as an independent antibody target remains limited.

Turning to antibody formats, the review identifies nanobodies as the most extensively studied novel format in PRRSV research. Derived from the variable domain of camelid heavy-chain antibodies, nanobodies are small, soluble, and structurally simple, and their elongated CDR3 loops can access recessed or glycan-masked epitopes that conventional antibodies may miss. The clearest viral-target evidence comes from GP4-targeting nanobodies Nb6, Nb31, and Nb85, which significantly reduced PRRSV infection at 10 micromolar, with Nb31 shown to interfere with both viral attachment and internalization during infection with the PRRSV-II XJSW2021 isolate. On the host side, nanobodies recognizing the SRCR5–6 or SRCR5–9 region of CD163 inhibited multiple PRRSV-2 isolates in vitro; nanobody Nb2 completely blocked PRRSV-II HuN4 infection at 100 micrograms per milliliter at an inoculum of 100 TCID50, and Nb1 and Nb2 at 50 micrograms per milliliter inhibited a broad panel of isolates including JXA1, HuN4, JK-100, ZJfh17, JS18-3, ZJnb16-2, and HNxx16. CD163-targeting nanobodies have also been reported to reduce virus-induced inflammatory signaling.

Single-chain variable fragments, or scFvs, offer a complementary recombinant format that preserves paired heavy- and light-chain antigen recognition in a smaller molecule. They can be generated by cloning variable region genes from hybridoma cells or by screening phage display libraries. The most advanced scFv evidence involves GP3: a GP3-specific scFv built from mAb-PN9cx3 showed antiviral activity in vitro, and a Lactobacillus plantarum-delivered construct reduced tissue viral loads and improved protection in piglets challenged with PRRSV-JXA1, with serum viral load falling to 1.4 × 10^4 copies per microliter at 10 days post-infection and a survival rate of 80 percent. Phage display-derived scFvs have so far been isolated mainly against nonstructural proteins such as NSP1β, which inhibited replication in vitro, leaving entry-directed candidates from this route largely unexplored.

Engineered antibodies extend these fragment formats by adding functional modules. Cell-penetrating strategies, such as TAT peptide fusion, have allowed nanobodies against the N protein and cell-penetrating scFvs against NSP1β to enter cells and suppress intracellular viral replication, although these post-entry approaches complement rather than replace surface entry blockade. Fc fusion offers another route: the NSP9-specific nanobody Nb6 fused to porcine IgG Fc showed improved antiviral activity in porcine alveolar macrophages compared with the unmodified nanobody, but Fc fusion reintroduces Fc receptor interactions, and Fcγ receptor-mediated antibody-dependent enhancement is documented in PRRSV, so Fc-modified fragments must be evaluated carefully. A further design fuses PRRSV-binding nanobodies with CD163-derived peptides to simultaneously engage viral particles and interfere with virus–receptor interactions, efficiently inhibiting infection in vitro and providing a conceptual basis for multivalent or bispecific antibodies that target both GP4 and CD163 to raise the genetic barrier to viral escape.

The review’s authors are candid about the remaining barriers. Most candidate antibodies have been evaluated only in vitro, and systematic animal studies are needed to confirm that cell-based activity translates into effective protection, reduced viral shedding, and improved clinical outcomes in pigs. Broad protection remains difficult given PRRSV’s marked genetic diversity and the strain-dependent activity of many neutralizing antibodies. Nanobodies and scFvs, typically around 15 to 30 kilodaltons, may undergo rapid renal clearance and short serum half-life, yet pharmacokinetic data for most PRRSV-targeted fragments are lacking. Manufacturing costs, formulation stability, gastrointestinal survival for orally delivered fragments, and potential immunogenicity of camelid-derived nanobodies in pigs all require attention. For host-directed strategies targeting CD163, antiviral efficacy must be balanced against risks to macrophage homeostasis, since CD163 participates in hemoglobin scavenging and immune regulation. The authors also note the absence of standardized head-to-head comparisons among antibody formats and call for matched assays against identical targets, particularly GP4 and CD163, measuring binding affinity, receptor-blocking activity, neutralization breadth, escape risk, pharmacokinetics, delivery efficiency, and safety. Their conclusion is that conserved determinants involved in receptor recognition, above all GP4 and CD163, should remain the priorities for next-generation antibody development against PRRSV entry.

Subject of Research: Engineered antibody strategies for blocking PRRSV entry in swine

Article Title: Engineered antibodies for the inhibition of PRRSV entry

Article References: Zhao, S., Yin, X., Xu, J., Qiu, H., Pratama, A. C., Yang, H., & He, F. (2026). Engineered antibodies for the inhibition of PRRSV entry. Animal Diseases, 6(1), Article 59. https://doi.org/10.1186/s44149-026-00274-2

Image Credits: AI Generated

DOI: 10.1186/s44149-026-00274-2

Keywords: PRRSV, swine, viral entry, CD163, GP4, nanobodies, scFv, engineered antibodies, porcine alveolar macrophages, neutralizing antibodies, antiviral strategy, Animal Diseases