Characterization of the IgA response to PRRS virus in pig oral fluids

Abstract

Porcine Reproductive and Respiratory Syndrome (PRRS) is a complex model of host/virus relationship. Disease control measures often includes "acclimatization", i.e. the exposure of PRRS-naïve gilts and sows to PRRSV-infected pigs and premises before the breeding period. In this respect, we had repeatedly observed an association between PRRSV-specific IgA responses in oral fluids (OF) of gilts and block of PRRSV spread. Therefore, we set out to investigate in vitro the inhibition of PRRSV replication by OF samples with different titers of PRRSV-specific IgA and IgG antibody, using Real-time RT PCR. PRRSV yield reduction in monocyte-derived macrophages was associated with the IgA content in OF samples, whereas the IgG-rich samples were sometimes associated with antibody-dependent enhancement (ADE) of replication. Accordingly, we could discriminate between ADE-positive and ADE-negative PRRSV strains. Next, we separated Ig isotypes in OF samples of PRRSV-infected pigs by means of protein A and size exclusion chromatography. The above results were confirmed by using separated Ig isotypes. Both dimeric and monomeric IgA were associated with the strongest reduction of PRRSV replication. The treatment of pig macrophages with separated OF antibodies before PRRSV infection was also associated with PRRSV yield reduction, along with clear changes of both CD163 and CD169 surface expression. Our results point at a role of mucosal IgA in the control of PRRSV replication by extra- and/or intracellular interaction with PRRSV, as well as by induction of signals leading to a reduced susceptibility of macrophages to PRRSV infection.

Fig 1. Yield reduction assays.

Five PRRSV strains were reacted with IgA-low, IgA-medium, IgA-high OF samples, and with medium only (no OF), respectively. The replication of PRRSV in macrophages from blood monocytes was measured by Real time RT-PCR for PRRSV ORF 7 and expressed in terms of PRRSV genome copies / ml. Panel A: experiment 1. Panel B: experiment 2 on macrophages from another pig. P< 0.05 for PRRSV strain in experiment 2.

Fig 2. Permissiveness to PRRSV of pig macrophages.

The replication of 5 PRRSV strains was investigated on macrophages from blood monocytes of three different pigs under the same culture conditions. The replication of PRRSV was measured by Real time RT-PCR for PRRSV ORF 7 and expressed in terms of PRRSV genome copies / ml. P< 0.05 for macrophage population.

Fig 3. Separation of Ig fractions in OF samples.

Dimeric IgA and total monomeric IgA + IgG were separated by means of gel filtration chromatography. Samples were run on a Superdex 200 10/300 GL column (GE Healthcare) under HPLC conditions in an AKTA Purifier apparatus (GE Healthcare). Twenty-six, 0.5-mL fractions were collected and tested by ELISA for IgA and IgG antibody, respectively. This enabled us to detect the fractions corresponding to dimeric IgA (12 to 16) and monomeric IgG+IgA (18 to 21), respectively.

Fig 4. Yield reduction assay in PAM, test 1.

Ig fractions were separated from a pool of antibody-positive OF samples (s/p IgA: 2.73; s/p IgG: 3.13) and used in a yield reduction assay on PAM. Results are shown in terms of mean genome copies/ml for PRRSV strains 433/5, 009/8 and 377 ± 1 standard deviation. Separated Ig significantly differed for antiviral activity (P< 0.001; P< 0.05 for non-IgG fraction vs. IgG, and monomeric IgA/IgG vs. IgG).

Fig 5. Yield reduction assay in PAM, test 2.

Ig fractions were separated from an OF pool showing little if any IgA response (s/p IgA: 0.63; s/p IgG: 1.54). These IgA-low fractions exerted by far less or no antiviral activity on strains 009/8, 377 and 957, compared with the IgG fraction, whereas strain 433/5 replicated very poorly (P< 0.1, tendency).

Fig 6. Surface expression of CD169 in PAM, test 4.

The direct interaction of IgG, monomeric IgA+IgG and non-IgG fractions with PAM was investigated. Panel A: gating strategy. PAM were gated first by a combination of forward and side scatter. Next, viable cells were selected by staining with PI (red fluorescence), and submitted to singlet analysis. CD169+ PAM were detected by monoclonal antibody 3B11/11 and Alexa Fluor® 488 F(ab')2 fragment of goat anti-mouse IgG, IgM (H+L). Panel B: prevalence of CD169-high PAM is depicted in a bar graph. The three Ig fractions caused up-regulation of CD169 in PAM (P< 0.001 with respect to control cells).