Antibody to Poly-N-acetyl glucosamine provides protection against intracellular pathogens: Mechanism of action and validation in horse foals challenged with Rhodococcus equi

Abstract

Immune correlates of protection against intracellular bacterial pathogens are largely thought to be cell-mediated, although a reasonable amount of data supports a role for antibody-mediated protection. To define a role for antibody-mediated immunity against an intracellular pathogen, Rhodococcus equi, that causes granulomatous pneumonia in horse foals, we devised and tested an experimental system relying solely on antibody-mediated protection against this host-specific etiologic agent. Immunity was induced by vaccinating pregnant mares 6 and 3 weeks prior to predicted parturition with a conjugate vaccine targeting the highly conserved microbial surface polysaccharide, poly-N-acetyl glucosamine (PNAG). We ascertained antibody was transferred to foals via colostrum, the only means for foals to acquire maternal antibody. Horses lack transplacental antibody transfer. Next, a randomized, controlled, blinded challenge was conducted by inoculating at ~4 weeks of age ~106 cfu of R. equi via intrabronchial challenge. Eleven of 12 (91%) foals born to immune mares did not develop clinical R. equi pneumonia, whereas 6 of 7 (86%) foals born to unvaccinated controls developed pneumonia (P = 0.0017). In a confirmatory passive immunization study, infusion of PNAG-hyperimmune plasma protected 100% of 5 foals against R. equi pneumonia whereas all 4 recipients of normal horse plasma developed clinical disease (P = 0.0079). Antibodies to PNAG mediated killing of extracellular and intracellular R. equi and other intracellular pathogens. Killing of intracellular organisms depended on antibody recognition of surface expression of PNAG on infected cells, along with complement deposition and PMN-assisted lysis of infected macrophages. Peripheral blood mononuclear cells from immune and protected foals released higher levels of interferon-γ in response to PNAG compared to controls, indicating vaccination also induced an antibody-dependent cellular release of this critical immune cytokine. Overall, antibody-mediated opsonic killing and interferon-γ release in response to PNAG may protect against diseases caused by intracellular bacterial pathogens.

In vitro evaluation of complement deposition and opsonophagocytic killing of Rhodococcus equi mediated by poly‐N‐acetyl glucosamine hyperimmune plasma compared to commercial plasma products

“…We conclude that PNAG‐HIP was superior to RE‐HIP at killing of R. equi in vitro. Our finding that PNAG‐HIP mediated significantly more killing than an equivalent volume of SP in vitro is consistent with the observation that PNAG‐HIP mediated significantly more protection than SP against intrabronchial infection of foals …”

“…The following plasma products were used in this study: non‐HIP (SP; High‐Glo Equine IgG, Mg Biologics, Inc, Ames, Iowa), plasma hyperimmune to R. equi (RE‐HIP; ReSolution Rhodococcus Equi Antibody, Mg Biologics, Inc), and plasma hyperimmune to PNAG (PNAG‐HIP; Mg Biologics, Inc). Concentrations of IgG for each plasma were measured by ELISA performed in duplicate using a commercial kit (Horse IgG ELISA Kit, Abcam, Cambridge, Massachusetts).…”

“…Concentrations of IgG for each plasma were measured by ELISA performed in duplicate using a commercial kit (Horse IgG ELISA Kit, Abcam, Cambridge, Massachusetts). The complement source was prepared from commercial equine serum (Sigma‐Aldrich, St. Louis, Missouri) that was adsorbed twice with R. equi (EIDL 5‐331) at 4°C for 30 minutes to remove R. equi ‐specific antibodies, as previously described . After adsorption, the complement solution was centrifuged, filter sterilized, diluted to a 40% concentration in phosphate‐buffered saline (PBS), and frozen until use.…”

“…The capsule‐like polysaccharide, β‐(1→6)‐linked poly‐ N ‐acetyl‐glucosamine (PNAG), is a highly conserved sugar molecule present on the surface of many pathogenic microbes including R. equi . Recently, our laboratory has demonstrated that vaccinating pregnant mares protected their foals against experimental infection with R. equi when challenged at approximately 4 weeks of age . Moreover, transfusion of plasma hyperimmune for PNAG (PNAG‐HIP) protected additional foals against a similar experimental infection .…”

“…Although these findings indicate that PNAG is a valid target for immunizing donors to prepare PNAG‐HIP, this study only compared foals transfused with standard non‐HIP (SP) to those treated with PNAG‐HIP; comparison of PNAG‐HIP with RE‐HIP was lacking. Protection against R. equi either by PNAG vaccination of mares or by transfusion of PNAG‐HIP to foals was mediated by anti‐PNAG antibodies that fix complement and mediate opsonophagocytic killing (OPK) by neutrophils in conjunction with complement . Our objective was to better characterize the protection provided by PNAG‐HIP relative to RE‐HIP by comparing complement deposition and OPK of PNAG‐HIP, RE‐HIP, and SP in vitro.…”