Date Published: July 19, 2018
Publisher: Public Library of Science
Author(s): Colette Cywes-Bentley, Joana N. Rocha, Angela I. Bordin, Mariana Vinacur, Safia Rehman, Tanweer S. Zaidi, Mark Meyer, Sarah Anthony, McKenzie Lambert, Daniel R. Vlock, Steeve Giguère, Noah D. Cohen, Gerald B. Pier, Thomas R. Hawn.
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.
Correlates of cellular and humoral immunity to major intracellular, non-viral pathogens capable of informing vaccine development are incompletely understood. It is unknown which ones can form the basis of a highly effective vaccine to prevent diseases such as tuberculosis (TB). Protection studies conducted to date, primarily in laboratory rodents and non-human primates, have not led to an effective human vaccine for such pathogens [1, 2] outside of the limited efficacy of the live Bacillus Calmette-Guerin whole-cell vaccine against TB [2–4]. Rhodococcus equi is a Gram-positive, facultative intracellular pathogen carrying an essential virulence plasmid that primarily infects alveolar macrophages of horse foals following inhalation. R. equi replicates within a modified phagocytic vacuole, with survival dependent on the virulence plasmid preventing phagosome-lysosome fusion, resulting in a granulomatous pneumonia that is pathologically similar to that caused by Mycobacterium tuberculosis infection in humans . R. equi also causes extrapulmonary disorders including osseous and intra-abdominal lymphadenitis [5–7]. The disease is of considerable importance to the equine industry [5, 7], and while some reports indicate vaccination and/or passive transfer of hyperimmune plasma using bactrin-based or virulence associated protein A vaccines can reduce the severity of R. equi pneumonia [8, 9], it is generally felt that most attempts to date to create an effective R. equi vaccine have been unsuccessful [10, 11]. There is no approved vaccine for R. equi in any animal species.
In this study we tested the hypotheses that antibody to the conserved surface microbial polysaccharide, PNAG, could mediate protection against a significant intracellular pathogen of horse foals, R. equi. Overall we supported this hypothesis by showing maternal immunization against the deacetylated glycoform PNAG induced antibodies that protected ~4-week-old foals from challenge with live, virulent R. equi. Mechanistically we found that vaccine-induced antibody to PNAG deposited complement component C1q onto the purified PNAG antigen, mediated opsonic killing of both extracellular and intracellular R. equi, and sensitized PBMC from vaccinated foals to release IFN-γ in response to PNAG. It appears that this spectrum of antibody activity induced by the 5GlcNH2-TT vaccine were all critical to the protective efficacy observed.