Profiling of local disease-sparing responses to bovine respiratory syncytial virus in intranasally vaccinated and challenged calves

Highlights

• Vaccination alters the pharyngeal tonsil proteome of BRSV-challenged calves

• Responses associated with antigen presentation, neutrophil degranulation, and reactive oxygen species detoxification

• Magnitude of proteome response enhanced by multivalent formulations

• Correlation between pharyngeal tonsil vaccination response and disease-sparing outcomes

Abstract

Bovine and human respiratory syncytial viruses (BRSV, HRSV) are primary causes of pneumonia in calves and children respectively, with vaccination offering protection via antibody and cellular immune responses. However, with no vaccines currently licensed for human use, evaluation of local responses to BRSV vaccination may provide insights to aid the design of effective safe HRSV vaccines. Calves received intranasal single component BRSV vaccine or “3-Way” vaccine (BRSV, Bovine Herpes Virus-1 (BHV-1), Bovine Parainfluenza Virus Type-3 (BPIV-3)), and were BRSV-challenged 42 days post-vaccination. All vaccinates exhibited reduced pulmonary lesioning with elevated anti-BRSV serum IgG, and higher nasal anti-BRSV IgA in 3-Way vaccinates. Thirty-nine proteins associated with homeostatic and immune processes were altered in vaccinates, with enhanced 3-Way vaccinate group proteins associated with Th1/Th2 balance and immunoglobulin class switching. Proteins altered in the pharyngeal tonsil of animals euthanized early related to anti-inflammatory responses and lymphoid tissue remodeling. These findings indicate that multivalent vaccines distinctly modulate local immune responses, with clear correlation between the pharyngeal tonsil proteome profile and resulting immune protection and disease-sparing. This suggests that the efficacy of low-antigenic subunit vaccine components for problematic pathogens such as HRSV could be enhanced by use in combination with existing safe live vaccines.

Significance

This study demonstrates that vaccine valency can alter post-challenge proteome responses within the pharyngeal tonsil, a sentinel site of primary immune responses, with the magnitude of response dependent on antigen formulation. Observed differential responses can be attributed to antigenic material and viral nucleic acid from multivalent formulations providing additional T-cell epitopes and PAMPS. These findings indicate that incorporation of subunits proteins within multivalent formulations containing live virus has the potential to induce/skew a favorable immune response, utilising the natural adjuvanting effects of safe proven live vaccines.

Introduction

Human and bovine respiratory syncytial viruses (HRSV, BRSV) are important paramyxoviral respiratory pathogens in their respective hosts, with very similar pathobiology [1,2]. Although both viruses can cause disease in all ages of hosts, clinical infections are generally most severe in the young, coincident with the decay of passive immunity, and can be fatal [1,2]. Given its recognized importance in veterinary medicine, a variety of mucosal and parenteral vaccines for BRSV have been commercially available and commonly used since the late 1970's [4]. Many of these vaccines have been shown to confer significant disease-sparing immunity in a challenge model that closely parallels the naturally occurring disease [4]. In contrast, despite years of effort, equivalent immunoprophylactics are still wanting for human pediatric patients [5] due to safety and reactogenicity issues related to formalin inactivated virus [6,7] and HRSV glycoprotein G vaccinia virus vaccines [8]. As such, human RSV vaccine development is predominately focused on nucleic acid and subunit-based vaccines with several Phase 2 and 3 clinical trials targeting Fusion glycoprotein (gF) as the major neutralizing antigen [7].

In the field of vaccinology, refining vaccine components (in the form of subunit, peptide, virus-like particle or DNA vaccines) is employed under the pretense of supplying the immune system only with the necessary components to generate a measurable (potentially protective) immune response (often assessed by antibody titers) and/or reducing unwanted reactogenic effects. However, it is often seen that such approaches have poor efficacy due to reduced antigenicity from the removal of pathogen associated molecular patterns (PAMPs), requiring adjuvant use to boost suboptimal immunogenicity. Furthermore, multivalent vaccine formulations are further frowned upon as they may dilute the immune response, potentially reducing protection against a single pathogen in favor of protecting against many. The effects of these additional vaccine components, or how such components may alter local immune memory (favorably or unfavorably) to modulate responses to challenge, are often not assessed. This is of particular interest as subunit vaccine formulations often turn to native PAMPS (e.g. TLR agonists) or cytokines to boost efficacy. However, combination vaccine formulations themselves are potentially self-adjuvanting, encompassing a more diverse PAMP profile than monovalent vaccine formulations, with the potential to induce trained innate immunity/memory, which being enhanced protects against reinfection and secondary infection through mechanisms independent of T/B cell adaptive responses [9]. Currently, there are few reports that address the effect of vaccine valency on local immune responses or examine the potential to favorably boost or skew subunit vaccine immune responses through combination with safe MLV commercial vaccines.

In the post-genomic era, the rapid growth in ‘omic’ technologies has led to a greater understanding of physiological processes within the body, allowing researchers to understand how a particular perturbation in a biological system affects up-stream and downstream biological processes [[10], [11], [12]]. Proteomics, in contrast to transcriptomics, has the capacity to highlight actual changes in functional protein levels and provide information not only on proteins produced within tissue of interest but also on those effector proteins originating from the circulatory system. Proteomics is therefore particularly suited to the investigation of immune responses to vaccination which incorporates not only local immune responses but also system responses derived from lymphatic tissue distributed throughout the body. Proteomic marker screening technologies offer the ability to assess potential diagnostic indicators of infection/disease [11] and the application of proteomics to human medicine has resulted in promising prospects for improved vaccinology [13,14]. The pharyngeal tonsil or adenoid, as it is otherwise known in humans, is unique among tonsils, being lined by pseudostratified columnar (respiratory) epithelium [15] in which viruses such as BRSV replicate [16]. This, together with its location between the nasal cavity and the upper airways, makes the pharyngeal tonsil a sentinel site of primary immune responses following natural exposure or mucosal vaccination [17], and therefore a highly relevant tissue for proteomic examination of local responses to vaccination and infection.

Documented disease-sparing has been associated with a variety of mucosal and systemic immune responses in vaccinated cattle including BRSV-specific neutralizing antibody, BRSV-specific total IgG as determined by ELISA, nasal BRSV-specific IgA antibody, and BRSV-specific antibody and cellular immune responses in bronchoalveolar lavage (BAL) fluid [4]. However, the role of the differing viral antigen components within multivalent formulations on disease sparing responses is not clearly understood. A greater understanding of the necessary components required to successfully elicit long-lasting protective immunity could provide significant benefits to design of vaccines for problematic pathogens such as HRSV. Using an established challenge model for BRSV, this study aims to employ proteomic analysis to investigate the effect of additional antigenic material from multivalent mucosal vaccine formulations on local immune responses in the pharyngeal tonsil, and identify potential mechanisms of disease sparing in animals responding favorably to post-vaccination viral challenge.