Inducible bronchus-associated lymphoid tissue elicited by a protein cage nanoparticle enhances protection in mice against diverse respiratory viruses

Abstract

Background: Destruction of the architectural and subsequently the functional integrity of the lung following pulmonary viral infections is attributable to both the extent of pathogen replication and to the host-generated inflammation associated with the recruitment of immune responses. The presence of antigenically disparate pulmonary viruses and the emergence of novel viruses assures the recurrence of lung damage with infection and resolution of each primary viral infection. Thus, there is a need to develop safe broad spectrum immunoprophylactic strategies capable of enhancing protective immune responses in the lung but which limits immune-mediated lung damage. The immunoprophylactic strategy described here utilizes a protein cage nanoparticle (PCN) to significantly accelerate clearance of diverse respiratory viruses after primary infection and also results in a host immune response that causes less lung damage.

Methodology/principal findings: Mice pre-treated with PCN, independent of any specific viral antigens, were protected against both sub-lethal and lethal doses of two different influenza viruses, a mouse-adapted SARS-coronavirus, or mouse pneumovirus. Treatment with PCN significantly increased survival and was marked by enhanced viral clearance, accelerated induction of viral-specific antibody production, and significant decreases in morbidity and lung damage. The enhanced protection appears to be dependent upon the prior development of inducible bronchus-associated lymphoid tissue (iBALT) in the lung in response to the PCN treatment and to be mediated through CD4+ T cell and B cell dependent mechanisms.

Conclusions/significance: The immunoprophylactic strategy described utilizes an infection-independent induction of naturally occurring iBALT prior to infection by a pulmonary viral pathogen. This strategy non-specifically enhances primary immunity to respiratory viruses and is not restricted by the antigen specificities inherent in typical vaccination strategies. PCN treatment is asymptomatic in its application and importantly, ameliorates the damaging inflammation normally associated with the recruitment of immune responses into the lung.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Representative diagram of assembled protein cage nanoparticle showing individual protein subunits.

Figure 2. Induction of pulmonary iBALT structures in response to treatment with PCN.

iBALT structures (arrows) form in association with airways and blood vessels of the lung in response to 5 PCN treatments (A), whereas no iBALT structures were induced in response to 5 PBS treatments (B and C). PCN-induced iBALT structures contained CD4+ T cells, B220+ B cells (D), and CD21+ follicular dendritic cells (E).

Figure 3. Resolution of PR8 influenza virus infection is enhanced in mice treated with PCN prior to infection.

A) Viral recovery from the lungs of mice treated with PCN 9x prior to infection is significantly enhanced; **, P = 0.002; dotted line is limit of assay detection. B) PCN-treated mice did not lose body weight during resolution of subsequent influenza virus infection; **, P = 0.0024; ***, P

Figure 4. Survival of PCN and PBS-treated mice following challenge with SARS-coronavirus.

A). All PCN-treated mice (▪) survived infection with SARS-coronavirus whereas all of the PBS-treated control mice (Δ) were dead by day 4 post-infection; ***, P

Figure 5. Resolution of a Coxiella burnetii infection in PCN-treated mice.

A) PCN-treated mice (▪) gained body weight following C. burnetii infection whereas PBS-treated control mice (Δ) initially lost weight but were able to recover by 2 weeks post-infection. B) No difference in the rate of clearance of the C. burnetii infection from the lungs of the PCN-treated (shaded bar) and PBS-treated (clear bar) mice was detected.

Figure 6. Kinetics of PR8 influenza-specific antibody response following infection in mice treated with PCN (filled symbols) or PBS (clear symbols) prior to infection.

PR8 influenza-specific antibody levels were measured on day 0 (square symbol), day 5 (diamond symbols), day 7 (circle symbols) and day 9 (triangle symbols) post-infection. A) PR8 influenza-specific serum IgG levels appear sooner and are significantly enhanced in PCN-treated mice relative to the PBS-treated control mice; ***, P

Figure 7. PCN-enhanced resolution of a PR8 influenza virus infection is antibody and CD4+ T cell dependent.

A). Viral burden in lungs of PCN-treated (clear bars) wild type mice were significantly less than in the PBS-treated (clear bar with lines) wild type mice at day 7 post-infection whereas no differences in viral burdens in the lungs of the PCN-treated (shaded bars) and PBS-treated (shaded bars with lines) μMT mice were detected at this time; **, P10. B). Equivalent levels of body weight loss were detected in the PBS-treated wild type (Δ) and μMT (○) whereas PCN-treated wild type (▴) and μMT (•) both gained equivalent amounts of body weight during the week following influenza infection; *, P<0.05; **, P<0.001 relative to their respective PBS-treated control. C). Recruitment of CD8 (triangle symbols) and CD4 (square symbols) T cells occurs sooner in PCN-treated mice (filled symbols) relative to the PBS-treated (clear symbols) control mice following influenza virus infection; *, P<0.05; **, P<0.01; ***, P<0.001. D). Viral recovery in PBS-treated (shaded bars) and PCN-treated (clear bars) wild type mice depleted of CD8 and/or CD4 T cells at 48 hours before and 4 days after influenza infection; *, P<0.05; **, P<0.01 relative to PBS-treated control; dotted line at 1.82 log10 is limit of assay detection.

Figure 8. Effect of PCN treatment endures and elicits iBALT structures that can exclusively resolve PR8 influenza infections.

A). Mice that had been treated with PCN (clear bars) or PBS (shaded bars) were rested for a designated period of time before being challenged with influenza virus. Assessment of viral clearance was carried out by plaque assay; *, P