Enhanced inflammation in New Zealand white rabbits when MERS-CoV reinfection occurs in the absence of neutralizing antibody

Abstract

The Middle East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic betacoronavirus that was first detected in humans in 2012 as a cause of severe acute respiratory disease. As of July 28, 2017, there have been 2,040 confirmed cases with 712 reported deaths. While many infections have been fatal, there have also been a large number of mild or asymptomatic cases discovered through monitoring and contact tracing. New Zealand white rabbits are a possible model for asymptomatic infection with MERS-CoV. In order to discover more about non-lethal infections and to learn whether a single infection with MERS-CoV would protect against reinfection, we inoculated rabbits with MERS-CoV and monitored the antibody and inflammatory response. Following intranasal infection, rabbits developed a transient dose-dependent pulmonary infection with moderately high levels of viral RNA, viral antigen, and perivascular inflammation in multiple lung lobes that was not associated with clinical signs. The rabbits developed antibodies against viral proteins that lacked neutralizing activity and the animals were not protected from reinfection. In fact, reinfection resulted in enhanced pulmonary inflammation, without an associated increase in viral RNA titers. Interestingly, passive transfer of serum from previously infected rabbits to naïve rabbits was associated with enhanced inflammation upon infection. We further found this inflammation was accompanied by increased recruitment of complement proteins compared to primary infection. However, reinfection elicited neutralizing antibodies that protected rabbits from subsequent viral challenge. Our data from the rabbit model suggests that people exposed to MERS-CoV who fail to develop a neutralizing antibody response, or persons whose neutralizing antibody titers have waned, may be at risk for severe lung disease on re-exposure to MERS-CoV.

Conflict of interest statement

The authors have declared that no competing interests exist

Figures

Fig 1. Schematic of rabbit infection studies.

Rabbits were inoculated intranasally with EMC/2012 strain of MERS-CoV (green arrows) and tissue samples were collected for viral titration and histopathology at necropsy (blue arrows). Three rabbits were necropsied at each time point. Numbers indicate days since virus administration for primary, (secondary), or [tertiary] infections.

Fig 2. Viral RNA titers in the respiratory tract following primary infection with MERS-CoV.

Viral RNA titers in the nasal turbinates (A) and lungs (B) of rabbits following infection with either 103 or 105 TCID50 of EMC/2012 strain of MERS-CoV through day 5 after infection. In a separate experiment, viral RNA titers were determined in the nasal turbinates (C) and lungs (D) following infection with 106.5 TCID50. n = 3 rabbits per group. Statistical significance was determined using one-way ANOVA with Tukey’s multiple comparisons test. p values *<0.05, ***<0.001.

Fig 3. Histopathology in the lungs following primary infection with EMC/2012 strain of MERS-CoV.

Images show H&E (left) and IHC against the MERS-CoV N protein (right) following infection with 105 TCID50 (A,D), 103 TCID50 (B,E), or a media only control (C,F). All images at 10x, (bar equivalent to 100μm) with 40x insets (bar equivalent to 20μm). Images shown are from day 3 post-infection for all groups.

Fig 4. Viral RNA titers and histopathology in the lungs of rabbits following reinfection with MERS-CoV.

Viral RNA titers in the lungs of rabbits following reinfection with EMC/2012 (A). Images show H&E (left) and IHC for the MERS-CoV N protein (right) following reinfection for the 103//105 TCID50 reinfection group (B,E) and 105//105 TCID50 reinfection group (C,F). The 105 TCID50//media control group was included to demonstrate that the observed inflammation was not residual from the primary infection (D,G). n = 3 rabbits per group. All images at 10x, (bar equivalent to 100μm) with 40x inset (bar equivalent to 20μm). Images from day 3 post-infection.

Fig 5. Viral RNA titers and histopathology in lungs following infection with MERS-CoV when neutralizing antibodies are present.

Viral RNA titers in the lungs following tertiary infection with EMC/2012 strain (A). Images show H&E staining (B) and IHC with an antibody against the MERS-CoV N protein (C) in the 105//105//105 TCID50 group. Images are representative of all rabbits following tertiary infection. All images at 10x, (bar equivalent to 100μm) with 40x inset (bar equivalent to 20μm). n = 3 rabbits per group. Statistical significance was determined using one-way ANOVA with Tukey’s multiple comparisons test. Images from day 3 post-infection. p values **<0.01, ***<0.001.

Fig 6. Viral RNA titers and histopathology in the lungs following MERS-CoV infection in rabbits that received passive transfer (PT) of serum from infected rabbits.

Viral RNA titers in the lungs upon infection with 105 TCID50 of MERS-CoV in rabbits either previously infected with a low dose of MERS-CoV (103 TCID50) four weeks prior or naïve rabbits following PT of post-infection sera at either a full dose or 1:10 dilution (A). Images show the H&E staining (left) and IHC with an antibody against the MERS-CoV N protein (right) following infection for the 103//105 TCID50 (reinfection) group (B,E), the group that received passive transfer of undiluted post-infection serum (C, F), and the group that received passive transfer of post-infection serum at 1:10 dilution (D,G). n = 3 rabbits per group. Images from day 3 post-infection at 10x, bar equivalent to 100μm.

Fig 7. Antibody-dependent enhancement (ADE) assay using rabbit sera throughout the infection series.

Sera from naïve rabbits (week 0), following primary infection (week 8), and following secondary infection (week 13) were collected from both the 103 and 105 infection schedules. Week 8 serum had no neutralizing activity while week 13 serum had neutralizing activity. Sera were tested in Vero81 cells (A), Raji cells (B) and THP-1 cells (C). None = virus only control. p values **<0.01, ***<0.001.

Fig 8. Detection of complement protein during primary infection and reinfection.

ELISA against C3a protein in rabbit lung homogenates (A) show an increase in C3a levels present during reinfection compared to primary infection. Immunofluorescence images show MERS-CoV N antigen (green) and complement (red) following secondary infection (B) and primary infection (C). Images from day 3 post-infection at 40x, bar equivalent to 20μm. n = 3 rabbits per group. Statistical significance was determined using one-way ANOVA with Dunnett’s multiple comparisons test. p values *

Fig 9. CD3+ cells in the lungs following primary infection and reinfection.

DAB images from primary infection (A) and reinfection (B). Immunofluorescence (IF) image of CD3 (green) and virus antigen (red) within the same perivascular region following reinfection (C). DAB images from day 3 post-infection at 10x, bar equivalent to 100μm. IF images at 40x, bar equivalent to 20μm.