Severe pandemic 2009 H1N1 influenza disease due to pathogenic immune complexes

Abstract

Pandemic influenza viruses often cause severe disease in middle-aged adults without preexisting comorbidities. The mechanism of illness associated with severe disease in this age group is not well understood. Here we find preexisting serum antibodies that cross-react with, but do not protect against, 2009 H1N1 influenza virus in middle-aged adults. Nonprotective antibody is associated with immune complex-mediated disease after infection. We detected high titers of serum antibody of low avidity for H1-2009 antigen, and low-avidity pulmonary immune complexes against the same protein, in severely ill individuals. Moreover, C4d deposition--a marker of complement activation mediated by immune complexes--was present in lung sections of fatal cases. Archived lung sections from middle-aged adults with confirmed fatal influenza 1957 H2N2 infection revealed a similar mechanism of illness. These observations provide a previously unknown biological mechanism for the unusual age distribution of severe cases during influenza pandemics.

Figure 1. Histopathology and virus titers in 2009 H1N1 disease

(a) Pulmonary histopathology in representative lung sections of fatal 2009 H1N1 and seasonal H1N1 virus infected patients (H&E). (b) Detection of 2009 H1N1 and seasonal H1N1 influenza viruses. Scale, 100μm. The boxes are details of pulmonary edema (2009 H1N1, H&E), peribronchiolar mononuclear cell infiltration (seasonal H1N1, H&E), and virus-infected cells (anti-H1N1 stains). (c) 2009 H1N1 RT-PCR C(t) values in NP secretions of fatal (black circles), ICU (black squares) and ambulatory (black triangles) patients; p=NS. (d) 2009 H1N1 RT-PCR C(t) values by days of symptoms before NP sampling.

Figure 2. Inflammation in influenza A 2009 H1N1 disease

IL-8(a), TNF-α (b), IL-6(c) and IL-1β (d) responses in respiratory secretions of patients infected with 2009 H1N1 influenza virus (tracheal aspirates, white circles; NP secretions, black squares) or seasonal influenza viruses (NP secretions of: H1N1, black up-pointing triangles; H3N2, black down-pointing triangles). Comparisons for NP secretions: IL-8, p=0.01; all other cytokines, p=NS. IL-1β (e), IL-6(f), IL-10 (g), and TNF-α (h) production by human monocytes incubated with a dose range of recombinant protein H1-1918 (black squares), H1-1999 (black triangles), H1-2009 (black circles), avian H5 (gray circles), hMPV F (white circles), and a control monoclonal human IgG against the Sa antigenic site of influenza HA (2D1; black diamond). Representative of three independent experiments. (i) Individual TLR activation in HEK293 cells. Dose: 5μg of H1-1918 (white bars), H1-1999 (yellow bars), H1-2009 (green bars), hMPV F (blue bars), and no ligand (red bars). Representative of two independent experiments.

Figure 3. Lymphopenia in influenza A 2009 H1N1 disease

(a)CD3⁺, (b) CD4⁺ and (c) CD8⁺ T lymphocyte counts in fatal and ICU patients infected with 2009 H1N1 influenza A virus on admission. Immunohistochemistry for (d)CD3⁺ T lymphocytes (red arrows), (e)CD8⁺ T lymphocytes(yellow arrows) in representative lung sections of fatal influenza 2009 H1N1 and seasonal infection. Positive controls are archived sections from different organs in unrelated subjects provided for every antibody. Negative controls are lung sections from a patient who died due to a non-pulmonary disease (scale, 100μm).

Figure 4. IC-mediated disease in 2009 H1N1 influenza infection

(a) Serum IgG endpoint titers against HA proteins by immunoassay in infants [mean age(range)=7.6 mo(6.1–11.8); n=10; white bars], middle-aged adults (n=16; gray bars), and elderly (n=12; black bars). (b) Protein-specific avidity of IgG after 6–9M urea wash in naïve adults (age range=25–43 year old; black squares) and elderly (age range= 75–97 years old; black diamonds); p<0.05. (c) Microneutralization titers for infants (black circles), adults (black squares) and elderly (black triangles); p value for elderly vs. both groups <0.05. (d) End point titer of serum IgG against H1-2009 antigens by immunoassay in adults with severe (n=12) vs mild (n=11) disease, p<0.05. (e) H1-2009-specific avidity of IgG after 7–9M urea wash in infected adults with severe (n=12) vs. mild (n=11) pandemic disease, p<0.05. (f) H1-2009-specific avidity of IgG from immune complexes after 8M urea wash in infected adults with severe (n=14) vs. mild (n=11) pandemic disease, p<0.05. (g) C4d detection in representative slides from lung sections of 2 of 6 fatal 2009 H1N1 infected middle-aged patients showing extensive peribronchiolar IC-mediated complement activation. Trace C4d deposition in representative lung section of fatal seasonal H1N1 virus from an elderly woman. Positive control: C4d deposition in a kidney from an adult patient with IC-mediated glomerulonephritis; negative control: lung section from an adult patient with solid tumor (scale, 100 μm). (h) Serum complement C3 levels in ICU adults (white circles), floor adults(black squares) and in infants (black circles) infected with H1N1 2009 influenza A virus, adults infected with seasonal influenza virus (black triangles) and adult patients with pulmonary diseases other than influenza in ICU and floor (white diamonds); p=0.036 (i) C4d detection in representative slides from lung sections of two fatal 1957 H2N2 influenza-infected patients showing extensive peribronchiolar deposition of complement cleavage product. Negative control from an archived sample with no infectious pulmonary disease. The box in the microphotograph is a detail of lung complement deposition.