Pandemic viruses may promote bacterial infections1
, injure the lungs2–4
, decrease type I interferon (IFN) levels5
, promote a cytokine storm, and induce apoptosis6
. Although all attractive hypotheses, the explanation for the enhanced severity of cases in middle-aged adults during pandemics7–10
In 2009, a novel H1N1 influenza A virus caused severe disease in naïve middle-aged individuals with preexisting immunity against seasonal strains11–13
. In contrast to seasonal disease, the elderly were relatively spared and young children had milder disease than middle-aged subjects11–16
. Preexisting neutralizing cross-reactive antibodies elicited by an H1N1 virus circulating before 1957 protected the elderly11,12
. Adults had been exposed repeatedly to seasonal influenza viruses leading to antibody production, while young children often lacked previous exposures12
. An antibody repertoire in adults shaped by seasonal infections may recognize, but fail to neutralize the new pandemic strain, leading to IC-mediated disease17,18
. In this manuscript, we characterized the pathogenesis of severe pandemic respiratory disease in middle-aged adults with no preexisting co-morbidities.
Tracheal (TA) and nasopharyngeal (NP) aspirates, and serum samples reflecting different disease severities in adult outpatients (n=21) and inpatients (n=54) infected with 2009 H1N1 were obtained. Median age of patients was 39 years (range=17–57). Twenty-three subjects died; 16 (69%) of refractory hypoxemia. Fifteen survivors required intensive care.
In addition, NP secretions from adults hospitalized with seasonal influenza A viruses (2007/08), and from infants and young children infected with 2009 H1N1 were analyzed. In Argentina, universal immunization against influenza in children was not recommended until 2010.
Lung sections of patients with fatal 2009 H1N1 showed widened inter-alveolar septa, interstitial hemorrhages, abundant intra-alveolar edema with deposition of hyaline membranes, and an infiltrate of mononuclear cells ()10,19
. Lungs evidenced hyperplasia and detachment of type II pneumocytes into the lumen. Fatal cases of seasonal H1N1 influenza also revealed interstitial edema, desquamation of type II pneumocytes, and mononuclear cell infiltration (). Influenza A 2009 H1N1 was detected mainly in epithelial cells of bronchioles; seasonal H1N1 was occasionally detected in respiratory epithelial cells from pre-exposed elders ().
Histopathology and virus titers in 2009 H1N1 disease
2009 H1N1 RNA (vRNA) expression was similar in outpatients, and inpatients requiring ICU or not surviving (; p=0.9). However, vRNA levels correlated with days of symptoms (p=0.027; ), and patients with severe disease (ICU+fatal) were sampled later than outpatients (median= 6 vs. 3 days; p= 0.02). Adjusting the relationship between vRNA levels and severity for days of symptoms did not reach statistical significance (p=0.3).
Analysis of type I IFN production showed lower TA than NP levels of IFN-α, with similar levels in 2009 H1N1 vs. seasonal influenza infections (p= NS; Suppl. Fig 1
). IFN-β was universally low (p=0.6 pandemic vs. seasonal; Suppl. Fig 1
Certain pandemic hemagglutinins (HA) are thought to cause a cytokine storm20
. NP secretions in 2009 H1N1 and seasonal infections evidenced similar levels of TNF-α, IL-6, IL-1β, IL-10, and IL-12 (, Suppl. Fig. 2
). IL-8 levels were higher in pandemic patients (p=0.01; ).
Inflammation in influenza A 2009 H1N1 disease
The effect of H1-2009, H1-1918 and H1-1999 proteins on human monocyte production of inflammatory cytokines was similar (). Higher levels of inflammatory cytokines were detected with avian H521,22
. Surprisingly, the inflammatory response against the human metapneumovirus fusion protein (hMPV-F) was higher than against influenza HAs. In fact, hMPV-F was a vigorous TLR2 and TLR4 agonist, while H1-2009 and H1-1918 were weak TLR2 and TLR4 agonists, respectively (). Seasonal H1-1999 activated TLR4. No HA protein activated other TLRs.
Patients with severe pandemic influenza presented profound lymphopenia ()10
. Both CD4+
T lymphocyte and CD8+
T lymphocyte counts were below normal ranges (). Lymphopenia associated with a lung T lymphocytosis (), likely explained by pandemic virus conservation of numerous T cell epitopes from seasonal strains23,24
. In fact, many CD8+
T lymphocytes were observed ()19
. No significant T lymphocyte apoptosis in the lungs was detected (Suppl. Fig. 3
Lymphopenia in influenza A 2009 H1N1 disease
We then asked whether lung lymphocytosis was associated with Th2-immunopathogenesis, a mechanism of immune-mediated viral respiratory illnesses25
. Analysis of IFN-γ (Th1), IL-4(Th2) and IL-17(Th17) showed few cytokine-positive cells in lung sections and low cytokine levels in secretions in pandemic patients(not shown).
2009 H1N1 virus shares 17% of its B cell epitopes against HA and NA with seasonal influenza A viruses24
. Therefore, we examined whether cross-reactive, non-protective antibodies against 2009 H1N1 were present in sera of naïve adults (). IgG against HA antigens was absent in infants, but detected in naïve adults and elderly (). However, antibody avidity for H1-2009 was lower in adults than in older patients (p<0.05; ). In fact, adults had higher avidity for H1-1999 than for H1-2009 (p=0.03; Suppl. Fig. 4
). Moreover, unlike elderly subjects, middle-aged adults lacked protective titers of neutralizing antibody against 2009 H1N1 ()11,12
IC-mediated disease in 2009 H1N1 influenza infection
IgG against H1-2009 and H1-1999 was present in adults <10 days after 2009 H1N1 infection (Suppl. Fig. 5
). Interestingly, anti-H1-2009 IgG titers were higher in severe vs. mildly ill adults (; p=0.02). Moreover, IgG avidity remained lower for H1-2009 than for H1-1999 in pandemic patients (p=0.03; Suppl. Fig. 5
), and severely ill patients had antibody of lower avidity for H1-2009 than mildly ill outpatients (p<0.05; ). Importantly, severe cases also had anti-H1-2009 IgG of lower avidity than mild cases in respiratory IC (p<0.05; ).
Non-protective antibody responses of low avidity have been associated with IC disease in other respiratory infections17,27
. We therefore stained lung sections for complement cleavage product C4d26
. Extensive C4d deposition was detected in bronchioles of patients infected with 2009 H1N1 (), matching distribution in IC-mediated diseases due to other viruses27
. Conversely, trace deposition of C4d was found in lung sections from patients infected with seasonal influenza.
ICs were detected using an anti-C1q assay in secretions of individuals infected with 2009 H1N1, but rarely in samples from patients infected with seasonal influenza (p=0.003 comparing floor admissions; Suppl. Fig. 6
). Furthermore, higher levels of ICs were detected in secretions of adults admitted with 2009 H1N1 to the ICU compared to those admitted to regular floors (Suppl. Fig. 6
Confirming our observations, most adults admitted to the ICU with pandemic influenza had low serum C3 levels, while C3 levels were higher in moderately ill subjects. Infants infected with 2009 H1N1, adults infected with seasonal influenza, and patients with other pulmonary diseases often had normal C3 levels (p=0.036; ).
Finally, we retrieved archived lung sections from adult patients who died during the 1957 H2N2 pandemic in Tennessee. Sections of sufficient quality to be processed and stained showed extensive C4d peribronchiolar deposition (). Presence of influenza A vRNA was confirmed by real time-PCR. A control archived lung section from an individual with no pulmonary infection evidenced no C4d deposition.
Taken together, these observations demonstrate that 2009 H1N1 influenza virus leads to IC-mediated disease in adults through high titers of low avidity non-protective antibody and IC-mediated complement activation in the respiratory tract. IC-mediated lung disease also contributed to fatal cases caused by 1957 H2N2 pandemic influenza. We speculate that this phenomenon contributes to severe symptoms in the adult population during all pandemics8
Young infants and children -as in previous pandemics-had high rates of infection with comparatively low mortality7,11
. This paradox is explained by absence of protective and pathogenic immunity in pediatric patients11–13
, particularly in countries where immunization of children against seasonal viruses was not recommended. Therefore, severe pediatric 2009 H1N1 illness associates with widespread infection in a naive population.
This study addresses several attractive hypotheses advanced to explain the pathogenesis of influenza viruses1–6
. While increased severity of 1918 and 2009 H1N1 pandemic viruses was presumptively associated with higher pulmonary virus titers2–4
, a dose-dependent effect on mortality was never described. Similarly, type I IFN modulation appears to play an important role in severe cases of influenza28
, but its role in the unusual age distribution of severe cases during pandemic flu remains to be determined.
Secondary bacterial infections were responsible for most deaths in 19181
. During 2009, most fatal cases were primary infections with refractory hypoxemia10,12
, and neutrophil lung infiltration was minimal19
. Interestingly, depletion of inflammatory cytokines or pretreatment with steroids did not affect mortality in a murine model of fatal H5N1 influenza infection29
. Moreover, the main neutralizing antigen of milder hMPV elicits significantly more inflammation in vitro
than influenza HAs.
However, other roles for innate immunity may be at play in pathogenesis. For example, S.pneumoniae
nasopharyngeal carriage may also affect illness severity during 2009 H1N1 infection30
. Indeed, several factors likely contribute to severe pandemic disease in adults and explain different outcomes in individuals of similar ages and backgrounds.
Certain limitations are inherent to a study of these characteristics. For instance, since we lacked determinations of vRNA levels over time, disregarding a role for viral injury in severity is not possible. However, the impact of pandemic flu in adults compared to frail infants and elderly argue against a preponderant pathogenic role for viral injury. Also, we rapidly identified a cross-reactive antibody against 2009 H1N1, known to recognize seasonal H1N1. But whether reactivity against both viruses is identical in our descriptive slides is unknown.
In summary, our study provides a novel biological explanation for the unusual age distribution of severe cases during pandemic influenza. The association of severe pandemic disease in adults with high titers of low avidity, non-protective antibody and complement activation by pulmonary IC opens a new paradigm for future therapeutic interventions.