ORIGINAL ARTICLE Low Titers of Serum Antibodies Inhibiting Hemagglutination Predict Fatal Fulminant Influenza A(H1N1) 2009 Infection Amelie ´ Guihot1,2,3, Charles-Edouard Luyt4, Antoine Parrot5, Dominique Rousset6,7,8, Jean-Marc Cavaillon9, David Boutolleau10, Catherine Fitting9, Priya Pajanirassa1,2, Alain Mallet11, Muriel Fartoukh5, Henri Agut10, Lucile Musset3, Rima Zoorob2,12, Amos Kirilovksy2,12, Behazine ´ Combadiere ` 1,2, Sylvie van der Werf 6,7,8, 1,2,3 1,2,3 Brigitte Autran *, and Guislaine Carcelain *; for the FluBAL Study Group† 1

Laboratory of Immunity and Infection, UPMC Univ Paris 06, UMR-S945, Paris, France; 2Laboratory of Immunity and Infection, INSERM, U945, Paris, France; 3Departement ´ d’Immunologie, 4Service de Reanimation, ´ and 11Service de Biostatistiques, URC, AP-HP, Hopital ˆ Pitie-Salp ´ etri ˆ ere, ` Paris, France; 5Service de Reanimation, ´ AP-HP, Hopital ˆ Tenon, Paris, France; 6Unite´ de Gen ´ etique ´ Moleculaire ´ des 9 Virus a` ARN, Departement ´ de Virologie, CNR du Virus Influenzae (Region-Nord), ´ and Unite´ Cytokines et Inflammation, Institut Pasteur, 7 8 10 Paris, France; CNRS, URA3015, Paris, France; Universite´ Paris Diderot Paris 7, Paris, France; UPMC Univ Paris 06, ER1 DETIV, Service de Virologie, Groupe Hospitalier Pitie-Salp ´ etri ˆ ere, ` AP-HP, Paris, France; and 12Universite´ Paris Sud, Paris-XI, Paris, France

Abstract Rationale: The biology of fatal pandemic influenza infection

remains undefined. Objectives: To characterize the virologic and immune parameters

associated with severity or death in patients who required mechanical ventilation for A(H1N1) 2009 pneumonia of various degrees of severity during the two waves of the 2009–2011 pandemic in Paris, France. Methods: This multicenter study included 34 unvaccinated patients

with very severe or fatal confirmed influenza A(H1N1) infections. It analyzed plasma A(H1N1) 2009 reverse-transcriptase polymerase chain reaction, hemagglutinin 222G viral mutation, and humoral and cellular immune responses to the virus, assessed in hemagglutination inhibition (HI), microneutralization, ELISA, lymphoproliferative, ELISpot IFN-g, and cytokine and chemokine assays. Measurements and Main Results: The patients’ median age was 35 years. Influenza A(H1N1) 2009 viremia was detected in 4 of 34 cases, and a 222G hemagglutinin mutation in 7 of 17 cases, all of them with sequential organ failure assessment greater than or equal to 8. HI

antibodies were detectable in 19 of 26 survivors and undetectable in all six fatal fulminant cases. ELISA and microneutralization titers were concordant. B-cell immunophenotyping and plasma levels of immunoglobulin classes did not differ between patients who survived and died. After immune complex dissociation, influenza ELISA serology became strongly positive in the bronchoalveolar lavage of the two fatal cases tested. H1N1-specific T-cell responses in lymphoproliferative and IFN-g assays were detectable in survivors’ peripheral blood, and lymphoproliferative assays were negative in the three fatal cases tested. Plasma levels of IL-6 and IL-10 were high in fatal cases and correlated with severity. Finally, a negative HI serology 4 days after the onset of influenza symptoms predicted death from fulminant influenza (P = 0.04). Conclusions: Early negative A(H1N1) 2009 HI serology can predict death from influenza. This negative serology in fatal cases in young adults reflects the trapping of anti-H1N1 antibodies in immune complexes in the lungs, associated with poor specific helper T-cell response. Clinical trial registered with www.clinicaltrials.gov (NCT 01089400). Keywords: influenza; serology; immune responses; H1N1; intensive

care unit

( Received in original form November 24, 2013; accepted in final form March 14, 2014 ) *These authors are senior coauthors. †

A complete list of members may be found before the beginning of the REFERENCES.

Supported by the Institut de Microbiologie et Maladies Infectieuses, INSERM. Author Contributions: A.G., A.P., M.F., S.v.d.W., J.-M.C., C.-E.L., H.A., G.C., and B.A. designed the study. C.-E.L., A.P., and M.F. included patients. D.B., D.R., H.A., and S.v.d.W. supervised virologic assays. A.G., L.M., B.C., R.Z., B.A., and G.C. supervised immunologic assays. A.K., P.P., and C.F. performed immunological assays. C.F., A.G., and J.-M.C. performed cytokine-chemokine assays and analysis. A.M. and A.G. performed statistical analysis. A.G., B.C., B.A., and G.C. wrote the manuscript. Correspondence and requests for reprints should be addressed to Guislaine Carcelain, M.D., Ph.D., Departement ´ d’Immunologie, UMR S945, Hopital ˆ Pitie´ Salpetri ˆ ere-83, ` boulevard de l’Hopital, ˆ 75013 Paris, France. E-mail: [email protected] This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org Am J Respir Crit Care Med Vol 189, Iss 10, pp 1240–1249, May 15, 2014 Copyright © 2014 by the American Thoracic Society Originally Published in Press as DOI: 10.1164/rccm.201311-2071OC on March 19, 2014 Internet address: www.atsjournals.org

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ORIGINAL ARTICLE

At a Glance Commentary Scientific Knowledge on the Subject: Despite knowledge about

hypercytokinemia linked to severity and mortality during previous H1N1 pandemic and H5N1/H7N9 avian influenza infections, the pathologic mechanisms underlying fatal pandemic influenza in young adults remain poorly understood. Laboratory tests predicting severity and fatality could help in managing future pandemics. Furthermore, elucidation of the immunopathology of severe forms of influenza is a key issue for understanding the pathogenesis of severe pandemic influenza infection and evaluating new treatments in future pandemics. What This Study Adds to the Field: In this work we report the largest series of cases of extremely severe A(H1N1) 2009 influenza pneumonia during the two waves of the 2009–2011 pandemic in 34 young to middle-aged adults in France and propose a mechanism that caused the deaths. Blood from all six fatal fulminant cases had consistently low serum anti-H1N1 antibody responses in hemagglutination-inhibition (HI) assays and low anti-H1N1 T-cell responses, although the local immune response to the virus was strong, even explosive. We propose a mechanism of lethality: the HI assay in bronchoalveolar lavage turned positive after immune complex dissociation; T cells in the lungs became hyperactivated; and a strong inflammatory profile developed, with significantly higher IL-6 and IL-10 levels than in survivors. These results suggest a strong specific immune response located in the lungs in fatal cases, associated with strong innate immune responses. Finally, a mathematical model showed that the absence of influenzaspecific serum HI antibodies by Day 4 after the first influenza symptoms predicts mortality. Multivariate analysis showed that neither cytokine levels nor anti-H1N1 titers were independently correlated with death. These results have important implications for clinical management of life-threatening influenza pneumonia, showing that a negative HI assay as early as Day 4 after the onset of influenza symptoms can predict death.

During the 2009 influenza A pandemic, most patients presented a flulike syndrome with a benign course (1). Acute life-threatening infections, chiefly of the lungs, occurred in a very small proportion of young adults who required mechanical ventilation or extracorporeal oxygenation. Preexisting chronic illness, obesity, and pregnancy were identified as conditions predisposing to severe H1N1 infection (2–6). Nevertheless, one-third of the severe cases occurred in immunocompetent young adults with no known predisposing factor. Reports describe mortality rates of 14–39% (2, 7). Cytokine storms observed during severe avian H5N1 infections pointed IL-6, IL-8, IFNg–induced protein-10 (IP-10), and MIG (monokine induced by IFN-g) as severityassociated cytokines and chemokines (8). During the more recent H7N9 outbreak, only IL-6 and IP-10 were linked with severity (9, 10). In more severe cases of pandemic A(H1N1) 2009 infection, peripheral blood was rich in IL6 and IL-10 (11–15) and IL-6 was reported to predict fatal outcome (16). Numerous other cytokines and chemokines (IL-1Ra, IL-6, tumor necrosis factor-a, IL-8, MIP1-b [macrophage inflammatory protein 1-b], monocyte chemotactic protein-1 [MCP-1], IP-10) were expressed in the lungs of fatal cases (17). These cases also presented various immunologic defects, including low levels of natural killer cells in blood (18, 19), CD4 and CD8 T cells (19), plasma IgG2 (20), Toll-like receptor 2 and 4 expression on blood lymphocytes and monocytes (14), and T-cell anergy (21). A few studies reported specific immune responses to the virus: A(H1N1) 2009–specific Th1 CD4 T cells were induced after infection (22), and blood IFN-g1 CD4 T-cell responses to internal A(H1N1) 2009 proteins were seen at higher magnitudes in severe than in mild forms (23). Anti-A(H1N1) IFNg1CD81 T cells against conserved epitopes were correlated to protection (24). Peribronchiolar deposition of immune complexes with presumed preformed antibodies of low avidity specific for other influenza strains was reported to be responsible for severe cases in young adults (25). Elucidation of the immunopathology of severe forms of influenza is a key issue for understanding the pathogenesis of severe pandemic influenza infection and evaluating new treatments in future pandemics. Furthermore, laboratory findings that predict severity or death soon after early influenza symptoms could help manage an influenza pandemic crisis.

We sought to characterize the virologic and immune parameters associated with severity or death in 34 patients (eight of whom died) who required mechanical ventilation for A(H1N1) 2009 pneumonia of various degrees of severity during the two waves of the 2009–2011 pandemic in Paris, France. This work was partially presented at the Options for the Control of Influenza VII symposium in Hong Kong, 2010 (26, 27).

Methods Patients and Samples

Patients were eligible for inclusion if they underwent invasive mechanical ventilation in an intensive care unit (ICU) for documented A(H1N1) 2009 influenza pneumonia. Clinical severity was determined as the worst sequential organ failure assessment (SOFA) score in the ICU (28). Additional details on inclusion criteria, extracorporeal membrane oxygenation (ECMO), and bronchoalveolar lavage (BAL) are provided in the METHODS section of the online supplement. The CPP Ile de France II, Paris institutional review board, approved this study. Virologic Investigations

A(H1N1) 2009 influenza genome detection in BAL fluid, nasopharyngeal aspirates, and plasma was performed by real-time reverse-transcriptase polymerase chain reaction (RT-PCR) assays from the French National Influenza Center, as previously described (29). Hemagglutinin mutations were tested when sufficient viral RNA was extracted. Additional details on the virologic methods included are provided in the METHODS section of the online supplement. H1N1 Serologic Procedures

Titers of antibodies against A/California/ 07/09 were determined by hemagglutination inhibition (HI), microneutralization assays, and ELISA assay, with standard techniques. Additional details about H1N1 serologic methods are provided in the METHODS section of the online supplement. For the dissociation of antibody-antigen immune complexes, serum was treated with dissociation buffer (1.5 M glycine [pH: 2.8]) and immune complexes were dissociated for 1 hour at 378 C. The reaction was stopped by adding neutralization buffer (1.5 M Tris-HCL [pH: 9.7]).

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ORIGINAL ARTICLE Immunoglobulin and IgG Subclass Determinations

Serum concentrations of immunoglobulin and IgG subclasses were determined by turbidimetry (SPA Plus) with reagents from the Binding Site (Birmingham, UK). Gene Expression Analysis

Gene expression profiles were evaluated from blood samples (PAXgene Blood RNA tube;

PreAnalytix, Hombrechtikon, Switzerland) and compared with those of three healthy blood donors. Details are provided in the METHODS section of the online supplement.

monovalent inactivated vaccine according to availability at reception of patients’ fresh blood samples. Details are provided in the METHODS section of the online supplement.

Lymphoproliferative Assays

ELISpot IFN-g Assays

Freshly isolated peripheral blood mononuclear cells (PBMCs) were stimulated as previously described (30) with recombinant A/California/ 2009 hemagglutinin or A(H1N1) 2009

Fresh PBMC or BAL lymphocytes were stimulated with 18-mer overlapping peptides covering the A(H1N1) 2009 hemagglutinin (n = 65), and 18-mer

Table 1: Patient Characteristics and Virologic Data Baseline Characteristics

Number Age Sex Winter 2009–2010 1 35 F 2 3

40 21

F M

4 5 6 7 8 9 10 11 12

27 26 34 33 39 42 33 30 25

F F F M F F F F F

13 14

14 64

F M

15

38

M

16 35 M 17 22 M Winter 2010–2011 18 46 M 19 53 M 20 35 F 21 17 F 22 34 M 23 62 M 24 52 F 25 17 M 26 23 F 27 49 M 28 46 M 29 21 F 30 27 F 31 54 M 32 34 M 33 34

42 35

Median

35

M F

Predisposing Conditions

Obesity, pregnancy None Steroids (myasthenia gravis) None Pregnancy None Obesity None Obesity, HBP Pregnancy Pregnancy Pregnancy

Characteristics at First Sample in ICU H1N1 Days Days from First 2009 with Pulmonary Flu Symptoms Virus Oseltamivir Coinfection

ECMO: Yes/No

Worst SOFA

Outcome

8

Not done

3

None

N

1

A

8 4

Not done Not done

6 1

Pneumococcus None

N N

3 3

A A

13 16 14 10 5 5 7 15 5

Not done Not done Not done Not done Not done Not done Viremia 222G Viremia, 222G None Not done

7 9 5 5 4 2 4 0 3

None None Streptococcus Staphylococcus None None None None Pneumococcus

N Y N Y Y Y Y Y Y

7 7 8 9 10 10 12 13 17

A A A A A A A A A

3 1

Staphylococcus None

Y Y

18 10

A D, NP

Viremia, 222G None 222G

1

Pneumococcus

Y

19

D, MOF

3 10

None Staphylococcus

N N

12 18

D, MOF D, MOF

3 5 4 0 6 ND ND 10 7 9 11 3 2 2 1

None None None None None None None None Staphylococcus Staphylococcus Aspergillus None Staphylococcus None None

Y Y Y N Y Y N Y Y Y Y Y Y Y Y

5 5 6 6 8 8 8 9 10 11 16 16 18 14 16

A A A A A A A A A A A A A D, MOF D, MOF

3 ND

None None

Y Y

17 19

D, NP D, MOF

25

10

None Obesity, Waldenstrom ¨ Steroids 1 myc. mofetil (AIA) Obesity None

4 14

7 10

Obesity Asthma Down syndrome None Obesity COPD Transplantation Asthma Pregnancy None Transplantation Down syndrome Steroids None None

8 9 12 4 17 9 8 17 11 12 21 7 6 12 6

None CVID

10 22

9

9

None None Not done Not done Not done None 222G Not done None None Not done Not done None 222G Viremia, 222G Not done Not done

4

Definition of abbreviations: 222G = hemagglutinin mutation conferring lower respiratory tract viral tropism; A = alive; AIA = autoimmune anemia; COPD = chronic obstructive pulmonary disease; CVID = common variable immunodeficiency; D = dead; ECMO = extracorporeal membrane oxygenation; F = female; HBP = high blood pressure; ICU = intensive care unit; M = male; MOF = multiorgan failure; NA = not available; ND = not done; NP = nosocomial pneumonia; SOFA = sequential organ failure assessment.

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ORIGINAL ARTICLE overlapping peptides covering the A (H1N1) 2007 nucleoprotein with 95% homology with the A(H1N1) 2009 nucleoprotein (n = 49), as described in more detail in the METHODS section of the online supplement.

for survival were based on the antibody titer at Day 4, estimated for each individual. Details on the statistical methods are provided in the METHODS section of the online supplement.

Results Cytokine and Chemokine Assays

Concentrations of IL-6, IL-10, CXCL10 (IP-10), and CCL2 (MCP-1) were determined in quantitative multiplex assays (LEGENDplex 16-plex human; BioLegend, San Diego CA) and transforming growth factor (TGF)-b1 concentrations were determined by ELISA (Duoset; R&D Systems, Minneapolis, MN). Details are provided in the METHODS section of the online supplement. Statistical Analysis

The Mann-Whitney U, Kruskal-Wallis, and Spearman rank correlation coefficient tests were used as appropriate with Prism 5 (Graph Pad, La Jolla, CA) software. Regression models

Patient and Virus Characteristics

This prospective study included 34 patients (16 men, 18 women, median age 35 years [interquartile range, 26–43]) with proven A (H1N1) 2009 infection and recruited in ICU during the two waves of the A(H1N1) 2009 pandemic in Paris, France (winters 2009–2010 and 2010–2011). Table 1 summarizes the patients’ characteristics. None had been vaccinated against pandemic influenza. Ten patients had a concomitant pulmonary bacterial infection at ICU entry, and 25 required ECMO. The worst SOFA scores, measuring severity, ranged from 1 (mildest) to 19 (median, 10; interquartile range, 7–16).

Eight patients died, six from multiple organ failure and fatal fulminant infection (median, 5 d after admission [range, 1–14]), and two from nosocomial pneumonia (17 and 33 d after admission). Three with lethal disease had underlying immunodepression: preexisting Waldenstr¨om macroglobulinemia (patient #14), current immunosuppressive treatment for autoimmune anemia, and untreated common variable immunodeficiency (CVID) (patients #15 and 34) (Table 1). A(H1N1) 2009 viremia was detected at first blood sample as defined in Table 1 (median, 9 d [4–22] of first flu symptoms), by RT-PCR in 4 of 34 cases, all with SOFA scores greater than 10 and including two fatal cases. Of 16 patients with sufficient viral RNA extracted from BAL or nasopharyngeal aspirates, 7 had A(H1N1) 2009 hemagglutinin 222G mutations. Of the 11 patients in the group with very severe infection (SOFA .10), 6 (54%) had a hemagglutinin mutation, when compared with one of the five patients

Figure 1. Low levels of hemagglutination-inhibition (HI) antibodies in fatal cases of influenza A(H1N1) 2009 infection. (A–D) Antibody (Ab) responses to the A(H1N1) 2009 virus in HI (A), microneutralization assay (MN, B), and ELISA assays (C), as described in the METHODS and online supplement METHODS sections. Each black dot represents a surviving patient, crosses represent patients with fatal fulminant infections, and gray dots fatal cases from nosocomial pulmonary infection. Dashed lines are positive thresholds, as defined in the METHODS. (D) Concordance between the three serologic assays used: ELISA, MN, and HI. (E and F) Antibody responses to the A(H1N1) 2009 virus in HI assay according to the time of the assay from first influenza symptoms in survivors (E), and in fatal cases (F). The dashed line is the positive threshold as defined in the METHODS.

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ORIGINAL ARTICLE with less severe infection (SOFA ,10) (20%; P = 0.19). To put this in perspective, from August 2009 to March 2011, we collected 129 influenza A(H1N1) 2009 cases from general practice (GROG- Groupes R´egionaux d’Observation de la Grippe) and found no case of 222G mutation. Note that the mutation was found in three of the H1N1 viruses from the four cases with viremia (Table 1). These results show that concomitant 222G hemagglutinin mutations and viremia were observed in the more severe forms of this infection. Low to Undetectable Serum HI Antibodies in Fatal Cases of the A(H1N1) Influenza Infection

We next studied the humoral responses to the virus. Depending on serum availability, three techniques were used for serologic assays on the first blood sample (times from first symptoms listed in Table 1): HI assays and microneutralization in 34 patients and ELISA for only 17. With the HI assay, antibody titers were above the positive threshold of 1:40 in only two of the eight (25%) patients who died, whereas 19 of the 26 (73%) survivors tested had antibodies above the threshold (P = 0.05) (Figure 1A). The seven surviving patients with negative serology (#1, 3, 13, 19, 24, 26, and 29) did not differ (age, clinical factors, viral mutation, viremia, bacterial infection, and so forth) from other surviving patients (Table 1). HI antibodies were below the positive threshold in all six patients with fatal fulminant forms (Figure 1A). We note that the cause of death of both patients with

positive HI serology was nosocomial pulmonary infection rather than fulminant influenza. The presence of a pulmonary bacterial infection did not impact the trend of the low HI profile in fatal cases when compared with survivors (see Figure E1A in the online supplement). In microneutralization and ELISA, a trend toward weaker anti-H1N1 antibody titers was observed in the fatal forms of influenza: median titers of 117 in microneutralization, compared with 960 for the survivors (P = 0.10) (Figure 1B), and median titers of 9.7 in ELISA compared with 16.3 in the survivors (P = 0.06) (Figure 1C). Results from the three serologic assays (HI, microneutralization, and ELISA) were therefore concordant (P = 0.05) (Figure 1D). These negative titers in the fulminant cases did not result from taking the blood sample too soon after the first antigen contact, because the time to testing did not differ between the patients with fatal fulminant forms and the survivors (Table 1, Figures 1E and 1F). HI serology for the 13 survivors who were tested after ICU discharge (median, 56 [44–156] d after first symptoms) showed high antibody titers for all (geometric mean of titers, 303) (Figure 1E). Taken together, these results show that levels of anti-H1N1 antibodies in the HI assay never reached a positive level in any patient who died of fulminant influenza, and contrast strongly with the high antibody titers in survivors.

Immunoglobulin Gene Transcription Levels Are Very Strong during Very Severe and Fatal Influenza

We then further investigated whether B-cell deficiencies might explain the failure of the HI assay to detect antibodies in the patients who died of fulminant influenza. B-cell diseases were indeed observed in three of those six cases (one CVID, one Waldenstr¨om disease, and one autoimmune anemia) (Table 1). B-cell numbers and differentiation, their immunoglobulin production capacities, and their transcriptional profiles were evaluated. First, the peripheral blood B-cell counts were similar in the patients who died (median, 267 per cubic millimeter) and the survivors (median, 264 per cubic millimeter; P = 0.66) (see Figure E2A). Second, the B-cell phenotype was similar in the fatal cases and survivors, with normal percentages of naive and switched memory B cells in both groups (73% vs. 66%, P = 0.42; and 12.8% vs. 20%, P = 0.18, respectively) (see Figures E2B and E2C), and detectable peripheral plasmablasts, as expected during acute viral infection (1.8 vs. 2%; P = 0.71) (see Figure E2D). Third, plasma levels of total immunoglobulin classes (see Figures E2E–E2G) and IgG subclasses (see Figures E2H–E2K) did not differ significantly between the two groups. Finally, we studied the host genome-wide transcription profiles in available whole blood cells from three patients with a low SOFA (,10) and three with a high SOFA (>10) score, including one fatal case. Surprisingly, in the three high SOFA patients, among the

Figure 2. Immunoglobulin gene transcription profile in very severe and fatal influenza infection. (A) Ten genes up-regulated most in whole blood from three very severe and fatal cases (sequential organ failure assessment [SOFA] > 10, including one fatal case), classified by fold-change in descending order. P values are indicated above. Ig g-2 chain C, immunoglobulin heavy constant g 2 (IGHG2); MZB1, marginal zone B- and B1-cell–specific protein; MCEM1, mast cell–expressed membrane protein 1 (C19ORF59); Ig heavy chain V-II, immunoglobulin heavy chain V-II region ARH-77; Ig heavy chain V-I, immunoglobulin heavy chain V-I region HG3; miscRNA, homo sapiens misc_RNA (LOC728178); RNASE 2 RNase, RNase A family, 2 (liver, eosinophil-derived neurotoxin); Ig kappa chain V-V, immunoglobulin kappa chain V-V region L7. (B) Heat map showing expression profiles of immunoglobulin genes and T cell–associated genes (CD40L, TCR) in three patients with low SOFA scores (,10) and in three patients with high SOFA scores (>10), including one fatal case.

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ORIGINAL ARTICLE 10 most up-regulated genes, 5 were associated with immunoglobulin production (Figure 2A). As shown on the heatmap, immunoglobulin genes were strongly up-regulated, with or without pulmonary bacterial superinfection, in blood of the three high SOFA patients (#26, 27, and 32) and, to a lesser extent, in the low SOFA patients (#18, 19, and 20) (Figure 2B). Conversely, genes related to T-cell responses, namely TCR-a and CD40L, were not upregulated. Taken together, these results do not suggest any underlying constitutive B-cell deficiency during severe or fatal influenza infection in young adults. Fatal Influenza Infection Is Associated with the Trapping in the Lungs of Influenza-Specific Immunoglobulins That Form Immune Complexes

Because of the discordance between the low levels of anti-H1N1 antibodies in the peripheral blood of fatal cases and the observation of an appropriate immunoglobulin production by peripheral B cells both at the proteic and transcriptomic levels, we tested whether anti-H1N1 antibodies might be undetectable in serum because trapped in immune complexes deposits in the lungs of fatal cases, as previously suggested (25). ELISA assays were performed in the available BAL fluid before and after immune complex dissociation. Antiinfluenza antibodies were barely detectable before dissociation, but increased above the detection threshold afterward in the two fatal cases tested (patients #31 and 33) (Figure 3A). It is noteworthy that immune complex dissociation induced a nearly twofold increase in the levels of BAL H1N1-specific antibodies in the two fatal cases tested and in only two of the seven survivors tested. In the non-H1N1 critically ill patients, these BAL levels remained far below the positive thresholds after immune complex dissociation (Figure 3B). ELISA assays were also performed in available serum samples from five fatal cases (#16, 31, 32, 33, and 34) and eight surviving patients (#5, 10, 11, 18, 19, 20, 26, and 27). Before immune complex dissociation, antibody titers were lowest in serum from fatal cases, as previously shown. After dissociation, influenza-specific antibody titers became positive in all H1N1 patient samples, although they remained weaker in samples from fatal cases (see Figure E3A). ELISA serology also became positive after dissociation in samples from surviving patients with low SOFA (#10, 18, 20, and 26). Serology also became positive in serum

Figure 3. Low serum anti-H1N1 antibody (Ab) titers are caused by immune complex formation in fatal cases and predict death from fatal fulminant infection as early as Day 4 after first flu symptoms. (A and B) Antiinfluenza ELISA antibody levels in bronchoalveolar lavage (BAL) fluid before (left, connected dots) and after (right, connected dots) immune complex dissociation (ICD) as defined in the METHODS section. BAL from nine H1N1 patients including two fatal cases (A, H1N1 pts), and from seven non-H1N1 critically ill patients (B, Critically ill pts) were studied as defined in METHODS. Each black dot represents a surviving patient; crosses represent patients with fatal fulminant infections. Dashed lines are positive ELISA thresholds as defined in the METHODS or online supplement METHODS sections. (C) Statistical model run with data from 76 HI assays for 35 patients as described in the METHODS or online supplement METHODS sections: A(H1N1) 2009-specific antibody titers measured by HIA 4 days after the onset of influenza symptoms predict the probability of death from fatal fulminant H1N1 infection (P = 0.04).

from all nine critically ill patients without H1N1 infection (see Figure E3B). Taken together, these results show that during fatal cases of pandemic influenza, the production of anti-H1N1 antibodies is not altered: the low titers observed in the peripheral blood reflect the trapping of specific immune complexes in the lungs. Pulmonary Translocation of Effector or Effector-Memory H1N1-Specific T Cells in Survivors, with No Memory T-Cell Response to the Virus in Blood from Fatal Cases

We then tested whether the low antibody titers observed in fatal cases were associated with a poor T-helper cellular response to the virus. Strong lymphoproliferative responses were detected against an A(H1N1) 2009 antigen in 9 of the 11 survivors tested (median stimulation

index, 22), but in none of the three fatal cases tested (median stimulation index, 4) (Figure 4A), two of whom had known immune abnormalities (CVID and Waldenstr¨om disease, patients #34 and 17). Global T-cell defects are nonetheless ruled out by the development of T-cell responses against control candidin antigens in two of the three samples from fatal cases tested (patients #14 and 34) (Figure 4A) and against cytomegalovirus in the third fatal case (patient #17, data not shown). In IFN-g ELISpot assay against hemagglutinin and nucleoprotein peptides, high spontaneous IFN-g production by BAL T cells (NS, nonstimulated cells) from the two fatal cases tested (patients #31 and 32) (Figure 4B) prevented interpretation after influenza virus antigen stimulation. With PBMCs, weak IFN-g–specific responses were

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ORIGINAL ARTICLE

Figure 4. Strong local pulmonary-specific T-cell responses to A(H1N1) 2009 antigens despite the severity of infection. (A) Peripheral blood mononuclear cells (PBMCs) lymphoproliferative T-cell responses to an A(H1N1) 2009 antigen (whole inactivated monovalent vaccine, triangles; or recombinant hemagglutinin [HA], circles; left panel) and to a candidin antigen (squares, right panel), as defined in the METHODS or online supplement METHODS sections in surviving patients (black symbols) or fatal cases (gray symbols). Results are expressed as stimulation index (SI). Closed symbols are positive responses (SI > 3 and count per minute [CPM] > 3000) and open symbols are negative responses, as described in the METHODS. Note that one fatal case had a negative response to the H1N1 antigen (CPM = 2,686) despite a high SI (128). Dashed lines are positive thresholds as defined in the METHODS. (B) Specific T-cell responses in ELISpot IFN-g assay to A(H1N1) 2009 HA and nucleoprotein (NP) peptides, as defined in the METHODS, in six surviving H1N1 patients (black dots), and two fatal cases (fulminant form, crosses), after background was subtracted. NS = nonstimulated cells. PHA = phytohemagglutinin tested as control. ELISpot assays were done with PBMC (left) or bronchoalveolar lavage (BAL) lymphocytes (right). Results are expressed as spot-forming cells (SFC) per 106 PBMCs. The dashed line is the positivity threshold, as defined in the METHODS.

observed (Figure 4B). In six survivors, weak responses were also detectable in PBMCs (median, 185 and 0 SFC/106 PBMCs against hemagglutinin and nucleoprotein peptides, respectively) (Figure 4B), in contrast to the very strong responses observed in BAL (median, 2,800 and 2,228 SFC/106 BAL lymphocytes) (Figure 4B). Thus, Figure 4B clearly shows that poor H1N1-Th1 specific T-cell responses in blood contrast with strong Th1 cell activation and IFN-g production in BAL with frequencies of specific cells against NP 2 logs above those in peripheral blood. Otherwise, despite those strong responses, BAL lymphocytosis was mild (median, 4%): the main cellular population was neutrophils (median, 53%), and did not differ regarding the presence of a bacterial pneumonia and as observed in most cases of adult respiratory distress syndrome (see Figure E4). 1246

These results show the pulmonary translocation of effector or effector-memory H1N1-specific T cells in survivors and its contrast to the absence of specific centralmemory cellular responses in the peripheral blood from the three fatal cases tested. The spontaneous IFN-g production in the lungs of fatal cases might also reflect a massive and effective translocation of specific T cells to the lungs. The Severity of H1N1 Infection Is Associated with the Strength of the Proinflammatory Response

Proinflammatory and antiinflammatory cytokines and chemokines were measured in first plasma and BAL samples, and their levels compared for survivors (n = 26) and fatal cases (n = 8). Plasma and BAL levels of IL-6, IL-10, and IP-10 were higher in fatal cases, whereas the TGF-b levels were similar

(Figures 5A and 5B). Both IL-6 and IL-10 plasma levels were positively correlated with the SOFA severity score, whereas TGF-b plasma levels were inversely correlated with it (Figure 5C). The IL-6/TGF-b ratio was analyzed to clarify the cytokine profile (proinflammatory or antiinflammatory); its positive correlation with the SOFA severity score reflects the more proinflammatory profile in patients with the most severe course (Figure 5C). Fatal outcome was also associated with higher levels of the chemokines IL-8, and MCP-1 in plasma but not BAL (see Figure E5). Plasma levels of these chemokines, like those of the cytokines, were positively correlated with the SOFA score (see Figure E5C). To assess if those chemokine and cytokine profiles were specific to influenza infection, we studied 14 critically ill patients (see Table E1). Among the inflammatory profile observed in survivors, only the BAL IP-10 levels were specifically increased in relationship with H1N1 infection because all other cytokine/chemokine levels were not significantly increased in H1N1 patients in both compartments (Figures 5A, 5B, and E5). Finally, neither bacterial pulmonary superinfection nor need for ECMO influenced the cytokine and chemokine profiles, which were identical for both groups in BAL and in plasma (see Figures E1B and E6). This finding confirms that the inflammatory profile we observed was closely correlated with the severity of the H1N1 infection. High Plasma IL-10 Levels, 222G Hemagglutinin Mutation, and Low HI Antibody Titers Predict Fatal Fulminant Influenza Infection

We then examined the various associations of the clinical, virologic, and immunologic parameters with death from fulminant influenza infection. Overall, 76 HI results were available at various time points from first symptoms in 34 patients, including the six fatal fulminant cases. Because the time from first symptoms to the serology assays varied greatly from patient to patient, we used smoothing splines to estimate the individual HI antibody titers at Day 4 from all available individual titers. Then, the number of the following clinical and biological predisposing conditions, described in Table 1, was included as a parameter in a univariate analysis of prognostic factors: bacterial pulmonary coinfection; H1N1 viremia detected by RT-PCR; 222G hemagglutinin viral mutation; plasma levels of IL-6, IL-10, TGF-b, and IP-10; specific lymphoproliferative

American Journal of Respiratory and Critical Care Medicine Volume 189 Number 10 | May 15 2014

ORIGINAL ARTICLE T-cell responses; anti-A(H1N1) 2009 microneutralizing antibody titers; and estimated (or actual) HI antibody titers at Day 4. Four prognostic factors were significantly associated with death from fulminant influenza: 222G mutation, plasma IL-10 and IP-10 levels, and Day 4 estimated HI antibody titers (P , 0.05) (Table 2). In the multivariate analysis, the two best prognostic factors were plasma IL-10 levels and HI antibody titers, although neither was independently significantly associated with death (P = 0.19 and P = 0.07) (Table 2). We then tested whether the HI assay, which was the best prognostic factor, could predict death from influenza infection. The influenza-specific log-antibody titer estimated at Day 4 after onset of H1N1 influenza symptoms predicted survival very accurately: a 1:40 titer predicted a 0.007% risk of death from fulminant H1N1 infection, but a low 1:2 titer at Day 4 after symptoms began increased the probability of death to 64%, and a 1:1 titer to 95% (P = 0.04) (Figure 3C). These data show that fatal fulminant influenza infection can be predicted by very low titers of HI antiH1N1 antibodies at Day 4 after onset of influenza symptoms.

Discussion

Figure 5. Cytokine and chemokine profile in plasma and bronchoalveolar lavage (BAL) during severe H1N1 pneumonia. (A and B) IL-6, IL-10, transforming growth factor (TGF)-b, and IP-10 levels in plasma (A) and BAL fluid (B) in surviving patients with severe H1N1 infection (black dots), fatal fulminant infection (crosses), and fatal nosocomial pulmonary infection (light gray dots), and in critically ill patients (dark gray dots). Dashed squares are normal values as defined in the METHODS. *P , 0.05, **P , 0.005, ***P , 0.0001, Mann-Whitney test. (C) IL-6, IL-10, TGF-b, IP-10, and IL-6/ TGF-b ratio of plasma levels correlated with the worst sequential organ failure assessment (SOFA) severity score in the intensive care unit (ICU) for each patient. The Spearman correlation test was used. All cytokine results are expressed in pg/ml. IP = IFN-g–induced protein; NS = nonsignificant.

Here we report a large series of severe-to-fatal influenza A(H1N1) 2009 for which extensive immunologic and virologic studies of both peripheral blood and BAL fluid are available. This study provides the identification of a biologic marker predicting death from fulminant infection (low anti-H1N1 antibody titers in serum Day 4 after first flu symptoms) and proposes a mechanism for this finding with indirect evidence of immune complex formation in the lung. We identify three immune characteristics during fatal cases of A(H1N1) 2009 infection in young adults. First, we showed systemic inflammation with high levels of IL-6, IL-10, IL-8, IP-10, and MCP-1, and with IL-6 and IP10 accumulating in the lungs. Only the high IP-10 BAL levels seemed to be specific to H1N1 infection since higher in the H1N11 patients and in line with the massive infiltration of H1N1-specific Th1 cells in the lungs (Figure 4B). Moreover, these cytokines are known to increase during very severe or fatal forms of avian H5N1 and H7N9 pulmonary infection (8–10), pandemic

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ORIGINAL ARTICLE Table 2: Univariate and Multivariate Analyses of Prognostic Factors for Death from Fulminant Influenza Infection

Prognostic Factor No. of clinical risk factors Bacteria in BAL (Y/N) Viral load (Y/N) H1N1 HA mutation 222 (D/G)† Plasma IL-6 Plasma IL-10 Plasma TGF-b Plasma IP-10 HA proliferation Neutralizing Ac IHA J4 (per twofold increase)

Parameter Estimate

Univariate Analysis Odds 95% CI of Ratio Odds Ratio P Value

21.01

0.36

0.06–2.09

0.26

20.12 1.79 21.44

0.89 6.00 0.05

0.13–5.85 0.65–55.6 0.004–0.81

0.90 0.11 0.03

0.044 0.028 20.094 0.11 20.24 20.33 22.28

1.04 1.03 0.91 1.12 0.78 0.72 0.10

1.00–1.09 1.001–1.06 0.76–1.09 0.004–0.81 0.53–1.17 0.43–1.19 0.07–0.93

0.05 0.04 0.31 0.03 0.23 0.19 0.04

AIC Criterion*

Parameter Estimate

Multivariate Analysis Odds 95% CI of Ratio Odds Ratio

P Value

34.1 26.1

0.03 (1.06)

1.03

0.99–1.07

0.19

22.36 (25.25)

0.09

0.01–1.28

0.07

34.1 22.2

Definition of abbreviations: AIC = Akaike information criterion; BAL = bronchoalveolar lavage; CI = confidence interval; HA = hemagglutinin; IHA = inhibition hemagglutination assay; IP-10 = IFN-g–induced protein-10; TGF = transforming growth factor. *Rescaled to account for missing data. † N = 16.

H1N1 infection (16, 31), and adult respiratory distress syndrome or septic shock. Second, we report a strong antibody response during very severe or fatal influenza infection (microarray data in six patients) located in the lungs where they are likely to form immune complexes, whereas systemic antibody levels are low. This lack of serum anti-H1N1 antibodies has not previously been reported (14, 25), perhaps because previous studies compared severe forms with mild disease and not with fatal cases, or were unable to study the kinetics of the immune response. The high ELISA antibody levels we observed after immune complex dissociation in BAL are reminiscent of the lung immune complex deposition reported in fatal cases of A(H1N1) 2009 infection by Monsalvo and coworkers (25), although they did not report either the specificity of the antibodies deposited. It is also reminiscent of mycoplasma chronic lymphatic and blood vessel remodeling related with immune complex–dependent inflammation (32). Third, we also observed a strong effector T-cell response localized in the lungs, with strong activation, that is, spontaneous IFN-g production and high IP-10 levels, together with an absence of a peripheral memory T-cell response. Another important aspect of our study is that it provides for the first time a biologic marker for a fatal case. The 1248

earliest influenza HI antibody response, detected 4 days after the onset of influenza symptoms, was the most predictive marker of death, compared with cytokine and chemokines levels and virologic factors. Indeed, half of patients with a negative serology die afterward. Although the beginning of influenza symptoms might not reflect exactly the time of infection, and although this was a predictive value and not real blood sample at Day 4, such a marker should be studied in future pandemics and proved to be useful in the clinical management of a severe flu pandemic. There are some limitations in our study. First, in a small group of patients, not all analysis was conducted: for example, we were unable to exhaustively study the immune mechanism leading to death in the fatal cases described. Second, the onset of influenza symptoms may not reflect the exact time of infection. Blood and BAL samples were collected late after the first symptoms of infection, whereas earlier serologies or BAL formula would be more pertinent. Third, it was not possible to analyze lung necropsies because this was not initially scheduled. However, we showed that after immune complex dissociation in BAL, the influenza serology became positive in the two lethal cases tested. In conclusion, we show here that in unvaccinated patients, low anti-H1N1 2009 antibody titers as early as Day 4 after the first influenza symptoms can predict death from

fulminant infection. This low level of specific antibodies in the blood does not reflect B-cell deficiencies or an inability to produce antibodies but reflects instead the trapping of antiinfluenza antibodies in immune complexes in the lungs. Those results encourage future works on the affinity of anti-H1N1 antibodies in fatal cases, and in animal models testing the efficacy of high-affinity antibody infusions, a first step before considering the possibility of using it in clinical practice. n

Author disclosures are available with the text of this article at www.atsjournals.org. Acknowledgment: The authors thank the T4T8 department, Melanie ´ Lavenu, Ben ´ edicte ´ Hoareau, Olivier Pelle, Anne Oudin, and Veronique Morin at INSERM UMR-S945 for technical assistance. They thank Vincent Enouf, Valerie ´ Caro, and their team at the Institut Pasteur. They also thank Drs. Marion Antona, Gael Mouillot, Antoine Roux, and Pascale Ghillani for medical assistance. They thank Christophe Combadiere, ` Bruno Lina, and Eric Oksenhendler for their advice, and Jo Ann Cahn for revising the English. The FluBAL Study Group: Henri Agut, Djillali Annane, Brigitte Autran, David Boutolleau, Christian Brun-Buisson, Guislaine Carcelain, Jean-Marc Cavaillon, Behazine ´ Combadiere, ` Christophe Combadiere, ` Jean-Luc Diehl, Alexandre Duguet, Muriel Fartoukh, Amelie ´ Guihot, Charles-Edouard Luyt, Alain Mallet, Eric Maury, Jean-Louis Pallot, Antoine Parrot, Didier Payen de la Garanderie, Dominique Rousset, Sylvie van der Werf, and Michel Wolff.

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