Host adaptive immunity deficiency in severe pandemic influenza
- Jesus F Bermejo-Martin1, 2Email author,
- Ignacio Martin-Loeches3,
- Jordi Rello4,
- Andres Antón5,
- Raquel Almansa1, 2,
- Luoling Xu6,
- Guillermo Lopez-Campos7,
- Tomás Pumarola5,
- Longsi Ran6,
- Paula Ramirez8,
- David Banner6,
- Derek Cheuk Ng6,
- Lorenzo Socias9,
- Ana Loza10,
- David Andaluz11,
- Enrique Maravi12,
- Maria J Gómez-Sánchez12,
- Mónica Gordón8,
- Maria C Gallegos13,
- Victoria Fernandez13,
- Sara Aldunate12,
- Cristobal León10,
- Pedro Merino14,
- Jesús Blanco14,
- Fernando Martin-Sanchez7,
- Lucia Rico1, 2,
- David Varillas1, 2,
- Veronica Iglesias1, 2,
- Maria Ángeles Marcos5,
- Francisco Gandía11,
- Felipe Bobillo11,
- Begoña Nogueira2,
- Silvia Rojo2,
- Salvador Resino15,
- Carmen Castro16,
- Raul Ortiz de Lejarazu1, 2 and
- David Kelvin6, 17, 18
© Bermejo-Martin et al.; licensee BioMed Central Ltd. 2010
Received: 14 June 2010
Accepted: 14 September 2010
Published: 14 September 2010
Pandemic A/H1N1/2009 influenza causes severe lower respiratory complications in rare cases. The association between host immune responses and clinical outcome in severe cases is unknown.
We utilized gene expression, cytokine profiles and generation of antibody responses following hospitalization in 19 critically ill patients with primary pandemic A/H1N1/2009 influenza pneumonia for identifying host immune responses associated with clinical outcome. Ingenuity pathway analysis 8.5 (IPA) (Ingenuity Systems, Redwood City, CA) was used to select, annotate and visualize genes by function and pathway (gene ontology). IPA analysis identified those canonical pathways differentially expressed (P < 0.05) between comparison groups. Hierarchical clustering of those genes differentially expressed between groups by IPA analysis was performed using BRB-Array Tools v.3.8.1.
The majority of patients were characterized by the presence of comorbidities and the absence of immunosuppressive conditions. pH1N1 specific antibody production was observed around day 9 from disease onset and defined an early period of innate immune response and a late period of adaptive immune response to the virus. The most severe patients (n = 12) showed persistence of viral secretion. Seven of the most severe patients died. During the late phase, the most severe patient group had impaired expression of a number of genes participating in adaptive immune responses when compared to less severe patients. These genes were involved in antigen presentation, B-cell development, T-helper cell differentiation, CD28, granzyme B signaling, apoptosis and protein ubiquitination. Patients with the poorest outcomes were characterized by proinflammatory hypercytokinemia, along with elevated levels of immunosuppressory cytokines (interleukin (IL)-10 and IL-1ra) in serum.
Our findings suggest an impaired development of adaptive immunity in the most severe cases of pandemic influenza, leading to an unremitting cycle of viral replication and innate cytokine-chemokine release. Interruption of this deleterious cycle may improve disease outcome.
Pandemic 2009 influenza A(H1N1)(p2009A(H1N1)) viral infections continues to be a public health threat . While the overall case fatality rate is low (< 0.5%), approximately 9 to 31% of hospitalized patients require admission to an intensive care unit (ICU), and 14 to 46% of these severe patients have a fatal outcome [2–5]. Understanding the pathogenic events leading to critical pandemic H1N1disease is important for designing better strategies for prevention and treatment of severe outcomes. Previous studies examining host immune responses in other emerging viruses such as severe acute respiratory syndrome (SARS)-associated coronavirus, suggest that severe disease is characterized by a malfunction of the switch from innate to adaptive immunity in response to the virus . Similar to severe infections caused by H5N1 influenza virus  dysregulated cytokine secretion have been described in severe cases of p2009A(H1N1) [8, 9]. Infection by pandemic 2009 influenza virus causes defective host responses to S. pneumoniae as showed in ex vivo cultured peripheral blood mononuclear cells from pandemic 2009 influenza (A/H1N1) patients . In ferrets infected with pandemic influenza virus, recovery from infection and improved clinical signs are paralleled by a switch between the innate and the adaptive phase of host immune responses .
The potential for the use of gene signatures to better assess the immunopathology and clinical management of severe viral infections has been widely demonstrated in the past [6, 12, 13]. By using a systems biology-based approach, we analyzed the response to viral infection following hospitalization of 19 p2009A(H1N1) critically ill patients admitted to seven Spanish intensive care units. Our results indicate that pandemic H1N1 patients with severe respiratory disease and poor outcome are characterized by an impaired activation of those genes participating in the development of the antiviral adaptive response.
Materials and methods
Study design, participants and sample collection
Nineteen patients attending the participants' ICUs with primary viral pneumonia during the acute phase of influenza virus illness with acute respiratory distress and unequivocal alveolar opacification involving two or more lobes with negative respiratory and blood bacterial cultures at admission were recruited from 1 November to 31 December 2009. Patients older than 65 years and younger than 18 years were excluded from the study to avoid immaturity/aging of the immune system as confusion factor in the analysis. Only those patients with confirmed H1N1 infection by real-time polymerase chain reaction (PCR) were included in the study (n = 19). Serial blood samples for plasma, serum and RNA were collected by using serum, ethylenediaminetetraacetic acid (EDTA) and PaxGene (BD) venous blood vacuum collection following the manufacturer's instructions at days 1, 3/5 and 7 after admission to the ICU, according to a unified protocol for all the participant centers. A pharyngeal sample was collected in parallel. Fifteen healthy volunteers of similar age to the patients were recruited between workers of the University of Valladolid, Spain. A standard survey was employed to collect the clinical data, including history and physical examination, oximetric measurement, hematological, biochemical, radiological and microbiological investigation in all the participant centers. Treatment decisions for all patients, including corticosteroid therapy, were not standardized and were decided by the attending physician. Informed consent was obtained directly from each patient or their legal representative and also from the healthy controls before enrollment. Patient and control identification remained anonymous. Approval of the study protocol in both the scientific and the ethical aspects was obtained from the Scientific Committees for Clinical Research of each one of the participant centers.
Samples were stored at -80°C until cytokine, antibody and RNAm profiling. Attending to timing of seroconversion (production of antibodies against p2009A(H1N1)), day 9 from onset of symptoms was considered as the border between the innate and the adaptive immune response in the patients, establishing two moments in the evolution of the disease: an early phase (from onset of symptoms (day 0) to day 8) and a late phase (from day 9 and above). Patients were divided into two groups, depending on their respiratory status. The MV group needed invasive mechanical ventilation following admission; the NMV group was composed of those patients not needing mechanical ventilation at any moment during hospitalization. Cytokines, gene expression and viral load of MV patients were compared with those of NMV patients in both early and late phases separately. The number of samples analyzed in each phase is detailed in the Additional file 1.
Viral diagnosis was performed on RNA from pharyngeal swabs in the Microbiology Services of the participant hospitals by reverse transcription-polymerase chain reaction (RT-PCR)-based methods using reagents provided free of charge by the Centers for Disease Control (CDC, Atlanta, GA, USA) or purchased from Roche (Basel, Switzerland) (H1N1 detection set). These samples were also assessed by multiplex PCR (Luminex) with the xTAG RVP kit from Luminex-Abbott for coinfection with respiratory syncytial virus, influenza B virus, parainfluenza viruses 1-4, human metapneumovirus, enteroviruses, rhinovirus, adenovirus, bocavirus and coronaviruses NL63, HKU1, 229E, OC43, in accordance with the manufacturer's instructions. Viral load was quantified in both pharyngeal swabs and plasma in the Virology Lab of the WHO-associated center at Hospital Clinic in Barcelona, Spain, as detailed in Additional file 1. Oseltamivir resistance was directly detected in the initial positive pharyngeal swab by RT-PCR and sequencing of a 1296-bp fragment of the neuraminidase gene for the presence of the mutation H274Y by using an ABI 3130XL Genetic Analyzer.
Hemagglutination inhibition assays (HAI)
HAI assays were performed on a 100-μl aliquot of the samples at University Health Network (UHN), Toronto, Ontario, Canada. The sera were treated with receptor-destroying enzyme (RDE) of V. cholerae by diluting one part serum with three parts enzyme and were incubated overnight in a 37°C water bath. The enzyme was inactivated by 30-min incubation at 56°C followed by the addition of six parts 0.85% physiological saline for a final dilution of 1/10. HI assays were performed in V-bottom 96-well microtiter plates (Corning Costar Co., Cambridge, MA, USA) with 0.5% turkey erythrocytes as previously described  using inactivated pandemic influenza A/California/07/2009(p2009A(H1N1)) antigens.
Microarrays were performed at University Health Network (UHN), Toronto, Ontario, Canada. More detailed explanation of microarray assays is provided in Additional file 1. Ingenuity Pathway Analysis 8.5 (IPA) (Ingenuity Systems, Redwood City, CA, USA) was used to select, annotate and visualize genes by function and pathway (gene ontology). IPA analysis identified those canonical pathways differentially expressed (P < 0.05) between comparison groups. Hierarchical clustering of those genes differentially expressed between groups by IPA analysis was performed using BRB-Array Tools v.3.8.1 stable release developed by Dr. Richard Simon and the BRB-array tools development team. Resulting microarray data sets have been uploaded at the GEO microarray data repository [GEO:GSE21802] . We verified changes in microarray gene expression using quantitative real-time PCR (QRT-PCR) for representative genes from our analysis (Figure S1 in Additional file 2). Primers specific for human GAPDH mRNA were used to normalize samples.
Immune mediator profiling
Immune mediator levels in serum were measured in patients and controls by using the multiplex Bio-Rad 27-plex assay (Hercules, CA, USA) in the Infection & Immunity Unit (Hospital Clínico Universitario-IECSCYL, Valladolid, Spain). This system allows for quantitative measurement of 27 different chemokines, cytokines, growth factors and immune mediators while consuming a small amount of biological material. A number of additional soluble mediators were measured by using enzyme-linked inmunosorbent assays (ELISAs): interferon α and β (Verikine kits purchased from Pbl Interferon Source, Piscataway, NJ, USA), IL-23, TGF-β1 (Quantikine kits purchased from R&D Systems, Minneapolis, MN, USA), IL28A (Legend Max kit purchased from BioLegend, San Diego, CA, USA). Immune mediator's concentration of each individual sample was normalized against the median of the concentration of the control group (n = 15), and the resultant ratios were compared between groups of patients.
The Mann-Whitney U test was employed for cytokine comparison purposes, since the Saphiro Wilk test evidenced absence of normal distribution of the data, and the Levene test demostrated absence of homogeneity of variance in both MV and NMV groups. Correlation studies between cytokine levels, gene expression levels, viral load and clinical parameters were done by calculating the Spearman correlation coefficients. All statistical tests were two-sided, and P < 0.05 was considered significant.
Clinical characteristics of p2009A (H1N1) Patients
Clinical and laboratory characteristics of the patients
MV (n= 12)
NMV (n= 7)
Caucasian (10/12), gipsy (1/12), India (1/12)
Caucasian (5/7), Black (1/7), Magreb (1/7)
Pandemic influenza vaccine
Chronic respiratory disease
Chronic renal disease
Obesity (BMI > 30)
1/12 (32 weeks)
2/7 (31 and 27 weeks)
Duration of symptoms at ICU admission
O2 saturation at admission
Days at ICU
Days at hospital
Days since onset to intubation
Duration of symptoms before oseltamivir
Days with oseltamivir (days)
Steroids at sampling
Infiltrates in chest X-ray
Progression of infiltrates to all 4 quadrants on chest X-ray
[Microbe, sample (resp culture-RC; hemoculture-HC),
days from ICU admission to first bacterial isolation,
days from symptoms onset to first bacterial isolation]
1. S. aureus, A. fumigatus, RC, E. faecalis, HC, 2,10
2. S. marcenses, HC, 7,10
3. P. aeruginosa, RC, 5,14
4. C. albicans, S. Marcenses, RC, 43,52
5. A. fumigatus, C. Albicans, RC, 6, 12
Lymphocytes x103 /mm3
Gene expression profiling
Immune mediators profiling
Here we examined host immune responses in severe patients requiring admission to the ICU. On the basis of the presence of anti-p2009A(H1N1) antibodies, we were able to identify two phases of disease in severe patients: an early phase characterized by the absence of antibodies (innate immunity phase) and a later phase defined by the presence of circulating anti-p2009A(H1N1) antibodies (adaptive immunity phase). Analysis of gene expression and cytokine profiles led to the characterization of signatures that are associated with disease severity and poor outcome in the late phase.
The impaired expression of a number of MHC class II (DM, DP, DQ, DR), MHC class I (HLA-C) genes, of T cell receptor-associated genes (CD4, CD8A, CD8B1) and also of a group of genes participating in dendritic cell maturation (CCR7, CD1C, IL18) points to the existence of a defective antigen presentation in the most severe group of patients (those who needed mechanical ventilation, MV) in the late phase of adaptive immunity. An adequate antigen presentation is needed to develop an effective adaptive immunity to influenza viruses . Under some circumstances, changes affecting antigen presentation more strongly impact viral kinetics in the host than other viral or immune factors . Disruption of antigen presentation prevents an effective adaptive immune response. Evidence on the potential role of an altered antigen presentation on the development of an appropriated adaptive response against the virus comes from the impaired expression of a group of genes pivotal to the activation and function of both T and B cells observed in the MV group in the late phase. Defective expression of CXCR5, MHC class II molecules, IL12RB1, IL21R and IL6R supports an impaired T helper cell differentiation signaling in this group of patients. Poor expression of CD4, FYN, GRB2, MHC class II molecules, ITPR3, MALT1, NFATC1, NFATC3, PDPK1, PIK3R1 and PLCG1 genes indicates a disruption in CD28 signaling in T helper cells, which is needed for effective primary T-cell expansion . Impaired expression of DFFA, ENDOG, NUMA1, PARP1 and PRKDC affects granzyme B signaling. This pathway is involved in the induction of apoptosis in virus-infected cells by cytotoxic T lymphocytes (CTLs) . Impaired T helper cell differentiation, CD28 and granzyme B signalling, along with the poor expression of T cell receptor associated genes (CD4, CD8A, CD8B1), supports a defective T cell response during the phase of adaptive immunity in the MV group. Moreover, the poor expression of genes related to B cell development and B cell receptor signaling (CD79A, CD79B, IL7R, MHC class II molecules, ABL1, CAMK2 D, MALT1, INPP5 D, HRAS, GRB2) points to an altered B cell function during this key period of the host response to the virus.
Additional clues on the existence of a defective adaptive response in severe pandemic influenza come from the impaired expression of a group of genes participating in the apoptosis signaling pathway (AIFM1, BIRC3, CAPN1, CAPN7, CAPNS1, CASP6, DFFA, ENDOG, HRAS, PARP1, PLCG1, TP 53). Since apoptosis is a recognized antiviral mechanism , a defect in apoptosis could translate into poor control of the virus. Additionally, defective expression of several ubiquitin-conjugating enzymes and ubiquitin-specific peptidases demonstrates that ubiquitination is also affected in severe pandemic influenza during the phase of adaptive response. Ubiquitination regulates the development of many phases of the immune response, including its initiation, propagation and termination . The alteration of this pathway in severe pandemic influenza could affect in consequence all the steps needed for the development of an appropriate response to the virus. The role of steroids or immunosuppressor drugs in the genesis of the impaired adaptive response should be very limited, since none of the patients of the most severe group were under immunosuppressor treatment by the admission date. In addition, as detailed in Table 1, the proportion of patients under steroid treatment at the moment of sample collection during the hospitalization period was very similar in both groups of patients (41.6% for MV and 57.1% in NMV); in consequence, steroid treatment should affect similarly both groups in terms of modulation of the immune response. The ability showed by the vast majority of the patients in the MV group to produce specific antibodies indicates that antibody generation was insufficient to overcome the infection. Our results on gene expression support a defect in cellular immunity on the basis of the poor control of the virus in this group. It is well known that T helper and CTL responses play a determinant role in the containment of influenza once infection has occurred [21–23]. Our group is now designing further studies aimed at clarifying the participation of cellular responses in the severe disease caused by p2009A(H1N1).
On the other hand, patients of the MV group showed higher expression levels of those genes participating of the IL-6 and IL-10 canonical pathways during the phase of adaptive immunity. These pathways play opposite roles: proinflammatory and anti-inflammatory, respectively. In addition, serum levels of both IL-6 and IL-10 proteins are the highest in the MV group in this phase group which showed also elevated levels of chemokines, Th1 cytokines and growth factors. The presence of hypercytokinemia has been recently reported during infection by p2009A(H1N1) . It has been described also during fatal H5N1 disease, severe SARS [6, 7], acute RSV bronchiolitis  and sepsis . Positive association between chemokines, cytokines and viral load in our study evidences that they are markers of ongoing viral replication as previously observed in SARS and H5N1 infection [6, 12]. The high levels of the immunomodulatory molecules IL-1ra and IL-10 could represent an attempt to prevent cytokine-driven inflammatory damage or alternatively a virus-induced evasion mechanism [26–29]. The positive correlation observed between IL-10, viral load and SOFA, and the negative correlations between this cytokine and the expression levels of the genes participating in the antigen presentation pathway, supports the role of this mediator in favoring viral replication. As detailed in Table 1, bacterial superinfections took place not in the early but in the late course of the disease. This supports the role of the impaired adaptive response and the release of immunosuppressory cytokines in the increased incidence of bacterial superinfection observed in severe disease following infection by p2009A(H1N1) .
The association between host immune responses and clinical outcome in severe pandemic 2009 influenza is poorly known. The potential for the use of gene signatures to better assess the immunopathology and clinical management of severe viral infections has been widely demonstrated in the past.
Previous studies examining host gene expression profiles in other emerging viruses such as SARS-associated coronavirus, suggest severe disease is characterized by a malfunction of the switch from innate to adaptive immunity in response to the virus. Similar to severe infections caused by H5N1 influenza virus, dysregulated cytokine secretion has been described in severe cases of p2009A(H1N1).
Pandemic H1N1 patients with severe respiratory disease and poor outcomes are characterized by an impaired activation of those genes participating in the development of the antiviral adaptive response and by persistence of the virus in the respiratory tract. These findings suggest a state of HAID that resembles the concept of immunoparalysis described for sepsis.
HAID coexists with a persistent release of cytokines in those patients with the poorest outcomes.
These results support the idea that HAID would lead to an unremitting cycle of viral replication and innate cytokine-chemokine release. Interruption of this deleterious cycle with antiviral and/or immunomodulatory therapies may lead to improved disease outcome.
human fibroblast growth factor-basic
granulocyte colony-stimulating factor
granulocyte macrophage colony-stimulating factor
host adaptive immunity deficiency
interleukin 1 receptor antagonist
monocyte chemoattractant protein-1
macrophage inflammatory protein-1α
macrophage inflammatory protein-1β
new variant of H1N1 influenza virus
platelet-derived growth factor
respiratory syncytial virus
severe acute respiratory syndrome
sepsis-related organ failure assessment
tumor necrosis factor α
vascular endothelial growth factor.
This work has been developed by an international team pertaining to the Spanish-Canadian Consortium for the Study of Influenza Immunopathogenesis. The authors would like to thank also to the Nursery teams who kindly collected the samples. The authors would like to thank Nikki Kelvin for language revision of this article. The study was scientifically sponsored by the Spanish Society for Critical Care Medicine (SEMICYUC). Funding: MICCIN-FIS/JCYL-IECSCYL-SACYL (Spain): Programa de Investigación Comisionada en Gripe, GR09/0021-EMER07/050- PI081236-RD07/0067. CIHR-NIH-Sardinia Recherché-LKSF Canada support DJK.
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