IL-17 response mediates acute lung injury induced by the 2009 pandemic influenza A (H1N1) virus

The 2009 flu pandemic involved the emergence of a new strain of a swine-origin H1N1 influenza virus (S-OIV H1N1) that infected almost every country in the world. Most infections resulted in respiratory illness and some severe cases resulted in acute lung injury. In this report, we are the first to describe a mouse model of S-OIV virus infection with acute lung injury and immune responses that reflect human clinical disease. The clinical efficacy of the antiviral oseltamivir (Tamiflu) administered in the early stages of S-OIV H1N1 infection was confirmed in the mouse model. Moreover, elevated levels of IL-17, Th-17 mediators and IL-17-responsive cytokines were found in serum samples of S-OIV-infected patients in Beijing. IL-17 deficiency or treatment with monoclonal antibodies against IL-17-ameliorated acute lung injury induced by the S-OIV H1N1 virus in mice. These results suggest that IL-17 plays an important role in S-OIV-induced acute lung injury and that monoclonal antibodies against IL-17 could be useful as a potential therapeutic remedy for future S-OIV H1N1 pandemics.     

Interleukin-6 is a potential biomarker for severe pandemic H1N1 influenza A infection

Pandemic H1N1 influenza A (H1N1pdm) is currently a dominant circulating influenza strain worldwide. Severe cases of H1N1pdm infection are characterized by prolonged activation of the immune response, yet the specific role of inflammatory mediators in disease is poorly understood. The inflammatory cytokine IL-6 has been implicated in both seasonal and severe pandemic H1N1 influenza A (H1N1pdm) infection. Here, we investigated the role of IL-6 in severe H1N1pdm infection. We found IL-6 to be an important feature of the host response in both humans and mice infected with H1N1pdm. Elevated levels of IL-6 were associated with severe disease in patients hospitalized with H1N1pdm infection. Notably, serum IL-6 levels associated strongly with the requirement of critical care admission and were predictive of fatal outcome. In C57BL/6J, BALB/cJ, and B6129SF2/J mice, infection with A/Mexico/4108/2009 (H1N1pdm) consistently triggered severe disease and increased IL-6 levels in both lung and serum. Furthermore, in our lethal C57BL/6J mouse model of H1N1pdm infection, global gene expression analysis indicated a pronounced IL-6 associated inflammatory response. Subsequently, we examined disease and outcome in IL-6 deficient mice infected with H1N1pdm. No significant differences in survival, weight loss, viral load, or pathology were observed between IL-6 deficient and wild-type mice following infection. Taken together, our findings suggest IL-6 may be a potential disease severity biomarker, but may not be a suitable therapeutic target in cases of severe H1N1pdm infection due to our mouse data.     

Differential Pathological and Immune Responses in Newly Weaned Ferrets Are Associated with a Mild Clinical Outcome of Pandemic 2009 H1N1 Infection

Young children are typically considered a high-risk group for disease associated with influenza virus infection. Interestingly, recent clinical reports suggested that young children were the smallest group of cases with severe pandemic 2009 H1N1 (H1N1pdm) influenza virus infection. Here we established a newly weaned ferret model for the investigation of H1N1pdm infection in young age groups compared to adults. We found that young ferrets had a significantly milder fever and less weight loss than adult ferrets, which paralleled the mild clinical symptoms in the younger humans. Although there was no significant difference in viral clearance, disease severity was associated with pulmonary pathology, where newly weaned ferrets had an earlier pathology improvement. We examined the immune responses associated with protection of the young age group during H1N1pdm infection. We found that interferon and regulatory interleukin-10 responses were more robust in the lungs of young ferrets. In contrast, myeloperoxidase and major histocompatibility complex responses were persistently higher in the adult lungs; as well, the numbers of inflammation-prone granulocytes were highly elevated in the adult peripheral blood. Importantly, we observed that H1N1pdm infection triggered formation of lung structures that resembled inducible bronchus-associated lymphoid tissues (iBALTs) in young ferrets which were associated with high levels of homeostatic chemokines CCL19 and CXCL13, but these were not seen in the adult ferrets with severe disease. These results may be extrapolated to a model of the mild disease seen in human children. Furthermore, these mechanistic analyses provide significant new insight into the developing immune system and effective strategies for intervention and vaccination against respiratory viruses.     

A protective role for complement C3 protein during pandemic 2009 H1N1 and H5N1 influenza A virus infection

Highly pathogenic H5N1 influenza infections are associated with enhanced inflammatory and cytokine responses, severe lung damage, and an overall dysregulation of innate immunity. C3, a member of the complement system of serum proteins, is a major component of the innate immune and inflammatory responses. However, the role of this protein in the pathogenesis of H5N1 infection is unknown. Here we demonstrate that H5N1 influenza virus infected mice had increased levels of C5a and C3 activation byproducts as compared to mice infected with either seasonal or pandemic 2009 H1N1 influenza viruses. We hypothesized that the increased complement was associated with the enhanced disease associated with the H5N1 infection. However, studies in knockout mice demonstrated that C3 was required for protection from influenza infection, proper viral clearance, and associated with changes in cellular infiltration. These studies suggest that although the levels of complement activation may differ depending on the influenza virus subtype, complement is an important host defense mechanism.     

Seasonal H1N1 influenza virus infection induces cross-protective pandemic H1N1 virus immunity through a CD8-independent, B cell-dependent mechanism

During the 2009 H1N1 influenza virus pandemic (pdmH1N1) outbreak, it was found that most individuals lacked antibodies against the new pdmH1N1 virus, and only the elderly showed anti-hemagglutinin (anti-HA) antibodies that were cross-reactive with the new strains. Different studies have demonstrated that prior contact with the virus can confer protection against strains with some degree of dissimilarity; however, this has not been sufficiently explored within the context of a pdmH1N1 virus infection. In this study, we have found that a first infection with the A/Brisbane/59/2007 virus strain confers heterologous protection in ferrets and mice against a subsequent pdmH1N1 (A/Mexico/4108/2009) virus infection through a cross-reactive but non-neutralizing antibody mechanism. Heterologous immunity is abrogated in B cell-deficient mice but maintained in CD8(-/-) and perforin-1(-/-) mice. We identified cross-reactive antibodies from A/Brisbane/59/2007 sera that recognize non-HA epitopes in pdmH1N1 virus. Passive serum transfer showed that cross-reactive sH1N1-induced antibodies conferred protection in naive recipient mice during pdmH1N1 virus challenge. The presence or absence of anti-HA antibodies, therefore, is not the sole indicator of the effectiveness of protective cross-reactive antibody immunity. Measurement of additional antibody repertoires targeting the non-HA antigens of influenza virus should be taken into consideration in assessing protection and immunization strategies. We propose that preexisting cross-protective non-HA antibody immunity may have had an overall protective effect during the 2009 pdmH1N1 outbreak, thereby reducing disease severity in human infections.     

Protection from the 2009 H1N1 Pandemic Influenza by an Antibody from Combinatorial Survivor-Based Libraries

Influenza viruses elude immune responses and antiviral chemotherapeutics through genetic drift and reassortment. As a result, the development of new strategies that attack a highly conserved viral function to prevent and/or treat influenza infection is being pursued. Such novel broadly acting antiviral therapies would be less susceptible to virus escape and provide a long lasting solution to the evolving virus challenge. Here we report the in vitro and in vivo activity of a human monoclonal antibody (A06) against two isolates of the 2009 H1N1 pandemic influenza virus. This antibody, which was obtained from a combinatorial library derived from a survivor of highly pathogenic H5N1 infection, neutralizes H5N1, seasonal H1N1 and 2009 “Swine” H1N1 pandemic influenza in vitro with similar potency and is capable of preventing and treating 2009 H1N1 influenza infection in murine models of disease. These results demonstrate broad activity of the A06 antibody and its utility as an anti-influenza treatment option, even against newly evolved influenza strains to which there is limited immunity in the general population.     

Pathogenesis and transmission of triple-reassortant swine H1N1 influenza viruses isolated before the 2009 H1N1 pandemic

The 2009 H1N1 pandemic influenza virus represents the greatest incidence of human infection with an influenza virus of swine origin to date. Moreover, triple-reassortant swine (TRS) H1N1 viruses, which share similar host and lineage origins with 2009 H1N1 viruses, have been responsible for sporadic human cases since 2005. Similar to 2009 H1N1 viruses, TRS viruses are capable of causing severe disease in previously healthy individuals and frequently manifest with gastrointestinal symptoms; however, their ability to cause severe disease has not been extensively studied. Here, we evaluated the pathogenicity and transmissibility of two TRS viruses associated with disease in humans in the ferret model. TRS and 2009 H1N1 viruses exhibited comparable viral titers and histopathologies following virus infection and were similarly unable to transmit efficiently via respiratory droplets in the ferret model. Utilizing TRS and 2009 H1N1 viruses, we conducted extensive hematologic and blood serum analyses on infected ferrets to identify lymphohematopoietic parameters associated with mild to severe influenza virus infection. Following H1N1 or H5N1 influenza virus infection, ferrets were found to recapitulate several laboratory abnormalities previously documented with human disease, furthering the utility of the ferret model for the assessment of influenza virus pathogenicity.     

Pre-Existing Immunity with High Neutralizing Activity to 2009 Pandemic H1N1 Influenza Virus in Shanghai Population

Pre-existing immunity is an important factor countering the pandemic potential of an emerging influenza virus strain. Thus, studying of pre-existing immunity to the 2009 pandemic H1N1 virus (2009 H1N1) will advance our understanding of the pathogenesis and epidemiology of this emerging pathogen. In the present study, sera were collected from 486 individuals in a hospital in Shanghai, China, before the 2009 H1N1 influenza pandemic. The serum anti-hemagglutinins (HA) antibody, hemagglutination inhibition (HI) antibody and neutralizing antibody against the 2009 H1N1 were assayed. Among this population, 84.2%, 14.61% and 26.5% subjects possessed anti-HA antibody, HI antibody and neutralizing antibody, respectively. Although neutralizing antibody only existed in those sera with detectable anti-HA antibody, there was no obvious correlation between the titers of anti-HA and neutralizing antibody. However, the titers of anti-HA and neutralizing antibody against seasonal H1N1 virus were highly correlated. In the same population, there was no correlation between titers of neutralizing antibody against 2009 H1N1 and seasonal H1N1. DNA immunization performed on mice demonstrated that antibodies to the HA of 2009 pandemic and seasonal H1N1 influenza viruses were strain-specific and had no cross-neutralizing activity. In addition, the predicted conserved epitope in the HA of 2009 H1N1 and recently circulating seasonal H1N1 virus, GLFGAIAGFIE, was not an immunologically valid B-cell epitope. The data in this report are valuable for advancing our understanding of 2009 H1N1 influenza virus infection.     

Preexisting influenza-specific CD4+ T cells correlate with disease protection against influenza challenge in humans

Protective immunity against influenza virus infection is mediated by neutralizing antibodies, but the precise role of T cells in human influenza immunity is uncertain. We conducted influenza infection studies in healthy volunteers with no detectable antibodies to the challenge viruses H3N2 or H1N1. We mapped T cell responses to influenza before and during infection. We found a large increase in influenza-specific T cell responses by day 7, when virus was completely cleared from nasal samples and serum antibodies were still undetectable. Preexisting CD4+, but not CD8+, T cells responding to influenza internal proteins were associated with lower virus shedding and less severe illness. These CD4+ cells also responded to pandemic H1N1 (A/CA/07/2009) peptides and showed evidence of cytotoxic activity. These cells are an important statistical correlate of homotypic and heterotypic response and may limit severity of influenza infection by new strains in the absence of specific antibody responses. Our results provide information that may aid the design of future vaccines against emerging influenza strains.     

Pulmonary immune cells and inflammatory cytokine dysregulation are associated with mortality of IL-1R1-/-mice infected with influenza virus (H1 N1)

Respirovirus infection can cause viral pneumonia and acute lung injury (ALI).The interleukin-1 (IL-1) family consists of proinflammatory cytokines that play essential roles in regulating immune and inflammatory responses in vivo.IL-1 signaling is associated with protection against respiratory influenza virus infection by mediation of the pulmonary anti-viral immune response and inflammation.We analyzed the infiltration lung immune leukocytes and cytokines that contribute to inflammatory lung pathology and mortality of fatal H1N1 virus-infected IL-1 receptor 1 (IL-1R1) deficient mice.Results showed that early innate immune cells and cytokine/chemokine dysregulation were observed with significantly decreased neutrophil infiltration and IL-6,TNF-α,G-CSF,KC,and MIP-2 cytokine levels in the bronchoalveolar lavage fluid of infected IL-1R1-/-mice in comparison with that of wild type infected mice.The adaptive immune response against the H1N1 virus in IL-1R1-/-mice was impaired with downregulated anti-viral Th1 cell,CD8+ cell,and antibody functions,which contributes to attenuated viral clearance.Histological analysis revealed reduced lung inflammation during early infection but severe lung pathology in late infection in IL-1R1-/-mice compared with that in WT infected mice.Moreover,the infected IL-1R1-/-mice showed markedly reduced neutrophil generation in bone marrow and neutrophil recruitment to the inflamed lung.Together,these results suggest that IL-1 signaling is associated with pulmonary anti-influenza immune response and inflammatory lung injury,particularly via the influence on neutrophil mobilization and inflammatory cytokine/chemokine production.     

Pregnant Women Infected with Pandemic H1N1pdm2009 Influenza Virus Displayed Overproduction of Peripheral Blood CD69+ Lymphocytes and Increased Levels of Serum Cytokines

The first pandemic of the 21^st century occurred in 2009 and was caused by the H1N1pdm influenza A virus. Severe cases of H1N1pdm infection in adults are characterized by sustained immune activation, whereas pregnant women are prone to more severe forms of influenza, with increased morbi-mortality. During the H1N1pdm09 pandemic, few studies assessed the immune status of infected pregnant women. The objective of this study was to evaluate the behavior of several immune markers in 13 H1N1pdm2009 virus-infected pregnant (PH1N1) women, in comparison to pregnant women with an influenza-like illness (ILI), healthy pregnant women (HP) and healthy non-pregnant women (HW). The blood leukocyte phenotypes and the serological cytokine and chemokine concentrations of the blood leukocytes, as measured by flow cytometry, showed that the CD69+ cell counts in the T and B-lymphocytes were significantly higher in the PH1N1 group. We found that pro-inflammatory (TNF-α, IL-1β, IL-6) and anti-inflammatory (IL-10) cytokines and some chemokines (CXCL8, CXCL10), which are typically at lower levels during pregnancy, were substantially increased in the women in the ILI group. Our findings suggest that CD69 overexpression in blood lymphocytes and elevated levels of serum cytokines might be potential markers for the discrimination of H1N1 disease from other influenza-like illnesses in pregnant women.     

Antiviral role of toll-like receptors and cytokines against the new 2009 H1N1 virus infection

People are generally susceptible to the 2009 new mutate of H1N1 influenza due to lack of appropriate immunity. Influenza H1N1 2009 infection triggers a massive inflammatory response that contributes to fever, lung impairment or other tissue damage, eventually leading to death. Infection with pathogenic influenza virus H1N1 induces severe pulmonary immune pathology. To date, more than 10,000 cases worldwide have died of the disease. It still has strong infectious ability although the mortality of influenza isn’t currently high. Therefore, to explore the pathogenesis of H1N1 influenza can help with the disease prevention, diagnosis and provide a theoretical basis and the new ideas of treatment. Laboratory confirmed cases of pandemic influenza H1N1 2009 were enrolled to collect general information on pre-clinical, clinical and laboratory data for analysis. Blood samples were obtained from patients with H1N1, healthy volunteers and patients with bacterial pneumonia. Serum were separated and collected. RT–PCR and ELISA methods were applied to detect the different expression of TLRs and cytokines. The young, pregnant and postpartum women and infant are highly susceptible to influenza H1N1 2009 infection; degree of susceptibility is not associated with BMI. Biochemical changes can be seen in the patients with influenza H1N1 2009 infection: ALT, AST, CK, LDH increased in varying degrees. TLR2, TLR3, TLR9 expression increased in the patients with influenza H1N1 2009 infection; no obvious changes of TLR4, TLR7, TLR8 can be detected. In pregnant and postpartum women group, only TLR9 expression increased. The expression of IL-2, IL-6, IFN-γ, TNF-α in the patients with influenza H1N1 2009 infection was significantly increased; while IL-10 expression decreased and IL-4 expression did not change. H1N1 influenza-infected pregnant and postpartum women group, only IL-2 and TNF-α expression expression increased, other cytokines decreased or didn’t change. TLR2, TLR3, TLR9 are the major members of TLR family in the recognition of the novel H1N1 virus to start the innate immune response and adaptive immune responses. TLR9 may be the key receptor among pattern recognized receptors to recognize and bind to H1N1 virus. Cellular immune responses induced by Th1 may participate in modulating the influenza H1N1 2009.     

Comparative analyses of pandemic H1N1 and seasonal H1N1, H3N2, and influenza B infections depict distinct clinical pictures in ferrets

Influenza A and B infections are a worldwide health concern to both humans and animals. High genetic evolution rates of the influenza virus allow the constant emergence of new strains and cause illness variation. Since human influenza infections are often complicated by secondary factors such as age and underlying medical conditions, strain or subtype specific clinical features are difficult to assess. Here we infected ferrets with 13 currently circulating influenza strains (including strains of pandemic 2009 H1N1 [H1N1pdm] and seasonal A/H1N1, A/H3N2, and B viruses). The clinical parameters were measured daily for 14 days in stable environmental conditions to compare clinical characteristics. We found that H1N1pdm strains had a more severe physiological impact than all season strains where pandemic A/California/07/2009 was the most clinically pathogenic pandemic strain. The most serious illness among seasonal A/H1N1 and A/H3N2 groups was caused by A/Solomon Islands/03/2006 and A/Perth/16/2009, respectively. Among the 13 studied strains, B/Hubei-Wujiagang/158/2009 presented the mildest clinical symptoms. We have also discovered that disease severity (by clinical illness and histopathology) correlated with influenza specific antibody response but not viral replication in the upper respiratory tract. H1N1pdm induced the highest and most rapid antibody response followed by seasonal A/H3N2, seasonal A/H1N1 and seasonal influenza B (with B/Hubei-Wujiagang/158/2009 inducing the weakest response). Our study is the first to compare the clinical features of multiple circulating influenza strains in ferrets. These findings will help to characterize the clinical pictures of specific influenza strains as well as give insights into the development and administration of appropriate influenza therapeutics.     

Immune protection induced on day 10 following administration of the 2009 A/H1N1 pandemic influenza vaccine

Background

The 2009 swine-origin influenza virus (S-OIV) H1N1 pandemic has caused more than 18,000 deaths worldwide. Vaccines against the 2009 A/H1N1 influenza virus are useful for preventing infection and controlling the pandemic. The kinetics of the immune response following vaccination with the 2009 A/H1N1 influenza vaccine need further investigation.

Methodology/Principal Findings

58 volunteers were vaccinated with a 2009 A/H1N1 pandemic influenza monovalent split-virus vaccine (15 µg, single-dose). The sera were collected before Day 0 (pre-vaccination) and on Days 3, 5, 10, 14, 21, 30, 45 and 60 post vaccination. Specific antibody responses induced by the vaccination were analyzed using hemagglutination inhibition (HI) assay and enzyme-linked immunosorbent assay (ELISA). After administration of the 2009 A/H1N1 influenza vaccine, specific and protective antibody response with a major subtype of IgG was sufficiently developed as early as Day 10 (seroprotection rate: 93%). This specific antibody response could maintain for at least 60 days without significant reduction. Antibody response induced by the 2009 A/H1N1 influenza vaccine could not render protection against seasonal H1N1 influenza (seroconversion rate: 3% on Day 21). However, volunteers with higher pre-existing seasonal influenza antibody levels (pre-vaccination HI titer ≥1∶40, Group 1) more easily developed a strong antibody protection effect against the 2009 A/H1N1 influenza vaccine as compared with those showing lower pre-existing seasonal influenza antibody levels (pre-vaccination HI titer <1∶40, Group 2). The titer of the specific antibody against the 2009 A/H1N1 influenza was much higher in Group 1 (geometric mean titer: 146 on Day 21) than that in Group 2 (geometric mean titer: 70 on Day 21).

Conclusions/Significance

Recipients could gain sufficient protection as early as 10 days after vaccine administration. The protection could last at least 60 days. Individuals with a stronger pre-existing seasonal influenza antibody response may have a relatively higher potential for developing a stronger humoral immune response after vaccination with the 2009 A/H1N1 pandemic influenza vaccine.     

Memory immune responses against pandemic (H1N1) 2009 influenza virus induced by a whole particle vaccine in cynomolgus monkeys carrying Mafa-A1*052:02

We made an H1N1 vaccine candidate from a virus library consisting of 144 (?=?16 HA×9 NA) non-pathogenic influenza A viruses and examined its protective effects against a pandemic (2009) H1N1 strain using immunologically na?ve cynomolgus macaques to exclude preexisting immunity and to employ a preclinical study since preexisting immunity in humans previously vaccinated or infected with influenza virus might make comparison of vaccine efficacy difficult. Furthermore, macaques carrying a major histocompatibility complex class I molecule, Mafa-A1*052:02, were used to analyze peptide-specific CD8(+) T cell responses. Sera of macaques immunized with an inactivated whole particle formulation without addition of an adjuvant showed higher neutralization titers against the vaccine strain A/Hokkaido/2/1981 (H1N1) than did sera of macaques immunized with a split formulation. Neutralization activities against the pandemic strain A/Narita/1/2009 (H1N1) in sera of macaques immunized twice with the split vaccine reached levels similar to those in sera of macaques immunized once with the whole particle vaccine. After inoculation with the pandemic virus, the virus was detected in nasal samples of unvaccinated macaques for 6 days after infection and for 2.67 days and 5.33 days on average in macaques vaccinated with the whole particle vaccine and the split vaccine, respectively. After the challenge infection, recall neutralizing antibody responses against the pandemic virus and CD8(+) T cell responses specific for nucleoprotein peptide NP262-270 bound to Mafa-A1*052:02 in macaques vaccinated with the whole particle vaccine were observed more promptly or more vigorously than those in macaques vaccinated with the split vaccine. These findings demonstrated that the vaccine derived from our virus library was effective for pandemic virus infection in macaques and that the whole particle vaccine conferred more effective memory and broader cross-reactive immune responses to macaques against pandemic influenza virus infection than did the split vaccine.     

Early adaptive humoral immune responses and virus clearance in humans recently infected with pandemic 2009 H1N1 influenza virus

Few studies on the humoral immune responses in human during natural influenza infection have been reported. Here, we used serum samples from pandemic 2009 H1N1 influenza infected patients to characterize the humoral immune responses to influenza during natural infection in humans. We observed for the first time that the pandemic 2009 H1N1 influenza induced influenza A-specific IgM within days after symptoms onset, whereas the unit of IgG did not changed. The magnitude of influenza A-specific IgM antibodies might have a value in predicting the rate of virus clearance to some degree. However, the newly developed IgM was not associated with hemagglutination inhibition (HI) activities in the same samples but correlated with HI activities of subsequently collected sera which were mediated by IgG antibodies, indicating that IgM was critical for influenza infection and influences subsequent IgG antibody responses. These findings provide new important insights on the human immunity to natural influenza infection.     

Implication of inflammatory macrophages, nuclear receptors, and interferon regulatory factors in increased virulence of pandemic 2009 H1N1 influenza A virus after host adaptation

While pandemic 2009 H1N1 influenza A viruses were responsible for numerous severe infections in humans, these viruses do not typically cause corresponding severe disease in mammalian models. However, the generation of a virulent 2009 H1N1 virus following serial lung passage in mice has allowed for the modeling of human lung pathology in this species. Genetic determinants of mouse-adapted 2009 H1N1 viral pathogenicity have been identified, but the molecular and signaling characteristics of the host response following infection with this adapted virus have not been described. Here we compared the gene expression response following infection of mice with A/CA/04/2009 (CA/04) or the virulent mouse-adapted strain (MA-CA/04). Microarray analysis revealed that increased pathogenicity of MA-CA/04 was associated with the following: (i) an early and sustained inflammatory and interferon response that could be driven in part by interferon regulatory factors (IRFs) and increased NF-κB activation, as well as inhibition of the negative regulator TRIM24, (ii) early and persistent infiltration of immune cells, including inflammatory macrophages, and (iii) the absence of activation of lipid metabolism later in infection, which may be mediated by inhibition of nuclear receptors, including PPARG and HNF1A and -4A, with proinflammatory consequences. Further investigation of these signatures in the host response to other H1N1 viruses of various pathogenicities confirmed their general relevance for virulence of influenza virus and suggested that lung response to MA-CA/04 virus was similar to that following infection with lethal H1N1 r1918 influenza virus. This study links differential activation of IRFs, nuclear receptors, and macrophage infiltration with influenza virulence in vivo.     

Cross-reactive T cells are involved in rapid clearance of 2009 pandemic H1N1 influenza virus in nonhuman primates

In mouse models of influenza, T cells can confer broad protection against multiple viral subtypes when antibodies raised against a single subtype fail to do so. However, the role of T cells in protecting humans against influenza remains unclear. Here we employ a translational nonhuman primate model to show that cross-reactive T cell responses play an important role in early clearance of infection with 2009 pandemic H1N1 influenza virus (H1N1pdm). To "prime" cellular immunity, we first infected 5 rhesus macaques with a seasonal human H1N1 isolate. These animals made detectable cellular and antibody responses against the seasonal H1N1 isolate but had no neutralizing antibodies against H1N1pdm. Four months later, we challenged the 5 "primed" animals and 7 naive controls with H1N1pdm. In naive animals, CD8+ T cells with an activated phenotype (Ki-67+ CD38+) appeared in blood and lung 5-7 days post inoculation (p.i.) with H1N1pdm and reached peak magnitude 7-10 days p.i. In contrast, activated T cells were recruited to the lung as early as 2 days p.i. in "primed" animals, and reached peak frequencies in blood and lung 4-7 days p.i. Interferon (IFN)-γ Elispot and intracellular cytokine staining assays showed that the virus-specific response peaked earlier and reached a higher magnitude in "primed" animals than in naive animals. This response involved both CD4+ and CD8+ T cells. Strikingly, "primed" animals cleared H1N1pdm infection significantly earlier from the upper and lower respiratory tract than the naive animals did, and before the appearance of H1N1pdm-specific neutralizing antibodies. Together, our results suggest that cross-reactive T cell responses can mediate early clearance of an antigenically novel influenza virus in primates. Vaccines capable of inducing such cross-reactive T cells may help protect humans against severe disease caused by newly emerging pandemic influenza viruses.     

Hemagglutinin 222D/G Polymorphism Facilitates Fast Intra-Host Evolution of Pandemic (H1N1) 2009 Influenza A Viruses

The amino acid substitution of aspartic acid to glycine in hemagglutinin (HA) in position 222 (HA-D222G) as well as HA-222D/G polymorphism of pandemic (H1N1) 2009 influenza viruses (A(H1N1)pdm09) were frequently reported in severe influenza in humans and mice. Their impact on viral pathogenicity and the course of influenza has been discussed controversially and the underlying mechanism remained unclarified. In the present study, BALB/c mice, infected with the once mouse lung- and cell-passaged A(H1N1)pdm09 isolate A/Jena/5258/09 (mpJena/5258), developed severe pneumonia. From day 2 to 3 or 4 post infection (p.i.) symptoms (body weight loss and clinical score) continuously worsened. After a short disease stagnation or even recovery phase in most mice, severity of disease further increased on days 6 and 7 p.i. Thereafter, surviving mice recovered. A 45 times higher virus titer maximum in the lung than in the trachea on day 2 p.i. and significantly higher tracheal virus titers compared to lung on day 6 p.i. indicated changes in the organ tropism during infection. Sequence analysis revealed an HA-222D/G polymorphism. HA-D222 and HA-G222 variants co-circulated in lung and trachea. Whereas, HA-D222 variant predominated in the lung, HA-G222 became the major variant in the trachea after day 4 p.i. This was accompanied by lower neutralizing antibody titers and broader receptor recognition including terminal sialic acid α-2,3-linked galactose, which is abundant on mouse trachea epithelial cells. Plaque-purified HA-G222-mpJena/5258 virus induced severe influenza with maximum symptom on day 6 p.i. These results demonstrated for the first time that HA-222D/G quasispecies of A(H1N1)pdm09 caused severe biphasic influenza because of fast viral intra-host evolution, which enabled partial antibody escape and minor changes in receptor binding.     

Pandemic influenza A H1N1 2009 infection versus vaccination: a cohort study comparing immune responses in pregnancy

Background

With the emergence of H1N1 pandemic (pH1N1) influenza, the CDC recommended that pregnant women be one of five initial target groups to receive the 2009 monovalent H1N1 vaccine, regardless of prior infection with this influenza strain. We sought to compare the immune response of pregnant women to H1N1 infection versus vaccination and to determine the extent of passive immunity conferred to the newborn.

Methods/Findings

During the 2009-2010 influenza season, we enrolled a cohort of women who either had confirmed pH1N1 infection during pregnancy, did not have pH1N1 during pregnancy but were vaccinated against pH1N1, or did not have illness or vaccination. Maternal and umbilical cord venous blood samples were collected at delivery. Hemagglutination inhibition assays (HAI) for pH1N1 were performed. Data were analyzed using linear regression analyses. HAIs were performed for matched maternal/cord blood pairs for 16 women with confirmed pH1N1 infection, 14 women vaccinated against pH1N1, and 10 women without infection or vaccination. We found that pH1N1 vaccination and wild-type infection during pregnancy did not differ with respect to (1) HAI titers at delivery, (2) HAI antibody decay slopes over time, and (3) HAI titers in the cord blood.

Conclusions

Vaccination against pH1N1 confers a similar HAI antibody response as compared to pH1N1 infection during pregnancy, both in quantity and quality. Illness or vaccination during pregnancy confers passive immunity to the newborn.