Individual Antibody and T Cell Responses to Vaccination and Infection with the 2009 Pandemic Swine-Origin H1N1 Influenza Virus

Introduction  

The 2009 swine-origin H1N1 influenza virus (swH1N1) provided an opportunity to study immune responses to a new influenza strain in the context of seasonal influenza vaccination. Our goals were: to assess whether analyzing multiple parameters of immune responsiveness to influenza has an advantage over evaluating hemagglutination inhibition (HAI) titer alone, to determine whether vaccination with the seasonal vaccine induced cross-reactive immunity to swH1N1 in some individuals, and to determine whether the immune response against swH1N1 is higher after infection than vaccination.     

Serologic Evidence of Pandemic Influenza Virus H1N1 2009 Infection in Cats in China

Infection of domestic cats with (H1N1) pandemic 2009 (pdm09) influenza A virus has recently been documented. In this paper, we report for the first time the sporadically current seroprevalence of (H1N1) pdm09 influenza A virus infection in cats in China. Thirteen of 1,080 sera were found positive by nucleoprotein (NP)-specific enzyme-linked immunosorbent assays (ELISAs) in different cat populations in southern China. It is very important to stress further surveillance of pandemic (H1N1) 2009 influenza A virus in cats in southern China.     

Influenza and HIV: Lessons from the 2009 H1N1 Influenza Pandemic

Influenza is a common respiratory disease in adults, including those infected with HIV. In the spring of 2009, a pandemic influenza A (H1N1) virus (pH1N1) emerged. In this article, we review the existing literature regarding pH1N1 virus infection in HIV-infected adults, which suggests that susceptibility to pH1N1 virus infection and severity of influenza illness are likely not increased in HIV-infected adults without advanced immunosuppression or comorbid conditions. The risk of influenza-related complications, however, may be increased in those with advanced immunosuppression or high-risk comorbid conditions. Prevention and treatment of high-risk comorbid conditions and annual influenza vaccination should continue to be part of HIV clinical care to help prevent influenza illness and complications. Additional information about pH1N1 vaccine immunogenicity and efficacy in HIV-infected patients would be useful to guide strategies to prevent influenza virus infection in this population.     

Development of an Immunochromatographic Assay Specifically Detecting Pandemic H1N1 (2009) Influenza Virus

The pandemic caused by a new type of influenza virus, pandemic H1N1 (2009) influenza virus A (AH1pdm), has had a major worldwide impact. Since hemagglutinin (HA) genes are among the most specific genes in the influenza virus genome, AH1pdm can be definitively diagnosed by viral gene analysis targeting the HA genes. This type of analysis, however, cannot be easily performed in clinical settings. While commercially available rapid diagnosis kits (RDKs) based on immunochromatography can be used to detect nucleoproteins (NPs) of influenza A and B viruses in clinical samples, there are no such kits that are specific for AH1pdm. We show here that an RDK using a combination of monoclonal antibodies against NP can be used to specifically detect AH1pdm. The RDK recognized AH1pdm virus isolates but did not recognize seasonal H1N1 and H3N2 and influenza B viruses, indicating that the specificity of the RDK is 100%. A parallel comparison of RDK with a commercial influenza A/B virus kit revealed that both types of kits had equal sensitivities in detecting their respective viruses. Preliminary evaluation of clinical samples from 5 individuals with PCR-confirmed human AH1pdm infection showed that the RDK was positive for all samples, with the same detection intensity as that of a commercial influenza A/B virus kit. This RDK, together with a new vaccine and the stockpiling of anti-influenza drugs, will make aggressive measures to contain AH1pdm infections possible.The pandemic caused by a new type of influenza virus, pandemic H1N1 (2009) influenza virus A (AH1pdm), has had a major worldwide impact. As of 27 September 2009, more than 4,100 deaths from AH1pdm infection have been reported to the World Health Organization (WHO) (http://www.who.int/csr/don/2009_10_02/en/index.html). Current methods used to diagnose AH1pdm virus in clinical specimens are based on viral RNA analysis targeting hemagglutinin (HA) genes, because the HA genes are among the most specific genes in the influenza virus genome. Although these methods are highly sensitive, they usually take more than 2 to 6 h to complete and require well-equipped laboratories with virologists or well-trained medical technicians and specialized tools for virus genome isolation and amplification (6, 8) (http://www.who.int/entity/csr/resources/publications/swineflu/CDCRealtimeRTPCR_SwineH1Assay-2009_20090430.pdf). Rapid diagnostic kits (RDKs) based on immunochromatography utilize antibodies (Abs) against antigens of interest. Although RDKs are usually less sensitive than genetic assays, they do not require the isolation of a viral genome, thus overcoming the intrinsic difficulties of viral gene analyses. RDKs for many infectious diseases (2, 4, 9, 11-14), including influenza viruses A and B (1), are commercially available. However, RDKs capable of distinguishing AH1pdm viruses from seasonal influenza viruses have yet to be implemented in a clinical setting.Nucleoproteins (NPs) of influenza A, B, and C viruses have important differences in their antigenicities that enable them to be distinguished from one another but are highly conserved within each major serotype. Thus, antibodies to NPs have been utilized in commercially available RDKs to distinguish between influenza A and B viruses (15). In a monoclonal antibody (MAb) preparation procedure targeting NPs derived from highly pathogenic H5N1 avian influenza (HPAI), we obtained 2 MAbs that reacted with NPs of AH1pdm as well as that of HPAI but not those of seasonal influenza A virus. We have therefore utilized these MAbs in the development of novel RDKs for AH1pdm, and we have validated these RDKs in laboratory environments.     

Evidence of Human-to-Swine Transmission of the Pandemic (H1N1) 2009 Influenza Virus in South Korea

As the pandemic (H1N1) 2009 influenza virus continues to infect human populations globally, reports on epidemiologically linked animal infections are also on the rise. Since December 2009, pandemic (H1N1) 2009-like viruses have been isolated in pigs from different swine farms of South Korea. Genetic and phylogenetic analyses of viral segments demonstrated several events of human-to-swine transmission with no apparent signs of reassortment. These events were also supported by serological surveillance in pig sera collected from April to December, suggesting that reverse transmission probably started between June and July with a drastic increase in prevalence the following months. Although molecular characterization indicates that the swine isolates are generally stable, some viruses are genetically evolving, most notably in their surface proteins. Animal studies (ferrets and mice) reveal that swine pandemic isolates epitomize biological properties attributed to the currently circulating human pandemic viruses, including replication kinetics and efficient transmission, indicating their potential to return to circulation among humans. Overall, these results indicate widespread human-to-animal transmission of pandemic (H1N1) 2009 influenza viruses in South Korea. With the significant role of pigs in the ecology of influenza viruses, these transmission events should be closely monitored and minimized to prevent the risk of generating viruses with greater human health concerns.In June 2009, a global pandemic was declared by the World Health Organization (WHO) for the emergence and rapid spread of a novel influenza A (H1N1) virus (6, 7). The causative virus strain, termed as the pandemic (H1N1) 2009 influenza virus, is highly transmissible among humans and contains a unique reassortment of gene segments derived from viruses of the triple reassortant swine North American lineage and the avian-like swine Eurasian lineage (12, 39). At present, the mortality rate due to infection with the pandemic virus is relatively low among humans, where the majority of laboratory-confirmed infections result in self-limiting, uncomplicated influenza (44). Fatal cases are largely often associated with preexisting medical conditions (40). Experts have already demonstrated that the virus is pathogenic in mammalian hosts like mice, ferrets, and nonhuman primates (18, 24, 26). Furthermore, pigs have been shown to be susceptible and can transmit the virus (3, 18, 20, 30).Accordingly, natural cases of reverse zoonosis into turkeys and primarily pigs have been increasing considerably in different continents since the first detection of the virus among pigs in a Canadian swine farm (16, 41), as reflected in reports through the weekly disease information of the Paris-based World Organization for Animal Health Information Database (28). Due to dual susceptibility to both human and animal influenza viruses, pigs are considered important intermediate hosts, acting as “mixing vessels” for genetic reassortment (4, 17, 23, 33). Such events may consequently lead to generation of novel reassortant influenza viruses which can cause human pandemics or, as in the current influenza pandemic, a reassortant virus with potentially enhanced pathogenicity and lethality.Here we report the detection and isolation of the pandemic (H1N1) 2009 influenza viruses isolated from various swine farms in South Korea. Virus isolates were genetically characterized to determine whether these swine viruses have undergone any evolutions that would significantly alter their overall phenotype. Subsequently, pathogenicity and transmissibility in ferrets were tested and compared with local Korean human pandemic viruses and a recent Korean swine H1N1 virus.     

Utility of Pandemic H1N1 2009 Influenza Virus Recombinant Hemagglutinin Protein-Based Enzyme-Linked Immunosorbent Assay for Serosurveillance

An enzyme-linked immunosorbent assay (ELISA) for the detection of IgG antibodies against the pandemic H1N1 2009 influenza A virus, employing a recombinant hemagglutinin protein of the virus, was compared to the hemagglutination inhibition (HI) test using 783 serum samples. The results showed a concordance of 98.4%, suggesting the utility of the ELISA in serosurveillance. Two hundred sixty-nine (100%) serum samples with an HI titer of ≥20 were ELISA reactive.Influenza viruses are negative-strand RNA viruses that belong to the family Orthomyxoviridae, which includes 4 genera, Influenzavirus A, B, and C and Thogotovirus. Influenza A viruses are widely distributed in nature and can infect a wide variety of mammals and birds. Based on the antigenicity of the two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA), influenza A viruses have been classified into 16 HA and 9 NA subtypes. Of these, H1, H2, and H3 HA subtypes and N1 and N2 NA subtypes have circulated in human populations. In recent years, H5N1 virus of avian origin was expected to be a pandemic-causing pathogen (4). However, the first pandemic of this century was caused by a novel H1N1 influenza A virus of swine origin that emerged in 2009 (hereinafter called p-H1N1-09) (3, 5).Due to the circulation of several influenza A virus subtypes, cross-reactivity is a major problem in influenza virus serology. The inhibition of hemagglutination (HI) caused by antibodies to the HA of the virus is routinely used for assessing the prevalence of a specific virus in a community or an animal population.India was affected by the pandemic H1N1 influenza during the latter half of 2009 (2). In order to understand the degree of exposure of different populations to the virus, an extensive serosurvey was undertaken by the National Institute of Virology, Pune, India. The test of choice, HI, was performed as described earlier (1). Considering the requirement of fresh red blood cells and time and the cumbersome protocol, it was thought important to evaluate the utility of a recombinant HA protein enzyme-linked immunosorbent assay (ELISA) for the detection of p-H1N1-09 IgG antibodies as evidence of exposure to this novel virus.The HA gene of the p-H1N1-09 influenza virus isolated at the National Institute of Virology (A/India-Blore/NIV310/2009, GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"GU292347","term_id":"273039286","term_text":"GU292347"}}GU292347) was PCR amplified, cloned into the pFastBac1 vector (Invitrogen) within the EcoRI and XhoI restriction sites, and expressed with a baculovirus expression system (Invitrogen) in an insect cell line. The sequence of the cloned HA was identical to that of the original isolate. The HA protein was purified by lentil lectin affinity chromatography (GE Healthcare) and used for ELISA.An indirect sandwich ELISA was performed. Briefly, a Maxisorb microtiter plate (Nunc) was coated with p-H1N1-09 HA protein, 2 μg/well, and incubated at 37°C for 2 h. The plate was blocked with phosphate-buffered saline (PBS) containing 10% donor calf serum, 0.5% Tween 20, 0.5% gelatin (blocking solution) at 37°C for 30 min. After washing 3 times with the wash solution (PBS containing 0.5% Tween 20), test serum samples and positive and negative controls diluted 1:100 in blocking solution were added to the previously designated wells of the coated assay plates and incubated at 37°C for 30 min. Following 4 washes with the wash solution, horseradish peroxidase-conjugated anti-human IgG (Sigma Chemicals, St. Louis, MO) was added to each well as the detector antibody and allowed to incubate for 30 min. The enzymatic reaction with the substrate (O-phenylenediamine and urea peroxide, 10 min) was stopped by the addition of 4 M H₂SO₄, and optical density (OD) values were determined at 492 nm. Human serum samples known to be positive and negative for HI antibodies against the pandemic influenza virus were included in every assay plate as controls. The cutoff values for IgG anti-p-H1N1-09 antibodies were calculated as the mean OD values for the results of 3 negative controls in triplicate. Samples with values greater than or equal to the cutoff values were considered antibody positive. Samples showing OD values within 10% of the cutoff value were considered borderline reactive and repeated.A total of 783 serum samples previously screened by the HI test for the presence of p-H1N1-09 antibodies were retested in the ELISA. As evident from the results (Table ​(Table1),1), the ELISA emerged as an excellent assay for the detection of virus-specific antibodies. Of the 397 HI-negative samples, 389 were scored negative in the ELISA, giving 98% specificity in the comparison to the gold-standard HI test. Importantly, all samples with HI titers of >20 (n = 269) were positive in the ELISA, documenting 100% sensitivity of the ELISA. A large number of samples (n = 117) exhibited low HI titers (1:10). Usually, reactivity at this dilution is not considered specific. Of these, only 4 were recorded as reactive in the ELISA. When HI-negative samples with titers of 10 were considered antibody negative, the ELISA specificity was 97.7% and the concordance between the HI and ELISA results was 98.5%. To examine the relationship of the HI and ELISA titers, 2-fold dilutions of the test samples were tested in the ELISA. The reciprocal of the highest dilution at which the OD value was greater than or equal to the cutoff value was considered the IgG anti-p-H1N1-09 HA titer of the serum. All HI-positive sera with titers ≥20 were titrated in the ELISA. When the HI and ELISA titers were compared, the Spearman''s rank correlation coefficient was estimated to be 0.864. Thus, a good correlation between the HI and ELISA titers was apparent. In addition, a linear relationship was noted when the log HI and ELISA titers were compared (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Relationship of log HI and ELISA titers. Each point represents the mean positive ELISA titer for a given HI titer value. Error bars represent standard errors of the means.

TABLE 1.

Relationship of HI titers and IgG positivity in ELISA against p-H1N1-09 virus

Sensitivity of Influenza Rapid Diagnostic Tests to H5N1 and 2009 Pandemic H1N1 Viruses

Simple and rapid diagnosis of influenza is useful for making treatment decisions in the clinical setting. Although many influenza rapid diagnostic tests (IRDTs) are available for the detection of seasonal influenza virus infections, their sensitivity for other viruses, such as H5N1 viruses and the recently emerged swine origin pandemic (H1N1) 2009 virus, remains largely unknown. Here, we examined the sensitivity of 20 IRDTs to various influenza virus strains, including H5N1 and 2009 pandemic H1N1 viruses. Our results indicate that the detection sensitivity to swine origin H1N1 viruses varies widely among IRDTs, with some tests lacking sufficient sensitivity to detect the early stages of infection when the virus load is low.Influenza is one of the primary infectious diseases affecting public health. The H1N1 and H3N2 subtypes of human influenza A and B viruses cause seasonal influenza with high morbidity and mortality, especially in pediatric, geriatric, and immunocompromised patients (25). In addition to the clinical aspects of these infections, influenza epidemics also have a significant impact on our social economy (14). Furthermore, viruses possessing variants of hemagglutinin (HA) and neuraminidase (NA) to which humans are immunologically naïve have the potential to cause global outbreaks, or “pandemics.”The rapid diagnosis of influenza during the early stage of infection allows physicians the opportunity to limit the infection and its sequelae by administering the appropriate antiviral drugs to the patient. NA inhibitors (i.e., oseltamivir and zanamivir), which are widely used to treat influenza, must be administered 36 to 48 h after the onset of symptoms for maximal therapeutic efficacy (18). It is, however, difficult to distinguish influenza from other acute respiratory disorders based purely on clinical signs and symptoms. The availability of a diagnostic test with high sensitivity that can accommodate the large volume of clinical specimens generated during influenza epidemics and pandemics and that is simple and quick is the Holy Grail of influenza diagnostics.Recently, many rapid tests have been made available to diagnose seasonal influenza in clinical practice. These influenza rapid diagnostic tests (IRDTs) may also help detect sporadic human infections with other influenza viruses (e.g., avian H5N1 viruses), which have the potential to cause a pandemic. In fact, the first case of swine origin pandemic (H1N1) 2009 virus infection in California was diagnosed as influenza A virus infection by the use of an IRDT (3). Although the sensitivity of some IRDTs has been experimentally evaluated (6, 10, 13, 22, 23), studies on the sensitivity of these tests for nonhuman influenza viruses are limited (24, 27). Furthermore, although the detection sensitivity of some IRDTs to pandemic (H1N1) 2009 viruses has been reported (2, 4, 5, 7, 8, 11, 12, 15, 26), no one has conducted an extensive side-by-side comparison of IRDT sensitivity with multiple isolates and clinical specimens. Here, we compared the sensitivity of 20 IRDTs for detection of H1N1, H3N2, and type B seasonal viruses, human and avian H5N1 viruses, other subtypes of avian viruses, and pandemic (H1N1) 2009 viruses. Our findings emphasize the importance of selecting the right IRDT for rapid diagnosis of nonseasonal influenza viruses, since the sensitivity of the IRDTs we tested varied by as much as 100-fold.     

Humans and Ferrets with Prior H1N1 Influenza Virus Infections Do Not Exhibit Evidence of Original Antigenic Sin after Infection or Vaccination with the 2009 Pandemic H1N1 Influenza Virus

The hypothesis of original antigenic sin (OAS) states that the imprint established by an individual''s first influenza virus infection governs the antibody response thereafter. Subsequent influenza virus infection results in an antibody response against the original infecting virus and an impaired immune response against the newer influenza virus. The purpose of our study was to seek evidence of OAS after infection or vaccination with the 2009 pandemic H1N1 (2009 pH1N1) virus in ferrets and humans previously infected with H1N1 viruses with various antigenic distances from the 2009 pH1N1 virus, including viruses from 1935 through 1999. In ferrets, seasonal H1N1 priming did not diminish the antibody response to infection or vaccination with the 2009 pH1N1 virus, nor did it diminish the T-cell response, indicating the absence of OAS in seasonal H1N1 virus-primed ferrets. Analysis of paired samples of human serum taken before and after vaccination with a monovalent inactivated 2009 pH1N1 vaccine showed a significantly greater-fold rise in the titer of antibody against the 2009 pH1N1 virus than against H1N1 viruses that circulated during the childhood of each subject. Thus, prior experience with H1N1 viruses did not result in an impairment of the antibody response against the 2009 pH1N1 vaccine. Our data from ferrets and humans suggest that prior exposure to H1N1 viruses did not impair the immune response against the 2009 pH1N1 virus.     

The 2009 H1N1 Pandemic Adds to Our Knowledge of Influenza Pathogenesis

This commentary discusses the pathology and pathogenesis of the 2009 H1N1 influenza A pandemic.The article by Shieh et al¹ on the pathology and pathogenesis of the 2009 pandemic H1N1 influenza A virus is a landmark work in the long evolution of our understanding of this ever-threatening public health challenge. During the intervals between the pandemics of 1889, 1918, 1957, 1968, and 2009, most of the public, including physicians, did not distinguish between colds and the flu. Influenza A virus is not well controlled in the overall population by antiviral drugs and vaccination, and even during these interpandemic periods, the toll from seasonal influenza in the United States was a remarkable 36,000 deaths annually. Therefore, pathologists have much to potentially contribute to the better understanding the pathogenesis of influenza.This study of 100 fatal cases of 2009 pandemic H1N1 influenza A is a remarkably large sample of cases that were etiologically well documented and were investigated with state-of-the-art contemporary methods. Immunohistochemical (IHC) analyses revealed that the distribution of the target cells of the 2009 cases differed from those of seasonal influenza and H5N1 avian influenza.^2,3 The 2009 H1N1 influenza virus infected not only the tracheobronchial epithelium but also the submucosal glands and alveolar lining cells, particularly type II pneumocytes, which were distinguished from alveolar macrophages and type I pneumocytes by antisurfactant IHC, as sloughed alveolar cells are not easily identified by morphology alone and could be mistaken on a purely morphological basis as alveolar macrophages.¹ The concordance of influenza viral antigen with diffuse alveolar damage and infection of type II pneumocytes is critical to understanding the pathogenesis of the 2009 pandemic disease. IHC and polymerase chain reaction were also used to definitively identify the agents of superinfecting bacterial pneumonia: Streptococcus pneumoniae, S. pyogenes, Hemophilus influenzae, Staphylococcus aureus, and even methicillin-resistant strains.This series of 100 cases adds significantly to the information in two other particularly excellent 2009 H1N1 influenza autopsy series. A series of 34 cases largely from the New York City medical examiner’s office provided correlations with computerized tomographic pulmonary lesions but in contrast had a higher rate of secondary bronchopneumonia (55% versus 29%), less infection of alveolar type I and II pneumocytes, and less apparent colocalization of diffuse alveolar damage and viral antigen.⁴ A Brazilian study of 21 pandemic H1N1 influenza autopsies described the same pathology (DAD, interstitial pneumonia, airway⁵ lesions, with extensive hemorrhage and/or microthrombi in some patients).⁵ IHC was used in this study to address aspects of immunity and immunopathogenesis, including identifying many CD8 T cells, granzyme B expressing cells, and expression of Toll-like receptor 3 (TLR3) and gamma interferon by macrophages and alveolar epithelium in these patients. Intriguing data from experimental mouse infections suggest that TLR3, the receptor for double-stranded RNA, the genetic material of influenza A virus not only plays a role in controlling viral infection but also contributes to pathogenic innate and adaptive responses that vary with the viral load.⁶Although fatal outcomes of influenza A are present as outliers in pandemics or in seasonal disease, H5N1 avian influenza cases provide an exception. Indeed, the current pandemic has been relatively mild. Thus, it is not surprising that the autopsy series has contained a high proportion of patients with comorbid conditions. Mechanisms governing the enhanced disease severity in obesity, asthma, and pregnancy remain as unanswered questions that will provide future directions of research. Interestingly, a study of diet-induced obese mice immune to seasonal H3N2 influenza and challenged with 2009 pandemic H1N1 virus revealed more severe illness, higher mortality and viral titers, and reduced levels in the lung of gamma interferon, virus-specific CD8 T cells, and memory CD8 T cell gamma interferon production than nonobese mice.⁷ It would be useful to pursue these leads in studies of humans with influenza.Other future avenues of research should pursue unanswered questions such as the mechanism of cell death, the pathogenic role of the immune response, the potential importance and pathogenesis of alveolar capillary thrombi, the variable occurrence and pathogenesis of myocarditis, myopathy, and acute encephalopathy, which have apparently not been features of the current pandemic, and the mechanisms of hematogenous dissemination, which seems to have been rare in humans during pandemics of the past century.An extensive picture of human pathology is critical, but often animal models are required to provide insights into human disease. The molecular basis of the attachment of influenza A virus by its receptor, hemagglutinin, to the sialic acid–containing host cell receptor is critical to understanding why the anatomical distribution of lesions of an experimental animal and humans do or do not coincide. The precise determination of the target cells in this study by Shieh et al provides the reality from which sialy-terminating oligosaccharide moieties can be investigated^8,9 and whether the predictions of studies in experimental animals such as ferrets can be relied on.^10,11,12Our knowledge of influenza in 2010 has left many stones unturned. The Centers for Disease Control Pathology Branch¹ has used the material in their hands yet again to contribute to knowledge of pathogenesis as well as pathology as they have done many times previously, such as with hantavirus pulmonary syndrome, the 2001 anthrax attacks, and coronavirus severe acute respiratory syndrome. Yet other gaps in knowledge could be closed if pathologists were organized to do a greater number of well-organized autopsy studies. Appropriately collected tissues could address questions ranging from molecular mechanisms to the pathological basis for radiological images. Even this straightforward anatomical problem would require harvesting the exact tissues of the lesions that had been imaged close to the time of death.What the dead have taught the living about influenza is clearly told in history. In 1889, Leichtenstern was convinced from clinical and anatomical evidence that a primary pneumonia was produced by the poison of influenza. As a result of careful bacteriological studies of autopsies in 1919 in which he isolated Hemophilus influenzae, from 23 of 28 cases, Wolbach was tempted to believe that the early deaths with abundant hyaline membranes and later bacterial pneumonia “simply represent different stages of the same process.”¹³ However, he considered that failure to reproduce influenza in human studies involving inoculation with pure cultures of H. influenzae¹⁴ was “strong argument against it being the cause of influenza.” After the isolation of influenza A virus from swine by Robert Shope in 1930 and from humans by Andrewes, Laidlaw, and Smith in 1933,¹⁵ Hers and Mulder were prepared in 1957 to identify by immunofluorescence staining that tracheobronchial ciliated epithelial cells and alveolar lining cells are the infected targets.¹⁶Influenza A virus circulates not only by human-to-human transmission with seasonal oscillation between the Northern and Southern Hemispheres but also resides in wild aquatic fowl. There are 16 hemagglutinin and nine neuraminidase types that may emerge by accidental spillover into swine or other routes and threaten to establish transmission to and among humans. The threat will not disappear, and we will face a more serious pandemic in the future. Thus, it is of paramount importance to learn as much about the present pandemic as possible to sufficiently meet the challenge of these future pandemics.     

Comparison of Immune Response by Virus Infection and Vaccination to 2009 Pandemic Influenza A/H1N1 in Children

We aimed to compare the immune response induced by natural infection with 2009 pandemic influenza A/H1N1 (pH1N1) virus and by monovalent pH1N1 vaccination in children and adolescents. This cross-sectional clinical study was conducted at 3 hospitals in Korea from February to May 2010. A total of 266 healthy subjects aged from 6 months to 18 yr were tested for the presence of the antibody against pH1N1 using hemagglutination inhibition (HI) test. Information about pH1N1 vaccination and laboratory-confirmed pH1N1 infection history was obtained. The overall rate of HI titers of ≥ 1:40 against pH1N1 was 38.7%, and the geometric mean titer (GMT) was 20.5. Immunogenicity of pH1N1 vaccination only was reflected by a 41.1% of seroprotection rate and a GMT of 22.5. Immunogenicity of natural infection only was reflected by a 61.0% of seroprotection rate and a GMT of 40.0. GMT was significantly higher in the subjects of natural infection group than in the subjects of pH1N1 vaccination group (P < 0.001). The immune responses induced by natural pH1N1 infection exceed those induced by pH1N1 vaccinations.     

Preexisting Antibody Response against 2009 Pandemic Influenza H1N1 Viruses in the Taiwanese Population

A novel pandemic influenza H1N1 (pH1N1) virus spread rapidly across the world in 2009. Due to the important role of antibody-mediated immunity in protection against influenza infection, we used an enzyme-linked immunosorbent assay-based microneutralization test to investigate cross-reactive neutralizing antibodies against the 2009 pH1N1 virus in 229 stored sera from donors born between 1917 and 2008 in Taiwan. The peak of cumulative geometric mean titers occurred in donors more than 90 years old and declined sharply with decreasing age. Sixteen of 27 subjects (59%) more than 80 years old had cross-reactive antibody titers of 160 or more against the 2009 pH1N1 virus, whereas none of the donors from age 9 to 49 had an antibody titer of 160 or more. Interestingly, 2 of 51 children (4%) from 6 months to 9 years old had an antibody titer of 40. We further tested the antibody responses in 9 of the 51 pediatric sera to three endemic seasonal influenza viruses isolated in 2006 and 2008 in Taiwan, and the results showed that only the 2 sera from children with antibody responses to the 2009 pH1N1 virus had high titers of neutralizing antibody against recent seasonal influenza virus strains. Our study shows the presence of some level of cross-reactive antibody in Taiwanese persons 50 years old or older, and the elderly subjects who may already have been exposed to the 1918 virus had high titers of neutralizing antibody to the 2009 pH1N1 virus. Our data also indicate that natural infection with the Taiwan 2006 and 2008 seasonal H1N1 viruses may induce a cross-reactive antibody response to the 2009 pH1N1 virus.Influenza A viruses have caused several pandemics during the past century and continue to cause epidemics around the world yearly. Pandemics are typically caused by the introduction of a virus with a hemagglutinin (HA) subtype that is new to human populations (14). In 2009, a novel pandemic influenza H1N1 (pH1N1) virus of swine origin spread rapidly and has caused variable disease globally via interhuman transmission (2, 3).The 2009 pH1N1 virus contains a unique combination of gene segments from both the North American and Eurasian swine lineages and is antigenically distinct from any known seasonal human influenza virus (14). Since H1N1 influenza A viruses have been circulating in human populations for decades, much of the world has encountered these viruses repeatedly, either through infection or through vaccination. Under the threat of a pandemic outbreak, however, a major concern is whether preexisting immunity can provide some protection from the novel 2009 pH1N1 virus.Recent reports from the United States suggested that 33% of individuals over the age of 60 years had neutralization antibodies to the novel 2009 pH1N1 virus, probably due to previous exposure to antigenically similar H1N1 viruses (1, 7). In Japan, however, appreciable neutralization antibodies against the 2009 pH1N1 virus were found only in individuals more than 90 years old (9). The differences in geographical location and vaccination programs against influenza in 1976 may account for the different age distributions of neutralization antibodies in the two countries. In the early 1900s, Taiwan had had a close relationship with Japan historically and geographically. The prevalence of influenza in Taiwan may be quite similar to that in Japan. In recent years, however, sequence analysis of epidemic influenza virus strains revealed that the Taiwanese strains usually circulate in Taiwan prior to their circulation in many other countries, including Japan. (16). The differences between the studies from United States and Japan, and the unique epidemic situation in Taiwan, highlight the need for us to assess the level of preexisting immunity in the Taiwanese population.In this study, we measured the titers of neutralizing antibodies against the 2009 pH1N1 virus in sera obtained from previous influenza infection or vaccination of different age groups. In addition, we also assessed the antibodies against the local seasonal H1N1 strains isolated in Taiwan in 2006 and 2008 (A/Taiwan/N86/06, A/Taiwan/N94/08, and A/Taiwan/N510/08) to evaluate whether there is a cross-reactive antibody response between recent local strains and the 2009 pH1N1 virus.     

Seasonal and Pandemic Human Influenza Viruses Attach Better to Human Upper Respiratory Tract Epithelium than Avian Influenza Viruses

Influenza viruses vary markedly in their efficiency of human-to-human transmission. This variation has been speculated to be determined in part by the tropism of influenza virus for the human upper respiratory tract. To study this tropism, we determined the pattern of virus attachment by virus histochemistry of three human and three avian influenza viruses in human nasal septum, conchae, nasopharynx, paranasal sinuses, and larynx. We found that the human influenza viruses—two seasonal influenza viruses and pandemic H1N1 virus—attached abundantly to ciliated epithelial cells and goblet cells throughout the upper respiratory tract. In contrast, the avian influenza viruses, including the highly pathogenic H5N1 virus, attached only rarely to epithelial cells or goblet cells. Both human and avian viruses attached occasionally to cells of the submucosal glands. The pattern of virus attachment was similar among the different sites of the human upper respiratory tract for each virus tested. We conclude that influenza viruses that are transmitted efficiently among humans attach abundantly to human upper respiratory tract, whereas inefficiently transmitted influenza viruses attach rarely. These results suggest that the ability of an influenza virus to attach to human upper respiratory tract is a critical factor for efficient transmission in the human population.Influenza is an important cause of morbidity and mortality in humans during seasonal, pandemic, and zoonotic outbreaks. Seasonal influenza is estimated to cause 250,000 to 500,000 deaths per year worldwide. Pandemic influenza viruses of the previous century resulted in an estimated 1 to 4 million deaths for the 1957 H2N2 (Asian flu) and the 1968 H3N2 (Hong Kong flu) influenza pandemics, and 20 to 50 million deaths for the 1918 H1N1 (Spanish flu) influenza pandemic.^1,2 The first influenza pandemic of the 21st century, the currently ongoing new H1N1 virus outbreak (Mexican flu), has caused at least 3486 deaths as of September 13, 2009 (http://www.who.int/csr/don/2009_09_18/en/index.html). The zoonotic highly pathogenic avian influenza virus (HPAIV) H5N1, which is causing an ongoing outbreak in poultry, only occasionally infects humans, but has a high mortality rate, with 262 deaths out of 400+ confirmed infections as of August 2009 (http://www.who.int/csr/disease/avian_influenza/country/cases_table_2009_08_11/en/index.html).The pandemic potential of an influenza virus depends largely on its efficiency of human-to-human transmission. Human influenza viruses, including seasonal H1N1 and H3N2 viruses, and the pandemic H1N1 virus, are transmitted efficiently.³ In contrast, the zoonotic HPAIV H5N1 is only rarely transmitted from human to human.⁴ However, the factors determining efficient virus transmission among humans are poorly understood.Tropism of influenza virus for the human upper respiratory tract (URT) has been speculated to be an important determinant for the efficiency of virus transmission, based both on receptor distribution and virus replication studies.^5,6 Based on lectin histochemistry, the human URT has abundant receptors for human influenza viruses, which are efficiently transmitted.^5,7 This fits with the ability for human influenza viruses to replicate in human URT tissues based on in vivo,⁸ ex vivo,⁷ and in vitro studies.^9,10,11 In contrast, the human URT has only limited receptors for avian influenza viruses.^5,7 This fits with the absence or rarity of HPAIV H5N1 transmission among humans.⁴ However, it is discordant with a study of Nicholls and others, who showed that HPAIV H5N1 can replicate in URT tissues. They explained this discordance by suggesting that HPAIV H5N1 attached to receptors not detected by the lectins used. Therefore, there is currently no consensus on the tropism of HPAIV H5N1 for the human URT. In addition, the studies to date have not studied the human URT systematically, and it is not known what the tropism of the new H1N1 virus is for the human URT.To address the question whether URT tropism of influenza viruses is linked to efficient transmission, we determined the pattern of attachment of selected influenza viruses in the human URT: human influenza viruses, including seasonal H1N1 and H3N2 viruses and pandemic H1N1 virus, which are transmitted efficiently, and avian influenza viruses, including a HPAIV H5N1, isolated from a fatal human case, which is not transmitted efficiently among humans. We measured the pattern of virus attachment by use of virus histochemistry instead of lectin histochemistry.⁶ Virus histochemistry measures the attachment of influenza virus to its host cell directly. Therefore, any receptors other than SA-α-2,3-Gal terminated saccharides and SA-α-2,6-Gal terminated saccharides also would be detected by virus histochemistry. We have used this technique previously to show that the pattern of attachment in the human lower respiratory tract is different for human and avian influenza viruses, which correlates with differences in primary disease.¹²     

Cardiac Tamponade and Heart Failure Due to Myopericarditis as a Presentation of Infection with the Pandemic H1N1 2009 Influenza A Virus

We describe a fatal case of myopericarditis presenting with cardiac tamponade in a previously healthy 11-year-old child. Pandemic H1N1 2009 influenza A virus sequences were identified in throat and myocardial tissues and pericardial fluid, suggesting damage of myocardial cells directly caused by the virus.     

Long-Term Immunogenicity of an Inactivated Split-Virion 2009 Pandemic Influenza A H1N1 Virus Vaccine with or without Aluminum Adjuvant in Mice

In 2009, a global epidemic of influenza A(H1N1) virus caused the death of tens of thousands of people. Vaccination is the most effective means of controlling an epidemic of influenza and reducing the mortality rate. In this study, the long-term immunogenicity of influenza A/California/7/2009 (H1N1) split vaccine was observed as long as 15 months (450 days) after immunization in a mouse model. Female BALB/c mice were immunized intraperitoneally with different doses of aluminum-adjuvanted vaccine. The mice were challenged with a lethal dose (10× 50% lethal dose [LD₅₀]) of homologous virus 450 days after immunization. The results showed that the supplemented aluminum adjuvant not only effectively enhanced the protective effect of the vaccine but also reduced the immunizing dose of the vaccine. In addition, the aluminum adjuvant enhanced the IgG antibody level of mice immunized with the H1N1 split vaccine. The IgG level was correlated to the survival rate of the mice. Aluminum-adjuvanted inactivated split-virion 2009 pandemic influenza A H1N1 vaccine has good immunogenicity and provided long-term protection against lethal influenza virus challenge in mice.     

Evidence of Renal Infection in Fatal Cases of 2009 Pandemic Influenza A (H1N1)

The 2009 pandemic influenza A (H1N1) caused significant morbidity and mortality. Acute lung injury is the hallmark of the disease, but multiple organ system dysfunction can develop and lead to death. Therefore, we sought to investigate whether there was postmortem evidence of H1N1 presence and virus-induced organ injury in autopsy specimens. Five cases in which patients died of influenza A (H1N1) virus infection were studied. The lungs of all patients showed macroscopic and microscopic findings already described for H1N1 (consolidation, edema, hemorrhage, alveolar damage, hyaline membrane, and inflammation), and H1N1 viruses were present in alveolar cells in immunochemical studies. Acute tubular necrosis was present in all cases, but there was no evidence of direct virus-induced kidney injury. Nevertheless, H1N1 viruses were found in the cytoplasm of glomerular macrophages in the kidneys of 4 patients. Therefore, our data provide strong evidence that H1N1 presence is not restricted to the lungs.