Cold-adapted pandemic 2009 H1N1 influenza virus live vaccine elicits cross-reactive immune responses against seasonal and H5 influenza A viruses

The rapid transmission of the pandemic 2009 H1N1 influenza virus (pH1N1) among humans has raised the concern of a potential emergence of reassortment between pH1N1 and highly pathogenic influenza strains, especially the avian H5N1 influenza virus. Here, we report that the cold-adapted pH1N1 live attenuated vaccine (CApH1N1) elicits cross-reactive immunity to seasonal and H5 influenza A viruses in the mouse model. Immunization with CApH1N1 induced both systemic and mucosal antibodies with broad reactivity to seasonal and H5 strains, including HAPI H5N1 and the avian H5N2 virus, providing complete protection against heterologous and heterosubtypic lethal challenges. Our results not only accentuate the merit of using live attenuated influenza virus vaccines in view of cross-reactivity but also represent the potential of CApH1N1 live vaccine for mitigating the clinical severity of infections that arise from reassortments between pH1N1 and highly pathogenic H5 subtype viruses.     

The responses of Aboriginal Canadians to adjuvanted pandemic (H1N1) 2009 influenza vaccine

Background:

Because many Aboriginal Canadians had severe cases of pandemic (H1N1) 2009 influenza, they were given priority access to vaccine. However, it was not known if the single recommended dose would adequately protect people at high risk, prompting our study to assess responses to the vaccine among Aboriginal Canadians.

Methods:

We enrolled First Nations and Métis adults aged 20–59 years in our prospective cohort study. Participants were given one 0.5-mL dose of ASO3-adjuvanted pandemic (H1N1) 2009 vaccine (Arepanrix, GlaxoSmithKline Canada). Blood samples were taken at baseline and 21–28 days after vaccination. Paired sera were tested for hemagglutination-inhibiting antibodies at a reference laboratory. To assess vaccine safety, we monitored the injection site symptoms of each participant for seven days. We also monitored patients for general symptoms within 7 days of vaccination and any use of the health care system for 21–28 days after vaccination.

Results:

We enrolled 138 participants in the study (95 First Nations, 43 Métis), 137 of whom provided all safety data and 136 of whom provided both blood samples. First Nations and Métis participants had similar characteristics, including high rates of chronic health conditions (74.4%–76.8%). Pre-existing antibody to the virus was detected in 34.3% of the participants, all of whom boosted strongly with vaccination (seroprotection rate [titre ≥ 40] 100%, geometric mean titre 531–667). Particpants with no pre-existing antibody also responded well. Fifty-eight of 59 (98.3%) First Nations participants showed seroprotection and a geometric mean titre of 353.6; all 30 Métis participants with no pre-existing antibody showed seroprotection and a geometric mean titre of 376.2. Pain at the injection site and general symptoms frequently occurred but were short-lived and generally not severe, although three participants (2.2%) sought medical attention for general symptoms.

Interpretation:

First Nations and Métis adults responded robustly to ASO3-adjuvanted pandemic (H1N1) 2009 vaccine. Virtually all participants showed protective titres, including those with chronic health conditions.

Trial registration:

ClinicalTrials.gov trial register no. NCT.01001026.During the first wave of the H1N1 pandemic in Canada in 2009, some First Nations communities were severely affected, with younger adults and children most at risk for severe disease.^1,2 Whereas Aboriginal Canadians make up 3.4% of the population (with 1.14 million people), they accounted for 16% of admissions to hospital during the first wave of the pandemic, and 43% of Aboriginal patients had underlying medical conditions.³ The increased rate of severe disease might have resulted from residential crowding, prevalence of chronic health conditions, delayed access to health care or suboptimal immune responses to infection.⁴ When a federally funded, ASO3-adjuvanted (squalene/tocopherol) pandemic vaccine became available for Canadians later in 2009,⁵ Aboriginal people were given priority access to it.³ However, dosing requirements at the time were tentative. Previous studies of an ASO3-adjuvanted influenza A (H5N1) vaccine established that two doses were needed for immunity in adults.⁶ Because the 2009 influenza (H1N1) pandemic occurred without warning, no prepandemic studies had been done with vaccines based on this novel swine-derived virus.⁷The ASO3-adjuvanted pandemic (H1N1) 2009 vaccine manufactured in Canada (Arepanrix, GlaxoSmithKline, Laval, Quebec) was released for public use as soon as it was available, unstudied, to mitigate morbidity during the pandemic’s second wave, which was already in progress. A single 3.75-μg dose of hemagglutinin was recommended for adults using the preliminary results of a European trial of another ASO3-adjuvanted vaccine (Pandemrix, GlaxoSmithKline, Rixensart, Belgium) given to 65 adults aged 18–60 years.⁸ The European product was believed to be equivalent to the Canadian-made vaccine, but this had not yet been shown.We wondered if the recommended single dose would be adequate for Aboriginal Canadian adults given their heightened risk of severe influenza during the first wave. We were unable to identify any previous studies of influenza vaccines involving Aboriginal Canadians to determine if their responses would be similar to other Canadians or to the healthy European study participants on whom the dosing recommendation was based. Consequently, we undertook a study involving First Nations and Métis adults to assess their responses to the pandemic 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.     

The seroprevalence of pandemic influenza H1N1 (2009) virus in China

Background

Mainland China experienced pandemic influenza H1N1 (2009) virus (pH1N1) with peak activity during November-December 2009. To understand the geographic extent, risk factors, and attack rate of pH1N1 infection in China we conducted a nationwide serological survey to determine the prevalence of antibodies to pH1N1.

Methodology/Principal Findings

Stored serum samples (n = 2,379) collected during 2006-2008 were used to estimate baseline serum reactogenicity to pH1N1. In January 2010, we used a multistage-stratified random sampling method to select 50,111 subjects who met eligibility criteria and collected serum samples and administered a standardized questionnaire. Antibody response to pH1N1 was measured using haemagglutination inhibition (HI) assay and the weighted seroprevalence was calculated using the Taylor series linearization method. Multivariable logistic regression analyses were used to examine risk factors for pH1N1 seropositivity. Baseline seroprevalence of pH1N1 antibody (HI titer ≥40) was 1.2%. The weighted seroprevalence of pH1N1 among the Chinese population was 21.5%(vaccinated: 62.0%; unvaccinated: 17.1%). Among unvaccinated participants, those aged 6-15 years (32.9%) and 16-24 years (30.3%) had higher seroprevalence compared with participants aged 25–59 years (10.7%) and ≥60 years (9.9%, P<0.0001). Children in kindergarten and students had higher odds of seropositivity than children in family care (OR: 1.36 and 2.05, respectively). We estimated that 207.7 million individuals (15.9%) experienced pH1N1 infection in China.

Conclusions/Significance

The Chinese population had low pre-existing immunity to pH1N1 and experienced a relatively high attack rate in 2009 of this virus. We recommend routine control measures such as vaccination to reduce transmission and spread of seasonal and pandemic influenza viruses.     

Sublingual administration of bacteria-expressed influenza virus hemagglutinin 1 (HA1) induces protection against infection with 2009 pandemic H1N1 influenza virus

Influenza viruses are respiratory pathogens that continue to pose a significantly high risk of morbidity and mortality of humans worldwide. Vaccination is one of the most effective strategies for minimizing damages by influenza outbreaks. In addition, rapid development and production of efficient vaccine with convenient administration is required in case of influenza pandemic. In this study, we generated recombinant influenza virus hemagglutinin protein 1 (sHA1) of 2009 pandemic influenza virus as a vaccine candidate using a well-established bacterial expression system and administered it into mice via sublingual (s.l.) route. We found that s.l. immunization with the recombinant sHA1 plus cholera toxin (CT) induced mucosal antibodies as well as systemic antibodies including neutralizing Abs and provided complete protection against infection with pandemic influenza virus A/CA/04/09 (H1N1) in mice. Indeed, the protection efficacy was comparable with that induced by intramuscular (i.m.) immunization route utilized as general administration route of influenza vaccine. These results suggest that s.l. vaccination with the recombinant non-glycosylated HA1 protein offers an alternative strategy to control influenza outbreaks including pandemics.     

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.     

Prevalence of seroprotection against the pandemic (H1N1) virus after the 2009 pandemic

Background

Before pandemic (H1N1) 2009, less than 10% of serum samples collected from all age groups in the Lower Mainland of British Columbia, Canada, showed seroprotection against the pandemic (H1N1) 2009 virus, except those from very elderly people. We reassessed this profile of seroprotection by age in the same region six months after the fall 2009 pandemic and vaccination campaign.

Methods

We evaluated 100 anonymized serum samples per 10-year age group based on convenience sampling. We measured levels of antibody against the pandemic virus by hemagglutination inhibition and microneutralization assays. We assessed geometric mean titres and the proportion of people with seroprotective antibody levels (hemagglutination inhibition titre ≥ 40). We performed sensitivity analyses to evaluate titre thresholds of 80, 20 and 10.

Results

Serum samples from 1127 people aged 9 months to 101 years were obtained. The overall age-standardized proportion of people with seroprotective antibody levels was 46%. A U-shaped age distribution was identified regardless of assay or titre threshold applied. Among those less than 20 years old and those 80 years and older, the prevalence of seroprotection was comparably high at about 70%. Seroprotection was 44% among those aged 20–49 and 30% among those 50–79 years. It was lowest among people aged 70–79 years (21%) and highest among those 90 years and older (88%).

Interpretation

We measured much higher levels of seroprotection after the 2009 pandemic compared than before the pandemic, with a U-shaped age distribution now evident. These findings, particularly the low levels of seroprotection among people aged 50–79 years, should be confirmed in other settings and closer to the influenza season.In a previous age-based survey of about 1000 anonymized serum samples collected before substantial pandemic (H1N1) 2009 activity in the Lower Mainland of the province of British Columbia, Canada, we found that less than 10% of children and adults under 70 years of age had seroprotective levels of antibody against the pandemic (H1N1) virus.¹ This proportion was slightly higher among people aged 70–79 years (27%) and substantially higher among those above 80 years of age (77%).¹The 2009 influenza pandemic and the broad and effective vaccination campaign introduced major changes to this population’s immune status. The first wave in the province, in the spring and summer months, was of limited activity and was followed by a second, more substantial and widespread wave in the fall that peaked during the last week of October and resolved by the end of 2009.² Meanwhile, a highly immunogenic adjuvanted vaccine was provided free of charge through a universal vaccination campaign that targeted all Canadians.³ Supply was limited initially, requiring sequenced rollout of the vaccine, starting with children under five years of age, pregnant women, and people under 65 years who had comorbidities.⁴ The uptake of the vaccine of about 35%–45% in the province overall^4–6 and 44% in the Lower Mainland (Dr. Monika Naus, BC Centre for Disease Control, Vancouver, BC: personal communication, 2010) was estimated to be moderate compared with rates of uptake in other provinces.To assess seroprotective antibody levels after the 2009 pandemic, we repeated our age-based survey of antibody levels against the pandemic (H1N1) 2009 virus in a further 1000 serum samples collected from people in the Lower Mainland in May and June 2010, more than six months after the last peak of the epidemic.     

Susceptibility of antiviral drugs against 2009 influenza A (H1N1) virus

The recent outbreak of the novel strain of influenza A (H1N1) virus has raised a global concern of the future risk of a pandemic. To understand at the molecular level how this new H1N1 virus can be inhibited by the current anti-influenza drugs and which of these drugs it is likely to already be resistant to, homology modeling and MD simulations have been applied on the H1N1 neuraminidase complexed with oseltamivir, and the M2-channel with adamantanes bound. The H1N1 virus was predicted to be susceptible to oseltamivir, with all important interactions with the binding residues being well conserved. In contrast, adamantanes are not predicted to be able to inhibit the M2 function and have completely lost their binding with the M2 residues. This is mainly due to the fact that the M2 transmembrane of the new H1N1 strain contains the S31N mutation which is known to confer resistance to adamantanes.     

Prevalence of 2009 pandemic influenza A (H1N1) virus antibodies, Tampa Bay Florida--November-December, 2009

Background

In 2009, a novel influenza virus (2009 pandemic influenza A (H1N1) virus (pH1N1)) caused significant disease in the United States. Most states, including Florida, experienced a large fall wave of disease from September through November, after which disease activity decreased substantially. We determined the prevalence of antibodies due to the pH1N1 virus in Florida after influenza activity had peaked and estimated the proportion of the population infected with pH1N1 virus during the pandemic.

Methods

During November-December 2009, we collected leftover serum from a blood bank, a pediatric children''s hospital and a pediatric outpatient clinic in Tampa Bay Florida. Serum was tested for pH1N1 virus antibodies using the hemagglutination-inhibition (HI) assay. HI titers ≥40 were considered seropositive. We adjusted seroprevalence results to account for previously established HI assay specificity and sensitivity and employed a simple statistical model to estimate the proportion of seropositivity due to pH1N1 virus infection and vaccination.

Results

During the study time period, the overall seroprevalence in Tampa Bay, Florida was 25%, increasing to 30% after adjusting for HI assay sensitivity and specificity. We estimated that 5.9% of the population had vaccine-induced seropositivity while 25% had seropositivity secondary to pH1N1 virus infection. The highest cumulative incidence of pH1N1 virus infection was among children aged 5–17 years (53%) and young adults aged 18–24 years (47%), while adults aged ≥50 years had the lowest cumulative incidence (11–13%) of pH1N1 virus infection.

Conclusions

After the peak of the fall wave of the pandemic, an estimated one quarter of the Tampa Bay population had been infected with the pH1N1 virus. Consistent with epidemiologic trends observed during the pandemic, the highest burdens of disease were among school-aged children and young adults.     

Severe pandemic 2009 H1N1 influenza disease due to pathogenic immune complexes

Pandemic influenza viruses often cause severe disease in middle-aged adults without preexisting comorbidities. The mechanism of illness associated with severe disease in this age group is not well understood. Here we find preexisting serum antibodies that cross-react with, but do not protect against, 2009 H1N1 influenza virus in middle-aged adults. Nonprotective antibody is associated with immune complex-mediated disease after infection. We detected high titers of serum antibody of low avidity for H1-2009 antigen, and low-avidity pulmonary immune complexes against the same protein, in severely ill individuals. Moreover, C4d deposition--a marker of complement activation mediated by immune complexes--was present in lung sections of fatal cases. Archived lung sections from middle-aged adults with confirmed fatal influenza 1957 H2N2 infection revealed a similar mechanism of illness. These observations provide a previously unknown biological mechanism for the unusual age distribution of severe cases during influenza pandemics.     

Replication and pathogenesis of the pandemic (H1N1) 2009 influenza virus in mammalian models

This study aimed to characterize the replication and pathogenic properties of a Korean pandemic (H1N1) 2009 influenza virus isolate in ferrets and mice. Ferrets infected with A/Korea/01/2009 (H1N1) virus showed mild clinical signs. The virus replicated well in lungs and slightly in brains with no replication in any other organs. Severe bronchopneumonia and thickening of alveolar walls were detected in the lungs. Viral antigens were detected in the bronchiolar epithelial cells, in peribronchial glands with severe peribronchitis and in cells present in the alveoli. A/Korea/01/2009 (H1N1) virus-infected mice showed weight loss and pathological lung lesions including perivascular cuffing, interstitial pneumonia and alveolitis. The virus replicated highly in the lungs and slightly in the nasal tissues. Viral antigens were detected in bronchiolar epithelial cells, pneumocytes and interstitial macrophages. However, seasonal H1N1 influenza virus did not replicate in the lungs of ferrets, and viral antigens were not detected. Thus, this Korean pandemic (H1N1) 2009 isolate infected the lungs of ferrets and mice successfully and caused more pathological lesions than did the seasonal influenza virus.     

Increased pathogenicity of a reassortant 2009 pandemic H1N1 influenza virus containing an H5N1 hemagglutinin

A novel H1N1 influenza virus emerged in 2009 (pH1N1) to become the first influenza pandemic of the 21st century. This virus is now cocirculating with highly pathogenic H5N1 avian influenza viruses in many parts of the world, raising concerns that a reassortment event may lead to highly pathogenic influenza strains with the capacity to infect humans more readily and cause severe disease. To investigate the virulence of pH1N1-H5N1 reassortant viruses, we created pH1N1 (A/California/04/2009) viruses expressing individual genes from an avian H5N1 influenza strain (A/Hong Kong/483/1997). Using several in vitro models of virus replication, we observed increased replication for a reassortant CA/09 virus expressing the hemagglutinin (HA) gene of HK/483 (CA/09-483HA) relative to that of either parental CA/09 virus or reassortant CA/09 expressing other HK/483 genes. This increased replication correlated with enhanced pathogenicity in infected mice similar to that of the parental HK/483 strain. The serial passage of the CA/09 parental virus and the CA/09-483HA virus through primary human lung epithelial cells resulted in increased pathogenicity, suggesting that these viruses easily adapt to humans and become more virulent. In contrast, serial passage attenuated the parental HK/483 virus in vitro and resulted in slightly reduced morbidity in vivo, suggesting that sustained replication in humans attenuates H5N1 avian influenza viruses. Taken together, these data suggest that reassortment between cocirculating human pH1N1 and avian H5N1 influenza strains will result in a virus with the potential for increased pathogenicity in mammals.     

Genetical features of influenza virus A (H1N1) strain that caused the 2009 pandemic

Genetical features of the A(H1N1) influenza virus strain that caused the 2009 pandemic are analyzed in the review. Mutations typical for this strain, unique and similar to influenza viruses of swine, avian and seasonal types, and phenotypic (pathologic) features associated with them, that are experimentally confirmed, are described. A possibility of reassortation of avian and swine influenza viruses and possible epidemiologic consequences are discussed.     

Correlates of severe disease in patients with 2009 pandemic influenza (H1N1) virus infection

Background

In the context of 2009 pandemic influenza (H1N1) virus infection (pandemic H1N1 influenza), identifying correlates of the severity of disease is critical to guiding the implementation of antiviral strategies, prioritization of vaccination efforts and planning of health infrastructure. The objective of this study was to identify factors correlated with severity of disease in confirmed cases of pandemic H1N1 influenza.

Methods

This cumulative case–control study included all laboratory-confirmed cases of pandemic H1N1 influenza among residents of the province of Manitoba, Canada, for whom the final location of treatment was known. Severe cases were defined by admission to a provincial intensive care unit (ICU). Factors associated with severe disease necessitating admission to the ICU were determined by comparing ICU cases with two control groups: patients who were admitted to hospital but not to an ICU and those who remained in the community.

Results

As of Sept. 5, 2009, there had been 795 confirmed cases of pandemic H1N1 influenza in Manitoba for which the final treatment location could be determined. The mean age of individuals with laboratory-confirmed infection was 25.3 (standard deviation 18.8) years. More than half of the patients (417 or 52%) were female, and 215 (37%) of 588 confirmed infections for which ethnicity was known occurred in First Nations residents. The proportion of First Nations residents increased with increasing severity of disease (116 [28%] of 410 community cases, 74 [54%] of 136 admitted to hospital and 25 [60%] of 42 admitted to an ICU; p < 0.001), as did the presence of an underlying comorbidity (201 [35%] of 569 community cases, 103 [57%] of 181 admitted to hospital and 34 [76%] of 45 admitted to an ICU; p < 0.001). The median interval from onset of symptoms to initiation of antiviral therapy was 2 days (interquartile range, IQR 1–3) for community cases, 4 days (IQR 2–6) for patients admitted to hospital and 6 days (IQR 4–9) for those admitted to an ICU (p < 0.001). In a multivariable logistic model, the interval from onset of symptoms to initiation of antiviral therapy (odds ratio [OR] 8.24, 95% confidence interval [CI] 2.82–24.1), First Nations ethnicity (OR 6.52, 95% CI 2.04–20.8) and presence of an underlying comorbidity (OR 3.19, 95% CI 1.07–9.52) were associated with increased odds of admission to the ICU (i.e., severe disease) relative to community cases. In an analysis of ICU cases compared with patients admitted to hospital, First Nations ethnicity (OR 3.23, 95% CI 1.04–10.1) was associated with increased severity of disease.

Interpretation

Severe pandemic H1N1 influenza necessitating admission to the ICU was associated with a longer interval from onset of symptoms to treatment with antiviral therapy and with the presence of an underlying comorbidity. First Nations ethnicity appeared to be an independent determinant of severe infection. Despite these associations, the cause and outcomes of pandemic HINI influenza may involve many complex and interrelated factors, all of which require further research and analysis.In April 2009, Canada’s first wave of pandemic influenza (H1N1) virus infections (pandemic H1N1 influenza) began. The highest burden of severe illness in Canada occurred in the province of Manitoba, where 45 Manitobans and 9 out-of-province patients were admitted to an intensive care unit (ICU). In this first wave, ICU staff and equipment were mobilized to expand bed capacity and ventilator capabilities to accommodate clinical need.Although many individuals presented with mild, self-limited symptoms and no sign of pulmonary involvement, some people required admission to an ICU and received maximal life support measures.^1–3 Predicting disease and mitigating hazard in at-risk populations is an important aim of public heath epidemiology, and in preparation for future waves of pandemic H1N1 influenza, determining correlates of the severity of disease may be very important. Initial reports have suggested that, in addition to many of the previously known risk factors for complications of seasonal influenza, obesity⁴ and other underlying comorbidities^3,5 may be risk factors for severe disease. The interval from onset of symptoms to initiation of antiviral therapy or other treatment and supportive care was also associated with adverse outcome in a recent case series.⁶ In a Canadian study of severe pandemic H1N1 influenza, First Nations people were proportionally overrepresented among patients in the ICU.² However, it is unclear if this association was independent of potential confounding factors. The ability to determine correlates of severe pandemic H1N1 disease and subsequent need for ICU resources in at-risk populations would provide opportunities for public and population health analysis and action, public education, strategic prioritization of vaccination efforts, efficient and equitable allocation and use of antiviral drugs, and development of infrastructure within the health system.The objectives of this study were to identify factors that were correlated with severity of disease in confirmed cases of pandemic H1N1 influenza. Our hypothesis, which was based on existing literature, was that obesity, First Nations ethnicity and longer interval from onset of symptoms to treatment would be important determinants of the severity of disease.     

T cell-mediated protection against lethal 2009 pandemic H1N1 influenza virus infection in a mouse model

Genetic mutation and reassortment of influenza virus gene segments, in particular those of hemagglutinin (HA) and neuraminidase (NA), that lead to antigenic drift and shift are the major strategies for influenza virus to escape preexisting immunity. The most recent example of such phenomena is the first pandemic of H1N1 influenza of the 21st century, which started in 2009. Cross-reactive antibodies raised against H1N1 viruses circulating before 1930 show protective activity against the 2009 pandemic virus. Cross-reactive T-cell responses can also contribute to protection, but in vivo support of this view is lacking. To explore the protection mechanisms in vivo, we primed mice with H1 and H3 influenza virus isolates and rechallenged them with a virus derived from the 2009 H1N1 A/CA/04/09 virus, named CA/E3/09. We found that priming with influenza viruses of both H1 and H3 homo- and heterosubtypes protected against lethal CA/E3/09 virus challenge. Convalescent-phase sera from these primed mice conferred no neutralization activity in vitro and no protection in vivo. However, T-cell depletion studies suggested that both CD4 and CD8 T cells contributed to the protection. Taken together, these results indicate that cross-reactive T cells established after initial priming with distally related viruses can be a vital component for prevention of disease and control of pandemic H1N1 influenza virus infection. Our results highlight the importance of establishing cross-reactive T-cell responses for protecting against existing or newly emerging pandemic influenza viruses.     

Novel antiviral characteristics of nanosized copper(I) iodide particles showing inactivation activity against 2009 pandemic H1N1 influenza virus

We investigated the antiviral activity of nanosized copper(I) iodide (CuI) particles having an average size of 160 nm. CuI particles showed aqueous stability and generated hydroxyl radicals, which were probably derived from monovalent copper (Cu(+)). We confirmed that CuI particles showed antiviral activity against an influenza A virus of swine origin (pandemic [H1N1] 2009) by plaque titration assay. The virus titer decreased in a dose-dependent manner upon incubation with CuI particles, with the 50% effective concentration being approximately 17 μg/ml after exposure for 60 min. SDS-PAGE analysis confirmed the inactivation of the virus due to the degradation of viral proteins such as hemagglutinin and neuraminidase by CuI. Electron spin resonance (ESR) spectroscopy revealed that CuI generates hydroxyl radicals in aqueous solution, and radical production was found to be blocked by the radical scavenger N-acetylcysteine. Taken together, these findings indicate that CuI particles exert antiviral activity by generating hydroxyl radicals. Thus, CuI may be a useful material for protecting against viral attacks and may be suitable for applications such as filters, face masks, protective clothing, and kitchen cloths.     

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.     

Distinct regulation of host responses by ERK and JNK MAP kinases in swine macrophages infected with pandemic (H1N1) 2009 influenza virus

Swine influenza is an acute respiratory disease in pigs caused by swine influenza virus (SIV). Highly virulent SIV strains cause mortality of up to 10%. Importantly, pigs have long been considered "mixing vessels" that generate novel influenza viruses with pandemic potential, a constant threat to public health. Since its emergence in 2009 and subsequent pandemic spread, the pandemic (H1N1) 2009 (H1N1pdm) has been detected in pig farms, creating the risk of generating new reassortants and their possible infection of humans. Pathogenesis in SIV or H1N1pdm-infected pigs remains poorly characterized. Proinflammatory and antiviral cytokine responses are considered correlated with the intensity of clinical signs, and swine macrophages are found to be indispensible in effective clearance of SIV from pig lungs. In this study, we report a unique pattern of cytokine responses in swine macrophages infected with H1N1pdm. The roles of mitogen-activated protein (MAP) kinases in the regulation of the host responses were examined. We found that proinflammatory cytokines IL-6, IL-8, IL-10, and TNF-α were significantly induced and their induction was ERK1/2-dependent. IFN-β and IFN-inducible antiviral Mx and 2'5'-OAS were sharply induced, but the inductions were effectively abolished when ERK1/2 was inhibited. Induction of CCL5 (RANTES) was completely inhibited by inhibitors of ERK1/2 and JNK1/2, which appeared also to regulate FasL and TNF-α, critical for apoptosis in pig macrophages. We found that NFκB was activated in H1N1pdm-infected cells, but the activation was suppressed when ERK1/2 was inhibited, indicating there is cross-talk between MAP kinase and NFκB responses in pig macrophages. Our data suggest that MAP kinase may activate NFκB through the induction of RIG-1, which leads to the induction of IFN-β in swine macrophages. Understanding host responses and their underlying mechanisms may help identify venues for effective control of SIV and assist in prevention of future influenza pandemics.