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.     

Vaccination against Human Influenza A/H3N2 Virus Prevents the Induction of Heterosubtypic Immunity against Lethal Infection with Avian Influenza A/H5N1 Virus

Annual vaccination against seasonal influenza viruses is recommended for certain individuals that have a high risk for complications resulting from infection with these viruses. Recently it was recommended in a number of countries including the USA to vaccinate all healthy children between 6 and 59 months of age as well. However, vaccination of immunologically naïve subjects against seasonal influenza may prevent the induction of heterosubtypic immunity against potentially pandemic strains of an alternative subtype, otherwise induced by infection with the seasonal strains.Here we show in a mouse model that the induction of protective heterosubtypic immunity by infection with a human A/H3N2 influenza virus is prevented by effective vaccination against the A/H3N2 strain. Consequently, vaccinated mice were no longer protected against a lethal infection with an avian A/H5N1 influenza virus. As a result H3N2-vaccinated mice continued to loose body weight after A/H5N1 infection, had 100-fold higher lung virus titers on day 7 post infection and more severe histopathological changes than mice that were not protected by vaccination against A/H3N2 influenza.The lack of protection correlated with reduced virus-specific CD8+ T cell responses after A/H5N1 virus challenge infection. These findings may have implications for the general recommendation to vaccinate all healthy children against seasonal influenza in the light of the current pandemic threat caused by highly pathogenic avian A/H5N1 influenza viruses.     

A contributing role for anti-neuraminidase antibodies on immunity to pandemic H1N1 2009 influenza A virus

Background

Exposure to contemporary seasonal influenza A viruses affords partial immunity to pandemic H1N1 2009 influenza A virus (pH1N1) infection. The impact of antibodies to the neuraminidase (NA) of seasonal influenza A viruses to cross-immunity against pH1N1 infection is unknown.

Methods and Results

Antibodies to the NA of different seasonal H1N1 influenza strains were tested for cross-reactivity against A/California/04/09 (pH1N1). A panel of reverse genetic (rg) recombinant viruses was generated containing 7 genes of the H1N1 influenza strain A/Puerto Rico/08/34 (PR8) and the NA gene of either the pandemic H1N1 2009 strain (pH1N1) or one of the following contemporary seasonal H1N1 strains: A/Solomon/03/06 (rg Solomon) or A/Brisbane/59/07 (rg Brisbane). Convalescent sera collected from mice infected with recombinant viruses were measured for cross-reactive antibodies to pH1N1 via Hemagglutinin Inhibition (HI) or Enzyme-Linked Immunosorbent Assay (ELISA). The ectodomain of a recombinant NA protein from the pH1N1 strain (pNA-ecto) was expressed, purified and used in ELISA to measure cross-reactive antibodies. Analysis of sera from elderly humans immunized with trivalent split-inactivated influenza (TIV) seasonal vaccines prior to 2009 revealed considerable cross-reactivity to pNA-ecto. High titers of cross-reactive antibodies were detected in mice inoculated with either rg Solomon or rg Brisbane. Convalescent sera from mice inoculated with recombinant viruses were used to immunize naïve recipient Balb/c mice by passive transfer prior to challenge with pH1N1. Mice receiving rg California sera were better protected than animals receiving rg Solomon or rg Brisbane sera.

Conclusions

The NA of contemporary seasonal H1N1 influenza strains induces a cross-reactive antibody response to pH1N1 that correlates with reduced lethality from pH1N1 challenge, albeit less efficiently than anti-pH1N1 NA antibodies. These findings demonstrate that seasonal NA antibodies contribute to but are not sufficient for cross-reactive immunity to pH1N1.     

Cross‐reactive antibody response to the pandemic A (H1N1) 2009 influenza virus induced by vaccination with a seasonal trivalent influenza vaccine: a longitudinal study of three influenza seasons in Japan

The cross‐reactivity of antibody to the swine‐origin pandemic influenza A (H1N1) 2009 virus induced by vaccination with a seasonal trivalent influenza vaccine was studied. Paired sera from a cohort of adult volunteers vaccinated with a trivalent seasonal influenza vaccine every year from 2006 to 2008 were collected each year and tested by hemagglutination inhibition (HI) for antibody against the pandemic influenza A (H1N1) 2009 virus. There was little increase in the geometric mean titer overall; a slight increase was detected in the sera obtained in the 2007–2008 season but not in the other two seasons. The proportion of individuals with HI antibody titers ≥ 1:40 did not change significantly from year to year. These results indicate that cross‐reactivity of the antibodies induced by a trivalent seasonal vaccine to the pandemic influenza A (H1N1) 2009 virus is marginal.     

H5N1 and 1918 pandemic influenza virus infection results in early and excessive infiltration of macrophages and neutrophils in the lungs of mice

Fatal human respiratory disease associated with the 1918 pandemic influenza virus and potentially pandemic H5N1 viruses is characterized by severe lung pathology, including pulmonary edema and extensive inflammatory infiltrate. Here, we quantified the cellular immune response to infection in the mouse lung by flow cytometry and demonstrate that mice infected with highly pathogenic (HP) H1N1 and H5N1 influenza viruses exhibit significantly high numbers of macrophages and neutrophils in the lungs compared to mice infected with low pathogenic (LP) viruses. Mice infected with the 1918 pandemic virus and a recent H5N1 human isolate show considerable similarities in overall lung cellularity, lung immune cell sub-population composition, and cellular immune temporal dynamics. Interestingly, while these similarities were observed, the HP H5N1 virus consistently elicited significantly higher levels of pro-inflammatory cytokines in whole lungs and primary human macrophages, revealing a potentially critical difference in the pathogenesis of H5N1 infections. Primary mouse and human macrophages and dendritic cells were also susceptible to 1918 and H5N1 influenza virus infection in vitro. These results together indicate that infection with HP influenza viruses such as H5N1 and the 1918 pandemic virus leads to a rapid cell recruitment of macrophages and neutrophils into the lungs, suggesting that these cells play a role in acute lung inflammation associated with HP influenza virus infection.     

The 2009 pandemic H1N1 and triple-reassortant swine H1N1 influenza viruses replicate efficiently but elicit an attenuated inflammatory response in polarized human bronchial epithelial cells

The pandemic H1N1 virus of 2009 (2009 H1N1) produced a spectrum of disease ranging from mild illness to severe illness and death. Respiratory symptoms were frequently associated with virus infection, with relatively high rate of gastrointestinal symptoms reported. To better understand 2009 H1N1 virus pathogenesis in humans, we studied virus and host responses following infection of two cell types: polarized bronchial and pharyngeal epithelial cells, which exhibit many features of the human airway epithelium, and colon epithelial cells to serve as a human intestinal cell model. Selected 2009 H1N1 viruses were compared to both seasonal H1N1 and triple-reassortant swine H1N1 influenza viruses that have circulated among North American pigs since before the 2009 pandemic. All H1N1 viruses replicated productively in airway cells; however, in contrast to seasonal H1N1 virus infection, infection with the 2009 H1N1 and triple-reassortant swine H1N1 viruses resulted in an attenuated inflammatory response, a weaker interferon response, and reduced cell death. Additionally, the H1N1 viruses of swine origin replicated less efficiently at the temperature of the human proximal airways (33°C). We also observed that the 2009 H1N1 viruses replicated to significantly higher titers than seasonal H1N1 virus in polarized colon epithelial cells. These studies reveal that in comparison to seasonal influenza virus, H1N1 viruses of swine origin poorly activate multiple aspects of the human innate response, which may contribute to the virulence of these viruses. In addition, their less efficient replication at human upper airway temperatures has implications for the understanding of pandemic H1N1 virus adaptation to humans.     

Infection with seasonal influenza virus elicits CD4 T cells specific for genetically conserved epitopes that can be rapidly mobilized for protective immunity to pandemic H1N1 influenza virus

In recent years, influenza viruses with pandemic potential have been a major concern worldwide. One unresolved issue is how infection or vaccination with seasonal influenza virus strains influences the ability to mount a protective immune response to novel pandemic strains. In this study, we developed a mouse model of primary and secondary influenza infection by using a widely circulating seasonal H1N1 virus and the pandemic strain of H1N1 that emerged in Mexico in 2009, and we evaluated several key issues. First, using overlapping peptide libraries encompassing the entire translated sequences of 5 major influenza virus proteins, we assessed the specificity of CD4 T cell reactivity toward epitopes conserved among H1N1 viruses or unique to the seasonal or pandemic strain by enzyme-linked immunospot (ELISpot) assays. Our data show that CD4 T cells reactive to both virus-specific and genetically conserved epitopes are elicited, allowing separate tracking of these responses. Populations of cross-reactive CD4 T cells generated from seasonal influenza infection were found to expand earlier after secondary infection with the pandemic H1N1 virus than CD4 T cell populations specific for new epitopes. Coincident with this rapid CD4 T cell response was a potentiated neutralizing-antibody response to the pandemic strain and protection from the pathological effects of infection with the pandemic virus. This protection was not dependent on CD8 T cells. Together, our results indicate that exposure to seasonal vaccines and infection elicits CD4 T cells that promote the ability of the mammalian host to mount a protective immune response to pandemic strains of influenza virus.     

Efficacy of vaccination with different combinations of MF59-adjuvanted and nonadjuvanted seasonal and pandemic influenza vaccines against pandemic H1N1 (2009) influenza virus infection in ferrets

Serum antibodies induced by seasonal influenza or seasonal influenza vaccination exhibit limited or no cross-reactivity against the 2009 pandemic swine-origin influenza virus of the H1N1 subtype (pH1N1). Ferrets immunized once or twice with MF59-adjuvanted seasonal influenza vaccine exhibited significantly reduced lung virus titers but no substantial clinical protection against pH1N1-associated disease. However, priming with MF59-adjuvanted seasonal influenza vaccine significantly increased the efficacy of a pandemic MF59-adjuvanted influenza vaccine against pH1N1 challenge. Elucidating the mechanism involved in this priming principle will contribute to our understanding of vaccine- and infection-induced correlates of protection. Furthermore, a practical consequence of these findings is that during an emerging pandemic, the implementation of a priming strategy with an available adjuvanted seasonal vaccine to precede the eventual pandemic vaccination campaign may be useful and life-saving.     

Complex patterns of human antisera reactivity to novel 2009 H1N1 and historical H1N1 influenza strains

Background

During the 2009 influenza pandemic, individuals over the age of 60 had the lowest incidence of infection with approximately 25% of these people having pre-existing, cross-reactive antibodies to novel 2009 H1N1 influenza isolates. It was proposed that older people had pre-existing antibodies induced by previous 1918-like virus infection(s) that cross-reacted to novel H1N1 strains.

Methodology/Principal Findings

Using antisera collected from a cohort of individuals collected before the second wave of novel H1N1 infections, only a minority of individuals with 1918 influenza specific antibodies also demonstrated hemagglutination-inhibition activity against the novel H1N1 influenza. In this study, we examined human antisera collected from individuals that ranged between the ages of 1 month and 90 years to determine the profile of seropositive influenza immunity to viruses representing H1N1 antigenic eras over the past 100 years. Even though HAI titers to novel 2009 H1N1 and the 1918 H1N1 influenza viruses were positively associated, the association was far from perfect, particularly for the older and younger age groups.

Conclusions/Significance

Therefore, there may be a complex set of immune responses that are retained in people infected with seasonal H1N1 that can contribute to the reduced rates of H1N1 influenza infection in older populations.     

Heterosubtypic antibody response elicited with seasonal influenza vaccine correlates partial protection against highly pathogenic H5N1 virus

Background

The spread of highly pathogenic avian influenza (HPAI) H5N1 virus in human remains a global health concern. Heterosubtypic antibody response between seasonal influenza vaccine and potential pandemic influenza virus has important implications for public health. Previous studies by Corti et al. and by Gioia et al. demonstrate that heterosubtypic neutralizing antibodies against the highly pathogenic H5N1 virus can be elicited with a seasonal influenza vaccine in humans. However, whether such response offers immune protection against highly pathogenic H5N1 virus remained to be determined.

Methodology/Principal Findings

In this study, using a sensitive influenza HA (hemagglutinin) and NA (neuraminidase) pseudotype-based neutralization (PN) assay we first confirmed that low levels of heterosubtypic neutralizing antibody response against H5N1 virus were indeed elicited with seasonal influenza vaccine in humans. We then immunized mice with the seasonal influenza vaccine and challenged them with lethal doses of highly pathogenic H5N1 virus. As controls, we immunized mice with homosubtypic H5N1 virus like particles (VLP) or PBS and challenged them with the same H5N1 virus. Here we show that low levels of heterosubtypic neutralizing antibody response were elicited with seasonal influenza vaccine in mice, which were significantly higher than those in PBS control. Among them 2 out of 27 whose immune sera exhibited similar levels of neutralizing antibody response as VLP controls actually survived from highly pathogenic H5N1 virus challenge.

Conclusions/Significance

Therefore, we conclude that low levels of heterosubtypic neutralizing antibody response are indeed elicited with seasonal influenza vaccine in humans and mice and at certain levels such response offers immune protection against severity of H5N1 virus infection.     

季节性流感疫苗对小鼠感染 H7N9 禽流感病毒的保护效力简

目的 评价季节性流感裂解疫苗对流感病毒H7N9的免疫保护效力.方法 用我国2012～2013年度季节性流感裂解疫苗,以腹腔注射方式免疫BALB/c小鼠,并设PBS免疫模型组,末次免疫14 d后以5 LD50 A/Anhui/1（H7N9）进行攻试验.感染后观察记录小鼠临床表现,体重变化,并分别于第2天和第4天每组处死3只小鼠,取肺组织和鼻甲骨测病毒滴度和载量.结果 感染后疫苗与模型组小鼠体重下降明显,疫苗组存活率为10%,模型组全部死亡.感染后第4天疫苗组鼻甲骨滴度显著低于模型组.血凝抑制试验及中和实验表明免疫小鼠血清无中和H7N9病毒抗体.结论 季节性流感疫苗在小鼠中对于H7N9流感病毒感染无明显保护作用.     

Seasonal FluMist vaccination induces cross-reactive T cell immunity against H1N1 (2009) influenza and secondary bacterial infections

T cell epitopes have been found to be shared by circulating, seasonal influenza virus strains and the novel pandemic H1N1 influenza infection, but the ability of these common epitopes to provide cross-protection is unknown. We have now directly tested this by examining the ability of live seasonal influenza vaccine (FluMist) to mediate protection against swine-origin H1N1 influenza virus infection. Naive mice demonstrated considerable susceptibility to H1N1 Cal/04/09 infection, whereas FluMist-vaccinated mice had markedly decreased morbidity and mortality. In vivo depletion of CD4(+) or CD8(+) immune cells after vaccination indicated that protective immunity was primarily dependent upon FluMist-induced CD4(+) cells but not CD8(+) T cells. Passive protection studies revealed little role for serum or mucosal Abs in cross-protection. Although H1N1 influenza infection of naive mice induced intensive phagocyte recruitment, pulmonary innate defense against secondary pneumococcal infection was severely suppressed. This increased susceptibility to bacterial infection was correlated with augmented IFN-γ production produced during the recovery stage of H1N1 influenza infection, which was completely suppressed in mice previously immunized with FluMist. Furthermore, susceptibility to secondary bacterial infection was decreased in the absence of type II, but not type I, IFN signaling. Thus, seasonal FluMist treatment not only promoted resistance to pandemic H1N1 influenza infection but also restored innate immunity against complicating secondary bacterial infections.     

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.     

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.     

Characterization of cross protection of Swine-Origin Influenza Virus (S-OIV) H1N1 and reassortant H5N1 influenza vaccine in BALB/c mice given a single-dose vaccination

Background

Influenza virus has antigen drift and antigen shift effect, vaccination with some influenza vaccine might not induce sufficient immunity for host to the threat of other influenza virus strains. S-OIV H1N1 and H5N1 influenza vaccines in single-dose immunization were evaluated in mice for cross protection to the challenge of A/California/7/2009 H1N1 or NIBRG-14 H5N1 virus.

Results

Both H1N1 and H5N1 induced significant homologous IgG, HAI, and microneutralization antibody responses in the mice, while only vaccines plus adjuvant produced significant heterogeneous IgG and HAI antibody responses. Both alum and MPLA adjuvants significantly reduced the S-OIV H1N1 vaccine dose required to elicit protective HAI antibody titers from 0.05 μg to 0.001 μg. Vaccines alone did not protect mice from challenge with heterogeneous influenza virus, while H5N1 vaccine plus alum and MPLA adjuvants did. Mouse body weight loss was also less significant in the presence of adjuvant than in the vaccine without adjuvant. Furthermore, both H1N1 and H5N1 lung viral titers of immunized mice were significantly reduced post challenge with homologous viruses.

Conclusion

Only in the presence of MPLA adjuvant could the H5N1 vaccine significantly reduce mouse lung viral titers post H1N1 virus challenge, and not vice versa. MPLA adjuvant induced cross protection with a single dose vaccination to the challenge of heterogeneous influenza virus in mice. Lung viral titer seemed to be a better indicator compared to IgG, neutralization antibody, and HAI titer to predict survival of mice infected with influenza virus.     

Protection of Mice against Lethal Challenge with 2009 H1N1 Influenza A Virus by 1918-Like and Classical Swine H1N1 Based Vaccines

The recent 2009 pandemic H1N1 virus infection in humans has resulted in nearly 5,000 deaths worldwide. Early epidemiological findings indicated a low level of infection in the older population (>65 years) with the pandemic virus, and a greater susceptibility in people younger than 35 years of age, a phenomenon correlated with the presence of cross-reactive immunity in the older population. It is unclear what virus(es) might be responsible for this apparent cross-protection against the 2009 pandemic H1N1 virus. We describe a mouse lethal challenge model for the 2009 pandemic H1N1 strain, used together with a panel of inactivated H1N1 virus vaccines and hemagglutinin (HA) monoclonal antibodies to dissect the possible humoral antigenic determinants of pre-existing immunity against this virus in the human population. By hemagglutinination inhibition (HI) assays and vaccination/challenge studies, we demonstrate that the 2009 pandemic H1N1 virus is antigenically similar to human H1N1 viruses that circulated from 1918–1943 and to classical swine H1N1 viruses. Antibodies elicited against 1918-like or classical swine H1N1 vaccines completely protect C57B/6 mice from lethal challenge with the influenza A/Netherlands/602/2009 virus isolate. In contrast, contemporary H1N1 vaccines afforded only partial protection. Passive immunization with cross-reactive monoclonal antibodies (mAbs) raised against either 1918 or A/California/04/2009 HA proteins offered full protection from death. Analysis of mAb antibody escape mutants, generated by selection of 2009 H1N1 virus with these mAbs, indicate that antigenic site Sa is one of the conserved cross-protective epitopes. Our findings in mice agree with serological data showing high prevalence of 2009 H1N1 cross-reactive antibodies only in the older population, indicating that prior infection with 1918-like viruses or vaccination against the 1976 swine H1N1 virus in the USA are likely to provide protection against the 2009 pandemic H1N1 virus. This data provides a mechanistic basis for the protection seen in the older population, and emphasizes a rationale for including vaccination of the younger, naïve population. Our results also support the notion that pigs can act as an animal reservoir where influenza virus HAs become antigenically frozen for long periods of time, facilitating the generation of human pandemic viruses.     

A single mutation in the PB1-F2 of H5N1 (HK/97) and 1918 influenza A viruses contributes to increased virulence

The proapoptotic PB1-F2 protein of influenza A viruses has been shown to contribute to pathogenesis in the mouse model. Expression of full-length PB1-F2 increases the pathogenesis of the influenza A virus, causing weight loss, slower viral clearance, and increased viral titers in the lungs. After comparing viruses from the Hong Kong 1997 H5N1 outbreak, one amino acid change (N66S) was found in the PB1-F2 sequence at position 66 that correlated with pathogenicity. This same amino acid change (N66S) was also found in the PB1-F2 protein of the 1918 pandemic A/Brevig Mission/18 virus. Two isogenic recombinant chimeric viruses were created with an influenza A/WSN/33 virus background containing the PB1 segment from the HK/156/97: WH and WH N66S. In mice infected with WH N66S virus there was increased pathogenicity as measured by weight loss and decreased survival, and a 100-fold increase in virus replication when compared to mice infected with the WH virus. The 1918 pandemic strain A/Brevig Mission/18 was reconstructed with a pathogenicity-reducing mutation in PB1-F2 (S66N). The resultant 1918 S66N virus was attenuated in mice having a 3-log lower 50% lethal dose and caused less morbidity and mortality in mice than the wild-type virus. Viral lung titers were also decreased in 1918 S66N-infected mice compared with wild-type 1918 virus-infected mice. In addition, both viruses with an S at position 66 (WH N66S and wt 1918) induced elevated levels of cytokines in the lungs of infected mice. Together, these data show that a single amino acid substitution in PB1-F2 can result in increased viral pathogenicity and could be one of the factors contributing to the high lethality seen with the 1918 pandemic virus.     

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.     

Impact of Prior Seasonal H3N2 Influenza Vaccination or Infection on Protection and Transmission of Emerging Variants of Influenza A(H3N2)v Virus in Ferrets

Influenza H3N2 A viruses continue to circulate in swine and occasionally infect humans, resulting in outbreaks of variant influenza H3N2 [A(H3N2)v] virus. It has been previously demonstrated in ferrets that A(H3N2)v viruses transmit as efficiently as seasonal influenza viruses, raising concern over the pandemic potential of these viruses. However, A(H3N2)v viruses have not acquired the ability to transmit efficiently among humans, which may be due in part to existing cross-reactive immunity to A(H3N2)v viruses. Although current seasonal H3N2 and A(H3N2)v viruses are antigenically distinct from one another, historical H3N2 viruses have some antigenic similarity to A(H3N2)v viruses and previous exposure to these viruses may provide a measure of immune protection sufficient to dampen A(H3N2)v virus transmission. Here, we evaluated whether prior seasonal H3N2 influenza virus vaccination or infection affects virus replication and transmission of A(H3N2)v virus in the ferret animal model. We found that the seasonal trivalent inactivated influenza virus vaccine (TIV) or a monovalent vaccine prepared from an antigenically related 1992 seasonal influenza H3N2 (A/Beijing/32/1992) virus failed to substantially reduce A(H3N2)v (A/Indiana/08/2011) virus shedding and subsequent transmission to naive hosts. Conversely, ferrets primed by seasonal H3N2 virus infection displayed reduced A(H3N2)v virus shedding following challenge, which blunted transmission to naive ferrets. A higher level of specific IgG and IgA antibody titers detected among infected versus vaccinated ferrets was associated with the degree of protection offered by seasonal H3N2 virus infection. The data demonstrate in ferrets that the efficiency of A(H3N2)v transmission is disrupted by preexisting immunity induced by seasonal H3N2 virus infection.     

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.