Recombinant Parainfluenza Virus 5 Expressing Hemagglutinin of Influenza A Virus H5N1 Protected Mice against Lethal Highly Pathogenic Avian Influenza Virus H5N1 Challenge

A safe and effective vaccine is the best way to prevent large-scale highly pathogenic avian influenza virus (HPAI) H5N1 outbreaks in the human population. The current FDA-approved H5N1 vaccine has serious limitations. A more efficacious H5N1 vaccine is urgently needed. Parainfluenza virus 5 (PIV5), a paramyxovirus, is not known to cause any illness in humans. PIV5 is an attractive vaccine vector. In our studies, a single dose of a live recombinant PIV5 expressing a hemagglutinin (HA) gene of H5N1 (rPIV5-H5) from the H5N1 subtype provided sterilizing immunity against lethal doses of HPAI H5N1 infection in mice. Furthermore, we have examined the effect of insertion of H5N1 HA at different locations within the PIV5 genome on the efficacy of a PIV5-based vaccine. Interestingly, insertion of H5N1 HA between the leader sequence, the de facto promoter of PIV5, and the first viral gene, nucleoprotein (NP), did not lead to a viable virus. Insertion of H5N1 HA between NP and the next gene, V/phosphorprotein (V/P), led to a virus that was defective in growth. We have found that insertion of H5N1 HA at the junction between the small hydrophobic (SH) gene and the hemagglutinin-neuraminidase (HN) gene gave the best immunity against HPAI H5N1 challenge: a dose as low as 1,000 PFU was sufficient to protect against lethal HPAI H5N1 challenge in mice. The work suggests that recombinant PIV5 expressing H5N1 HA has great potential as an HPAI H5N1 vaccine.     

Virus-Like Particle Vaccine Protects against 2009 H1N1 Pandemic Influenza Virus in Mice

Background

The 2009 influenza pandemic and shortages in vaccine supplies worldwide underscore the need for new approaches to develop more effective vaccines.

Methodology/Principal Findings

We generated influenza virus-like particles (VLPs) containing proteins derived from the A/California/04/2009 virus, and tested their efficacy as a vaccine in mice. A single intramuscular vaccination with VLPs provided complete protection against lethal challenge with the A/California/04/2009 virus and partial protection against A/PR/8/1934 virus, an antigenically distant human isolate. VLP vaccination induced predominant IgG2a antibody responses, high hemagglutination inhibition (HAI) titers, and recall IgG and IgA antibody responses. HAI titers after VLP vaccination were equivalent to those observed after live virus infection. VLP immune sera also showed HAI responses against diverse geographic pandemic isolates. Notably, a low dose of VLPs could provide protection against lethal infection.

Conclusion/Significance

This study demonstrates that VLP vaccination provides highly effective protection against the 2009 pandemic influenza virus. The results indicate that VLPs can be developed into an effective vaccine, which can be rapidly produced and avoid the need to isolate high growth reassortants for egg-based production.     

Immunization with a Live Attenuated H7N9 Influenza Vaccine Protects Mice against Lethal Challenge

The emergence of severe cases of human influenza A (H7N9) viral infection in China in the spring of 2003 resulted in a global effort to rapidly develop an effective candidate vaccine. In this study, a cold-adapted (ca), live attenuated monovalent reassortant influenza H7N9 virus (Ah01/AA ca) was generated using reverse genetics that contained hemagglutinin (HA) and neuraminidase (NA) genes from a 2013 pandemic A H7N9 isolate, A/Anhui/01/2013 virus (Ah01/H7N9); the remaining six backbone genes derived from the cold-adapted influenza H2N2 A/Ann Arbor/6/60 virus (AA virus). Ah01/AA ca virus exhibited temperature sensitivity (ts), ca, and attenuation (att) phenotypes. Intranasal immunization of female BALB/c mice with Ah01/AA ca twice at a 2-week interval induced robust humoral, mucosal, and cell-mediated immune responses in a dose-dependent manner. Furthermore, the candidate Ah01/AA ca virus was immunogenic and offered partial or complete protection of mice against a lethal challenge by the live 2013 influenza A H7N9 (A/Anhui/01/2013). Protection was demonstrated by the inhibition of viral replication and the attenuation of histopathological changes in the challenged mouse lung. Taken together, these data support the further evaluation of this Ah01/AA ca candidate vaccine in primates.     

硒对甲型H1N1流感病毒感染小鼠的保护作用

目的:通过研究硒对流感病毒悬液滴鼻处理的小鼠的体重变化、死亡率、血清硒水平和细胞因子的影响,探讨硒对感染流感病毒小鼠的保护作用。方法:将60只昆明小鼠分为5组,每组12只,分别为缺硒组(0 mg/kg)、正常给硒组(0.2 mg/kg)、补充硒组(0.3 mg/kg)、补充硒组(0.4 mg/kg)、补充硒组(0.5 mg/kg)。给5周龄的小鼠滴鼻接种50μL的A/NWS/33(H1N1)病毒悬液并观察21天,监测每组小鼠的体重变化和死亡率;并在接种病毒后的第3天、第5天,检测小鼠的血清硒、TNF-α和IFN-γ水平。结果:缺硒组小鼠的死亡率高于正常给硒组和补充硒组(P0.05);缺硒组小鼠的血清硒水平明显低于正常给硒组和补充硒组(P0.05);在病毒感染第5天,缺硒组小鼠的TNF-α和IFN-γ含量低于正常给硒组和补充硒组(P0.05),差异均有统计学意义。结论:硒可以提高机体对抗流感病毒的免疫反应。     

Corticosteroid Treatment Ameliorates Acute Lung Injury Induced by 2009 Swine Origin Influenza A (H1N1) Virus in Mice

Background

The 2009 influenza pandemic affected people in almost all countries in the world, especially in younger age groups. During this time, the debate over whether to use corticosteroid treatment in severe influenza H1N1 infections patients resurfaced and was disputed by clinicians. There is an urgent need for a susceptible animal model of 2009 H1N1 infection that can be used to evaluate the pathogenesis and the therapeutic effect of corticosteroid treatment during infection.

Methodology/Principal Findings

We intranasally inoculated two groups of C57BL/6 and BALB/c mice (using 4- or 6-to 8-week-old mice) to compare the pathogenesis of several different H1N1 strains in mice of different ages. Based on the results, a very susceptible 4-week-old C57BL/6 mouse model of Beijing 501 strain of 2009 H1N1 virus infection was established, showing significantly elevated lung edema and cytokine levels compared to controls. Using our established animal model, the cytokine production profile and lung histology were assessed at different times post-infection, revealing increased lung lesions in a time-dependent manner. In additional,the mice were also treated with dexamethasone, which significantly improved survival rate and lung lesions in infected mice compared to those in control mice. Our data showed that corticosteroid treatment ameliorated acute lung injury induced by the 2009 A/H1N1 virus in mice and suggested that corticosteroids are valid drugs for treating 2009 A/H1N1 infection.

Conclusions/Significance

Using the established, very susceptible 2009 Pandemic Influenza A (H1N1) mouse model, our studies indicate that corticosteroids are a potential therapeutic remedy that may address the increasing concerns over future 2009 A/H1N1pandemics.     

Protection from H1N1 Influenza Virus Infections in Mice by Supplementation with Selenium: A Comparison with Selenium-Deficient Mice

The present paper describes protective effects of supplemental selenium in mice infected with influenza virus. The effects of supplemental selenium on serum selenium levels, mortality, lung virus titers, and cytokine titers were investigated in mice inoculated intranasally with suspensions of influenza virus. Whereas the mortality of the virus-infected Se-deficient mice was 75%, along with a marked reduction in body weight, lower levels of TNF-α and IFN-γ and lower serum selenium concentrations, the mortality of mice maintained on feed containing 0.5 mg Se/kg in the form of sodium selenite was 25%.There were no significantly differences, however, in viral titer between the Se-adequate and the selenium-supplemented groups. The data indicate that selenium supplementation may provide a feasible approach to improving the immune response to viral infections, such as lethal influenza 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.     

Monoclonal Antibodies against the Fusion Peptide of Hemagglutinin Protect Mice from Lethal Influenza A Virus H5N1 Infection

The HA2 glycopolypeptide (gp) is highly conserved in all influenza A virus strains, and it is known to play a major role in the fusion of the virus with the endosomal membrane in host cells during the course of viral infection. Vaccines and therapeutics targeting this HA2 gp could induce efficient broad-spectrum immunity against influenza A virus infections. So far, there have been no studies on the possible therapeutic effects of monoclonal antibodies (MAbs), specifically against the fusion peptide of hemagglutinin (HA), upon lethal infections with highly pathogenic avian influenza (HPAI) H5N1 virus. We have identified MAb 1C9, which binds to GLFGAIAGF, a part of the fusion peptide of the HA2 gp. We evaluated the efficacy of MAb 1C9 as a therapy for influenza A virus infections. This MAb, which inhibited cell fusion in vitro when administered passively, protected 100% of mice from challenge with five 50% mouse lethal doses of HPAI H5N1 influenza A viruses from two different clades. Furthermore, it caused earlier clearance of the virus from the lung. The influenza virus load was assessed in lung samples from mice challenged after pretreatment with MAb 1C9 (24 h prior to challenge) and from mice receiving early treatment (24 h after challenge). The study shows that MAb 1C9, which is specific to the antigenically conserved fusion peptide of HA2, can contribute to the cross-clade protection of mice infected with H5N1 virus and mediate more effective recovery from infection.Highly pathogenic avian influenza (HPAI) virus H5N1 strains are currently causing major morbidity and mortality in poultry populations across Asia, Europe, and Africa and have caused 385 confirmed human infections, with a fatality rate of 63.11% (37, 39). Preventive and therapeutic measures against circulating H5N1 strains have received a lot of interest and effort globally to prevent another pandemic outbreak. Influenza A virus poses a challenge because it rapidly alters its appearance to the immune system by antigenic drift (mutating) and antigenic shift (exchanging its components) (5). The current strategies to combat influenza include vaccination and antiviral drug treatment, with vaccination being the preferred option. The annual influenza vaccine aims to stimulate the generation of anti-hemagglutinin (anti-HA) neutralizing antibodies, which confer protection against homologous strains. Current vaccines have met with various degrees of success (31). The facts that these strategies target the highly variable HA determinant and that predicting the major HA types that pose the next epidemic threat is difficult are significant limitations to the current antiviral strategy. In the absence of an effective vaccine, therapy is the mainstay of control of influenza virus infection.Therefore, therapeutic measures against influenza will play a major role in case a pandemic arises due to H5N1 strains. Currently licensed antiviral drugs include the M2 ion-channel inhibitors (rimantidine and amantidine) and the neuraminidase inhibitors (oseltamivir and zanamivir). The H5N1 viruses are known to be resistant to the M2 ion-channel inhibitors (2, 3). Newer strains of H5N1 viruses are being isolated which are also resistant to the neuraminidase inhibitors (oseltamivir and zanamivir) (5, 17). The neuraminidase inhibitors also require high doses and prolonged treatment (5, 40), increasing the likelihood of unwanted side effects. Hence, alternative strategies for treatment of influenza are warranted.Recently, passive immunotherapy using monoclonal antibodies (MAbs) has been viewed as a viable option for treatment (26). The HA gene is the most variable gene of the influenza virus and also the most promising target for generating antibodies. It is synthesized as a precursor polypeptide, HA0, which is posttranslationally cleaved to two polypeptides, HA1 and HA2, linked by a disulfide bond. MAbs against the HA1 glycopolypeptide (gp) are known to neutralize the infectivity of the virus and hence provide good protection against infection (12). However, they are less efficient against heterologous or mutant strains, which are continuously arising due to antigenic shift and, to an extent, drift. Recent strategies for alternative therapy explore the more conserved epitopes of the influenza virus antigens (18, 33), which not only have the potential to stimulate a protective immune response but are also conserved among different subtypes, so as to offer protection against a broader range of viruses.The HA2 polypeptide represents a highly conserved region of HA across influenza A virus strains. The HA2 gp is responsible for the fusion of the virus and the host endosomal membrane during the entry of the virus into the cell (16). Previously, anti-HA MAbs that lacked HA inhibition activity were studied and were found to reduce the infectivity of non-H5 influenza virus subtypes by inhibition of fusion during viral replication (14). They are known to block fusion of the virus to the cell membrane at the postbinding and prefusion stage, thereby inhibiting viral replication. Furthermore, in vivo studies show that anti-HA2 MAbs that exhibit fusion inhibition activity contribute to protection and recovery from H3N2 influenza A virus infection (8). It is interesting that although the HA2 gp is generally conserved, the fusion peptide represents the most conserved region of the HA protein. So far, there have been no studies on the possible therapeutic effects of MAbs, specifically against the fusion peptide of HA, on lethal HPAI H5N1 infections.Previous studies have suggested that HA2 could contain a potential epitope responsible for the induction of antibody-mediated protective immunity (9). In the present study, a panel of MAbs against HA2 gp was characterized for their respective epitopes by epitope mapping. The therapeutic and prophylactic efficacies of these MAbs were evaluated in mice challenged with HPAI H5N1 virus infection.     

Mx1 Gene Protects Mice Against the Highly Lethal Human H5N1 Influenza Virus

We investigated the importance of the host Mx1 gene in protection against highly pathogenic H5N1 avian influenza virus. Mice expressing the Mx1 gene survived infection with the lethal human H5N1 isolate A/Vietnam/1203/04 and with reassortants combining its genes with those of the non-lethal virus A/chicken/Vietnam/C58/04, while all Mx1–/– mice succumbed. Mx1-expressing mice showed lower organ virus titers, fewer lesions, and less pulmonary inflammation. Our data support the hypothesis that Mx1 expression protects mice against the high pathogenicity of H5N1 virus through inhibition of viral polymerase activity ultimately resulting in reduced viral growth and spread. Drugs that mimic this mechanism may be protective in humans.     

Cytotoxic T Lymphocytes Established by Seasonal Human Influenza Cross-React against 2009 Pandemic H1N1 Influenza Virus

While few children and young adults have cross-protective antibodies to the pandemic H1N1 2009 (pdmH1N1) virus, the illness remains mild. The biological reasons for these epidemiological observations are unclear. In this study, we demonstrate that the bulk memory cytotoxic T lymphocytes (CTLs) established by seasonal influenza viruses from healthy individuals who have not been exposed to pdmH1N1 can directly lyse pdmH1N1-infected target cells and produce gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α). Using influenza A virus matrix protein 1 (M1_58-66) epitope-specific CTLs isolated from healthy HLA-A2⁺ individuals, we further found that M1_58-66 epitope-specific CTLs efficiently killed both M1_58-66 peptide-pulsed and pdmH1N1-infected target cells ex vivo. These M1_58-66-specific CTLs showed an effector memory phenotype and expressed CXCR3 and CCR5 chemokine receptors. Of 94 influenza A virus CD8 T-cell epitopes obtained from the Immune Epitope Database (IEDB), 17 epitopes are conserved in pdmH1N1, and more than half of these conserved epitopes are derived from M1 protein. In addition, 65% (11/17) of these epitopes were 100% conserved in seasonal influenza vaccine H1N1 strains during the last 20 years. Importantly, seasonal influenza vaccination could expand the functional M1_58-66 epitope-specific CTLs in 20% (4/20) of HLA-A2⁺ individuals. Our results indicated that memory CTLs established by seasonal influenza A viruses or vaccines had cross-reactivity against pdmH1N1. These might explain, at least in part, the unexpected mild pdmH1N1 illness in the community and also might provide some valuable insights for the future design of broadly protective vaccines to prevent influenza, especially pandemic influenza.Since its first identification in North America in April 2009, the novel pandemic H1N1 2009 (pdmH1N1) virus has been spreading in humans worldwide, giving rise to the first pandemic in the 21st century (13, 18). The pdmH1N1 virus contains a unique gene constellation, with its NA and M gene segments being derived from the Eurasian swine lineage while the other gene segments originated from the swine triple-reassortant H1N1 lineage. The triple-reassortant swine viruses have in turn derived the HA, NP, and NS gene segments from the classical swine lineage (20). The 1918 pandemic virus gave rise to both the seasonal influenza H1N1 and the classical swine H1N1 virus lineages (41). Evolution in different hosts during the subsequent 90 years has led to increasing antigenic differences between recent seasonal H1N1 viruses and swine H1 viruses (42). Thus, younger individuals have no antibodies that cross neutralize pdmH1N1, while those over 65 years of age are increasingly likely to have cross-neutralizing antibodies to pdmH1N1 (10, 25).Currently available seasonal influenza vaccines do not induce cross-reactive antibodies against this novel virus in any age group (10, 25). In animal models, it has been shown that pdmH1N1 replicated more efficiently and caused more severe pathological lesions than the current seasonal influenza virus (28). However, most patients with pdmH1N1 virus infection show a mild illness comparable to seasonal influenza (9, 42). The incidence of severe cases caused by pdmH1N1 was not significantly higher than that caused by human seasonal influenza viruses (43). These findings imply that seasonal influenza A virus-specific memory T cells preexisting in previously infected individuals may have cross-protection to this novel pdmH1N1.Cross-reactivity of influenza A virus-specific T-cell immunity against heterosubtypic strains which are serologically distinct has been demonstrated (5, 29, 33, 47). Humans who have not been exposed to avian influenza A (H5N1) virus do have cross-reactive memory CD4 and CD8 T cells to a wide range of H5N1 peptides (33, 47). More recently, one study also showed that some seasonal influenza A virus-specific memory T cells in individuals without exposure to prior pdmH1N1 infection can recognize pdmH1N1 (24). However, the results in most of these studies were determined by the gamma interferon (IFN-γ) responses to influenza virus peptides. Although the recalled IFN-γ response is commonly used to detect memory CD4 and CD8 T cells, the activated T cells that bind major histocompatibility complex (MHC)-presented peptide are not necessarily capable of lysing the target cells (6). In addition, the peptides, but not the whole virus, may not be able to fully represent the human cross-response against the virus as a whole. Therefore, in addition to cytokine production, the demonstration of direct antigen-specific cytotoxicity of cytotoxic T lymphocytes (CTLs) against both peptide-pulsed and virus-infected target cells is needed for better understanding of human CTL responses against pdmH1N1 virus.In this study, using bulk memory CTLs and epitope-specific CTLs established by seasonal influenza A viruses and epitope-specific peptide from healthy individuals, respectively, we evaluated their cross-cytotoxicity and cytokine responses to pdmH1N1. We also examined the expression of chemokine receptors CXCR3 and CCR5, which could help CTLs to migrate to the site of infection. In addition, to understand whether the seasonal influenza vaccines have benefit for people who have not been exposed to pdmH1N1, we further examined the ability of seasonal influenza vaccines to induce the conserved M1_58-66 epitope-specific CTLs in HLA-A2-seropositive healthy individuals.     

Designing Inhibitors of M2 Proton Channel against H1N1 Swine Influenza Virus

Background

M2 proton channel of H1N1 influenza A virus is the target protein of anti-flu drugs amantadine and rimantadine. However, the two once powerful adamantane-based drugs lost their 90% bioactivity because of mutations of virus in recent twenty years. The NMR structure of the M2 channel protein determined by Schnell and Chou (Nature, 2008, 451, 591–595) may help people to solve the drug-resistant problem and develop more powerful new drugs against H1N1 influenza virus.

Methodology

Docking calculation is performed to build the complex structure between receptor M2 proton channel and ligands, including existing drugs amantadine and rimantadine, and two newly designed inhibitors. The computer-aided drug design methods are used to calculate the binding free energies, with the computational biology techniques to analyze the interactions between M2 proton channel and adamantine-based inhibitors.

Conclusions

1) The NMR structure of M2 proton channel provides a reliable structural basis for rational drug design against influenza virus. 2) The channel gating mechanism and the inhibiting mechanism of M2 proton channel, revealed by the NMR structure of M2 proton channel, provides the new ideas for channel inhibitor design. 3) The newly designed adamantane-based inhibitors based on the modeled structure of H1N1-M2 proton channel have two pharmacophore groups, which act like a “barrel hoop”, holding two adjacent helices of the H1N1-M2 tetramer through the two pharmacophore groups outside the channel. 4) The inhibitors with such binding mechanism may overcome the drug resistance problem of influenza A virus to the adamantane-based drugs.     

H1N1亚型猪流感病毒的拯救

利用8质粒拯救系统成功拯救出了猪流感病毒毒株A/Swine/TianJin/01/2004(H1N1)(A/S/TJ/04)。将猪流感病毒8个基因节段经RT-PCR合成cDNA后, 分别克隆到RNA聚合酶I/II双向表达载体PHW2000中, 构建成8个重组质粒。用8个重组质粒共转染COS-1细胞, 30 h后加入TPCK-胰酶至终浓度0.5 mg/mL。共转染48小时后收获COS-1细胞及其上清, 经尿囊腔接种9日龄SPF鸡胚。收获死亡鸡胚尿囊液并继续用SPF鸡胚传3代, 得到有感染性的病毒。经血凝、血凝抑制验、测序分析、电镜观察等均证实了A/S/TJ/04猪流感病毒的成功拯救。这是目前国内首次报道拯救出H1N1亚型猪流感病毒, 为进一步研究猪流感病毒基因组结构与功能的关系、流感跨种传播的机制以及构建新型猪流感疫苗株奠定了基础。     

H1N1亚型猪流感病毒的拯救

利用8质粒拯救系统成功拯救出了猪流感病毒毒株A/Swine/TianJin/01/2004(H1N1)(A/S/TJ/04)。将猪流感病毒8个基因节段经RT-PCR合成cDNA后, 分别克隆到RNA聚合酶I/II双向表达载体PHW2000中, 构建成8个重组质粒。用8个重组质粒共转染COS-1细胞, 30 h后加入TPCK-胰酶至终浓度0.5 mg/mL。共转染48小时后收获COS-1细胞及其上清, 经尿囊腔接种9日龄SPF鸡胚。收获死亡鸡胚尿囊液并继续用SPF鸡胚传3代, 得到有感染性的病毒。经血凝、血凝抑制验、测序分析、电镜观察等均证实了A/S/TJ/04猪流感病毒的成功拯救。这是目前国内首次报道拯救出H1N1亚型猪流感病毒, 为进一步研究猪流感病毒基因组结构与功能的关系、流感跨种传播的机制以及构建新型猪流感疫苗株奠定了基础。     

2009甲型H1N1流感病毒研究进展

2009年3月在美国和墨西哥爆发的新型甲型H1N1流感在很短的时间内便扩散到世界多个国家,形成了流感的大流行,引起世界卫生组织和各国的高度重视。综述新型甲型H1N1流感病毒的基因组来源、目前主要的检测手段,并对预防和治疗的方法进行简单介绍。     

Differential Activation of NK Cells by Influenza A Pseudotype H5N1 and 1918 and 2009 Pandemic H1N1 Viruses

Natural killer (NK) cells are the effectors of innate immunity and are recruited into the lung 48 h after influenza virus infection. Functional NK cell activation can be triggered by the interaction between viral hemagglutinin (HA) and natural cytotoxicity receptors NKp46 and NKp44 on the cell surface. Recently, novel subtypes of influenza viruses, such as H5N1 and 2009 pandemic H1N1, transmitted directly to the human population, with unusual mortality and morbidity rates. Here, the human NK cell responses to these viruses were studied. Differential activation of heterogeneous NK cells (upregulation of CD69 and CD107a and gamma interferon [IFN-γ] production as well as downregulation of NKp46) was observed following interactions with H5N1, 1918 H1N1, and 2009 H1N1 pseudotyped particles (pps), respectively, and the responses of the CD56^dim subset predominated. Much stronger NK activation was triggered by H5N1 and 1918 H1N1 pps than by 2009 H1N1 pps. The interaction of pps with NK cells and subsequent internalization were mediated by NKp46 partially. The NK cell activation by pps showed a dosage-dependent manner, while an increasing viral HA titer attenuated NK activation phenotypes, cytotoxicity, and IFN-γ production. The various host innate immune responses to different influenza virus subtypes or HA titers may be associated with disease severity.Influenza is a contagious, acute respiratory disease caused by influenza viruses and has caused substantial human morbidity and mortality over the past century (24, 27). The 1918-1919 pandemic caused by influenza virus type A H1N1 was responsible for an estimated 50 million deaths (21). In recent years, novel subtype influenza viruses, such as H5N1 and the 2009 pandemic H1N1, have been transmitted directly from animals to the human population. These infections were characterized by unusually high rates of severe respiratory disease and mortality among young patients (8, 18). Various genetic shifts have occurred in these viruses, allowing them to evade the host protective effects of specific antihemagglutinin (HA) or antineuraminidase (NA) antibodies (27). Therefore, host innate immunity in the early phase of infection, which includes a variety of pattern recognition molecules, inflammatory cytokines, and immune cells, such as macrophages and natural killer (NK) cells, plays a critical role in host defense.NK cells are bone marrow-derived, large, granular lymphocytes and are key effector cells in innate immunity for host defense against invading infectious pathogens and malignant transformation through cytolytic activity and production of cytokines, such as gamma interferon (IFN-γ) (10, 28, 43, 51). In humans, NK cells account for approximately 10% of all blood lymphocytes and are identified by their expression of the CD56 surface antigen and their lack of CD3. Two distinct subsets of human NK cells have been defined according to the cell surface density of CD56 expression (10). The majority (∼90% in blood) of human NK cells are CD56^dim, and a minor population (∼10% in blood) is CD56^bright. These NK subsets are functionally distinct, with the immunoregulatory CD56^bright cells producing abundant cytokines and the cytotoxic CD56^dim cells probably functioning as efficient effectors of natural and antibody-dependent target cell lysis (11).Many lines of evidence suggest that NK cells can be functionally activated by the interaction between natural cytotoxicity receptors (NCRs) on the cell surface and influenza virus HA protein or stress-induced proteins from infected cells (2, 13, 33, 44, 46). On the other hand, influenza virus is able to evade host immunity by infecting NK cells and triggering cell apoptosis or by attenuating NK cell lysis of H3N2-infected cells, owing to alterations in HA binding properties (35, 39). The infiltration of macrophages and lymphocytes into the lung and strong inflammatory responses were detected in H5N1 and the 1918 and 2009 pandemic H1N1 infections. Nevertheless, little is known about the precise roles of NK cells in these infections.In this study, the responses of NK cells to 1918 H1N1, 2009 H1N1, and H5N1 influenza A viruses were evaluated using three strains of influenza A virus pseudotyped particles (pps). Our findings may aid in understanding the pathogenicity of influenza viruses and its correlation with clinical severity.     

流感病毒基质蛋白DNA疫苗的免疫保护作用研究

流感病毒基质蛋白(matrix protein,M)在病毒复制和毒力方面有重要作用.编码基质蛋白的M1基因和M2基因胞外域序列是A型流感病毒的保守序列,是研究具有交叉保护能力流感疫苗的候选基因.我们构建了真核表达质粒pCAGGSP7/M1和pCAGGSP7/M2,用质粒DNA免疫小鼠以观察其免疫原性.分别在M1DNA免疫2、3、4、5、6次或M2 DNA免疫4、5、6次7 d后,用致死量同源流感病毒A/PR/8攻击小鼠,通过检测小鼠血清抗体滴度、肺部病毒量和小鼠存活率来观察质粒DNA的保护效果.结果表明,随着免疫次数增加,M1 DNA免疫组在病毒攻击后小鼠存活率增高,而M2 DNA免疫组小鼠攻毒后全部死亡.说明M1 DNA多次免疫后能提供抗流感病毒的部分保护,M2 DNA没有免疫保护作用.     

New Orf Virus (Parapoxvirus) Recombinant Expressing H5 Hemagglutinin Protects Mice against H5N1 and H1N1 Influenza A Virus

Previously we demonstrated the versatile utility of the Parapoxvirus Orf virus (ORFV) as a vector platform for the development of potent recombinant vaccines. In this study we present the generation of new ORFV recombinants expressing the hemagglutinin (HA) or nucleoprotein (NP) of the highly pathogenic avian influenza virus (HPAIV) H5N1. Correct foreign gene expression was examined in vitro by immunofluorescence, Western blotting and flow cytometry. The protective potential of both recombinants was evaluated in the mouse challenge model. Despite adequate expression of NP, the recombinant D1701-V-NPh5 completely failed to protect mice from lethal challenge. However, the H5 HA-expressing recombinant D1701-V-HAh5n mediated solid protection in a dose-dependent manner. Two intramuscular (i.m.) injections of the HA-expressing recombinant protected all animals from lethal HPAIV infection without loss of body weight. Notably, the immunized mice resisted cross-clade H5N1 and heterologous H1N1 (strain PR8) influenza virus challenge. In vivo antibody-mediated depletion of CD4-positive and/or CD8-posititve T-cell subpopulations during immunization and/or challenge infection implicated the relevance of CD4-positive T-cells for induction of protective immunity by D1701-V-HAh5n, whereas the absence of CD8-positive T-cells did not significantly influence protection. In summary, this study validates the potential of the ORFV vectored vaccines also to combat HPAIV.