Circulating Avian Influenza Viruses Closely Related to the 1918 Virus Have Pandemic Potential

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禽流感病毒与人流感的关系

禽流感病毒 (AIV)是甲 (A)型流感病毒 ,常引起禽类全身性感染或主要限于呼吸器官传染病 ,带来巨大的经济损失并严重威胁人类健康。对AIV的基因组、所编码的蛋白质及其功能、AIV毒力变异的分子基础、禽流感疫苗以及AIV与人流感的关系等进行概述。     

禽流感病毒新型疫苗的研究进展

疫苗免疫是禽流感防控的主要措施之一,随着生物技术的不断发展,基因工程亚单位疫苗、活载体疫苗、DNA疫苗等新型疫苗得以研究和开发,这为禽流感的防控提供了新的手段。新型疫苗除具有传统疫苗的保护效果外,在生物安全和普遍防控等方面也具有广泛的优势,是禽流感疫苗发展的新方向。     

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.     

Protection against a Lethal Avian Influenza A Virus in a Mammalian System

The question of how best to protect the human population against a potential influenza pandemic has been raised by the recent outbreak caused by an avian H5N1 virus in Hong Kong. The likely strategy would be to vaccinate with a less virulent, laboratory-adapted H5N1 strain isolated previously from birds. Little attention has been given, however, to dissecting the consequences of sequential exposure to serologically related influenza A viruses using contemporary immunology techniques. Such experiments with the H5N1 viruses are limited by the potential risk to humans. An extremely virulent H3N8 avian influenza A virus has been used to infect both immunoglobulin-expressing (Ig^+/+) and Ig^−/− mice primed previously with a laboratory-adapted H3N2 virus. The cross-reactive antibody response was very protective, while the recall of CD8⁺ T-cell memory in the Ig^−/− mice provided some small measure of resistance to a low-dose H3N8 challenge. The H3N8 virus also replicated in the respiratory tracts of the H3N2-primed Ig^+/+ mice, generating secondary CD8⁺ and CD4⁺ T-cell responses that may contribute to recovery. The results indicate that the various components of immune memory operate together to provide optimal protection, and they support the idea that related viruses of nonhuman origin can be used as vaccines.     

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.     

禽流感疫苗研究进展

禽流感是由正黏病毒科流感病毒属的A型流感病毒引起的 ,发生于各种家禽和野鸟的一种急性传染病。由于其重要的经济和公共卫生学意义 ,使得禽流感的防治显得突出重要。疫苗的使用是控制禽流感的主要手段。目前实际应用中仍以禽流感全病毒灭活疫苗为主 ,但由于其潜在的缺点使得人们将目光转向其它类型疫苗的研制。从常规疫苗、新型疫苗和交叉保护性疫苗三个方面对禽流感疫苗研究进展加以阐述。常规疫苗包括基因工程亚单位疫苗和重组活载体疫苗 :新型疫苗主要有冷适应流感弱毒疫苗 ,基因工程活流感病毒疫苗 ,复制缺陷型病毒疫苗 ,DNA疫苗 ,RNA复制子疫苗 ,表位疫苗等 :交叉保护性疫苗主要依据流感病毒表面的保守蛋白M和NP的特性 ,构建疫苗来达到交叉保护的目的。     

人呼吸道禽流感病毒受体的分布趋势

禽类流感病毒和人类流感病毒具有很强的受体识别特异性,分别与唾液酸α-2,3Gal和α-2,6Gal受体分子结合而感染各自的宿主细胞．这种受体结合特异性是流感病毒在禽类和人类之间跨种属传递的主要障碍．应用凝集素组织化学染色技术,探讨人呼吸道各解剖学部位流感病毒唾液酸受体的分布特征．结果显示,唾液酸α-2,3Gal受体, 即禽类流感受体,主要分布在下呼吸道的呼吸部即呼吸细支气管和肺泡, 而在主气管、支气管和细支气管仅少量分布．相反,人类流感病毒受体,唾液酸α-2,6Gal受体在气管、支气管呈高密度分布,随着支气管分级逐渐降低分布减少,至肺泡分布最少．但比较人呼吸道发育成熟过程中,唾液酸α-2,3Gal和α-2,6Gal受体的表达,未发现明显差别．禽流感H5N1病毒体外感染人呼吸道组织试验结果表明,肺泡上皮较支气管和气管上皮易感染,与唾液酸α-2,3Gal受体分布特点相符合．结果提示,人呼吸道可被禽流感病毒感染,目前H5N1病毒极少发生人传人的特点,可能与个体间上呼吸道唾液酸α-2,3Gal受体表达差异有关．     

禽流感病毒感染人类及其致病机制研究

禽流感病毒感染禽类可引发呼吸系统到全身不同程度的病变,严重的可导致败血症、休克、多脏器功能衰竭,甚至死亡。上世纪末,禽流感病毒开始跨种向人类传播,其感染引起急性肺损伤、多器官衰竭等,具有较高的致病率和致死率,危害极大,引起了研究者的广泛关注。目前禽流感病毒感染人类及其致病机制尚不明确,本文就此做一综述,为其防治提供参考。     

Live Attenuated Influenza Viruses Containing NS1 Truncations as Vaccine Candidates against H5N1 Highly Pathogenic Avian Influenza

Due to the high mortality associated with recent, widely circulating strains of H5N1 influenza virus in poultry, the recurring introduction of H5N1 viruses from birds to humans, and the difficulties in H5N1 eradication by elimination of affected flocks, an effective vaccine against HPAI (highly pathogenic avian influenza) is highly desirable. Using reverse genetics, a set of experimental live attenuated vaccine strains based on recombinant H5N1 influenza virus A/Viet Nam/1203/04 was generated. Each virus was attenuated through expression of a hemagglutinin protein in which the polybasic cleavage site had been removed. Viruses were generated which possessed a full-length NS1 or a C-terminally truncated NS1 protein of 73, 99, or 126 amino acids. Viruses with each NS genotype were combined with a PB2 polymerase gene which carried either a lysine or a glutamic acid at position 627. We predicted that glutamic acid at position 627 of PB2 would attenuate the virus in mammalian hosts, thus increasing the safety of the vaccine. All recombinant viruses grew to high titers in 10-day-old embryonated chicken eggs but were attenuated in mammalian cell culture. Induction of high levels of beta interferon by all viruses possessing truncations in the NS1 protein was demonstrated by interferon bioassay. The viruses were each found to be highly attenuated in a mouse model. Vaccination with a single dose of any virus conferred complete protection from death upon challenge with a mouse lethal virus expressing H5N1 hemagglutinin and neuraminidase proteins. In a chicken model, vaccination with a single dose of a selected virus encoding the NS1 1-99 protein completely protected chickens from lethal challenge with homologous HPAI virus A/Viet Nam/1203/04 (H5N1) and provided a high level of protection from a heterologous virus, A/egret/Egypt/01/06 (H5N1). Thus, recombinant influenza A/Viet Nam/1203/04 viruses attenuated through the introduction of mutations in the hemagglutinin, NS1, and PB2 coding regions display characteristics desirable for live attenuated vaccines and hold potential as vaccine candidates in poultry as well as in mammalian hosts.     

Effect of AcHERV-GmCSF as an Influenza Virus Vaccine Adjuvant

Introduction

The first identification of swine-originated influenza A/CA/04/2009 (pH1N1) as the cause of an outbreak of human influenza accelerated efforts to develop vaccines to prevent and control influenza viruses. The current norm in many countries is to prepare influenza vaccines using cell-based or egg-based killed vaccines, but it is difficult to elicit a sufficient immune response using this approach. To improve immune responses, researchers have examined the use of cytokines as vaccine adjuvants, and extensively investigated their functions as chemoattractants of immune cells and boosters of vaccine-mediated protection. Here, we evaluated the effect of Granulocyte-macrophage Colony-Stimulating Factor (GmCSF) as an influenza vaccine adjuvant in BALB/c mice.

Method and Results

Female BALB/c mice were immunized with killed vaccine together with a murine GmCSF gene delivered by human endogenous retrovirus (HERV) envelope coated baculovirus (1×10⁷ FFU AcHERV-GmCSF, i.m.) and were compared with mice immunized with the killed vaccine alone. On day 14, immunized mice were challenged with 10 median lethal dose of mouse adapted pH1N1 virus. The vaccination together with GmCSF treatment exerted a strong adjuvant effect on humoral and cellular immune responses. In addition, the vaccinated mice together with GmCSF were fully protected against infection by the lethal influenza pH1N1 virus.

Conclusion

Thus, these results indicate that AcHERV-GmCSF is an effective molecular adjuvant that augments immune responses against influenza virus.     

Generation of High-yield Vaccine Strain Wholly Derived from Avian Influenza Viruses by Reverse Genetics

Highly pathogenic avian influenza A (HPAI) viruses of the H5N1 subtypes caused enormous economical loss to poultry farms in China and Southeastern Asian countries. The vaccination program is a reliable strategy in controlling the prevalence of these disastrous diseases. The six internal genes of the high-yield influenza virus A/Goose/Dalian/3/01 (H9N2), the haemagglutinin (HA) gene of A/Goose/HLJ/QFY/04 (H5N1) strain, and the neuraminidase gene from A/Duck/Germany/1215/73 (H2N3) reference strain were amplified by RT-PCR technique. The HA gene was modified by the deletion of four basic amino acids of the connecting peptide between HA1 and HA2. Eight gene expressing plasmids were constructed, and the recombinant virus rH5N3 were generated by cell transfection. The infection of chicken embryos and the challenge tests involving chickens demonstrated that the recombinant H5N3 (rH5N3) influenza virus is avirulent. The allantoic fluids of rH5N3-infected eggs contain high-titer influenza viruses with haemagglutination unit of 1:2 048, which are eight times those of the parental H5N1 virus. The rH5N3 oil-emulsified vaccine could induce haemagglutination inhibition (HI) antibodies in chickens in 2 weeks post-vaccination, and the maximum geometric mean HI-titers were observed 4–5 weeks post-vaccination and were kept under observation for 18 weeks. The rH5N3-vaccinated chickens were fully protected against morbidity and mortality of the lethal challenge of the H5N1 HPAI viruses, A/Goose/Guangdong/1/96 and A/Goose/HLJ/QFY/04, which had 8 years expansion and differences among multiple amino acids in HA protein. The N3 neuraminidase protein marker makes it possible to distinguish between H5N1-infected and H5N3-vaccinated animals.     

利用反向遗传学技术构建H5亚型禽流感高产疫苗株

采用RT-PCR技术分别扩增了鹅源高产禽流感病毒的6条内部基因片段,近期分离的H5N1亚型禽流感病毒的血凝素基因以及N3亚型参考毒株的神经氨酸酶基因,分别构建了8个基因的转录与表达载体,利用反向遗传学技术拯救出了全部基因都源于禽源的重组流感病毒疫苗株rH5N3。通过对血凝素蛋白HA1和HA2连接肽处的5个碱性氨基酸(R-R-R-K-K)基因缺失与修饰,从而消除了病毒基因的毒力相关序列,拯救的rH5N3疫苗株对鸡和鸡胚均无致病性,病毒在鸡胚尿囊液和细胞培养上清的HA效价得到极大提高,分别为12048和1512。制备的禽流感疫苗免疫动物后4~5周即可诱导产生高效价的HI抗体,鸡免疫后18周依然保持高水平的HI抗体。重组疫苗不论是对于国内早期分离的禽流感病毒A/Goose/Guangdong/1/96还是近期分离的A/Goose/HLJ/QFY/04都能够产生完全的免疫保护作用,免疫鸡攻毒后不发病、不排毒、不死亡。带有N3鉴别诊断标记禽流感疫苗株的研制为H5N1高致病性禽流感的防治提供了新的技术保障。