Stability of neuraminidase in inactivated influenza vaccines

Influenza vaccines are effective in protecting against illness and death caused by this seasonal pathogen. Antibodies that block the function of either hemagglutinin (HA) or neuraminidase (NA) contribute to vaccine efficacy, however vaccine potency is based only on HA content. NA protein content in vaccines varies from season to season due to differences in the relative amounts of HA and NA in influenza A, H1N1 and H3N2, and influenza B viruses that are selected for each manufacturing campaign. This, as well as potential inherent differences in NA immunogenicity, may result in varying responses from year to year. Moreover, the antigenic stability of NA is likely to dictate whether similar antibody responses will be obtained to this antigen throughout the shelf-life of the vaccine. To address this factor, we subjected NAs of influenza A (subtypes N1 and N2) and B viruses to denaturing conditions to evaluate the stability of enzyme activity. Each NA type/subtype had unique sensitivity to denaturing conditions. The N2 enzyme activity was more thermostable than that of N1 or influenza B, while the NA activity of influenza B was most resistant to detergent. N1 enzyme activity was most resistant of the three NAs to freeze–thaw cycling. In these experiments, enzyme activity was indicative of the immunogenicity of NA, but was strain-dependent, with greater neuraminidase inhibiting (NI) antibody titers elicited following immunization with the 2009 H1N1 pandemic virus A/California/7/2009, than the previously circulating seasonal H1N1 strain, A/Brisbane/59/2007. Robust NI antibody titers against both N1 and N2 components were induced following vaccination of mice with a trivalent inactivated influenza vaccine. When stored under recommended conditions, the NA of both N1 and N2 subtypes remained immunogenic well after the vaccine expiry date.     

Immunization with influenza A virus hemagglutinin and neuraminidase produced in recombinant baculovirus results in a balanced and broadened immune response superior to conventional vaccine

Influenza A virus hemagglutinin (HA) and neuraminidase (NA) from A/Nanchang/933/95 were expressed by recombinant baculovirus-infected insect cell lines. HA and NA were chromatographically purified then combined in a single vaccine preparation. Immunization of mice with this preparation resulted in high titers of antibodies to both HA and NA equivalent for each antigen to titers in animals immunized with either antigen alone. Anti-NA antibody titers, measured by either enzyme linked immunoabsorbant assay or neuraminidase inhibition test were higher in the combined recombinant vaccine than in conventional monovalent inactivated vaccine. There was no difference in the anti-HA antibody titers between these two vaccine preparations. Homotypic and closely related heterotypic infections were suppressed and greater reduction in viral replication was observed following a distantly related heterotypic infectious challenge than was observed with conventional inactivated vaccine. The combined HA and NA vaccine takes advantage of the equivalent immunogenicity of dissociated HA and NA, to produce a broader and more balanced immune response to both antigens, without the HA-dominant antigenic competition that occurs with natural infection or immunization with conventional vaccine. Additionally, the recombinant baculovirus expression system offers a reliable rapid production system without the use of massive numbers of embryonated chicken eggs. These studies in a mouse model system suggest that production of a combined HA and NA vaccine from recombinant baculovirus offers an improved alternative to conventional inactivated influenza vaccine.     

Generation and evaluation of the trivalent inactivated reassortant vaccine using human, avian, and swine influenza A viruses

Reassortant technology was used to obtain three interspecific reassortant influenza viruses using three influenza viruses of A/Puerto Rico/8/34(H1N1), A/swine/Hebei/1/2005(H3N2) and A/chicken/Guangdong/126/2002(H9N2). The high-growth reassortant strains were H9/PR8, H3/H9N2 and H1/H9N2 that contained hemagglutinin (HA) and neuraminidase (NA) genes from the inactivated parental viruses and the other 6 internal genes from the live parental viruses. The trivalent formalin-inactivated vaccine, containing H1, H3 and H9 subtype antigens from human, swine and avian influenza viruses respectively, was prepared using these reassortant viruses. Animal studies showed that the vaccine was safe and immunogenic. Two-dosing regimen of the influenza vaccine induced high titers of hemagglutination inhibiting (HI) antibodies and influenza-specific IgG antibodies without antigenic cross-interference. It protected 100% chickens from challenge of A/chicken/Guangdong/126/2002 virus and protected 100% mice against challenges with different combinations of the three infective parental viruses. These results indicated that the trivalent vaccine could offer multi-protection against multi-influenza viruses synchronously. This kind of multivalent inactivated reassortant influenza vaccine maybe enlightens the pandemic influenza preparedness as the emergency measure.     

Generation of an attenuated H5N1 avian influenza virus vaccine with all eight genes from avian viruses

In the face of disease outbreaks in poultry and the potential pandemic threat to humans caused by the highly pathogenic avian influenza viruses (HPAIVs) of H5N1 subtype, improvement in biosecurity and the use of inactivated vaccines are two main options for the control of this disease. Vaccine candidates of influenza A viruses of H5N1 subtype have been generated in several laboratories by plasmid-based reverse genetics with hemagglutinin (HA) and neuraminidase (NA) genes from the epidemic strains of avian viruses in a background of internal genes from the vaccine donor strain of human strains, A/Puerto Rico/8/34 (PR8). These reassortant viruses containing genes from both avian and human viruses might impose biosafety concerns, also may be do if C4/F AIV would be a live attenuated vaccine or cold-adaptive strain vaccine. In order to generate better and safer vaccine candidate viruses, we genetically constructed attenuated reassortant H5N1 influenza A virus, designated as C4/F AIV, by plasmid-based reverse genetics with all eight genes from the avian strains. The C4/F AIV virus contained HA and NA genes from an epidemic strain A/Chicken/Huadong/04 (H5N1) (C4/H5N1) in a background of internal genes derived from a low pathogenic strain of A/Chicken/F/98(H9N2). The reassortant virus was attenuated by removal of the multibasic amino acid motif in the HA gene by mutation and deletion (from PQRERRRKKR (downward arrow) G to PQIETR (downward arrow) G). The intravenous pathogenicity index (IVPI) of C4/F AIV virus was 0, whereas that of the donor virus C4/H5N1 was 3.0. The virus HA titer of C4/H5N1 in the allantoic fluid from infected embryonated eggs was as high as 1:2048. The inactivated vaccine prepared from the reassortant virus C4/F AIV-induced high HI titer in vaccinated chickens and gave 100% protection when challenged with highly pathogenic avian influenza virus of H5N1 subtype.     

A high dosage influenza vaccine induced significantly more neuraminidase antibody than standard vaccine among elderly subjects

Antibody to the neuraminidase (NA) antigen of influenza viruses has been shown to correlate with immunity to influenza in humans and animal models. In a previous report, we showed that an inactivated influenza vaccine containing 60 μg of the hemagglutinin (HA) of each strain induced significantly more serum anti-HA antibody among elderly persons than did the standard vaccine containing 15 μg of the HA of each component. We developed a lectin-based assay for anti-NA antibody and used it to measure anti-NA antibody responses among subjects who had participated in that study. The high dosage vaccine contained eight times as much NA activity as the standard vaccine and induced a significantly higher frequency of antibody responses and higher mean postvaccination anti-NA titers to the N1 and N2 of the A/H1N1 and A/H3N2 viruses in the vaccines than did the standard vaccine. Ensuring an increased antibody response to the NA antigen in inactivated influenza virus vaccines should increase the protection against influenza. An increased quantity of the NA antigen in the vaccine will ensure an increased response.     

Randomized comparative study of the serum antihemagglutinin and antineuraminidase antibody responses to six licensed trivalent influenza vaccines

Background

Serum antibody to the hemagglutinin (HA) surface protein of influenza virus induced by influenza vaccination is a correlate of protection against influenza. The neuraminidase (NA) protein is also on the surface of the virus; antibody to it has been shown to impair virus release from infected cells and to reduce the intensity of influenza infections in animal models and in humans challenged with infectious virus. Recently we have shown that NA inhibiting antibody can independently contribute to immunity to naturally-occurring influenza immunity in the presence of antibody to the HA.

Purpose

The present study was conducted to evaluate induction of antibody to the NA and the HA by commercially available influenza vaccines.

Methods

Healthy young adults were vaccinated with one of five commercially available trivalent inactivated vaccines or live influenza vaccine. Frequencies of serum antibody and fold geometric mean titer (GMT) increases four weeks later were measured to each of the three vaccine viruses (A/H1N1, A/H3N2, B) in hemagglutination-inhibition (HAI) and neutralization (neut) assays. Frequency and fold GMT increase in neuraminidase-inhibition (NI) antibody titers were measured to the influenza A viruses (A/H1N1, A/H3N2).

Results

No significant reactogenicity occurred among the vaccinated subjects. The Fluvirin inactivated vaccine induced more anti-HA antibody responses and a higher fold GMT increase than the other inactivated vaccines but there were no major differences in response frequencies or fold GMT increase among the inactivated vaccines. Both the frequency of antibody increase and fold GMT increase were significantly lower for live vaccine than for any inactivated vaccine in HAI and neut assays for all three vaccine viruses. Afluria inactivated vaccine induced more N1 antibody and Fluarix induced more N2 antibody than the other vaccines but all inactivated vaccines induced serum NI antibody. The live vaccine failed to elicit any NI responses for the N2 NA of A/H3N2 virus and frequencies were low for the N1 of A/H1N1 virus.

Conclusions

Trivalent inactivated influenza vaccines with similar HA dosage induce similar serum anti-HA antibody responses in healthy adults. Current inactivated vaccines all induce serum anti-NA antibody to the N1 and N2 NA proteins but some are better than others for N1 or N2. The live vaccine, Flumist, was a poor inducer of either anti-HA or anti-NA serum antibody compared to inactivated vaccine in the healthy adults. In view of the capacity for contributing to immunity to influenza in humans, developing guidelines for NA content and induction of NA antibody is desirable.     

Influenza virus-like particles comprised of the HA, NA, and M1 proteins of H9N2 influenza virus induce protective immune responses in BALB/c mice

Avian influenza viruses represent a growing threat for an influenza pandemic. To develop recombinant vaccine for avian influenza of the H9N2 subtype, we expressed in insect cells virus-like particles (VLPs) consisting of three structural proteins of influenza A/Hong Kong/1073/99 (H9N2) virus. Upon infection of Sf9 cells with recombinant baculoviruses, the hemagglutinin (HA), neuraminidase (NA), and matrix (M1) proteins were co-expressed in the infected cells, self-assembled, and released into the culture medium as VLPs of 80–120 nm in diameter. VLPs exhibited functional characteristics of influenza virus including hemagglutination and neuraminidase activities. In BALB/c mice, VLPs elicited serum antibodies specific for influenza A/Hong Kong/1073/99 (H9N2) virus and inhibited replication of the influenza virus after challenge. Thus, VLPs represent a potential strategy for the development of human vaccines against avian influenza H9N2 viruses.     

Recombinant H1N1 virus-like particle vaccine elicits protective immunity in ferrets against the 2009 pandemic H1N1 influenza virus

The pandemic virus of 2009 (2009 H1N1) continues to cause illness worldwide, especially in younger age groups. The widespread H1N1 virus infection further emphasizes the need for vaccine strategies that are effective against emerging pandemic viruses and are not dependent on the limitations of traditional egg-based technology. This report describes a recombinant influenza virus-like particle (VLP) vaccine consisting of hemagglutinin (HA), neuraminidase (NA), and matrix (M1) proteins of influenza A/California/04/2009 (H1N1) virus. Influenza H1N1 VLPs with a diameter of approximately 120 nm were released into the culture medium from Sf9 insect cells infected with recombinant baculovirus coexpressing HA, NA, and M1 proteins. Purified recombinant H1N1 VLPs morphologically resembled influenza virions and exhibited biological characteristics of influenza virus, including HA and NA activities. In the ferret challenge model, 2009 influenza H1N1 VLPs elicited high-titer serum hemagglutination inhibition (HI) antibodies specific for the 2009 H1N1 virus and inhibited replication of the influenza virus in the upper and lower respiratory tract tissues following A/Mexico/4482/09 (H1N1) virus challenge. Moreover, a single 15 μg dose of H1N1 VLPs resulted in complete virus clearance in the ferret lung. These results provide support for the use of recombinant influenza VLP vaccine as an effective strategy against pandemic H1N1 virus.     

VaxArray for hemagglutinin and neuraminidase potency testing of influenza vaccines

Practical methods to measure the potency of influenza vaccines are needed as alternatives for the standard single radial immunodiffusion (SRID) assay. VaxArray assays for influenza hemagglutinin (HA) and neuraminidase (NA) have been developed to address this need. In this report, we evaluate the use of these assays to assess the potency of HA and NA of an A/H3N2 subunit vaccine by determining the correlation between the amounts measured by VaxArray and the immunogenicity in mice. The antibody response after one and two doses of five formulations of the vaccine ranging from 5 µg/mL to 80 µg/mL of HA, was measured by hemagglutination inhibition (HAI) and neuraminidase inhibition (NAI) assays. For hemagglutinin, vaccine potency determined by VaxArray was equivalent to potency measured SRID and these amounts were predictive of immunogenicity, with excellent correlation between potency measured by VaxArray and the HAI geometric mean titers (GMT). Likewise, the amount of NA measured by VaxArray was predictive of the NAI GMT. The VaxArray NA assay reported non-detectable levels of intact NA for a sample that had been heat degraded at 56 °C for 20 h, demonstrating that the assay measures the native, active form of NA. Similarly, the HA potency measured by VaxArray in this heat-treated sample was very low when a monoclonal antibody was used to detect the amount of antigen bound. Importantly, the force degraded sample induced low HAI titers and the NAI titers were not measurable, supporting the conclusion that the VaxArray HA and NA assays measure the immunogenic forms of these A/H3N2 antigens. This study indicates that VaxArray assays can be used to assess the potency of HA and NA components in influenza vaccines as a proxy for immunogenicity.     

H5N1 VLP vaccine induced protection in ferrets against lethal challenge with highly pathogenic H5N1 influenza viruses

In this study, recombinant virus-like particles (VLPs) were evaluated as a candidate vaccine against emerging influenza viruses with pandemic potential. The VLPs are composed of the hemagglutinin (HA), neuraminidase (NA), and matrix 1 (M1) proteins of the H5N1 A/Indonesia/05/2005 (clade 2.1; [Indo/05]) virus, which were expressed using baculovirus in Spodoptera frugiperda (Sf9) cells. Ferrets received either 2 injections of the VLP vaccine at escalating doses (based on HA content), recombinant HA, or were mock vaccinated. Vaccinated ferrets were then challenged with either H5N1 Indo/05 or H5N1 A/Viet Nam 1203/2004 (VN/04) wild-type viruses. All ferrets that received the VLP vaccine survived regardless of the VLP dose or challenge strain, whereas seven of eight mock vaccinated ferrets died. The VLP vaccine induced HAI antibodies against the homologous H5N1 clade 2.1 strain, as well as heterologous strains from H5N1 clades 1, 2.2, and 2.3. The magnitude of the HAI titers correlated with VLP dose. Neutralizing antibody responses against the Indo/05 and VN/04 strains showed a similar pattern. Affinity of the anti-HA antibodies raised by the H5N1 Indo/05 VLPs had a higher association rate to the homologous clade 2.1 HA than to the clade 1 (VN/04) HA; however, once bound, antibodies had similar slow disassociation rates. These results provide support for continued development of the H5N1 VLPs as a candidate vaccine against pandemic influenza. Exploration of immunologic correlates of protection for H5N1 vaccines beyond HAI and neutralizing antibody responses is warranted.     

The rapid identification of human influenza neuraminidase N1 and N2 subtypes by ELISA

An ELISA assay was developed to allow the rapid and accurate identification of human influenza A N1 and N2 neuraminidases. Initial testing using a fetuin pre-coating of wells correctly identified 81.7% of the neuraminidase type from a series of human A(H1N1), A(H1N2) and A(H3N2) viruses. This result could be improved to detect the neuraminidase subtype of almost all human influenza A viruses from a large panel of viruses isolated from 2000 to 2005, if the fetuin pre-coating was removed and the viruses were coated directly onto wells. This method is simple, rapid and can be used to screen large numbers of currently circulating human influenza A viruses for their neurraminidase subtype and is a good alternative to RT-PCR.     

Generation of reassortant influenza vaccines by reverse genetics that allows utilization of a DIVA (Differentiating Infected from Vaccinated Animals) strategy for the control of avian influenza

Vaccination of poultry with inactivated influenza vaccine can be an effective tool in the control of avian influenza (AI). One major concern of using inactivated vaccine is vaccine-induced antibody interference with serologic surveillance and epidemiology. In the United States, low pathogenicity H5 and H7 subtype AI viruses have caused serious economic losses in the poultry industry. Most of these viruses also have the accompanying N2 subtype and no H5N1 or H7N8 subtype AI viruses have been identified in poultry in the US. In order to allow the Differentiation of Infected from Vaccinated Animals (DIVA) while maintaining maximum efficacy of the vaccine, we generated reassortant viruses by reverse genetics that contained the same H5 and H7 hemagglutinin (HA) gene as the challenge virus, but a heterologous N1 or N8 neuraminidase (NA) gene. In vaccination-challenge experiments in 2-week-old specific pathogen free chickens, reassortant influenza vaccines (rH5N1 and rH7N8) demonstrated similar antibody profiles and comparable protection rates as vaccines prepared with parent H5N2 and H7N2 viruses. Further, we were able to differentiate the sera from infected and vaccinated birds by neuraminidase inhibition test and indirect immunofluorescent antibody assay on the basis of different antibodies elicited by their NA proteins. These results demonstrate the usefulness of a reverse genetics system for the rapid generation of reassortant AI virus that allows utilization of the DIVA strategy for the control of AI infections in poultry.     

2017年深圳地区A/H3N2亚型流感病毒HA和NA基因的分子进化特征

  目的  了解深圳市2017年上半年流行的甲型H3N2亚型流感病毒HA和NA基因的分子进化特征,为预测流感病毒流行和变异提供科学依据。  方法  利用DNAStar、MEGA 7.0等生物信息学软件对研究分离的40株H3N2流感病毒的HA和NA基因及其编码的氨基酸序列进行比对,构建分子进化树,分析基因特征和变异情况。  结果  深圳分离株的同源性均达到97.8%~100.0%,位于亚洲北美人源分支。与世界卫生组织（World Health Organization,WHO）推荐的疫苗株A/Switzerland/9715293/2013（H3N2）和A/Hong Kong/4801/2014（H3N2）相比,有较高的序列相似性;与当年疫苗株对比发现,HA发生6个抗原位点和2个受体结合位点的改变,NA出现第151位酶活性位点的改变;HA和NA的潜在N-糖基化位点在数量和位置上也发生变化。  结论  深圳市2017年上半年甲型H3N2亚型流感病毒在流行过程中尚未发生明显变异,目前推荐的疫苗株仍对深圳地区人群有一定保护作用,HA和NA多个氨基酸位点的变异提示仍需加强H3N2亚型流感病毒分子水平的动态监测。     

2015-2017年盐城市甲型H3N2流感病毒HA1、NA基因分子特征

目的 从分子流行病学角度对盐城市2015-2017年流行的甲型H3N2流感病毒血凝素（hemagglutinin,HA）和神经氨酸酶（neuraminidase,NA）基因的分子特征进行研究。方法 收集盐城市2015-2017年流感监测哨点医院以及流感爆发点采集的3 891例流感样病例标本进行核酸检测和病毒分离,从中筛选出20株经血凝抑制试验（hemagglutination inhibition,HI）验证为甲型H3N2亚型的毒株,采用反转录聚合酶链式反应方法扩增其HA1和NA基因并进行测序。结果 2015-2017年流行的20株甲型H3N2毒株HA1和NA基因各自分支的聚类关系基本一致。盐城市A/Jiangsu-YC/1474/2015、A/Jiangsu-YC/1725/2015两株分离株与疫苗株A/Switzerland/9715293/2013和A/Hongkong/4801/2014的进化距离较近,而其余18株分离株与国内部分省市相近年份毒株亲缘关系相近,并与疫苗株A/Switzerland/9715293/2013和A/Hongkong/4801/2014进化距离较远。该20株毒株HA1基因编码区抗原表位、受体结合位点和糖基化位点均发生了一定程度变异。从基因变异角度进行分析,疫苗株A/Switzerland/9715293/2013对盐城地区流感流行的保护效果要弱于疫苗株A/Hongkong/4801/2014。结论 2015-2017年盐城市流行的甲型H3N2毒株HA1和NA基因已逐渐发生变异,可能导致流感病毒发生实质性的抗原性漂移,降低与流感疫苗株的匹配度,减弱流感疫苗的保护作用。     

Immunoinformatic comparison of T-cell epitopes contained in novel swine-origin influenza A (H1N1) virus with epitopes in 2008–2009 conventional influenza vaccine

In March 2009 a novel swine-origin influenza A (H1N1) virus (S-OIV) emerged in Mexico and the Western United States. Vaccination with conventional influenza vaccine (CIV) does not result in cross-reactive antibodies, however, the disproportionate number of cases (37%) occurring among persons younger than 50 years old suggested that adaptive immune memory might be responsible for the relative lack of virulence in older, healthy adults. Using EpiMatrix, a T-cell epitope prediction and comparison tool, we compared the sequences of the three hemagglutinin (HA) and neuraminidase (NA) proteins contained in 2008–2009 CIV to their counterparts in A/California/04/2009 (H1N1) looking for cross-conserved T-cell epitope sequences. We found greater than 50% conservation of T helper and CTL epitopes between novel S-OIV and CIV HA for selected HLA. Conservation was lower among NA epitopes. Sixteen promiscuous helper T-cell epitopes are contained in the S-OIV H1N1 HA sequence, of which nine (56%) were 100% conserved in the 2008–2009 influenza vaccine strain; 81% were either identical or had one conservative amino acid substitution. Fifty percent of predicted CTL epitopes found in S-OIV H1N1 HA were also found in CIV HA sequences. Based on historical performance, we expect these epitope predictions to be 93–99% accurate. This in silico analysis supports the proposition that T-cell response to cross-reactive T-cell epitopes, due to vaccination or exposure, may have the capacity to attenuate the course of S-OIV H1N1 induced disease—in the absence of cross-reactive antibody response. The value of the CIV or live-attenuated influenza vaccine containing the 2008–2009 vaccine strains, as defense against H1N1, could be further tested by evaluating human immune responses to the conserved T-cell epitopes using PBMC from individuals infected with H1N1 and from CIV vaccinees.     

AF03-adjuvanted and non-adjuvanted pandemic influenza A (H1N1) 2009 vaccines induce strong antibody responses in seasonal influenza vaccine-primed and unprimed mice

Pandemic influenza vaccines have been manufactured using the A/California/07/2009 (H1N1) strain as recommended by the World Health Organization. We evaluated in mice the immunogenicity of pandemic (H1N1) 2009 vaccine and the impact of prior vaccination against seasonal trivalent influenza vaccines (TIV) on antibody responses against pandemic (H1N1) 2009. In naïve mice, a single dose of unadjuvanted H1N1 vaccine (3 μg of HA) was shown to elicit hemagglutination inhibition (HI) antibody titers >40, a titer associated with protection in humans against seasonal influenza. A second vaccine dose of pandemic (H1N1) 2009 vaccine strongly increased these titers, which were consistently higher in mice previously primed with TIV than in naïve mice. At a low immunization dose (0.3 μg of HA), the AF03-adjuvanted vaccine elicited higher HI antibody titers than the corresponding unadjuvanted vaccines in both naïve and TIV-primed animals, suggesting a potential for antigen dose-sparing. These results are in accordance with the use in humans of a split-virion inactivated pandemic (H1N1) 2009 vaccine formulated with or without AF03 adjuvant to protect children and young adults against influenza A (H1N1) 2009 infection.     

2010-2012年长沙市流感流行情况及B型流感病毒基因特性分析

目的分析2010-2012年长沙市流感流行情况,并探讨B型流感病毒分离株血凝素(HA)和神经氨酸酶(NA)基因的分子流行病学特征。方法采集长沙市流感网络监测哨点医院及暴发点流感样病例(ILI)咽拭子样本,狗肾传代细胞(MDCK)进行病毒分离,血凝和血凝抑制实验进行型别和亚型鉴定;提取B型流感病毒核酸,一步法RT-PCR扩增HA和NA基因片段,双向测定扩增产物核苷酸序列,对基因序列和氨基酸序列进行分析。结果 2010-2012年间,长沙市甲型H1N1、H3N2、B型流感交替流行,流行高峰为冬春季。3年间共分离到B型流感病毒78株,其中79.5%为Victoria系,20.5%为Yamagata系,以Victoria系为主。与WHO推荐疫苗株B/Brisbane/60/2008相比,HA基因的同源性在87.2%~98.4%之间,NA基因的同源性在94.5%~97.5%之间。氨基酸序列分析发现,与疫苗株相比,HA蛋白主要存在单个氨基酸的突变;种系进化分析发现,所有毒株HA和NA蛋白位于相应的谱系内,无抗原重排发生。结论 2010-2012年间长沙市甲型H1N1、H3N2、B型流感交替流行,B型流感病毒HA基因出现抗原漂移,但无重组毒株的出现。     

Increased hemagglutinin content in a reassortant 2009 pandemic H1N1 influenza virus with chimeric neuraminidase containing donor A/Puerto Rico/8/34 virus transmembrane and stalk domains

The glycoproteins, heamagglutinin (HA) and neuraminidase (NA) of influenza virus confer host protective immune responses during vaccination, which is the most effective approach for preventing influenza-associated morbidity and mortality. Since the functional balance between the HA and NA proteins may affect viral receptor binding and replication, a pandemic influenza A virus (H1N1 pdm09), strain A/Texas/05/2009, was optimized to elevate its HA antigen content by modifying the NA gene. In this study, we have constructed two 2:6 reassortant viruses between pdmH1N1 (A/Texas/05/2009) and A/Puerto Rico/8/34 (PR8), in which the NA gene of A/Texas/05/2009 was modified to contain part of the NA gene from PR8. One chimeric NA virus has the PR8 transmembrane (TM) region (HNtm 2:6) and the other contains both the PR8 NA TM and stem regions (HNst 2:6). Using quantitative reverse phase-HPLC (RP-HPLC) analysis, we observed that the HNst2:6 virus contains a higher HA1 content than HN2:6 wild type. In addition, this mutant virus displays a higher HA1 to nucleoprotein (NP) ratio, based on gel electrophoresis densitometry analysis. Furthermore, the neuraminidase activity of purified HNst 2:6 virus is approximately 30% lower than that of HN2:6 virus, which is suggestive of a lower incorporation of NA into the viral envelope. Therefore, we propose that the reduction of NA packaging in the virion may lead to a compensatory increase of HA. Such an improvement in HA yield is possibly beneficial to H1N1 pdm09 vaccine production.     

Analysis of the immunogenicity and bioactivities of a split influenza A/H7N9 vaccine mixed with MF59 adjuvant in BALB/c mice

The H7N9 influenza virus caused significant mortality and morbidity in humans during an outbreak in China in 2013. A recombinant H7N9 influenza seed with hemagglutinin (HA) and neuraminidase (NA) gene segments from A/Zhejiang/DTID-ZJU01/2013(H7N9) and six internal protein gene segments from A/Puerto Rico/8/34(H1N1; PR8) were generated using reverse genetics. We sought to determine the immunogenic, protective properties, and mechanisms of a split avian influenza A/H7N9 vaccine mixed with MF59 adjuvant in comparison to vaccines that included other adjuvant. BALB/c mice were vaccinated with two doses of different amounts and combinations of this novel A/ZJU01/PR8/2013 split vaccine with adjuvant. Mice were subsequently challenged with A/Zhejiang/DTID-ZJU01/2013(H7N9) by intranasal inoculation. We verified that MF59 enhanced the HI, MN, and IgG antibody titers to influenza antigens. Compared with alum, MF59 could more potentially induce humoral immune responses and Th2 cytokine production after virus infection, while both MF59 and alum can slightly increase NK cell activity. This split H7N9 influenza vaccine with MF59 adjuvant could effectively induce antibody production and protect mice from H7N9 virus challenge. We have selected this vaccine for manufacture and future clinical studies to protect humans from H7N9 virus infection.     

2017 - 2018年度陕西省甲型H1N1流感病毒基因特征分析

目的 了解陕西省2017 - 2018年度甲型H1N1流感病毒的血凝素（HA）和神经氨酸酶（NA）基因变异情况。方法 选取2017 - 2018年度分离的6株甲型H1N1流感毒株进行全基因测序,利用生物信息学软件对分离株的HA和NA基因进行进化和变异分析,并与北半球疫苗株A/Michigan/45/2015进行对比;采用MEGA（5.05） 软件NJ法构建基因进化树,进行系统进化分析。结果 陕西省2017 - 2018年度甲型H1N1流感病毒阳性占比36.85%,流行高峰为2017年12月和2018年1月。6株甲型H1N1流感病毒测序得到的HA、NA序列与疫苗株比对,分别有12、11个氨基酸位点发生变异,其中HA有7个位点变异发生在抗原决定簇,受体结合位点、潜在糖基化位点比较稳定;NA序列没有发现耐药位点变异。结论 2017 - 2018年度陕西省甲型H1N1流感病毒为优势流行株。目前甲型H1N1流感病毒与WHO推荐的新疫苗株高度同源。