Protective effect of nasal immunization of influenza virus hemagglutinin with recombinant cholera toxin B subunit as a mucosal adjuvant in mice

To develop an efficient nasal influenza vaccine, influenza A and B virus HA with rCTB as a mucosal adjuvant were administered to mice intranasally. Serum anti-HA IgG and IgA antibody responses for both HA vaccines were significantly increased in the presence of rCTB. Higher HI and neutralizing antibody titers and higher mucosal IgA antibody responses in the respiratory tract were detected when rCTB was added than without rCTB. When mice were immunized with HA vaccine with or without rCTB and challenged by intranasal administration of mouse-adapted pathogenic influenza A virus, all mice immunized with HA plus rCTB survived for seven days without any inflammatory changes in the lungs, while not all the mice immunized with HA without rCTB survived, and all of them had lung consolidations. These results demonstrate that intranasal co-administration of rCTB as a mucosal adjuvant with influenza virus HA is necessary not only for the induction of systemic and mucosal HA antibodies, but also for the protection of mice from morbidity and mortality resulting from virus infection.     

Immunologic response to the influenza virus neuraminidase is influenced by prior experience with the associated viral hemagglutinin. I. Studies in human vaccinees

Analysis of an earlier study of H3N2 and H7N2 inactivated influenza vaccines in schoolchildren demonstrated a greater viral neuraminidase (NA) immunogenicity of the vaccine containing the H7 hemagglutinin (HA) antigen to which they had not been primed, despite the lesser NA antigen content of that vaccine. Thus, prior experience with the influenza viral HA appeared to have a negative influence on immune response to NA, the associated external glycoprotein, presumably on the basis of intermolecular antigenic competition. In a second study, sequential immunologic response to influenza viral NA was compared in college students who were immunized with either conventional commercial vaccine or an antigenic reassortant H7N1 vaccine, and who subsequently experienced natural infection with an H1N1 influenza virus. Although both vaccines were only marginally immunogenic in inducing NA antibody response in seronegative subjects, in vaccinees initially seropositive for HA antibody significant NA antibody titer increases occurred with H7N1 vaccine. Subsequent natural infection boosted NA antibody less effectively in the population previously primed by natural infection than in initially seronegative subjects primed by H7N1 vaccination. It is suggested that primary immunization monospecific for influenza viral NA may alter the subsequent pattern of immune response to one more favorable to the induction of NA antibody when virus is encountered.     

广州2006年乙型流感病毒血凝素和神经氨酸酶基因特性分析

为了解2006年广州地区流行的乙型流感病毒株血凝素(HA)和神经氨酸酶(NA)的基因特性,选择病原学监测病毒株和暴发性疫情病毒株,提取病毒RNA并逆转录为cDNA,通过PCR方法扩增乙型流感病毒HA和NA全长基因,将扩增的DNA片段接入T-A克隆载体进行测序,并使用DNAStar软件对测序结果进行分析。结果显示:不同来源的流感病毒株HA的同源性为99%以上,都属于Victoria系;不同来源的病毒株NA同源性为98%以上。HA和NA的种系发生树分析表明:病原学监测毒株同源性更接近,而暴发性疫情毒株的同源性则相对较为分散。所有毒株与WHO推荐的2005~2006年度疫苗株B/Shanghai/361/2002的同源性只有88.9%~89.7%,说明该年度的流感疫苗对乙型流感不能提供最佳的保护。     

Balancing the influenza neuraminidase and hemagglutinin responses by exchanging the vaccine virus backbone

Virions are a common antigen source for many viral vaccines. One limitation to using virions is that the antigen abundance is determined by the content of each protein in the virus. This caveat especially applies to viral-based influenza vaccines where the low abundance of the neuraminidase (NA) surface antigen remains a bottleneck for improving the NA antibody response. Our systematic analysis using recent H1N1 vaccine antigens demonstrates that the NA to hemagglutinin (HA) ratio in virions can be improved by exchanging the viral backbone internal genes, especially the segment encoding the polymerase PB1 subunit. The purified inactivated virions with higher NA content show a more spherical morphology, a shift in the balance between the HA receptor binding and NA receptor release functions, and induce a better NA inhibitory antibody response in mice. These results indicate that influenza viruses support a range of ratios for a given NA and HA pair which can be used to produce viral-based influenza vaccines with higher NA content that can elicit more balanced neutralizing antibody responses to NA and HA.     

H5N1 virus-like particle vaccine elicits cross-reactive neutralizing antibodies that preferentially bind to the oligomeric form of influenza virus hemagglutinin in humans

Transmission of pathogenic avian influenza viruses (AIV) from wild birds to domestic poultry and humans is continuing in multiple countries around the world. In preparation for a potential AIV pandemic, multiple vaccine candidates are under development. In the case of H5N1 AIV, a clear shift in transmission from clade 1 to clade 2 viruses occurred in recent years. The virus-like particle (VLP) represents an economical approach to pandemic vaccine development. In the current study, we evaluated the humoral immune response in humans vaccinated with H5N1 A/Indonesia/05/2005 (clade 2.1) VLP vaccine manufactured in Sf9 insect cells. The VLPs were comprised of the influenza virus hemagglutinin (HA), neuraminidase (NA), and matrix 1 (M1) proteins. In an FDA-approved phase I/II human clinical study, two doses of H5N1 VLPs at 15, 45, or 90 μg HA/dose resulted in seroconversion and production of functional antibodies. Moreover, cross-reactivity against other clade 2 subtypes was demonstrated using virus neutralization assays. H5N1 whole-genome fragment phage display libraries (GFPDL) were used to elucidate the antibody epitope repertoire in postvaccination human sera. Diverse epitopes in HA1/HA2 and NA were recognized by postvaccination sera from the two high-dose groups, including large segments spanning the HA1 receptor binding domain. Importantly, the vaccine elicited sera that preferentially bound to an oligomeric form of recombinant HA1 compared with monomeric HA1. The oligomeric/monomeric HA1 binding ratios of the sera correlated with the virus neutralizing titers. Additionally, the two high-dose VLP vaccine groups generated NA-inhibiting antibodies that were associated with binding to a C-terminal epitope close to the sialic acid binding site. These findings represent the first report describing the quality of the antibody responses in humans following AIV VLP immunization and support further development of such vaccines against emerging influenza virus strains.     

Evaluation of the sublingual route for administration of influenza H5N1 virosomes in combination with the bacterial second messenger c-di-GMP

Avian influenza A H5N1 is a virus with pandemic potential. Mucosal vaccines are attractive as they have the potential to block viruses at the site of entry, thereby preventing both disease and further transmission. The intranasal route is safe for the administration of seasonal live-attenuated influenza vaccines, but may be less suitable for administration of pandemic vaccines. Research into novel mucosal routes is therefore needed. In this study, a murine model was used to compare sublingual administration with intranasal and intramuscular administration of influenza H5N1 virosomes (2 μg haemagglutinin; HA) in combination with the mucosal adjuvant (3',5')-cyclic dimeric guanylic acid (c-di-GMP). We found that sublingual immunisation effectively induced local and systemic H5N1-specific humoral and cellular immune responses but that the magnitude of response was lower than after intranasal administration. However, both the mucosal routes were superior to intramuscular immunisation for induction of local humoral and systemic cellular immune responses including high frequencies of splenic H5N1-specific multifunctional (IL-2+TNF-α+) CD4+ T cells. The c-di-GMP adjuvanted vaccine elicited systemic haemagglutination inhibition (HI) antibody responses (geometric mean titres ≥ 40) both when administered sublingually, intranasally and inramuscularly. In addition, salivary HI antibodies were elicited by mucosal, but not intramuscular vaccination. We conclude that the sublingual route is an attractive alternative for administration of pandemic influenza vaccines.     

共表达H5亚型禽流感病毒HA和NA基因的重组鸡痘病毒及其免疫效力

为了构建更为安全有效地抵抗高致病性H5亚型禽流感病毒的基因工程疫苗,将H5亚型禽流感病毒分离株的血凝素(HA)基因和神经氨酸酶(NA)基因定向插入鸡痘病毒转移载体p11S中,H5A和NA基因的启动子分别为PS和PE/L,获得用不同的启动子启动不同的外源基因且两基因盒方向为背向串联的重组转移载体p11SH5ANA。将p11SH5ANA转染至已感染鸡痘病毒282E4疫苗株(wt-FPV)的鸡胚成纤维细胞(CEF)中。p11SH5ANA与wt-FPV基因组DNA之间的同源重组产生了重组鸡痘病毒rFPV-11SH5ANA。通过在含X-Gal的营养琼脂上连续挑选蓝色病毒蚀斑,获得纯化的重组病毒。经传代证实该重组病毒具有良好的遗传稳定性。用105PFU的rFPV-11SH5NA免疫无特定病原体(SPF)鸡,能激发机体产生有效的血凝抑制(HI)抗体。初步的动物试验表明,该重组病毒能使经肌肉注射攻毒的SPF鸡抵抗H5亚型AIV的致死性攻击,保护率为100%,显示出一定的应用前景。     

Influenza A viruses possessing type B hemagglutinin and neuraminidase: potential as vaccine components

A licensed live attenuated influenza vaccine is available as a trivalent mixture of types A (H1N1 and H3N2) and B vaccine viruses. Thus, interference among these viruses could restrict their replication, affecting vaccine efficacy. One approach to overcoming this potential problem is to use a chimeric virus possessing type B hemagglutinin (HA) and neuraminidase (NA) in a type A vaccine virus background. We previously generated a type A virus possessing a chimeric HA in which the entire ectodomain of the type A HA molecule was replaced with that of the type B HA, and showed that this virus protected mice from challenge by a wild-type B virus. In the study described here, we generated type A/B chimeric viruses carrying not only the chimeric (A/B) HA, but also the full-length type B NA instead of the type A NA, resulting in (A/B) HA/NA chimeric viruses possessing type B HA and NA ectodomains in the background of a type A virus. These (A/B) HA/NA chimeric viruses were attenuated in both cell culture and mice as compared with the wild-type A virus. Our findings may allow an effective live influenza vaccine to be produced from a single master strain, providing a model for the design of future live influenza vaccines.     

重配技术构建高产H1N1型流感疫苗病毒株

目的以经典重配技术制备高产H1N1流感疫苗病毒株。方法以野生型A1/云南昆明/03/2009(H1N1)作为HA及NA基因的供体株,以WHO疫苗株A/Perth/16/2009(H3N2)作为高产基因供体株,共同感染SPF鸡胚,经抗H3及抗N2血清中和筛选法及终末稀释法筛选高产重配H1N1病毒。结果获得一株重配H1N1流感病毒株,病毒血凝滴度为1∶4 096,病毒滴度为7.8 lg EID50/mL,显示为鸡胚高产病毒株;血凝抑制结果为1∶1 024,单向免疫扩散试验结果为阳性,证明抗原性与野生株一致;基因测序结果表明重配株的HA及NA基因序列与野生株序列一致。结论构建了高产重配H1H1流感疫苗病毒株,并应用经典重配技术建立了制备高产流感疫苗病毒株的技术平台。     

Expression of Functional Recombinant Hemagglutinin and Neuraminidase Proteins from the Novel H7N9 Influenza Virus Using the Baculovirus Expression System

The baculovirus expression system is a powerful tool for expression of recombinant proteins. Here we use it to produce correctly folded and glycosylated versions of the influenza A virus surface glycoproteins - the hemagglutinin (HA) and the neuraminidase (NA). As an example, we chose the HA and NA proteins expressed by the novel H7N9 virus that recently emerged in China. However the protocol can be easily adapted for HA and NA proteins expressed by any other influenza A and B virus strains. Recombinant HA (rHA) and NA (rNA) proteins are important reagents for immunological assays such as ELISPOT and ELISA, and are also in wide use for vaccine standardization, antibody discovery, isolation and characterization. Furthermore, recombinant NA molecules can be used to screen for small molecule inhibitors and are useful for characterization of the enzymatic function of the NA, as well as its sensitivity to antivirals. Recombinant HA proteins are also being tested as experimental vaccines in animal models, and a vaccine based on recombinant HA was recently licensed by the FDA for use in humans. The method we describe here to produce these molecules is straight forward and can facilitate research in influenza laboratories, since it allows for production of large amounts of proteins fast and at a low cost. Although here we focus on influenza virus surface glycoproteins, this method can also be used to produce other viral and cellular surface proteins.     

Pathogenicity of influenza viruses with genes from the 1918 pandemic virus: functional roles of alveolar macrophages and neutrophils in limiting virus replication and mortality in mice

The Spanish influenza pandemic of 1918 to 1919 swept the globe and resulted in the deaths of at least 20 million people. The basis of the pulmonary damage and high lethality caused by the 1918 H1N1 influenza virus remains largely unknown. Recombinant influenza viruses bearing the 1918 influenza virus hemagglutinin (HA) and neuraminidase (NA) glycoproteins were rescued in the genetic background of the human A/Texas/36/91 (H1N1) (1918 HA/NA:Tx/91) virus. Pathogenesis experiments revealed that the 1918 HA/NA:Tx/91 virus was lethal for BALB/c mice without the prior adaptation that is usually required for human influenza A H1N1 viruses. The increased mortality of 1918 HA/NA:Tx/91-infected mice was accompanied by (i) increased (>200-fold) viral replication, (ii) greater influx of neutrophils into the lung, (iii) increased numbers of alveolar macrophages (AMs), and (iv) increased protein expression of cytokines and chemokines in lung tissues compared with the levels seen for control Tx/91 virus-infected mice. Because pathological changes in AMs and neutrophil migration correlated with lung inflammation, we assessed the role of these cells in the pathogenesis associated with 1918 HA/NA:Tx/91 virus infection. Neutrophil and/or AM depletion initiated 3 or 5 days after infection did not have a significant effect on the disease outcome following a lethal 1918 HA/NA:Tx/91 virus infection. By contrast, depletion of these cells before a sublethal infection with 1918 HA/NA:Tx/91 virus resulted in uncontrolled virus growth and mortality in mice. In addition, neutrophil and/or AM depletion was associated with decreased expression of cytokines and chemokines. These results indicate that a human influenza H1N1 virus possessing the 1918 HA and NA glycoproteins can induce severe lung inflammation consisting of AMs and neutrophils, which play a role in controlling the replication and spread of 1918 HA/NA:Tx/91 virus after intranasal infection of mice.     

Influenza subunit vaccine: antibody responses to one and two doses of vaccine and length of response,with particular reference to the elderly.

Antibody responses to subunit influenza vaccine prepared against A2/England/42/72 (h3n2) were studied in 69 volunteers aged 60 and over and 231 aged 59 and below over 12 months in 1973 and 1974. After two doses of vaccine seroconversion frequencies and geometric mean haemagglutination-inhibition (HI) titres were higher in the elderly, but no differences were observed between the two groups in the length of their responses. Sixteen (23%) of the elderly volunteers seroconverted only after receiving a second dose of vaccine or seroconverted twice after receiving both doses of vaccine. It was considered justifiable, therefore, to recommend the continuation of a two-dose schedule for patients in a high-risk category. Within 30 weeks of vaccination 87 (29%) volunteers had considerably reduced HI titres (less than 48), which might indicate potential susceptibility to influenza during an epidemic, and the number had risen to 132 (44%) by 50 weeks. It was suggested that high-risk patients should receive annnual vaccination two to four months before the possible epidemic period.     

Phase 1 Evaluation of Intranasal Virosomal Influenza Vaccine with and without Escherichia coli Heat-Labile Toxin in Adult Volunteers

Virosomal vaccines were prepared by extracting hemagglutinin (HA) and neuraminidase from influenza virus and incorporating it in the membranes of liposomes composed of phosphatidylcholine. Two intranasal spray vaccine series were prepared: one series comprised 7.5 micrograms of HA of each of three strains recommended by the World Health Organization and 1 microgram of Escherichia coli heat-labile toxin (HLT), and the other contained the HA without HLT. In addition, a third vaccine preparation contained 15 micrograms of HA and 2 micrograms of HLT. The parenteral virosomal vaccine contained 15 micrograms of HA without additional adjuvant. The immunogenicity of a single spray vaccination (15 micrograms of HA and 2 micrograms of HLT) was compared with that of two vaccinations (7.5 micrograms of HA with or without 1 microgram of HLT) with an interval of 1 week in 60 healthy working adults. Twenty volunteers received one parenteral virosomal vaccine. Two nasal spray vaccinations with HLT-adjuvanted virosomal influenza vaccine induced a humoral immune response which was comparable to that with a single parenteral vaccination. A significantly higher induction of influenza virus-specific immunoglobulin A was noted in the saliva after two nasal applications. The immune response after a single spray vaccination was significantly lower. It could be shown that the use of HLT as a mucosal adjuvant is necessary to obtain a humoral immune response comparable to that with parenteral vaccination. All vaccines were well tolerated.     

Modified pulmonary surfactant is a potent adjuvant that stimulates the mucosal IgA production in response to the influenza virus antigen

The intranasal administration of influenza hemagglutinin (HA) vaccine with Surfacten, a modified pulmonary surfactant free of antigenic c-type lectins, as a mucosal adjuvant induced the highest protective mucosal immunity in the airway. The intranasal immunization of mice with HA vaccine (0.2 microg)-Surfacten (0.2 microg) selectively induced the neutralizing anti-HA IgA, but not IgG, and conferred nearly maximal protection in the airway, without inducing a systemic response. In contrast, intranasal inoculation of vaccine with 0.2 microg of the potent mucosal adjuvant cholera toxin B* (CT-B*), prepared by adding 0.2% native CT to the B subunit of CT, induced both anti-HA IgA and IgG in the airway and in the serum. The intranasal administration of HA vaccine alone induced a limited amount of mucosal IgA against influenza virus. Although the s.c. administration of HA vaccine prominently induced serum IgG and IgA, Surfacten and CT-B* did not enhance their induction, and the concentrations of Abs leaking into the airways were insufficient to prevent viral multiplication. The intranasal administration of HA-Surfacten stimulated the expression of MHC class II, CD40, and CD86 molecules in the CD11c-positive cells isolated from the nasal mucosa, but not the expression of cells from the lungs or spleens. Lymphocytes isolated from the airway mucosa after intranasal HA-Surfacten immunization prominently induced TGF-beta1 which, compared with inoculation without Surfacten, promoted an Ag-specific mucosal IgA response. Surfacten alone, however, did not induce TGF-beta1. Our observations suggest that Surfacten, by mimicking the natural surfactant, is an effective mucosal adjuvant in the process of airway immunization.     

H5N1 influenza marker vaccine for serological differentiation between vaccinated and infected chickens

Using plasmid-based reverse genetics, we generated a molecularly altered virus, H5N1/PR8-5B19, containing modified HA and NA genes from A/Goose/Guangdong/1/96 (GS/GD/1/96). In the H5N1/PR8-5B19 virus, the HA cleavage site was modified to resemble that of low-pathogenic avian strains and a portion of the NA stalk region was replaced by the immunodominant 5B19 epitope of the S2 glycoprotein of murine hepatitis virus (MHV). H5N1/PR8-5B19 is not lethal to embryonated eggs or chickens. Chickens immunized with the H5N1/PR8-5B19 inactivated vaccine produced high levels of HI antibody and a measurable antibody response against the MHV 5B19 epitope, and were fully protected against subsequent challenge with different highly pathogenic H5N1 avian influenza viruses. H5N1/PR8-5B19 is therefore an attractive marker vaccine candidate, eliciting a strong, protective antibody response and enabling serological discrimination between vaccinated and wild-type virus-infected chickens.     

Expression and Characterization of Recombinant,Tetrameric and Enzymatically Active Influenza Neuraminidase for the Setup of an Enzyme-Linked Lectin-Based Assay

Developing a universal influenza vaccine that induces broad spectrum and longer-term immunity has become an important potentially achievable target in influenza vaccine research and development. Hemagglutinin (HA) and neuraminidase (NA) are the two major influenza virus antigens. Although antibody responses against influenza virus are mainly directed toward HA, NA is reported to be more genetically stable; hence NA-based vaccines have the potential to be effective for longer time periods. NA-specific immunity has been shown to limit the spread of influenza virus, thus reducing disease symptoms and providing cross-protection against heterosubtypic viruses in mouse challenge experiments.The production of large quantities of highly pure and stable NA could be beneficial for the development of new antivirals, subunit-based vaccines, and novel diagnostic tools. In this study, recombinant NA (rNA) was produced in mammalian cells at high levels from both swine A/California/07/2009 (H1N1) and avian A/turkey/Turkey/01/2005 (H5N1) influenza viruses. Biochemical, structural, and immunological characterizations revealed that the soluble rNAs produced are tetrameric, enzymatically active and immunogenic, and finally they represent good alternatives to conventionally used sources of NA in the Enzyme-Linked Lectin Assay (ELLA).     

Novel function of plasminogen-binding activity of the NA determines the pathogenicity of influenza A virus

Because cleavage of the hemagglutinin (HA) molecule by proteases is a prerequisite for infectivity of influenza A viruses, this molecule is a major determinant of viral pathogenicity. Although well documented in the pathogenicity of avian influenza viruses, the role of HA cleavage in the pathogenicity of mammalian viruses is not well understood. Therefore, we studied a mouse-adapted human isolate A/WSN/33 (WSN), a neurovirulent influenza virus strain that causes systemic infection when inoculated intranasally into mice. We found a novel mechanism of HA cleavage for WSN virus: the neuraminidase (NA) of WSN virus binds and sequesters plasminogen on the cell surface, leading to enhanced cleavage of the HA. The structural basis of this novel function of the NA molecule appears to be the presence of a carboxyl-terminal lysine and the absence of an oligosaccharide side chain at position 146. To obtain direct evidence that the plasminogen-binding activity of the NA enhances the pathogenicity of WSN virus, we generated mutant viruses that are deficient in plasminogen-binding activity by reverse genetics. The mutant viruses showed attenuated growth in mice and failed to grow at all in the brains of these animals. Therefore, we concluded that the novel function of plasminogen-binding activity of the NA determines the pathogenicity of WSN virus in mice.     

Influenza virus hemagglutinin and neuraminidase glycoproteins stimulate the membrane association of the matrix protein.

We have analyzed the mechanism by which the matrix (M1) protein associates with cellular membranes during influenza A virus assembly. Interaction of the M1 protein with the viral hemagglutinin (HA) or neuraminidase (NA) glycoprotein was extensively analyzed by using wild-type and transfectant influenza viruses as well as recombinant vaccinia viruses expressing the M1 protein, HA, or NA. Membrane binding of the M1 protein was significantly stimulated at the late stage of virus infection. Using recombinant vaccinia viruses, we found that a relatively small fraction (20 to 40%) of the cytoplasmic M1 protein associated with cellular membranes in the absence of other viral proteins, while coexpression of the HA and the NA stimulated membrane binding of the M1 protein. The stimulatory effect of the NA (>90%) was significant and higher than that of the HA (>60%). Introduction of mutations into the cytoplasmic tail of the NA interfered with its stimulatory effect. Meanwhile, the HA may complement the defective NA and facilitate virus assembly in cells infected with the NA/TAIL(-) transfectant. In conclusion, the highly conserved cytoplasmic tails of the HA and NA play an important role in virus assembly.     

Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle and the induction of protective immune responses^1,2. As the main target for neutralizing antibodies, HA is currently used as the influenza vaccine potency marker and is measured by single radial immunodiffusion (SRID)³. However, the dependence of SRID on the availability of the corresponding subtype-specific antisera causes a minimum of 2-3 months delay for the release of every new vaccine. Moreover, despite evidence that NA also induces protective immunity⁴, the amount of NA in influenza vaccines is not yet standardized due to a lack of appropriate reagents or analytical method⁵. Thus, simple alternative methods capable of quantifying HA and NA antigens are desirable for rapid release and better quality control of influenza vaccines.Universally conserved regions in all available influenza A HA and NA sequences were identified by bioinformatics analyses^6-7. One sequence (designated as Uni-1) was identified in the only universally conserved epitope of HA, the fusion peptide⁶, while two conserved sequences were identified in neuraminidases, one close to the enzymatic active site (designated as HCA-2) and the other close to the N-terminus (designated as HCA-3)⁷. Peptides with these amino acid sequences were synthesized and used to immunize rabbits for the production of antibodies. The antibody against the Uni-1 epitope of HA was able to bind to 13 subtypes of influenza A HA (H1-H13) while the antibodies against the HCA-2 and HCA-3 regions of NA were capable of binding all 9 NA subtypes. All antibodies showed remarkable specificity against the viral sequences as evidenced by the observation that no cross-reactivity to allantoic proteins was detected. These universal antibodies were then used to develop slot blot assays to quantify HA and NA in influenza A vaccines without the need for specific antisera^7,8. Vaccine samples were applied onto a PVDF membrane using a slot blot apparatus along with reference standards diluted to various concentrations. For the detection of HA, samples and standard were first diluted in Tris-buffered saline (TBS) containing 4M urea while for the measurement of NA they were diluted in TBS containing 0.01% Zwittergent as these conditions significantly improved the detection sensitivity. Following the detection of the HA and NA antigens by immunoblotting with their respective universal antibodies, signal intensities were quantified by densitometry. Amounts of HA and NA in the vaccines were then calculated using a standard curve established with the signal intensities of the various concentrations of the references used. Given that these antibodies bind to universal epitopes in HA or NA, interested investigators could use them as research tools in immunoassays other than the slot blot only. Download video file.^{(80M, mov)}     

共表达H5N1流感病毒M1和HA基因的DNA疫苗与腺病毒载体疫苗的小鼠免疫评价

为评价在小鼠体内表达流感病毒M1和HA基因诱导的免疫反应,制备共表达H5N1亚型禽流感病毒 (A/Anhui/1/2005) 全长基质蛋白1 (M1) 基因和血凝素 (HA) 基因的重组DNA疫苗pStar-M1/HA和重组腺病毒载体疫苗Ad-M1/HA,将其按初免-加强程序免疫BALB/c小鼠,共免疫4次,每次间隔14 d。第1、3次用DNA疫苗,第2、4次用重组腺病毒载体疫苗,每次免疫前及末次免疫后14 d采集小鼠血清用于检测体液免疫应答,末次免疫后14 d采集小鼠脾淋巴细胞用于检测细胞免疫应答。血凝