Antigenic variants of influenza viruses: marked differences in the frequencies of variants selected with different monoclonal antibodies

Monoclonal antibodies against the hemagglutinin of two influenza A viruses and one influenza B virus were used to analyze the frequencies of antigenic variant subpopulations in cloned virus seeds. Antigenic variants selected with monoclonal antibodies specific for different antigenic determinants were obtained over a wide range of frequencies. With one monoclonal antibody directed to the hemagglutinin of A/PR/8/34 virus antigenic variants were isolated at a frequency of 10-^4.4, whereas another monoclonal antibody, specific for the hemagglutinin of X-31 virus, completely neutralized all infectious virus in the X-31 virus seeds examined, indicating a frequency of antigenic variation, for one seed at least, of less than 10^?8.1.     

Characterization of variable-region genes and shared crossreactive idiotypes of antibodies specific for antigens of various influenza viruses

Several syngeneic monoclonal anti-idiotypic antibodies were obtained against PY206, a monoclonal antibody specific for X-31 (H3N2) influenza virus hemagglutinin. This idiotype was found in the sera of BALB/c mice immunized with various influenza viruses. Adsorption experiments indicated that the PY206 Id was borne by antibodies specific for viral hemagglutinin (HA) and/or neuraminidase (NA). This idiotype was identified on other monoclonal antibodies specific for various influenza HAs (H3 and H1). Study of the variable-region (V) genes of these monoclonal antibodies showed that its expression is independent of variable kappa (VK)21 light-chains and that the heavy-chains of the strongly idiotype-positive hybridomas derive from either the variable heavy (VH) J558 or VH 7183 family. Finally, Western blot analysis demonstrated that PY206 idiotypic determinants are located exclusively on the heavy chain.     

Antigenic activity of a synthetic peptide comprising the "loop" region of influenza virus hemagglutinin

A peptide representing the linear sequence 123–151 (the “loop”) within the hemagglutinin of X-31 (H3N2) influenza virus was synthesized and its immunogenic and antigenic properties investigated. When inoculated into rabbits, the synthetic peptide alone, or conjugated to high-molecular-weight carriers, elicited the production of antibody which bound to the peptide in a solid phase radioimmunoassay. No significant binding was detected between antipeptide antibodies and X-31 virus particles. However, antibodies raised against X-31 virus did exhibit some binding to the synthetic peptide.     

Searching for MHC-restricted anti-viral antibodies: antibodies recognizing the nucleoprotein of influenza virus dominate the serological response of C57BL/6 mice to syngeneic influenza-infected cells

An attempt has been made to generate monoclonal antibodies which recognize the same target structures on influenza-infected cells as those seen by cytotoxic T lymphocyte (CTL) receptors. Such antibodies, if they mimicked the T cell receptor specificity, would be expected to be both virus specific and restricted in their binding by the major histocompatibility complex (MHC) antigens. Approximately 200 hybridomas from C57BL/6 (H-2b) mice primed and boosted with influenza virus (X-31)-infected EL4 (a C57BL/6 T cell lymphoma) were screened for reactivity on infected and uninfected cells of different MHC haplotypes. Of the 10 hybridoma antibodies which were identified as being reactive with X-31-infected EL4, but not uninfected EL4, all reacted equally well with X-31-infected cells of H-2b, H-2d and H-2k haplotypes, indicating a lack of MHC restriction in their recognition of the infected cells. Unexpectedly, 7 of the 10 monoclonal antibodies were found to react specifically with the purified influenza virus nucleoprotein (NP), a predominant viral antigen in CTL recognition of infected cells. Fluorescence-activated flow cytometry confirmed that these antibodies were able to recognize NP serological determinants on the surface of viable, infected cells, but the anti-NP antibodies were unable to block the lytic activity of an NP-specific CTL clone.     

Use of Recombinant Nucleoproteins in Enzyme-Linked Immunosorbent Assays for Detection of Virus-Specific Immunoglobulin A (IgA) and IgG Antibodies in Influenza Virus A- or B-Infected Patients

The nucleoprotein genes of influenza virus A/Netherlands/018/94 (H3N2) and influenza virus B/Harbin/7/94 were cloned into the bacterial expression vector pMalC to yield highly purified recombinant influenza virus A and B nucleoproteins. With these recombinant influenza nucleoproteins, enzyme-linked immunosorbent assays (ELISAs) were developed for the detection of influenza virus A- and B-specific immunoglobulin A (IgA) and IgG serum antibodies. Serum samples were collected at consecutive time points after the onset of clinical symptoms from patients with confirmed influenza virus A or B infections. Nucleoprotein-specific IgA antibodies were detected in 41.2% of influenza virus A-infected patients and in 66.7% of influenza virus B-infected patients on day 6 after the onset of clinical symptoms. In serum samples taken on day 21 (influenza virus A-infected patients) or day 28 (influenza virus B-infected patients), nucleoprotein-specific IgA antibodies could be detected in 58.8 and 58.3% of influenza virus A- and B-infected patients, respectively. At the same time, IgG antibody rises were detected in 88.2% of influenza virus A-infected patients and in 95.8% of influenza virus B-infected patients. On comparison, hemagglutination inhibition assays detected antibody titer rises in 81.3 and 72.7% of patients infected with influenza viruses A and B, respectively. In contrast to the detection of nucleoprotein-specific IgG antibodies or hemagglutination-inhibiting antibodies, the detection of nucleoprotein-specific IgA antibodies does not require paired serum samples and therefore can be considered an attractive alternative for the rapid serological diagnosis of influenza.     

Recognition of influenza A virus nucleoprotein by an H-2-restricted cytotoxic T-cell clone

Cytotoxic T-cell clones raised against X-31 (H3N2) influenza virus in C57BL/6 mice can be directed against an influenza A virus subtype specific determinant (1). A representative T-cell clone (A3.1) has been used in combination with a set of genetically typed recombinant viruses, to show that the A/PR/8/34 nucleoprotein can be responsible for cytotoxic T-lymphocyte recognition of infected target cells.     

Immunoglobulin G subclass antibody responses in influenza A and parainfluenza type 1 virus infections

Antibody responses in immunoglobulin G1, G2, G3, G4, A (IgA1) and M isotypes were studied in 10 patients with an acute influenza A and in another 10 patients with a parainfluenza type 1 virus infection using radioimmunoassay with standardized monoclonal anti-immunoglobulins. A four-fold or greater increase of antibody in patients have an acute influenza A virus infection, were found in IgG1 (all 10 cases), IgG3 (seven cases), IgG4 (eight cases) and in IgA1 (six cases) whereas IgG2 and IgM responses were observed only in one and three cases, respectively. The antibody titre values were converted to immunoglobulin units by multiplying the titre by a pre-determined correction coefficient compensating for the varying affinity of the individual monoclonal anti-immunoglobulins. These units were then used to calculate the actual proportions of each isotype. In the convalescent phase, 78% of total anti-influenza A antibodies were estimated to be of IgG1 isotype and other immunoglobulin isotypes varied from 3 to 7% of total. Similar results in parainfluenza virus antibodies were obtained with serum pairs from patients with an acute parainfluenza virus infection.     

Cross-protection against influenza A virus infection by passively transferred respiratory tract IgA antibodies to different hemagglutinin molecules.

Mice that were intranasally immunized with different influenza A virus hemagglutinins (HA), derived from PR8 (H1N1), A/Yamagata (H1N1) or A/Fukuoka (H3N2) virus, together with cholera toxin B subunit as an adjuvant, were examined for protection against PR8 infection; PR8 HA and A/Yamagata HA immunization conferred complete protection, while A/Fukuoka HA immunization failed to confer protection. In parallel with protection, PR8 HA-, A/Yamagata HA-, and A/Fukuoka HA-immunized mice produced a high, a moderate and a low level of PR8 HA-reactive IgA in the respiratory tract, respectively. These IgA antibodies were not only higher in content in the nasal secretions, but also more cross-reactive than IgG. The purified IgA antibodies from respiratory tract washings of PR8 HA-immunized mice, which contained the HA-specific IgA corresponding to the amount detected in the nasal wash, were able to protect mice from PR8 challenge when transferred to the respiratory tract of naive mice. The transfer of IgA from A/Yamagata HA-immunized mice also afforded cross-protection against PR8 infection, whereas the IgA from A/Fukuoka HA-immunized mice failed to provide protection. The ability of transferred IgA to prevent viral infection was dependent on the amount of HA-reactive IgA remaining in the respiratory tract of the host at the time of infection. These experiments directly demonstrate that IgA antibodies to influenza A virus HA by themselves play a pivotal role in defence not only against homologous virus infection, but also against heterologous drift virus infection at the respiratory mucosa, the portal of entry for the viruses.     

IgA subclasses of human colostral antibodies specific for microbial and food antigens

The distribution of total and antigen-specific IgA1 and IgA2 antibodies in human colostrum was determined by ELISA using subclass-specific monoclonal reagents. In 18 samples of colostrum the mean ratio of total IgA1 to IgA2 was found to be 53:47, respectively, but significant individual variations were observed. In two samples we found unusually low levels of IgA1, while IgA2 was in the normal range. IgA1 and IgA2 antibody activities were determined against the following antigens: bovine gamma-globulin and beta-lactoglobulin, tetanus toxoid, protein antigen I/II of Streptococcus mutans, influenza virus vaccine, polysaccharides of pneumococcal, meningococcal and Haemophilus influenzae type b origin, and lipopolysaccharide (LPS) from Escherichia coli K235. The IgA antibody activity directed against the polysaccharides was almost equally distributed between the two subclasses. However, antibody activity specific for protein antigens was found predominantly in the IgA1 subclass while anti-LPS activity was mostly of the IgA2 subclass.     

Identification of PR8 M1 protein in influenza virus high-yield reassortants by M1-specific monoclonal antibodies

A panel of monoclonal antibodies to the M1 protein of A/PR8/34 (H1N1) (PR8) influenza A virus was found to distinguish in ELISA high-yielding reassortant viruses derived from reassortment of PR8 and X-31 (H3N2) viruses with recently prevalent field strains of H1N1 or H3N2 subtype. These findings are concordant with results of genotyping that demonstrated the presence of PR8 RNA 7 or M1 protein in high-yield reassortants by RNA or protein PAGE. All high-yield vaccine candidate reassortants Application of the M1 monoclonal antibody panel facilitates the isolation of high-yield vaccine candidate reassortants bearing the PR8 M1 gene, and should aid in epidemiologic strain tracking as well.     

Subclass distribution and molecular form of immunoglobulin A hemagglutinin antibodies in sera and nasal secretions after experimental secondary infection with influenza A virus in humans

Serum and nasal wash specimens from 13 human volunteers undergoing experimental secondary infection with influenza A/Peking/2/79 (H3N2) wild-type virus were examined for the molecular form and subclass distribution of immunoglobulin A (IgA) antibodies to the viral hemagglutinin (HA). Nasal IgA antibodies were polymeric and did not bind radiolabeled secretory component, indicating that they were secretory IgA antibodies. Both IgA1 and IgA2 antibodies were detected; however, IgA1 accounted for most of the rise in IgA anti-HA levels seen after infection. In serum virtually all of the IgA HA antibodies were of the IgA1 subclass. Furthermore, the serum antibodies were predominantly polymeric and were capable of binding radiolabeled secretory component. These results suggested that the serum IgA antibodies to HA were of mucosal origin and that influenza A virus HA preferentially stimulates an IgA1 response.     

Cell-mediated immune responses to the hemagglutinin and neuraminidase antigens of influenza A virus after immunization in humans.

Humoral and cell-mediated immunity (CMI) were evaluated in groups of school children after immunization with inactivated influenza virus vaccines. A conventional biphasic strain (H3ChN2Ch) of Port Chalmers influenza virus (X-41), a recombinant influenza virus specific for the neuraminidase antigen (Heq1N2Ch) of Port Chambers influenza A virus (X-42), and a placebo were employed for immunization. The techniques of hemagglutination inhibition and neuraminidase inhibition were used to determine serum antibody titers. The CMI responses were evaluated by the in vitro lymphocyte transformation assay employing HavN2Ch, Heq1Neq1, H3ChNeq1, and H3ChN2Ch influenza A virus strains as stimulants. Specific HAI antibody and CMI responses to H3Ch were observed in X-41 but not in X-42 vaccinees. Specific anti-neuraminidase antibodies and CMI responses to N2Ch were manifested by both X-41 and X-42 vaccinees. Immunization with the placebo resulted in no influenza-specific immune responses. The CMI response was first detectable 10 days after immunization and then declined. These observations demonstrate the induction of CMI responses to the HA and NA influenza surface antigens after immunization. These responses may be important in antiviral immunity and the recovery from influenza infection.     

Use of monoclonal antibodies for rapid detection of influenza a viras in nasopharyngeal secretions

Two monoclonal antibodies against influenza A virus were assessed for use as diagnostic reagents in an indirect immunofluorescence assay (IFA) of nasopharyngeal secretions. Monoclonal antibody IA-52, directed at an internal antigen, reacted with all influenza A tested. The high stability of this epitope permitted its use in a rapid IFA test, which gave results comparable to those obtained with polyclonal antibodies and viral isolation. The second monoclonal antibody, IA-279 was directed at a surface epitope (hemagglutinin); it reacted with almost all H₃ subtype strains. Positive IFA using these monoclonal antibodies permitted rapid preliminary differentiation between the current two major subtypes of influenza A virus (H₁N₁,H₃N₂).     

禽流感病毒非结构蛋白1单克隆抗体的制备及其特异性分析

为研制禽流感病毒(H5N1)非结构蛋白1(NS1)的特异性单克隆抗体(mAb),并鉴定其特异性,本研究在分别表达了具有良好抗原性的A/Vietnam/1194/04(H5N1)-NS1和A/HongKong/486/97(H5N1)-NS1重组蛋白基础上,用A/Viet-nam/1194/04(H5N1)-NS1蛋白免疫BALB/c小鼠,取其脾细胞与小鼠骨髓瘤细胞进行融合,间接ELISA筛选阳性的杂交瘤细胞,并结合免疫荧光和免疫印迹对抗体的特异性进行鉴定,通过竞争抑制实验对单抗识别的抗原位点进行分析。结果共获得19株能识别4个H5N1-NS1蛋白不同抗原位点的mAb,亚类测定显示,5株为IgG2a、1株为IgG2b,另外13株为IgG1。这些mAb均与A/Vietnam/1194/04(H5N1)-NS1和A/HongKong/486/97(H5N1)-NS1重组蛋白特异性结合,免疫荧光检测均与A型流感病毒(H1N1和H3N2)有交叉反应,而与B型流感病毒无交叉现象。表明成功获得特异性针对H5N1-NS1蛋白的mAb,为进一步研究禽流感病毒NS1蛋白的结构与功能奠定基础。     

Cross-protection in mice infected with influenza A virus by the respiratory route is correlated with local IgA antibody rather than serum antibody or cytotoxic T cell reactivity

Mice previously infected with an aerosol of A/Rec 31 influenza virus were strongly protected against an aerosol challenge with A/Vic influenza as judged by lung virus titers recovered 2 days after the challenge infection. Such complete homotypic immunity was not achieved by priming with live Rec 31 virus injected i.v. or UV-inactivated Rec 31 virus administered s.c. together with Al(OH)₃ and saponin. The reason for the superior protective effect of the natural infection was investigated. The protection induced by respiratory infection with Rec 31 virus was specific for influenza A viruses. It was not correlated with specific serum hemagglutination inhibition antibody titer or cross-reactive cytotoxic T (T_c) cell reactivity. Moreover, the transfer of splenic and lymphoid T cell populations with strong secondary T_c activity did not significantly reduce lung virus titers in recipient mice 3 days after infection. The protection however occurred in parallel with the presence of cross-reactive IgA antibody in the lung washings. It thus appears that local secretory IgA plays a causal role in the prevention of cross-infection by influenza A virus. Serum antibody and T_c cells, on the other hand, may be crucial for recovery from such infection. All mice primed with live Rec 31 virus, administered i.v. or by aerosol and expressing equally high levels of T_c reactivity, survived a lethal challenge with A/PR8 virus. The same challenge, however, killed half of the mice immunized s.c. with inactivated Rec 31 virus which induced only a low level of T_c reactivity.     

Electroelution for purification of influenza A matrix protein for use in immunoassay

A new preparative method for isolation of matrix protein from type A influenza virus was developed. Commercially available whole virus or split virus vaccines were lysed, and the soluble proteins separated by electrophoresis on polyacrylamide gel. The matrix protein was located on the gel by precipitation with KCl, and recovered by electroelution. The method was technically simple and required little direct supervision during the two-step recovery process. Yields of A matrix were consistently high, averaging 68.1% in five trials with A/Brazil/X-71. The method was also successful with other A viruses, although not with influenza B virus. Isolated A matrix had less than 0.5% contamination by hemagglutinin or nucleoprotein, as determined by immunoblotting and ELISA. Matrix protein was immunoreactive in Western blots and was detectable in concentrations as low as 1 ng/ml with ELISA. The isolated matrix provided a suitable standard for detection of matrix protein in nasal washes from patients with influenza A virus infection, and could also be used to detect anti-matrix antibodies, including monoclonal antibodies in tissue culture supernatants. The advantages of electroelution for separation of matrix protein compared to other methods were its technical simplicity, applicability to formalin-fixed influenza virus in commercially available vaccines, its consistently high yield, and its very high level of purification.     

H5N1流感病毒M1蛋白免疫制备的单克隆抗体的研究

目的 制备抗人流感病毒H5N1株M1蛋白的单克隆抗体,为流感的快速诊断和研究提供新的工具.方法 应用在大肠埃希菌中表达的人H5N1亚型禽流感病毒(A/Anhui/1/2005)株M1蛋白,以纯化的表达产物免疫BALB/c小鼠,取脾细胞与sp2/0细胞系作细胞融合后,间接ELISA法筛选阳性的杂交瘤细胞,并应用间接免疫荧光法对抗体的特异性进行鉴定.结果 获得3株能稳定分泌抗禽流感病毒M1抗原的McAb杂交瘤细胞株,交叉反应试验及间接免疫荧光检测表明,三株McAb具有型特异性.结论 用H5N1禽流感病毒M1蛋白免疫制备的单克隆抗体,具有一定的交叉反应性,可用于多种亚型甲型流感病毒的检测.     

Characterization and evaluation of monoclonal antibodies developed for typing influenza A and influenza B viruses.

Monoclonal antibodies that are broadly reactive with influenza A or influenza B viruses were produced as stable reagents for typing influenza viruses. Monoclonal antibodies to influenza A were specific for either matrix protein or nucleoprotein. The antibodies to influenza B were specific for nucleoprotein or hemagglutinin protein. In an enzyme immunoassay procedure, influenza A antibodies detected H1N1, H2N2, and H3N2 influenza A virus strains collected between 1934 and 1984. Each of the influenza B antibodies detected influenza B reference viruses collected between 1940 and 1984. Pools of either influenza A or influenza B monoclonal antibodies were used to detect influenza viruses reisolated from clinical specimens in tissue culture. At 48 h after inoculation, the influenza A monoclonal antibodies detected 64% of H1N1 and 94% of H3N2 influenza A specimens, and the influenza B monoclonal antibodies detected 79% of the influenza B specimens. The results of this study suggest that the monoclonal antibodies described should provide useful diagnostic reagents for workers in virology laboratories who wish to isolate and identify influenza virus but have been unable to obtain consistent supplies of animal sera specific for influenza A or B viruses.     

甲型流感病毒NS1抗原捕获ELISA的建立

目的 建立基于单克隆抗体的甲型流感病毒非结构蛋白1(NS1)抗原检测的酶联免疫吸附(ELISA)法.方法 用甲型流感病毒NS1特异性单克隆抗体,通过抗体的优化组合,建立双抗体夹心抗原捕获ELISA,检测不同来源的流感病毒及副流感病毒.结果 对多种抗体组合进行反复筛选,最终确定了特异性检测到甲型流感病毒的NS1蛋白,而与乙型流感病毒和副流感病毒不发生交叉反应的最佳抗体组合.该方法 检测重组H5N1-NS1[A/HongKong/486/97(H5N1)-NS1和A/Vietnam/1194/04(H5N1)-NS1]蛋白的灵敏度最低检测值分别为15.6 ng/ml和240 pg/ml.结论 成功建立了甲型流感病毒NS1抗原捕获ELISA,为建立甲型流感病毒感染早期诊断新方法 奠定基础.     

甲型流感病毒NS1抗原捕获ELISA的建立

目的 建立基于单克隆抗体的甲型流感病毒非结构蛋白1(NS1)抗原检测的酶联免疫吸附(ELISA)法.方法 用甲型流感病毒NS1特异性单克隆抗体,通过抗体的优化组合,建立双抗体夹心抗原捕获ELISA,检测不同来源的流感病毒及副流感病毒.结果 对多种抗体组合进行反复筛选,最终确定了特异性检测到甲型流感病毒的NS1蛋白,而与乙型流感病毒和副流感病毒不发生交叉反应的最佳抗体组合.该方法 检测重组H5N1-NS1[A/HongKong/486/97(H5N1)-NS1和A/Vietnam/1194/04(H5N1)-NS1]蛋白的灵敏度最低检测值分别为15.6 ng/ml和240 pg/ml.结论 成功建立了甲型流感病毒NS1抗原捕获ELISA,为建立甲型流感病毒感染早期诊断新方法 奠定基础.