Application of the single radial diffusion test for assay of antibody to influenza type A viruses.

Single radial diffusion (SRD) tests for antibodies to influenza type A hemagglutinin, neuraminidase, nucleoprotein, and matrix protein antigens were compared with conventional hemagglutination inhibition, neuraminidase inhibition, and complement fixation tests. Sera used in this study were obtained in 1968-1969 from volunteers before and after vaccination and before and after an ensuing epidemic of Hong Kong influenza. The SRD test compared favorably with conventional tests for assessment of vaccine- or infection-induced rises in antibody titers to influenza type A viruses. Little linear relationshiip was seen between zone areas with SRD and titers with conventional tests, suggesting that the SRD test may detect antibody of different quality or specificity. The SRD seemed equal to the hemagglutination inhibition test for predicting susceptibility to influenza. SRD is a simple test for the recognition of antibody to various antigenic components of the influenza virus and could prove to be a valuable epidemiological tool.     

Comparison of new triton X-100- and tween-ether-treated split-treated vaccines in children.

Split-product vaccines (SPVs) combine the desirable properties of no systemic reactogenicity and adequate immunogenicity when two doses are given. We compared a new Triton X-100 SPV (Connaught Laboratories, Inc.) with the commercially available Tween-ether SPV (Parke-Davis & Co.) in 76 children and young adults 2 to 25 years old; there were 39 and 37, respectively, in each vaccine group. Both vaccines contained influenza A/Brazil/78, A/Texas/77, and B/Hong Kong/72 (7 microgram of hemagglutinin for each strain); two doses were administered 1 month apart. Among persons seronegative by the hemagglutination inhibition test, the geometric mean antibody titers rose to approximately 100 after the first vaccination for influenza A/Brazil/78 and A/Texas/77. For B/Hong Kong/72, however, seronegative recipients developed lower geometric mean titers of approximately 32 after one immunization. Against the new B/Singapore/79 strain neither SPV stimulated adequate cross-reacting hemagglutination inhibition antibody (geometric mean titers of approximately 10). In conclusion, the new Triton X-100 SPV appears to be comparable to the ether-treated SPV in primed subjects. Further studies in unprimed children should be done to confirm this impression. In addition, it would be advisable to study other dosage regimens in unprimed children with these SPVs.     

Enzyme-linked immunosorbent assay for detection of antibodies to influenza A and B and parainfluenza type 1 in sera of patients.

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of antibodies to influenza A/Hong Kong/1/68, influenza A/Victoria/3/75, influenza B/Hong Kong/5/72, and parainfluenza type 1 viruses. Development and standardization of the method indicated that an acetone-ethyl alcohol mixture was a suitable fixative for the preparation of the solid-phase coupled antigen. The addition of sodium azide to the enzyme-conjugated solution and the concentrations of the enzyme-conjugate antiglobulin and test sera employed were all critical factors in the success of the ELISA procedure. The ELISA test was specific; there was no cross-reaction between influenza A and B or parainfluenza type 1 viruses. The concordance between ELISA and hemagglutination inhibition results suggested that both tests probably detected the same type of antibodies. The ELISA procedure was 8 to 64 times more sensitive than complement fixation and/or hemagglutination inhibition tests. Low levels of antibody in patients' sera were detected only by the ELISA test. During the course of the testing period false positive reactions were not encountered. The results of ELISA could be obtained within 3 h. The ELISA test required a very small amount of serum and, therefore, offered an opportunity to detect the presence of maternal antibodies to influenza viruses in blood collected from infants by heel prick.     

Inactivated Influenza Vaccine Efficacy: Diminished Antigenicity of Split-Product Vaccines in Mice

Groups of 60 to 120 mice were given a single intraperitoneal inoculation of varying dilutions of commercially prepared and licensed bivalent (A/England and B/Mass) and monovalent (A/Aichi or B/Hong Kong) inactivated influenza vaccines, and their antibody responses at 14 days were quantitated by hemagglutination inhibition tests. Split-product vaccines prepared by the treatment of A/England, B/Mass, and B/Hong Kong whole virus with Tween-80 and either tributylphosphate or ether produced significantly lower mean antibody titers than did equivalent whole-virus preparations. The rates of seroconversion (<1:8 to >/=1:8) at the various dilutions tested were also significantly reduced when these split-product vaccines were given. When the antigen content of all vaccines was quantitated by the chick cell agglutination test, between 10 and 100 times more split-product antigen than whole-virus antigen was required to produce seroconversion in 50% of the mice tested. Differences between split-product and whole-virus A/Aichi vaccines were less marked. These data point out the need to consider factors other than hemagglutinin content alone in determining the immunogenicity of inactivated influenza vaccines.     

Antibody response in humans to influenza virus type B host-cell-derived variants after vaccination with standard (egg-derived) vaccine or natural infection.

Hemagglutination inhibition (HI) and neutralization tests were used to determine antibody responses to egg-derived and Madin-Darby canine kidney (MDCK)-derived influenza B virus (B/England/222/82) in paired sera from persons naturally infected with influenza B and in persons vaccinated with standard egg-derived inactivated influenza vaccine. When tested by HI, the MDCK-derived antigen gave significantly higher (8- to 12-fold) geometric mean titers (GMT) in convalescent-phase sera from persons naturally infected during community outbreaks, as well as more 4-fold titer rises, than did tests with egg-derived antigen. When tested by neutralization, however, the convalescent-phase sera GMTs were only threefold higher with the MDCK-derived antigen and an equivalent number of fourfold titer rises were detected with both antigens. With postvaccine sera, the MDCK-derived antigen gave GMTs that were threefold higher than those obtained with egg-derived antigen in both the HI and neutralization tests and both antigens detected an equivalent number of fourfold titer rises in HI and neutralization tests. Sucrose gradient-fractionated egg-derived antigen showed a single peak of hemagglutinin activity corresponding to whole virions, whereas MDCK-derived antigen contained two distinct peaks of hemagglutinin activity, one of which had a lower sedimentation rate. The overall findings indicate that the egg-derived antigen in the vaccine induced HI and neutralizing antibody to both egg- and MDCK-derived variants and suggest that titers of antibody to MDCK-derived virus may be affected by the physical form of the hemagglutinin antigen.     

Characterization of human serum strain-specific antihemagglutinin antibody to A/Port Chalmers/73 (H3N2) influenza virus by radioimmunoprecipitation assays.

We performed radioimmunoprecipitation assays in which iodinated preparations of A/Port Chalmers/73 (A/PC/73) hemagglutinin were used as the test antigens and high concentrations of unlabeled A/Hong Kong/68 viral protein were used to inhibit the binding of cross-reactive antibodies to quantitate strain-specific antibody responses in postvaccination sera. Strain-specific antibodies comprised 8 to 48% (mean, 20%) of the total A/PC/73 antigen-binding capacity of the sera tested. Competition radioimmunoprecipitation assays in which disrupted preparations of purified whole virus representative of several of the H3N2 variants were used indicated that the A/PC/73 strain-specific antibody that was present after adsorption of serum by A/Hong Kong/68 antigen was capable of reacting with A/England/72 and A/Victoria/75 hemagglutinins, but generally with lower avidity than with A/PC/73 hemagglutinin. A comparison of the A/PC/73 antibody titers measured by radioimmunoprecipitation and hemagglutination inhibition tests before and after adsorption with A/Hong Kong/68 whole virus suggested that cross-reactive and strain-specific antibodies were comparable in efficiency of inhibiting viral hemagglutination. These data indicated that vaccines containing later variants within a subtype could induce antihemagglutinin antibodies of restricted specificity, but that these antibodies may not be directed against unique antigenic determinant(s).     

Increased sensitivity and reduced specificity of hemagglutination inhibition tests with ether-treated influenza B/Singapore/222/79

Hemagglutination inhibition (HI) tests against whole virus (WV) influenza B/Singapore/222/79 antigen detected prevaccination serum antibody in only 15 (20%) of 50 predominantly elderly volunteers and fourfold or greater titer rises in only three (6%) after they received 1981-1982 trivalent influenza vaccine containing antigens of this virus. HI titers against ether-treated (ET) B/Singapore/222/79 were about eightfold higher than those against WV antigen and were comparable to microneutralization titers against this virus. The ET HI detected prevaccination antibody in 84%, a postvaccination titer rise in 32%, and a final titer of 80 or higher in 66%. Among 51 additional persons with known or presumed influenza B virus infections early in 1982, ET B/Singapore/222/79 was also more sensitive than WV for serodiagnosis (69 versus 49%), but eight persons with both WV and ET B/Singapore/222/79 HI responses also had an HI titer rise to WV A/Brazil/11/78 (H1N1) antigen. Conversely, among 14 college students with febrile, culture-proven influenza A (H1N1) infections early in 1982, 6 (43%) developed HI titer rises to ET B/Singapore/222/79 with no other serological evidence of influenza B virus infection. Moreover, young adult volunteers with mild experimental influenza A (H1N1) infections also exhibited a 17% (3 of 18) incidence of ET B/Singapore/222/79 HI titer rises, versus none in matched, uninfected volunteers. These data indicate that ET B/Singapore/222/79 virus has increased sensitivity but reduced specificity compared to WV as an HI antigen and that caution is needed in interpretation of a single HI test for serodiagnosis, whether with WV or ET antigen.     

Immunization of elderly people with two doses of influenza vaccine.

A total of 104 elderly patients were immunized with one or two doses of the commercial 1985-1986 inactivated influenza vaccine formulation. Two types of vaccines (split virus [SV] vaccine and whole virus [WV] vaccine) and one or two doses 1 month apart were given. No difference in local or systemic reactions was noted among the four groups. The reciprocal geometric mean hemagglutination inhibition antibody titers against influenza A/Philippines/82 (H3N2) after one or two doses were: 78 for SV vaccine (one dose), 65 for SV vaccine (two doses), 55 for WV vaccine (one dose), and 51 for WV vaccine (two doses). Similar nonsignificant differences were observed for the other two antigens contained in the vaccine. The percentage with a hemagglutination inhibition titer of greater than or equal to 1:40 also did not differ after one or two doses. We then compared the postvaccination hemagglutination inhibition titers in young and old patients from previous studies in which apparent differences had appeared. We retested all sera simultaneously on the same day with the same reagents. No significant differences were apparent among age groups. In summary, the humoral immune response to inactivated influenza vaccine in healthy ambulatory elderly patients who have been previously immunized may not differ significantly from that of children and young adults. A booster dose 1 month after the first dose does not enhance immune responses in the elderly.     

Detection of antibodies to influenza virus M protein by an enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay test system was developed in which purified influenza virus M protein was used for the detection of M antibody in human sera. Antibody levels to influenza A virus M protein were monitored in sera from a vaccine study population by using an enzyme-linked immunosorbent assay technique with purified M protein as the adsorbent antigen. A 10-fold variation in titers of preexisting M antibody was observed in this population of young adults. Increases of anti-M titer of 7- to 24-fold were observed upon immunization with Formalin-inactivated vaccine or after natural infection. The antibody response to M protein was dissociated from the response to the hemagglutinin or neuraminidase antigens. The M antibody response preceded or was coincident with the antibody response to H1 hemagglutinin upon natural exposure to circulating virus.     

Use of the enzyme-linked immunosorbent assay to detect serum antibody responses of volunteers who received attenuated influenza A virus vaccines.

Sera from volunteers who received live influenza A wild-type or ts recombinant virus were tested by hemagglutination inhibition (HI) assay, neuraminidase inhibition (NI) assay, and the enzyme-linked immunosorbent assay (ELISA) to determine which assay system was the most sensitive in detecting an immunological response to infection. The ELISA was performed with inactivated whole virus antigen, and the optical density at each of five serial twofold dilutions of pre- and postimmunization sera was measured. The difference in the amount of ELISA antibody in pre- and postinoculation serum specimens was taken to be proportional to the area between the respective titration curves. The ELISA was more sensitive than the HI or NI test in detecting a seroresponse in volunteers infected with A/Hong Kong/123/77 (H1N1), A/New Jersey/8/76 (Hswine N1), or A/Alaska/6/77 (H3N2) ts recombinant virus. These results suggest that the ELISA should be used to determine the frequency of infection with attenuated viruses as well as the 50% human infectious dose of candidate live influenza A vaccine viruses.     

Improved colorimetric assay for detecting influenza B virus neutralizing antibody responses to vaccination and infection.

An automated neutralization test for influenza B virus is described in which antibody titers are determined according to the release of neutral red from infected or uninfected cells of the Madin-Darby canine kidney line. Endpoints are determined in a standard enzyme-linked immunosorbent assay reader. The test requires no expensive immunologic reagents and was used to evaluate responses to both vaccination and natural infection against influenza B virus. Overall responses to vaccination were comparable with those obtained by hemagglutination inhibition, using Tween-ether-split influenza B/Ann Arbor/1/86 virus as the antigen (the HI-TE test). The sensitivities of neutralization responses compared with those obtained by the HI-TE test for two vaccines were 88 and 89%; the specificities were lower at 61 and 60%, respectively. Responses to vaccination, measured by hemagglutination inhibition, were significantly higher with split virus compared with whole virus. However, seroconversion by both the HI-TE and neutralization tests was observed in 5 of 10 individuals from whom virus was detected by either culture of nasal or throat washings or the presence of antigen from immunofluorescence in cells from nasal washings.     

Evaluation of the Single Radial Hemolysis Test for Measuring Hemagglutinin- and Neuraminidase-Specific Antibodies to H3N2 Influenza Strains and Antibodies to Influenza B

Antibodies to the H3 hemagglutinin of influenza A virus could be specifically measured by single radial hemolysis (SRH) when test antigens were recombinant viruses containing the relevant H3 hemagglutinin antigen and irrelevant Neq1 neuraminidase of A/equine/Prague/1/56 virus. Antibodies to influenza B virus could also be measured by the SRH technique. Antibody rises to influenza A or B virus measured by SRH agreed with results of hemagglutination inhibition (HI) tests for about 80% of the sera tested, including sera from volunteers receiving killed influenza vaccine and sera from patients naturally infected with influenza. Correlation between antibody titers measured by SRH and HI was also good. Antibodies to the N2 neuraminidase of influenza A virus could be specifically measured by SRH when test antigens were recombinant viruses containing the relevant N2 neuraminidase antigen and irrelevant Heq1 hemagglutinin of A/equine/Prague/1/56 virus. The SRH test for neuraminidase antibodies was more strain specific than was the SRH test for hemagglutinin antibodies. Probably for this reason, agreement between neuraminidase antibody determinations in human sera by the SRH test and by the neuraminidase inhibition test was poorer than agreement between the SRH test for hemagglutinin antibodies and the HI test.     

甲型H1N1流感患者血清流感病毒血凝抑制抗体滴度变化分析

目的 了解甲型H1N1流感患者发病后体内流感病毒特异性血凝抑制抗体滴度变化.方法 采集28例甲型H1N1流感患者发病后不同时间的血清,采用血凝抑制试验测定其抗体滴度并分析.结果 患者距发病1、5、15、22、37、49天和58天的血凝抑制抗体滴度分别为5.36、9.39、39.02、57.99、137.92、55.19和57.99,患者的最高抗体滴度几何均数为148.55,其中96.4％( 27/28)的患者在病程中产生保护性抗体和发生抗体血清转换.结论 甲型H1N1流感患者能有效产生保护性水平的抗体,有较好的免疫反应.     

Antibody reactive in antibody-dependent cell-mediated cytotoxicity following influenza virus vaccination

The antibody reactive in antibody-dependent, cell-mediated cytotoxicity (ADCC) to influenza virus-infected cells was measured in two groups of seven volunteers each, before and after immunization with inactivated or live attenuated A/Victoria/3/75 influenza virus vaccines. Age-matched controls were seven adult individuals who experienced natural influenza infection due to A/Victoria/3/75-like virus strain. After inactivated whole influenza virus immunization all the subjects showed a significant rise of the antibody reactive in ADCC (from a mean value of 4.7% to 17.1% cytotoxicity, before and 5 weeks after immunization, respectively) as well as of hemagglutination inhibition (HI) antibody (fourfold or greater increase). These immune responses were similar to those observed among naturally infected controls. After live attenuated virus vaccination, no significant increase in titer of antibody reactive in ADCC was detected, even though the vaccine induced significant increase of HI antibody titer. Little correlation was found between ADCC and HI antibody rises in sera of recipients of inactivated virus vaccine and of naturally infected individuals, while, in live attenuated influenza virus vaccinees, the rise of HI antibody titer did not correspond to a significant increase of ADCC antibody titer; several subjects who developed a significant rise in ADCC antibody titer did not show significant variation in antibody to neuraminidase and/or to complement fixation influenza virus antigens.     

Cross-Protection in Mice After Immunization with H2N2, H3N2, and Heq2Neq2 Influenza Virus Strains

Mice were vaccinated with the influenza viruses A/Japan/57 (H2N2), A/Hong Kong/68 (H3N2), and A/Equi/Miami/63 (Heq2Neq2) and the hemagglutinin and neuraminidase recombinants derived from these viruses. After infection with the parent viruses, protection was compared with serological findings. It was found that influenza vaccine protects not only against infection with a strain identical or closely related to the vaccine strain, but against heterologous strains as well. Vaccination with Hong Kong/68 and its neuraminidase recombinant resulted in a heterologous neuraminidase inhibition titer against Japan/57 and in a protection against infection with Japan/57. By contrast, after vaccination with Japan/57 and its neuraminidase recombinant, no relevant heterologous neuraminidase inhibition titer against Hong Kong/68 was observed, whereas a protection against infection with Hong Kong/68 did exist. A cross-protection between Hong Kong/68 and Miami/63, but no relationship in the hemagglutination or neuraminidase inhibition tests, was established in the preinfection sera. A one-way antigenic relationship between these viruses was confirmed by the rise of hemagglutinin or neuraminidase antibodies against Hong Kong/68 in the postinfection sera. No cross-protection or serological relationship existed between Miami/63 and Japan/57. Besides the hemagglutinin and neuraminidase, a third factor, the “mouse-protecting antigen,” was considered to contribute to the protection obtained. According to the protection observed, the mouse-protecting antigen of Hong Kong/68 virus is related to that of Japan/57 as well as Miami/63 virus. The mouse-protecting antigens of both Japan/57 and Miami/63 are related to that of Hong Kong/68.     

Immunological potency of a subunit influenza vaccine in adults

Summary The immunological potency of a subunit influenza vaccine (from A/England/42/72 virus) and of two commercial whole-virus vaccines (containing either A/England/42/72 or A/Hong Kong/68 virus) was studied in adults in an industrial plant. Serum samples were taken in vaccinated and control non-vaccinated subjects prior to and three weeks and five months after the vaccination. Most of the vaccinees developed high levels of hemagglutination inhibiting antibodies against all the type A influenza viruses employed in the test; these antigens included a new strain (A/Dunedin/73) that had not previously circulated in Czechoslovakia. The antibody response after the subunit vaccine was somewhat better than after the whole-virus vaccines administration. The whole-virus vaccine from the A/England/42/72 virus was more efficient in inducing antibody response against the more recent isolates than the A/Hong Kong/68 virus vaccine.     

流行性感冒病毒裂解疫苗的安全性和免疫原性研究

目的 研究流行性感冒(流感)病毒裂解疫苗的安全性和免疫原性。方法 根据国家食品药品监督管理局2002SL0043号《新药临床研究申请批件》的要求，选择876例观察对象按随机双盲法分成试验组和对照组，观察局部反应和全身反应。用微量血凝抑制(H1)试验检验抗体应答反应，计算抗体阳转率、保护率及几何平均滴度(GMT)增长倍数。结果 试验疫苗和对照疫苗间全身和局部不良反应发生率差异无显著意义；两组间H1抗体阳转率、保护率及H3N2亚型和B型H1抗体GMT增长倍数差异无显著意义；然而，两种疫苗H1N1亚型的H1抗体GMT增长倍数间差异有显著意义。结论 两种疫苗均具有很好的安全性和免疫原性。     

Impaired serum antibody response to inactivated influenza A and B vaccine in renal transplant recipients.

Before and after vaccination with a commercial inactivated influenza vaccine containing A/Aichi/2/68 (H3N2) and B/Massachusetts/1/71 antigens, the serum hemagglutination inhibition antibody titers to homologous and heterologous strains of A and B influenza viruses were measured in 45 renal transplant patients and 66 healthy controls (62 for the B strains). At least a fourfold titer rise to the homologous A strain occurred in 14 of 45 transplant patients (31%) versus 37 of 66 controls (56%). Fourfold or greater heterologous A rises occurred in only 8 of 45 transplant patients (18%) compared with 40 of 60 controls (61%). In both the homologous and heterologous B responses, at least fourfold hemagglutination inhibition titer rises were seen in significantly fewer transplant patients than control subjects. In the transplant group, no correlation was observed between degree of antibody response and age, previous influenza vaccination, percentage of patients initially seronegative, time since transplantation, dose of immunosuppressive drugs, level of renal function, or nature of original renal disease.     

Immunity to influenza virus infection induced by heterologous,inactivated vaccines

The ability of inactivated influenza A vaccines to induce serum HI antibody and immunity to challenge infection was studied in hamsters and in volunteers. Groups of hamsters were immunized with 200 IU of influenza virus A/Scotland/74, A/Port Chalmers/73, A/England/72, or A/Hong Kong/68. The serum HI antibody response of animals to, and immunity to challenge infection was directly related to the known relationship between the vaccine and test viruses. Thus, hamsters given A/Hong Kong/68 or A/England/72 vaccine produced serum HI antibody and immunity to A/Hong Kong virus infection, and animals given A/Scotland/74, A/Port Chalmers/73, and A/England/72 produced antibody and immunity to A/Scotland infection.In a volunteer study, groups of students were immunized with 400 IU of the same vaccines as used above. The ability to infect these volunteers with WRL 105 virus given 4 weeks later was directly related to the vaccine-induced serum HI antibody to the challenge virus. The highest titers of serum HI antibody to A/Scotland virus were found in volunteers inoculated with homologous vaccine, lower titers were found in volunteers given A/Port Chalmers or A/England/ 72 vaccine and the lowest levels were seen in volunteers given A/Hong Kong/68 vaccine: the largest number of infections by the challenge virus was seen in volunteers given A/Hong Kong/68 vaccine, less were observed in volunteers given A/England/72 vaccine, and least were found in groups given A/Port Chalmers or A/Scotland/74 vaccine. Compared with the incidence of infection in volunteers given B/Hong Kong/73 vaccine, all groups given heterologous influenza A vaccines showed some immunity to challenge infection.     

Comparison of the hemagglutination inhibition procedure and an enzyme-linked immunosorbent assay for detection of specific antibodies to pneumonia virus of mice in experimentally infected laboratory rats.

An enzyme-linked immunosorbent assay (ELISA) and the hemagglutination inhibition (HI) test were used to evaluate the response of laboratory rats to experimental infection with pneumonia virus of mice. The ELISA procedure was more sensitive than the HI test and detected very low levels of antibodies early in the course of infection. At 5 days postinfection, ELISA detected antibody increases in five of five animals, whereas only two of five increases were detected by the HI test. At 9 days postinfection, the HI test failed to detect one titer increase measured by ELISA. Later during the course of infection, increases were detected by both tests. The ELISA procedure was, in general, more sensitive for detecting low levels of antibody than was the HI test, but was equal in sensitivity when high titers of antibody were measured.