Detection of antibody to avian influenza A (H5N1) virus in human serum by using a combination of serologic assays

From May to December 1997, 18 cases of mild to severe respiratory illness caused by avian influenza A (H5N1) viruses were identified in Hong Kong. The emergence of an avian virus in the human population prompted an epidemiological investigation to determine the extent of human-to-human transmission of the virus and risk factors associated with infection. The hemagglutination inhibition (HI) assay, the standard method for serologic detection of influenza virus infection in humans, has been shown to be less sensitive for the detection of antibodies induced by avian influenza viruses. Therefore, we developed a more sensitive microneutralization assay to detect antibodies to avian influenza in humans. Direct comparison of an HI assay and the microneutralization assay demonstrated that the latter was substantially more sensitive in detecting human antibodies to H5N1 virus in infected individuals. An H5-specific indirect enzyme-linked immunosorbent assay (ELISA) was also established to test children's sera. The sensitivity and specificity of the microneutralization assay were compared with those of an H5-specific indirect ELISA. When combined with a confirmatory H5-specific Western blot test, the specificities of both assays were improved. Maximum sensitivity (80%) and specificity (96%) for the detection of anti-H5 antibody in adults aged 18 to 59 years were achieved by using the microneutralization assay combined with Western blotting. Maximum sensitivity (100%) and specificity (100%) in detecting anti-H5 antibody in sera obtained from children less than 15 years of age were achieved by using ELISA combined with Western blotting. This new test algorithm is being used for the seroepidemiologic investigations of the avian H5N1 influenza outbreak.     

Sensitivity and specificity of serologic assays for detection of human infection with 2009 pandemic H1N1 virus in U.S. populations

Swine origin 2009 H1N1 influenza virus has spread globally to cause the first influenza pandemic of the 21st century. Serological studies can improve our understanding of the extent of human infection and risk factors associated with the transmission of this pandemic virus. The "gold standard" for serodiagnosis of human influenza virus infection is the detection of seroconversion between acute- and convalescent-stage samples. However, the timing of seroepidemiological investigations often precludes the collection of truly acute-phase sera, requiring development of serological criteria for evaluating convalescent-phase sera that optimize detection of true positives and true negatives. To guide seroepidemiological investigations into the spread of the novel 2009 pandemic H1N1 virus, we characterized serum antibody responses to 2009 H1N1 virus in 87 individuals with confirmed viral infection and 227 nonexposed U.S. individuals using microneutralization (MN) and hemagglutination inhibition (HI) assays. Sensitivity and specificity were determined for each assay alone and in combination for detection of 2009 H1N1 virus-specific antibodies in convalescent-phase sera. Although the HI assay was more specific for detecting antibody to 2009 H1N1, the MN assay was more sensitive, particularly for detecting low-titer seroconversions. A combination of titers (MN ≥ 40 and HI ≥ 20) provided the highest sensitivity (90%) and specificity (96%) for individuals aged <60 years and 92% specificity for adults aged ≥ 60 years for detection of serologically confirmed 2009 H1N1 infections in U.S. populations during the first pandemic waves. These studies provide an approach to optimize timely serological investigations for future pandemics or outbreaks of novel influenza viruses among humans.     

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.     

Increased sensitivity for detecting avian influenza-specific antibodies by a modified hemagglutination inhibition assay using horse erythrocytes

The hemagglutination inhibition (HI) assay is a widely used serological method to measure the levels of protective antibody responses against influenza viruses. However, the traditional HI assay which uses chicken erythrocytes is not sufficiently sensitive for detecting HI antibodies specific to avian influenza viruses. Previously, it was demonstrated that employing an assay using horse erythrocytes was able to increase the sensitivity of HI assay. The current report describes further optimization of this modified HI assay. It was shown that this method was able to increase detection of HI activities in rabbit sera immunized with H5 HA antigens, and proved that this increased sensitivity is useful in dissecting the strain specificity of HI antibody responses. In addition, the modified HI assay using horse erythrocytes increased the sensitivity of detecting HI antibodies specific for three major serotypes of avian influenza viruses, H5, H7 and H9, in people who may have asymptomatic infection with avian influenza viruses. Based on these results, the optimized use of horse erythrocytes should be standard practice for detecting HI activities against avian influenza viruses.     

Serum antibody responses in naturally occurring influenza A virus infection determined by enzyme-linked immunosorbent assay, hemagglutination inhibition, and complement fixation

Serum antibody responses to influenza A virus infection were examined in 388 normal subjects during a trial of chemoprophylaxis in an outbreak of influenza A in 1980-1981 in which both A/H1N1 and A/H3N2 viruses circulated. Paired serum specimens obtained over a 6-week period were tested for antibodies to both A/H1N1 and A/H3N2 viruses by conventional hemagglutination inhibition, complement fixation, and an enzyme-linked immunosorbent assay (ELISA). Antibody responses detected by ELISA were determined by calculation of the area generated between titration curves of paired sera (area method), as well as by a conventional endpoint dilution method (endpoint method). Forty-two significant antibody rises were detected; 42 by ELISA (area method), 33 by ELISA (endpoint method), 32 by hemagglutination inhibition, and 13 by complement fixation. ELISA (area method) detected rises more frequently than either ELISA (endpoint method) (P less than 0.01), hemagglutination inhibition (P less than 0.005), or complement fixation (P less than 0.001). Another sensitive assay, the microneutralization test, detected significantly fewer rises (33, P less than 0.025) than the ELISA (area method). In the 42 subjects with ELISA (area method) rises, corroborating evidence of influenza A infection by other techniques (virus isolation, microneutralization, hemagglutination inhibition, or complement fixation tests) were available for 39 (93%). ELISA (area method) rises were subtype specific in all serum pairs in which other documentation of subtype-specific infection was available (38 of 38). Thus, ELISA (area method) was the single most sensitive assay for detection of serum antibody rises in this setting and possessed a high degree of subtype specificity.     

ts P1 and P3 genes are responsible for satisfactory level of attenuation of ts-1A2 recombinants bearing H1N1 or H3N2 surface antigens of influenza a virus

To produce live, temperature-sensitive (ts) influenza A virus vaccines, the A/Udorn/72-ts-1A2 donor strain, which has is lesions in the genes coding for the P3 and P1 polymerase proteins, was mated with three wild-type viruses: A/Victoria/3/75 (H3N2), A/Alaskal6/77 (H3N2), and A/Hong Kong/123/77 (H1N1). From each mating a vaccine strain was selected which contained the two ts genes from the 1A2 parent and the hemagglutinin and neuraminidase genes from the wild-type parent. The three ts-1A2 recombinant viruses were given to adult seronegative volunteers and each was found to be satisfactorily attenuated. The parental origin of genes in each ts-1A2 recombinant was identified to determine which genes from the A/Udorn/72-ts-1A2 parent were responsible for the attenuation of the wild-type influenza A viruses. Only the ts-1A2 P3 and P1 genes were required to produce a satisfactory level of attenuation of the wild-type viruses for adults.     

A contribution of cellular immunity to protection against influenza in man

The degree of lymphocyte transformations and leukocyte migration inhibition (LMI) in the presence of inactivated A/Scotland/74 (H3N2) influenza virus vaccine was measured in blood samples collected from 56 medical student volunteers. At the same time the volunteers were skin tested, using the same vaccine. Using the antigenically similar WRL 105 (H3N2), recombinant influenza virus, the level of haemagglutination-inhibiting (HI) antibodies in serum, and neutralizing antibodies in nasal washings collected from the volunteers, were also determined. Each volunteer was then inoculated with live, attenuated WRL 105 influenza virus vaccine and infections demonstrated by virus isolations and serology.Correlations between the ability to infect the volunteers and the various parameters of humoral and cellular immunity were then determined. The results showed a good correlation between the level of serum HI antibody and infection. Thus 16 of 20 volunteers with serum HI antibody titres of 110, but only 6 of 20 volunteers with antibody levels of 130, showed evidence of infection. No direct correlation was observed between any of the other parameters measured and infection by WRL 105 virus. However, when the LMI and serum HI antibody levels were considered together, a contribution of cellular immunity, as measured by the LMI test, could be found. Of 19 volunteers with low serum HI antibody and low LMI levels, 16 were infected, whereas of 13 volunteers with low HI antibody, but with high LMI levels, only 6 showed evidence of infection with WRL 105 influenza virus.     

Serological survey of antibodies to influenza A viruses in a group of people without a history of influenza vaccination

A serological survey for antibodies to influenza viruses was performed in China on a group of people without a history of influenza vaccination. Using the haemagglutination inhibition (HI) assay, we found seropositivity rates for seasonal H3N2 to be significantly higher than those for seasonal H1N1. Samples positive for antibodies to the pandemic (H1N1) 2009 virus increased from 0.6% pre-outbreak to 4.5% (p <0.01) at 1 year post-outbreak. Interestingly, HI and neutralization tests showed that 1.4% of people in the group have antibodies recognizing H9N2 avian influenza viruses, suggesting that infection with this subtype may be more common than previously thought.     

Genetic and Antigenic Typing of Seasonal Influenza Virus Breakthrough Cases from a 2008-2009 Vaccine Efficacy Trial

Estimations of the effectiveness of vaccines against seasonal influenza virus are guided by comparisons of the antigenicities between influenza virus isolates from clinical breakthrough cases with strains included in a vaccine. This study examined whether the prediction of antigenicity using a sequence analysis of the hemagglutinin (HA) gene-encoded HA1 domain is a simpler alternative to using the conventional hemagglutination inhibition (HI) assay, which requires influenza virus culturing. Specimens were taken from breakthrough cases that occurred in a trivalent influenza virus vaccine efficacy trial involving >43,000 participants during the 2008-2009 season. A total of 498 influenza viruses were successfully subtyped as A(H3N2) (380 viruses), A(H1N1) (29 viruses), B(Yamagata) (23 viruses), and B(Victoria) (66 viruses) from 603 PCR- or culture-confirmed specimens. Unlike the B strains, most A(H3N2) (377 viruses) and all A(H1N1) viruses were classified as homologous to the respective vaccine strains based on their HA1 domain nucleic acid sequence. HI titers relative to the respective vaccine strains and PCR subtyping were determined for 48% (182/380) of A(H3N2) and 86% (25/29) of A(H1N1) viruses. Eighty-four percent of the A(H3N2) and A(H1N1) viruses classified as homologous by sequence were matched to the respective vaccine strains by HI testing. However, these homologous A(H3N2) and A(H1N1) viruses displayed a wide range of relative HI titers. Therefore, although PCR is a sensitive diagnostic method for confirming influenza virus cases, HA1 sequence analysis appeared to be of limited value in accurately predicting antigenicity; hence, it may be inappropriate to classify clinical specimens as homologous or heterologous to the vaccine strain for estimating vaccine efficacy in a prospective clinical trial.     

Evaluation of live avian-human reassortant influenza A H3N2 and H1N1 virus vaccines in seronegative adult volunteers.

An avian-human reassortant influenza A virus deriving its genes coding for the hemagglutinin and neuraminidase from the human influenza A/Washington/897/80 (H3N2) virus and its six "internal" genes from the avian influenza A/Mallard/NY/6750/78 (H2N2) virus (i.e., a six-gene reassortant) was previously shown to be safe, infectious, nontransmissible, and immunogenic as a live virus vaccine in adult humans. Two additional six-gene avian-human reassortant influenza viruses derived from the mating of wild-type human influenza A/California/10/78 (H1N1) and A/Korea/1/82 (H3N2) viruses with the avian influenza A/Mallard/NY/78 virus were evaluated in seronegative (hemagglutination inhibition titer, less than or equal to 1:8) adult volunteers for safety, infectivity, and immunogenicity to determine whether human influenza A viruses can be reproducibly attenuated by the transfer of the six internal genes of the avian influenza A/Mallard/NY/78 virus. The 50% human infectious dose was 10(4.9) 50% tissue culture infectious doses for the H1N1 reassortant virus and 10(5.4) 50% tissue culture infectious doses for the H3N2 reassortant virus. Both reassortants were satisfactorily attenuated with only 5% (H1N1) and 2% (H3N2) of infected vaccines receiving less than 400 50% human infectious doses developing illness. Consistent with this level of attenuation, the magnitude of viral shedding after inoculation was reduced 100-fold (H1N1) to 10,000-fold (H3N2) compared with that produced by wild-type virus. The duration of virus shedding by vaccines was one-third that of controls receiving wild-type virus. At 40 to 100 50% human infectious doses, virus-specific immune responses were seen in 77 to 93% of volunteers. When vaccinees who has received 10(7.5) 50% tissue culture infectious doses of the H3N2 vaccine were experimentally challenged with a homologous wild-type human virus only 2 of 19 (11%) vaccinees became ill compared with 7 of 14 (50%) unvaccinated seronegative controls ( P < 0.025; protective efficacy, 79%). Thus, three different virulent human influenza A viruses have been satisfactorily attenuated by the acquisition of the six internal genes of the avian influenza A/Mallard/NY/78 virus. The observation that this donor virus can reproducibly attenuate human influenza A viruses indicates that avian-human influenza A reassortants should be further studied as potential live influenza A virus vaccines.     

Antigenic variation of H1N1, H1N2 and H3N2 swine influenza viruses in Japan and Vietnam

The antigenicity of the influenza A virus hemagglutinin is responsible for vaccine efficacy in protecting pigs against swine influenza virus (SIV) infection. However, the antigenicity of SIV strains currently circulating in Japan and Vietnam has not been well characterized. We examined the antigenicity of classical H1 SIVs, pandemic A(H1N1)2009 (A(H1N1)pdm09) viruses, and seasonal human-lineage SIVs isolated in Japan and Vietnam. A hemagglutination inhibition (HI) assay was used to determine antigenic differences that differentiate the recent Japanese H1N2 and H3N2 SIVs from the H1N1 and H3N2 domestic vaccine strains. Minor antigenic variation between pig A(H1N1)pdm09 viruses was evident by HI assay using 13 mAbs raised against homologous virus. A Vietnamese H1N2 SIV, whose H1 gene originated from a human strain in the mid-2000s, reacted poorly with post-infection ferret serum against human vaccine strains from 2000-2010. These results provide useful information for selection of optimal strains for SIV vaccine production.     

Serological diagnosis of influenza A/USSR/77 H1N1 infection: value of ELISA compared to other antibody techniques

Serologic diagnosis of influenza is an important but imperfect tool. During an outbreak of natural H1N1 A/USSR/77 infection, volunteers who received either amantadine, rimantadine, or placebo were tested to determine serologic response to infection by four different antibody techniques. Hemagglutination inhibition (HAI) and complement fixation (CF) were least sensitive, detecting only about half of the virus-positive subjects, whereas neutralization detected 81% and enzyme-linked immune peroxidase (ELISA) detected 95%. Failure to detect significant antibody response was associated with a higher titer of antibody in acute serum specimens and with a history of receipt of A/New Jersey/76 Hsw1N1 vaccine. Although antibody response measured by ELISA was of lower magnitude in vaccinees, it still was sufficient to be diagnostic. Thus, in situations where there is no access to viral isolation facilities, ELISA antibody techniques appear to be an excellent measure of assessing the rate of influenza infection.     

Suboptimal Humoral Immune Response against Influenza A(H7N9) Virus Is Related to Its Internal Genes

Influenza A(H7N9) virus pneumonia is associated with a high case fatality rate in humans. Multiple viral factors have been postulated to account for the high virulence of the virus. It has been reported that patients with influenza A(H7N9) virus infection have relatively low titers of neutralizing antibodies compared to those with seasonal influenza virus infections. In this study, we compared serum hemagglutination inhibition (HI) and microneutralization (MN) antibody titers of mice challenged with wild-type A(H7N9) viruses [H7N9(Anhui) and H7N9(Zhejiang)], an A(H1N1)pdm09 virus [pH1N1(2009)], and a recombinant A(H7N9) virus with PR8/H1N1 internal genes (rg-PR8-H7-N9). All mice infected by H7N9(Anhui) and H7N9(Zhejiang) developed serum HI antibodies at 14 days postinfection (dpi) but no detectable MN antibodies, even at 28 dpi. A low level of neutralizing activity was detected in H7N9(Anhui)- and H7N9(Zhejiang)-infected mice using fluorescent focus MN assay, but convalescent-phase serum samples obtained from H7N9(Anhui)-infected mice did not reduce the mortality of naive mice after homologous virus challenge. Reinfection with homologous A(H7N9) virus induced higher HI and MN titers than first infection. In contrast, pH1N1(2009) virus infection induced robust HI and MN antibody responses, even during the first infection. Moreover, rg-PR8-H7-N9 induced significantly higher HI and MN antibody titers than H7N9(Zhejiang). In conclusion, the internal genes of A(H7N9) virus can affect the humoral immune response against homologous viral surface proteins, which may also contribute to the virulence of A(H7N9) virus.     

Profiling of humoral immune responses to influenza viruses by using protein microarray

The emergence of pandemic A(H1N1) 2009 influenza showed the importance of rapid assessment of the degree of immunity in the population, the rate of asymptomatic infection, the spread of infection in households, effects of control measures, and ability of candidate vaccines to produce a response in different age groups. A limitation lies in the available assay repertoire: reference standard methods for measuring antibodies to influenza virus are haemagglutination inhibition (HI) assays and virus neutralization tests. Both assays are difficult to standardize and may be too specific to assess possible partial humoral immunity from previous exposures. Here, we describe the use of antigen-microarrays to measure antibodies to HA1 antigens from seven recent and historical seasonal H1, H2 and H3 influenza viruses, the A(H1N1) 2009 pandemic influenza virus, and three avian influenza viruses. We assessed antibody profiles in 18 adult patients infected with A(H1N1) 2009 influenza virus during the recent pandemic, and 21 children sampled before and after the pandemic, against background reactivity observed in 122 persons sampled in 2008, a season dominated by seasonal A(H1N1) influenza virus. We show that subtype-specific and variant-specific antibody responses can be measured, confirming serological responses measured by HI. Comparison of profiles from persons with similar HI response showed that the magnitude and broadness of response to individual influenza subtype antigens differs greatly between individuals. Clinical and vaccination studies, but also exposure studies, should take these findings into consideration, as they may indicate some level of humoral immunity not measured by HI assays.     

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.     

Immunity to influenza in ferrets

Ferrets were infected with recombinant influenza A viruses which possessed either the haemagglutinin or neuraminidase antigens of A/Hong Kong/68 influenza virus. After five weeks the immunity of the animals was challenged by infection with A/HK/68 virus. Immunity to challenge infection was greatest in those ferrets with serum HI antibody to A/HK/68; the presence of NI antibody conferred a measurably lower degree of immunity. A small degree of heterotypic immunity was observed following challenge infection of ferrets previously infected with influenza virus A/PR/8/34, although the surface antigens of this virus are completely different from those of A/HK/68. Experiments in which ferrets were infected with A/HK/68 virus and subsequently challenged with the recombinant viruses confirmed the results of the first experiment.     

Temperature-Sensitive Mutants of Influenza A Virus: Transfer of the Two Temperature-Sensitive Lesions in the Udorn/72-ts-1A2 Virus to the A/Hong Kong/123/77 (H1N1) Wild-Type Virus

The influenza A/Udorn/72-ts-1A2 virus possesses temperature-sensitive mutations in the genes coding for the P1 and P3 polymerase proteins. It is being evaluated as a donor of its attenuating temperature-sensitive genes to produce recombinant live vaccine strains of epidemic variants of influenza A virus. Transfer of the P1 and P3 genes to two viruses within the H3N2 subtype of influenza A virus (i.e., the A/Victoria/3/75 and A/Alaska/6/77 viruses) conferred on each variant the following properties: (i) 37°C shutoff temperature for plaque formation, (ii) almost complete restriction of viral replication in the lungs, (iii) a 100-fold restriction of viral replication in the nasal turbinates, and (iv) genetic stability after replication in hamsters. This study was undertaken to determine whether the transfer of the two ts-1A2 temperature-sensitive genes into a virus belonging to the H1N1 subtype (i.e., the A/Hong Kong/123/77 virus) would result in a restriction of replication in vitro and in vivo comparable to that observed with the previously studied H3N2 recombinant viruses in hamsters. This was found to be the case. In addition, infection of hamsters with the A/Hong Kong/77-ts-1A2 virus induced significant resistance to infection with wild-type A/Hong Kong/77 virus. Thus, the two ts-1A2 temperature-sensitive genes attenuated influenza A viruses belonging to two distinct subtypes to a specific and predictable level. An unexpected genetic interaction was observed between several A/Hong Kong/77-ts-1A2 segregants bearing the group 5 (P1) temperature-sensitive lesion. One interpretation of these results is that intracistronic complementation occurred between these segregants.     

Establishment of sandwich ELISA for detecting the H7 subtype influenza A virus

Avian H7N9 subtype influenza virus infects human with high case-fatality rate since it emerged in 2013. Although the vaccination has been rapidly used in poultry due to the emergence of highly pathogenic strain, this virus remains prevalent in this region. Thus, rapid diagnosis both in poultry and human clinic is critically important for the control and prevention of H7N9 infection. In this study, a batch of H7 subtype-specific monoclonal antibodies (mAbs) were developed and a pair of mAb, 2B6, and 5E9 were used to establish a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) to quantify H7 protein and detect influenza A virus baring H7 subtype HA. The lowest detection limit for the recombinant H7 protein was 10 ng/mL and 0.5 HAU/50 μL of A/Guangdong/17SF003/2016(H7N9), 2 HAU/50 μL of A/Netherlands/219/2003(H7N7) and A/Anhui/1/2013(H7N9) for live virus, respectively. The ELISA could not only detect the prevailing H7N9 virus, but also antigenic drift H7 subtype viruses, showing excellent sensitivity and high specificity. Hence, it could serve as a valuable approach to diagnose H7 subtype virus which showed great potential to cause pandemic, as well as antigen quantification.