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.     

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.     

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.     

E-rosette forming cells and humoral antibody titres in humans after vaccination with three different inactivated influenza virus vaccines A/USSR/92/77 (H1N1)

The number of E-rosette forming cells and the serum haemagglutination inhibition (HI) antibody titres were examined in 37 volunteers immediately before and 14, 28, 35 and 63 days after immunization with three inactivated influenza virus vaccines A/USSR/92/77 (H1N1)--NIB 6 and in 11 non-vaccinated controls. From the former, 10 volunteers were immunized with 1000 haemagglutinin (HA) IU per dose, 11 volunteers with the NIB 6 adsorbate vaccine (340 HA IU/dose) and 16 volunteers with a bivalent vaccine composed of 180 HA IU/dose NIB 6 and 180 HA IU/dose of influenza virus A/Bangkok X-73 (H3N2). The percentage of E-rosette forming cells was decreased in all vaccinated volunteers 14 days after vaccination; later on the values reached normal level of non-vaccinated controls or of subjects before vaccination. The number of E-rosette forming cells was in correlation with the applied virus vaccine dose, i.e. for the 1000 HA IU/dose: 29.95 +/- 11.74%, p less than 0.001 and for the 340 HA IU/dose: 47.75 +/- 11.15%, p less than 0.005; however, after administration of 180 HA IU/dose of NIB 6 in the bivalent vaccine, the value 58.65 +/- 11.5% was not significantly decreased in comparison to non-vaccinated donors. The serum HI antibody titres reached the highest level 14 days after vaccination and remained constant during the next 6 weeks. There was a correlation between decreased E-rosette values and increased serum antibody titres (p less than 0.05). The current study indicates that the number of E-rosette forming cells may serve as a further laboratory criterion for controlling the effect of inactivated influenza virus vaccines on the immune system of man.     

Quantitative estimation of Newcastle disease virus antibody levels in chickens and turkeys by ELISA

A quantitative single-well ELISA for estimation of Newcastle disease (ND) virus antibodies in chickens and turkeys was developed using purified antigen from PMV-1/Chicken/Ulster 2C/71. The test was standardised using sera from 20-week-old chickens or 20- 30-week-old turkeys. Absorbance values for negative sera in chickens increased with the age of the birds but overall was lower than the cut-off for the test. ND haemagglutination inhibition (HI) positive field sera were always positive by ELISA and the mean was significantly higher than that of the negative population. Standard antisera to four of seven of the other PMV serotypes (including PMV-3) gave positive reactions in ELISA and three were also positive at low level by PMV-1 HI test. Absorbance values remained negative in turkeys given two inoculations of PMV-3 vaccine in spite of good PMV-3 HI responses. Doubling dilutions of chicken and turkey sera were tested by ELISA and end-point titres calculated. Standard curves relating ELISA titre and absorbance of each sample at 1/100 dilution were constructed and used to determine titres of test samples from single well absorbance values. A significant positive correlation between ELISA titre and HI titre for chicken and turkey sera was demonstrated. Sensitivity of the test was investigated using birds experimentally infected with PMV-1/Chicken/Ulster 2C/71 or a pigeon PMV-1 isolate. Seroconversion was detected at the same time by ELISA and HI. In experiments to estimate the ELISA titre required to protect birds against virulent ND, five groups of chickens were vaccinated twice with one of four commercial ND vaccines (three inactivated; one live) on two occasions and challenged with a virulent ND strain (PMV-1/ Chicken/Antrim/73). Two of the groups vaccinated with inactivated vaccines were protected against challenge. In another group also given inactivated vaccine, clinical signs were seen in one bird and ELISA proved a better indicator of immune status than HI. In the groups given living vaccine, no signs of ND were seen and ELISA indicated a much higher level of vaccinal antibody than HI test. In turkeys given two inoculations with inactivated vaccine, antibody levels were boosted to acceptable levels by ELISA and HI indicating that vaccinal antibody levels were adequate.     

Immunity following intranasal administration of an inactivated,freeze-dried A/England/42/72 vaccine

A group of 23 student volunteers were each inoculated intranasally with 400 IU of inactivated, freeze-dried A/England/42/72 vaccine. Only one volunteer showed a four-fold rise in serum HI antibody following immunization, and the mean increase in serum HI antibody (gmt) for all volunteers did not increase two-fold. Thirteen of the volunteers developed detectable levels of nasal wash neutralizing antibody after immunization; local antibody was most commonly found in volunteers who produced a detectable but less than four-fold fise in serum antibody titre, and who produced nasal washings with relatively high concentrations of protein and secretory IgA. Four weeks after immunization, the vaccinees and a matched group of control subjects were inoculated with attenuated A/England/42/72 (MRC-7) virus. Evidence of infection was found in 14 of 23 (61 per cent) of control subjects and in seven of 23 (30 per cent) of immunized volunteers. This result showed a significant protection (P = 0.04) against challenge virus infection for volunteers given intranasal vaccine.     

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.     

Serodiagnosis of La Crosse virus infections in humans by detection of immunoglobulin M class antibodies.

Sera from 92 humans with illnesses clinically compatible with those caused by California serogroup virus infections were tested for antibody to La Crosse (LAC) virus by using the immunoglobulin M (IgM) antibody-capture enzyme-linked immunosorbent assay (MAC ELISA), the IgG ELISA, and the hemagglutination inhibition (HI), complement fixation and serum dilution-plaque reduction neutralization tests. On the reported day of onset of illness in 18 individuals, 94% had IgM antibody, 50% had neutralization antibody, 33% had HI antibody, and 11% had IgG antibody. Neutralization, HI, and IgG antibody prevalence rates increased thereafter, whereas IgM antibody prevalence remained high (92% 2 or more weeks after the onset of illness). It was concluded that the MAC ELISA is a sensitive test for the presence of antibody to LAC virus. The sensitivity of the MAC ELISA and the rapidity with which it can be performed appear to provide a powerful tool for the clinically relevant serodiagnosis of LAC virus infections in humans.     

Oral Fluids as a Live-Animal Sample Source for Evaluating Cross-Reactivity and Cross-Protection following Intranasal Influenza A Virus Vaccination in Pigs

In North American swine, there are numerous antigenically distinct H1 influenza A virus (IAV) variants currently circulating, making vaccine development difficult due to the inability to formulate a vaccine that provides broad cross-protection. Experimentally, live-attenuated influenza virus (LAIV) vaccines demonstrate increased cross-protection compared to inactivated vaccines. However, there is no standardized assay to predict cross-protection following LAIV vaccination. Hemagglutination-inhibiting (HI) antibody in serum is the gold standard correlate of protection following IAV vaccination. LAIV vaccination does not induce a robust serum HI antibody titer; however, a local mucosal antibody response is elicited. Thus, a live-animal sample source that could be used to evaluate LAIV immunogenicity and cross-protection is needed. Here, we evaluated the use of oral fluids (OF) and nasal wash (NW) collected after IAV inoculation as a live-animal sample source in an enzyme-linked immunosorbent assay (ELISA) to predict cross-protection in comparison to traditional serology. Both live-virus exposure and LAIV vaccination provided heterologous protection, though protection was greatest against more closely phylogenetically related viruses. IAV-specific IgA was detected in NW and OF samples and was cross-reactive to representative IAV from each H1 cluster. Endpoint titers of cross-reactive IgA in OF from pigs exposed to live virus was associated with heterologous protection. While LAIV vaccination provided significant protection, LAIV immunogenicity was reduced compared to live-virus exposure. These data suggest that OF from pigs inoculated with wild-type IAV, with surface genes that match the LAIV seed strain, could be used in an ELISA to assess cross-protection and the antigenic relatedness of circulating and emerging IAV in swine.     

The immune response of hamsters to purified haemagglutinins and whole influenza virus vaccines following live influenza virus infection

The ability of several, live type A influenza viruses to enhance the serum haemagglutination-inhibiting (HI) antibody response of hamsters to subsequent immunization with inactivated, heterotypic influenza virus vaccines was examined. Live influenza viruses were found to vary in their priming ability for a given vaccine, and a given virus was not able to prime for all inactivated vaccines to an equal extent. Common determinants in the haemagglutinin antigens of the priming virus and the vaccine virus were suggested as responsible for the enhancement of the antibody response to some of the vaccines, but for other pairs of viruses the haemagglutinin antigens were distinct. Thus, enhancement in these instances cannot be due to cross-reacting haemagglutinins. Pre-infection of hamsters by several influenza type A viruses was employed in an attempt to enhance the serum HI antibody response to purified, haemagglutinin antigens prepared from A/PR/8/34 and the MRC-2 recombinant strain of A/England/42/72 viruses. Although prior infection enhanced the antibody response to whole virus, this was not demonstrable for the purified haemagglutinin components of the virus. The possible reasons for this are discussed.     

Enzyme-linked immunosorbent assay for measurement of antibody against cytomegalovirus and rubella virus in a single serum dilution.

Enzyme-linked immunosorbent assays (ELISA) were developed for quantifying cytomegalovirus (CMV) and rubella antibodies using a single serum dilution (1/800) in conjunction with a standard curve. A near linear relation was found between the logarithms of absorbance values of sera at a dilution of 1/800 and the titres as determined by an end point dilution ELISA. The reproducibility of the single dilution ELISA was good; the within-test coefficients of variation averaged 7.5% for CMV antibody and 12.4% for rubella antibody. A close correlation was found between ELISA and complement-fixing (CF) antibody titres to CMV and between ELISA and haemagglutination-inhibition (HI) antibody titres to rubella virus. The titres in ELISA were 200 to 1000 times higher than in CF for CMV and 50 to 100 times higher than in HI for rubella virus.     

Hemagglutination-Inhibition Test in Rhinovirus Infections of Volunteers

The hemagglutination-inhibition (HI) test for antirhinovirus antibody was carried out on paired sera from volunteers inoculated with rhinovirus type 3 or type 4 (RV4). The HI test gave results which paralleled the neutralization test and was at least as sensitive as a microneutralization method for detection of serotype-specific antibody. Although high levels of HI antibody in the serum were associated with protection from infection, in the case of RV4 low serum HI antibody levels did not necessarily imply susceptibility to challenge with small doses of virus. HI activity could be measured in concentrated nasal-washing fluids, and this antibody also seemed relevant to protection against infection.     

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.     

Comparison of the enzyme-linked immunosorbent assay (elisa), haemagglutination inhibition test and agar gel precipitation test for detection of antibodies to avian infectious bronchitis virus

The immune response after vaccination with infectious bronchitis virus (IBV) under field conditions was measured by the ELISA, haemagglu-tination-inhibition (HI) and agar-gel precipitin (AGP), tests. Vaccinations were performed in three flocks and one experimental group via the drinking water with the vaccine strains H 120 and H 52. In each flock 40 random serum samples were taken every 2 weeks and tested individually. In the experimental group blood samples were collected every week from each of the 10 chickens. The primary vaccination with H 120 resulted in a rapid increase of antibody titre as detected by ELISA followed by a slow decrease over the next few weeks. By the HI and AGP tests no antibody responses could be seen after this primary vaccination. Revaccination with the H 52 strain provoked a further increase in ELISA titres. In the experimental group, and in flock W, a similar increase occurred by the HI test and precipitating antibodies appeared. The formation of HI antibodies in flock T (nipple waterers) was somewhat retarded and precipitating antibodies were just detectable. In flock F revaccination did not result in the immediate production of HI and AGP antibodies. However, 6 weeks after revaccination a significant rise in ELISA, HI and AGP antibodies was observed, probably as the result of a field infection. It was demonstrated that, based on the higher sensitivity, the ELISA test is more suitable than HI and AGP to monitor antibody responses to vaccination against infectious bronchitis. Strain specificity in the HI test is discussed as a reason for its failure to detect antibodies after primary vaccination with the highly attenuated vaccine strain H 120.     

Determination of antibody response to influenza virus surface glycoproteins by kinetic enzyme-linked immunosorbent assay.

We modified an existing enzyme-linked immunosorbent assay (ELISA) to be able to use new spectrophotometers which can measure the rate of color development in microtiter wells. This new kinetic-based ELISA (KELISA) required only a single dilution of specimen rather than the multiple dilutions required with endpoint ELISA. In addition, 10- to 100-fold-less specimen was required to perform the KELISA than the ELISA. The level of serum or nasal wash antibody against surface glycoproteins of influenza A or influenza B viruses determined by KELISA was reproducible and correlated highly with the results of endpoint ELISA or hemagglutination inhibition tests. The difference between the KELISA rates, which indicated than an antibody response to infection had occurred, was defined and was analogous to a 2.2-fold rise in titer for serum and a 3.4-fold rise in titer for nasal wash determined by endpoint ELISA. The KELISA was similar to endpoint ELISAs in its ability to detect rises in antibody level in paired serum or nasal wash specimens obtained from volunteers who received live attenuated influenza A reassortant virus vaccines. By eliminating the need for multiple dilutions, the use of KELISA offers the advantage of increasing the number of assays that can be performed by the same personnel compared with endpoint ELISA, while it maintains sensitivity and specificity.     

Antibody response after application of a new live attenuated mumps vaccine (Pariorix®) measured by enzyme-linked immunosorbent assay (ELISA)

Mumps virus specific antibody concentrations were determined by enzyme-linked immunosorbent assay (ELISA), hemolysis-in-gel (HIG) test, and hemagglutination-inhibition (HI) test in 17 adult volunteers before and after vaccination with a new live mumps vaccine, Pariorix®. The results of the present study indicate that the strain Urabe-Am 9 of the mumps virus (Pariorix) induces antibodies in 100% of seronegative adults when measured by ELISA and in 70.6% and 64.7% in HIG and HI, respectively. ELISA is therefore a very sensitive and reliable method for the demonstration of sero-conversion after vaccination. The vaccine was well tolerated and clinically acceptable in the group of adult volunteers.     

Comparison of immunofluorescence and hemagglutination inhibition tests and enzyme-linked immunosorbent assay for detection of serum antibody in rats infected with hemorrhagic fever with renal syndrome virus.

Serum antibodies against the B-1 strain of hemorrhagic fever with renal syndrome (HFRS) virus in wild and experimental rats were investigated by the indirect immunofluorescent antibody (IF) test, the hemagglutination inhibition (HI) test, and an enzyme-linked immunosorbent assay (ELISA). In the newly developed ELISA, monoclonal antibodies to the B-1 strain were applied as coating antibody. In sera from wild rats, the results obtained by the ELISA agreed quite well with those obtained by the HI test, but some serum samples that gave a negative reaction in the HI test gave a positive reaction in the IF test, although their IF titers were very low. In serum samples from experimental rats that had been kept in an animal house infected with HFRS virus, a group with high titers and a group with low or negative titers were clearly differentiated by all the tests.     

Impact of influenza vaccine formulation with a detailed analysis of the cytokine response

Vaccination provides the most effective method of limiting the impact of influenza. Inactivated influenza vaccines are available in three formulations and more information needs to be generated on how antigen presented in different vaccine formulations influences the subsequent immune response. In the present study, we have investigated the effect of two different influenza vaccine formulations on the resulting antibody and cytokine responses and compared these responses with influenza infection. Mice were vaccinated intramuscularly with one or two doses of whole or split virus vaccine or alternatively intranasally infected with influenza virus. Lymphocytes were isolated from spleen cells and stimulated in vitro for 24 or 72 h for analysis of cytokine profile at the gene expression and at the protein level. Additionally, whole blood was collected and the serum antibody response investigated by haemagglutination inhibition (HI) and enzyme-linked immunosorbent assay (ELISA). We found that one dose of whole virus vaccine induced higher antibody and cytokine responses and thus was more immunogenic in unprimed mice than split virus vaccine. Whole virus vaccine induced a strong IFN-gamma (type 1) immune response after one dose of vaccine and a more mixed cytokine response after the second dose. Split virus vaccine induced a type 2 response, particularly after two vaccine doses. Our results show that two doses of vaccine (both vaccine formulation) were more effective in induction of Th2 type of cytokines and thus indicate that both the formulation and also the number of vaccine doses substantially influences the magnitude and quality of the immune response.     

The use of homologous virus in the haemagglutination-inhibition assay after vaccination with Newcastle disease virus strain La Sota or Clone30 leads to an over estimation of protective serum antibody titres.

We evaluated the influence of the use of the Newcastle disease virus (NDV)-strains Ulster and La Sota in the haemagglutination inhibition (HI) assay for the measurement of antibody titres after NDV vaccination. The use of the homologous La Sota antigen in the HI assay after Clone30 and La Sota vaccination of SPF-chickens, resulted in significantly higher titres than the use of the heterologous Ulster virus. The mean difference was 1.4 on a log 2 scale (2.6-fold). A significant difference was also found in virus neutralization (VN) assays. The virus strain in the HI or VN test did not influence the resulting titres after Ulster vaccination. When HI antibody titres after vaccination were related to VN titres measured with virulent Herts NDV or to survival after virulent challenge, it was found that the use of La Sota antigen in the HI assay after vaccination with Clone30 or La Sota resulted in an overestimation of protective serum antibody titres. Also in commercially derived White Leghorns vaccinated with Clone30, significantly higher HI titres were obtained when La Sota antigen was used in the HI test. Our data have direct implications for potency testing of inactivated vaccines as the European Pharmacopeia does not prescribe the antigen to be used in the HI test.     

Exacerbation of Mycoplasma gallisepticum infection in turkeys by rhinotracheitis virus

Groups of 1-day-old turkey poults from a parent flock free of antibodies to turkey rhinotracheitis virus (TRTV) and the pathogenic mycoplasmas, were infected by eyedrop with virulent TRTV, with Mycoplasma gallisepticum (Mg) or with both agents together. Dual infection resulted in increased morbidity compared with those groups given single infections. The presence of the Mg in the dual infection had no apparent effect on the pathogenesis of the virus, but the virus caused the Mycoplasma to be more invasive. Mg infection caused a transient depression in TRTV ELISA antibody titres at 29 days post-inoculation. At 14 days post-infection Mg haemagglutination inhibition (HI) and rapid serum agglutination (RSA) titres were higher (P <0.01) in the mixed infection group compared with those infected with Mg alone, but there was no significant difference between ELISA antibody titres of these two groups.