Live tissue culture influenza vaccine for oral administration. I. Specific immune response to monovaccine A (H3N2) in volunteers.

Oral immunization of volunteers with a live tissue culture influenza A (H3N2) vaccine induced an increase in virus neutralizing (VN), haemagglutination inhibiting (HI) and neuraminidase inhibiting (NI) antibody. The dynamics of antibody and IgM and IgG immunoglobulin formation in the blood depended to a great extent on their prevaccination levels. The highest per cent of seroconversions was observed after the 3rd vaccination: a 4-fold or greater rise of VN antibody was found in 80% (titre increase by 3.0 log2 units), of HI antibody in 70% (titre increase by 2.6 log2 units) and NI antibody in 73% (titre increased by 2.5 log2 units) of the volunteers. The level of IgG increased after each vaccination but its highest level did not coincide in time with the maximum antibody production. High titres of the antibodies studied were recorded 1-2 months after the 3rd vaccination, irrespective of their prevaccination levels.     

Mumps-specific immunoglobulin M and G antibodies in natural mumps infection as measured by enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay for the demonstration of mumps immunoglobulin G (IgG ELISA) and immunoglobulin M antibodies (IgM ELISA) in serum was compared with complement fixation (CF), hemagglutination inhibition (HI), and hemolysis-in-gel (HIG) tests. The antibody levels measured by IgG ELISA had a high positive correlation with the CF and HIG tests, whereas only a moderate correlation was found between IgG ELISA and HI. Similar patterns of antibody response were observed with IgG ELISA, CF, and HIG: the antibody titres increased rapidly after the onset of symptoms and reached the maximal values in about three weeks. The HI antibodies developed more slowly during the first week of disease, after which the titres increased rapidly up to the fourth week. IgM antibodies measured by ELISA developed soon after onset of symptoms; most patients had IgM antibodies from the second day, and the highest titres were reached within the first week. The antibody response in mumps parotitis did not differ from that in mumps meningitis/encephalitis, while relatively higher antibody titres were found in patients with orchitis/epididymitis. The diagnostic efficiencies of the methods were compared with serum specimens from 33 patients who had a serologically verified mumps infection by at least one of the five methods used (rising antibody titres in paired sera or detectable IgM): IgM ELISA detected all 33 cases, IgG ELISA 29, HIG 28, HI 23, and CF 13. In 27 cases, IgM antibodies were already present in the acute phase serum specimens. It was concluded that mumps IgM ELISA is a more rapid and sensitive means for the serological diagnosis of mumps infection than the conventional tests.     

Attempts at the detection of antibodies to Newcastle disease virus by haemofusion-inhibition test

Two modifications of a haemofusion-inhibition test (HFI-1 and HFI-2) were applied for the titration of antibodies to Newcastle disease virus (NDV) in chicken sera. Statistical analysis revealed a positive correlation of the HFI-1 antibody titres with those measured by the standard haemagglutination-inhibition (HI), virus neutralization (VN) and haemolysis-inhibition (HLI) tests. The same appeared true when the HFI-2 antibody titres were compared with the HI, VN, and HLI tests. Except for the several sera collected from birds immunized with formalin-inactivated vaccine, the HFI-2 antibody titres of individual serum samples were usually lower than those determined by HFI-1. The interpretation of these differences as well as some advantages and disadvantages of the proposed test are discussed.     

Levels of immunoglobulins and antibodies to haemaglutinin and neuraminidase of influenza virus in nasal secretions after natural infection.

Nasal washings (NW) from 16 influenza patients in the course of an epidemic in November and December, 1974 were examined for the presence of influenza virus, immunoglobulins (Ig) and titres of haemagglutination inhibiting (HI) and neuraminidase inhibiting (NI) antibodies. Influenza virus identical with A/Port Chalmers/1/73 (H3N2), increased levels of IgA and occasionally IgG, and specific antibodies were detected in the NW. The dynamics of HI and NI antibody formation did not differ substantially, but there were individual differences in titres and persistence of antibodies. Convalescent sera always contained increased levels of HI and NI antibodies. In some cases, the titres of antibody to viral ribonucleoprotein did not increase.     

The immune response to influenza virus: III. Changes in the avidity and specificity of early IgM and IgG antibodies

Serum samples taken from rabbits 5 days after vaccination with SW influenza virus by the intravenous route contained high levels of IgM antibodies. IgG antibodies were either not detected or were present at very low levels. By the 10th day after vaccination both IgM and IgG antibodies were present in the serum. The early IgM antibodies were of high avidity while the early IgG antibodies were of very low avidity. The presence of low avidity IgG antibodies in whole serum caused a decrease in the average avidity of the antibodies in whole serum from the 5th to the 10th day post vaccination. The avidity of the IgM antibodies remained fairly constant for the first 20 days of the immune response but a slight increase was detected after secondary vaccination. The avidity of the early IgG antibodies increased during the test period of 20 days. The early IgM and IgG antibodies were heterogeneous with respect to avidity.

The highly avid IgM antibodies showed high cross-reactivity with related influenza viruses, i.e. they were of low specificity. The early IgG antibodies that were of low avidity cross-reacted with only one other influenza virus out of the four tested, i.e. they were more specific; as the avidity of the IgG antibodies increased so did their cross-reactivity.

     

Radioimmunoassay of measles virus antibodies in SSPE

A sensitive radioimmunological assay (RIA) was introduced for detection of measles virus IgG and IgM antibodies. The hyperimmune response to measles virus could be demonstrated more precisely by RIA than by haemagglutination inhibition (HI). The ratio between RIA and HI antibody titres was decidedly higher in sera and cerebrospinal fluids (CSF) of patients with subacute sclerosing panencephalitis (SSPE) than in those of other groups tested.     

Contribution to laboratory diagnosis of mumps and parainfluenza

Specific IgM and IgG antibodies to mumps virus (MV) were detected in sera of mumps-patients by ELISA in agreement with the results obtained by indirect immunofluorescence (IF). Of given sera 37.5% contained IgM reacting in indirect ELISA also with the antigens of parainfluenza virus (PiV) T3. In all patients with respiratory illness over 2 years of age, the significant increase of antibodies to PiV in haemagglutination inhibition (HI) test was in good correlation with serum IgM and IgG antibody levels to PiV T3 determined by ELISA; but, in addition, 30.7% of these sera cross-reacted with MV antigens. The cross-reactions were eliminated by using MV-nucleocapsid antigen in indirect ELISA, or in direct ELISA using the peroxidase-labelled whole virion antigen. In some children under two years of age a discrepancy was observed between the significant increase of serum antibodies in HI and the inability to detect specific IgM antibodies by means of ELISA in their sera. The low-avidity antibodies appearing after primary PiV infection were probably washed off during the ELISA procedure.     

Reactions of chicken biliary immunoglobulin A with avian mycoplasmas

Chicken bile was examined for mycoplasma by culture and for antibody against mycoplasma by indirect immunoperoxidase assay (IIPA) detecting chicken either IgA or IgG and IgM, as well as by rapid plate agglutination (RPA), haemagglutination-inhibition (HI) and double immuno-diffusion (DID). Cultures indicated the presence of M. gallisepticum (Mg) and Ai. synoviae (Ms) in bile and their isolations were positively correlated with those from upper respiratory tract. In 45 chickens from five flocks examination of biliary samples with IIPA detected considerably higher rates of Mg and Ms antibodies than the same assay performed with sera especially those of IgA class of antibody. In chickens with a strong serological response to Mg, titres of specific agglutinins and HI antibody in biliary samples were significantly higher than those found in sera. Only biliary fractions containing Ig exhibited antibody activity.     

The Occurrence and Sources of Natural Antibody in Human Bile and Serum Against the O Antigens of Two Escherichia coli Serotypes

Paired serum and bile samples from normal subjects as well as patients with biliary disease were tested for natural antibody to two individual Escherichia coli O antigens by ELISA. Serum antibody was most commonly of IgM and IgG class. Antibody was less frequently detected in bile and was more commonly IgM than IgA, with IgG activity detected infrequently. Little relation was apparent between antibody in paired samples; activity could be present in both serum and bile or in either fluid alone. Titres in paired samples also did not correspond when 'normalized' with respect to the concentrations of relevant isotypes; bile was frequently enriched for natural antibody as a proportion of total immunoglobulin compared with serum. Secretory component-bound antibody was detectable in some biles that contained IgA and/or IgM activity and in the serum of 33% of subjects with biliary disorders but not in normal sera. A series of paired samples taken from three individuals was also examined for antibody against each subject's own intestinal commensal E. coli. Serum IgM and IgG activity was present in all samples, but antibody in bile was less frequent and was of IgM or IgA class. These results suggest that natural antibody in human bile occurs independently of antibody in serum and that it is substantially derived from local sources.     

Relevance of detection of immunoglobulin M antibody response in birds used for arbovirus surveillance.

Young chickens were inoculated with 5,000 PFU of eastern equine encephalitis (EEE) virus and bled at intervals thereafter for determinations of hemagglutination-inhibiting (HI), neutralizing (N), immunoglobulin M (IgM), and IgG antibodies. HI, N, and IgM antibodies were first detected 4 days after infection, and IgG was detected 7 days after infection. All four antibodies persisted through day 90 after infection. HI, N, and IgM antibody titers remained elevated and were not cross-reactive with the related alphavirus western equine encephalitis (WEE) virus. IgG antibody titers also remained high, but heterologous reactivity to WEE virus increased with time after infection. Serum samples from sentinel chickens and wild birds infected in nature with EEE, WEE, or St. Louis encephalitis virus and submitted to this laboratory from state and local health departments were tested for IgM antibody by using anti-chicken IgM for capture and for IgG antibodies to the EEE and WEE viruses. There was essentially complete correlation between HI, N, and either IgM (indicating recent infections) or IgG (indicating more remote infections) antibody. We conclude that the IgM antibody capture enzyme immunoassay can be used as a specific and sensitive assay to replace the routinely used HI test for detecting antibody in sentinel chickens and in young, wild birds used for arbovirus surveillance. The test is rapid and relatively inexpensive and can be performed in essentially all adequately supplied laboratories.     

Indirect and reverse radioimmunoassays and their apparent specificities in the detection of antibodies to enteroviruses in human sera

Indirect radioimmunoassays (RIAs) of IgM and IgG antibodies to enteroviruses have been developed, using coxsackieviruses B1 and B3, and echoviruses 11 and 30. The titres of IgM and IgG were assayed in paired sera from patients infected with one of these viruses or coxsackieviruses A7, A9, A16, B2, B4, B5 or echoviruses 4, 17, or 25. Both IgM and IgG were found in almost all serum pairs with each of the four viruses used as an antigen, and there were no certain differences between titres obtained with homologous and heterologous antigens. The convalescent phase specimens contained significantly higher titres compared with the acute phase specimens, the difference being most pronounced for IgG. Of the specimens from patients with nonenterovirus infections, a relatively high percentage contained IgM and IgG against enterovirus antigen. However, no increases in titres were seen between acute and convalescent specimens. When specimens from younger patients, aged 2 days to 22 months, without evidence of enterovirus infections, were assayed with enterovirus antigen, the frequency of IgM titres was seen to increase with age. Almost all specimens from newborns were negative, whereas the specimens from 12- to 22-month-old children showed a high frequency of IgM titres. In specimens from patients aged 2 days to 8 months, the ratio between IgM and IgG titres increased with age, probably due to a loss of maternal IgG. The IgG titres in specimens from 8.5- to 22-month-old children were similar to the titres of specimens from the patients with nonenterovirus infections. A reverse IgM assay was also developed, using the same viruses and serum specimens as for the indirect assays. In contrast to the indirect IgM assay, the reverse IgM assay was apparently type specific, provided that the amount of labeled virus was carefully standardized. The reverse IgM RIA detected and identified antibody responses better than the neutralization test. Attempts to develop a reverse IgG assay were promising concerning the specificity, but the sensitivity was low.     

Characterization of the antibody response in vaccinated mice protected against Coxsackievirus B3-induced myocarditis

Adolescent male CD-1 mice can be rendered resistant to coxsackievirus B3 (CVB3m)-induced myocarditis following inoculation as neonates with a single dose of a temperature-sensitive mutant virus (ts1), derived from the prototype parent virus (CVB3m). Previously, anti-CVB3 neutralizing antibodies were not detected in sera of adolescent ts1 vaccinees by a standard plaque-reduction assay (Gauntt et al 1983. Infect. Immun. 39:851). However, a more sensitive cytopathic effects-reduction assay permitted detection of low titers of anti-CVB3m neutralizing antibodies of the IgG class prior to challenge with CVB3m. Following CVB3m challenge, serum anti-CVB3m neutralizing antibody titers of ts1 vaccines declined on days 1-2 post-inoculation (p.i.) then increased over the next 6 days. The neutralizing antibodies were of both the IgG and IgM classes. Normal mice challenged with CVB3 did not produce detectable serum anti-CVB3m neutralizing antibody until day 4 p.i. and by 8 days p.i. the neutralizing antibody was only of the IgM class. Thus, adolescent murine ts1 vaccines mount a secondary antibody response to CVB3m in both neutralizing IgG and IgM, but resistance to CVB3m-induced myocarditis is due to the presence of low levels of anti-CVB3m IgG neutralizing antibody in serum at the time of challenge with CVB3m.     

Enzyme immunoassay, complement fixation and hemagglutination inhibition tests in the diagnosis of influenza A and B virus infections. Purified hemagglutinin in subtype-specific diagnosis

The efficacy of enzyme immunoassay (EIA) in detecting diagnostic antibody rises to influenza A and B viruses was compared with complement fixation (CF) and hemagglutination inhibition (HI) tests in 455 patients with an acute respiratory infection. EIA and HI detected significantly more diagnostic antibody rises against influenza A than the CF method (96 and 87 vs. 47, respectively). In the case of influenza B significantly more diagnostic influenza B antibody rises were observed by EIA than by CF or HI (59 vs. 37 and 40, respectively). In most of the cases antibody rises in EIA were found in both IgG and IgA isotypes whereas increases in IgM antibodies were seen less frequently. Purified hemagglutinins (HA) were prepared from influenza A HI- and H3-subtypes and from influenza B viruses and used as antigens in EIA and the results were compared with those of HI. Infections caused by influenza A HI-subtype showed good homologous antibody responses in EIA but heterologous antibody responses to H3-subtype and influenza B HAs were frequently observed. Heterologous responses were clearly less frequent in patients with infections caused by the H3-subtype. Influenza B infections occasionally raised HA antibodies against influenza A H1-subtype but not to the H3-subtype. Interestingly, HI detected these heterologous responses at least as frequently as EIA. When whole viruses were used as antigens in EIA, subtype specificity was not observed and cross-reactions between influenza A and B virus antibodies were found. These observations suggest that, although EIA can show greater diagnostic efficacy over HI and CF methods, HI is still the serological method of choice in determining the causative subtype of influenza A virus infection.     

Serological investigations on Indian kala-azar.

Detailed serological investigations were carried out in forty-nine active kala-azar (KA) cases in North Bihar, India. Various classes of immunoglobulin (IgG, IgA, and IgM) and third component of complement (C3) levels were determined in these sera and results were compared with those obtained in normal controls. Antibody titres were determined by the indirect haemagglutination (IHA) method using soluble Leishmania antigen. Immunoglobulin G and M class-specific antibody titres were also determined separately by the enzyme-linked immunosorbent assay (ELISA) method. Polyclonal hypergammaglobulinaemia with marked increase in serum IgG (and to a lesser extent in IgM) level was a rather common feature in the majority of these sera. Much of this immunoglobulin increase, however, appeared to be non-specific in nature and no absolute correlation could be noted between serum IgG or IgM levels and corresponding IgG or IgM antibody titres. Significant decrease in serum C3 level was observed in KA and this decrease was found to be independent of immunoglobulin levels or specific antibody titres. A fairly good correlation between aldehyde test and serum IgG level was evident from this study. Aldehyde-positive KA sera usually gave higher antibody titres than aldehyde-negative ones. Anti-leishmanial antibodies belonged mostly to IgG class although some IgM antibodies were also demonstrable. The latter class of antibodies probably appeared early in KA infection although their serological specificity was poorer to IgG antibodies. Out of forty-nine KA sera examined in this study thirty-six (73.5%) gave positive IHA titres while forty-six (93.9%) were positive by IgG-ELISA which appeared to be a highly specific and sensitive serodiagnostic method particularly for the early detection of KA cases.     

Measles antibodies in nasal secretions and sera of children with measles.

Measles antibodies were determined, in the course of measles, in sera and nasal secretions of 54 and 27 children, respectively. The examinations were performed on the 3rd or 4th day (1st period) and between the 10th and 14th day (2nd period) after onset of rash in both sera and nasal secretions and after 25 to 60 days (3rd period) in sera only. Geometric mean titres of antibodies in sera determined by haemmagglutination inhibition (HI) and neutralization tests in the 1st period were 126.3 and 115.3 respectively. For the 2nd period the respective figures were 318.4 and 396.1 and for the 3rd period--388.0 and 445.6. Fractionation on Sephadex G-200 of sera from the 1st period revealed HI and neutralizaing antibodies associated mainly with the IgM serum globulin class. Measles HI and neutralizing antibodies were also found in nasal secretions of all 27 children, but their titres were much lower than in serum. The antibodies determined by indirect immunofluorescence in nasal secretions were associated with IgA in 26 and with IgG immunoglobulin in 15 of the 27 subjects. No IgM antibodies were found.     

A serological survey of chickens, Japanese quail, pigeons, ducks and crows for antibodies to chicken anaemia virus (CAV) in Japan.

The chicken has been considered as the natural host for chicken anaemia virus (CAV). To examine the prevalence of CAV in domestic and wild birds in Japan, we analyzed serum samples collected from 211 chickens, 168 Japanese quail, 105 pigeons, 113 ducks and 116 crows for the presence of antibodies to CAV by a micro-scale virus neutralization (VN) test. Nine of the 13 chicken flocks (69.2%) were found to be positive for VN antibodies to CAV. Of the 211 individual chicken serum samples, 127 (60.2%) were positive. VN antibodies were detected in 103 of the 168 (61.3%) quail samples with titres ranging from 1:20 to >/= 1:2560. Serum samples collected from quail in 1992 were found to possess a lower rate of antibody-positive sera (7.7%) and lower antibody titres (1:20 to 1:80) than those collected in 1995 (90.2% and 1:20 to >/= 1:2560, respectively). None of the pigeon, duck and crow samples neutralized CAV at a 1:20 dilution. These results indicate that quail might be one of the hosts of CAV or CAV-like virus in Japan. This is the first report of VN antibodies to CAV in a species other than chicken.     

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.     

Bile immunoglobulin of the duck (Anas platyrhynchos). I. Preliminary characterization and ontogeny

Immunoglobulin (Ig) of a single class was found in duck (Anas platyrhynchos) bile. Its molecular weight was 890,000; serum IgM was 800,000. Heavy chains were 75,000 for bile Ig, 86,000 for IgM, 67,000 for 7.8S IgG and 37,000 for 5.7S IgG. Antigenic comparison showed that bile Ig resembled IgM but carried additional determinants. The ontogeny of bile Ig was distinct from that of serum IgM and IgG. Thus, duck bile Ig appears to be an IgM-like molecule secreted independently of serum Ig.     

Enzyme immunoassay for detection of antibodies against eastern equine encephalomyelitis virus in sentinel chickens

We developed an enzyme immunoassay (EIA) for the detection of immunoglobulin M (IgM) and IgG subclass antibodies directed against eastern equine encephalomyelitis (EEE) virus in chickens. The assays were compared with the serum plaque reduction neutralization test (PRNT) and the hemagglutination inhibition (HI) test for ability to detect antibodies against EEE virus in laboratory-infected birds. No cross-reactivity was detected in serum from chickens inoculated with St. Louis encephalitis or Highlands J virus. The interval after infection when EEE virus-specific antibodies were first detected by IgM and IgG EIAs was found to be similar to that determined by the PRNT and HI tests: 2 to 4 days. The IgG EIA, PRNT, and HI test detected antibodies to EEE virus for at least 27 to 30 days after inoculation. In contrast, serum from five of seven chickens did not contain detectable IgM 30 days after infection. Similarly, in all three naturally infected sentinel chickens from Maryland, IgM class antibody was undetectable 1 to 5 weeks after IgM was initially detected. EIAs provide simple and rapid alternatives to traditional tests for monitoring EEE virus infections in sentinel chicken flocks. Moreover, the IgM EIA provides a means to separate recently infected chickens from those infected greater than or equal to 1 month earlier.     

Measurement of heterophil antibody and antibodies to EB viral capsid antigen IgG and IgM in suspected cases of infectious mononucleosis.

The EBV IgG titres in acute and convalescent specimens from 97 cases of infectious mononucleosis were compared with titres from acute and convalescent sera from 96 students with illnesses resembling infectious mononucleosis but without heterophil antibody, EB IgM or EB IgG seroconversion; and also with titres from 91 healthy students known to have had EB IgG antibody for at least six months. These titres were related to the titre of the Research Standard A.66/235 for infectious mononucleosis serum prepared by the National Institute for Biological Standards and Control. Serial sera were tested for heterophil antibody and EBVCA specific IgG and IgM from 61 university students with infectious mononucleosis. The period of persistence of heterophil antibody and EBV IgM after illness was outlined from the results of the tests. Single sera from 406 patients in hospital or general practice sent to the diagnostic laboratory for heterophil antibody tests were also tested for EBV antibodies without prior knowledge of the heterophil antibody result. The close agreement between heterophil antibody and EBV IgM results is shown. False positive EB IgM results were correlated with the presence of rheumatoid factor.