Typing Herpesvirus hominis Antibodies and Isolates by Inhibition of the Indirect Hemagglutination Reaction

Inhibition of the indirect hemagglutination reaction (IHA inhibition) was compared to several other methods for type-specific identification of Herpesvirus hominis (HVH) antibodies and isolates. The method appears to have the greatest value for typing antibodies for HVH type 1 and HVH type 2 in human sera; identification of antibody type was relatively simple and results were definitive. The IHA-inhibition test permitted serological diagnosis of HVH type 2 infection in three young adults with meningoencephalitis, thus extending the mounting evidence that nervous system involvement with this virus type is not limited to neonatal infections. II/I indexes of neutralizing or IHA antibody gave an accurate indication of the presence of HVH type 2 antibody in those sera containing type 2 antibody by IHA inhibition, but they indicated the presence of HVH type 2 antibody in one-half or more of the sera shown to contain only HVH type 1 antibody by IHA inhibition. For typing HVH isolates, the IHA-inhibition test gave results identical to those obtained by direct fluorescent-antibody staining using cross-absorbed conjugates, but the IHA-inhibition test was much more cumbersome and time-consuming to perform than was direct fluorescent-antibody staining. A microneutralization technique for virus typing also gave results identical to those obtained with direct fluorescent-antibody staining and IHA inhibition. However, typing HVH isolates by plaque size or the differential effect of incubation temperature was found to be less definitive and accurate.     

A rapid fluorescent focus-inhibition test for determining the neutralizing-antibody response to lymphocytic choriomeningitis virus.

Levels of neutralizing antibody to lymphocytic choriomeningitis (LCM) virus in the sera of 66 infected persons were assayed by a rapid fluorescent focus-inhibition test (RFFIT). The test was more sensitive than the mouse-neutralization (MN) test and could be completed in less than 24 h. The RFFIT titers were compared with titers obtained by the indirect fluorescent-antibody (IFA) and complement-fixation (CF) tests. Neutralizing antibody detected by the RFFIT remained positive after IRA, CF and MN antibodies had disappeared. The RFFIT for detection of LCM antibody is specific and reproducible and seems especially useful for determining the incidence and epidemiology of LCM virus infections.     

Problems posed by the preparation of specific anti-cytomegalovirus immunoglobulins

The authors describe the preparation of a first batch of intravenous cytomegalovirus (CMV) immune globulin at the Nancy Regional blood transfusion centre. Immune plasmas were selected from 3 640 healthy volunteer blood donors on the basis of CF antibody titers to CMV (Kolmer's method modified) of, at least, 1:8; plasmas from approximately 10% of the donors were therefore selected. The 68 liters of pooled immune plasma had à CF antibody titer of 1:16 (CMV antibody titers of 1: 10 000 and 1: 640 when tested in the ELISA assay and passive hemagglutination assay respectively). Intravenous immune globulin was produced from pooled plasma by Cohn fractionation and treatment with pepsin at pH 4; 4.8 liters of immune globulin were prepared and divided in 96 doses of 50 ml each. The final product was found to have a CMV antibody titer of 1: 32 (CF) 1: 50 000 (ELISA) or 1: 2 560 (passive hemagglutination). Recent reports on the preparation of CMV immune globulin are briefly reviewed.     

Antigenic analysis of human cytomegalovirus isolates from Japanese women and infants.

The antigenic differences among human cytomegalovirus (CMV), two laboratory strains (Davis, AD 169), isolates from pregnant women's cervical secretions, mother's milk, infants' throat swabs and urine were analyzed by means of the plaque reduction assay using human sera which were assumed to contain monotypic antibodies by primary infection and boosted antibodies by reinfection or reactivation of the latent virus. In the cross-neutralization tests, there were no remarkable differences among the CMV strains examined. Moreover, in the neutralization kinetics, normalized kappa values among the strains constantly exceeded 80. Therefore, it is suggested that the human CMV strains examined in the study were serologically identical or very closely related.     

Antibody-dependent cellular cytotoxicity-inducing antibodies against human immunodeficiency virus. Presence at different clinical stages

The presence of antibodies mediating antibody-dependent cellular cytotoxicity (ADCC) against human immunodeficiency virus (HIV)-infected target cells was investigated with 170 sera from patients with varying severity of HIV infection. Approximately 40% of sera from individuals representing all stages of infection were ADCC-positive when tested against HTLV-IIIB infected 0937 clone 2 target cells. The positive sera had higher HIV antibody titers as measured by enzyme-linked immunosorbent assay compared with ADCC-negative sera. ADCC titers were lower in patients with acquired immune deficiency syndrome than in asymptomatic carriers. This decline in ADCC titer was not correlated with a general decrease of HIV antibodies. No correlation between the CD4:CD8 lymphocyte ratio and ADCC activity was found. The possible beneficial effect of ADCC-inducing antibodies early in infection is discussed in relation to the effect of ADCC-inducing antibodies in other retrovirus systems and to the nature of lentivirus infections.     

Serological follow-up study on the antibody levels to Epstein-Barr virus-determined nuclear antigen (EBNA) patients with nasopharyngeal carcinoma (NPC) after radiation therapy

Serological follow-up studies for up to 4 years on the levels of IgG antibody to EBV-determined nuclear antigen (EBNA) were carried out on 36 patients with nasopharyngeal carcinoma (NPC). The serum levels of IgA antibody specific to EBV capsid antigen (VCA) were also measured in some of the patients. The titers of EBNA antibody were measured by enzyme-linked immunosorbent assay (ELISA) and those of IgA antibody to VCA were measured by the indirect immunofluorescence method. The EBNA antibody titers in most sera from the patients before radiation therapy were found to be at least 4 times the mean values in the sera of healthy control adults. Within 2 to 8 months after completion of therapy by 4-MV liniac X-ray irradiation with total doses of 60 to 80 Gy, the titers of EBNA antibody in the sera of 6 patients had returned to normal levels, and low levels of EBNA antibody were maintained for a long time after therapy. These serological data were associated with a good clinical prognosis without recurrence or metastases. But in 6 patients, the patterns of change in the EBNA antibody levels were different: the levels remained high after therapy or first decreased to the normal level and then rose to at least 4 times this level. These 6 patients showed recurrence or metastases. The patterns of change in the EBNA antibody levels were well correlated with those of change in the levels of IgA antibody specific to VCA.(ABSTRACT TRUNCATED AT 250 WORDS)     

The influence of various adjuvants on antibody synthesis following immunization with an hapten.

For the production of specific antibodies to the hapten MATP (4-Amino-1,2,2-trimethyl-phenylphosphonate) in Balb/c mice various non-toxic adjuvants were compared to Freund's complete adjuvant (FCA). For immunization the hapten MATP was coupled to the carrier human serum albumin (HSA). The immunostimulating effect of the synthetic lipopeptides Pam3Cys-OH, Pam3Cys-Ser-Ser-Asn-Ala and different concentrations of the lipohexapeptide Pam3Cys-Ser-(Lys)4 (Pam3Cys = S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-N- palmitoyl-(R)-cysteine as well as of aluminium hydroxide were tested. IgG antibody titers in serum were determined in ELISA. In dose-response studies 50 micrograms Pam3Cys-Ser-(Lys)4 per mouse was the most effective dose with a long period of high antibody levels after the second booster. Pam3Cys-Ser-Ser-Asn-Ala provoked only low antibody titers. Immunostimulation with Pam3Cys--OH did not result in an increased production of specific antibodies. Compared to the control group an enhanced antibody synthesis could be provoked with aluminium hydroxide. However, the increase was much smaller than by using FCA. The lipopeptide Pam3Cys-Ser-(Lys)4 turned out to be a very potent adjuvant. One week after booster injection into mice 50 micrograms of this substance helped to elicit a higher antibody titer than FCA. Hence, as far as the degree of antibody production is concerned, Pam3Cys-Ser--(Lys)4 represents an alternative adjuvant to FCA.     

Higher Throughput Quantification of Neutralizing Antibody to Herpes Simplex Viruses

We report a rapid, higher throughput method for measuring neutralizing antibody to herpes simplex virus (HSV) in human sera. Clinical isolates and sera from the Herpevac Trial for Women were used in a colorimetric assay in which infection of tissue culture (lack of neutralization) was indicated by substrate metabolism by beta-galactosidase induced in the ELVIS cell line. The neutralization assay was optimized by addition of guinea pig complement, which particularly enhanced neutralizing antibody titers to HSV-2. Higher neutralizing antibody titers were also achieved using virus particles isolated from the supernatant of infected cells rather than lysate of infected cells as the source of virus. The effect of assay incubation time and incubation time with substrate were also optimized. We found that incubating with substrate until a standard optical density of 1.0 was reached permitted a better comparison among virus isolates, and achieved reliable measurement of neutralizing antibody activity. Interestingly, in contrast to results in the absence of complement, addition of complement allowed sera from HSV-2 gD-vaccinated subjects to neutralize HSV-1 and HSV-2 clinical and laboratory isolates with equal potency.     

Acquisition of serum antibodies to specific viral glycoproteins of parainfluenza virus 3 in children.

A radioimmunoprecipitation assay was used to study antibody responses to parainfluenza virus 3 glycoproteins in human sera. The method was not only more sensitive than the neutralization test for the detection of antibody but also provided semiquantitative assessments of the antibody response to both glycoproteins in a single assay system. Anti-hemagglutinin-neuraminidase titers were consistently higher than anti-fusion levels in the same serum specimen. Thirteen children were monitored serologically and virologically from birth until 12 months or more after their primary infection with parainfluenza virus 3. At 1 to 3 months after infection, a significant increase in the level of antibody to the hemagglutinin-neuraminidase protein developed in 12 children; of these, 9 showed rises in the level of fusion protein. In 11 of the children, antibody titers continued to rise and the geometric mean titers to the hemagglutinin-neuraminidase protein was highest in sera collected 8 to 10 months after primary infection. Reinfection as the reason for these progressive increases in antibody levels could only be confirmed for four of the children. Three other children had reinfections after the 10-month sera were obtained; in each instance the only antibody responses were to the fusion protein.     

脆弱类杆菌的免疫原性研究及其应用

脆弱类杆菌ATCC 25285和CDC14462分别经甲醛、超声破碎和热酚等处理,制得全菌抗原(WCA)、外膜抗原(OMA)和脂多糖抗原(LPS),其免疫血清的凝集效价以WCA抗血清最高,OMA抗血清次之,LPS抗血清最低。三种抗血清以间接免疫荧光抗体技术(IFA)检定35株脆弱类杆菌,仍以WCA抗血清检出率最高。WCA免疫原性强,免疫产生抗体效价高,能检出更多的同种菌株,且制备简便,值得选用。     

Serum antibodies to human fetal antigens in patients with systemic lupus erythematosus (SLE)

Serum antibodies to human fetal antigens were measured by a radiolabeled anti-immunoglobulin binding assay by using human fetal fibroblasts (Flow cell line No. 1000) as target cells. High titers of IgG antibody to the fetal cells were found in sera of patients with systemic lupus erythematosus (SLE). The antibody reacted with surface membrane antigens shared by various fetal tissues of human and murine origin but not by adult tissues. The reaction of the SLE antibody to the fetal cells was inhibited by heterologous antiserum to the Flow 1000 cells and antiserum to murine embryonic fibroblasts, but not by antiserum to human alpha-fetoprotein or human fibronectin. Absorption of SLE serum with isolated nuclei did not abolish the reaction indicating that these were not anti-nuclear antibodies. The antibody activity was found to reside in the F(ab')2 fragment. The serum titer of the anti-fetal antibody was higher in SLE patients with active disease than those in clinical remission.     

Cell-mediated and humoral immunity in mice: cross reaction between lysozyme and S-carboxymethylated lysozyme studied by a modified footpad test.

The mouse sensitized by subcutaneous (sc) injection of lysozyme in emulsion of Freund's complete adjuvant (FCA) was shown by a modified footpad test to develop three kinds of hypersensitivities. Injecting lysozyme in 2.5-mul emulsion of Freund's incomplete adjuvant (FIA) into the footpad elicited strong footpad swelling in 30 min (anaphylactic reaction), in 3 hr (Arthus-type reaction) and in 24 hr (delayed-type hypersensitivity; DTH). The mice showing anaphylactic reaction in the footpad test manifested severe active systemic anaphylaxis, and the sera of these animals showed high IgG1 antibody titers with only sparingly detectable or no IgE antibody titers. In the sensitizing system with the use of FCA, the antigenicity of S-carboxymethylated lysozyme (CM-lysozyme) devoid of the three-dimensional conformation of lysozyme was compared with that of the native molecule. CM-lysozyme and lysozyme completely cross-reacted to each other in DTH, but not at all in the anaphylactic or Arthus-type reaction or in IgG1 antibody production. CM-lysozyme was shown also to have the ability to bestow immunological memory for the induction of humoral immunity against lysozyme; intravenous (iv) injection of lysozyme in saline or sc injection of CM-lysozyme-FCA alone failed to induce immediate hypersensitivities and IgG1 antibody production against lysozyme, but pre-sensitization by sc injection of CM-lysozyme-FCA enabled the animal to induce these responses to significant levels when iv injection of lysozyme in saline was given as a booster.     

Complement-mediated enhancement of antibody function for neutralization of pseudotype virus containing hepatitis C virus E2 chimeric glycoprotein

We previously reported a number of features of hepatitis C virus (HCV) chimeric glycoproteins related to pseudotype virus entry into mammalian cells. In this study, pseudotype virus was neutralized by HCV E2 glycoprotein-specific antibodies and infected human sera. Neutralization (50% reduction of pseudotype virus plaque formation) was observed with two human immunoglobulin G1 monoclonal antibodies (MAbs) at concentrations of between 2.5 and 10 microg/ml. A hyperimmune rabbit antiserum to an E2 hypervariable region 1 (HVR1) mimotope also exhibited an HCV E2 pseudotype virus neutralization titer of approximately 1/50. An E1 pseudotype virus used as a negative control was not neutralized to a significant level (<1/10) by these MAbs or rabbit antiserum to E2 HVR1. Since HCV probably has a lipid envelope, the role of complement in antibody-mediated virus neutralization was examined. Significant increases in the neutralization titers of the human MAbs (approximately 60- to 160-fold higher) and rabbit antiserum to HVR1 mimotopes (approximately 10-fold higher) were observed upon addition of guinea pig complement. Further, these studies suggested that complement activation occurred primarily by the classical pathway, since a deficiency in the C4 component led to a significant decrease in the level of virus neutralization. This same decrease was not observed with factor B-deficient complement. We also determined that 9 of 56 HCV-infected patient sera (16%) had detectable pseudotype virus neutralization activity at serum dilutions of between 1/20 and 1/50 and that complement addition enhanced the neutralization activity of some of the HCV-infected human sera. Taken together, these results suggest that during infection, HCV E2 glycoprotein induces a weak neutralizing antibody response, that those antibodies can be measured in vitro by the surrogate pseudotype virus plaque reduction assay, and that neutralization function can be augmented by complement.     

Measurement of Neutralizing Serum Antibodies of Patients Vaccinated with Human Papillomavirus L1 or L2-Based Immunogens Using Furin-Cleaved HPV Pseudovirions

Antibodies specific for neutralizing epitopes in either Human papillomavirus (HPV) capsid protein L1 or L2 can mediate protection from viral challenge and thus their accurate and sensitive measurement at high throughput is likely informative for monitoring response to prophylactic vaccination. Here we compare measurement of L1 and L2-specific neutralizing antibodies in human sera using the standard Pseudovirion-Based Neutralization Assay (L1-PBNA) with the newer Furin-Cleaved Pseudovirion-Based Neutralization Assay (FC-PBNA), a modification of the L1-PBNA intended to improve sensitivity towards L2-specific neutralizing antibodies without compromising assay of L1-specific responses. For detection of L1-specific neutralizing antibodies in human sera, the FC- PBNA and L1-PBNA assays showed similar sensitivity and a high level of correlation using WHO standard sera (n = 2), and sera from patients vaccinated with Gardasil (n = 30) or an experimental human papillomavirus type 16 (HPV16) L1 VLP vaccine (n = 70). The detection of L1-specific cross-neutralizing antibodies in these sera using pseudovirions of types phylogenetically-related to those targeted by the L1 virus-like particle (VLP) vaccines was also consistent between the two assays. However, for sera from patients (n = 17) vaccinated with an L2-based immunogen (TA-CIN), the FC-PBNA was more sensitive than the L1-PBNA in detecting L2-specific neutralizing antibodies. Further, the neutralizing antibody titers measured with the FC-PBNA correlated with those determined with the L2-PBNA, another modification of the L1-PBNA that spacio-temporally separates primary and secondary receptor engagement, as well as the protective titers measured using passive transfer studies in the murine genital-challenge model. In sum, the FC-PBNA provided sensitive measurement for both L1 VLP and L2-specific neutralizing antibody in human sera. Vaccination with TA-CIN elicits weak cross-protective antibody in a subset of patients, suggesting the need for an adjuvant.     

Multiplexed measurement of serum antibodies using an array biosensor

The array biosensor provides the capability for simultaneously measuring titers of antibody against multiple antigens. Human antibodies against four different targets, tetanus toxin, diphtheria toxin, staphylococcal enterotoxin B (SEB) and hepatitis B, were measured simultaneously in sera from eight different donors in a single assay and titers were determined. The assays could measure amounts of bound antibody as low as approximately 100 fg. Each individual serum exhibited a different pattern of reactivity against the four target antigens. Applications of this biosensor capability include monitoring for exposure to pathogens and for efficacy of vaccination.     

Heterophile Hanganutziu-Deicher antibodies in sera of patients with Kawasaki diseases

The heterophile antibody levels in sera from patients with Kawasaki disease (49 sera from 39 cases) were measured by sheep erythrocytes (SRBC) agglutination and radioimmunoassay in microplates coated with Hanganutziu-Deicher (H-D) antigen-active glycosphingolipid, equine hematoside. The antibody levels were low in the first week of illness, increased rapidly in the 2nd week, and thereafter gradually decreased. The SRBC agglutination titers and H-D antibody titers of sera from patients with Kawasaki disease from week 2 to 8 of illness were significantly higher than those of healthy children (44 sera) and normal cord blood (13 sera).     

Characterization of serum and mucosal antibody responses in white sturgeon (Acipenser transmontanus Richardson) following immunization with WSIV and a protein hapten antigen

Serum and cutaneous mucus antibodies were monitored in white sturgeon for 15 weeks following intraperitoneal immunization. Ten fish were immunized (50 microg) with white sturgeon iridovirus (WSIV) or white sturgeon gonad (WSGO) tissue culture cells emulsified with or without FCA. An additional group was immunized with FITC:KLH+FCA. Fish were booster immunized at 6 weeks. Fish immunized with FITC:KLH+FCA produced significant serum antibodies to FITC by 6 weeks and this response peaked at 12 weeks (average titer 31,000). Mucosal antibodies to FITC were first detected at 12 weeks and significantly elevated by 15 weeks (average titer 18). Anti-WSIV antibody titers were detected in the serum by 9 weeks in fish immunized with WSIV and WSIV+FCA, but only a small number responded to immunization. At 15 weeks, four fish immunized with WSIV produced serum antibodies (average titer 838) and one fish immunized with WSIV+FCA had a serum titer of 1600. Mucosal anti-WSIV antibody titers of 8 and 16 were observed in two fish from the WSIV group at 12 weeks while four different fish from this group responded at 15 weeks (average titer 4). Western Blot using a monoclonal antibody confirmed immunoglobulin in mucus, and specificity to WSIV was further demonstrated by immunocytochemistry using serum from fish immunized with WSIV. Specific antibody was not detected in mucus of fish immunized with WSIV+FCA, WSGO, or WSGO+FCA. Collectively, these experiments demonstrate that white sturgeon can generate a specific antibody response following immunization, and is the first report showing mucosal immunoglobulin is present in this species.     

Heterologous Prime-Boost Vaccination with MF59-Adjuvanted H5 Vaccines Promotes Antibody Affinity Maturation towards the Hemagglutinin HA1 Domain and Broad H5N1 Cross-Clade Neutralization

In an open label clinical study (2007), MF59-adjuvanted hemagglutinin (HA) vaccine from H5N1-A/Vietnam/1194/2004 (clade 1) was administered to subjects previously vaccinated (primed) with clade 0 H5N3 (A/duck/Singapore/97) vaccine at least 6 years earlier (in 1999 or 2001). The primed individuals responded rapidly and generated high neutralizing antibody titers against the H5N1-Vietnam strain within 7 days of a single booster vaccination. Furthermore, significant cross-neutralization titers were measured against H5N1 clade 0, 1, and 2 viruses. In the current study, the impact of MF59 adjuvant during heterologous priming on the quality of humoral polyclonal immune response in different vaccine arms were further evaluated using real time kinetics assay by surface plasmon resonance (SPR). Total anti-H5N1 HA1 polyclonal sera antibody binding from the heterologous prime-boost groups after a single MF59-H5N1 boost was significantly higher compared with sera from unprimed individuals that received two MF59-H5N1 vaccinations. The antigen-antibody complex dissociation rates (surrogate for antibody affinity) of the polyclonal sera against HA1 of H5N1-A/Vietnam/1194/2004 from the MF59-H5N3 primed groups were significantly higher compared to sera from unadjuvanted primed groups or unprimed individuals that received two MF59-H5N1 vaccines. Furthermore, strong inverse correlations were observed between the antibody dissociation off-rates of the immune sera against HA1 (but not HA2) and the virus neutralization titers against H5 vaccine strains and heterologous H5N1 strains. These findings supports the use of oil-in-water-adjuvanted pandemic influenza vaccines to elicit long term memory B cells with high affinity BCR capable of responding to potential variant pandemic viruses likely to emerge and adapt to human transmissions.