Seropositive Sera Research Articles

Lack of autoantibody production associated with cytomegalovirus infection

To confirm an association between cytomegalovirus (CMV) infection and the presence of antibodies to Smith (Sm), to ribonucleoprotein (RNP), and to a component of the U1 ribonucleoproteins (U1-70 kD), we measured antibodies to these protein antigens using an enzyme immunoassay and an immunoblot. The antibodies were measured in the sera of 80 healthy subjects, one-half of whom were naturally CMV seropositive and one-half were CMV seronegative, and in eight subjects immunized with a live attenuated strain of CMV. None of the vaccinees developed antibodies to Sm, to RNP, or to U1-70 kD at either 4 or 12 months after immunization. Additionally, there was no statistically significant association between levels of antibodies to Sm or to RNP and between sera obtained from vaccinees, natural CMV seropositive individuals, and CMV seronegative individuals. One CMV seropositive serum and one CMV seronegative serum tested positive for antibodies to U1-70 kD. These data indicate that neither wild-type infection nor the live-attenuated Towne vaccine frequently induce autoantibody production.

Prevalence of hepatitis B virus infections in nonhuman primates.

The aim of this study was to determine the prevalence of hepatitis B virus (HBV) infection in nonhuman primates. Serum samples from Europe, Thailand and Vietnam were analyzed. Sera obtained from 262 apes and 454 monkeys were tested for HBV infection serologically and for HBV DNA using nested PCR (nPCR). A total number of 198 ape sera and all but one (Cercopithecus aethiops) of the 4543 monkey sera had no serological signs of HBV infection. Among the 64 of 262 (24.4%) seropositive ape sera, we found, as in humans, different stages of HBV infection: very early HBV infection, active infection with high level of infectivity, virus carriers with low infectivity, and passed HBV infection. In the cases with passed infection, 47.8% harbored HBV DNA in the presence of protective antibodies to the HBV surface antigen (HBsAb). This indicates HBV persistence in apes despite immune control. In contrast to apes, in monkeys HBV infection is a very rare event.

Chlamydia Pneumonia Seropositivity Correlates with Serum Fibrinogen and Lipoprotein a Levels: Any Role in Atherosclerosis?

The aim of the study is to determine the impact of Chlamydial seropositivity on atherosclerosis in a group of patient requiring coronary and/or carotid revascularization. A population of 30 diabetic patients (group 3) and 26 nondiabetic patients (group 2) with angiographically documented coronary and/or carotid artery disease were enrolled for the study. Volunteers from the relatives of hospital staff with no known disease (n=29; group 1) were included as the control group. Serum samples from the participants were assayed for cardiovascular risk factors including total serum cholesterol, triglyceride and lipoprotein levels, fibrinogen, Hb A1c levels and IgG titers for Chlamydia pneumonia (C. pneumonia). Chlamydial seropositivity was analysed further to determine a possible impact on atherogenesis. Serum LDL cholesterol levels revealed statistically significant difference between groups 1 and 2 (p=0.001). There was no difference between groups 2 and 3 regarding LDL cholesterol levels. There was no significant difference among the groups with respect to C. pneumonia seropositivity and the other atherosclerotic risk factors. Chlamydial seropositivity was found to be more frequent in males than in females (p=0.008). In the C. pneumonia seropositive group, serum fibrinogen and lipoprotein a levels were found to be significantly higher than the seronegative group (p=0.0001 and p=0.001, respectively). Other atherogenic risk factors were similar in the seropositive and negative groups. The causal role of Chlamydial infections in atherosclerotic plaque formation might be due to their influence on the serum fibrinogen and lipoprotein a levels.

Development of an immunoenzymatic assay for the detection of human antibodies against Trypanosoma cruzi calreticulin, an immunodominant antigen

We have developed an indirect immunoenzymatic assay (ELISA) for the detection of human antibodies against calreticulin (formerly known as Tc45), a dimorphic Trypanosoma cruzi antigen, described in our laboratory. PVC microtitration plates were sensitized with the monoclonal anti-calreticulin antibody (MoAb) and reacted with calreticulin present in a partially purified preparation. The presence of anti- T. cruzi calreticulin IgG in sera from infected individuals was tested. The data generated with this assay were validated by correlation, in a regression analysis, with those obtained by an indirect immunoradiometric assay (IRMA). From the 12 seropositive sera (as defined by a commercial test), eight came out positive and four negative in both assays. The 12 human sera were also analyzed in direct immunometric assays (ELISA and IRMA), where the solid phase was sensitized with a whole parasite extract. The direct ELISA and IRMA correlated positively ( P<0.01). Further validation of this ELISA was achieved with an indirect immunofluorescense assay. The high degree of significance obtained when the indirect IRMA and ELISA systems were compared, indicated that the relatively small sample number used (12) was statistically satisfactory for the purposes of this investigation. Thus, the IRMA can be replaced by the ELISA, with advantages mainly derived from the cumbersome manipulation of radioactive wastes. The MoAb used as an antigen capture agent in the ELISA proposed here, recognizes a homologous protein in Trypanosoma rangeli, suggesting that individuals infected with this parasite might have crossreactive antibodies. However, the system retains its diagnostic interest, given the facts that the MoAb does not recognize a homologous protein in Leishmania mexicana, Leishmania donovani, or Crithidia fasciculata.

Anomalous colloidal stability of protein coated polystyrene latex beads studied by small angle light scattering

Results of small angle laser light scattering experiments carried out on polystyrene latex beads coated with lysate of Plasmodium falciparum (Pf) antigen (Ag) and human seropositive sera (10:1 ratio) are reported for various NaCl concentrations (0–300 mM). The protein coated beads showed time-dependent coagulation. The normalised intensity of scattered light I s( t)/ I 0 showed I s( t)/ I 0=1+( Γt) δ behaviour with the coagulation rate, Γ and exponent, δ showing anomalous dependence on NaCl concentration. The coagulation rate exhibited strong increase up to NaCl concentration of 50 mM, above this and up to 300 mM the coagulation rate was found to remain independent of NaCl concentration yielding non-DLVO behaviour. The same was true for δ which increased from 1.04±0.06 to 6.94±0.07 as NaCl concentration was raised from 0 to 50 mM. Above 50 mM it remained constant with δ=6.94±0.07. Results are discussed through Smoluchowski aggregation kinetics and theoretical construction of interparticle interaction potentials relevant to our problem.

Hepatitis C virus genotypes in a northeastern area of Brazil.

We used a reverse transcription-polymerase chain reaction (RT-PCR) to obtain the genotypes of circulating hepatitis C virus (HCV) in patients from a Gastro-Hepatology Unit in the city of Salvador (Bahia State) in northeastern Brazil. Viral RNA was detected in 83 (65.4%) of 127 anti-HCV seropositive serum samples. Positivity was significantly associated with alterations in levels of aspartate aminotransferase and alanine aminotransferase (P < 0.05). Genotyping of HCV was performed by RT-PCR using genotype-specific primers from the core region: 24.1% were infected with subtype 1a, 38.6% with 1b, 3.6% with 2, 21.7% with 3a, and 12.0% with a mixed genotype. There was no difference in genotype distribution when compared with results from other Brazilian locations. Surprisingly, the high frequency of genotype 3 in Brazilian samples continues to be different from that reported around the world and warrants further investigation.

Detection of borna disease virus-reactive antibodies from patients with psychiatric disorders and from horses by electrochemiluminescence immunoassay.

The prevalence of Borna disease virus (BDV)-specific antibodies among patients with psychiatric disorders and healthy individuals has varied in several reports using several different serological assay methods. A reliable and specific method for anti-BDV antibodies needs to be developed to clarify the pathological significance of BDV infections in humans. We developed a new electrochemiluminescence immunoassay (ECLIA) for the antibody to BDV that uses two recombinant proteins of BDV, p40 and p24 (full length). Using this ECLIA, we examined 3,476 serum samples from humans with various diseases and 917 sera from blood donors in Japan for the presence of anti-BDV antibodies. By ECLIA, 26 (3.08%) of 845 schizophrenia patients and 9 (3.59%) of 251 patients with mood disorders were seropositive for BDV. Among 323 patients with other psychiatric diseases, 114 with neurological diseases, 75 with chronic fatigue syndrome, 85 human immunodeficiency virus-infected patients, 50 with autoimmune diseases including rheumatoid arthritis and systemic lupus erythematosis and 17 with leprosy, there was no positive case except one case each with alcohol addiction, AIDS, and dementia. Although 19 (1.36%) of 1,393 patients with various ocular diseases, 10 (1.09%) of 917 blood donors, and 3 (4.55%) of 66 multitransfused patients were seropositive for BDV-specific antigen, high levels of seroprevalence in schizophrenia patients and young patients (16 to 59 years old) with mood disorders were statistically significant. The immunoreactivity of seropositive sera could be verified for specificity by blocking with soluble p40 and/or p24 recombinant protein. Anti-p24 antibody was more frequent than p40 antibody in most cases, and in some psychotic patients antibody profiles showed only p40 antibody. Although serum positive for both p40 and p24 antibodies was not found in this study, the p40 ECLIA count in schizophrenia patients was higher than that of blood donors. Furthermore, we examined 90 sera from Japanese feral horses. Antibody profiles of control human samples are similar to that of naturally BDV-infected feral horses. We concluded that BDV infection was associated in some way with psychiatric disorders.

Performance of a novel combination of recombinant antigens for the detection of CMV IgG antibodies in key diagnostic populations

We have developed an indirect immunoenzymatic assay (ELISA) for the detection of human antibodies against calreticulin (formerly known as Tc45), a dimorphic Trypanosoma cruzi antigen, described in our laboratory. PVC microtitration plates were sensitized with the monoclonal anti-calreticulin antibody (MoAb) and reacted with calreticulin present in a partially purified preparation. The presence of anti-T. cruzi calreticulin IgG in sera from infected individuals was tested. The data generated with this assay were validated by correlation, in a regression analysis, with those obtained by an indirect immunoradiometric assay (IRMA). From the 12 seropositive sera (as defined by a commercial test), eight came out positive and four negative in both assays. The 12 human sera were also analyzed in direct immunometric assays (ELISA and IRMA), where the solid phase was sensitized with a whole parasite extract. The direct ELISA and IRMA correlated positively (P<0.01). Further validation of this ELISA was achieved with an indirect immunofluorescense assay. The high degree of significance obtained when the indirect IRMA and ELISA systems were compared, indicated that the relatively small sample number used (12) was statistically satisfactory for the purposes of this investigation. Thus, the IRMA can be replaced by the ELISA, with advantages mainly derived from the cumbersome manipulation of radioactive wastes. The MoAb used as an antigen capture agent in the ELISA proposed here, recognizes a homologous protein in Trypanosoma rangeli, suggesting that individuals infected with this parasite might have crossreactive antibodies. However, the system retains its diagnostic interest, given the facts that the MoAb does not recognize a homologous protein in Leishmania mexicana, Leishmania donovani, or Crithidia fasciculata.

The association of skin diseases with human herpesvirus 8 infection in HIV carriers.

The seroprevalence to Kaposi's sarcoma-associated herpesvirus (KSHV) or human herpesvirus type 8 (HHV-8) was surveyed in human immunodeficiency virus type 1 (HIV-1) carriers with or without skin diseases, and also in HIV-1 negative individuals in Thailand. Using an immunofluorescence assay, the seropositive rates to lytic antigens of HHV-8 in HIV-1 carriers with or without skin diseases were 25% and 7.4%, respectively, but none of HIV-1 negative individuals had antibody. The seroprevalence to HHV-8 antigens was high in HIV positive individuals with low CD4/CD8 ratio, suggesting that HHV-8 is reactivated during the immunosuppressive state. Several polypeptides with apparent molecular weights of 34-38,000 and 40,000, which were specific to HHV-8, were identified by the immunoprecipitation test using the seropositive sera. Our results suggested that HHV-8 co-existed with HIV in HIV-1 carriers and the existence of HHV-8 may be associated with clinical features in the skin.

Passive transfer, rate of decay, and protein specificity of antibodies against equine arteritis virus in horses from a Standardbred herd with high seroprevalence

Objective— To determine rate of decay of passively acquired antibodies in Standardbred foals on a farm with a high seroprevalence to equine arteritis virus (EAV) and to determine whether vertical or horizontal transmission of the virus was responsible for infection on the farm. Design— Repeated-measures study. Animals— 46 Standardbred horses (15 brood mares and their foals, 5 stallions, and 11 young horses). Procedure— Serum samples obtained from horses on the farm were evaluated by serum neutralization and western immunoblot analysis to detect EAV-specific antibodies. The half-life of passively acquired antibodies in foals was estimated by use of regression analysis. Results— Most (14/15) of the mares evaluated were seropositive to EAV. After suckling, their foals were also seropositive. Mean biological half-life for passively acquired antibodies in serum samples obtained from foals was 32 days (r2 = 0.61). The foal born to a seronegative dam and all 11 young horses from the farm were seronegative to EAV. At least 2 of 5 stallions on the farm were persistently infected carriers that were shedding virus in their semen. Immunoblot analysis of seropositive serum samples most consistently recognized the Μ protein of EAV. Clinical Implications— Analysis of these data indicated that a modified-live EAV vaccine can be administered to foals after they are 8 months old without risk of interference from maternal antibodies, regardless of serologic status of the foal's dam. Horizontal transmission of EAV via the respiratory tract apparently was uncommon on the farm, indicating that mares primarily were infected by venereal transmission of virus from carrier stallions. (J Am Vet Med Assoc 1998;213:839-842)

Mechanisms of Resistance of HIV-1 Primary Isolates to Complement-Mediated Lysis

Previous studies suggested that HIV-1 primary isolates (PI) were resistant to complement-mediated lysis (CML), while virus produced in certain T cell lines and virus taken directly from the plasma of HIV+persons were both susceptible to CML. The purpose of this study was to investigate the mechanism(s) of PI resistance. PI were resistant to CML using pooled seropositive serum as an antibody source. Additionally, PI obtained from two patients at several times over 2 years were resistant to CML using autologous antibody. PI were also resistant to CML induced by monoclonal antibodies which neutralize a broad range of PI. Resistance to CML was associated with low binding of antibody to PI but was not due to low gp120 levels. Cell-line-derived virus and PI were equally sensitive to CML induced by antibody to host-cell proteins, suggesting that PBMC do not contribute properties to virions which make them more physically resistant to CML in general but that PI resistance is restricted to CML induced by antiviral antibody. These studies show that PI are resistant to CML mediated by various antiviral antibodies and indicate that low binding of antibody to virus is an important factor contributing to resistance.

Reactivities of Human Sera with Human Herpesvirus-8-Infected BCBL-1 Cells and Identification of HHV-8-Specific Proteins and Glycoproteins and the Encoding cDNAs

The reactivates of human sera with uninduced and phorbol ester (TPA)-induced human herpesvirus-8 (HHV-8)-infected BCBL-1 cells were examined by immunofluorescence assay (IFA) and by radioimmunoprecipitation reactions (RIP). The seroprevalence of HHV-8 infections is low in the United States general population and only low levels of HHV-8 antibodies were detected in the seropositive sera. In contrast, high levels of antibodies against HHV-8 lytic and latent antigens were detected by IFA in the sera from HIV+Kaposi's sarcoma (KS)-positive individuals. These sera recognized several proteins and glycoproteins from BCBL-1 cells in RIP reactions. Two types of antibody responses were detected in the sera from HIV+KS−homosexual men. In majority of the sera with and without detectable HHV-8 DNA in the peripheral blood mononuclear cells (PBMC), significantly low levels of HHV-8 antibodies were detected by IFA. These sera recognized only a subset of HHV-8 proteins and glycoproteins in RIP reactions. In contrast, in a subgroup of sera from HIV+KS−homosexual men, higher levels of IFA antibodies against HHV-8 lytic and latent antigens were detected. These sera also recognized several viral proteins and glycoproteins in RIP reactions. These results suggest that antibody response profiles to HHV-8 infection vary significantly and serologic assays to detect antibody responses to a panel of both lytic and latent antibodies may be required for maximum sensitivity. Screening of a cDNA library from TPA-induced BCBL-1 cells with an HIV+KS+serum identified cDNAs encoding 12 HHV-8 proteins. Further characterization of these HHV-8 proteins would define the HHV-8 antigens useful for seroepidemiological studies and in discriminating lytic, latent, past, and/or reactivation infections.

Optimal conditions of immune complex transfer enzyme immunoassays for antibody IgGs to HIV‐1 using recombinant p17, p24, and reverse transcriptase as antigens

The immune complex transfer enzyme immunoassays for antibody IgGs to p17, p24, and reverse transcriptase (RT) of HIV-1 were tested under various conditions. Antibody IgGs to HIV-1 were reacted for up to 20 hr with 2,4-dinitrophenyl-bovine serum albumin-recombinant HIV-1 protein conjugates and recombinant HIV-1 protein-β-D-galactosidase conjugates, and the immune complexes formed, comprising the three components, were trapped onto polystyrene beads coated with (anti-2,4-dinitrophenyl group) IgG by incubation at 4–30°C for up to 2 hr with shaking and were transferred onto polystyrene beads coated with (antihuman IgG γ-chain) IgG in the presence of excess of ϵN-2,4-dinitrophenyl-L-lysine by incubation at 4–30°C for up to 2 hr with shaking. When serum randomly collected from an HIV-1 seropositive subject and serum included in an Western blot kit were tested, the formation of the immune complex was almost completed within 1 hr for antibody IgG to 17, within 1–2 hr for antibody IgG to p24 and within 4 hr for antibody IgG to RT. Even for antibody IgG to p17, however, the immune complex continued to be formed for at least 2 hr, when serum samples at early stages of HIV-1 infection were tested. Trapping and transferring of the immune complexes were faster at higher temperatures and were almost completed within 0.5–1.5 hr, although the amount of the immune complexes trapped and transferred at 25 and/or 30°C increased for 0.5–1 hr, but subsequently tended to decline. When the formation, trapping, and transferring of the immune complexes were performed for 0.5, 1, and 1 hr, respectively, with shaking followed by 1 hr assay of bound β-D-galactosidase activity, the sensitivities for antibody IgGs to p17, p24, and RT using 10 μl of serum samples were similar to or significantly higher than those of the corresponding previous immune complex transfer enzyme immunoassays using 10 μl of serum samples, in which the formation, trapping, and transferring of the immune complexes were performed for 3, 16, and 3 hr, respectively, without shaking, followed by 2.5 hr assay of bound β-D-galactosidase activity, and the sensitivities for antibody IgGs to p17, p24, and RT using 100 μl of serum samples were 21–22-fold, 5.5–6.3-fold, and 5.3–6.0-fold, respectively, higher. When each period of time for the formation, trapping, and transferring of the immune complexes was prolonged to up to 4 hr, the sensitivities for antibody IgGs to p17, p24, and RT using 100 μl of serum samples were improved 88–93-fold, 15–17-fold and 20–24-fold, respectively, as compared with those of the previous ones. J. Clin. Lab. Anal. 12:98–107, 1998. © 1998 Wiley-Liss, Inc.

Optimal conditions of immune complex transfer enzyme immunoassays for antibody IgGs to HIV-1 using recombinant p17, p24, and reverse transcriptase as antigens

The immune complex transfer enzyme immunoassays for antibody IgGs to p17, p24, and reverse transcriptase (RT) of HIV-1 were tested under various conditions. Antibody IgGs to HIV-1 were reacted for up to 20 hr with 2,4-dinitrophenyl-bovine serum albumin-recombinant HIV-1 protein conjugates and recombinant HIV-1 protein-beta-D-galactosidase conjugates, and the immune complexes formed, comprising the three components, were trapped onto polystyrene beads coated with (anti-2,4-dinitrophenyl group) IgG by incubation at 4-30 degrees C for up to 2 hr with shaking and were transferred onto polystyrene beads coated with (antihuman IgG gamma-chain) IgG in the presence of excess of epsilon N-2,4-dinitrophenyl-L-lysine by incubation at 4-30 degrees C for up to 2 hr with shaking. When serum randomly collected from an HIV-1 seropositive subject and serum included in an Western blot kit were tested, the formation of the immune complex was almost completed within 1 hr for antibody IgG to p17, within 1-2 hr for antibody IgG to p24 and within 4 hr for antibody IgG to RT. Even for antibody IgG to p17, however, the immune complex continued to be formed for at least 2 hr, when serum samples at early stages of HIV-1 infection were tested. Trapping and transferring of the immune complexes were faster at higher temperatures and were almost completed within 0.5-1.5 hr, although the amount of the immune complexes trapped and transferred at 25 and/or 30 degrees C increased for 0.5-1 hr, but subsequently tended to decline. When the formation, trapping, and transferring of the immune complexes were performed for 0.5, 1, and 1 hr, respectively, with shaking followed by 1 hr assay of bound beta-D-galactosidase activity, the sensitivities for antibody IgGs to p17, p24, and RT using 10 microliters of serum samples were similar to or significantly higher than those of the corresponding previous immune complex transfer enzyme immunoassays using 10 microliters of serum samples, in which the formation, trapping, and transferring of the immune complexes were performed for 3, 16, and 3 hr, respectively, without shaking, followed by 2.5 hr assay of bound beta-D-galactosidase activity, and the sensitivities for antibody IgGs to p17, p24, and RT using 100 microliters of serum samples were 21-22-fold, 5.5-6.3-fold, and 5.3-6.0-fold, respectively, higher. When each period of time for the formation, trapping, and transferring of the immune complexes was prolonged to up to 4 hr, the sensitivities for antibody IgGs to p17, p24, and RT using 100 microliters of serum samples were improved 88-93-fold, 15-17 fold and 20-24-fold, respectively, as compared with those of the previous ones.

Markers of human T lymphotropic virus type I in patients with cancer of uterine cervix in Amazon, Brazil

Human T lymphotropic virus type I(HTLV-I) has been implicated in various human diseases. Serum samples of 390 Brazilian Amazonians with cancer of various types were tested for HTLV-I antibodies by Gelatin particle agglutination test, Enzyme linked immunosorbent assay and Western blotting. Of 134 sera from patients with cancer of uterine cervix, 4 were positive by all the methods. Three of these were from non-transfused patients. DNA was extracted from 2 of 4 seropositive sera that gave strong reactions and were analyzed by PCR-SSCP for HTLV-I sequences. One was positive for all HTLV-I genes tested while the other one was positive for LTR and tax and negative for gag. In view of a possible pathway of the virus by sexual contact, the involvement of HTLV-I in cervical cancer warrants further studies.

A search for hepatitis C virus polymerase chain reaction-positive but seronegative subjects among blood donors with elevated alanine aminotransferase.

Previous studies reported the existence of hepatitis C virus (HCV) polymerase chain reaction (PCR)-positive but seronegative sera. This is not surprising in the case of window-phase specimens, because PCR can detect HCV RNA many weeks before the appearance of antibody. To determine whether such sera can also be found in chronically infected subjects, a high-risk population of blood donors with elevated alanine aminotransferase was studied. Freshly frozen plasma from 301 donors with alanine aminotransferase > 100 IU per L was tested with PCR assays that were rigidly controlled for specificity and contamination, and with current and newer versions of assays for anti-HCV. Sera were classified as seropositive if positive in two screening assays and one supplemental assay or if positive in two screening assays and PCR. New versions of screening assays detected 100 percent of seropositive samples. A second-generation immunoblot assay detected 98 percent of seropositive sera, a second-generation recombinant immunoblot assay detected 96 percent, and an enzyme immunoassay for antibody to the envelope protein of HCV detected 98 percent. Fifty-one of 54 seropositive sera were PCR positive. None of the 247 seronegative samples was reproducibly positive on PCR. No PCR-positive but seronegative donors were found in this high-risk donor population. The possible benefit of PCR screening of blood donors can be determined only by large-scale comparative testing of donor populations and may be limited to the detection of window-phase infections.

Optimisation of a peptide-based indirect ELISA for the detection of antibody in the serum of HIV-1 seropositive patients

A modified peptide-based indirect ELISA technique for the detection of HIV-1 specific antibodies in the sera of HIV-1 seropositive individuals is described. We found that the reduction of non-specific binding of HIV-1 seropositive sera to the ELISA plate was essential for the reliable detection of serum antibodies in the peptide based indirect ELISA. Optimal results were obtained using Immulon microtitre plates, different concentrations of denatured, purified grade of casein in the blocking (1%) and washing (0.25%) solutions and by diluting HIV-1 seropositive sera 1 in 1600. These conditions reduced non-specific binding and improved assay sensitivity. We show that the inclusion of a control peptide is essential to reducing the incidence of false positive and false negative results. Taken together, the modifications described in this report improve reliability of the peptide-based indirect ELISA without compromising its sensitivity and have particular relevance for those wishing to apply the peptide-based indirect ELISA technique to serum samples which exhibit high levels of non-specific binding. To illustrate this, levels of antibody in the sera of HIV-1 seropositive and seronegative donors that are specific for peptides derived from a conserved region of HIV-1 gp120 sharing homology with the FAS apoptosis antigen were analysed using this technique.

Glycoprotein H of human cytomegalovirus is a major antigen for the neutralizing humoral immune response.

A recombinant baculovirus expressing glycoprotein H (gpUL75) of human cytomegalovirus was used to examine the humoral immune response in naturally infected individuals. Recombinant baculovirus infected insect cells produced two forms of gH with molecular masses of 78-82 kDa and 94 kDa. The 94 kDa polypeptide was modified by high mannose oligosaccharide side-chains as shown by reduction in molecular mass after treatment with endoglycosidases H and F. The 78-82 kDa protein represented the non-glycosylated precursor which was resistant to the enzymes. In contrast to gH expressed in mammalian cells, the recombinant baculovirus expressed gH was transported to the cell surface. Glycoprotein H produced in insect cells was reactive with human convalescent sera and all tested neutralizing monoclonal antibodies recognizing either linear or conformational epitopes. Antibodies reacting with insect cell derived gH were detected in 96 percent of HCMV seropositive human sera. Using insect cells infected with the gH expressing recombinant baculovirus as immunoabsorbent, between 0 percent and 58 percent of the total virus neutralizing activity was removed from sera of individuals with a past HCMV infection. gH must therefore be considered a major antigen for the induction of neutralizing antibodies during natural infection.

Mechanisms of opsonized HIV entry in normal B lymphocytes

Using our in vitro model of normal B cell infection that functions with low doses of HIV but requires virus opsonization by seropositive patient serum, and complement, we analyzed what receptors allowed virus entry. Here, we show that HIV infection of B cells occurs through 2 major receptors: the CD4 antigen and the CR1/CR2 complex. These 2 pathways work independently since a complete inhibition of virus entry requires both CD4 and CD21/CD35 blockade on CD4 dim tonsillar B cells whereas only the latter is critical on CD4-negative B cells.

Phosphonoacid prodrugs with greatly increased antiviral activity in HCMV-infected cells, in vitro: K.Y. Hostetler, K.A. Aldern, G.D. Kini, C.J. Wheeler and C.N. Sridhar, University of California, San Diego, La Jolla, CA 92093-0676; Veterans Affairs Medical Center, San Diego, CA. 92161; and Vical Inc., San Diego, CA., 92121

Human cytomegalovirus (HCMV) is a leading cause of congenital infection that can result in serious disabilities in affected children. To facilitate HCMV vaccine development, a microscale neutralization assay based on reverse transcription quantitative PCR (RT-qPCR) was developed to quantify HCMV-neutralizing antibodies. Our approach relies on the generation of crude lysates from virus-infected cells that are amenable to direct analysis by RT-qPCR, thereby circumventing rate-limiting procedures associated with sample RNA extraction and purification. By serial passaging of the laboratory HCMV strain AD169 in epithelial cells (ARPE-19), a revertant virus with restored epithelial cell tropism, designated AD169wt131, was obtained. AD169 and AD169wt131 were evaluated in both epithelial cells (ARPE-19) and fibroblasts (MRC-5) by one-step RT-qPCR targeting the immediate-early gene IE1 transcript of HCMV. Expression kinetics indicated that RT-qPCR assessment could be conducted as early as 6 h post-infection. Human serum samples (n = 30) from healthy donors were tested for HCMV-specific IgG using a commercially available ELISA and for HCMV-neutralizing activity using our RT-qPCR-based neutralization assay. In agreement with the ELISA results, higher neutralizing activity was observed in the HCMV IgG seropositive group when compared with the HCMV IgG seronegative group. In addition, HCMV IgG seropositive human sera exhibited higher neutralizing titers using epithelial cells compared with using fibroblasts (geometric mean titers of 344 and 8 in ARPE-19 cells and MRC-5 cells, respectively). Our assay was robust to variation in input virus dose. In addition, a simple lysis buffer containing a non-ionic detergent was successfully demonstrated to be a less costly alternative to commercial reagents for cell-lysate preparation. Thus, our rapid HCMV neutralization assay may be a straightforward and flexible high-throughput tool for measuring antibody responses induced by vaccination and natural infection.