Epitope mapping of the HIV-1 gag region with monoclonal antibodies.

A new type of immunochemical mapping of the human immunodeficiency virus type 1 (HIV-1) gag region was performed. By use of native HIV-1 viral lysates or the gag recombinant p24-15 antigen, a new set of monoclonal antibodies (Mabs) to the gag region proteins was generated. Synthetic HIV-1 peptides covering the entire gag region were used to specifically localize the continuous epitopes by direct binding to the Mabs and by blocking the Mab immunoreactivity. The identified immunogenic epitopes were localized between the gag amino acids (aa) 108-127, 203-217, 208-222, 248-282, 273-302, 288-307, 308-322, 331-354 and 408-422. These continuous epitopes formed seven immunogenic regions. One strongly p17-reactive Mab appeared to react with a discontinuous epitope, the components of which were 110 aa distant in the linear sequence: aa 23-27 and 128-132. The synthetic peptides appeared to be more congruent with the Mab-reactive sites in solution than when coated to a solid phase.

IgG-subclass-specific CMV reactivity in bone marrow transplant recipients.

IgG subclasses of cytomegalovirus (CMV) antiviral antibodies were determined in 37 donor-recipient pairs of bone marrow transplants (BMT). Bone marrow transplant recipients, like healthy persons, have a restricted immune reactivity, producing mainly two types of anti-CMV IgG: IgG1 and IgG3. Passively transfused specific antibody subclasses were readily measurable. Take of the transplant could be detected from the production of subclass IgG antiviral antibody 1-3 months after BMT of seronegative recipients with marrow from seropositive donors. Patients with protracted CMV infections or other severe diseases initially also produced CMV IgG1 and IgG3, but anti-CMV subclass titers then decreased. In severe disease, CMV was isolated from blood cells as well as from urine. In moderate infections, in which the patients recovered, CMV was isolated from urine but usually not from blood, and a strong and durable antiviral subclass response was measured.

Cytomegalovirus DNA in peripheral blood leukocytes and plasma from bone marrow transplant recipients.

Granulocytes, monocytes, and T- and B-lymphocytes were separated from 28 blood samples collected from 5 bone marrow transplant (BMT) recipients. About 40% of granulocyte, monocyte, and B-lymphocyte samples were CMV DNA-positive by polymerase chain reaction in recipients with cytomegalovirus (CMV) infection. CMV DNA was rarely detected in separated T-lymphocytes. Within each of the simultaneously separated paired samples, there were several with single positive cell subtypes. Monocytes, granulocytes, and B-lymphocytes were the single positive samples in some instances. Thus, it is important to have all of the different cell subtypes present in samples for detection of CMV DNA in peripheral blood. We also studied the appearance of CMV DNA in plasma and peripheral blood leukocytes (PBLs) from 351 blood samples collected from 30 BMT recipients during a follow-up period of at least 3 months after BMT. All cell subtypes were represented in the PBL samples. In the 13 recipients who developed symptoms possibly associated with CMV infection or CMV disease, a correlation with the detection of CMV DNA in < or = 2 x 10(5) PBLs was found. In PBLs from 11 of the 13 BMT recipients, CMV DNA was detected before the onset of symptoms. CMV DNA was not detected in < or = 2 x 10(5) PBLs from recipients without CMV infection. The virus load in PBLs decreased during ganciclovir treatment. Nine of the 13 recipients displayed PCR-positive plasma samples, and CMV DNA was detected frequently after the onset of symptoms.

Human polyspecific immunoglobulin for therapeutic use induces p21/WAF-1 and Bcl-2, which may be responsible for G1 arrest and long-term survival.

High-dose intravenous immunoglobulin (IVIg) is used as therapy in an increasing number of immune mediated disorders including infections and autoimmune conditions. IVIg exerts profound effects both in vivo as well as in vitro on humoral and cell-mediated immunity. In this study we investigated whether IVIg could alter the pattern of apoptosis and apoptosis related proteins including Bcl-2, Bax, p53, CD95, and p21/WAF-1, a protein well known to arrest cells in G1 phase of the cell cycle and finally proliferation marker Ki-67 on peripheral blood mononuclear cells (PBMC). The cells were cultured either unstimulated or with mitogen in the presence or absence of different IVIg preparations. A dual effect by IVIg was found. The incidence of apoptosis was elevated in activated Ki-67 and CD95 positive PBMC, whereas it was lower in small, nonactivated cells. The cells that survived were associated with a striking increase in the expression of p21/WAF-1 suggesting G1 arrest. A concomitant upregulation of Bcl-2 was also obtained by IVIg exposition resulting in long-term survival. We suggest that these abilities of IVIg to alter cell cycle progression and apoptosis could explain some of the beneficial effects obtained in vivo with IVIg therapy.

HIV antigen-reactive T cells detected by antigen-induced interferon gamma secretion.

The T cell repertoire to human immunodeficiency virus (HIV) was studied in HIV-infected patients of different clinical stages by the detection and enumeration of cells that secreted interferon gamma (IFN-gamma) in short-term cultures of blood mononuclear cells after stimulation in vitro with the HIV recombinant antigens pB1, p121, p24-15, gp160bac, and the HIV V3 loop peptide. T cell reactivities to cytomegalovirus (CMV) and Mycobacterium tuberculosis-purified protein derivative (PPD) were examined in parallel. Among 29 patients with HIV infection, 48% had blood cells recognizing one or more of the five HIV antigens. The mean numbers of HIV antigen-reactive T cells varied between 1/approximately 6000 blood cells for pB1 and 1/approximately 20,000 cells for p24-15. None of the five HIV antigens studied was identified as an immunodominant T cell epitope in HIV infection. T cells from 20% of the patients responded to all five HIV antigens in parallel, but the antigen preferentially recognized varied from patient to patient. Those with more advanced disease had a tendency to lower numbers of HIV antigen-reactive T cells. Most HIV-infected patients had both CMV- and PPD-reactive T cells, but numbers were significantly lower in more advanced disease. It should be possible to adopt the present method to evaluate fine specificities of the T cell repertoire to other antigens and to study the involvement of other cytokines besides IFN-gamma, for example, the Th2 cell-related cytokine interleukin 4.

Solid phase ELISA for determination of the virus dose dependent sensitivity of human cytomegalovirus to antiviral drugs in vitro.

The main problems in determining the true in vivo susceptibility of human cytomegalovirus (CMV) to antiviral compounds are the influence of the size of the viral inoculum, the variation in the replication capacity of different CMV strains and the subjective evaluation of the inhibition of viral growth in the plaque assay. In this study, a specific assay was developed which reproducibly determines the sensitivity of primary isolates of CMV. The assay includes simultaneous virus titration and determination of the antiviral sensitivity. When individual virus doses were evaluated, the IC50 was generally dependent on the virus dose, except for resistant isolates, where the IC50 did not change irrespective of the dose of virus. The novel method of IC50 calculation takes into account all values derived from the linear part of the inhibition curve. This may better reflect the in vivo conditions, where the antiviral drug encounters different amounts of virus in different organs. Two human fibroblast-derived cell lines showed similar results.

Frequency and specificity of varicella zoster virus IgM response.

A direct ELISA was developed for determination of IgM antibody to varicella zoster virus (VZV). With this sensitive method VZV IgM antibodies were detected in all patients with a varicella and in 84% with a herpes zoster infection. All but one of 28 renal allograft recipients had previously had varicella. A primary infection was seen in the last patient, and reactivated infections in 11 of the others. The VZV IgM response seems to be specific since patients with a herpes simplex virus (HSV) infection and a heterotypic VZV IgG titer rise did not have detectable VZV IgM. An indirect enzyme-linked immunosorbent assay (ELISA) for detection of IgG antibodies has been used for serodiagnosis of VZV infections and to determine the immune status. After injection of zoster immune globulin, it was possible to measure passively transferred antibodies.

An interchangeable ELISA for cytomegalovirus antigen and antibody.

An enzyme-linked immunosorbent assay (ELISA) was developed to demonstrate cytomegalovirus (CMV) antibodies and antigen. A nuclear antigen from CMV-infected cells was used for detection of IgG and IgM antibodies. Significant rises of CMV-IgG and significant levels of CMV-IgM were found in sera from patients with acute CMV only, and not with other herpesvirus infections. CMV antigens were demonstrated in cells and in culture medium by a sandwich or by an inhibition ELISA technique. In the sandwich technique, the plates were coated with monkey anti-CMV IgG and rabbit anti-CMV IgG was used as the second antibody. Both early and late CMV antigens were identified with this method. Treatment of CMV containing samples using freeze--thawing or detergent reduced the infectivity, but the antigenicity was not affected. There was no cross-reactivity with herpes simplex or varicella-zoster virus.

Lymphocyte proliferation and IgG production with herpesvirus antigens in solid phase.

A technique for simultaneous specific lymphocyte proliferation and IgG production with herpesvirus antigens in solid phase was developed. The cell mediated immune response was highly specific. Stronger cellular responses were found after stimulation with nucleocapsid antigens than with membrane antigens. Varicella-zoster virus (VZV) and herpes simplex virus (HSV) specific antibody production in vitro were found in almost 100% of seropositive individuals, while cytomegalovirus (CMV) antibody production was detectable in 70%. A higher specific IgG production was found after stimulation with CMV and VZV membrane antigens than with CMV nucleocapsid and VZV cell antigens. No differences in IgG production were found with the two types of HSV antigens. A method for viral IgG subclass determination in vitro was also developed. These methods may be used for monitoring immunosuppressed patients for immunological responsiveness to herpesvirus infections.

Computer-based virus sensitivity assay and neutralization method. Applications for herpesviruses.

A method for computerized analysis of the antiviral efficiency of drugs and immunoglobulins was developed. The sensitivities of herpes simplex virus (HSV), cytomegalovirus (CMV) and varicella-zoster virus (VZV) to antiviral drugs were determined. A new type of neutralization assay could be performed rapidly and accurately with HSV and CMV. Combining immunological methods for herpesvirus antigen detection with a computerized analysis permitted rapid, sensitive measurement of drug sensitivities, comparison of antiviral activities at various virus concentrations and selection of potent neutralizing antisera.

Cytomegalovirus DNA detection of an immediate early protein gene with nested primer oligonucleotides.

A rapid and sensitive polymerase chain reaction (PCR) was developed to detect conserved sequences from the immediate early gene of human cytomegalovirus (HCMV). The primers sequences were from EcoRI J fragment of Ad169. The first primer set was selected to amplify a 242 bp fragment and the next primer set was nested within the first and amplified a 146 bp fragment. With the single PCR system it was possible to detect 100 fg HCMV DNA but with double PCR 5-10 fg were detectable. Specific amplification was seen in urines from patients with HCMV infections. 20 urine samples were analysed by single PCR, double PCR and virus cultivation. The double PCR was the most sensitive method. Urines from healthy seropositive persons and cells infected with other members of the herpes virus family were negative with all three methods. This suggests that specific amplification by double PCR is sensitive and can be used for rapid detection of HCMV DNA in cases with activated infection.

An enzyme-linked immunosorbent assay for IgG antibodies to human herpes virus 6.

An indirect enzyme-linked immunosorbent assay (ELISA) with human herpes virus 6 (HHV6) membrane antigen was compared with indirect immunofluorescence assay (IFA) for measurement of HHV6 IgG antibodies. Five hundred serum samples from 403 Swedish patients with suspected symptomatic Epstein-Barr virus (EBV) infections were examined. The specificity of the ELISA compared with IFA was 98.7% and the sensitivity was 98.4%. In 90% of the patients, IgG antibodies to HHV6 were detected with both assays. The highest HHV6 IgG titers were found mainly in patients with EBV or CMV infections, but HHV6 mononucleosis was not diagnosed. The same HHV6 antigen was assessed for IgM ELISA but was found to be of limited value due to high IgM reactivity with the control antigen. The HHV6 IgM ELISA requires further investigation. The IgG ELISA described is a reliable alternative to IFA for measurement of HHV6 IgG antibodies and for large scale epidemiological studies.

Acid hydrolysis of serum samples to increase detection of HIV antigen.

Sera from 32 HIV-infected patients and 20 controls were assayed for HIV antigen (HIV-Ag) and antibodies following acid hydrolysis. Acid hydrolysis, followed by neutralization immediately prior to the assay, was found to be a simple means to solubilize immune complexes and allowed recovery of 40-50% of complexed antigen. Following acid hydrolysis, HIV-Ag levels rose or became detectable in 22/32 patients and HIV IgG1-4 levels rose in 17/20 patients studied. The results show that complexed HIV-Ag may evade detection in HIV-infected patients.

A novel method for determining the sensitivity of herpes simplex virus to antiviral compounds.

A rapid and sensitive assay was developed to analyse the sensitivity of wild type HSV-1 and HSV-2 isolates with respect to a battery of antiviral substances. In the viral sensitivity assay, human embryonic lung fibroblasts are incubated with the virus isolate and different concentrations of the antivirals. After 1-3 days, the cells are disrupted and analysed for HSV type 1 or 2 antigens by an enzyme-linked immunosorbent assay. Antigens corresponding to 17 plaque-forming units were detectable after 1 day of incubation. After 3 days, HSV antigens derived from less than one plaque-forming unit were measurable. The sensitivities of 22 HSV-1 and 19 HSV-2 primary isolates from untreated patients were tested against adenosine arabinoside, acyclovir, phosphonoformic acid and iododeoxyuridine. Each isolate was found to have an individual pattern of sensitivities to the different antivirals. Five isolates were judged to be relatively resistant to one or more of the drugs tested.

Sensitive analytic ELISAs for subclass herpes virus IgG.

The subclass distribution of antiviral antibodies to three herpes viruses was studied in a population of healthy blood donors. Subclassification by monoclonal antibodies led to the identification of certain viral IgG patterns. IgG1 appeared to be formed in response to almost all CMV, HSV-1 and VZV infections. A higher frequency of virus-specific IgG3 to CMV and HSV-1 suggested that these infections may be reactivated subclinically more often than VZV. The presence of CMV and VZV IgG4 showed a familial relationship, while IgG4 responses to HSV-1 were common. Persons with IgG4 as the only subclass-reactive antibody to CMV showed cell-related reactivities in a high frequency. Patients with leukemias, myelomas and Crohn's disease had a near-normal subclass pattern to the herpes viruses.

Combination ELISAs for antiviral antibodies in CSF and serum in patients with neurological symptoms and in healthy controls.

A combination enzyme-linked immunosorbent assay (ELISA) was designed to improve the estimation of serum: cerebrospinal fluid (CSF) antiviral IgG ratios. Microplate wells were coated with five different virus antigens. Serum and CSF from 66 patients referred for CSF serology and from 21 healthy controls were studied. In a comparison with serum: CSF IgG titre ratios, absorbance ratios were found to be suitable for the evaluation of intrathecal antiviral IgG synthesis. A slight blood-brain barrier (BBB) disturbance affected only the passage of albumin over the BBB, whereas a more pronounced BBB defect resulted in increased IgG levels in the CSF. Intrathecal antiviral IgG synthesis was demonstrated in 15 patients with viral CNS infections or inflammatory diseases. Very high serum: CSF antiviral IgG titre ratios and absorbance ratios, found in six patients, were interpreted as signs of diminished IgG passage over the BBB due to impaired CSF circulation.

Total, anti-viral, and anti-myelin IgG subclass reactivity in inflammatory diseases of the central nervous system.

Total IgG subclass levels, anti-viral, anti-myelin basic protein (anti-MBP), and anti-ganglioside 1 (anti-GM1) IgG subclass levels were measured in 6 patients with herpes simplex virus encephalitis (HSVE), 16 with borreliosis, 8 with other bacterial infections, 12 with multiple sclerosis (MS), 13 with subacute sclerosing panencephalitis (SSPE), 5 with glioblastoma and 12 controls. Total IgG1 levels were elevated in cerebrospinal fluid (CSF) from all patient groups (but not in the controls), IgG2 in bacterial infections, IgG3 in HSVE and borreliosis and IgG4 in some SSPE patients. The anti-viral (anti-measles, varicella zoster virus and rubella) IgG antibodies in MS were restricted to IgG1, anti-measles IgG to IgG1 and sometimes IgG4 in SSPE, anti-borrelia IgG to IgG1, IgG2 and IgG3. In contrast to anti-viral antibodies, anti-MBP and GM1 antibodies belonged to IgG1, IgG3 or IgG4 in MS. The nature of the immunological activation appears to be reflected in the subclass patterns elicited in the central nervous system. Different IgG subclass patterns in infectious diseases and MS suggest a difference between antigen-specific and non-specific B-cell activation.

T and B cell responses to cytomegalovirus antigens in healthy blood donors and bone marrow transplant recipients.

We measured the production of interferon-gamma (IFN-gamma) from single T cells and the T cell proliferative response to different cytomegalovirus (CMV) antigens in healthy blood donors and bone marrow transplant recipients. The antigens consisted of a CMV nuclear antigen (CMV na) containing the pp65-kDa matrix protein and the immediate early antigens but lacking CMV glycoproteins, and an antigen comprising native CMV glycoproteins (CMV gp). We also measured the IgG antibodies to CMV na and CMV gp. The T cells reacted to CMV na in CMV seropositive blood donors both with the production of IFN-gamma and with proliferation, while bone marrow transplant recipients had a deficient T cell response. After stimulation with CMV gp, no T cell response could be observed in CMV seropositive subjects. IgG antibodies to CMV na coexisted in plasma with similar levels of antibodies to CMV gp.

Anti-herpes IgG and IgG subclass antibodies in Bell's palsy.

To ascertain if there is an association between Bell's palsy and infection by viruses of the herpes group, 78 patients and 59 controls were investigated. The specific antiviral IgG subclass pattern in serum against cytomegalovirus (CMV), herpes simplex virus type 1 (HSV-1) and varicella zoster virus (VZV) was analysed using an enzyme-linked immunosorbent assay (ELISA) combined with monoclonal antibodies directed against the four subclasses of human IgG. Additional ELISA assays of IgG and IgM antibodies were also used. The mean titres of IgG antibodies against HSV-1 were higher in the acute and convalescent stages compared with controls. The frequency of values greater than 0.2 for all subclasses was raised in the patients, but not significantly so. The mean values for the subclasses were alike in patients and controls. The mean titres of IgG antibodies against CMV and VZV were similar throughout the palsy and also in controls. In the patients, the pattern of IgG subclasses was different from the controls, but not statistically so. The patients and controls were not seropositive for IgM against CMV and VZV. Four patients in the acute phase, 4 in the convalescent phase and 3 controls were positive for IgM against HSV-1. While the subclass pattern of IgG antibodies against HSV-1 is not diagnostic of reactivation of the virus, the raised IgG antibodies may suggest reactivation of a disease process and/or a superadded infection.

Induction of MHC class I antigen expression following infection of a human esthesioneuroblastoma cell line with cytomegalovirus and human immunodeficiency virus.

Productive infections with cytomegalovirus (CMV) and human immunodeficiency virus (HIV) were established in the Tp41ON cell line derived from a human esthesioneuroblastoma. HIV antigen expression was highest in cultures coinfected with CMV and HIV. Viral infection caused increased MHC class I antigen expression while class II and CD4 antigens remained undetectable using immunofluorescence methods. Uninfected cultures showed 10% and coinfected cultures 80% class I antigen positive cells. In coinfected cultures, CMV and HIV antigens were detected in 4% and 8% of the cells, respectively. The detection of CMV antigens in some multinucleated cells suggests coinfection with both viruses in these cells, as multinucleated cells were not found in cultures infected with CMV only. The study shows that a cell line showing neuronal differentiation in vitro can be infected with CMV and HIV and that this infection increases MHC class I antigen expression.