Wolfgang Lüke

Analysis of the Systemic and Intrathecal Humoral Immune Response in Progressive Multifocal Leukoencephalopathy

Progressive multifocal leukoencephalopathy (PML) is a subacute viral infection of oligodendrocytes by JC virus occurring almost exclusively in immunocompromised patients. By use of partially purified recombinant VP1 as antigen, the IgG response was analyzed by a quantitative ELISA of paired cerebrospinal fluid (CSF) and serum samples. An intrathecal immune response to VP1, defined as an antibody-specificity index of CSF to serum antibody titers > or =1.5, was found in 76% of PML patients (47/62) but in only 3.2% of controls (5/155) (P < .001). Intra-blood-brain barrier synthesis of VP1-specific IgG antibodies is 76% sensitive and 96.8% specific for the diagnosis of PML. Furthermore, the excellent correlation (r = .985) between the plasma cell count in brain tissue and the humoral intrathecal immune response to VP1 in PML patients suggests a role for B cells in this disorder.

Highly Active Antiretroviral Therapy and Progressive Multifocal Leukoencephalopathy: Effects on Cerebrospinal Fluid Markers of JC Virus Replication and Immune Response

Cerebrospinal fluid (CSF) samples were examined from 7 patients infected with human immunodeficiency virus type 1 (HIV-1) who had progressive multifocal leukoencephalopathy (PML). Samples were obtained both before and after 35-365 days of highly active antiretroviral therapy (HAART). By polymerase chain reaction, JC virus (JCV) DNA was found in 6 of 7 patients at baseline but in only 1 patient after HAART. In contrast, in 25 historical control patients from whom sequential CSF specimens were obtained, no reversion from detectable to undetectable JCV DNA was observed. By use of enzyme-linked immunosorbent assay, intrathecal production of antibody to JCV-VP1 was shown in only 1 of 4 HAART recipients at baseline but in 5 of 5 patients after treatment. The neuroradiological picture improved or had stabilized in all patients after 12 months of HAART, and all were alive after a median of 646 days (range, 505-775 days). Prolonged survival after HAART for PML is associated with JCV clearance from CSF. JCV-specific humoral intrathecal immunity may play a role in this response.

Cellular and humoral immune response in progressive multifocal leukoencephalopathy.

Progressive multifocal leukoencephalopathy (PML) is a fatal, demyelinating disease caused by JC virus (JCV) in patients with severe immunosuppression. We studied the JCV‐specific cellular and humoral immune response in 7 healthy donors (HD), 6 human immunodeficiency virus‐1 (HIV‐1)‐infected patients without PML (HIV), 4 HIV‐1‐negative patients with PML (PML), and 8 HIV‐1‐positive patients with PML (HIV/PML). As antigens, recombinant virus‐like particles of the major structural protein VP1 (VP1‐VLP) of JCV, tetanus toxoid (TT), or the mitogen phytohemagglutinin (PHA) were used. Proliferation of peripheral blood mononuclear cells (PBMC) after stimulation with the VP1‐VLP was significantly suppressed in PML and HIV/PML patients compared to HD. After antigen stimulation the production of interferon‐γ (IFN‐γ) was reduced in PML, in HIV/PML, and in HIV patients. The production of interleukin‐10 (IL‐10), however, was elevated in HIV/PML patients. Neither proliferation nor cytokine production correlated with the presence of JCV DNA in PBMC. The immunoglobulin G serum antibody titer to the VP1‐VLP was slightly elevated in HIV, elevated in PML, and highly elevated in HIV/PML patients compared to HD. The development of PML appears to coincide with a general impairment of the Th1‐type T‐helper cell function of cell‐mediated immunity.

Progressive multifocal leukoencephalopathy diagnosed by amplification of JC virus-specific DNA from cerebrospinal fluid.

ObjectiveTo study the diagnostic sensitivity and specificity of polymerase chain reaction (PCR) for the non-invasive diagnosis of progressive multifocal leukoencephalopathy (PML) in HIV-1-infected individuals. DesignRetrospective analysis of stored cerebrospinal fluid (CSF) samples by PCR of HIV-1-infected patients. MethodsResults of the PCR analysis of the CSF of three AIDS patients with autopsy-proven PML were compared with the results in 15 neurologically asymptomatic HIV-1-infected patients and with 15 AIDS patients with other opportunistic infections of the central nervous system (CNS). A polyclonal antiserum to simian virus 40 (SV40) cross-reacting with JC virus (JCV) late antigens was used for immunocytochemical confirmation of the diagnosis. Two different primer pairs, one taken from the VP1/large T gene and the other from the large T gene, were used to amplify JCV-specific DNA sequences from CSF. ResultsFive CSF samples were analysed and JCV-specific DNA found in three patients with autopsy-proven PML. No JCV-specific DNA was detected in 47 CSF samples, including serial samples from 14 of the 30 non-PML patients. The diagnosis of PML was confirmed in all three cases by immunocytochemistry. Conclusion: PML can be diagnosed by PCR analysis of CSF. The sensitivity and specificity of the method depends on the sensitivity of the primers used for amplification. Using a primer pair from the large T gene, JCV-specific DNA was amplified in three cases with PML as early as the day of presentation with the first neurological symptom of PML.

Experimental infection of macaques with HIV-2ben, a novel HIV-2 isolate.

Ten rhesus (Macaca mulatta) and six fascicularis (Macaca fascicularis) macaques were inoculated with HIV-2ben using three different virus preparations and two routes of inoculation. Thirteen of the 16 inoculated macaques seroconverted 2-6 weeks after infection. Three M. mulatta remained seronegative. The seroconverted animals developed antibody titres from 80 to 40,000. Their antibodies reacted with gp160 and gp130 and, in varying degrees, with gp32 and core proteins. Virus could be re-isolated from 11 of the 16 macaques. M. mulatta were transiently viraemic 6-14 weeks after infection whereas all M. fascicularis were persistently viraemic 2-7 weeks after infection onwards. In the 6-18 months after infection one M. mulatta lost 20% of its body weight and two M. fascicularis showed transient lymphadenopathy and splenomegaly; the other animals remained clinically normal. A re-isolated virus from a M. mulatta was indistinguishable from the inoculated HIV-2ben by genomic restriction enzyme analysis. M. mulatta and M. fascicularis are infectable by a single intravenous injection of cell-free HIV-2ben. Persistent viraemia in M. fascicularis represents a valuable and reliable parameter for studies on antivirals and vaccines.

Clinical implications of nucleic acid amplification methods for the diagnosis of viral infections of the nervous system

Amplification of viral nucleic acids from the cerebrospinal fluid (CSF) has considerably improved the diagnosis of several acute, subacute and chronic viral infections of the nervous system. In herpes simplex virus (HSV) encephalitis (HSE) the polymerase chain reaction (PCR) has become the method of choice for the rapid, non invasive diagnosis. Other herpes virus associated diseases which can now be reliably diagnosed are encephalitis, ventriculoencephalitis, polymyeloradiculitis, myelitis and an inflammatory polyradiculoneuropathy caused by cytomegalovirus (CMV), HSV, varicella-zoster virus (VZV) or Epstein-Barr virus (EBV), EBV associated primary B-cell-lymphoma of the brain, acute aseptic meningitis in young adults allied with VZV, and meningoencephalitis with recurrent seizures due to human herpes virus type 6 (HHV-6). In AIDS patients, PCR has helped to differentiate lesions either due to the human immunodeficiency virus (HIV) itself or to opportunistic infections such as progressive multifocal leukoencephalopathy (PML) caused by JC virus (JCV) or CMV related complications. HIV can be detected early in the course of infection in the CSF and the amount of proviral DNA in CSF cells seems to be correlated with the severity and/or progression of neurological signs and symptoms. Acute epidemic aseptic meningitis caused by enterovirus infections can now be reliably diagnosed and typed by reverse transcriptase PCR (RT-PCR). Meningitis cases caused by vaccination with the Jeryl Lynn and Urabe vaccine strain of mumps virus have been identified using RT-PCR and sequencing of the amplified products (amplicon).

Packaging of small molecules into VP1-virus-like particles of the human polyomavirus JC virus

Recombinantly expressed VP1-virus-like particles (VP1-VLP) of human polyomavirus JC virus (JCV) were described recently as a new DNA transporter system. It was shown that DNA molecules could be packaged into VP1-VLP during a controlled chemical reassociation/dissociation process. In the present study VP1-VLP were studied as carriers for pharmaceutical substances. Propidium iodide (PI) was packaged into VP1-VLP as a reporter molecule. The PI-containing VP1-VLP could be detected directly by flow cytometry. The fluorescence intensity of the VP1-VLP depended strongly on the initial PI concentration. This packaging method is easy to handle and applicable to viruses and VP1-VLP which can be dissociated and reassociated chemically.

Immunization with tween-ether-treated SIV adsorbed onto aluminum hydroxide protects monkeys against experimental SIV infection.

In order to examine the efficiency of an AIDS vaccine potentially acceptable for human use we have investigated a split vaccine. Since such vaccines are safe and efficient, they have been in use for many years to protect man against enveloped RNA viruses, e.g., influenza and measles. Seven rhesus monkeys were immunized at Week 0, 4, 8, and 16 by im injection of 2 ml of vaccine containing 140 micrograms of Tween-ether-disrupted SIVmac251/32H adsorbed onto aluminum hydroxide. The immunized animals and three nonvaccinated control monkeys were challenged 2 weeks after the last immunization by iv injection of 10 to 50 minimal monkey infectious doses of SIVmac251/32H. Four of seven immunized animals did not show any signs of virus replication and therefore appeared to be protected. Nonvaccinated control animals and the vaccine failures showed a rise in their urinary neopterin concentrations 1 to 2 weeks after infection. At the end of the second week and thereafter, cocultures and polymerase chain reaction of their peripheral blood lymphocytes were positive. After the challenge, control animals and infected vaccinees showed a primary or secondary antibody response while antibody titers declined in virus-negative animals. Specific cytotoxic T-lymphocytes were not present prior to challenge, but were present in some animals thereafter. Therefore, these seem to reflect a response to viral replication rather than to immunization. Prior to challenge the CD4-positive lymphocytes of the peripheral blood of the four virus-negative animals only proliferated after exposure to the immunizing antigen. Thus, this reaction appears to predict protection.

Protection of monkeys by a split vaccine against SIVmac depends upon biological properties of the challenge virus.

ObjectiveTo investigate the role of the anti-cellular immune response in the protection of rhesus macaques against infection with the simian immunodeficiency virus SIVmac. To determine the biological differences between SIV challenge stocks grown either on human T-cell lines or on monkey peripheral blood mononuclear cells (MPBMC). DesignA protective SIVmac split vaccine was administered to rhesus macaques and their anti-, B- and T-cell response monitored. Vaccinees and controls were challenged with SIVmac grown either on human or on monkey cells. The in vivo replication rate of, and the immune response to, the two viruses was compared. MethodsFive rhesus macaques were immunized with a total of 2 mg each of purified SIVmac251/32H grown on the human C8166 T-cell line. The antibody and proliferative T-cell responses were evaluated by enzyme-linked immunosorbent assay and T-cell proliferation assay, respectively. Four protected animals and four controls were reboosted and challenged with MPBMC-grown SIVmac251 (SIVmac251/MPBMC). Cell-free virus load was determined by titration of plasma for SIV infectivity on C8166 cells and antigen with a core antigen capture assay. ResultsProtection from virus challenge with C8166-grown SIVmac251/32H or SIVmac251/ MPBMC did not correlate with anti-cellular antibodies or proliferative T-cell reactivities. Control animals infected with SIVmac251/MPBMC showed high persistent antigenaemia and high plasma virus titres. Both were absent in controls infected with complement C8166-grown SIVmac251/32H. Whereas the latter always seroconverted against the full panel of viral polypeptides, SIVmac251/MPBMC-infected animals showed a drastically decreased antibody response. ConclusionsNeither the antibody nor the proliferative T-cell response to SIVmac correlates with protection from virus challenge. In contrast to SIVmac251/32H grown on C8166 cells, the MPBMC-grown challenge virus SIVmac251 appears to belong to the ‘rapid-high’ phenotype, possibly explaining the lack of protection against this SIV.

Repeated exposure of rhesus macaques to low doses of simian immunodeficiency virus (SIV) did not protect them against the consequences of a high-dose SIV challenge

As part of an in vivo titration study of the macaque simian immunodeficiency virus (SIVmac) strain 251/spl, macaques were inoculated intravenously with various dilutions of this infectious SIVmac. Seven animals received dilutions from 10(-3) to 10(-6) of SIVmac251/spl. Two monkeys infected with the 10(-3) dilution of SIVmac exhibited a productive infection as indicated by seroconversion, detection of genomic RNA and proviral DNA and positive virus isolation. These animals showed a cytotoxic T cell (CTL) response against different SIVmac proteins without any measurable T cell proliferation. The five macaques receiving higher virus dilutions did not seroconvert and were negative for both viral RNA and for infectious virus, although proviral DNA was detected in their peripheral blood mononuclear cells. In contrast to the animals receiving the 10(-3) virus dilution, these five silently infected monkeys developed an SIV-specific proliferative T cell response but SIV-specific CTL could not be observed. The SIV-specific T cell proliferation of the silently infected animals could be boosted by a second low-dose exposure with a 10(-4) or 10(-5) dilution of SIVmac251/spl. The virological status of the animals was not changed following this second virus inoculation. Four months later these macaques were challenged intravenously with 2 ml of a 10(-4) dilution of SIVmac251/32H containing 10 monkey ID50. After this challenge all SIV-pre-exposed animals and three naive controls became productively infected. In addition, all infected animals developed typical signs of an immunodeficiency within 6 months after infection. These observations indicate that macaques infected silently by a low-dose exposure to infectious virus generated a virus-specific cellular immune response. However, SIV-specific T cell proliferation alone could not protect the monkeys against an intravenous challenge with SIVmac and the subsequent development of AIDS-like symptoms.