Prions and protein-folding diseases.

Prions represent a group of proteins with a unique capacity to fold into different conformations. One isoform is rich in beta-pleated sheets and can aggregate into amyloid that may be pathogenic. This abnormal form propagates itself by imposing its confirmation on the homologous normal host cell protein. Pathogenic prions have been shown to cause lethal neurodegenerative diseases in humans and animals. These diseases are sometimes infectious and hence referred to as transmissible spongiform encephalopathies. In the present review, the remarkable evolution of the heterodox prion concept is summarized. The origin of this phenomenon is based on information transfer between homologous proteins, without the involvement of nucleic acid-encoded mechanisms. Historically, kuru and Creutzfeldt-Jakob disease (CJD) were the first infectious prion diseases to be identified in man. It was their relationship to scrapie in sheep and experimental rodents that allowed an unravelling of the particular molecular mechanism that underlie the disease process. Transmission between humans has been documented to have occurred in particular contexts, including ritual cannibalism, iatrogenic transmission because of pituitary gland-derived growth hormone or the use in neurosurgical procedures of dura mater from cadavers, and the temporary use of a prion-contaminated protein-rich feed for cows. The latter caused a major outbreak of bovine spongiform encephalopathy, which spread to man by human consumption of contaminated meat, causing approximately 200 cases of variant CJD. All these epidemics now appear to be over because of measures taken to curtail further spread of prions. Recent studies have shown that the mechanism of protein aggregation may apply to a wider range of diseases in and possibly also outside the brain, some of which are relatively common such as Alzheimer's and Parkinson's diseases. Furthermore, it has become apparent that the phenomenon of prion aggregation may have a wider physiological importance, but a full understanding of this remains to be defined. It may involve maintaining neuronal functions and possibly contributing to the establishment of long-term memory.

Four distinct antigenic regions are present in the primary structure of HIV-1 and HIV-2 proteinases.

OBJECTIVE: The purpose of this study was to assay reactivity of antibody-positive sera to different parts of the HIV-1 and HIV-2 proteinase (PR) proteins. DESIGN: Since the majority of HIV-1-antibody-positive sera react to the proteinase, but the antigenic determinants on the protein have not been identified, we attempted to identify these determinants. INTERVENTIONS: We synthesized 18 peptides representing the PR of HIV-1 and HIV-2 in order to map serum reactivity to the PR protein. RESULTS: Both HIV-1- and HIV-2-antibody-positive sera recognized four distinct antigenic regions in the HIV-1 and HIV-2 PR. CONCLUSIONS: Correlation between our results and the crystallographic structure of the protein revealed that the antigenic regions are positioned at the surface of the HIV-1 PR. Although the structure of HIV-2 PR has not yet been characterized, our results indicate that the folding of the HIV-1 and HIV-2 PR may be very similar.

Identification of four antibody-binding sites in the envelope proteins of simian immunodeficiency virus, SIVsm.

OBJECTIVE: To identify antigenic regions in the envelope glycoproteins of the simian immunodeficiency virus isolate, SIVsm. METHODS: Thirty-eight peptides were synthesized and used in site-directed enzyme-linked immunosorbent assays with sera from experimentally infected macaques. RESULTS: Four antibody-binding regions were identified, corresponding to the second variable region [V2; amino acids (aa) 170-196], the region homologous to V3 in HIV-1 (aa 313-346), the carboxy terminus of gp120 (aa 514-537) and the amino terminus of the transmembrane protein (aa 608-638). Serum reactivity to the V2 region was higher in surviving monkeys than in animals with an early development of simian AIDS. The antigenicity of the peptide appears to be conformationally dependent. CONCLUSIONS: The majority of antigenic sites identified in the envelope proteins of SIV correspond to sites identified in HIV-1 and HIV-2, which further supports the use of the simian model in vaccine development. The pattern of reactivity to the V2 region suggests that absence of antibodies directed to this site might correlate with disease progression.

Infection of cynomolgus monkeys with HIV-2 protects against pathogenic consequences of a subsequent simian immunodeficiency virus infection.

Simian immunodeficiency virus (SIV) infection in cynomolgus macaques leads to severe immunodeficiency with a fatal outcome. In contrast, HIV-2 infects these primates without apparently causing any immunological abnormalities. In this study three cynomolgus monkeys were experimentally infected with HIV-2 strain SBL-K135 and 168 days later challenged with 10-100 animal infectious doses of the closely related SIV strain SM to study protective immunity. At the time of SIV challenge the HIV-2-infected monkeys had neutralizing antibodies against HIV-2, but virus could no longer be recovered from their peripheral blood mononuclear cells (PBMCs) and no clinical symptoms or decrease in CD4+ lymphocytes were observed. Follow-up for 9 months after challenge with SIV showed that the HIV-2-infected monkeys were protected against SIV-induced immunodeficiency (no decrease of CD4+ lymphocytes) and lymphadenopathy. However, they were not resistant to SIV infection since virus could be recovered from their PBMCs and they developed anamnestic antibody responses. Four naive control monkeys which were inoculated with the same dose of SIV became persistently infected and developed a decrease of the absolute numbers of CD4+ cells and showed a marked lymphadenopathy. Two out of four control animals died 58-265 days postinfection with an immunosuppressive disease. Immunohistochemical examination showed abundant viral antigen in lymph-node biopsies from the SIV-infected control monkeys but absence of SIV or HIV-2 antigens in the biopsies from the three HIV-2-preinfected and SIV-superinfected monkeys. The present study demonstrates possibilities for induction of immunity against immunodeficiency induced by a primate lentivirus, a concept with application also to HIV infection and AIDS in man.

Simian immunodeficiency virus (SIVsm) isolation from blood and brain of experimentally infected macaques.

The possibility of using simian immunodeficiency virus (SIV)-infected macaques to study pathogenic events linked to HIV infection of the brain prompted us to investigate some of the virological features in SIV-infected macaques. Nine cynomolgus macaques were inoculated with SIVsm and killed at different times. We successfully isolated virus from the blood of all the animals and from the brains of eight. These results point to the early and regular spread of this lentivirus to the brain. Neutralizing activity was studied in the serum and cerebrospinal fluid specimens obtained from these macaques against a selected group of isolates. Cerebrospinal fluid did not show any neutralizing activity. Our findings integrate the observations from HIV-1 infection in man and indicate that SIV infection of macaques is a useful model for studying pathogenic events of brain infection.

Site-directed enzyme-linked immunosorbent assay with a synthetic simian immunodeficiency virus SIVmac peptide identifying antibodies against the HIV-2 transmembrane glycoprotein.

A 23-amino-acid-long peptide (AIEKYLEDQAQLNAWGCAFRQVC) representing the transmembranous protein gp32 in SIVmac was used in site-directed enzyme-linked immunosorbent assay (ELISA) for detection of HIV-2-specific antibodies in 567 sera from Bissau, Guinea Bissau. Ninety out of the 567 sera were identified to contain HIV-2 antibodies by whole antigen ELISA and Western blot assays. The peptide ELISA correctly identified 89 out of these 90 seropositives (sensitivity 98.9%). Three sera falsely interpreted to be positive were encountered (specificity 99.4%). The HIV-2 peptide was also used for testing of 93 HIV-1-positive Swedish sera. None of these sera reacted. Site-directed serology employing synthetic peptides should be considered for application as a screening assay.

PIP: HIV-1 is predominant in Central and East Africa, while HIV-2 is predominant in West Africa. The HIV-2 virus, originally described as human T-lymphotropic virus type IV, is identical to the simian immunodeficiency virus SIV-mac. Whole-antigen enzyme-linked immunosorbent assay (ELISA) can detect heterotypic antigens in 80% of sera, but separate tests are required for detecting HIV-1 and HIV-2. Site-directed ELISA using amino acids 586-620 of the synthetic transmembranous protein gp41 as antigen has been used for type-specific antibody determination in HIV-1. The homologous site on the 23-amino-acid-long peptide AIEKYLEDQAQLNAWGCAFRQVC representing the transmembranous protein gp32 in the simian immunodeficiency virus SIV-mac is WGCAFR, which can be used in site-directed serology of HIV-2. This approach was tested on sera from 567 Africans in Guinea-Bissau and 49 suspected AIDS patients. None of these sera had HIV-1 antibodies. 93 HIV-1-positive sera from Sweden were used as controls. The HIV-2 peptide ELISA correctly identified 89 of the 90 HIV-2 seropositives out of the sample of 567. The peptide ELISA also correctly identified all of the 93 HIV-1-positive sera as HIV-2-negative. Use of HIV-2-specific ELISA in combination with an HIV-1-specific ELISA can efficiently screen blood for both types of HIV.

Measles encephalitis in rodents: defective expression of viral proteins.

Synthesis of measles virus proteins in rodent brains and Vero cell cultures infected with the hamster neurotropic (HNT), and for comparison the LEC strain, was studied by use of monoclonal antibodies against five structural components. In the brains of HNT-infected adult BALB/C mice two proteins, the nucleocapsid (NP) and phosphoprotein (P) were detected. Suckling hamster brains in addition expressed demonstrable hemagglutinin (HA) protein. In cell cultures all structural components except the matrix (M) protein were detected. In contrast, all five proteins were found in LEC strain-infected suckling hamster brains and cell cultures. The restriction in HNT viral replication observed may be caused by a primary defectiveness in M-protein expression, but the possibility that this restriction is secondary to cellular suppression remains to be explored. Minimal inflammation was seen in the brains of HNT-infected adult mice and viral antigen was primarily located in the cerebral cortex. A selective necrosis of the pyramidal cell layer of the hippocampus was observed. This change did not seem to correlate with virus replication.

Non-lethal infection of aminergic reticular core neurons: age-dependent spread of ts mutant vesicular stomatitis virus from the nose.

In order to induce a non-lethal infection restricted to central aminergic neurons projecting to the olfactory bulbs a series of temperature sensitive (ts) and G-protein monoclonal antibody escape mutants of vesicular stomatitis virus (VSV) were instilled into the nasal cavity of mice. In three-week (wk)-old NMRI mice four monoclonal antibody escape mutants caused an extensive infection of the olfactory epithelium and, like a wild type strain, a lethal brain infection after spread along olfactory pathways. Three ts mutant strains showed an attenuated pathogenic potential. Strain G31 caused a lethal infection with a somewhat prolonged course while the strain G11 failed to invade the nervous system. Strain G41 showed minimal invasion of central nervous system in three-wk-old mice and caused a lethal infection in newborn and one-wk-old mice. In contrast, two-wk-old mice survived infection with this mutant, which spread along olfactory pathways and rather selectively affected aminergic reticular core neurons in the diagonal band, the locus ceruleus and the raphe nuclei in the brainstem. Thus, an age-dependent virus infection of the olfactory pathways can cause restricted lesions in the brain providing a model for studies of virus-induced changes in aminergic neurotransmission.

Mumps virus infection of the developing mouse brain--appearance of structural virus proteins demonstrated with monoclonal antibodies.

Newborn mice and hamsters were inoculated intracerebrally with mumps virus strains of high and low neurovirulence, Kilham and RW, respectively and with an egg-adapted patient isolate. The presence of viral antigen in brain tissue was analyzed with the immunofluorescence technique employing monoclonal antibodies against nucleoprotein (NP), polymerase (P), matrix (M), hemagglutinin-neuraminidase (HN) and fusion (F) mumps virus components. As expected, hamsters developed a fatal encephalitis eight to nine days after infection with the Kilham strain and synthesis of all five structural viral antigens was identified. In contrast, mice infected with any of the virus strains did not develop signs of disease, but in brain material collected on days nine and 12 after infection viral antigen was present in many neurons. However, only NP and P antigens were demonstrable and no infectious virus was present. The antibody response in mice developed later than in hamsters. Neurons in the mouse brain may exert a host cell restriction on the virus maturation, and mice offer a suitable host for the establishment of defective, persistent mumps virus infections.

Measles virus hemagglutinin. Removal of the initiator methionine in the mature protein, and evidence for further processing to produce a 'ragged' end.

Measles virus hemagglutinin has been isolated by immunoadsorption. The total composition of the protein and its N-terminal amino acid sequence give data matching the structure indirectly deduced from cDNA. However, direct analysis of the hemagglutinin also shows that the mature protein is proteolytically processed and has a partly heterogeneous N-terminus. The initiator Met is removed, and non-stoichiometrically also the second residue.

A novel approach to the study of glycolipid receptors for viruses. Binding of Sendai virus to thin-layer chromatograms.

A method for the binding of virus to a silica gel thin-layer chromatogram is presented. After development the chromatogram is overlayed with the 125I-labelled virus and the bound virus is autoradiographed. Alternatively, the unlabelled virus may be detected after exposure to monoclonal antibody and labelled anti-antibody. The Sendai virus strain used did not bind to brain gangliosides earlier proposed to be receptors, but bound to human erythrocyte gangliosides. This finding may be explained by the existence of Sendai virus variants with different receptor specificities.

Immunoglobulin abnormalities and measles antibody response in chronic myelopathy.

Patients with chronic myelopathy of unknown origin were separated into two groups on the basis of presence or absence of oligoclonal immunoglobulin G (lgG) in cerebrospinal fluid (CSF). Thirty-nine of 48 patients (81%) with oligoclonal lgG in CSF had measles virus antibodies in the CSF and 31 (65%) showed reduced serum/CSF measles virus antibody ratios, in comparison with the corresponding ratios of adenovirus, group-specific, penton hemagglutination-enhancement and poliovirus neutralization-enhancement antibodies. Of 25 patients with myelopathy, but without oligoclonal lgG in their CSF, three had detectable titers of measles CSF antibodies and one of these had a greatly reduced serum/CSF ratio. The conditions of patients with chronic myelopathy of unknown origin and oligoclonal lgG in CSF may be diagnosed as probable multiple sclerosis (MS), in contrast to patients with this disease who lack oligoclonal lgG IN CSF. However, the clinical features of the disease in these two groups do not differ substantially.

Antiviral effects of 3'-fluorothymidine and 3'-azidothymidine in cynomolgus monkeys infected with simian immunodeficiency virus.

An acute infection with simian immunodeficiency virus (SIVSM) in cynomolgus monkeys was used to evaluate the antiviral effects of 3'-fluorothymidine (FLT) and 3'-azidothymidine [zidovudine (ZDV)]. Neither compound prevented the infection despite dosing prior to virus inoculation. FLT was about ten times more potent than ZDV in delaying the appearance of SIVSM antigen in the monkeys. The serum half-life of FLT was longer than that of ZDV and ZDV was bound to plasma proteins to about 60% while FLT was virtually unbound. It is proposed that the in vivo difference in potency between ZDV and FLT could, at least partly, be explained as the combined effects of a longer plasma half-life and a higher free concentration of FLT and possibly a higher intracellular concentration of the triphosphate of FLT.

Identification of B-cell antigenic sites on HIV-2 gp125.

Synthetic peptides were used to identify continuous antigenic sites on the external envelope glycoprotein gp125 of human immunodeficiency virus (HIV)-2. Initially, seven HIV-2-positive human serum samples were screened with 172 sequential nonapeptides containing a six-amino-acid overlap. This represents the entire gp125 molecule of HIV-2ISY. The antibody reactivity was found to be mainly restricted to 14 regions within gp125. Following these results, 33 longer peptides, 15-24 amino acids in length, were synthesized and tested against a larger number of samples. Eleven antigenic regions were thus identified. Two of these were detected within a region corresponding to the C1 region and four others within a region corresponding to the C2 region of HIV-1. The highest frequency of reactivity (90%) of 31 HIV-2 seropositive human serum samples was elicited by three peptides from a region corresponding to the V3 region of HIV-1. The C-terminal portion of this region was recognized by almost 80% of the samples. Reactive regions corresponding to the V4, V5, and N-terminal portion of V4 were also identified. A mouse monoclonal antibody reacting with gp125 was mapped to the N-terminal region of the molecule and was found to react with the sequence DVWNLFETS. The peptides were used to evaluate the antibody response of monkeys immunized with whole killed HIV-2 or simian immunodeficiency virus (SIV). The monkeys showed a pattern of reactivity similar to HIV-2-infected human serum samples. Postinfection samples from monkeys inoculated with HIV-2 or SIV reacted mainly to peptides from the V3 region. Two peptides were used to detect the seroconversion of two SIV-infected monkeys. Thus, we have demonstrated that human seroreactivity to HIV-2 gp125 occurs at a few distinct linear antigenic sites distributed at similar positions on the molecule as those in HIV-1 gp120.

Experimental infection of cynomolgus monkeys (Macaca fascicularis) with simian immunodeficiency virus (SIVsm).

Five healthy cynomolgus monkeys were inoculated intravenously with simian immunodeficiency virus (SIVsm) propagated in human lymphocytes. All five animals became infected. Virus was recovered from blood mononuclear cells and viral antigen was detected in serum 12 days postinoculation (PI) in all inoculated animals. Virus was also isolated in all five animals tested 74 to 226 days PI. Antibodies to different structural proteins of SIV and HIV-2 were demonstrated by ELISA, Western blot, and radioimmunoprecipitation assay from day 31 PI concomitantly with a reduction of viral proteins in the serum. Reappearance of antigen accompanied by a fall in antibody to gag products (p26) was observed in two monkeys 69 days PI. All SIV-infected monkeys showed a pronounced decrease in CD4+ lymphocytes demonstrable already 12 days PI. They also developed persistent lymphadenopathy. Thus, infection of cynomolgus monkeys with SIVsm mimics events in human immunodeficiency virus infection in humans but the course of evolution of pathogenic events in the monkey is markedly compressed. This experimental model will be useful for evaluation of HIV vaccines and antiviral testing.

Long-standing protection of macaques against cell-free HIV-2 with a HIV-2 iscom vaccine.

We investigated the capacity of two immunostimulating-complex (iscom) formulations including inactivated native HIV-2 viral proteins and selected peptides to induce protective immunity against HIV-2 in a nonhuman primate. Four cynomolgus monkeys were first immunized with five i.m. injections of purified detergent-disrupted HIV-2 virions (total dose, 0.7 mg) in iscoms over a period of 16 months. At months 18 and 20, all four macaques were given booster immunizations with iscom-coupled V3-derived synthetic peptides representing a dominating neutralizing region of HIV-2 gp125. Two weeks after the final dose of vaccine, the four vaccinated animals, together with four controls, were challenged i.v. with 10 monkey infectious doses (MID50) of monkey-cell-grown homologous cell-free virus, HIV-2SBL-6669/H5. After the challenge, the four control animals became readily infected; however, three of four vaccinated animals were protected as shown by repeated negative virus isolations and negative polymerase chain reaction for viral DNA and by failure to transmit HIV-2 infection with whole blood and lymph node cells into naive cynomolgus macaques. One of three protected animals showed an anamnestic antibody response to a dominating antigenic site, indicating possible limited virus replication. The vaccine-protected monkeys were subsequently resistant to rechallenge infection at 12, 15, and 18 months after the first challenge, suggesting that a reasonable duration of protective immunity had been induced by the vaccine.

Viruses and behavioural changes: a review of clinical and experimental findings.

This review focuses on behavioural neurovirology. Profound changes in behaviour are observed following infection of the central nervous system by some viruses. Irritability, insomnia, hyperactivity and learning disability are some of the behavioural disturbances that have been described in both humans and animals with central nervous system infection. The reticular core neurons which innervate the entire brain play an important role in regulating behaviour. Some of these neurons--locus coeruleus, raphe and diagonal bands--send projections to the olfactory bulbs and can be targets for exogenous agents attacking the olfactory epithelium. In infant rats, vesicular stomatitis virus is transported along the olfactory pathway by retrograde transport and reaches the reticular core neurons causing destruction of raphe, diagonal bands and, to a lesser extent, the locus coeruleus. As the neurons degenerate, the viral antigens disappear and the animals sustain severe deficits in neurotransmitter levels and behaviour. Such a "hit and run" effect of the virus suggests the possibility that a similar mechanism may be operating in some human disorders. Apart from their intrinsic interest as possible aetiological factors, viruses may provide valuable tools in experimental work seeking to correlate behaviour, morphology and neurotransmitter function.

Electrophoresis of immunoglobulin G. Facilitated migration of minute amounts in agarose.

Migration of very small amounts of immunoglobulin (20 ng) is restricted in agarose electrophoresis. Incorporation of a stable protein matrix (rabbit gamma globulin 1 mg/ml) in the agarose permits unrestricted migration so that immunoelectrophoresis of this quantity of radiolabelled antibody is possible. Very small amounts of radiolabelled and non-radiolabelled antibody were subjected to successful crossed immunoelectrophoresis through barriers of antigen under conditions which provide favorable ratios of antibody to antigen. These methods should be useful for studies of antibody eluted from tissue in acquired and autoimmune diseases associated with tissue bound immunoglobulin.

Gamma interferon expression and major histocompatibility complex induction during measles and vesicular stomatitis virus infections of the brain.

Lymphocytic interferon gamma (IFN-gamma) production and major histocompatibility complex (MHC) antigen induction were studied in experimental measles and vesicular stomatitis virus infections in the brain. Fifteen-day-old Sprague-Dawley rats injected intracerebrally with the HNT strain of measles virus showed already within 1 day after infection an increased number of cells producing IFN-gamma in the spleen, cervical lymph nodes and leptomeninges. These rats recovered after a transient neuronal infection in the brain. Rats infected intracerebrally with vesicular stomatitis virus, on the other hand, all succumbed after 2 days and showed no IFN-gamma production in lymphoid cells. Immunohistochemically MHC class I antigen appeared in infected and uninfected cells in the brain during replication of both viruses. A role for the recently discovered nerve fibres with IFN-gamma-like immunoreactivity, which are normally present in the brain, in the MHC antigen induction is discussed.

Induction of class I and class II transplantation antigens in rat brain during fatal and non-fatal measles virus infection.

Measles virus induced a marked increase in the expression of MHC-coded class I and class II antigens as detected by immunostaining during both fatal and non-fatal brain infections in rats. The distribution of these molecules in the brain was much more widespread than the occurrence of viral antigen suggesting a soluble factor for their induction. In 14-day-old rats with a non-fatal infection there was a marked infiltration of T lymphocytes of 'cytotoxic/suppressor' phenotype in the brain parenchyma, whereas T 'helper' cell phenotypes mainly were located perivascularly. In brains from newborn rats with a fatal infection no or only few lymphocytes were detected.