Vaccination for neuroprotection in the mouse optic nerve: implications for optic neuropathies.

T-cell autoimmunity to myelin basic protein was recently shown to be neuroprotective in injured rat optic nerves. In the present study, using the mouse optic nerve, we examined whether active immunization rather than passive transfer of T-cells can be beneficial in protecting retinal ganglion cells (RGCs) from post-traumatic death. Before severe crush injury of the optic nerve, SJL/J and C3H.SW mice were actively immunized with encephalitogenic or nonencephalitogenic peptides of proteolipid protein (PLP) or myelin oligodendrocyte glycoprotein (MOG), respectively. At different times after the injury, the numbers of surviving RGCs in both strains immunized with the nonencephalitogenic peptides pPLP 190-209 or pMOG 1-22 were significantly higher than in injured controls treated with the non-self-antigen ovalbumin or with a peptide derived from beta-amyloid, a non-myelin-associated protein. Immunization with the encephalitogenic myelin peptide pPLP 139-151 was beneficial only when the disease it induced, experimental autoimmune encephalomyelitis, was mild. The results of this study show that survival of RGCs after axonal injury can be enhanced by vaccination with an appropriate self-antigen. Furthermore, the use of nonencephalitogenic myelin peptides for immunization apparently allows neuroprotection without incurring the risk of an autoimmune disease. Application of these findings might lead to a promising new approach for treating optic neuropathies such as glaucoma.

Rhesus monkeys are highly susceptible to experimental autoimmune encephalomyelitis induced by myelin oligodendrocyte glycoprotein: characterisation of immunodominant T- and B-cell epitopes.

Eight rhesus monkeys with different MHC backgrounds were immunized with myelin oligodendrocyte glycoprotein (MOG). All developed severe experimental autoimmune encephalomyelitis associated with large inflammatory foci and extensive demyelination. T-cell autoreactivity to MOG was directed against three main epitopes encompassed within amino acids 4-20, 35-50 and 94-116, of which two are also immunodominant epitopes for the autoimmune T cell response to MOG in patients with MS. A strong B cell response to MOG was observed in all monkeys and major epitopes recognized were located within amino acids 4-26, 24-46 and 44-66/54-76.

The central nervous system-specific myelin oligodendrocytic basic protein (MOBP) is encephalitogenic and a potential target antigen in multiple sclerosis (MS).

Uncovering primary target antigens in multiple sclerosis (MS) is of major significance for understanding the etiology and pathophysiology of the disease, and for designing immunospecific therapy. In this study, a synthetic peptide representing a predicted T cell epitope on myelin oligodendrocytic basic protein (MOBP) was found to be encephalitogenic in C3H.SW mice, inducing experimental autoimmune encephalomyelitis with an abrupt onset. Two separate preliminary studies with MOBP peptides indicated that autoreactivity to MOBP occurs in MS. These data strongly suggest that MOBP is a highly relevant target in MS and further point to the complexity of antigen specificities in MS.

Mice overexpressing Bcl-2 in their neurons are resistant to myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE).

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS) characterized by destruction of myelin. Recent studies have indicated that axonal damage is involved in the pathogenesis of the progressive disability of this disease. To study the role of axonal damage in the pathogenesis of MS-like disease induced by myelin oligodendrocyte glycoprotein (MOG), we compared experimental autoimmune encephalomyelitis (EAE) in wild-type (WT) and transgenic mice expressing the human bcl-2 gene exclusively in neurons under the control of the neuron-specific enolase (NSE) promoter. Our study shows that, following EAE induction with pMOG 35-55, the WT mice developed significant clinical manifestations with complete hind-limb paralysis. In contrast, most of the NSE-bcl-2 mice (16/27) were completely resistant, whereas the others showed only mild clinical signs. Histological examination of CNS tissue sections showed multifocal areas of perivascular lymphohistiocytic inflammation with loss of myelin and axons in the WT mice, whereas only focal inflammation and minimal axonal damage were demonstrated in NSE-bcl-2 mice. No difference could be detected in the immune potency as indicated by delayed-type hypersensitivity (DTH) and T-cell proliferative responses to MOG. We also demonstrated that purified synaptosomes from the NSE-bcl-2 mice produce significantly lower level of reactive oxygen species (ROS) following exposure to H2O2 and nitric oxide (NO) than WT mice. In conclusion, we demonstrated that the expression of the antiapoptotic gene, bcl-2, reduces axonal damage and attenuates the severity of MOG-induced EAE. Our results emphasize the importance of developing neuroprotective therapies, in addition to immune-specific approaches, for treatment of MS.

Human immunodeficiency virus types 1 and 2 differ in the predominant mechanism used for selection of genomic RNA for encapsidation.

Retroviral RNA encapsidation is a highly selective process mediated through recognition by the viral Gag proteins of cis-acting RNA packaging signals in genomic RNA. This RNA species is also translated, producing the viral gag gene products. The relationship between these processes is poorly understood. Unlike that of human immunodeficiency virus type 1 (HIV-1), the dominant packaging signal of HIV-2 is upstream of the major splice donor and present in both unspliced and spliced viral RNAs, necessitating additional mechanisms for preferential packaging of unspliced genomic RNA. Encapsidation studies of a series of HIV-2-based vectors showed efficient packaging of viral genomes only if the unspliced, encapsidated RNA expressed full-length Gag protein, including functional nucleocapsid. We propose a novel encapsidation initiation mechanism, providing selectivity, in which unspliced HIV-2 RNA is captured in cis by the Gag protein. This has implications for the use of HIV-2 and other lentiviruses as vectors.

Nonreciprocal packaging of human immunodeficiency virus type 1 and type 2 RNA: a possible role for the p2 domain of Gag in RNA encapsidation.

The ability of human immunodeficiency virus types 1 (HIV-1) and 2 (HIV-2) to cross-package each other's RNA was investigated by cotransfecting helper virus constructs with vectors derived from both viruses from which the gag and pol sequences had been removed. HIV-1 was able to package both HIV-1 and HIV-2 vector RNA. The unspliced HIV-1 vector RNA was packaged preferentially over spliced RNA; however, unspliced and spliced HIV-2 vector RNA were packaged in proportion to their cytoplasmic concentrations. The HIV-2 helper virus was unable to package the HIV-1 vector RNA, indicating a nonreciprocal RNA packaging relationship between these two lentiviruses. Chimeric proviruses based on HIV-2 were constructed to identify the regions of the HIV-1 Gag protein conferring RNA-packaging specificity for the HIV-1 packaging signal. Two chimeric viruses were constructed in which domains within the HIV-2 gag gene were replaced by the corresponding domains in HIV-1, and the ability of the chimeric proviruses to encapsidate an HIV-1-based vector was studied. Wild-type HIV-2 was unable to package the HIV-1-based vector; however, replacement of the HIV-2 nucleocapsid by that of HIV-1 generated a virus with normal protein processing which could package the HIV-1-based vector. The chimeric viruses retained the ability to package HIV-2 genomic RNA, providing further evidence for a lack of reciprocity in RNA-packaging ability between the HIV-1 and HIV-2 nucleocapsid proteins. Inclusion of the p2 domain of HIV-1 Gag in the chimera significantly enhanced packaging.

Development of cell lines stably expressing human immunodeficiency virus type 1 proteins for studies in encapsidation and gene transfer.

Experiments were done to test cell lines for their capacity to express human immunodeficiency virus type 1 (HIV-1) proteins in a stable manner. Marked differences were seen in the ability to stably express and export viral Gag and Pol proteins. Two cell lines, one suspension (MDS) and one monolayer (SW480), were established which exported these proteins at high level. Two other cell lines, HeLa and THP-1, showed poorer expression and very limited particle release. Single cell cloning was used to select the optimal producing clones from the lines. These produced large quantities of viral core particles pelletable from the supernatants. Cell lines were constructed from these clones which stably expressed in addition either the HIV-1 Envelope or a packageable HIV-based vector. The vector was shown to be packaged within the viral core particles. Transient transfection of envelope expressing constructs into a gag-pol plus vector cell line, or the vector into a gag-pol plus envelope expressing cell line resulted in gene transfer to CD4+ target cells. These cell lines provide useful tools with which to study the assembly and export of viral proteins and RNA, for assay of alternative envelope proteins to pseudotype HIV cores, for assessment of antiviral drugs and as a source of correctly processed proteins for immunological studies.

trans-acting proteins involved in RNA encapsidation and viral assembly in human immunodeficiency virus type 1.

The human immunodeficiency virus type 1 gag gene product Pr55gag self-assembles when expressed on its own in a variety of eukaryotic systems. Assembly in T lymphocytes has not previously been studied, nor is it clear whether Pr55gag particles can package genomic RNA or if the Gag-Pol polyprotein is required. We have used a series of constructs that express Gag or Gag-Pol proteins with or without the viral protease in transient transfections in COS-1 cells and also expressed stably in CD4+ T cells to study this. Deletion of the p6 domain at the C terminus of protease-negative Pr55gag did not abolish particle release, while truncation of the nucleocapsid protein reduced it significantly, particularly in lymphocytes. Gag-Pol polyprotein was released from T cells in the absence of Pr55gag but did not encapsidate RNA. Pr55gag encapsidated human immunodeficiency virus type 1 RNA whether expressed in a protease-positive or protease-negative context. p6 was dispensable for RNA encapsidation. Marked differences in the level of RNA export were noted between the different cell lines.

cis-acting sequences involved in human immunodeficiency virus type 1 RNA packaging.

We have previously described a series of human immunodeficiency virus type 1-based vectors in which efficient RNA encapsidation appeared to correlate with the presence of a 1.1-kb env gene fragment encompassing the Rev-responsive element (RRE). In this report, we explore in detail the role of the RRE and flanking env sequences in vector expression and RNA encapsidation. The analysis of a new series of vectors containing deletions within the env fragment failed to identify a discrete packaging signal, although the loss of certain sequences reduced packaging efficiency three- to fourfold. Complete removal of the env fragment resulted in a 100-fold decrease in the vector transduction titer but did not abolish RNA encapsidation. We conclude that the RRE and 3' env sequences are not essential for human immunodeficiency virus type 1 vector encapsidation but may be important in vectors in which a heterologous gene has been placed adjacent to the 5' packaging signal, potentially disrupting its structure.

Helper virus-free transfer of human immunodeficiency virus type 1 vectors.

Recombinant vectors based on the type 1 human immunodeficiency virus (HIV-1) can be used to deliver genes into cells expressing the HIV receptor, CD4. We have used a transient RNA packaging system to compare the safety and efficacy of HIV-1 vector transduction by wild-type and replication-deficient helper viruses. Helper virus-free vector transfer was consistently achieved when the helper virus gag-pol and env genes were expressed from separate plasmids such that two recombination events were required to form an infectious genome. Other forms of attenuation, such as deletion of the 5' phi region, were inadequate to prevent helper virus transmission. Vector transduction by the wild-type and non-replicating helper viruses occurred with comparable efficiency except in instances where efficient vector RNA expression was dependent upon transactivating factors supplied by the helper virus. These data demonstrate the feasibility of safe gene transfer using HIV-1 vectors.

Glycoprotein H of human cytomegalovirus (HCMV) forms a stable complex with the HCMV UL115 gene product.

The human cytomegalovirus (HCMV) UL75 gene product is the homologue of herpes simplex virus type 1 (HSV-1) glycoprotein H (gH), a virion glycoprotein that is essential for infectivity and which is conserved among members of the alpha-, beta- and gamma-herpesviruses. It has previously been shown that HSV-1 gH forms a stable complex with HSV-1 gL, the product of the UL1 gene, and the formation of this complex facilitates the cell surface expression of gH. None of the open reading frames within the HCMV genome encode a product with discernible sequence homology with HSV-1 gL, but an examination of the arrangement of conserved genes in HCMV suggested that the UL115 gene is a 'positional homologue' of HSV-1 UL1 which, like UL1, encodes a small secreted glycoprotein. Co-expression of HCMV gH (the UL75 gene product) and the UL115 gene product revealed that these proteins form a disulphide-linked complex and that the formation of this complex results in cell surface expression of gH. This complex is analogous to the gH:gL complex of HSV-1 and the HCMV UL115 gene product is therefore the functional homologue of HSV-1 gL.