Ashok K. Srivastava

A purified inactivated Japanese encephalitis virus vaccine made in vero cells

A second generation, purified, inactivated vaccine (PIV) against Japanese encephalitis (JE) virus was produced and tested in mice where it was found to be highly immunogenic and protective. The JE-PIV was made from an attenuated strain of JE virus propagated in certified Vero cells, purified, and inactivated with formalin. Its manufacture followed current GMP guidelines for the production of biologicals. The manufacturing process was efficient in generating a high yield of virus, essentially free of contaminating host cell proteins and nucleic acids. The PIV was formulated with aluminum hydroxide and administered to mice by subcutaneous inoculation. Vaccinated animals developed high-titered JE virus neutralizing antibodies in a dose dependent fashion after two injections. The vaccine protected mice against morbidity and mortality after challenge with live, virulent, JE virus. Compared with the existing licensed mouse brain-derived vaccine, JE-Vax, the Vero cell-derived JE-PIV was more immunogenic and as effective as preventing encephalitis in mice. The JE-PIV is currently being tested for safety and immunogenicity in volunteers.

Mice immunized with a dengue type 2 virus E and NS1 fusion protein made in Escherichia coli are protected against lethal dengue virus infection

A gene fragment encoding the C-terminal 204 amino acids (AA) from the structural envelope glycoprotein (E) and the N-terminal 65 AA from non-structural protein one (NS1) of dengue type 2 virus (DEN-2) was expressed in Escherichia coli (E. coli) as a fusion protein with staphylococcal protein A. The recombinant fusion protein was purified and analysed for its antigenicity, its immunogenicity and its ability to protect mice against lethal challenge with live DEN-2 virus. The recombinant protein was found to be reactive with anti-DEN-2 polyclonal and monoclonal antibodies. Mice immunized with the purified fusion protein made anti-DEN-2 antibodies measured by the hemagglutination-inhibition (HI) and neutralization (N) tests, and were protected against lethal challenge with DEN-2 virus administered by intracranial inoculation.

Role of immune activation and cytokine expression in HIV-1-associated neurologic diseases

Central nervous system (CNS) involvement is common during human immunodeficiency virus type-1 (HIV-1) infection. The neurologic disease of the CNS most frequently observed during acquired immunodeficiency syndrome (AIDS) is HIV-1-associated cognitive/motor complex or AIDS dementia complex (ADC), which is most likely a direct consequence of HIV-1 infection of the CNS. The peripheral nervous system (PNS) is also affected in HIV-1-infected individuals and there are several features of immune- and cytokine-related pathogenesis in both the CNS and PNS that are reviewed. Several lines of evidence demonstrate aspects of immune activation in the CNS and peripheral nervous system (PNS) of HIV-1-infected individuals. The relative paucity of HIV-1 expression in contrast to widespread functional and pathologic changes in the CNS and PNS of AIDS patients, and the lack of evidence of productive infection of HIV-1 in neuronal cells in vivo lead to the possibility of indirect or immunopathogenic mechanisms for HIV-1-related neurologic diseases. Proposed mechanisms of neuronal and glial cell damage are injury of oligodendrocytes by tumor necrosis factor-alpha (TNF-alpha) released from activated macrophage/microglia, calcium-dependent excitoneurotoxicity induced by gp120 HIV-1 envelope protein, N-methyl-D-aspartate (NMDA) receptor-mediated neurotoxicity by quinolinic acid (a product of activated macrophages), cell injury by HIV-1-specific cytotoxic T cells, and apoptosis of oligodendrocytes or neurons triggered by interaction between cell surface receptors and HIV-1 gp120 protein. Common to those mechanisms is the dependence on cellular activation with expression of proinflammatory cytokines (TNF-alpha, interleukin-1). Amplification of activation signals through the cytokine network by macrophage/astrocyte/endothelial cell interactions, and cell-to-cell contact between activated macrophages and neural cells by upregulation of adhesion molecules dramatically enhances the toxic effect of macrophage products. Expression of immunosuppressive cytokines such as interleukin-4, interleukin-6, and transforming growth factor-beta is also increased in the CNS and PNS of HIV-1-infected patients. This may serve as neuroprotective and regenerative mechanism against insults to nervous system tissue.

HIV-1 Heterogeneity and Cytokines

Mild manifestations (HIV-1 associated minor cognitive/motor disorder), severe manifestations (HIV-1 associated dementia complex and HIV-1 associated myelopathy), and sensory neuropathy are consequences of HIV-1 infection. Our goal is to elucidate the role of HIV-1 in the complications of AIDS including cytokine immunopathology and HIV-1 DNA sequence variants. We have examined the brain and sensory ganglia from 60 AIDS patients and 20 seronegative controls using PCR, DNA sequencing of the HIV-1 envelope protein (env), in situ hybridization (ISH), and immunohistochemistry (IHC). Using our combined ISH-IHC technique, we could identify different types of cells and HIV-1 simultaneously in cryostat and paraffin sections. We found HIV-1 predominantly in macrophage/microglia in brain. In dorsal root ganglia (DRG) we found rare macrophages infected with HIV-1 and neurons and interstitial cells (including macrophages) which were apoptotic. Cytokines were detected in mononuclear and endothelial cells near neurons. We achieved single copy sensitivity detecting HIV-1 in nervous tissue using nested PCR. We sequenced HIV-1, DNA from 3 intravenous drug users (IDUs): from brain, CSF, and blood. PCR amplification was followed by cloning and then sequencing the HIV-1 insert: V1-V5 regions of the envelope (env) gene. We found that the env genes had increased sequence variation compared to the literature, cDNA sequences derived from RNA were less heterogeneous than clones derived from DNA from the same specimens, clones derived from brain are more closely related (show restricted heterogeneity) compared to clones from blood and CSF from the same patients. Patient 149 clones we examined to date did not correspond to any of the designated subtypes (A-F) of HIV-1 based on the DNA sequences of the C2-V3 regions. Finally, the HIV-1 RNA produced in these tissues is derived from a minority of DNA clones. Although HIV-1 infected macrophages are not entirely responsible for pathology in the brain and less so in sensory ganglia, some of the products of infection, cytokines, are more widespread in these tissues. Furthermore, HIV-1 strains infecting the brain appear to exhibit restricted heterogeneity compared to autologous CSF and blood and these strains may be associated with cytokines and pathology. HIV-1 strains that infect nervous tissue and cytokines produced in this tissue may effect neuropathogenesis, in vivo, in spite of low levels of local HIV-1 infection. We attempt to delineate, here, common sequence variations in HIV-1 isolates in the hope of developing future therapeutic strategies.

Hiv-1 Neuropathogenesis and Abused Drugs

HIV Associated Dementia (HAD) and peripheral neuropathy result from HIV-1 infection and several parameters impact on their initiation and course. These parameters include properties of the virus such as sequence heterogeneity of the envelope protein and virus and host gene expression in infected and uninfected macrophages in HIV infected individuals. The infection of brain cells such as macrophage/microglia may influence the course and duration of HAD through the elaboration of toxic molecules and viral host by-products of infection. Drugs of abuse complicate our interpretation of the resulting pathologies and affect the molecular and biochemical processes involved in HIV-1 infection and its life cycle. Furthermore, abused drugs may modulate the biology and function of macrophage/microglia as well as other cells in brain. Total HIV-1 virus load in the brain may also impact the clinical consequences of infection. Although specific strains of HIV-1 may inflict HAD by interactions at the molecular level, other mechanisms may operate in the initiation and development of peripheral neuropathy. An important aspect of these studies is the determination of their accuracy, specificity, sensitivity, and reproducibility. We describe important caveats involved in the performance, analysis, and interpretation of the results.

Dementia and the Neurovirulence of HIV-1.

Infection with human immunodeficiency virus type 1 (HIV-1) leads rapidly to infection of the brain and subsequent neuropsychological impairment, including subclinical impairment, minor cognitive-motor disorder, and HIV-1-associated dementia (HAD). This article reviews HAD and the factors involved in its pathogenesis; the effectiveness of antiretroviral therapy; the prevalence of HIV-1 and subtypes; and the role of chemokines and cytokines as the capstones associated with neuropathology due to inflammation.

Preparation of a purified, inactivated Japanese encephalitis (JE) virus vaccine in Vero cells

An attenuated Japanese encephalitis (JE) virus SA14-14-2 (PDK) was adapted to Vero cells, a continuous cell line that has been licensed for human vaccine production, by serial passages. The resulting virus was purified by tangential flow ultrafiltration followed by sucrose density gradient ultracentrifugation, giving 2.3 mg purified virus per liter of culture supernatant. Treatment with 0.05% formalin for 4 days at 22 °C completely inactivated viral infectivity while preserving its antigenicity. The purified, inactivated JE virus was formulated with alum hydroxide and administered to mice by intraperitoneal route. In terms of its ability to induce anti-JE neutralizing antibody and to protect the immunized animal against neurovirulent virus challenge, the purified, inactivated JE virus formulated with alum was equivalent to the exiting commercial mouse brain-derived vaccine (JE-VAX, Aventis Pasteur Inc.).

Viral and Host Determinants of Neurovirulence of HIV-1 Infection

HIV-1 infection of the brain is often associated with impairment in cognition, motor performance, and behavior in adults, and in addition, slowed milestones of brain growth and development in children(33,42,79). These may range in severity from subtle, mild deficits to conspicuous decline in performance diagnosed as HIV-1 associated dementia (HAD) and may occur in about 25% of AIDS patients independently of opportunistic infections, neoplasms, or metabolic derangements(44,85,104,120). However, close correlations between the clinical course of HAD and laboratory findings are not yet established(43,l40).