Biomedical subjects

M J Hariharan

Publications and source records attributed to M J Hariharan.

6 recordsLinked to original sources

PubMed2001

Heat shock protein-peptide complexes elicit cytotoxic T-lymphocyte and antibody responses specific for bovine herpesvirus 1.

Epitope-based vaccines offer a promising alternative to modified live vaccines against viruses such as herpesviruses which give rise to latent infections, and induce immunosuppression. The success of this approach depends on the ability to direct the CTL epitopes to the MHC class I antigen presentation pathway. The objective of this study was to evaluate the potential of the heat shock protein gp96 in this regard. A group of BALB/c mice was injected with three murine CTL epitope peptides of bovine herpesvirus 1 (BHV-1) complexed in vitro with bovine gp96 (gp96-peptides). Three other groups were injected with either the peptides alone, gp96 alone, or the peptides complexed with BSA. CTLs from mice immunized with gp96-peptides specifically lysed the peptide-pulsed syngeneic targets, as well as BHV-1-infected targets. CTLs from the other three groups did not lyse these targets. To further evaluate the utility of this approach, groups of BALB/c mice were immunized with gp96 isolated from a syngeneic cell-line transduced with BHV-1 glycoprotein D (BC-gD). Mice immunized with gp96 from BC-gD developed CTLs, as well as Abs specific for BHV-1 gD. Furthermore, in vitro stimulation of naive bovine PBMCs with gp96 from BC-gD resulted in CTLs specific for BHV-1. These results demonstrate the feasibility of using gp96-peptide complexes isolated from cells expressing BHV-1 proteins to induce CTL and Ab responses against BHV-1, without the prior knowledge of the CTL and Ab epitope sequences.

Amino Acid Sequence↗

PubMed1999

Stable alphavirus packaging cell lines for Sindbis virus and Semliki Forest virus-derived vectors.

Alphavirus vectors are being developed for possible human vaccine and gene therapy applications. We have sought to advance this field by devising DNA-based vectors and approaches for the production of recombinant vector particles. In this work, we generated a panel of alphavirus vector packaging cell lines (PCLs). These cell lines were stably transformed with expression cassettes that constitutively produced RNA transcripts encoding the Sindbis virus structural proteins under the regulation of their native subgenomic RNA promoter. As such, translation of the structural proteins was highly inducible and was detected only after synthesis of an authentic subgenomic mRNA by the vector-encoded replicase proteins. Efficient production of biologically active vector particles occurred after introduction of Sindbis virus vectors into the PCLs. In one configuration, the capsid and envelope glycoproteins were separated into distinct cassettes, resulting in vector packaging levels of 10(7) infectious units/ml, but reducing the generation of contaminating replication-competent virus below the limit of detection. Vector particle seed stocks could be amplified after low multiplicity of infection of PCLs, again without generating replication-competent virus, suggesting utility for production of large-scale vector preparations. Furthermore, both Sindbis virus-based and Semliki Forest virus-based vectors could be packaged with similar efficiency, indicating the possibility of developing a single PCL for use with multiple alphavirus-derived vectors.

Alphavirus↗

PubMed1998

DNA immunization against herpes simplex virus: enhanced efficacy using a Sindbis virus-based vector.

Previously we reported the development of a plasmid DNA expression vector system derived from Sindbis virus (T. W. Dubensky, Jr., et al., J. Virol. 70:508-519, 1996). In vitro, such vectors exhibit high-level heterologous gene expression via self-amplifying cytoplasmic RNA replication. In the present study, we demonstrated the in vivo efficacy of the Sindbis virus-based pSIN vectors as DNA vaccines. A single intramuscular immunization of BALB/c mice with pSIN vectors expressing the glycoprotein B of herpes simplex virus type 1 induced a broad spectrum of immune responses, including virus-specific antibodies, cytotoxic T cells, and protection from lethal virus challenge in two different murine models. In addition, dosing studies demonstrated that the pSIN vectors were superior to a conventional plasmid DNA vector in the induction of all immune parameters tested. In general, 100- to 1,000-fold-lower doses of pSIN were needed to induce the same level of responsiveness as that achieved with the conventional plasmid DNA vector. In some instances, significant immune responses were induced with a single dose of pSIN as low as 10 ng/mouse. These results indicate the potential usefulness of alphavirus-based vectors for DNA immunization in general and more specifically as a herpes simplex virus vaccine.

Animals↗

PubMed1997

Bovine herpesvirus 1 downregulates the expression of bovine MHC class I molecules.

The mechanism of immunosuppression induced by bovine herpesvirus 1 (BHV-1) was investigated by studying the effects of the virus on the expression of major histocompatibility complex (MHC) class I molecules. After infection with the virus, the expression of class I molecules was detected by flow cytometry and pulse-chase analysis. A selective downregulation of expression of class I molecules was seen in the infected cells, while the class II expression remained unaffected. The reduction in surface expression was evident as early as 8 hours postinfection, reaching significant levels by 12 hours. The downregulation was seen with a multiplicity of infection as low as 0.1. A modified live vaccine strain of BHV-1 also induced the downregulation of class I expression. Analysis of the viral proteins(s) involved in this downregulation with metabolic inhibitors (cycloheximide or phosphonoacetic acid), suggested that the immediate early and/or early proteins of the virus mediate this effect. Pulse-chase analysis revealed that the synthesis of the class I heavy chain, and the assembly/transport of class I molecules were affected by the virus infection. These results suggest that BHV-1 interferes with the molecular mechanisms involved in the synthesis, and assembly/transport of MHC-class I molecules. This interference with the class I antigen processing pathway might help the virus to evade the cytotoxic T-lymphocyte response of the host.

Animals↗

PubMed1993

Down regulation of murine MHC class I expression by bovine herpesvirus 1.

The objective of this study was to investigate the effect(s) of bovine herpesvirus 1 (BHV-1) infection on the expression of MHC class I molecules in murine fibroblasts. L-cells were infected with BHV-1 at a multiplicity of infection (m.o.i.) of 10 plaque forming units (PFU) per cell, and the expression of MHC class I molecules was analyzed by flow cytometry and immunoprecipitation. Temporal studies revealed a reduction in class I expression beginning at 8 h post infection (p.i.) which reached a maximum between 10 to 16 h p.i. The loss of class I expression was restored in the presence of phosphonoacetic acid (30 micrograms/ml), a late herpesviral protein synthesis inhibitor. However, addition of cycloheximide, a total protein synthesis inhibitor (100 micrograms/ml), did not result in any difference in class I expression between virus-infected and mock-infected cells. These results suggest that the reduced class I expression is a direct consequence of BHV-1 infection, and that the late viral gene product(s) may be involved in this process. Similar phenomena may occur in natural BHV-1 infection in cattle, and this may be one of the mechanisms of immune suppression by BHV-1.

Animals↗

PubMed1991

Bovine monoclonal anti-idiotypes induce antibodies specific for a synthetic peptide bearing a neutralizing epitope of bovine herpesvirus 1 glycoprotein gI (gB).

Bovine monoclonal anti-Id mimicking a neutralizing epitope of bovine herpesvirus-1 (BHV-1) glycoprotein gI were developed. An epitope present on the 74K subunit of gI identified by a murine mAb 1E11 was selected for this study. Bovine lymphocytes from the prefemoral lymph node of a heifer immunized with mAb 1E11 were fused with SP-2/0, a nonsecreting murine cell-line. Two bovine x murine hybridomas secreting bovine monoclonal anti-Id specific for the Id of 1E11 were stabilized. These anti-Id inhibited the binding of 1E11 to purified glycoprotein gI in a dose-dependent fashion. Naive mice immunized with the anti-Id produced anti-anti-Id (Ab3) that reacted with BHV-1 glycoprotein gI in a RIA, and neutralized BHV-1 infection in vitro. The Ab3 also showed reactivity to the 74K subunit of authentic gI glycoprotein in a Western blot analysis, and to the synthetic peptide bearing the 1E11 epitope in a RIA. These results substantiate the presence of the population of anti-Ab2 that functionally resemble antibodies specific for the immunizing Ag BHV-1 in Ab3, and demonstrate the ability of these anti-Id to elicit BHV-1-specific antibody response.

Animals↗