Thandavarayan Nagashunmugam

Viral interference with antibody and complement.

Abstract Viruses have evolved strategies to evade immunity mediated by antibody and complement. Herpesviruses and coronaviruses encode IgG Fc binding proteins that inhibit IgG activity, enabling the virus or infected cell to escape antibody attack. Herpesviruses, vaccinia virus and HIV-1 have the capacity to interfere with complement, either by incorporation of cellular complement regulatory proteins into the virion envelope or cell membrane, or by expression of viral molecules that mimic functions of complement regulatory proteins. The structure and biological activities of herpes simplex virus type 1 (HSV-1) glycoproteins gE, gI and gC are described. These glycoproteins protect HSV from immune attack; HSV-1 gE/gI form a complex that binds the Fc domain of IgG while gC is a C3b binding complement regulatory protein, providing a survival advantage to the virusin vitroandin vivoby inhibiting immune functions.

Human Submandibular Saliva Inhibits Human Immunodeficiency Virus Type 1 Infection by Displacing Envelope Glycoprotein gp120 from the Virus

Human submandibular saliva reduces human immunodeficiency virus type 1 (HIV-1) infection in vitro. To define the mechanism of inhibition, virus was incubated with saliva or medium, velocity sucrose gradient centrifugation was performed, and fractions were analyzed for p24 and gp120. The results show that after incubation with saliva, the envelope glycoprotein was displaced from both a laboratory-adapted and a low-passage clinical HIV-1 isolate. To identify the salivary protein(s) responsible, submandibular saliva was fractionated by anion- exchange chromatography. Protein fractions containing anti-HIV activity were assayed for their ability to strip gp120 from virus. The partially purified active fractions contained two high-molecular-weight sialyated glycoproteins identified as salivary agglutinin and mucin, as well as several lower-molecular-weight proteins. It thus appears that specific salivary proteins interact with HIV-1 to strip gp120 from the virus with a resultant decrease in infectivity.

HIV-1 viral protein R (Vpr) regulates viral replication and cellular proliferation in T cells and monocytoid cells in vitro

Among the putative accessory genes of HIV‐1, the 96‐amino‐acid virion‐associated vpr gene product has been described to have several novel biological activities. These include cytoplasmic‐to‐nuclear translocation, which empowers HIV to infect and replicate in non‐dividing cells and to increase viral replication, particularly in macrophages. Along with these viral effects, we found that HIV‐1 Vpr induces dramatic biological changes in the target cells of HIV infection, including induction of changes in transcriptional patterns, morphological changes, and complete inhibition of proliferation, which collectively was termed differentiation. These changes occur in the absence of other viral gene products, suggesting that Vpr mediates its proviral effects partially or perhaps solely through modulation of the state of the target cell rather than directly on the virus. The inhibition of proliferation in T cell lines has been extended by several groups to demonstrate that the inhibition of proliferation is through G2 cell cycle arrest, further supporting the idea that Vpr acts directly on cellular targets. We have recently described a role for Vpr in modulating the glucocorticoid pathway, which is involved in the regulation of the state of the cell, in cytoplasmic‐to‐nuclear translocation, and in modulation of host cell transcription. It is important to note that certain anti‐glucocorticoid compounds modulate Vpr activity in vitro. These results support the idea that the host cell contains specific receptor molecule(s) through which Vpr mediates its activity. Consequently, Vpr represents a unique target for anti‐HIV drug development and has significance for HIV‐1 disease progression. J. Leukoc. Biol. 62: 93–99; 1997.

Herpes Simplex Virus Type 1 Glycoprotein E Domains Involved in Virus Spread and Disease

ABSTRACT Herpes simplex virus type 1 (HSV-1) glycoprotein E (gE) functions as an immunoglobulin G (IgG) Fc binding protein and is involved in virus spread. Previously we studied a gE mutant virus that was impaired for IgG Fc binding but intact for spread and another that was normal for both activities. To further evaluate the role of gE in spread, two additional mutant viruses were constructed by introducing linker insertion mutations either outside the IgG Fc binding domain at gE position 210 or within the IgG Fc binding domain at position 380. Both mutant viruses were impaired for spread in epidermal cells in vitro; however, the 380 mutant virus was significantly more impaired and was as defective as gE null virus. gE mutant viruses were inoculated into the murine flank to measure epidermal disease at the inoculation site, travel of virus to dorsal root ganglia, and spread of virus from ganglia back to skin to produce zosteriform lesions. Disease at the inoculation and zosteriform sites was reduced for both mutant viruses, but more so for the 380 mutant virus. Moreover, the 380 mutant virus was highly impaired in its ability to reach the ganglia, as demonstrated by virus culture and real-time quantitative PCR. The results indicate that the domain surrounding amino acid 380 is important for both spread and IgG Fc binding and suggest that this domain is a potential target for antiviral therapy or vaccines.

Submandibular Salivary Proteases: Lack of a Role in Anti-HIV Activity:

Whole human saliva contains a number of proteolytic enzymes, mostly derived from white blood cells and bacteria in the oral cavity. However, less information is available regarding proteases produced by salivary glands and present in salivary secretions. In the present study, we have analyzed submandibular saliva, collected without contaminating cells, and identified multiple proteolytic activities. These have been characterized in terms of their susceptibility to a series of protease inhibitors. The submandibular saliva proteases were shown to be sensitive to both serine and acidic protease inhibitors. We also used protease inhibitors to determine if salivary proteolytic activity was involved in the inhibition of HIV infectivity seen when the virus is incubated with human saliva. This anti-HIV activity has been reported to occur in whole saliva and in ductal saliva obtained from both the parotid and submandibular glands, with highest levels of activity present in the latter fluid. Protease inhibitors, at concentrations sufficient to block salivary proteolytic activity in an in vitro infectivity assay, did not block the anti-HIV effects of saliva, suggesting that the salivary proteases are not responsible for the inhibition of HIV-1 infectivity.

Development of genetic vaccines for pathogenic genes : construction of attenuated vif DNA immunization cassettes

Objective:To develop a putative immunization cassette using HIV-1 vif accessory gene derived from HIV-1 clinical specimens as a component of a DNA vaccine for HIV-1. Methods:vif genes were cloned from HIV-1-infected patients and the sequence variation present within the patients was analyzed. Prototypic genetic variants were selected and the ability of these clones to induce humoral and cellular immune responses was studied in animals. The selected protective genetic variants were biologically characterized through transcomplementation assays using primary cells infected with a vif-defective HIV-1 proviral clone. Results:Analysis of vif variants from different patients revealed that vif is highly conserved with the open reading frame remaining intact in vivo. It was shown that attenuated vif clones from HIV-1-infected subjects can effectively induce both humoral and cellular responses against Vif protein in mice. Evaluation of the cellular responses in vitro using human cellular targets infected with a clinical HIV-1 isolate showed that vif clones could induce cellular responses capable of destroying the virus. Conclusions:The vif variants developed in this study exhibited non-productive phenotypes, yet were capable of inducing specific immune responses against HIV-1. These constructs could be used as part of a DNA vaccine strategy for HIV-1. This vaccine adaptation strategy could be used for the development of immunogens for any pathogen resulting in cross-reactive immunity and attenuated gene pathogenesis.

Construction of attenuated HIV-1 accessory gene immunization cassettes

Delivery of genetic expression cassettes into animals can effectively induce both humoral and cellular immunity to the expressed gene product. Previously, we used this strategy to immunize against HIV-1 structural and enzymatic proteins in mice, non-human primates and in humans. In contrast, the use of the accessory genes including vif, vpr, vpu and nef as immunotherapeutic vaccine targets has not been well characterized. Our goal is to design an effective genetic HIV vaccine, which includes the accessory genes as part of a multi-component immunogen. In order to develop accessory genes as genetic vaccines, we have molecularly cloned and analysed the sequence variation and immunogenic potential present in these genes derived from viral isolates obtained from HIV-1 infected patients and laboratory isolates. Prototype genetic variants were selected and their ability to induce humoral and cellular immune responses was studied in animal models. We observed that attenuated accessory genes can effectively induce both humoral and cellular responses in mice and the resulting immune response is directly correlated with DNA concentrations delivered and the number of boosts. This strategy can be used generally to develop an effective, safe DNA vaccine for any pathogen.