Nagendra R. Hegde

Inhibition of HLA-DR Assembly, Transport, and Loading by Human Cytomegalovirus Glycoprotein US3: a Novel Mechanism for Evading Major Histocompatibility Complex Class II Antigen Presentation

ABSTRACT Human cytomegalovirus (HCMV) establishes persistent lifelong infections and replicates slowly. To withstand robust immunity, HCMV utilizes numerous immune evasion strategies. The HCMV gene cassette encoding US2 to US11 encodes four homologous glycoproteins, US2, US3, US6, and US11, that inhibit the major histocompatibility complex class I (MHC-I) antigen presentation pathway, probably inhibiting recognition by CD8+ T lymphocytes. US2 also inhibits the MHC-II antigen presentation pathway, causing degradation of human leukocyte antigen (HLA)-DR-α and -DM-α and preventing recognition by CD4+ T cells. We investigated the effects of seven of the US2 to US11 glycoproteins on the MHC-II pathway. Each of the glycoproteins was expressed by using replication-defective adenovirus vectors. In addition to US2, US3 inhibited recognition of antigen by CD4+ T cells by a novel mechanism. US3 bound to class II α/β complexes in the endoplasmic reticulum (ER), reducing their association with Ii. Class II molecules moved normally from the ER to the Golgi apparatus in US3-expressing cells but were not sorted efficiently to the class II loading compartment. As a consequence, formation of peptide-loaded class II complexes was reduced. We concluded that US3 and US2 can collaborate to inhibit class II-mediated presentation of endogenous HCMV antigens to CD4+ T cells, allowing virus-infected cells to resist recognition by CD4+ T cells.

Endogenous human cytomegalovirus gB is presented efficiently by MHC class II molecules to CD4 CTL

Human cytomegalovirus (HCMV) infects endothelial, epithelial, and glial cells in vivo. These cells can express MHC class II proteins, but are unlikely to play important roles in priming host immunity. Instead, it seems that class II presentation of endogenous HCMV antigens in these cells allows recognition of virus infection. We characterized class II presentation of HCMV glycoprotein B (gB), a membrane protein that accumulates extensively in endosomes during virus assembly. Human CD4+ T cells specific for gB were both highly abundant in blood and cytolytic in vivo. gB-specific CD4+ T cell clones recognized gB that was expressed in glial, endothelial, and epithelial cells, but not exogenous gB that was fed to these cells. Glial cells efficiently presented extremely low levels of endogenous gB—expressed by adenovirus vectors or after HCMV infection—and stimulated CD4+ T cells better than DCs that were incubated with exogenous gB. Presentation of endogenous gB required sorting of gB to endosomal compartments and processing by acidic proteases. Although presentation of cellular proteins that traffic into endosomes is well known, our observations demonstrate for the first time that a viral protein sorted to endosomes is presented exceptionally well, and can promote CD4+ T cell recognition and killing of biologically important host cells.

Viral inhibition of MHC class II antigen presentation

T cells by at leasttwo mechanisms. First, and perhaps foremost, virusesinhibit induction of the expression of MHC class II genesby blocking interferon-g(IFN-g) signal transduction andexpression of the MHC class II transactivator (CIITA).Second, viruses inhibit the MHC class II antigen pre-sentation pathway by affecting the stability or intra-cellular sorting of class II proteins. It appears that thisviral inhibition is designed to prevent presentation ofendogenous viral antigens in virus-infected host cells,rather than presentation of exogenous antigens in‘professional’ antigen-presenting cells.Increasing evidence suggests that viral proteins normallyfound in the cytoplasm and exocytic compartments can beefficiently presented by MHC class II proteins. Thisapparently occurs in cells where viruses replicate, andsome viruses prevent recognition of infected cells by CD4

The Role of BiP in Endoplasmic Reticulum-associated Degradation of Major Histocompatibility Complex Class I Heavy Chain Induced by Cytomegalovirus Proteins

Human cytomegalovirus (HCMV1) US11 and US2 proteins cause rapid degradation of major histocompatibility complex (MHC) molecules, apparently by ligating cellular endoplasmic reticulum (ER)-associated degradation machinery. Here, we show that US11 and US2 bind the ER chaperone BiP. Four related HCMV proteins, US3, US7, US9, and US10, which do not promote degradation of MHC proteins, did not bind BiP. Silencing BiP reduced US11- and US2-mediated degradation of MHC class I heavy chain (HC) without altering the synthesis or translocation of HC into the ER or the stability of HC in the absence of US11 or US2. Induction of the unfolded protein response (UPR) did not affect US11-mediated HC degradation and could not explain the stabilization of HC when BiP was silenced. Unlike in yeast, BiP did not act by maintaining substrates in a retrotranslocation-competent form. Our studies go beyond previous observations in mammalian cells correlating BiP release with degradation, demonstrating that BiP is functionally required for US2- and US11-mediated HC degradation. Further, US2 and US11 bound BiP even when HC was absent and degradation of US2 depended on HC. These data were consistent with a model in which US2 and US11 bridge HC onto BiP promoting interactions with other ER-associated degradation proteins.

Inhibition of the MHC class II antigen presentation pathway by human cytomegalovirus

Human cytomegalovirus (HCMV) causes serious disease in immunocompromised individuals. Normally, anti-HCMV immune response controls virus replication following reactivation from latency. However, HCMV, like other large herpesviruses, encodes immune evasion proteins that allow the virus to replicate, for a time or in specific tissues, and produce viral progeny in the face of robust host immunity. HCMV glycoproteins US2, US3, US6 and US11 all inhibit different stages of the MHC class I antigen presentation pathway and can reduce recognition by CD8+ T lymphocytes. Here, we discuss two novel inhibitors of the MHC class II antigen presentation pathway, HCMV glycoproteins US2 and US3. Both US2 and US3 can inhibit presentation of exogenous protein antigens to CD4+ T lymphocytes in in vitro assays. US2 causes degradation of MHC class II molecules: HLA-DR-alpha and HLA-DM-alpha, as well as class I heavy chain (HC), but does not affect DR-beta or DM-beta chains. Mutant forms of US2 have been constructed that can bind to DR-alpha and class I HC but do not cause their degradation, separating the binding step from other processes that precede degradation. We also found evidence that US2-induced degradation of class I and II proteins involves a cellular component, other than Sec61, that is limiting in quantity. Unlike US2, US3 binds newly synthesized class II alpha/beta complexes, reducing the association with the invariant chain (Ii) and causing mislocalization of class II complexes in cells. US3 expression reduces accumulation of class II complexes in peptide-loading compartments and loading of peptides. Since US2 and US3 are expressed solely within HCMV-infected cells, it appears that these viral proteins have evolved to inhibit presentation of endogenous, intracellular viral antigens to anti-HCMV CD4+ T cells. This is different from how the MHC class II pathway is normally viewed, as a pathway for presentation of exogenous, extracellular proteins. The existence of these proteins indicates the importance of class II-mediated presentation of endogenous antigens in signalling virus infection to CD4+ T cells.

Herpes Simplex Virus with Highly Reduced gD Levels Can Efficiently Enter and Spread between Human Keratinocytes

ABSTRACT The rapid spread of herpes simplex virus type 1 (HSV-1) in mucosal epithelia and neuronal tissue depends primarily on the ability of the virus to navigate within polarized cells and the tissues they constitute. To understand HSV entry and the spread of virus across cell junctions, we have previously characterized a human keratinocyte cell line, HaCaT. These cells appear to reflect cells infected in vivo more accurately than many of the cultured cells used to propagate HSV. HSV mutants lacking gE/gI are highly compromised in spread within epithelial and neuronal tissues and also show defects in cell-to-cell spread in HaCaT cells, but not in other, nonpolarized cells. HSV gD is normally considered absolutely essential for entry and cell-to-cell spread, both in cultured cells and in vivo. Here, an HSV-1 gD mutant virus, F-US6kan, was found to efficiently enter HaCaT cells and normal human keratinocytes and could spread from cell to cell without gD provided by complementing cells. By contrast, entry and spread into other cells, especially highly transformed cells commonly used to propagate HSV, were extremely inefficient. Further analyses of F-US6kan indicated that this mutant expressed extraordinarily low (1/500 wild-type) levels of gD. Neutralizing anti-gD monoclonal antibodies inhibited entry of F-US6kan, suggesting F-US6kan utilized this small amount of gD to enter cells. HaCaT cells expressed high levels of an HSV gD receptor, HveC, and entry of F-US6kan into HaCaT cells could also be inhibited with antibodies specific for HveC. Interestingly, anti-HveC antibodies were not fully able to inhibit entry of wild-type HSV-1 into HaCaT cells. These results help to uncover important properties of HSV and human keratinocytes. HSV, with exceedingly low levels of a crucial receptor-binding glycoprotein, can enter cells expressing high levels of receptor. In this case, surplus gD may be useful to avoid neutralization by anti-gD antibodies.

Adenovirus Vectors Expressing Hantavirus Proteins Protect Hamsters against Lethal Challenge with Andes Virus

ABSTRACT Hantaviruses infect humans following aerosolization from rodent feces and urine, producing hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome. Due to the high rates of mortality and lack of therapies, vaccines are urgently needed. Nonreplicating adenovirus (Ad) vectors that express Andes hantavirus (ANDV) nucleocapsid protein (AdN) or glycoproteins (AdGN and AdGC) were constructed. Ad vectors were tested for their ability to protect Syrian hamsters from a lethal ANDV infection that mimics the pulmonary disease seen in humans. When administered once, all three Ad vectors, individually or in combination, elicited a robust immune response that protected hamsters. No vaccinated animal died, and there were no obvious clinical signs of disease. Further, hantavirus RNA was not detected by sensitive reverse transcription-PCR in tissues and blood of hamsters immunized with both AdGN and AdGC. Cellular immunity appeared to be important for protection because the AdN vector completely protected animals. All three Ad vectors produced strong cytotoxic T-lymphocyte responses directed to hantavirus proteins in mice. Moreover, hamsters vaccinated with AdN, AdGN, or AdGC produced no detectable neutralizing antibodies yet were protected. These Ad vectors represent the first vaccines that prevent lethal hantavirus disease and, in some instances (AdGN and AdGC), provide sterile immunity. These observations set the stage for a more detailed characterization of the types of immunity required to protect humans from hantavirus infections.

Human Cytomegalovirus US2 Causes Similar Effects on Both Major Histocompatibility Complex Class I and II Proteins in Epithelial and Glial Cells

ABSTRACT The human cytomegalovirus (HCMV) glycoprotein US2 specifically binds to major histocompatibility complex (MHC) class I heavy chain (HC) and class II proteins DRα and DMα, triggering their degradation by proteasomes. Effects of US2 on class II proteins were originally characterized in HCMV- or adenovirus vector-infected U373 astroglioma cells. Here, we have extended characterization of US2-mediated degradation of class II DRα to two other cell lines, including biologically relevant epithelial cells. Comparison of the effects of US2 in cells expressing both class I and II proteins demonstrated only a slight preference for class I HC. Moreover, US2 caused degradation of DRα and DMα when these proteins were expressed by transfection without DRβ, invariant chain (Ii), or DMβ. Therefore, US2 binds to α chains of DR and DM and triggers endoplasmic reticulum degradation without formation of class II DR αβ/Ii or DM αβ complexes. Similar levels of degradation of class II α were observed in cells expressing vastly different amounts of class II, suggesting that cellular factors, other than class II, were limiting. We concluded that US2 has broad effects in a variety of cells that express both class I and II proteins and is relevant to HCMV infection in vivo.

Full-Genome Sequencing as a Basis for Molecular Epidemiology Studies of Bluetongue Virus in India.

Since 1998 there have been significant changes in the global distribution of bluetongue virus (BTV). Ten previously exotic BTV serotypes have been detected in Europe, causing severe disease outbreaks in naïve ruminant populations. Previously exotic BTV serotypes were also identified in the USA, Israel, Australia and India. BTV is transmitted by biting midges (Culicoides spp.) and changes in the distribution of vector species, climate change, increased international travel and trade are thought to have contributed to these events. Thirteen BTV serotypes have been isolated in India since first reports of the disease in the country during 1964. Efficient methods for preparation of viral dsRNA and cDNA synthesis, have facilitated full-genome sequencing of BTV strains from the region. These studies introduce a new approach for BTV characterization, based on full-genome sequencing and phylogenetic analyses, facilitating the identification of BTV serotype, topotype and reassortant strains. Phylogenetic analyses show that most of the equivalent genome-segments of Indian BTV strains are closely related, clustering within a major eastern BTV ‘topotype’. However, genome-segment 5 (Seg-5) encoding NS1, from multiple post 1982 Indian isolates, originated from a western BTV topotype. All ten genome-segments of BTV-2 isolates (IND2003/01, IND2003/02 and IND2003/03) are closely related (>99% identity) to a South African BTV-2 vaccine-strain (western topotype). Similarly BTV-10 isolates (IND2003/06; IND2005/04) show >99% identity in all genome segments, to the prototype BTV-10 (CA-8) strain from the USA. These data suggest repeated introductions of western BTV field and/or vaccine-strains into India, potentially linked to animal or vector-insect movements, or unauthorised use of ‘live’ South African or American BTV-vaccines in the country. The data presented will help improve nucleic acid based diagnostics for Indian serotypes/topotypes, as part of control strategies.

Recent advances in the administration of vaccines for infectious diseases: microneedles as painless delivery devices for mass vaccination

Despite remarkable progress in the control of infectious diseases through vaccination, better delivery systems have been poorly explored. There is renewed interest in the discovery of novel vaccines and adjuvants owing to emerging and reemerging diseases and the burden and complexity of chronic infectious diseases. Conversely, the need for rapid local, regional, mucosal or parenteral bioavailability has led to advances in delivery systems and devices. Here, we present recent developments in the field of non-invasive cutaneous delivery of vaccines for infectious diseases. Transdermal delivery using microneedles could revolutionize the way prophylactic interventions for infectious diseases are carried out in the future.