Ann E. Campbell

Age-related impaired type 1 T cell responses to influenza: reduced activation ex vivo, decreased expansion in CTL culture in vitro, and blunted response to influenza vaccination in vivo in the elderly.

The objective of this study was to analyze the changes in the type 1 T cell response, including the CD4+ Th1 and CD8+ T cell responses, to influenza in the elderly compared with those in young adults. PBMC activated ex vivo with influenza virus exhibited an age-related decline in type 1 T cell response, shown by the decline in the frequency of IFN-γ-secreting memory T cells specific for influenza (IFN-γ+ ISMT) using ELISPOT or intracellular cytokine staining. The reduced frequency of IFN-γ+ ISMT was accompanied by a reduced level of IFN-γ secretion per cell in elderly subjects. Tetramer staining, combined with IFN-γ ELISPOT, indicated that the decline in IFN-γ+, influenza M1-peptide-specific T cells was not due to attrition of the T cell repertoire, but, rather, to the functional loss of ISMT with age. In addition, the decline in type 1 T cell response was not due to an increase in Th2 response or defects in APCs from the elderly. The expansion of influenza-specific CD8+ T cells in CTL cultures was reduced in the elderly. Compared with young subjects, frail elderly subjects also exhibited a blunted and somewhat delayed type 1 T cell response to influenza vaccination, which correlated positively with the reduced IgG1 subtype and the total Ab response. Taken together, these data demonstrate that there is a decline in the type 1 T cell response to influenza with age that may help explain the age-related decline in vaccine efficacy and the increases in influenza morbidity and mortality.

A mouse cytomegalovirus glycoprotein, gp34, forms a complex with folded class I MHC molecules in the ER which is not retained but is transported to the cell surface

Murine cytomegalovirus (MCMV) interferes with antigen presentation by means of retaining major histocompatibility complex (MHC) class I molecules in the endoplasmic reticulum (ER). Here we identify and characterize an MCMV‐encoded glycoprotein, gp34, which tightly associates with properly conformed MHC class I molecules in the ER. Gp34 is synthesized in large quantities during MCMV infection and it leaves the ER only in association with MHC class I complexes. Many but not all class I molecules are retained in the ER during the early phase of MCMV infection, and we observe an inverse correlation between amounts of gp34 synthesized during the course of infection and class I retention. An MCMV deletion mutant lacking several genes, including the gene encoding gp34, shows increased class I retention. Thus, MCMV gp34 may counteract class I retention, perhaps to decrease susceptibility of infected cells to recognition by natural killer cells.

Role of Murine Cytomegalovirus US22 Gene Family Members in Replication in Macrophages

ABSTRACT The large cytomegalovirus (CMV) US22 gene family, found in all betaherpesviruses, comprises 12 members in both human cytomegalovirus (HCMV) and murine cytomegalovirus (MCMV). Conserved sequence motifs suggested a common ancestry and related functions for these gene products. Two members of this family, m140 and m141, were recently shown to affect MCMV replication on macrophages. To test the role of all US22 members in cell tropism, we analyzed the growth properties in different cell types of MCMV mutants carrying transposon insertions in all 12 US22 gene family members. When necessary, additional targeted mutants with gene deletions, ATG deletions, and ectopic gene revertants were constructed. Mutants with disruption of genes M23, M24, m25.1, m25.2, and m128 (ie2) showed no obvious growth phenotype, whereas growth of M43 mutants was reduced in a number of cell lines. Genes m142 and m143 were shown to be essential for virus replication. Growth of mutants with insertions into genes M36, m139, m140, and m141 in macrophages was severely affected. The common phenotype of the m139, m140, and m141 mutants was explained by an interaction at the protein level. The M36-dependent macrophage growth phenotype could be explained by the antiapoptotic function of the gene that was required for growth on macrophages but not for growth on other cell types. Together, the comprehensive set of mutants of the US22 gene family suggests that individual family members have diverged through evolution to serve a variety of functions for the virus.

The salivary glands as a privileged site of cytomegalovirus immune evasion and persistence

The salivary glands (SG) provide a haven for persistent cytomegalovirus replication, and in this regard are a privileged site of virus immune evasion. The murine cytomegalovirus (MCMV) model has provided insight into the immunological environment of the SG and the unqiue virus–host relationship of this organ. In response to MCMV infection, a robust T cell-mediated immune response is elicited, comprised predominantly of CD8+ T cells that phenotypically and functionally appear activated. However, they fail to clear virus by an unknown evasion mechanism that is independent of inhibitory NKG2A- or Programmed Death 1-mediated signaling. Virus is eventually eliminated from the SG by effector CD4+ T cells expressing antiviral cytokines. However, this mechanism is severely dampened by high levels of the immunosuppressive cytokine IL-10, selectively expressed by SG CD4+ T cells.

Vigorous Innate and Virus-Specific Cytotoxic T-Lymphocyte Responses to Murine Cytomegalovirus in the Submaxillary Salivary Gland

ABSTRACT To better understand the immunological mechanisms that permit prolonged shedding of murine cytomegalovirus (MCMV) from the salivary gland, the phenotypic and functional characteristics of leukocytes infiltrating the submaxillary gland (SMG) were analyzed in infected BALB/c mice. A robust innate immune response, comprised of CD11c+ major histocompatibility complex class II+ CD11b− CD8α+ dendritic cells and γ/δ T-cell receptor-bearing CD3+ T cells was prominent through at least 28 days postinfection. Concurrently, a dramatic increase in pan-NK (DX5+) CD3+ and CD8+ T cells was observed, while CD4+ T cells, known to be essential for viral clearance from this tissue, increased slightly. The expression particularly of gamma interferon but also of interleukin-10 and CC chemokines was extraordinarily high in the SMG in response to MCMV infection. The gamma interferon was produced primarily by CD4+ and CD8+ T lymphocytes and DX5+ CD3+ T cells. The SMG CD8+ T cells were highly cytolytic ex vivo, and a significant proportion of these cells were specific to an immunodominant MCMV peptide. These peptide-specific clones were not exhausted by the presence of high virus titers, which persisted in the SMG despite the strength of the cell-mediated responses. In contrast, MCMV replication was efficiently cleared from the draining cervical and periglandular lymph nodes, a tissue displaying a substantially weaker antiviral response. Our data indicated that vigorous innate and acquired immune responses are elicited, activated, and retained in response to mucosal inflammation from persistent MCMV infection of the submaxillary gland.

Squalamine as a broad-spectrum systemic antiviral agent with therapeutic potential

Antiviral compounds that increase the resistance of host tissues represent an attractive class of therapeutic. Here, we show that squalamine, a compound previously isolated from the tissues of the dogfish shark (Squalus acanthias) and the sea lamprey (Petromyzon marinus), exhibits broad-spectrum antiviral activity against human pathogens, which were studied in vitro as well as in vivo. Both RNA- and DNA-enveloped viruses are shown to be susceptible. The proposed mechanism involves the capacity of squalamine, a cationic amphipathic sterol, to neutralize the negative electrostatic surface charge of intracellular membranes in a way that renders the cell less effective in supporting viral replication. Because squalamine can be readily synthesized and has a known safety profile in man, we believe its potential as a broad-spectrum human antiviral agent should be explored.

Products of US22 Genes M140 and M141 Confer Efficient Replication of Murine Cytomegalovirus in Macrophages and Spleen

ABSTRACT Efficient replication of murine cytomegalovirus (MCMV) in macrophages is a prerequisite for optimal growth and spread of the virus in its natural host. Simultaneous deletion of US22 gene family members M139, M140, and M141 results in impaired replication of MCMV in macrophages and mice. In this study, we characterized the proteins derived from these three genes and examined the impact of individual gene deletions on viral pathogenesis. The M139, M140, and M141 gene products were identified as early proteins that localize to both the nucleus and cytoplasm in infected cells. Gene M139 encodes two proteins, of 72 and 61 kDa, while M140 and M141 each encode a single protein of 56 (pM140) and 52 (pM141) kDa, respectively. No role for the M139 proteins in MCMV replication in macrophages or mice was determined in these studies. In contrast, deletion of either M140 or M141 resulted in impaired MCMV replication in macrophages and spleen tissue. Replication of the M140 deletion mutant was significantly more impaired than that of the virus lacking M141. Further analyses revealed that the absence of the pM140 adversely affected pM141 levels by rendering the latter protein unstable. Since the replication defect due to deletion of M140 was more profound than could be explained by the reduced half-life of pM141, pM140 must exert an additional, independent function in mediating efficient replication of MCMV in macrophages and spleen tissue. These data indicate that the US22 genes M140 and M141 function both cooperatively and independently to regulate MCMV replication in a cell type-specific manner and, thus, to influence viral pathogenesis.

Murine cytomegalovirus-induced suppression of antigen-specific cytotoxic T lymphocyte maturation

Antigen-specific cytotoxic T lymphocyte (CTL) maturation was inhibited in mice acutely infected with murine cytomegalovirus (MCMV). When immunization with Simian virus 40 (SV40) either preceded or followed infection with MCMV by 1 day, the frequency of SV40-specific CTL precursors among lymph node cells (LNC) was significantly reduced compared to noninfected mice. Replication of the herpesvirus in LNC could not be detected; however, MCMV rendered noninfectious by heat treatment was not suppressive to CTL development. Lymph node cells from nonimmunized, MCMV-infected mice contained a cell(s) present in low frequency which suppressed in vitro maturation of SV40 CTL in immune lymph nodes from mice not infected with MCMV. These suppressor cells did not affect the antigen- and interleukin-2-dependent growth nor cytotoxic activity of a mature, SV40-specific CTL clone. These results indicate that MCMV interferes with immunoregulatory functions required for development of antigen-specific CTL precursors to mature effector CTL. The immunosuppression is mediated at least in part by the MCMV-induced suppressor cell(s).

Inhibition of norovirus replication by morpholino oligomers targeting the 5'-end of the genome

Abstract Noroviruses are an important cause of non-bacterial epidemic gastroenteritis, but no specific antiviral therapies are available. We investigated the inhibitory effect of phosphorodiamidiate morpholino oligomers (PMOs) targeted against norovirus sequences. A panel of peptide-conjugated PMOs (PPMOs) specific for the murine norovirus (MNV) genome was developed, and two PPMO compounds directed against the first AUG of the ORF1 coding sequence near the 5′-end of the genome proved effective in inhibiting MNV replication in cells. A consensus PPMO (designated Noro 1.1), designed to target the corresponding region of several diverse human norovirus genotypes, decreased the efficiency of protein translation in a cell-free luciferase reporter assay and inhibited Norwalk virus protein expression in replicon-bearing cells. Our data suggest that PPMOs directed against the relatively conserved 5′-end of the norovirus genome may show broad antiviral activity against this genetically diverse group of viruses.

Complex Formation among Murine Cytomegalovirus US22 Proteins Encoded by Genes M139, M140, and M141

ABSTRACT The murine cytomegalovirus (MCMV) proteins encoded by US22 genes M139, M140, and M141 function, at least in part, to regulate replication of this virus in macrophages. Mutant MCMV having one or more of these genes deleted replicates poorly in macrophages in culture and in the macrophage-dense environment of the spleen. In this report, we demonstrate the existence of stable complexes formed by the products of all three of these US22 genes, as well as a complex composed of the products of M140 and M141. These complexes form in the absence of other viral proteins; however, the pM140/pM141 complex serves as a requisite binding partner for the M139 gene products. Products from all three genes colocalize to a perinuclear region of the cell juxtaposed to or within the cis-Golgi region but excluded from the trans-Golgi region. Interestingly, expression of pM141 redirects pM140 from its predominantly nuclear residence to the perinuclear, cytoplasmic locale where these US22 proteins apparently exist in complex. Thus, complexing of these nonessential, early MCMV proteins likely confers a function(s) independent of each individual protein and important for optimal replication of MCMV in its natural host.