Megan Steain

Characterization of the Host Immune Response in Human Ganglia after Herpes Zoster

ABSTRACT Varicella-zoster virus (VZV) causes varicella (chicken pox) and establishes latency in ganglia, from where it reactivates to cause herpes zoster (shingles), which is often followed by postherpetic neuralgia (PHN), causing severe neuropathic pain that can last for years after the rash. Despite the major impact of herpes zoster and PHN on quality of life, the nature and kinetics of the virus-immune cell interactions that result in ganglion damage have not been defined. We obtained rare material consisting of seven sensory ganglia from three donors who had suffered from herpes zoster between 1 and 4.5 months before death but who had not died from herpes zoster. We performed immunostaining to investigate the site of VZV infection and to phenotype immune cells in these ganglia. VZV antigen was localized almost exclusively to neurons, and in at least one case it persisted long after resolution of the rash. The large immune infiltrate consisted of noncytolytic CD8+ T cells, with lesser numbers of CD4+ T cells, B cells, NK cells, and macrophages and no dendritic cells. VZV antigen-positive neurons did not express detectable major histocompatibility complex (MHC) class I, nor did CD8+ T cells surround infected neurons, suggesting that mechanisms of immune control may not be dependent on direct contact. This is the first report defining the nature of the immune response in ganglia following herpes zoster and provides evidence for persistence of non-latency-associated viral antigen and inflammation beyond rash resolution.

Apparent Expression of Varicella-Zoster Virus Proteins in Latency Resulting from Reactivity of Murine and Rabbit Antibodies with Human Blood Group A Determinants in Sensory Neurons

ABSTRACT Analyses of varicella-zoster virus (VZV) protein expression during latency have been discordant, with rare to many positive neurons detected. We show that ascites-derived murine and rabbit antibodies specific for VZV proteins in vitro contain endogenous antibodies that react with human blood type A antigens in neurons. Apparent VZV neuronal staining and blood type A were strongly associated (by a χ2 test, α = 0.0003). Adsorption of ascites-derived monoclonal antibodies or antiserum with type A erythrocytes or the use of in vitro-derived VZV monoclonal antibodies eliminated apparent VZV staining. Animal-derived antibodies must be screened for anti-blood type A reactivity to avoid misidentification of viral proteins in the neurons of the 30 to 40% of individuals who are blood type A.

Human Cytomegalovirus Interleukin-10 Polarizes Monocytes toward a Deactivated M2c Phenotype To Repress Host Immune Responses

ABSTRACT Several human cytomegalovirus (HCMV) genes encode products that modulate cellular functions in a manner likely to enhance viral pathogenesis. This includes UL111A, which encodes homologs of human interleukin-10 (hIL-10). Depending upon signals received, monocytes and macrophages become polarized to either classically activated (M1 proinflammatory) or alternatively activated (M2 anti-inflammatory) subsets. Skewing of polarization toward an M2 subset may benefit the virus by limiting the proinflammatory responses to infection, and so we determined whether HCMV-encoded viral IL-10 influenced monocyte polarization. Recombinant viral IL-10 protein polarized CD14+ monocytes toward an anti-inflammatory M2 subset with an M2c phenotype, as demonstrated by high expression of CD163 and CD14 and suppression of major histocompatibility complex (MHC) class II. Significantly, in the context of productive HCMV infection, viral IL-10 produced by infected cells polarized uninfected monocytes toward an M2c phenotype. We also assessed the impact of viral IL-10 on heme oxygenase 1 (HO-1), which is an enzyme linked with suppression of inflammatory responses. Polarization of monocytes by viral IL-10 resulted in upregulation of HO-1, and inhibition of HO-1 function resulted in a loss of capacity of viral IL-10 to suppress tumor necrosis factor alpha (TNF-α) and IL-1β, implicating HO-1 in viral IL-10-induced suppression of proinflammatory cytokines by M2c monocytes. In addition, a functional consequence of monocytes polarized with viral IL-10 was a decreased capacity to activate CD4+ T cells. This study identifies a novel role for viral IL-10 in driving M2c polarization, which may limit virus clearance by restricting proinflammatory and CD4+ T cell responses at sites of infection.

HIV-1 co-infection superinfection and recombination.

As the human immunodeficiency virus (HIV) pandemic progresses, an increasing number of recombinant viruses have been identified and in many geographical regions they are now the predominating strain. These recombinants are formed when an individual has acquired a co-infection or superinfection with more than one HIV-1 strain or subtype. Thus, dually infected individuals provide opportunities for studying HIV recombinants and viral interactions between infecting strains in vivo. The possible epidemiological, clinical and therapeutic implications of dual infections and recombination are many. Recombination may result in the emergence of more pathogenic and virulent HIV strains with altered fitness, tropism, and resistance to multiple drugs, and may hamper the development of subtype-based vaccines. This review is aimed at providing a more thorough understanding of dual infections (both co-infection and super-infection) and the possible consequences of the emergence of recombinant HIV-1 strains.

Detection of influenza A H1N1 and H3N2 mutations conferring resistance to oseltamivir using rolling circle amplification

In the event of an influenza pandemic, the use of oseltamivir (OTV) will undoubtedly increase and therefore it is more likely that OTV-resistant influenza strains will also arise. OTV-resistance genotyping using sequence-based testing on viruses isolated in cell culture is time consuming and less likely to detect the low-level presence of drug-resistant virus populations. We have developed a novel rolling circle amplification (RCA) method to achieve the sensitive detection of OTV-resistant viruses from clinical specimens. Using artificially created templates, RCA could detect the presence of OTV-resistant mutations (N2: 119V, 292K, N1: 274Y) even if the population carrying the mutations was <1% of the total. By applying RCA to clinical samples, we identified the emergence of the 274Y mutation in one OTV-treated patient, as well as in seven individuals who were treatment-naïve (confirming community transmission of 274Y-containing resistant influenza A H1N1). These results were further confirmed by neuraminidase region sequencing. In conclusion, RCA technology can provide rapid (<24 h), high-throughput diagnosis of OTV resistance mutations with a high specificity and sensitivity.

Upregulation of CXCL10 in Human Dorsal Root Ganglia during Experimental and Natural Varicella-Zoster Virus Infection

ABSTRACT Varicella-zoster virus (VZV) reactivation causes herpes zoster, which is accompanied by an influx of lymphocytes into affected ganglia, but the stimulus for this infiltrate is not known. We report that VZV infection of ganglia leads to increased CXCL10 production in vitro, in an explant ganglion model and in naturally infected dorsal root ganglia (DRG) during herpes zoster. Lymphocytes expressing the receptor for CXCL10, CXCR3, were also observed throughout naturally infected ganglia during herpes zoster, including immediately adjacent to neurons. This study identifies VZV-induced CXCL10 as a potential driver of T lymphocyte recruitment into DRG during herpes zoster.

ANALYSIS OF T CELL RESPONSES DURING ACTIVE VARICELLA ZOSTER VIRUS REACTIVATION IN HUMAN GANGLIA

ABSTRACT Varicella-zoster virus (VZV) is responsible for both varicella (chickenpox) and herpes zoster (shingles). During varicella, the virus establishes latency within the sensory ganglia and can reactivate to cause herpes zoster, but the immune responses that occur in ganglia during herpes zoster have not previously been defined. We examined ganglia obtained from individuals who, at the time of death, had active herpes zoster. Ganglia innervating the site of the cutaneous herpes zoster rash showed evidence of necrosis, secondary to vasculitis, or localized hemorrhage. Despite this, there was limited evidence of VZV antigen expression, although a large inflammatory infiltrate was observed. Characterization of the infiltrating T cells showed a large number of infiltrating CD4+ T cells and cytolytic CD8+ T cells. Many of the infiltrating T cells were closely associated with neurons within the reactivated ganglia, yet there was little evidence of T cell-induced neuronal apoptosis. Notably, an upregulation in the expression of major histocompatibility complex class I (MHC-I) and MHC-II molecules was observed on satellite glial cells, implying these cells play an active role in directing the immune response during herpes zoster. This is the first detailed characterization of the interaction between T cells and neuronal cells within ganglia obtained from patients suffering herpes zoster at the time of death and provides evidence that CD4+ and cytolytic CD8+ T cell responses play an important role in controlling VZV replication in ganglia during active herpes zoster. IMPORTANCE VZV is responsible for both varicella (chickenpox) and herpes zoster (shingles). During varicella, the virus establishes a life-long dormant infection within the sensory ganglia and can reawaken to cause herpes zoster, but the immune responses that occur in ganglia during herpes zoster have not previously been defined. We examined ganglia obtained from individuals who, at the time of death, had active herpes zoster. We found that specific T cell subsets are likely to play an important role in controlling VZV replication in ganglia during active herpes zoster.

Differentiated Neuroblastoma Cells Provide a Highly Efficient Model for Studies of Productive Varicella-Zoster Virus Infection of Neuronal Cells

ABSTRACT Varicella-zoster virus (VZV) is a highly species-specific herpesvirus that targets sensory ganglionic neurons. This species specificity has limited the study of many aspects of VZV pathogenesis, including neuronal infection. We report development of a highly efficient neuroblastoma cell model to study productive VZV infection of neuronal cells. We show that differentiation of SH-SY5Y neuroblastoma cells yields a homogenous population of neuron-like cells that are permissive to the full VZV replicative cycle. These cells supported productive infection by both laboratory and clinical VZV isolates, including the live varicella vaccine. This model may enable rapid identification of genetic determinants facilitating VZV neurotropism.

Human Cytomegalovirus-Encoded Human Interleukin-10 (IL-10) Homolog Amplifies Its Immunomodulatory Potential by Upregulating Human IL-10 in Monocytes

ABSTRACT The human cytomegalovirus (HCMV) gene UL111A encodes cytomegalovirus-encoded human interleukin-10 (cmvIL-10), a homolog of the potent immunomodulatory cytokine human interleukin 10 (hIL-10). This viral homolog exhibits a range of immunomodulatory functions, including suppression of proinflammatory cytokine production and dendritic cell (DC) maturation, as well as inhibition of major histocompatibility complex (MHC) class I and class II. Here, we present data showing that cmvIL-10 upregulates hIL-10, and we identify CD14+ monocytes and monocyte-derived macrophages and DCs as major sources of hIL-10 secretion in response to cmvIL-10. Monocyte activation was not a prerequisite for cmvIL-10-mediated upregulation of hIL-10, which was dose dependent and controlled at the transcriptional level. Furthermore, cmvIL-10 upregulated expression of tumor progression locus 2 (TPL2), which is a regulator of the positive hIL-10 feedback loop, whereas expression of a negative regulator of the hIL-10 feedback loop, dual-specificity phosphatase 1 (DUSP1), remained unchanged. Engagement of the hIL-10 receptor (hIL-10R) by cmvIL-10 led to upregulation of heme oxygenase 1 (HO-1), an enzyme linked with suppression of inflammatory responses, and this upregulation was required for cmvIL-10-mediated upregulation of hIL-10. We also demonstrate an important role for both phosphatidylinositol 3-kinase (PI3K) and STAT3 in the upregulation of HO-1 and hIL-10 by cmvIL-10. In addition to upregulating hIL-10, cmvIL-10 could exert a direct immunomodulatory function, as demonstrated by its capacity to upregulate expression of cell surface CD163 when hIL-10 was neutralized. This study identifies a mechanistic basis for cmvIL-10 function, including the capacity of this viral cytokine to potentially amplify its immunosuppressive impact by upregulating hIL-10 expression. IMPORTANCE Human cytomegalovirus (HCMV) is a large, double-stranded DNA virus that causes significant human disease, particularly in the congenital setting and in solid-organ and hematopoietic stem cell transplant patients. A prominent feature of HCMV is the wide range of viral gene products that it encodes which function to modulate host defenses. One of these is cmvIL-10, which is a homolog of the potent immunomodulatory cytokine human interleukin 10 (hIL-10). In this study, we report that, in addition to exerting a direct biological impact, cmvIL-10 upregulates the expression of hIL-10 by primary blood-derived monocytes and that it does so by modulating existing cellular pathways. This capacity of cmvIL-10 to upregulate hIL-10 represents a mechanism by which HCMV may amplify its immunomodulatory impact during infection.

T-Cell Infiltration Correlates with CXCL10 Expression in Ganglia of Cynomolgus Macaques with Reactivated Simian Varicella Virus

ABSTRACT Ganglia of monkeys with reactivated simian varicella virus (SVV) contained more CD8 than CD4 T cells around neurons. The abundance of CD8 T cells was greater less than 2 months after reactivation than that at later times and correlated with that of CXCL10 RNA but not with those of SVV protein or open reading frame 61 (ORF61) antisense RNA. CXCL10 RNA colocalized with T-cell clusters. After SVV reactivation, transient T-cell infiltration, possibly mediated by CXCL10, parallels varicella zoster virus (VZV) reactivation in humans.