Varicella-zoster virus (VZV) is a highly species-specific herpesvirus that infects up to 90% of the human population (6). During primary infection, VZV is responsible for the predominantly childhood disease varicella (chicken pox). Following resolution of primary infection by the host immune system, the virus establishes a lifelong, latent infection in the dorsal root ganglia of the host. Reactivation from this site may occur many years later, resulting in herpes zoster (shingles), a condition which can be complicated by prolonged pain associated with postherpetic neuralgia (6, 37).The induction of VZV-specific T-cell immunity is critical for host recovery from varicella, and both major histocompatibility complex (MHC) class I-restricted CD8 ϩ T lymphocytes and MHC class II-restricted CD4 ϩ T lymphocytes are sensitized to viral antigens during primary infection (5). The role of VZVspecific T lymphocytes in maintaining the equilibrium between the host and virus during latency is implied by the association between a decline in the frequency of circulating VZV-specific T lymphocytes and an increased risk of VZV reactivation, causing herpes zoster (26). However, like several other herpesviruses, VZV has the capacity to interfere with the expression of MHC class I and MHC class II molecules (2, 3). VZV-encoded immunomodulatory mechanisms that limit the presentation of VZV peptides by MHC class I or MHC class II pathways to effector T lymphocytes are likely to play an important role in the pathogenesis of VZV disease and persistence of the virus in the human population (1).Several human viruses have evolved alternative strategies of evading immune recognition by selectively infecting and altering the function of specialized immune cells involved in host immune surveillance. For example, T lymphocytes play a critical role in adaptive immunity, and viruses such as human immunodeficiency virus (HIV) and measles virus can infect and destroy these cells, which may result in significant immunosuppression of the host (7,16,17).Dendritic cells (DCs) are potent antigen-presenting cells critical for the initiation of a successful antiviral immune response through the stimulation of immunologically naïve T lymphocytes (8,34). DCs located in the periphery exist as immature cells, expressing low levels of MHC class I and MHC class II molecules and costimulatory molecules such as CD80 and CD86. Immature DCs readily take up antigen and are induced to migrate to the secondary lymphoid organs, where they undergo maturation and present processed antigens to antigen-specific T lymphocytes (8,34,35). Maturation of DCs results in the downregulation of antigen uptake and processing properties and the upregulation of MHC class I and MHC class II molecules; increased surface expression of costimulatory molecules CD80, CD86, and CD40 and the maturation molecule CD83; and upregulation of adhesion molecules such as ICAM-1 (CD54) (8,14,36,(39)(40)(41)(42). The ability of mature DCs to efficiently activate naïve T lymphocytes which subsequently e...

During primary varicella-zoster virus (VZV) infection, it is presumed that virus is transmitted from mucosal sites to regional lymph nodes, where T cells become infected. The cell type responsible for VZV transport from the mucosa to the lymph nodes has not been defined. In this study, we assessed the susceptibility of human monocyte-derived dendritic cells to infection with VZV. Dendritic cells were inoculated with the VZV strain Schenke and assessed by flow cytometry for VZV and dendritic cell (CD1a) antigen expression. In five replicate experiments, 34.4% ؎ 6.6% (mean ؎ SEM) of CD1a ؉ cells were also VZV antigen positive. Dendritic cells were also shown to be susceptible to VZV infection by the detection of immediate-early (IE62), early (ORF29), and late (gC) gene products in CD1a؉ dendritic cells. Infectious virus was recovered from infected dendritic cells, and cell-to-cell contact was required for transmission of virus to permissive fibroblasts. VZV-infected dendritic cells showed no significant decrease in cell viability or evidence of apoptosis and did not exhibit altered cell surface levels of major histocompatibility complex (MHC) class I, MHC class II, CD86, CD40, or CD1a. Significantly, when autologous T lymphocytes were incubated with VZV-infected dendritic cells, VZV antigens were readily detected in CD3؉ T lymphocytes and infectious virus was recovered from these cells. These data provide the first evidence that dendritic cells are permissive to VZV and that dendritic cell infection can lead to transmission of virus to T lymphocytes. These findings have implications for our understanding of how virus may be disseminated during primary VZV infection.

DNA priming has previously been shown to elicit augmented immune responses when administered by electroporation (EP) or codelivered with a plasmid encoding interleukin-12 (pIL-12). We hypothesized that the efficacy of a DNA prime and recombinant adenovirus 5 boost vaccination regimen (DNA/rAd5) would be improved when incorporating these vaccination strategies into the DNA priming phase, as determined by pathogenic simian immunodeficiency virus SIVmac239 challenge outcome. The whole SIVmac239 proteome was delivered in 5 separate DNA plasmids (pDNA-SIV) by EP with or without pIL-12, followed by boosting 4 months later with corresponding rAd5-SIV vaccine vectors. Remarkably, after repeated low-dose SIVmac239 mucosal challenge, we demonstrate 2.6 and 4.4 log reductions of the median SIV peak and set point viral loads in rhesus macaques (RMs) that received pDNA-SIV by EP with pIL-12 compared to the median peak and set point viral loads in mock-immunized controls (P < 0.01). In 5 out of 6 infected RMs, strong suppression of viremia was observed, with intermittent "blips" in virus replication. In 2 RMs, we could not detect the presence of SIV RNA in tissue and lymph nodes, even after 13 viral challenges. RMs immunized without pIL-12 demonstrated a typical maximum of 1.5 log reduction in virus load. There was no significant difference in the overall magnitude of SIV-specific antibodies or CD8 T-cell responses between groups; however, pDNA delivery by EP with pIL-12 induced a greater magnitude of SIV-specific CD4 T cells that produced multiple cytokines. This vaccine strategy is relevant for existing vaccine candidates entering clinical evaluation, and this model may provide insights into control of retrovirus replication.

Herpes simplex viruses (HSV) infect human and murine dendritic cells (DCs) and interfere with their immunostimulatory functions in culture. HSV-2 infection increases human immunodeficiency virus (HIV) spread in patients, and DCs also promote HIV infection. We have studied these topics in rhesus macaque monocyte-derived DCs (moDCs) to set the stage for future studies of these issues in animals. We provide the first evidence that IntroductionDendritic cells (DCs) are involved in innate and adaptive immunity. [1][2][3][4][5] DCs survey for pathogens to which they respond innately while also processing pathogens and presenting antigenic determinants to induce adaptive immune responses. DCs need to be activated or matured to stimulate potent adaptive immunity. 6 Maturation involves the up-regulation of molecules on the DC surface and secretion of cytokines and chemokines that encourage the DC-T cell interactions needed to elicit strong immunity. Many pathogens trigger these pathways, modifying DC functions to encourage effective immune activation and clearance of infection. [7][8][9][10] Yet, other pathogens like immunodeficiency viruses (human [HIV] and simian [SIV]) [11][12][13][14][15] and herpes simplex viruses (HSV) [16][17][18] exploit DC biology to facilitate infection and elicit immune responses incapable of preventing or eradicating infection. Moreover, there is a strong correlation between genital HSV (HSV-2) infection and the probability of acquiring HIV. 19 Understanding how this might be orchestrated at the DC level is central to developing strategies to prevent DC-driven HIV spread. 12 Primary HSV-2 infection, occurring at the mucosal surfaces, is typically followed by the establishment of latency in the sacral root ganglia. 20 Neutralizing antibodies (Abs) and antiviral CD4 ϩ and CD8 ϩ T cells are induced, 20 which ultimately restrict virus replication at local sites to resolve (primary and reactivated) lesions. 21,22 DCs likely play a key role by orchestrating responses to HSV, 21,23-25 although low-level productive infection of DCs might also contribute to virus spread. 18 HSV infection of human monocytederived DCs (moDCs) is cytopathic 23,26,27 and results in the down-modulation of several surface markers involved in the activation of T cells. [16][17][18] While this would result in mediocre anti-HSV immunity allowing the establishment of HSV infection (but not preventing infection), it must be sufficient to clear virus upon reactivation. 24,25 Herpetic lesions also comprise activated leukocyte infiltrates and enable direct blood contact, providing mechanisms for increased HIV spread. It is possible that HSV-2 infection of immature DCs additionally alters innate DC responses and compromises the ability of DCs to elicit potent adaptive responses to other pathogens, thereby further exacerbating HIV infection.The initial step toward dissecting this biology in a relevant animal model was to validate HSV-2 infection of macaque DCs. Supported by National Institutes of Health (NIH) grants R01 AI040877 an...

The epithelial surface acts as an effective barrier against HIV. The various mucosal surfaces possess specific mechanisms that help prevent the transmission of virus. Yet, HIV manages to cross these barriers to establish infection, and this is enhanced in the presence of physical trauma or preexisting sexually transmitted infections. Once breached, the virus accesses numerous cells such as dendritic cells, T cells, and macrophages present in the underlying epithelia. Although these cells should contribute to innate and adaptive immunity to infection, they also serve as permissive targets to HIV and help in the initiation and dissemination of infection. Understanding how the various mucosal surfaces, and the cells within them, respond to the presence of HIV is essential in the design of therapeutic agents that will help to prevent HIV transmission.

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The epithelial surface acts as an effective barrier against HIV. The various mucosal surfaces possess specific mechanisms that help prevent the transmission of virus. Yet, HIV manages to cross these barriers to establish infection, and this is enhanced in the presence of physical trauma or pre-existing sexually transmitted infections. Once breached, the virus accesses numerous cells such as dendritic cells, T cells, and macrophages present in the underlying epithelia. Although these cells should contribute to innate and adaptive immunity to infection, they also serve as permissive targets to HIV and help in the initiation and dissemination of infection. Understanding how the various mucosal surfaces, and the cells within them, respond to the presence of HIV is essential in the design of therapeutic agents that will help to prevent HIV transmission.

Results from recent HIV-1 vaccine studies have indicated that high serum antibody (Ab) titers may not be necessary for Ab-mediated protection, and that Abs localized to mucosal sites might be critical for preventing infection. Enzyme-linked immunosorbent assay (ELISA) has been used for decades as the gold standard for Ab measurement, though recently, highly sensitive microsphere-based assays have become available, with potential utility for improved detection of Abs. In this study, we assessed the Bio-Plex® Suspension Array System for the detection of simian immunodeficiency virus (SIV)-specific Abs in rhesus macaques (RMs) chronically infected with SIV, whose serum or mucosal SIV-specific Ab titers were negative by ELISA. We developed a SIVmac239-specific 4-plex bead array for the simultaneous detection of Abs binding to Env, Gag, Pol, and Nef. The 4-plex assay was used to quantify SIV-specific serum IgG and rectal swab IgA titers from control (SIV-naive) and SIVmac239-infected RMs. The Bio-Plex assay specifically detected anti-SIV Abs in specimens from SIV-infected animals for all four analytes when compared to SIV-naive control samples (p≤0.04). Furthermore, in 70% of Env and 79% of Gag ELISA-negative serum samples, specific Ab was detected using the Bio-Plex assay. Similarly, 71% of Env and 48% of Gag ELISA-negative rectal swab samples were identified as positive using the Bio-Plex assay. Importantly, assay specificity (i.e., probability of true positives) was comparable to ELISA (94%–100%). The results reported here indicate that microsphere-based methods provide a substantial improvement over ELISA for the detection of Ab responses, aid in detecting specific Abs when analyzing samples containing low levels of Abs, such as during the early stages of a vaccine trial, and may be valuable in attempts to link protective efficacy of vaccines with induced Ab responses.