No abstract

Dendritic cells (DCs) are important APCs able to induce both tolerance and immunity. Therefore, DCs are attractive targets for immune intervention. However, the ex vivo generation and manipulation of DCs at sufficient numbers and without changing their original phenotypic and functional characteristics are major obstacles. To manipulate DCs in vivo, we developed a novel DC-specific self-inactivating lentiviral vector system using the 5′ untranslated region from the DC-STAMP gene as a putative promoter region. We show that a gene therapy approach with these DC-STAMP-lentiviral vectors yields long-term and cell-selective transgene expression in vivo. Furthermore, transcriptionally targeted DCs induced functional, Ag-specific CD4 and CD8 T cell tolerance in vivo, which could not be broken by viral immunization. Tolerized CTL were unable to induce autoimmune diabetes in a murine autoimmune model system. Therefore, delivering transgenes specifically to DCs by using viral vectors might be a promising tool in gene therapy.

Adeno-associated virus (AAV) has previously been shown to inhibit the replication of its helper virus herpes simplex virus type 1 (HSV-1), and the inhibitory activity has been attributed to the expression of the AAV Rep proteins. In the present study, we assessed the Rep activities required for inhibition of HSV-1 replication using a panel of wild-type and mutant Rep proteins lacking defined domains and activities. We found that the inhibition of HSV-1 replication required Rep DNA-binding and ATPase/helicase activities but not endonuclease activity. The Rep activities required for inhibition of HSV-1 replication precisely coincided with the activities that were responsible for induction of cellular DNA damage and apoptosis, suggesting that these three processes are closely linked. Notably, the presence of Rep induced the hyperphosphorylation of a DNA damage marker, replication protein A (RPA), which has been reported not to be normally hyperphosphorylated during HSV-1 infection and to be sequestered away from HSV-1 replication compartments during infection. Finally, we demonstrate that the execution of apoptosis is not required for inhibition of HSV-1 replication and that the hyperphosphorylation of RPA per se is not inhibitory for HSV-1 replication, suggesting that these two processes are not directly responsible for the inhibition of HSV-1 replication by Rep.Adeno-associated virus (AAV) is a widespread, nonpathogenic human parvovirus with a unique biphasic life cycle. In the absence of a helper virus, AAV establishes a latent infection in the host cell mediated either by site-specific integration of the viral genome into human chromosome 19 or by episomal persistence of circularized virus genomes (reviewed in reference 53). In the presence of helper viruses such as a herpesvirus, adenovirus (Ad), or papillomavirus, AAV is rescued from latency and undergoes lytic replication. The AAV genome is a single-stranded DNA (ssDNA) of 4,680 nucleotides, which is packaged into an icosahedral capsid with a diameter of 20 nm. The AAV genome harbors two open reading frames (ORFs), rep and cap, which are flanked by two inverted terminal repeats (ITRs) containing viral origins of DNA replication. The cap ORF is transcribed from the p40 promoter and encodes the capsid proteins VP1, VP2, and VP3, which differ in their N termini due to alternative start codons. The rep ORF encodes the Rep proteins, which are expressed in four different forms due to transcription from two different promoters, p5 and p19, and alternative splicing at an intron at the C-terminal end. The different Rep proteins are termed Rep40, Rep52, Rep68, and Rep78 according to their apparent molecular weight. The Rep proteins are involved in diverse processes in the viral life cycle, such as DNA replication, regulation of gene expression, genome packaging, and site-specific integration (reviewed in reference 56). The biochemical activities of Rep required for AAV DNA metabolism include site-specific DNA-binding and endonuclease activities, as well as non-site-sp...

Adeno-associated virus type 2 (AAV2) is a human parvovirus that relies on a helper virus for efficient replication. Herpes simplex virus 1 (HSV-1) supplies helper functions and changes the environment of the cell to promote AAV2 replication. In this study, we examined the accumulation of cellular replication and repair proteins at viral replication compartments (RCs) and the influence of replicating AAV2 on HSV-1-induced DNA damage responses (DDR). We observed that the ATM kinase was activated in cells coinfected with AAV2 and HSV-1. We also found that phosphorylated ATR kinase and its cofactor ATR-interacting protein were recruited into AAV2 RCs, but ATR signaling was not activated. DNA-PKcs, another main kinase in the DDR, was degraded during HSV-1 infection in an ICP0-dependent manner, and this degradation was markedly delayed during AAV2 coinfection. Furthermore, we detected phosphorylation of DNA-PKcs during AAV2 but not HSV-1 replication. The AAV2-mediated delay in DNA-PKcs degradation affected signaling through downstream substrates. Overall, our results demonstrate that coinfection with HSV-1 and AAV2 provokes a cellular DDR which is distinct from that induced by HSV-1 alone.A deno-associated virus type 2 (AAV2) is a small, nonenveloped parvovirus with a single-stranded DNA genome of 4.7 kb (52). In the absence of a helper virus, AAV2 establishes a latent infection characterized by site-specific integration of the viral genome into the AAVS1 site on human chromosome 19 (72). In the presence of a helper virus, AAV2 can replicate productively in the host cell nucleus. AAV2 DNA replication occurs at discrete sites in the nucleus, termed replication compartments (RCs). During the course of infection, several small RCs rapidly expand and fuse to large structures, which displace the cellular chromatin and fill the entire cell nucleus (28,35,37,79,91). AAV2 RCs contain AAV2 proteins, as well as defined helper virus proteins and cellular proteins (3,35,63,65,75,79,90,91). Replicating AAV2 has inhibitory effects on both the host cell (9,41,68,71,73,74,100,101) and the helper virus (5,30,31,34,40,44,61,84,100).One of the helper viruses for AAV2 replication is herpes simplex virus 1 (HSV-1) (14). The minimal HSV-1 helper factors for AAV2 replication from plasmid substrates include the helicaseprimase complex encoded by UL5, UL8, and UL52 and the major DNA binding protein ICP8 (3) (90). Besides viral helper factors, the fate of AAV2 replication also depends on cellular proteins. Recently, cellular proteins have been identified that interact with AAV2 Rep78/68 in adenovirus (Ad)-or HSV-1-supported AAV2 replication (63,65). Of these, the largest functional categories correspond to cellular proteins which are involved in DNA metabolism, including DNA replication, repair, and chromatin modification.There is accumulating evidence that the DNA damage response (DDR) pathways play central roles in viral replication (92). Control of DDR signaling may be a mechanism to prevent apoptosis and/or stop cell cycle progression (92)...

B cell activating factor receptor (BAFFR)-/- mice have a profound reduction in mature B cells, but unlike μMT mice, they have normal numbers of newly formed, immature B cells. Using a West Nile virus (WNV) challenge model that requires antibodies (Abs) for protection, we found that unlike wild-type (WT) mice, BAFFR-/- mice were highly susceptible to WNV and succumbed to infection within 8 to 12 days after subcutaneous virus challenge. Although mature B cells were required to protect against lethal infection, infected BAFFR-/- mice had reduced WNV E-specific IgG responses and neutralizing Abs. Passive transfer of immune sera from previously infected WT mice rescued BAFFR-/- and fully B cell-deficient μMT mice, but unlike μMT mice that died around 30 days post-infection, BAFFR-/- mice survived, developed WNV-specific IgG Abs and overcame a second WNV challenge. Remarkably, protective immunity could be induced in mature B cell-deficient mice. Administration of a WNV E-anti-CD180 conjugate vaccine 30 days prior to WNV infection induced Ab responses that protected against lethal infection in BAFFR-/- mice but not in μMT mice. Thus, the immature B cells present in BAFFR-/- and not μMT mice contribute to protective antiviral immunity. A CD180-based vaccine may promote immunity in immunocompromised individuals.

No abstract

Cross-presentation is an important mechanism to elicit both immune defenses and tolerance. Although only a few DC subsets possess the machinery required for crosspresentation, little is known about differences in cross-presenting capabilities of DCs belonging to the same subpopulation but localized in different lymphoid organs. In this study, we demonstrate that steady-state thymic CD81 DCs can efficiently cross-prime naïve CD8 1 T cells in the absence of costimulation. Surprisingly, cross-priming by splenic CD8 1 DCs was dependent on licensing factors such as GM-CSF. In the absence of GM-CSF,antigen-MHC-class-I complexes were detected on thymic but not on splenic CD8 1 DCs, indicating that the cross-presentation capacity of the thymic subpopulation was higher. The observed cross-priming differences between thymic and splenic CD8 1 DCs did not correlate with differential antigen capture or costimulatory molecules found on the surface of DCs. Moreover, we did not detect overall impairment of antigen presentation, as peptide-loaded splenic CD8 1 DCs were able to induce CD8 1 T-cell proliferation. The observation that thymic CD8 1 DCs are more efficient than splenic CD8 1 DCs in T-cell cross-priming in the absence of licensing factors indicates that the requirements for efficient antigen presentation differ between these cells.

Delivering antigens in vivo by coupling them to mAbs specific for unique receptors on antigen-presenting cells (APCs) is a promising approach for modulating immune responses. Antigen delivery to receptors found on myeloid dendritic cell (DC) subsets, plasmacytoid DCs and B cells has shown them all to be viable targets to stimulate either the cellular or humoral arms of the immune system. It is now evident that antigen-targeting approaches can also be used to invoke antigen-specific inhibition of immune responses. The outcome of activation versus inhibition is determined by a combination of factors that include the choice of APC, the receptor that is targeted, whether to include an adjuvant and, if so, which adjuvant to employ. In addition to their use as a means to modulate immune responses, antigen-targeting systems are also a useful method to investigate the function of DC subsets and the early mechanistic events that underlie the initiation of both cellular and humoral immune responses. In this review, we focus on the literature surrounding the control of B-cell responses when antigen is delivered to various APC subsets.