Pathogen recognition and development of particulate vaccines: does size matter?

The use of particulate carriers holds great promise for the development of effective and affordable recombinant vaccines. Rational development requires a detailed understanding of particle up-take and processing mechanisms to target cellular pathways capable of stimulating the required immune responses safely. These mechanisms are in turn based on how the host has evolved to recognize and process pathogens. Pathogens, as well as particulate vaccines, come in a wide range of sizes and biochemical compositions. Some of these also provide 'danger signals' so that antigen 'senting cells (APC), usually dendritic cells (DC), acquire specific stimulatory activity. Herein, we provide an overview of the types of particles currently under investigation for the formulation of vaccines, discuss cellular uptake mechanisms (endocytosis, macropinocytosis, phagocytosis, clathrin-dependent and/or caveloae-mediated) for pathogens and particles of different sizes, as well as antigen possessing and presentation by APC in general, and DC in particular. Since particle size and composition can influence the immune response, inducing humoral and/or cellular immunity, activating CD8 T cells and/or CD4 T cells of T helper 1 and/or T helper 2 type, particle characteristics have a major impact on vaccine efficacy. Recently developed methods for the formulation of particulate vaccines are presented in this issue of Methods, showcasing a range of "cutting edge" particulate vaccines that employ particles ranging from nano to micro-sized. This special issue of Methods further addresses practical issues of production, affordability, reproducibility and stability of formulation, and also includes a discussion of the economic and regulatory challenges encountered in developing vaccines for veterinary use and for common Third World infectious diseases.     

High-resolution immunocytochemical labeling of replicas with ultrasmall gold.

The use of 10-15-nm gold probes in freeze-fracture immunocytochemistry sometimes results in poor immunogold labeling. Replica sites are labeled with only one or two gold particles, making it unlikely that the labeling depicts the true distribution of antigen. In this study, the feasibility of using ultrasmall ( approximately 1.4-nm) gold probes for immunocytochemical labeling of replicas was examined. When HLA Class I in neutrophil membrane replicas was labeled with various sized immunogold particles as the secondary detection system, the apparent distribution density was inversely related to the size of the particles (1.4-nm > 5-nm >10-nm >15-nm). Indeed, the density of the apparent distribution of HLA Class I labeled with 1.4-nm gold particles was about sevenfold greater than when labeling was carried out with the 10-nm gold particles. Similar results were obtained with CD16, another neutrophil membrane protein. Silver enhancement was required to visualize the 1.4-nm gold particles, but this procedure did not adversely affect replica membranes. These results suggest that, when followed by silver enhancement, 1.4-nm gold particles are effective probes for achieving high-resolution immunocytochemical labeling of replicas.(J Histochem Cytochem 47:569-573, 1999)     

Fluorescence-intensity multiplexing: simultaneous seven-marker, two-color immunophenotyping using flow cytometry.

BACKGROUND: Conventional immuno-based multiparameter flow cytometric analysis has been limited by the requirement of a dedicated detection channel for each antibody-fluorophore set. To address the need to resolve multiple biological targets simultaneously, flow cytometers with as many as 10-15 detection channels have been developed. In this study, a new Zenon immunolabeling technology is developed that allows for multiple antigen detection per detection channel using a single fluorophore, through a unique method of fluorescence-intensity multiplexing. By varying the Zenon labeling reagent-to-antibody molar ratio, the fluorescence intensity of the antibody-labeled cellular targets can be used as a unique identifier. Although demonstrated in the present study with lymphocyte immunophenotyping, this approach is broadly applicable for any immuno-based multiplexed flow cytomety assay. METHODS: Lymphocyte immunophenotyping of 38 clinical blood specimens using CD3, CD4, CD8, CD16, CD56, CD19, and CD20 antibodies was performed using conventional flow cytometric analysis and fluorescence-intensity multiplexing analysis. Conventional analysis measures a single antibody-fluorophore per photomultiplier tube (PMT). Fluorescence-intensity multiplex analysis simultaneously measures seven markers with two PMTs, using Zenon labeling reagent-antibody complexes in a single tube: CD19, CD4, CD8, and CD16 antibodies labeled with Zenon Alexa Fluor 488 Mouse IgG(1) labeling reagent and CD56, CD3, and CD20 antibodies labeled with Zenon R-Phycoerythrin (R-PE) Mouse IgG(1) or IgG(2b) labeling reagents. RESULTS: The lymphocyte immunophenotyping results from fluorescence-intensity multiplexing using Zenon labeling reagents in a single tube were comparable to results from conventional flow cytometric analysis. CONCLUSIONS: Simultaneous evaluation of multiple antigens using a single fluorophore can be performed using antibodies labeled with varying ratios of a Zenon labeling reagent. Labeling two sets of antibodies with different Zenon labeling reagents can generate characteristic and distinguishable multivariate patterns. Combining multiple antibodies and fluorescent labels with fluorescence intensity multiplexing enables the resolution of more cellular targets than detection-channels, allowing sophisticated multiparameter flow cytometric studies to be performed on less complex 2- or 3-detection-channel flow cytometers. For typical biological samples, approximately 2-4 cellular targets per detection channel can be resolved using this technique.     

High-efficiency intracellular magnetic labeling with novel superparamagnetic-Tat peptide conjugates

A biocompatible, dextran coated superparamagnetic iron oxide particle was derivatized with a peptide sequence from the HIV-tat protein to improve intracellular magnetic labeling of different target cells. The conjugate had a mean particle size of 41 nm and contained an average of 6.7 tat peptides. Derivatized particles were internalized into lymphocytes over 100-fold more efficiently than nonmodified particles, resulting in up to 12.7 x 10(6) particles/cell. Internalized particles localized in cytoplasm and nuclear compartments as demonstrated by fluorescence microscopy and immunohistochemistry. Labeled cells were highly magnetic, were detectable by NMR imaging, and could be retained on magnetic separation columns. The described method has potential applications for in vivo tracking of magnetically labeled cells by MR imaging and for recovering intracellularly labeled cells from organs.     

Functional assessment of human dendritic cells labeled for in vivo 19F magnetic resonance imaging cell tracking

Background aimsDendritic cells (DC) are increasingly being used as cellular vaccines to treat cancer and infectious diseases. While there have been some promising results in early clinical trials using DC-based vaccines, the inability to visualize non-invasively the location, migration and fate of cells once adoptively transferred into patients is often cited as a limiting factor in the advancement of these therapies. A novel perflouropolyether (PFPE) tracer agent was used to label human DC ex vivo for the purpose of tracking the cells in vivo by ¹⁹F magnetic resonance imaging (MRI). We provide an assessment of this technology and examine its impact on the health and function of the DC.MethodsMonocyte-derived DC were labeled with PFPE and then assessed. Cell viability was determined by examining cell membrane integrity and mitochondrial lipid content. Immunostaining and flow cytometry were used to measure surface antigen expression of DC maturation markers. Functional tests included bioassays for interleukin (IL)-12p70 production, T-cell stimulatory function and chemotaxis. MRI efficacy was demonstrated by inoculation of PFPE-labeled human DC into NOD-SCID mice.ResultsDC were effectively labeled with PFPE without significant impact on cell viability, phenotype or function. The PFPE-labeled DC were clearly detected in vivo by ¹⁹F MRI, with mature DC being shown to migrate selectively towards draining lymph node regions within 18 h.ConclusionsThis study is the first application of PFPE cell labeling and MRI cell tracking using human immunotherapeutic cells. These techniques may have significant potential for tracking therapeutic cells in future clinical trials.     

Binding of human immunodeficiency virus type 1 to immature dendritic cells can occur independently of DC-SIGN and mannose binding C-type lectin receptors via a cholesterol-dependent pathway

Interactions of human immunodeficiency virus type 1 (HIV-1) with immature dendritic cells (DC) are believed to be multifactorial and involve binding to the CD4 antigen, DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN), mannose binding C-type lectin receptors (MCLR), and heparan sulfate proteoglycans (HSPG). In this study we assessed the relative contributions of these previously defined virus attachment factors to HIV binding and accumulation in DC and the subsequent transfer of the bound virus particle to CD4(+) T cells. Using competitive inhibitors of HIV-1 attachment to DC, we have identified the existence of DC-SIGN-, MCLR-, and HSPG-independent mechanism(s) of HIV attachment and internalization. Furthermore, virus particles bound by DC independently of CD4, DC-SIGN, MCLR, and HSPG are efficiently transmitted to T cells. Treatment of virus particles with the protease subtilisin or treatment of immature DC with trypsin significantly reduced virus binding, thus demonstrating the role of HIV envelope glycoprotein interactions with unidentified DC-surface factor(s). Finally, this DC-mediated virus binding and internalization are dependent on lipid rafts. We propose that pathways to HIV-1 attachment and uptake in DC exhibit functional redundancy; that is, they are made up of multiple independent activities that can, at least in part, compensate for one another.     

Use of immunogold preembedding technique to detect hepatitis A viral antigen in infected cells

Localization of virus and viral antigen in cell cultures infected with a rapidly replicating isolate of strain HM-175 of hepatitis A virus (HAV; pHM-175) was accomplished by using immunogold probes. Cells infected under one-step growth curve conditions were prefixed with 2% paraformaldehyde and 0.1-0.001% saponin at appropriate times postinfection for detection of maximum virus and viral antigen. An indirect labeling technique was employed using monoclonal antibody to HAV followed by 5 nm gold-antimouse IgG conjugate. Cells were then fixed by standard electron microscopy techniques and thin sectioned. This prefixation technique allowed penetration of the immunogold probes and moderate preservation of ultrastructure. Within infected cell cytoplasm, numerous antigenic sites were labeled with six to 200 gold particles. Two types of cells were infected with HAV and somewhat different results were obtained with the two cell types. In BS-C-1 cells, where a cytopathic effect (CPE) was not observed, myelin figures were immunogold labeled or frequently were located near immunogold-labeled sites. Vesicles containing viruslike particles (14-22 nm) were also observed. A significant observation in infected FRhK-4 cells was the presence of multivesicular bodies labeled with immunogold. Microfilaments were commonly seen near the multivesicular bodies. Our results demonstrate that the choice of prefixation method for immunogold labeling should be empirically determined for the cell type and condition.     

Targeting dendritic cells for priming cellular immune responses

The cardinal role of dendritic cells (DC) in priming adaptive immunity and in orchestrating immune responses against all classes of pathogens and also against tumors is well established. Their unique potential both to maintain self-tolerance and to initiate protective immune responses against foreign and/or dangerous structures is based on the functional diversity and flexibility of these cells. Tissue DC lining antigenic portals such as mucosal surfaces and the skin are specialized to take up a wide array of compounds including proteins, lipids, carbohydrates, glycoproteins, glycolipids and oligonucleotides, particles carrying such structures and apoptotic or necrotic cells. This process is facilitated by specialized receptors with high endocytic capacity, which provides potential targets for delivering designed molecules. The best route for targeting B- and/or T cell epitopes, however, is still the subject of intense investigation. Immature DC, which reside in various tissues, can be activated by pathogens, stress and inflammation or modified metabolic products, which induce mobilization of cells to draining lymph nodes where they act as highly potent professional antigen presenting cells. This is brought about by the ability to present their accumulated intracellular content for both CD4+ helper (Th) and CD8+ cytotoxic/cytolytic T lymphocytes (Tc/CTL). Engulfed proteins are processed intracellularly and their peptide fragments are transported to the cell surface in the context of major histocompatibility complex encoded class I and II molecules for presentation to Th cells and CTLs, respectively. The T cell priming capacity of DC, however, depends not only on antigen presentation but also on other features of DC. Human monocyte-derived DC provide an excellent tool to study the internalizing, antigen-presenting and T cell-activating functions of DC at their immature and activated differentiation states. These biological activities of DC, however, are highly dependent on their migratory potential from the peripheral non-lymphoid tissues to the lymph nodes, on the expression of adhesion molecules, which support the interaction of DC with T lymphocytes, and the cytokines secreted by DC, which polarize immune responses to Th1-mediated cellular or Th2-mediated antibody responses. These results altogether demonstrate that monocyte-derived DC are useful candidates for in vitro or in vivo targeting of antigens to induce efficient adaptive immune responses against pathogens and also against tumors.     

Ultrastructural localization of the circulating anodic antigen and the circulating cathodic antigen in the liver of mice infected with Schistosoma mansoni: a sequential study

The present study describes the ultrastructural localization of two important circulating schistosome antigens--the circulating anodic antigen (CAA) and the circulating cathodic antigen (CCA)--in livers of mice at various time intervals after infection with Schistosoma mansoni. For the demonstration of these antigens at the electron microscope level use was made of a direct, double immunogold labeling procedure, in which CAA-specific monoclonal antibodies, labeled with 5-nm gold particles, and CCA-specific monoclonal antibodies, labeled with 15-nm gold particles, were used. Both antigens were localized in granules and in inclusion bodies of Kupffer cells and granuloma macrophages and it was found that in these compartments the degree of 5- and 15-nm gold labeling increased with the duration of the infection. Sometimes gold particles were also encountered on the cell surface and in endocytotic vesicles of these cells, in endothelial cells, and in the space of Disse. From these data it was concluded that in the liver CAA and CCA were primarily accumulated in granules and inclusion bodies of Kupffer cells and granuloma macrophages. It is discussed whether at these locations both antigens are degraded by lysosomal enzymes and whether these antigens are complexed with antibodies.     

A novel CD4-CD8alpha+CD205+CD11b- murine spleen dendritic cell line: establishment, characterization and functional analysis in a model of vaccination to toxoplasmosis

Dendritic cells (DCs) play an essential role in the induction of immune responses to pathogen infections. Native DCs are difficult to obtain in large numbers and consequently the vast majority of DCs employed in all experiments are derived from bone marrow progenitors. In an attempt to solve this problem, we have established a novel CD8alpha(+) DC line (H-2(k)) from spleen, which we have named SRDC line, and which is easy to culture in vitro. These cells display similar morphology, phenotype and activity to CD4(-)CD8alpha(+)CD205(+)CD11b(-) DCs purified ex vivo. Toxoplasma gondii antigen was shown to be taken up by these cells and to increase class I and class II major histocompatibility complex (MHC), CD40, CD80 and CD86 surface expression. We report that vaccination with T. gondii antigen-pulsed SRDCs, which synthesize large amounts of interleukin-12, induced protective immune responses against this intracellular pathogen in syngeneic CBA/J mice. This protection was associated with strong cellular and humoral immune responses at systemic and intestinal levels. Spleen and mesenteric lymph node cell proliferations were correlated with a Th1/Th2-type response and a specific SRDC homing to spleen and intestine was observed. The SRDC or CD4(-)CD8alpha(+)CD205(+)CD11b(-) DC line can be expected to be a very useful tool for immunobiology studies of DC.     

Replication-defective viruses modulate immune responses.

By immunizing inbred mice with purified replication-competent, defective virus particles, or an admixture of the two, differential effects on the cellular immune system have been uncovered. Defective virus, exemplified by the vesicular stomatitis virus (VSV) defective interfering particle (DI 0.33), induced in BALB/c mice low levels of proliferating, IL-2 secreting, and cytolytic Ag-specific T lymphocytes. This was not caused by a dominant suppressor cell response, or by a failure to stimulate lymphokine-secreting cells, but appeared to reflect a reduced efficiency of priming as compared with standard virus. Mice primed with a mixture of wt and DI virus showed reduced proliferation compared with mice primed with wt virus. When histocompatible target cells were sensitized by pure DI particles, they were neither recognized nor lysed by CD8+ CTL. Cells co-infected with wt and DI particles were not as readily lysed by CD8+ CTL as cells infected by VSV alone. The extent of this reduction was dependent on the concentration of DI particles. This suggests that DI particles may have prevented the proper presentation of endogenously synthesized Ag for recognition by CD8+ CTL. Metabolic labeling studies indicated that the presence of DI particles suppressed the synthesis of viral proteins in dually infected cells. However, CD4+ T lymphocyte clones recognized and efficiently lysed histocompatible Ia+ cells infected with DI particles alone or co-infected with replication-competent and defective virus.     

Localization of a proteasomal antigen in human spermatozoa: immunohistochemical electron microscopic study

The ultrastructural localization of a proteasomal antigen in human spermatozoa was studied by means of immunolabeling with the MPC21 monoclonal antibody and secondary gold labeled antibody with 1.4 nm gold particles in combination with silver enhancement reaction using pre-embedding technique. The labeling was found in the acrosomal and postacrosomal regions, in the connecting-piece (neck) and, in some cases, in the middle-piece and also in the residual bodies. There was no significant reaction in condensed chromatin. In some abnormal forms of spermatozoa, in which the chromatin was not well condensed, the labeling in nuclei was present. The nuclear vacuoles with looser chromatin were usually strongly labeled. The nuclei of cells representing different stages of spermatogenesis, that were present in semen samples, were also labeled.     

Monoclonal antibodies conjugated with superparamagnetic iron oxide particles allow magnetic resonance imaging detection of lymphocytes in the mouse brain

We investigated the potential of antibody-vectorialized superparamagnetic iron oxide (SPIO) particles as cellular specific magnetic resonance contrast agents to image lymphocyte populations within the central nervous system (CNS), with the final goal of obtaining a reliable tool for noninvasively detecting and tracking specific cellular populations in vivo. We used superparamagnetic particles bound to a monoclonal antibody. The particle is the contrast agent, by means of its T?* relaxation properties; the antibody is the targeting vector, responsible for homing the particle to target a surface antigen. To investigate the efficiency of particle vectorialization by these antibodies, we compared two types of antibody-vectorialized CD3-specific particles in vivo. We successfully employed vectorialized SPIO particles to image B220? cells in a murine model of B-cell lymphoma. Likewise, we were able to identify CD3? infiltrates in a murine model of multiple sclerosis. The specificity of the technique was confirmed by immunohistochemistry and electron microscopy of corresponding sections. Our findings suggest that indirect binding of the antibody to a streptavidinated particle allows for enhanced particle vectorialization compared to covalent binding of the antibody to the particle.     

Human immunodeficiency virus gag and pol-specific CD8 T cells in perinatal HIV infection.

BACKGROUND: Binding of fluorochrome-conjugated MHC class I tetramers is a powerful means to detect antigen-specific CD8 T lymphocytes. In human immunodeficiency virus (HIV) infection, cellular immune response is essential in curtailing HIV disease progression but gaps persist in our understanding of HIV-specific cells during the disease course. In this study, we evaluated tetramer binding HIV-specific CD8 T cells in HIV-infected children. METHODS: Fluorescently labeled tetramers for HIV gag and pol were utilized to quantify antigen-specific cells by flow cytometry using a whole blood labeling method in a cohort of 19 HLA-A2+ HIV-infected children (age range 1 month to 17 years). RESULTS: Fourteen children had detectable gag (median 0.4%) and pol (median 0.1%) binding CD8 T cells, three children had gag binding cells only, and two had neither. Numbers of gag and pol binding cells correlated with each other and each correlated independently with total CD8 T cells and total CD4 T cells. CONCLUSIONS: HIV gag and pol-specific CD8 T cells are maintained during the chronic phase of HIV infection in children and CD4 lymphocytes appear to be important for sustaining their levels.     

Dendritic cells: Potential role in cancer therapy

Dendritic cells (DC) are extremely potent antigen presenting cells, uniquely capable of sensitizing naive T cells to protein antigens and eliciting antigen specific immune responses. Studies of human DC isolated from peripheral blood indicate that these cells can be used to stimulate and expand antigen specific CD4+ and CD8+ T cells, in vitro. On the basis of these findings we have initiated pilot clinical studies to investigate the ability of DC pulsed ex vivo with tumor associated proteins to stimulate host anti-tumor immunity when re-infused as a vaccine. In the first such study DC pulsed with tumor derived idiotype protein were infused into patients with low grade malignant B cell lymphoma who had failed conventional chemotherapy. The majority of treated patients developed T cell mediated anti-idiotype immune responses and some of the patients experienced tumor regression. These results suggest that DC based immunotherapy is a potentially useful approach to B cell lymphoma and raises the possibility that the approach may prove useful in the treatment of other tumors as well. This revised version was published online in June 2006 with corrections to the Cover Date.     

Therapeutic immune response induced by electrofusion of dendritic and tumor cells

To elicit a therapeutic antitumor immune response, dendritic cells (DCs) have been employed as a cellular adjuvant. Among various DC-based approaches, fusion of DCs and tumor cells potentially confers not only DC functionality, but also a continuous source of unaltered tumor antigens. We have recently demonstrated successful generation of fusion hybrids by a large-scale electrofusion technique. The immunogenicity and therapeutic potential of fusion hybrids were further analyzed in a model system of a murine melanoma cell line expressing beta-galactosidase (beta-gal) as a surrogate tumor antigen. A single vaccination with fusion hybrids plus IL-12 induced a therapeutic immune response against 3-day established pulmonary metastases. This immunotherapy was beta-gal specific and involved both CD4 and CD8 T cells. In vitro, fusion hybrids stimulated specific IFN-gamma secretion from both CD4 and CD8 immune T cells. They also nonspecifically induced IL-10 secretion from CD4 but not CD8 T cells. Compared to other DC loadings, our results demonstrate the superior immunogenicity of fusion. The current technique of electrofusion is adequately developed for clinical use in cancer immunotherapy.     

Linkage of CD40L to a self-tumor antigen enhances the antitumor immune responses of dendritic cell-based treatment

CD40-CD40 ligand (CD40L) interaction is an important costimulatory signal in the interaction between T cells and antigen-presenting cells (APC). In the present study, we determined whether the linkage of CD40L to the tumor-specific idiotype (Id) derived from a murine B-cell lymphoma, 38C13, could enhance its immunogenicity when presented by dendritic cells (DC). We showed that bone marrow-derived DC pulsed with Id-CD40L upregulated the expression of CD40, CD80, CD86, and major histocompatibility complex (MHC) class II molecules with the increased production of interleukin-12 (IL-12). Mice immunized with DC loaded with Id-CD40L showed high levels of anti-Id antibody response of both IgG2a and IgG1 isotypes. In addition, nylon wool-enriched T cells from these immunized mice showed a tumor-specific T-cell proliferative response upon stimulation with Id protein. Mice immunized with DC pulsed with Id alone failed to show any of these immune responses. Immunization with DC pulsed with Id-CD40L showed increased resistance to the challenge by 38C13 tumor, and tumor growth was significantly retarded. Together, these results show that linkage of CD40L to a self-tumor antigen enhances the anti-tumor immune response in DC-based treatment.     

Multiple dendritic cell populations activate CD4+ T cells after viral stimulation

Dendritic cells (DC) are a heterogeneous cell population that bridge the innate and adaptive immune systems. CD8alpha DC play a prominent, and sometimes exclusive, role in driving amplification of CD8(+) T cells during a viral infection. Whether this reliance on a single subset of DC also applies for CD4(+) T cell activation is unknown. We used a direct ex vivo antigen presentation assay to probe the capacity of flow cytometrically purified DC populations to drive amplification of CD4(+) and CD8(+) T cells following infection with influenza virus by different routes. This study examined the contributions of non-CD8alpha DC populations in the amplification of CD8(+) and CD4(+) T cells in cutaneous and systemic influenza viral infections. We confirmed that in vivo, effective immune responses for CD8(+) T cells are dominated by presentation of antigen by CD8alpha DC but can involve non-CD8alpha DC. In contrast, CD4(+) T cell responses relied more heavily on the contributions of dermal DC migrating from peripheral lymphoid tissues following cutaneous infection, and CD4 DC in the spleen after systemic infection. CD4(+) T cell priming by DC subsets that is dependent upon the route of administration raises the possibility that vaccination approaches could be tailored to prime helper T cell immunity.     

Dendritic Cell Migration Limits the Duration of CD8+ T-Cell Priming to Peripheral Viral Antigen

CD8⁺ T cells (T_CD8⁺) play a crucial role in immunity to viruses. Antiviral T_CD8⁺ are initially activated by recognition of major histocompatibility complex (MHC) class I-peptide complexes on the surface of professional antigen-presenting cells (pAPC). Migration of pAPC from the site of infection to secondary lymphoid organs is likely required during a natural infection. Migrating pAPC can be directly infected with virus or may internalize antigen derived from virus-infected cells. The use of experimental virus infections to assess the requirement for pAPC migration in initiation of T_CD8⁺ responses has proven difficult to interpret because injected virus can readily drain to secondary lymphoid organs without the need for cell-mediated transport. To overcome this ambiguity, we examined the generation of antigen-specific T_CD8⁺ after immunization with recombinant adenoviruses that express antigen driven by skin-specific or ubiquitous promoters. We show that the induction of T_CD8⁺ in response to tissue-targeted antigen is less efficient than the response to ubiquitously expressed antigen and that the resulting T_CD8⁺ fail to clear all target cells pulsed with the antigenic peptide. This failure to prime a fully functional T_CD8⁺ response results from a reduced period of priming to peripherally expressed antigen versus ubiquitously expressed antigen and correlated with a brief burst of pAPC migration from the skin, a requirement for induction of the response to peripheral antigen. These results indicate that a reduced duration of pAPC migration after virus infection likely reduces the amplitude of the T_CD8⁺ response, allowing persistence of the peripheral virus.The induction of effector CD8⁺ T cells (T_CD8⁺) is a vital step in the eradication or control of many viral infections. The induction of antiviral T_CD8⁺ requires the presentation of virally derived peptides in complex with major histocompatibility complex (MHC) class I on the surface of specialized professional antigen-presenting cells (pAPC), most commonly a subset of dendritic cells (DC) that bear the CD8α chain (1, 29). The CD8α⁺ DC reside only in secondary lymphoid organs and not in the tissues, implying that cell-mediated transport or drainage of virus particles to a lymph node is required for initiation of a T_CD8⁺ response. Partial inhibition of DC migration from the skin can impair the initiation of a T_CD8⁺ response (2). After influenza infection in the lungs, there is a burst of DC migration, followed by a refractory period in which no DC migration occurs (19). The functional consequences of this refractory period of DC migration have not been explored.A number of viruses, particularly human papillomaviruses, infect the skin and are ignored by the immune response for extended periods of time (31). We sought to explore the possibility that, after a low-level peripheral virus infection of the skin, changes in DC migration may limit the availability of antigen in the draining lymph node and thus the induction of a T_CD8⁺ response. There are a number of confounding factors that make the study of DC migration in the initiation of an antiviral T_CD8⁺ response difficult. Virus particles may directly drain to the lymph node within seconds (11, 13, 25). In addition, many viruses will alter DC functions, including migration, after infection of the DC itself. This may occur via specific viral modulation of DC function (16) or via nonspecific shut down of host protein synthesis (26), both of which will affect migration. Thus, it is often not possible to distinguish between the effects of virus infection upon DC migration, drainage of virus directly to the lymph node, and the natural response that follows migration of DC responding to a peripheral virus infection.There is currently no mouse model of a peripheral virus infection that is confined to the skin, as no natural mouse papillomavirus has ever been isolated. Therefore, to address these issues, we have made use of another small DNA virus, namely, an adenovirus vector that is replication deficient (rAd). These vectors express influenza virus nucleoprotein (NP) under the control of a ubiquitous (cytomegalovirus [CMV] immediate-early) or tissue-targeted promoter (K14, targeted to keratinocytes, the site of papillomavirus replication). Antigen driven by the K14 promoter is expressed only in skin cells, so only uninfected DC can present antigen in this system, removing the need to account for modulation of the function of virus-infected DC.We demonstrate that when antigen is expressed in only keratinocytes in the skin, the efficiency of T_CD8⁺ induction is reduced and the time period for which antigen is available to prime effector cells is reduced dramatically. DC-mediated transport is required for antigen to reach the lymph node where a T_CD8⁺ response is initiated. The reduced time period of antigen presentation is the result of a transient blockade in DC migration from the site of infection. The blockade in DC migration reduced the delivery of viral antigen to the lymph node needed to induce a T_CD8⁺ response. The resulting T_CD8⁺ response to peripheral viral antigen is not capable of clearing all target cells presenting a viral peptide, thus allowing the persistence of peripheral virus-infected cells. These results provide a potential mechanism for the long-term evasion of the immune response by papillomaviruses following natural infection and also have important implications for tissue targeted gene therapy vectors.     

Comparative evaluation of CD8+CTL responses following gene gun immunization targeting the skin with intracutaneous injection of antigen-transduced dendritic cells

The skin is an attractive target for antigen-specific vaccination. Particle bombardment of the epidermis with plasmid DNA using the gene gun results in antigen expression in keratinocytes of the epidermis leading to antigen presentation in the draining lymph nodes by migratory dendritic cells (DC). In order to better understand the role of the skin in stimulating antigen-specific CD8+cytotoxic T cells (CTL), we compared gene gun immunization with intracutaneous injections of antigen-transduced DC. A single intracutaneous injection of antigen-transduced DC was able to induce in vivo expansion of CD8+CTL specific for the model antigen chicken ovalbumin while four simultaneous shots with the gene gun were not effective. Antigen-transduced DC were much more efficient than particle bombardment of the epidermis in stimulating adoptively transferred TCR-transgenic CD8+CTL in the draining lymph nodes. Employing the novel technique of in vivo bioluminescence imaging, we demonstrated efficient gene transfer to the skin following gene gun bombardment and confirmed that a similar amount of antigen reached the lymph node when compared with injection of antigen-transduced DC. Our results suggest that direct transfection of the skin does not optimally reach and activate appropriate antigen-presenting DC. We believe that this reflects the immunological function of the epidermis which must balance immunity and tolerance to foreign antigens. Further investigations will have to address the role of Langerhans cells for the activation of cellular immunity in the skin.