Immunotherapy of cancer is attractive because of its potential for specificity and limited side effects. The efficacy of this approach may be improved by providing adjuvant signals and an inflammatory environment for immune cell activation. We evaluated antitumor immune responses in mice after treatment of OVA-expressing B16-F0 tumors with single (15 Gy) or fractionated (5 × 3 Gy) doses of localized ionizing radiation. Irradiated mice had cells with greater capability to present tumor Ags and specific T cells that secreted IFN-γ upon peptide stimulation within tumor-draining lymph nodes than nonirradiated mice. Immune activation in tumor-draining lymph nodes correlated with an increase in the number of CD45+ cells infiltrating single dose irradiated tumors compared with nonirradiated mice. Similarly, irradiated mice had increased numbers of tumor-infiltrating lymphocytes that secreted IFN-γ and lysed tumor cell targets. Peptide-specific IFN-γ responses were directed against both the class I and class II MHC-restricted OVA peptides OVA257–264 and OVA323–339, respectively, as well as the endogenous class I MHC-restricted B16 tumor peptide tyrosinase-related protein 2180–188. Adoptive transfer studies indicated that the increased numbers of tumor Ag-specific immune cells within irradiated tumors were most likely due to enhanced trafficking of these cells to the tumor site. Together these results suggest that localized radiation can increase both the generation of antitumor immune effector cells and their trafficking to the tumor site.

A better understanding of the pathogenesis of dengue hemorrhagic fever and dengue shock syndrome requires the precise identification of dengue virus (DV) permissive target cells. To examine the relative DV permissiveness among cell subsets, we inoculated unfractionated human peripheral blood mononuclear cells with DV2-16681 in the presence or absence of pooled DV-immune human sera (PHS), and assessed infection with fluorescent dye labeled DV-specific monoclonal antibody and cell surface markers using flow cytometry. We found significantly higher levels of DV antigen staining on DVinfected than mock-infected primary monocytes (3.54 AE 3.42% vs. 0.50 AE 0.38%; P ¼ 0.001). The magnitude of infection was markedly enhanced in the presence of highly diluted PHS (10.04 AE 6.10% vs. 3.54 AE 3.42%; P ¼ 0.015). Under identical experimental conditions, primary T or B cells were not infected either with or without the addition of PHS (0.06 AE 0.04% and 0.44 AE 0.22% for T and B cells, respectively). Furthermore, depletion of CD14þ monocytes prior to DV inoculation abrogated the detection of infected cells, and the addition of monoclonal antibodies to either FcgRI (CD64) or FcgRII (CD32) led to a 50-70% reduction in antibody-dependent enhancement (ADE) of DV infection. Collectively, these results provide further support to the notion that primary monocytes and FcgRs expressed on these cells may be important in the initial steps of immune enhancement observed in some patients with natural DV infection. They also demonstrate that using modern experimental technology, DV infection, and neutralization and enhancement of DV infection can be easily assessed simultaneously in multiple cell types.

Fc␥ receptor (Fc␥R)-mediated entry of infectious dengue virus immune complexes into monocytes/macrophages is hypothesized to be a key event in the pathogenesis of complicated dengue fever. Fc␥RIA (CD64) and Fc␥RIIA (CD32), which predominate on the surface of such dengue virus-permissive cells, were compared for their influence on the infectivity of dengue 2 virus immune complexes formed with human dengue virus antibodies. A signaling immunoreceptor tyrosine-based activation motif (ITAM) incorporated into the accessory ␥-chain subunit that associates with Fc␥RIA and constitutively in Fc␥RIIA is required for phagocytosis mediated by these receptors. To determine whether Fc␥RIA and Fc␥RIIA activation functions are also required for internalization of infectious dengue virus immune complexes, we generated native and signaling-incompetent versions of each receptor by site-directed mutagenesis of ITAM tyrosine residues. Plasmids designed to express these receptors were transfected into COS-7 cells, and dengue virus replication was measured by plaque assay and flow cytometry. We found that both receptors mediated enhanced dengue virus immune complex infectivity but that Fc␥RIIA appeared to do so far more effectively. Abrogation of Fc␥RIA signaling competency, either by expression without ␥-chain or by coexpression with ␥-chain mutants, was associated with significant impairment of phagocytosis and of dengue virus immune complex infectivity. Abrogation of Fc␥RIIA signaling competency was also associated with equally impaired phagocytosis but had no discernible effect on dengue virus immune complex infectivity. These findings point to fundamental differences between Fc␥RIA and Fc␥RIIA with respect to their immune-enhancing capabilities and suggest that different mechanisms of dengue virus immune complex internalization may operate between these Fc␥Rs.

Human papillomavirus-like particles (HPV VLPs) have shown considerable promise as a parenteral vaccine for the prevention of cervical cancer and its precursor lesions. Parenteral vaccines are expensive to produce and deliver, however, and therefore are not optimal for use in resource-poor settings, where most cervical HPV disease occurs. Transgenic plants expressing recombinant vaccine immunogens offer an attractive and potentially inexpensive alternative to vaccination by injection. For example, edible plants can be grown locally and can be distributed easily without special training or equipment. To assess the feasibility of an HPV VLP-based edible vaccine, in this study we synthesized a plant codon-optimized version of the HPV type 11 (HPV11) L1 major capsid protein coding sequence and introduced it into tobacco and potato. We show that full-length L1 protein is expressed and localized in plant cell nuclei and that expression of L1 in plants is enhanced by removal of the carboxy-terminal nuclear localization signal sequence. We also show that plant-expressed L1 self-assembles into VLPs with immunological properties comparable to those of native HPV virions. Importantly, ingestion of transgenic L1 potato was associated with activation of an anti-VLP immune response in mice that was qualitatively similar to that induced by VLP parenteral administration, and this response was enhanced significantly by subsequent oral boosting with purified insect cell-derived VLPs. Thus, papillomavirus L1 protein can be expressed in transgenic plants to form immunologically functional VLPs, and ingestion of such material can activate potentially protective humoral immune responses.Infection with specific human papillomavirus (HPV) genotypes is a necessary factor in the development of cervical cancer and its precursor lesions (7, 43). Multiple sexually transmitted "high-risk" genotypes (e.g., types 16,18,31,33,35, 45, 51, 52, 58, and 59) have been associated with malignant disease, while other sexually transmitted types (notably 6 and 11) primarily cause only benign disease (e.g., anogenital warts) (20). Recombinant virus-like particles (VLPs) are promising immunogens for controlling HPV-associated diseases because they induce high-titer neutralizing antibody responses that have been associated with protection from infectious challenge (27, 36). Results from phase I studies in human subjects indicate that VLPs are safe, well-tolerated, and immunogenic when administered parenterally (12,16). Encouraging data from a recent phase II study suggest that VLPs may be highly efficacious for HPV prophylaxis (18).Vaccination for disease prevention is a major success of modern medicine; however, the full potential of this strategy to reduce human suffering has not yet been realized. For example, several so-called "vaccine-preventable" diseases continue to affect the lives of millions of individuals who live mostly in economically disadvantaged regions. With few exceptions, recommended immunizations rely on parenteral administration, and in...

The Li coat protein of human papillomavirus type 11 (HPV-11) was expressed in Sf-9 insect cells with the recombinant baculovirus vector AcllLl. Viruslike particles (VLPs) were identified by electron microscopy in the nucleus and cytoplasm of Sf-9 cells infected with AclILl. The Li protein was purified from AcliLlinfected insect cells. The purified protein spontaneously assembled in vitro into various aggregates, including particles appearing similar to empty virions. Reaction of VLP-containing insect cell extracts with antisera directed against either denatured or nondenatured capsid epitopes in Western blot (immunoblot) and immuno-dot blot assays suggested that conformational epitopes present in native HPV-11 infectious virions were also present on the baculovirus-produced HPV-11 VLPs. Immuno-dot blot assays using human sera obtained from individuals with biopsy-proven condyloma accuminatum correlated closely with results previously obtained in HPV-11 whole virus particle-based enzyme-linked immunosorbent assays. These morphologic and immunologic similarities to native HPV-11 virions suggest that recombinant VLPs produced in the baculovirus system may be useful in seroepidemiology and pathogenesis studies of genital HPV infection and that they may also be potential candidates for vaccine development.