Robert C. Rose

Local Radiation Therapy of B16 Melanoma Tumors Increases the Generation of Tumor Antigen-Specific Effector Cells That Traffic to the Tumor

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 Phase 1 Study of a Recombinant Viruslike Particle Vaccine against Human Papillomavirus Type 11 in Healthy Adult Volunteers

Viruslike particles (VLPs) produced from the L1 protein of several papillomaviruses have induced protection from infection after live challenge in animal models. In the present study, the safety and immunogenicity of a human papillomavirus (HPV)--11 L1 VLP candidate vaccine were measured in a phase 1, dose-finding trial in humans. The vaccine was well tolerated and induced high levels of both binding and neutralizing antibodies. Marked increases in lymphoproliferation to HPV--11 L1 antigens were noted after the second vaccination. In addition, lymphoproliferation was induced after vaccination in peripheral blood mononuclear cells (PBMC) stimulated with heterologous L1 VLP antigens of HPV types 6 and 16. Statistically significant increases in HPV antigen--specific interferon--gamma and interleukin-5 production were measured from PBMC culture supernatants. This candidate HPV VLP vaccine induced robust B and T cell responses, and T cell helper epitopes appear to be conserved across HPV types.

Oral Immunogenicity of Human Papillomavirus-Like Particles Expressed in Potato

ABSTRACT 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.

Human Papillomavirus Virus-Like Particles Are Efficient Oral Immunogens when Coadministered with Escherichia coli Heat-Labile Enterotoxin Mutant R192G or CpG DNA

ABSTRACT Certain human papillomaviruses (HPVs) cause most cervical cancer, which remains a significant source of morbidity and mortality among women worldwide. HPV recombinant virus-like particles (VLPs) are promising vaccine candidates for controlling anogenital HPV disease and are now being evaluated as a parenteral vaccine modality in human subjects. Vaccines formulated for injection generally are more costly, more difficult to administer, and less acceptable to recipients than are mucosally administered vaccines. Since oral delivery represents an attractive alternative to parenteral injection for large-scale human vaccination, the oral immunogenicity of HPV type 11 (HPV-11) VLPs in mice was previously investigated; it was found that a modest systemic neutralizing antibody response was induced (R. C. Rose, C. Lane, S. Wilson, J. A. Suzich, E. Rybicki, and A. L. Williamson, Vaccine 17:2129–2135, 1999). Here we examine whether VLPs of other genotypes may also be immunogenic when administered orally and whether mucosal adjuvants can be used to enhance VLP oral immunogenicity. We show that HPV-16 and HPV-18 VLPs are immunogenic when administered orally and that oral coadministration of these antigens with Escherichia coli heat-labile enterotoxin (LT) mutant R192G (LT R192G) or CpG DNA can significantly improve anti-VLP humoral responses in peripheral blood and in genital mucosal secretions. Our results also suggest that LT R192G may be superior to CpG DNA in this ability. These findings support the concept of oral immunization against anogenital HPV disease and suggest that clinical studies involving this approach may be warranted.

Serological differentiation of human papillomavirus types 11, 16 and 18 using recombinant virus-like particles.

The L1 major capsid protein-coding sequences of human papillomavirus (HPV) types 11, 16 and 18 were expressed in the baculovirus system. Virus-like particles (VLPs) were purified from recombinant-infected Spodoptera frugiperda Sf9 cells and cell-free culture supernatants. Rabbits immunized with purified VLPs developed antibodies that reacted only with the specific VLP type used as the immunogen. In addition, rabbit antibodies raised against infectious HPV-11 virions only reacted with HPV-11 L1 VLPs and not with VLPs derived from either HPV-16 or HPV-18. These results suggest that HPV-11, HPV-16 and HPV-18 virions are antigenically distinct from one another. This observation should be considered in future studies of immune responses to HPV.

Differential Enhancement of Dengue Virus Immune Complex Infectivity Mediated by Signaling-Competent and Signaling-Incompetent Human FcγRIA (CD64) or FcγRIIA (CD32)

ABSTRACT Fcγ receptor (FcγR)-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. FcγRIA (CD64) and FcγRIIA (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 γ-chain subunit that associates with FcγRIA and constitutively in FcγRIIA is required for phagocytosis mediated by these receptors. To determine whether FcγRIA and FcγRIIA 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 FcγRIIA appeared to do so far more effectively. Abrogation of FcγRIA signaling competency, either by expression without γ-chain or by coexpression with γ-chain mutants, was associated with significant impairment of phagocytosis and of dengue virus immune complex infectivity. Abrogation of FcγRIIA 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 FcγRIA and FcγRIIA with respect to their immune-enhancing capabilities and suggest that different mechanisms of dengue virus immune complex internalization may operate between these FcγRs.

Primary Human Splenic Macrophages, but Not T or B Cells, Are the Principal Target Cells for Dengue Virus Infection In Vitro

ABSTRACT Understanding the pathogenesis of dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) requires the precise identification of dengue virus (DV)-permissive target cells. In a previous study using unfractionated human peripheral blood mononuclear cells, we found that monocytes, but not B or T cells, were the principal DV-permissive cells in the absence of DV-immune pooled human sera (PHS) and the major mediators of antibody-dependent enhancement in the presence of PHS. To further identify DV-permissive target cells in other tissues and organs, we isolated human splenic mononuclear cells (MNCs), inoculated them with DV type 2 (strain 16681) in the presence or absence of PHS, and assessed their infection either directly using flow cytometry and reverse transcription-PCR (RT-PCR) assays or indirectly by plaque assay. We found that in the absence of PHS, a small proportion of splenic macrophages appeared to be positive for DV antigens in comparison to staining controls by the flow cytometric assay (0.77% ± 1.00% versus 0.18% ± 0.12%; P = 0.07) and that viral RNA was detectable by the RT-PCR assay in MNCs exposed to DV. Additionally, supernatants from cultures of DV-exposed MNCs contained infectious virions that were readily detectable by plaque assay. The magnitude of infection was significantly enhanced in splenic macrophages in the presence of highly diluted PHS (5.41% ± 3.53% versus 0.77% ± 1.00%; P = 0.001). In contrast, primary T and B cells were not infected in either the presence or absence of PHS. These results provide evidence, for the first time, that human primary splenic macrophages, rather than B or T cells, are the principal DV-permissive cells in the spleen and that they may be uniquely important in the initial steps of immune enhancement that leads to DHF/DSS in some DV-infected individuals.

Oral vaccination of mice with human papillomavirus virus-like particles induces systemic virus-neutralizing antibodies

To assess whether oral vaccination against human papillomavirus (HPV) may be feasible, we administered HPV virus-like particles (VLPs) to mice by gavage. Enzyme-linked immunosorbent assay (ELISA) results indicated that serum anti-VLP immunoglobulin G (IgG) and IgA antibodies were induced after oral vaccination, and these responses demonstrated antigenic specificities that were conformationally dependent and restricted according to HPV genotype. Importantly, orally induced postimmune sera were found to neutralize HPV-11 virions in vitro. These results indicated that the VLPs were antigenically stable in the environment of the gastrointestinal tract and were able to engage in potentially useful immune system interactions. These findings support the concept of oral vaccination against anogenital HPV disease, and suggest the possibility that this may be a useful approach to the immunization of large populations against cervical cancer and other HPV associated diseases.

Expression of Human papillomavirus type 16 major capsid protein in transgenic Nicotiana tabacum cv. Xanthi

Summary. The production of vaccine antigens in plants is a safe and potentially very cost-effective alternative to traditional expression systems. We investigated the possibility of transgenic plant expression of the Human papillomavirus (HPV) type 16 L1 major capsid protein, with and without nuclear localisation signals, in Nicotiana tabacum cv. Xanthi plants. The genes were stably integrated into the N. tabacum genome, and both the expressed proteins were capable of assembling into capsomers and virus-like particles. The proteins in concentrated leaf extracts (L1Tr) were tested for antigenicity using a panel of characterised monoclonal antibodies (Mabs). Neutralising and conformation-specific Mabs (H16:V5 and H16:E70) were shown to bind to both types of the plant-produced particles. We estimated the L1Tr product yield to be 2–4 µg per kg of fresh leaf material. Rabbits immunised with small doses of plant-produced particles elicited a weak anti-HPV-16 L1 immune response. Our results support the feasibility of using transgenic plants for the production of HPV vaccines.

Prevalence of Human Papillomavirus Genotypes and Related Abnormalities of Cervical Cytological Results among HIV-1–Infected Women in Rochester, New York

Women with human immunodeficiency virus (HIV) infection have higher rates of concurrent human papillomavirus (HPV) infection and cervical dysplasia than do HIV-uninfected women. They are also more commonly infected with multiple HPV types simultaneously. To determine the prevalence of different HPV genotypes in a group of HIV-infected women and to correlate these findings with cervical cytological results, we studied a group of 229 women attending a university-based HIV clinic during a 7-year period. When cervicovaginal lavage specimens, the reverse line-blot assay, and DNA sequencing were used, the most commonly detected HPV types (in decreasing order of frequency) were 56, 53, 16, 58, 52, MM7, MM8, and 33. These results contrast sharply with similar studies of HIV-uninfected women, in whom HPV-16 and -18 generally predominate. In our study, the HPV types most commonly associated with low-grade squamous intraepithelial lesions (SILs) were 56 and 53. Types most commonly associated with high-grade SILs were 52 and 58. High-risk HPV types other than 16 and 18 are often found in HIV-infected women and are frequently associated with abnormal cervical cytological results in this setting. These observations have implications for the design of future HPV vaccines.