Induction of HPV16 capsid protein-specific human T cell responses by virus-like particles

It has been postulated that upon binding to a cell surface receptor, papilloma virus-like particles (VLPs) gain entry into the cytosol of infected cells and the capsid proteins L1 and L2 can be processed in the MHC class I presentation pathway. Vaccination of mice with human papilloma virus-like particles consisting of capsid proteins L1 and L2 induced a CD8-mediated and perforin dependent protective immune response against a tumor challenge with human papilloma virus transformed tumor cells, which express only minute amounts of L1 protein. Here we show that HPV16 capsid proteins stimulate a MHC class I restricted CTL response with human peripheral blood lymphocytes (PBL) in vitro. The vigorous response was specific for VLP-infected target cells and was MHC class I restricted. Moreover we show the presence of at least one HLA-A*0201 restricted CTL epitope within the HPV-16 capsid proteins by using a VLP-'infected' HLA-A*0201 transfected human cell line as target cells. These results demonstrated that VLPs can induce a HPV16 capsid protein-specific immune response in humans, allowing the monitoring of immune responses induced by vaccines based on chimeric VLPs carrying additional immunogenic peptides or proteins in therapeutical applications in human patients.     

Nasal Immunization of Mice with Human Papillomavirus Type 16 (HPV-16) Virus-Like Particles or with the HPV-16 L1 Gene Elicits Specific Cytotoxic T Lymphocytes in Vaginal Draining Lymph Nodes

Human papillomavirus type 16 (HPV-16) infects the genital tract and is closely associated with the development of cervical cancer. HPV-16 initiates infection at the genital mucosal surface; thus, mucosal immune responses are likely to contribute to defense against HPV-16 infection. However, little information is available regarding the induction of immune responses in the genital tract mucosa. In this study, we evaluated the potential of intranasally administered papillomavirus vaccines to elicit both systemic and vaginal immune responses. HPV-16 virus-like particles (VLPs) produced by self-assembly of L1 protein and the HPV-16 L1 gene cloned into a mammalian expression vector were used as vaccines. Intranasally administered VLPs induced serum immunoglobulin G (IgG) and vaginal IgA secretory antibodies. Very weak serum IgG and vaginal IgA responses were found after DNA immunization. Both splenic and vaginal lymphocytes could be activated by intranasal immunization with VLPs and the HPV-16 L1 gene. Activated CD4(+) Th1-like T cells were shown to synthesize gamma interferon, and activated CD8(+) T cells were demonstrated to be cytotoxic.     

Gene transfer using recombinant rabbit hemorrhagic disease virus capsids with genetically modified DNA encapsidation capacity by addition of packaging sequences from the L1 or L2 protein of human papillomavirus type 16

The aim of this study was to produce gene transfer vectors consisting of plasmid DNA packaged into virus-like particles (VLPs) with different cell tropisms. For this purpose, we have fused the N-terminally truncated VP60 capsid protein of the rabbit hemorrhagic disease virus (RHDV) with sequences which are expected to be sufficient to confer DNA packaging and gene transfer properties to the chimeric VLPs. Each of the two putative DNA-binding sequences of major L1 and minor L2 capsid proteins of human papillomavirus type 16 (HPV-16) were fused at the N terminus of the truncated VP60 protein. The two recombinant chimeric proteins expressed in insect cells self-assembled into VLPs similar in size and appearance to authentic RHDV virions. The chimeric proteins had acquired the ability to bind DNA. The two chimeric VLPs were therefore able to package plasmid DNA. However, only the chimeric VLPs containing the DNA packaging signal of the L1 protein were able efficiently to transfer genes into Cos-7 cells at a rate similar to that observed with papillomavirus L1 VLPs. It was possible to transfect only a very limited number of RK13 rabbit cells with the chimeric RHDV capsids containing the L2-binding sequence. The chimeric RHDV capsids containing the L1-binding sequence transfer genes into rabbit and hare cells at a higher rate than do HPV-16 L1 VLPs. However, no gene transfer was observed in human cell lines. The findings of this study demonstrate that the insertion of a DNA packaging sequence into a VLP which is not able to encapsidate DNA transforms this capsid into an artificial virus that could be used as a gene transfer vector. This possibility opens the way to designing new vectors with different cell tropisms by inserting such DNA packaging sequences into the major capsid proteins of other viruses.     

Do-it-yourself: construction of a custom cDNA macroarray platform with high sensitivity and linear range

Background

Human papillomavirus 16 (HPV-16) L1 protein has the capacity to self-assemble into capsomers or virus-like particles (VLPs) that are highly immunogenic, allowing their use in vaccine production. Successful expression of HPV-16 L1 protein has been reported in plants, and plant-produced VLPs have been shown to be immunogenic after administration to animals.

Results

We investigated the potential of HPV-16 L1 to act as a carrier of two foreign epitopes from Influenza A virus: (i) M2e_2-24, ectodomain of the M2 protein (M2e), that is highly conserved among all influenza A isolates, or (ii) M2e_2-9, a shorter version of M2e containing the N-terminal highly conserved epitope, that is common for both M1 and M2 influenza proteins. A synthetic HPV-16 L1 gene optimized with human codon usage was used as a backbone gene to design four chimeric sequences containing either the M2e_2-24 or the M2e_2-9 epitope in two predicted surface-exposed L1 positions. All chimeric constructs were transiently expressed in plants using the Cowpea mosaic virus-derived expression vector, pEAQ-HT. Chimeras were recognized by a panel of linear and conformation-specific anti HPV-16 L1 MAbs, and two of them also reacted with the anti-influenza MAb. Electron microscopy showed that chimeric proteins made in plants spontaneously assembled in higher order structures, such as VLPs of T = 1 or T = 7 symmetry, or capsomers.

Conclusions

In this study, we report for the first time the transient expression and the self-assembly of a chimeric HPV-16 L1 bearing the M2e influenza epitope in plants, representing also the first record of a successful expression of chimeric HPV-16 L1 carrying an epitope of a heterologous virus in plants. This study further confirms the usefulness of human papillomavirus particles as carriers of exogenous epitopes and their potential relevance for the production in plants of monovalent or multivalent vaccines.     

Th cell-independent immune responses to chimeric hemagglutinin/simian human immunodeficiency virus-like particles vaccine

CD4(+) Th cells are believed to be essential for the induction of humoral and cellular immune responses. In this study we tested the effect and possible mechanisms of the major antigenic component in influenza, hemagglutinin (HA), in helping HIV Env to induce immune responses in CD4(+) T cell knockout (CD4 KO) mice. Simian HIV virus-like particles (SHIV VLPs) or phenotypically mixed chimeric influenza HA/SHIV VLPs were used as immunogens to immunize CD4 KO mice either i.p. or intranasally (i.n.). We found that chimeric HA/SHIV VLPs significantly induced a greater IgG Ab response in both i.p. and i.n. immunized mice and a greater IgA Ab response in mucosal washes in i.n. immunized mice compared with SHIV VLPs. Importantly, chimeric HA/SHIV VLPs induced approximately 3-fold higher neutralizing Ab titers against HIV 89.6 than SHIV VLPs in the absence of CD4(+) T cell help. There was also approximately 40% more specific lysis of the HIV Env-expressing target cells in chimeric HA/SHIV VLP-immunized than in SHIV VLP-immunized CD4 KO mouse splenocytes. Moreover, we have found that chimeric HA/SHIV VLPs could efficiently bind and activate dendritic cells and stimulate the activated dendritic cells to secret TNF-alpha and IFN-gamma. Therefore, chimeric HA/SHIV VLPs could efficiently prime and activate APCs, which could, in turn, induce immune responses in a CD4(+) T cell-independent manner. This study suggests a novel adjuvant role of influenza HA as well as a new strategy to develop more effective therapeutic vaccines for AIDS patients with low CD4(+) T cell counts.     

Papillomavirus virus like particle-based therapeutic vaccine against human papillomavirus infection related diseases: immunological problems and future directions

Chronic infection with certain types of human papillomaviruses (HPV), especially HPV-16 and HPV-18, leads to the development of cervical cancer. Prophylactic HPV vaccines based on HPV virus like particles (VLPs) have now been developed. The commercial vaccines, Gardasil and Cervarix are clinically effective in preventing HPV infection but do not have a therapeutic effect against existing chronic HPV infections. However, papillomavirus (PV) VLPs elicit strong cytotoxic T cell (CTL) responses and PV VLPs without any adjuvant have therapeutic effects in animal PV infection model. Alum in Gardasil, Alum and 3-O-deacylated-4′-monophosphoryl lipid A (ASO4) in Cervarix may stimulate IL10 production and inhibit the Th1, CTL immune response of immunized individuals. PV VLPs also stimulate the production of IL10 by CD4⁺ T cells, which prevent their CTL generation effect as a therapeutic vaccine. Neutralizing IL10 at the time of PV VLPs immunization increases cytotoxic T cell responses. PV VLPs incorporating PV early protein E2, 6 and 7, together with immune stimulator that promote strong type 1 responses, and at the same time blocking the effect of IL10 may have therapeutic effect against HPV infection related diseases and are worth further basic and clinical investigation.     

Virus-like particles of SARS-like coronavirus formed by membrane proteins from different origins demonstrate stimulating activity in human dendritic cells

The pathogenesis of SARS coronavirus (CoV) remains poorly understood. In the current study, two recombinant baculovirus were generated to express the spike (S) protein of SARS-like coronavirus (SL-CoV) isolated from bats (vAcBS) and the envelope (E) and membrane (M) proteins of SARS-CoV, respectively. Co-infection of insect cells with these two recombinant baculoviruses led to self-assembly of virus-like particles (BVLPs) as demonstrated by electron microscopy. Incorporation of S protein of vAcBS (BS) into VLPs was confirmed by western blot and immunogold labeling. Such BVLPs up-regulated the level of CD40, CD80, CD86, CD83, and enhanced the secretion of IL-6, IL-10 and TNF-alpha in immature dendritic cells (DCs). Immune responses were compared in immature DCs inoculated with BVLPs or with VLPs formed by S, E and M proteins of human SARS-CoV. BVLPs showed a stronger ability to stimulate DCs in terms of cytokine induction as evidenced by 2 to 6 fold higher production of IL-6 and TNF-alpha. Further study indicated that IFN-gamma+ and IL-4+ populations in CD4+ T cells increased upon co-cultivation with DCs pre-exposed with BVLPs or SARS-CoV VLPs. The observed difference in DC-stimulating activity between BVLPs and SARS CoV VLPs was very likely due to the S protein. In agreement, SL-CoV S DNA vaccine evoked a more vigorous antibody response and a stronger T cell response than SARS-CoV S DNA in mice. Our data have demonstrated for the first time that SL-CoV VLPs formed by membrane proteins of different origins, one from SL-CoV isolated from bats (BS) and the other two from human SARS-CoV (E and M), activated immature DCs and enhanced the expression of co-stimulatory molecules and the secretion of cytokines. Finding in this study may provide important information for vaccine development as well as for understanding the pathogenesis of SARS-like CoV.     

Generation and neutralization of pseudovirions of human papillomavirus type 33.

Since human papillomaviruses (HPV) cannot be propagated in cell culture, the generation of infectious virions in vitro is a highly desirable goal. Here we report that pseudovirions can be generated by the assembly of virus-like particles (VLPs) in COS-7 cells containing multiple copies of a marker plasmid. Using recombinant vaccinia viruses, we have obtained spherical VLPs of HPV type 33 (HPV-33) which fractionate into heavy and light VLPs in cesium chloride density gradients. VLPs in the heavy fraction (1.31 g/cm3) carry the plasmid in DNase-resistant form and are capable of transferring the genetic marker located on the plasmid to COS-7 cells in a DNase-resistant way (pseudoinfection). The minor capsid protein L2 is not required for encapsidation but is essential for efficient pseudoinfection. Antiserum to HPV-33 VLPs inhibits VLP-mediated DNA transfer with high efficiency. Antisera to VLPs of HPV-18 and HPV-16 are not neutralizing, although the HPV-16 antiserum exhibited some cross-reactivity with HPV-33 VLPs in an enzyme-linked immunosorbent assay. In a cell binding assay, the titer of the HPV-33 VLP antiserum was 1:200 compared to the neutralization titer of 1:10(5). This indicates that neutralization is essentially due to the inhibition of cellular processes after VLP binding to cells. The encapsidation of marker plasmids into VLPs provides a sensitive and fast assay for the evaluation of neutralizing potentials of antisera against papillomavirus infections.     

Characterization of a major neutralizing epitope on human papillomavirus type 16 L1

Persistent infection with human papillomavirus type 16 (HPV-16) is strongly associated with the development of cervical cancer. Neutralizing epitopes present on the major coat protein, L1, have not been well characterized, although three neutralizing monoclonal antibodies (MAbs) had been identified by using HPV-16 pseudovirions (R. B. Roden et al., J. Virol. 71:6247-6252, 1997). Here, two of these MAbs (H16.V5 and H16.E70) were demonstrated to neutralize authentic HPV-16 in vitro, while the third (H16.U4) did not. Binding studies were conducted with the three MAbs and virus-like particles (VLPs) composed of the reference L1 sequence (114K) and three variant L1 sequences: Rochester-1k (derived from viral stock DNA), GU-1 (derived from cervical biopsy DNA), and GU-2 (derived from biopsy DNA, but containing some sequence changes likely to be artifactual). While all three MAbs bound to 114K and Rochester-1k VLPs, GU-1 VLPs were not recognized by H16.E70, and both H16.E70 and H16.V5 failed to bind to GU-2 VLPs. Site-directed mutagenesis was used to replace disparate amino acids in the GU-2 L1 with those found in the 114K L1. Alteration of the amino acid at position 50, from L to F, completely restored H16.V5 binding and partially restored H16.E70 binding, while complete restoration of H16.E70 binding occurred with GU-2 VLPs containing both L50F and T266A alterations. Immunization of mice with L1 variant VLPs revealed that GU-2 VLPs were poorly immunogenic. The L50F mutant of GU-2 L1, in which the H16.V5 epitope was restored, elicited HPV-16 antibody responses comparable to those obtained with 114K VLPs. These results demonstrate the importance of the H16.V5 epitope in the generation of potent HPV-16 neutralizing antibody responses.     

Papillomavirus-like particles induce acute activation of dendritic cells

The role of viral structural proteins in the initiation of adaptive immune responses is poorly understood. To address this issue, we focused on the effect of noninfectious papillomavirus-like particles (VLPs) on dendritic cell (DC) activation. We found that murine bone marrow-derived dendritic cells (BMDCs) effectively bound and rapidly internalized bovine papillomavirus VLPS: Exposure to fully assembled VLPs of bovine papillomavirus, human papillomavirus (HPV)16 or HPV18, but not to predominately disordered HPV16 capsomers, induced acute phenotypic maturation of BMDCS: Structurally similar polyomavirus VLPs bound to the DC surface and were internalized, but failed to induce maturation. DCs that had incorporated HPV16 VLPs produced proinflammatory cytokines IL-6 and TNF-alpha; however, the release of these cytokines was delayed relative to LPS activation. Production of IL-12p70 by VLP-exposed DCs required the addition of syngeneic T cells or rIFN-gamma. Finally, BMDCs pulsed with HPV16 VLPs induced Th1-dominated primary T cell responses in vitro. Our data provide evidence that DCs respond to intact papillomavirus capsids and that they play a central role in VLP-induced immunity. These results offer a mechanistic explanation for the striking ability of papillomavirus VLP-based vaccines to induce potent T and B cell responses even in the absence of adjuvant.     

Yeast-derived human immunodeficiency virus type 1 p55(gag) virus-like particles activate dendritic cells (DCs) and induce perforin expression in Gag-specific CD8(+) T cells by cross-presentation of DCs

To evaluate the immunogenicity of human immunodeficiency virus (HIV) type 1 p55(gag) virus-like particles (VLPs) released by budding from yeast spheroplasts, we have analyzed the effects of yeast VLPs on monocyte-derived dendritic cells (DCs). Yeast VLPs were efficiently incorporated into DCs via both macropinocytosis and endocytosis mediated by mannose-recognizing receptors, but not the mannose receptor. The uptake of yeast VLPs induced DC maturation and enhanced cytokine production, notably, interleukin-12 p70. We showed that yeast membrane components may contribute to DC maturation partly through Toll-like receptor 2 signaling. Thus, Gag particles encapsulated by yeast membrane may have an advantage in stimulating Gag-specific immune responses. We found that yeast VLPs, but not the control yeast membrane fraction, were able to activate both CD4(+) and CD8(+) T cells of HIV-infected individuals. We tested the effect of cross-presentation of VLP by DCs in two subjects recruited into a long-term nonprogressor-slow progressor cohort. When yeast VLP-loaded DCs of these patients were cocultured with peripheral blood mononuclear cells for 7 days, approximately one-third of the Gag-specific CD8(+) T cells were activated and became perforin positive. However, some of the Gag-specific CD8(+) T cells appeared to be lost during in vitro culture, especially in a patient with a high virus load. Our results suggest that DCs loaded with yeast VLPs can activate Gag-specific memory CD8(+) T cells to become effector cells in chronically HIV-infected individuals, but there still remain unresponsive Gag-specific T-cell populations in these patients.     

Human Papillomavirus Type 16 (HPV-16) Virus-Like Particle L1-Specific CD8+ Cytotoxic T Lymphocytes (CTLs) Are Equally Effective as E7-Specific CD8+ CTLs in Killing Autologous HPV-16-Positive Tumor Cells in Cervical Cancer Patients: Implications for L1 Dendritic Cell-Based Therapeutic Vaccines

Papillomavirus-like particles (VLPs) based on L1 capsid protein represent a promising prophylactic vaccine against human papillomavirus (HPV) infections. However, cell-mediated immune responses against this antigen are believed to be of limited therapeutic value in established HPV-infected cervical lesions and, for this reason, have not been intensively investigated in cervical cancer patients. In this study we analyzed and quantified by real-time PCR (RT-PCR) the RNA expression levels of E6, E7, and L1 genes in flash-frozen HPV-16 cervical carcinomas. In addition, the kinetics of expression of E6, E7, and L1 in HPV-16-infected primary cell lines established as long-term cultures in vitro was also evaluated at RNA and protein levels. Finally, in order to evaluate the therapeutic potential of L1-specific CD4⁺ and CD8⁺ T lymphocytes responses in cervical cancer patients, L1 VLP-loaded dendritic cells (DCs) were used to stimulate peripheral blood lymphocytes from cervical cancer patients and such responses were compared to those elicited by the E7 oncoprotein. We show that 22 of 22 (100%) flash-frozen cervical biopsy samples collected from HPV-16-positive cervical cancer patients harbor L1, in addition to E6 and E7 RNA, as detected by RT-PCR. E7 RNA copy number (mean, 176.2) was significantly higher in HPV-16-positive cervical cancers compared to the E6 RNA copy number (mean, 47.3) and the L1 copy number (mean, 58.3) (P < 0.0001 and P < 0.001, respectively). However, no significant differences in expression levels between E6 and L1 were found. Kinetic studies of E6, E7, and L1 RNA and protein expression levels in primary tumors showed a sharp reduction in L1 expression after multiple in vitro passages compared to E6 and E7. Autologous DCs pulsed with HPV-16 VLPs or recombinant full-length E7 elicited strong type 1 L1- and E7-specific responses in CD4⁺ and CD8⁺ T cells from cervical cancer patients. Importantly, L1 VLP-specific CD8⁺ T lymphocytes expressed strong cytolytic activity against autologous tumor cells and were as effective as E7-specific cytotoxic T lymphocytes in lysing naturally HPV-16-infected autologous tumor cells. Taken together, these data demonstrate a consistent expression of L1 in primary cervical tumors and the possibility of inducing effective L1/tumor-specific CD4⁺ and CD8⁺ T-lymphocyte responses in patients harboring HPV-infected cervical cancer. These results may have important implications for the treatment of patients harboring established HPV-infected lesions with L1 VLPs or combined E7/L1 DC-based vaccinations.Human papillomavirus (HPV) infection represents the most important risk factor for the development of cervical cancer. Although more than 100 distinct HPV genotypes have been described, and at least 20 are associated with cervical cancer, HPV type 16 (HPV-16) is by far the most frequently detected in cervical neoplasia regardless of the geographical origin of the patients (4). In the last few years significant advances have been made in the development of candidate prophylactic vaccine against cervical cancer and HPV-related infections. In several large prospective randomized studies, virus-like particles consisting of the HPV-16 and HPV-18 major capsid protein L1 (L1-VLPs) have shown promise in protecting young healthy females against persistent infection with HPV-16 and HPV-18 and their associated cervical intraepithelial neoplasia (reviewed in reference 12). These data strongly suggest that the implementation of large-scale L1-VLP-based prophylactic vaccinations have the potential to dramatically reduce worldwide cervical cancer rates in the years to come.Unfortunately, because HPV infection is endemic in humans and there is a long latency from HPV infection to the development of invasive cervical cancer in women, even if prophylactic L1-based vaccinations are implemented on a worldwide scale today it would take decades to perceive any significant benefit. Consistent with this view, an estimated 5 million cervical cancer deaths will occur in the next 20 years due to existing HPV infections (4, 12). Thus, the current development of therapeutic vaccines for protection against persistent HPV infections, cervical cancer, and its precursor lesions remains an area of great interest.Although the interactions between the host immune system and HPV-infected cells are still not completely understood, several lines of evidence suggest that protection against HPV-related infections by L1-VLP-based vaccines is likely conferred by the generation of high levels of neutralizing antibodies (12, 38). Nevertheless, a potential crucial role of L1-specific T-cell responses and the involvement of T cells in mediating the production of neutralizing antibodies and antiviral effect in infected hosts has been previously hypothesized (8, 24). This point may be particularly noteworthy in patients harboring HPV-infected cervical lesions because several studies have demonstrated the critical importance of both cytotoxic (CD8⁺) and helper (CD4⁺) T cells in achieving clinical responses (1, 5, 16-18, 20, 23). However, limited information is currently available to evaluate whether cell-mediated immune responses to L1-VLP may have any significant therapeutic effect in cervical cancer patients harboring HPV-16 positive tumors. Furthermore, to our knowledge, no direct comparison of the therapeutic efficacy of L1 and E7-specific immune responses against naturally HPV-16-infected cervical cancer have been yet reported in human patients.In the present study we have analyzed and quantified by highly sensitive real-time PCR (RT-PCR) the RNA levels of E6, E7, and L1 in flash-frozen biopsy specimens obtained from HPV-16-infected cervical carcinomas and in short- and long-term primary cultures of HPV-16-positive cervical tumors. In addition, we have studied the kinetics of expression of these genes and proteins during the establishment of HPV-16-positive primary tumors in vitro. Finally, using completely autologous systems of naturally infected HPV-16-positive human tumors, we have carefully studied the phenotype and function of L1-specific CD4⁺ and CD8⁺ T-lymphocyte responses generated by VLP-loaded dendritic cells (DCs) and compared their therapeutic potential to those elicited by DC loaded with the E7 oncoprotein.     

Enhanced effector and memory CTL responses generated by incorporation of receptor activator of NF-kappa B (RANK)/RANK ligand costimulatory molecules into dendritic cell immunogens expressing a human tumor-specific antigen

The outcome of dendritic cell (DC) presentation of Ag to T cells via the TCR/MHC synapse is determined by second signaling through CD80/86 and, importantly, by ligation of costimulatory ligands and receptors located at the DC and T cell surfaces. Downstream signaling triggered by costimulatory molecule ligation results in reciprocal DC and T cell activation and survival, which predisposes to enhanced T cell-mediated immune responses. In this study, we used adenoviral vectors to express a model tumor Ag (the E7 oncoprotein of human papillomavirus 16) with or without coexpression of receptor activator of NF-kappaB (RANK)/RANK ligand (RANKL) or CD40/CD40L costimulatory molecules, and used these transgenic DCs to immunize mice for the generation of E7-directed CD8(+) T cell responses. We show that coexpression of RANK/RANKL, but not CD40/CD40L, in E7-expressing DCs augmented E7-specific IFN-gamma-secreting effector and memory T cells and E7-specific CTLs. These responses were also augmented by coexpression of T cell costimulatory molecules (RANKL and CD40L) or DC costimulatory molecules (RANK and CD40) in the E7-expressing DC immunogens. Augmentation of CTL responses correlated with up-regulation of CD80 and CD86 expression in DCs transduced with costimulatory molecules, suggesting a mechanism for enhanced T cell activation/survival. These results have generic implications for improved tumor Ag-expressing DC vaccines, and specific implications for a DC-based vaccine approach for human papillomavirus 16-associated cervical carcinoma.     

RNA-containing adenovirus/polyethylenimine transfer complexes effectively transduce dendritic cells and induce antigen-specific T cell responses

BACKGROUND: Dendritic cells (DCs) are the most potent antigen-presenting cells in initiating primary immune responses. Given the unique properties of DCs, gene-modified DCs represent a particularly attractive approach for immunotherapy of diseases such as cancer. METHODS: Gene-modified DCs were obtained by a receptor-mediated gene delivery system using adenovirus (Ad) particles as ligand and RNA or DNA condensed by polyethylenimine (PEI). In vitro transcribed polyadenylated or non-polyadenylated RNA was used. RNA-transduced DCs were generated expressing chicken ovalbumin (OVA) or chimeric constructs thereof, and compared with DNA-transduced DCs. RESULTS: Ad/PEI transfection complexes efficiently delivered RNA into DCs. Such RNA-transduced DCs induced OVA-specific T cell responses more effectively than DNA-transduced DCs. Furthermore, DCs transduced with polyadenylated RNA were more potent in stimulating CD4(+) and CD8(+) T cell responses than DCs transduced with non-polyadenylated RNA and this was particularly important for CD4(+) T cell responses. CONCLUSIONS: Ad/PEI/RNA transfection is an efficient means for generating RNA-transduced DCs and for stimulating antigen-specific T cell responses. Polyadenylation of RNA enhances CD8(+) T cell responses and is essential for CD4(+) T cell responses.     

Requirement of mature dendritic cells for efficient activation of influenza A-specific memory CD8+ T cells

It is critical to identify the developmental stage of dendritic cells (DCs) that is most efficient at inducing CD8+ T cell responses. Immature DCs can be generated from monocytes with GM-CSF and IL-4, while maturation is accomplished by the addition of stimuli such as monocyte-conditioned medium, CD40 ligand, and LPS. We evaluated the ability of human monocytes and immature and mature DCs to induce CD8+ effector responses to influenza virus Ags from resting memory cells. We studied replicating virus, nonreplicating virus, and the HLA-A*0201-restricted influenza matrix protein peptide. Sensitive and quantitative assays were used to measure influenza A-specific immune responses, including MHC class I tetramer binding assays, enzyme-linked immunospot assays for IFN-gamma production, and generation of cytotoxic T cells. Mature DCs were demonstrated to be superior to immature DC in eliciting IFN-gamma production from CD8+ effector cells. Furthermore, only mature DCs, not immature DCs, could expand and differentiate CTL precursors into cytotoxic effector cells over 7 days. An exception to this was immature DCs infected with live influenza virus, because of the virus's known maturation effect. Finally, mature DCs pulsed with matrix peptide induced CTLs from highly purified CD8+ T cells without requiring CD4+ T cell help. These differences between DC stages were independent of Ag concentrations or the number of immature DCs. In contrast to DCs, monocytes were markedly inferior or completely ineffective stimulators of T cell immunity. Our data with several qualitatively different assays of the memory CD8+ T cell response suggest that mature cells should be considered as immunotherapeutic adjuvants for Ag delivery.     

Two Listeria monocytogenes vaccine vectors that express different molecular forms of human papilloma virus-16 (HPV-16) E7 induce qualitatively different T cell immunity that correlates with their ability to induce regression of established tumors immortalized by HPV-16.

Two recombinant Listeria monocytogenes (rLm) strains were produced that secrete the human papilloma virus-16 (HPV-16) E7 protein expressed in HPV-16-associated cervical cancer cells. One, Lm-E7, expresses and secretes E7 protein, whereas a second, Lm-LLO-E7, secretes E7 as a fusion protein joined to a nonhemolytic listeriolysin O (LLO). Lm-LLO-E7, but not Lm-E7, induces the regression of the E7-expressing tumor, TC-1, established in syngeneic C57BL/6 mice. Both recombinant E7-expressing rLm vaccines induce measurable anti-E7 CTL responses that stain positively for H-2D(b) E7 tetramers. Depletion of the CD8+ T cell subset before treatment abrogates the ability of Lm-LLO-E7 to impact on tumor growth. In addition, the rLm strains induce markedly different CD4+ T cell subsets. Depletion of the CD4+ T cell subset considerably reduces the ability of Lm-LLO-E7 to eliminate established TC-1 tumors. Surprisingly, the reverse is the case for Lm-E7, which becomes an effective anti-tumor immunotherapeutic in mice lacking this T cell subset. Ab-mediated depletion of TGF-beta and CD25+ cells improves the effectiveness of Lm-E7 treatment, suggesting that TGF-beta and CD25+ cells are in part responsible for this suppressive response. CD4+ T cells from mice immunized with Lm-E7 are capable of suppressing the ability of Lm-LLO-E7 to induce the regression of TC-1 when transferred to tumor-bearing mice. These studies demonstrate the complexity of L. monocytogenes-mediated tumor immunotherapy targeting the human tumor Ag, HPV-16 E7.     

Enterovirus-71 Virus-Like Particles Induce the Activation and Maturation of Human Monocyte-Derived Dendritic Cells through TLR4 Signaling

Enterovirus 71 (EV71) causes seasonal epidemics of hand-foot-and-mouth disease and has a high mortality rate among young children. We recently demonstrated potent induction of the humoral and cell-mediated immune response in monkeys immunized with EV71 virus-like particles (VLPs), with a morphology resembling that of infectious EV71 virions but not containing a viral genome, which could potentially be safe as a vaccine for EV71. To elucidate the mechanisms through which EV71 VLPs induce cell-mediated immunity, we studied the immunomodulatory effects of EV71 VLPs on human monocyte-derived dendritic cells (DCs), which bind to and incorporate EV71 VLPs. DC treatment with EV71 VLPs enhanced the expression of CD80, CD86, CD83, CD40, CD54, and HLA-DR on the cell surface; increased the production of interleukin (IL)-12 p40, IL-12 p70, and IL-10 by DCs; and suppressed the capacity of DCs for endocytosis. Treatment with EV71 VLPs also enhanced the ability of DCs to stimulate naïve T cells and induced secretion of interferon (IFN)-γ by T cells and Th1 cell responses. Neutralization with antibodies against Toll-like receptor (TLR) 4 suppressed the capacity of EV71 VLPs to induce the production of IL-12 p40, IL-12 p70, and IL-10 by DCs and inhibited EV71 VLPs binding to DCs. Our study findings clarified the important role for TLR4 signaling in DCs in response to EV71 VLPs and showed that EV71 VLPs induced inhibitor of kappaB alpha (IκBα) degradation and nuclear factor of kappaB (NF-κB) activation.     

An antigen chimera of poliovirus induces antibodies against human papillomavirus type 16.

It has been established that the surface of poliovirus type 1 can be extensively modified to incorporate antigenic domains from other poliovirus serotypes and from unrelated viruses. The fact that the modified (chimeric) viruses exhibit dual antigenicity and immunogenicity led us to explore the possibility of using the Sabin vaccine strain of poliovirus type 1 as a vector for the presentation of antigenic domains from human papillomavirus type 16 (HPV-16), a virus associated with the development of cervical carcinoma. We report here the construction and characterization of a chimeric poliovirus containing a 16-residue sequence derived from the major capsid protein (L1) of HPV-16. This virus chimera stimulated the production in rabbits of antibodies which recognized the HPV-16-derived peptide and an L1 fusion protein synthesized in Escherichia coli and detected HPV-16 in human biopsy material by immunoperoxidase staining. The possibility that poliovirus-HPV chimeras could be used as vaccines against HPV-16 is discussed.