Pulmonary infection of mice with human metapneumovirus induces local cytotoxic T-cell and immunoregulatory cytokine responses similar to those seen with human respiratory syncytial virus.

Human metapneumovirus (hMPV) is a major cause of upper and lower respiratory-tract infection in infants, the elderly and immunocompromised individuals. Virus-directed cellular immunity elicited by hMPV infection is poorly understood, in contrast to the phylogenetically and clinically related pathogen human respiratory syncytial virus (hRSV). In a murine model of acute lower respiratory-tract infection with hMPV, we demonstrate the accumulation of gamma interferon (IFN-gamma)-producing CD8+ T cells in the airways and lungs at day 7 post-infection (p.i.), associated with cytotoxic T lymphocytes (CTLs) directed to an epitope of the M2-1 protein. This CTL immunity was accompanied by increased pulmonary expression of Th1 cytokines IFN-gamma and interleukin (IL)-12 and antiviral cytokines (IFN-beta), as well as chemokines Mip-1alpha, Mip-1beta, Mig, IP-10 and CX3CL1. There was also a moderate increase in Th2-type cytokines IL-4 and IL-10 compared with uninfected mice. At 21 days p.i., a strong CTL response could be recalled from the spleen. A similar pattern of CTL induction to the homologous M2-1 CTL epitope of hRSV, and of cytokine/chemokine induction, was observed following infection with hRSV, highlighting similarities in the cellular immune response to the two related pathogens.

HBsAg-vectored vaccines simultaneously deliver CTL responses to protective epitopes from multiple viral pathogens.

We have previously demonstrated that the potent immunogenicity of hepatitis B surface antigen (HBsAg) may be exploited to deliver foreign antigens for cytotoxic T-lymphocyte (CTL) induction. Here we demonstrate that a single low-dose immunization with rHBsAg DNA is sufficient to prime for CTL responses against encoded foreign epitope and that the responses may be recalled many months after immunization. We show that simultaneous disease-protective CTL responses restricted through a diversity of MHC class I haplotypes are elicited by recombinant (r) HBsAg DNA containing multiple viral epitopes appended as a C'-terminal polyepitope or encoded individually within the HBsAg polypeptide. CTL responses delivered by rHBsAg DNA were elicited in the presence of HBsAg-directed antibody. These studies vindicate the use of HBsAg as a powerful vector to deliver CTL responses to foreign antigen and have implications for a multidisease vaccine applicable to an MHC-polymorphic population.

Both treated and untreated tumors are eliminated by short hairpin RNA-based induction of target-specific immune responses.

RNA interference (RNAi) for cancer treatment relies on the ability to directly kill cancer cells via down-regulation of target genes, but issues of delivery and efficacy have limited clinical adoption. Furthermore, current studies using immune-deficient animal models disregard potential interactions with the adaptive immune system. It has previously been observed that certain viral antigens appear to be more rapidly presented to the immune system than normal proteins due to the production of defective ribosomal products by the virus. Given that RNAi could potentially result in the generation of truncated mRNAs, we wondered whether a similar mechanism of immune presentation of a target gene was possible. Here we show that RNAi-cleaved mRNAs can be translated into incomplete protein, and if cleavage was downstream of cytotoxic T cell epitopes, resulted in increased presentation of target protein and the generation of a tumor-protective immune response. We show that mice inoculated with tumor cells treated with such short hairpin RNAs (shRNAs) were protected from subsequent challenge with untreated tumors. However, protection was only found if shRNAs were targeted downstream of the dominant cytotoxic T cell (CTL) epitope. Our work suggests that RNAi can alter immunity to targets and shows that not all tumor cells require direct RNAi exposure for treatment to be effective in vivo, pointing the way to a new class of RNAi-based therapy.

Major histocompatibility complex class I cytotoxic T lymphocyte immunity to human metapneumovirus (hMPV) in individuals with previous hMPV infection and respiratory disease.

Recently identified human metapneumovirus (hMPV) is an important respiratory pathogen in children and adults worldwide. Little is known about cytotoxic T lymphocyte (CTL) responses that may control hMPV infection in humans. To address this, we evaluated major histocompatibility complex (MHC) class I T cell immunity in 7 patients with previous hMPV respiratory disease. CTL responses were evident in most patients and to most proteins of hMPV. Individual patients had responses to at least 2 hMPV proteins (particularly the M protein) and had multiallele responses. In addition, we identified 9 CTL epitopes that are presented by human leukocyte antigen alleles of the most common MHC "supertypes." Many of these CTL epitopes are conserved across hMPV types, and there is epitope similarity between hMPV and human respiratory syncytial virus. This study provides the first report of MHC class I T cell immunity to hMPV in humans. These findings have significance for understanding cellular immunity to hMPV infection and for future vaccine development.

Multiple copies of a tumor epitope in a recombinant hepatitis B surface antigen (HBsAg) vaccine enhance CTL responses, but not tumor protection.

We propose the replacement of endogenous epitopes with foreign epitopes to exploit the highly immunogenic hepatitis B surface antigen (HBsAg) as a vaccine vector to elicit disease-protective cytotoxic T-lymphocyte (CTL) responses. Locations were defined within the HBsAg gene where replacements of DNA encoding HBsAg epitopes may be made to generate functional recombinant (r) HBsAg DNA vaccines. We demonstrate that rHBsAg DNA vaccines encoding multiple copies of a model tumor epitope from human papillomavirus (HPV) elicit enhanced CTL responses compared to rHBsAg DNA vaccines encoding a single copy. We show that rHBsAg DNA vaccines elicit a marked prophylactic and long-lived therapeutic protection against epitope expressing tumor, although protective efficacy was not improved by increasing the number of copies of the tumor epitope DNA. These results demonstrate the efficacy of HBsAg as a vector for the delivery of foreign CTL epitopes using the epitope replacement strategy, and have implications for rHBsAg vaccine design. The results also have implications for the derivation of a therapeutic vaccine for HPV-associated squamous carcinoma.

Co-immunisation with DNA encoding RANK/RANKL or 4-1BBL costimulatory molecules does not enhance effector or memory CTL responses afforded by immunisation with a tumour antigen-encoding DNA vaccine.

T cell mediated immune responses are induced following interaction of MHC-presented epitope on professional antigen presenting cells such as dendritic cells (DCs) with cognate T cell receptor. Up-regulation of receptor-ligand pairs of costimulatory molecules linking DC to T cell enhances the resulting T cell responses. This 'second signalling' occurs through the B7 molecules CD80/86 expressed by DCs, and importantly through members of the TNF ligand/TNF receptor superfamilies. We have previously shown that co-expression of RANK/RANKL or 41BB-L in addition to tumour antigen in dendritic cells augmented cognate effector and memory tumour antigen-directed cytotoxic T cell responses when the DCs were used to immunise mice. Here, we examined whether co-immunisation with naked plasmid DNAs encoding antigen and these costimulatory molecule(s), would enhance antigen specific T cell responses. We demonstrate that co-immunisation with DNAs encoding tumour antigen and costimulatory molecules failed to enhance antigen-directed CTL responses, or tumour protection, afforded by immunisation with DNA encoding tumour antigen alone.

Hepatitis B surface antigen vector delivers protective cytotoxic T-lymphocyte responses to disease-relevant foreign epitopes.

Although hepatitis B surface antigen (HBsAg) per se is highly immunogenic, its use as a vector for the delivery of foreign cytotoxic T-lymphocyte (CTL) epitopes has met with little success because of constraints on HBsAg stability and secretion imposed by the insertion of foreign sequence into critical hydrophobic/amphipathic regions. Using a strategy entailing deletion of DNA encoding HBsAg-specific CTL epitopes and replacement with DNA encoding foreign CTL epitopes, we have derived chimeric HBsAg DNA immunogens which elicited effector and memory CTL responses in vitro, and pathogen- and tumor-protective responses in vivo, when the chimeric HBsAg DNAs were used to immunize mice. We further show that HBsAg DNA recombinant for both respiratory syncytial virus and human papillomavirus CTL epitopes elicited simultaneous responses to both pathogens. These data demonstrate the efficacy of HBsAg DNA as a vector for the delivery of disease-relevant protective CTL responses. They also suggest the applicability of the approach of deriving chimeric HBsAg DNA immunogens simultaneously encoding protective CTL epitopes for multiple diseases. The DNAs we tested formed chimeric HBsAg virus-like particles (VLPs). Thus, our results have implications for the development of vaccination strategies using either chimeric HBsAg DNA or VLP vaccines. HBsAg is the globally administered vaccine for hepatitis B virus infection, inviting its usage as a vector for the delivery of immunogens from other diseases.

Cytotoxic T-lymphocyte epitope vaccination protects against human metapneumovirus infection and disease in mice.

Human metapneumovirus (hMPV) has emerged as an important human respiratory pathogen causing upper and lower respiratory tract infections in young children and older adults. In addition, hMPV infection is associated with asthma exacerbation in young children. Recent epidemiological evidence indicates that hMPV may cocirculate with human respiratory syncytial virus (hRSV) and mediate clinical disease similar to that seen with hRSV. Therefore, a vaccine for hMPV is highly desirable. In the present study, we used predictive bioinformatics, peptide immunization, and functional T-cell assays to define hMPV cytotoxic T-lymphocyte (CTL) epitopes recognized by mouse T cells restricted through several major histocompatibility complex class I alleles, including HLA-A*0201. We demonstrate that peptide immunization with hMPV CTL epitopes reduces viral load and immunopathology in the lungs of hMPV-challenged mice and enhances the expression of Th1-type cytokines (gamma interferon and interleukin-12 [IL-12]) in lungs and regional lymph nodes. In addition, we show that levels of Th2-type cytokines (IL-10 and IL-4) are significantly lower in hMPV CTL epitope-vaccinated mice challenged with hMPV. These results demonstrate for the first time the efficacy of an hMPV CTL epitope vaccine in the control of hMPV infection in a murine model.

A polytope DNA vaccine elicits multiple effector and memory CTL responses and protects against human papillomavirus 16 E7-expressing tumour.

Vaccine-induced CD8 T cells directed to tumour-specific antigens are recognised as important components of protective and therapeutic immunity against tumours. Where tumour antigens have pathogenic potential or where immunogenic epitopes are lost from tumours, development of subunit vaccines consisting of multiple individual epitopes is an attractive alternative to immunising with whole tumour antigen. In the present study we investigate the efficacy of two DNA-based multiepitope ('polytope') vaccines containing murine (H-2b) and human (HLA-A*0201)-restricted epitopes of the E7 oncoprotein of human papillomavirus type 16, in eliciting tumour-protective cytotoxic T-lymphocyte (CTL) responses. We show that the first of these polytopes elicited powerful effector CTL responses (measured by IFN-gamma ELISpot) and long-lived memory CTL responses (measured by functional CTL assay and tetramers) in immunised mice. The responses could be boosted by immunisation with a recombinant vaccinia virus expressing the polytope. Responses induced by immunisation with polytope DNA alone partially protected against infection with recombinant vaccinia virus expressing the polytope. Complete protection was afforded against challenge with an E7-expressing tumour, and reduced growth of nascent tumours was observed. A second polytope differing in the exact composition and order of CTL epitopes, and lacking an inserted endoplasmic reticulum targeting sequence and T-helper epitope, induced much poorer CTL responses and failed to protect against tumour challenge. These observations indicate the validity of a DNA polytope vaccine approach to human papillomavirus E7-associated carcinoma, and underscore the importance of design in polytope vaccine construction.

Recombinant Kunjin virus replicon vaccines induce protective T-cell immunity against human papillomavirus 16 E7-expressing tumour.

The persistence of the E7 oncoprotein in transformed cells in human papillomavirus (HPV)-associated cervical cancer provides a tumour-specific antigen to which immunotherapeutic strategies may be directed. Self-replicating RNA (replicon) vaccine vectors derived from the flavivirus Kunjin (KUN) have recently been reported to induce T-cell immunity. Here, we report that inclusion of a CTL epitope of HPV16 E7 protein into a polyepitope encoded by a KUN vector induced E7-directed T-cell responses and protected mice against challenge with an E7-expressing epithelial tumour. We found replicon RNA packaged into virus-like particles to be more effective than naked replicon RNA or plasmid DNA constructed to allow replicon RNA transcription in vivo. Protective immunity was induced although the E7 CTL epitope was subdominant in the context of other CTL epitopes in the polyepitope. The results demonstrate the efficacy of the KUN replicon vector system for inducing protective immunity directed towards a virally encoded human tumour-specific antigen, and for inducing multi-epitopic CTL responses.

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.

Immunogenicity of recombinant HBsAg/HCV particles in mice pre-immunised with hepatitis B virus-specific vaccine.

Due to their spatial structure virus-like particles (VLPs) generally induce effective immune responses. VLPs derived from the small envelope protein (HBsAg-S) of hepatitis B virus (HBV) comprise the HBV vaccine. Modified HBsAs-S VLPs, carrying the immunodominant hypervariable region (HVR1) of the hepatitis C virus (HCV) envelope protein E2 within the exposed 'a'-determinant region (HBsAg/HVR1-VLPs), elicited HVR1-specific antibodies in mice. A high percentage of the human population is positive for anti-HBsAg antibodies (anti-HBs), either through vaccination or natural infection. We, therefore, determined if pre-existing anti-HBs could influence immunisation with modified VLPs. Mice were immunised with a commercial HBV vaccine, monitored to ensure an anti-HBs response, then immunised with HBsAg/HVR1-VLPs. The resulting anti-HVR1 antibody titre was similar in mice with or without pre-existing anti-HBs. This suggests that HBsAg/HVR1-VLPs induce a primary immune response to HVR1 in anti-HBs positive mice and, hence, they may be used successfully in individuals already immunised with the HBV vaccine.

Provision of 4-1BB ligand enhances effector and memory CTL responses generated by immunization with dendritic cells expressing a human tumor-associated antigen.

Up-regulation of receptor-ligand pairs during interaction of an MHC-presented epitope on dendritic cells (DCs) with cognate TCR may amplify, sustain, and drive diversity in the ensuing T cell immune response. Members of the TNF ligand superfamily and the TNFR superfamily contribute to this costimulatory molecule signaling. In this study, we used replication deficient adenoviruses to introduce a model tumor-associated Ag (the E7 oncoprotein of human papillomavirus 16) and the T cell costimulatory molecule 4-1BBL into murine DCs, and monitored the ability of these recombinant DCs to elicit E7-directed T cell responses following immunization. Splenocytes from mice immunized with DCs expressing E7 alone elicited E7-directed effector and memory CTL responses. Coexpression of 4-1BBL in these E7-expressing DCs increased effector and memory CTL responses when they were used for immunization. 4-1BBL expression up-regulated CD80 and CD86 second signaling molecules in DCs. We also report an additive effect of 4-1BBL and receptor activator of NF-kappa B/receptor activator of NF-kappa B ligand coexpression in E7-transduced DC immunogens on E7-directed effector and memory CTL responses and on MHC class II and CD80/86 expression in DCs. Additionally, expression of 4-1BBL in E7-transduced DCs reduced nonspecific T cell activation characteristic of adenovirus vector-associated immunization. The results have generic implications for improved or tumor Ag-expressing DC vaccines by incorporation of exogenous 4-1BBL. There are also specific implications for an improved DC-based vaccine for human papillomavirus 16-associated cervical carcinoma.

Paucity of functional CTL epitopes in the E7 oncoprotein of cervical cancer associated human papillomavirus type 16.

Many specific antiviral and antitumour immune responses have been attributed to the protective effects of antigen-specific CD8+ cytotoxic T lymphocytes (CTL). Recognition of virus infected or tumour cells by CTL requires presentation of at least one peptide epitope from a virus or tumour-specific antigen by the relevant MHC Class I molecule. Viral genes with mutations which remove CTL epitopes may thus be favoured for survival. Human cervical cancers are caused by papillomavirus infection, and these cancers consistently express the E7 protein of the oncogenic papillomavirus. We therefore investigated the MHC Class I restricted T cell epitopes of the human papillomavirus type 16 E7 oncoprotein using mice of five different genetic backgrounds, and an IFN-gamma ELISPOT assay, to determine the frequency with which MHC Class I epitopes might be expected in this small oncoprotein (98 amino acids). No MHC Class I restricted responses were detected in E7 immunized BALB/c (H-2d), CBA/CaH (H-2 k), FVB/N (H-2q) or A2KbH2b human HLA2.1 transgenic mice. In C57BL/6 J (H-2b) mice, a previously identified single antigenic epitope was detected. Therefore, we conclude that there is a paucity of MHC Class I restricted T cell epitopes in HPV16 E7 protein because of its small size. This might be advantageous to the virus. Furthermore here we present a quick and easy method to exhaustively determine CD8 T cell epitopes in proteins using a unique set of overlapping 8, 9 and 10 mer synthetic peptides.

Generation of CTL responses using Kunjin replicon RNA.

The Kunjin replicon was used to express a polytope that consisted of seven hepatitis C virus cytotoxic T lymphocyte epitopes and one influenza cytotoxic T lymphocyte epitope for vaccination studies. The self-replicating nature of, and expression from, the ribonucleic acid was confirmed in vitro. Initial vaccinations with one dose of Kun-Poly ribonucleic acid showed that an influenza-specific cytotoxic T lymphocyte response was elicited more efficiently by intradermal inoculation compared with intramuscular delivery. Two micrograms of ribonucleic acid delivered in the ear pinnae of mice was sufficient to elicit a detectable cytotoxic T lymphocyte response 10 days post-vaccination. Further vaccination studies showed that four of the seven hepatitis C virus cytotoxic T lymphocyte epitopes were able to elicit weak cytotoxic T lymphocyte responses whereas the influenza epitope was able to elicit strong, specific cytotoxic T lymphocyte responses following three doses of Kun-Poly ribonucleic acid. These studies vindicate the use of the Kunjin replicon as a vector to deliver encoded proteins for the development of cell-mediated immune responses.

Limitations of HLA-transgenic mice in presentation of HLA-restricted cytotoxic T-cell epitopes from endogenously processed human papillomavirus type 16 E7 protein.

We investigated the use of mice transgenic for human leucocyte antigen (HLA) A*0201 antigen-binding domains to test vaccines composed of defined HLA A*0201-restricted cytotoxic T-lymphocyte (CTL) epitopes of human papillomavirus (HPV) type 16 E7 oncoprotein. HPV is detected in >90% of cervical carcinomas. HPV16 E7 oncoprotein transforms cells of the uterine cervix and functions as a tumour-associated antigen to which immunotherapeutic strategies may be directed. We report that although the HLA A*0201 E7 epitope peptides function both to prime for E7 CTL responses, and to sensitize target cells for E7-directed CTL killing in situations where antigen processing is not required, the epitopes are not processed out of either endogenously expressed or immunization-introduced E7, by the mouse antigen-processing and presentation machinery. Thus (1) CTL induced by HLA A*0201 peptide immunization killed E7 peptide-pulsed target cells, but did not kill target cells expressing whole E7; (2) immunization with whole E7 protein did not elicit CTL directed to HLA A*0201-restricted E7 CTL epitopes; (3) HLA A*0201-restricted CTL epitopes expressed in the context of a DNA polytope vaccine did not activate E7-specific T cells either in 'conventional' HLA A*0201 transgenic (A2.1Kb) mice, or in HHD transgenic mice in which expression of endogenous H-2 class 1 is precluded; and (4) HLA A*0201 E7 peptide epitope immunization was incapable of preventing the growth of an HLA A*0201- and E7-expressing tumour. There are generic implications for the universal applicability of HLA-class 1 transgenic mice for studies of human CTL epitope presentation in murine models of human infectious disease where recognition of endogenously processed antigen is necessary. There are also specific implications for the use of HLA A2 transgenic mice for the development of E7-based therapeutic vaccines for cervical cancer.

Immune evasion in human papillomavirus-associated cervical cancer.

Tumour-associated viruses produce antigens that, on the face of it, are ideal targets for immunotherapy. Unfortunately, these viruses are experts at avoiding or subverting the host immune response. Cervical-cancer-associated human papillomavirus (HPV) has a battery of immune-evasion mechanisms at its disposal that could confound attempts at HPV-directed immunotherapy. Other virally associated human cancers might prove similarly refractive to immuno-intervention unless we learn how to circumvent their strategies for immune evasion.