Nonspecific down-regulation of CD8+ T-cell responses in mice expressing human papillomavirus type 16 E7 oncoprotein from the keratin-14 promoter.

The E7 oncoprotein of human papillomavirus 16 (HPV16) transforms basal and suprabasal cervical epithelial cells and is a tumor-specific antigen in cervical carcinoma, to which immunotherapeutic strategies aimed at cytotoxic T-lymphocyte (CTL) induction are currently directed. By quantifying major histocompatibility complex class I tetramer-binding T cells and CTL in mice expressing an HPV16 E7 transgene from the keratin-14 (K14) promoter in basal and suprabasal keratinocytes and in thymic cortical epithelium, we show that antigen responsiveness of both E7- and non-E7-specific CD8+ cells is down-regulated compared to non-E7 transgenic control mice. We show that the effect is specific for E7, and not another transgene, expressed from the K14 promoter. Down-regulation did not involve deletion of CD8+ T cells of high affinity or high avidity, and T-cell receptor (TCR) Vbeta-chain usage and TCR receptor density were similar in antigen-responsive cells from E7 transgenic and non-E7 transgenic mice. These data indicate that E7 expressed chronically from the K14 promoter nonspecifically down-regulates CD8+ T-cell responses. The in vitro data correlated with the failure of immunized E7 transgenic mice to control the growth of an E7-expressing tumor challenge. We have previously shown that E7-directed CTL down-regulation correlates with E7 expression in peripheral but not thymic epithelium (T. Doan et al., J. Virol. 73:6166-6170, 1999). The findings have implications for the immunological consequences of E7-expressing tumor development and E7-directed immunization strategies. Generically, the findings illustrate a T-cell immunomodulatory function for a virally encoded human oncoprotein.

Peripheral T-cell tolerance defined through transgenic mouse studies.

Selection in the thymus restricted by MHC and self-peptide shapes the diverse reactivities of the T-cell population which subsequently seeds into the peripheral tissues, in anticipation of the universe of pathogen antigens to which the organism may be exposed. A necessary corollary is the potential for T-cell self-reactivity (autoimmunity) in the periphery. Transgenic mouse models in which transgene expression in the thymus is prevented or excluded, have been particularly useful for determining the immunological outcome when T-cells encounter transgene-encoded 'self' antigen in peripheral tissues. Data suggest that non-mutually exclusive mechanisms of T-cells 'ignoring' self-antigen, T-cell deletion, T-cell anergy and T-cell immunoregulation have evolved to prevent self-reactivity while maintaining T-cell diversity. The peripheral T-cell repertoire, far from being static following maturation through the thymus, is in a dynamic stated determined by these peripheral selective and immunoregulatory influences. This article reviews the evidence with particular reference to CD8+ive T-cells.

Low level expression of human papillomavirus type 16 (HPV16) E6 in squamous epithelium does not elicit E6 specific B- or T-helper immunological responses, or influence the outcome of immunisation with E6 protein.

Mice transgenic for E6/E7 oncogenes of Human Papillomavirus type 16 display life-long expression of E6 in lens and skin epithelium, and develop inflammatory skin disease late in life, which progresses to papillomata and squamous carcinoma in some mice. We asked whether endogenous expression of E6 induced a specific immunological outcome, i.e. immunity or tolerance, or whether the mice remained immunologically naïve to E6. We show that prior to the onset of skin disease, E6 transgenic mice did not develop a spontaneous E6-directed antibody response, nor did they display T-cell proliferative responses to dominant T-helper epitope peptides within E6. In contrast, old mice in which skin disease had arisen, developed antibodies to E6. We also show that following immunisation with E6, specific antibody responses did not differ significantly among groups of E6-transgenic mice of different ages (and therefore of different durations and amounts of exposure to endogenous E6), and non-transgenic controls. Additionally, E6 immunisation-induced T-cell proliferative responses were similar in E6-transgenic and non-transgenic mice. These data are consistent with the interpretation that unimmunised E6-transgenic mice that have not developed inflammatory skin disease remain immunologically naïve to E6 at the B- and Th levels. There are implications for E6-mediated tumorigenesis in humans, and for the development of putative E6 therapeutic vaccines.

Peripheral tolerance to human papillomavirus E7 oncoprotein occurs by cross-tolerization, is largely Th-2-independent, and is broken by dendritic cell immunization.

The E7 oncoprotein of human papillomavirus 16 functions as a tumor-specific antigen in transformed epithelial cells of the uterine cervix to which immunotherapeutic strategies aimed at CTL induction may be directed. We previously have shown in mice transgenic for the E7 gene driven off an epithelial specific (keratin-14) promoter, that expression of E7 protein in peripheral epithelium is sufficient to tolerize E7-directed CTL precursors (pCTL; Doan et al, J. Virol., 73: 6166-1670, 1999). Here we show that E7 is presented to T cells for tolerization by cells of bone marrow origin ("cross-tolerization"). We demonstrate that tolerization of E7-directed pCTLs occurs within 2 weeks of exposure to E7 in epithelium. It is maintained in the near absence of CD4+ cells and in the absence of the thymus, and is independent of a coexisting E7-directed Th2-type antibody response. Tolerance was broken by immunization with E7 CTL epitope-pulsed dendritic cells. These findings have implications for immunotherapy of patients with human papillomavirus 16-associated cervical carcinoma, whose immune systems may have experienced long-term exposure to E7-expressing epithelial cells.

Differences in the effectiveness of delivery of B- and CTL-epitopes incorporated into the hepatitis B core antigen (HBcAg) c/e1-region.

Work from a number of laboratories including our own has shown that foreign B-epitopes inserted into the c/e1-region of Hepatitis B core antigen (HBcAg) elicit powerful antibody responses when mice are immunised with the recombinant core particles. In the present study, we wished to take advantage of the immunodominance of the c/e1-region to deliver cytotoxic T-lymphocyte (CTL) epitopes as a recombinant HBcAg vaccine. Our results indicated that recombinant HBcAg containing CTL epitopes of the E7 protein of human papillomavirus failed to prime E7-directed CTL responses when used to immunise mice for antigen processing through either the endogenous pathway via a Salmonella typhimurium vector, or through the exogenous pathway by parenteral immunisation with recombinant core. Hydropathicity plots predict that the presumed surface location of the hydrophilic c/e1-region within the core particle may alter following insertion of hydrophobic residues constituting the CTL epitopes, thereby compromising their presentation to the afferent immune system. Our data indicate that while the c/e1-region has a powerful adjuvanting effect for inserted B-epitopes, it does not serve this function for inserted CTL epitopes. These findings have generic implications for the development of CTL inducing vaccines using HBcAg as a vaccine vehicle.

Potential strategies utilised by papillomavirus to evade host immunity.

The co-evolution of papillomaviruses (PV) and their mammalian hosts has produced mechanisms by which PV might avoid specific and non-specific host immune responses. Low level expression of PV proteins in infected basal epithelial cells, together with an absence of inflammation and of virus-induced cell lysis, restricts the opportunity for effective PV protein presentation to immunocytes by dendritic cells. Additionally, PV early proteins, by a range of mechanisms, may restrict the efficacy of antigen presentation by these cells. Should an immune response be induced to PV antigens, resting keratinocytes (KC) appear resistant to interferon-gamma-enhanced mechanisms of cytotoxic T-lymphocyte (CTL)-mediated lysis, and expression of PV antigens by resting KC can tolerise PV-specific CTL. Thus, KC, in the absence of inflammation, may represent an immunologically privileged site for PV infection. Together, these mechanisms play a part in allowing persistence of PV-induced proliferative skin lesions for months to years, even in immunocompetent hosts.

T-helper epitopes identified within the E6 transforming protein of cervical cancer-associated human papillomavirus type 16.

The E6 oncoprotein of human papillomavirus type 16 (HPV16 E6) produced by tumor cells of HPV16-associated cervical carcinoma is poorly immunogenic in patients, but nonetheless is a tumor-specific antigen to which therapeutic vaccine strategies may be directed. To investigate the subunit immunogenicity of E6 protein at the T-helper cell level, we immunized mice with overlapping peptides spanning the entire 158 amino acid sequence. Two peptides recalled a proliferative response in lymph node cells (LNC) from C57BL/6 (H-2b)-immunized mice. One of these peptides also recalled proliferative responses in the context of 5/5 other major histocompatibility complex (MHC) class II haplotypes, indicating a "promiscuous" T-epitope. Minimal consensus motif analysis identified the epitopes as 60VYRDGNPYA68 and 98GYNKPLCDLL107. LNC from mice immunized with T-epitope proliferated in response to challenge with whole E6 protein. Immunization with E6 T-epitopes linked to B-epitopes of HPV16 E7 protein elicited specific antibody indicating that T-cells recognizing the T-epitopes provided cognate "help" for B-cells. LNC from mice co-immunized with E6 T-epitope and the major T-helper epitope of HPV16 E7 (48DRAHYNI54) proliferated comparably when challenged with the peptides individually indicating co-dominance of the two T-epitopes. The findings have implications for incorporation of E6 into a therapeutic vaccine.

Capture ElISA and in vitro cell binding assay for the detection of antibodies to human papillomavirus type 6b virus-like particles in patients with anogenital warts.

To investigate human papillomavirus (HPV) virus-like particle (VLP)-specific antibody responses among anogenital warts patients, a VLP-based capture ELISA was established. Twenty-six percent (35/134) of control subjects and 50.0% (39/78) of patients with current anogenital warts showed IgG seropositivity to HPV 6b VLPs. HPV 6b VLP-specific antibody responses recognised native VLPs only, and had no cross-reaction with HPV type 16 VLPs. No differences in reactivity were observed between L1 and L1 + L2 VLPs, suggesting that L2 contributes little to the total immunogenicity of the papillomavirus virion. A VLP-cell binding assay was also established. Some sera from patients with anogenital warts specifically inhibited VLP binding to the surface of epithelial cells, suggesting that these antibodies might be functionally neutralising. These data show that serological responses to HPV 6b VLPs were induced among some but not all patients with anogenital warts, and give a proportional estimate of infection in the community.

Vaccines for human papillomavirus-associated anogenital disease and cervical cancer: practical and theoretical approaches.

The association of genital warts, cervical dysplasia and cervical cancer with certain human papillomavirus (HPV) types indicates that vaccine strategies that target the virus could be effective in controlling disease onset and progression. Three vaccine strategies are available. Firstly, a prophylactic approach of immunisation with HPV virus-like particles to elicit neutralising antibody would prevent infection. Secondly, vaccination targeting replicating virus in suprabasal cells of infected anogenital epithelium would be an effective therapy for infection and early dysplasias. Thirdly, immunotherapy directed to the oncoprotein products of the HPV E6 and E7 open reading frames would be effective in the control of cervical carcinoma. We examine how these strategies may be augmented by contemporary vaccine technologies, in particular through the use of live recombinant vaccine vectors, specific targeting of antigen processing pathways, dendritic cell and 'polytope' approaches, to produce 'designer' vaccines of maximum specificity and efficacy. How these approaches are being exploited by vaccine manufacturers and in clinical trials is discussed.

Split tolerance to a viral antigen expressed in thymic epithelium and keratinocytes.

When expressed as a transgene from the keratin 14 (K14) promoter in an MHC class II-deficient mouse, I-Ab expressed in thymic cortical epithelium promotes positive but not negative selection of I-Ab-restricted CD4+ T cells (Laufer, T. M. et al., Nature 1996. 383:81-85). Transgenic mice expressing the E7 protein of human papilloma virus 16 from the K14 promoter were studied to determine the consequence of expression of a cytoplasmic/ nuclear protein from the K14 promoter. K14E7-transgenic mice express E7 in the thymus and skin without evidence for autoimmunity to E7. Repeated immunization of FVB(H-2q) or F1(C57BL/6JxFVB) mice with E7 elicited similar antibody responses to the defined B cell epitopes of E7 in K14E7-transgenic and non-transgenic animals. In contrast, for each genetic background, a single immunization with E7 elicited demonstrable T cell proliferative responses to the major promiscuous T helper epitope of E7 in the transgenic but not the non-transgenic animals. Further, E7-immunized non-transgenic F1 (FVBxC57BL/6J) animals developed strong E7-specific cytotoxic T lymphocyte (CTL) responses and were protected against challenge with E7+ tumors, whereas similarly immunized K14E7-transgenic animals had a markedly reduced CTL response to E7 and no E7-specific tumor protection was observed, although the antibody and CTL response to ovalbumin was normal. Expression of E7 protein as a transgene from the K14 promoter in the skin and thymus thus induces E7-specific tolerance in the cytotoxic T effector repertoire, together with expansion of the E7-specific T helper repertoire. These findings demonstrate that limited tissue distribution of an autoantigen may result in "split" tolerance to that autoantigen.

Th2-type CD4+ cells neither enhance nor suppress antitumor CTL activity in a mouse tumor model.

Many cervical cancers express the E7 protein of human papillomavirus 16 as a tumor-specific Ag (TSA). To establish the role of E7-specific T cell help in CD8+ CTL-mediated tumor regression, C57BL/6J mice were immunized with E7 protein or with a peptide (GF001) comprising a minimal CTL epitope of E7, together with different adjuvants. Immunized mice were challenged with an E7-expressing tumor cell line, EL4.E7. Growth of EL4.E7 was reduced following immunization with E7 and Quil-A (an adjuvant that induced a Th1-type response to E7) or with GF001 and Quil-A. Depletion of CD8+ cells, but not CD4+ cells, from an immunized animal abrogated protection, confirming that E7-specific CTL are necessary and sufficient for TSA-specific protection in this model. Immunization with E7 and Algammulin (an alum-based adjuvant) induced a Th2-like response and provided no tumor protection. To investigate whether a Th2 T helper response to E7 could prevent the development of an E7-specific CTL-mediated protection, mice were simultaneously immunized with E7/Algammulin and GF001/Quil-A or, alternatively, were immunized with GF001/Quil-A 8 wk after immunization with E7/Algammulin. Tumor protection was observed in each case. We conclude that an established Th2 response to a TSA does not prevent the development of TSA-specific tumor protective CTL.

Vaccine-induced Th1-type responses are dominant over Th2-type responses in the short term whereas pre-existing Th2 responses are dominant in the longer term.

The effect of adjuvant on induction of human papillomavirus type 16 E7 protein-specific cytotoxic T lymphocytes (CTL) and immunoglobulin G (IgG)2a antibody was studied in C57BL/6 J mice immunized with various adjuvants and E7 protein. Quil-A adjuvant, but not complete Freund's adjuvant (CFA) or Algammulin, induced a T-helper 1 (Th1)-type response to E7, which was characterized by CTL activity against a tumour cell line transfected with E7 protein and by E7-specific IgG2a. All tested adjuvants elicited comparable levels of E7-specific IgG1. The longest duration and greatest magnitude of CTL response was seen following two immunizations with the highest dose of E7 and Quil-A. Simultaneous immunization with a Th1 and a T helper 2 (Th2)-promoting adjuvant gave a Th1-type response. However, E7 and Quil-A were unable to induce a Th1-type response (as measured by the inability to generate anti-E7 IgG2a antibody) in animals with a pre-existing Th2-type response to E7. These results suggest that saponin adjuvants may be suitable for immunotherapy in humans where a Th1-type response is sought, provided that there is no pre-existing Th2-type response to the antigen.

Presentation of the HPV16E7 protein by skin grafts is insufficient to allow graft rejection in an E7-primed animal.

The E7 transforming protein of Human Papillomavirus type 16 (HPV16) is expressed in the skin of a line of FVB mice transgenic for the E6 and E7 open reading frames of HPV16 driven from the alpha A crystallin promoter (FVB alpha AcryHPV16E6E7). We have transferred skin from FVB alpha AcryHPV16E6E7 mice to naive or E7-primed syngeneic FVB recipients to assess whether the E7 protein of HPV16 can function as a minor transplantation antigen (MTA) and promote skin graft rejection. FVB mice did not reject E7 expressing tail or flank skin grafts. E7 immunized FVB x C57BL/6J mice recipients of FVB alpha-AcryHPV16E6E7 x C57BL/6J skin generated humoral and DTH responses to E7 in vivo and E7-specific CTL precursors in the spleen, but failed to reject E7 expressing tail skin grafts by 100 days posttransfer. Thus although HPV16 E7 + ve mesenchymal and endodermal tumors can be eliminated by an E7-specific immune response, the same protein is unable to act as a MTA and promote graft rejection when expressed in skin cells. Lack of rejection of grafts expressing MTAs such as E7 may be relevant to the immunology of epithelial tumors expressing tumor-specific antigens and to our understanding of the immunology of diseases of the skin.

E7 oncoprotein of human papillomavirus type 16 expressed constitutively in the epidermis has no effect on E7-specific B- or Th-repertoires or on the immune response induced or sustained after immunization with E7 protein.

A line of FVB (H-2q) mice transgenic for the E6/E7 open reading frames of Human Papillomavirus type 16 driven from the alpha-A crystallin promoter expresses E7 mRNA in lens and skin epithelium. E7 protein is detectable in adult skin, coinciding with the development of inflammatory skin disease, which progresses to papillomata and squamous carcinomata in some mice. By examining the outcome of parenteral immunization with E7 protein, we sought to determine whether endogenous expression of E7 in skin had induced a preexisting immune outcome, i.e., specific immunity or tolerance, or whether the mice remain naive ("ignorant") to E7. Our data show that the antibody response to defined E7 B-epitopes, the proliferative response to Th epitopes, and the delayed-type hypersensitivity (DTH) response to whole E7 did not differ between groups of young and old E6/E7 transgenic mice (likely having different degrees of lifetime exposure to E7 protein) or between E6/E7-transgenic and nontransgenic parental strain control mice. Although an E7-specific CTL response could not be induced in the H-2q background of these mice, incorporation of a Db allele into the genome allowed comparison of Db-restricted CTL responses in E6/E7 transgenic and nontransgenic mice. Experiments indicated that the E7-immunization-induced CTL response did not differ significantly between E6/E7 transgenic and nontransgenic mice. We interpret these results to indicate that in spite of expression of E7 protein in adult skin, E6/E7 transgenic mice remain immunologically naive (ignorant) of E7 epitopes presented by immunization.

Immunomanipulative strategies for the control of human papillomavirus associated cervical disease.

Three vaccine strategies that target human papillomavirus (HPV) are likely to be effective in the control of HPV-associated preneoplastic and neoplastic lesions of the uterine cervix. 1. Immunotherapy for HPV-associated cervical cancer targeted at two nonstructural PV proteins expressed in cancer cells (E6 and E7). 2. Vaccines against existing HPV infection and early premalignant lesions targeted at early viral proteins expressed in suprabasal stem cells of infected anogenital epithelium. 3. Prophylactic vaccines to prevent HPV infection involving immunization with genetically engineered virus-like particles to elicit neutralizing antibody. Strategies 1 and 2 will need to evoke cytotoxic T-cell (CTL) mediated responses.

Human papillomavirus vaccines for cervical cancer.

The association of carcinoma of the uterine cervix with human papillomavirus indicates that vaccine strategies which target the virus could be useful in the control of disease progression. Recent advances have centered on directing the immune response to prevention of infection, to virus-infected (but nontransformed) cells and to virally transformed cells, with favourable results.

Immunisation of mice using Salmonella typhimurium expressing human papillomavirus type 16 E7 epitopes inserted into hepatitis B virus core antigen.

Live vaccines based on BRD509, an attenuated S. typhimurium (aroA, aroD) strain, were constructed that directed the expression of hepatitis B core antigen particles (HBcAg) (BRD969) or HBcAg harbouring human papillomavirus type 16 E7 protein sequences (BRD974), under the control of the in vivo inducible nirB promoter. These strains were used to orally or intravenously immunise different inbred mouse strains and humoral, secretory and cellular anti-E7 and anti-HBcAg responses were monitored. Both BRD969 and BRD974 induced anti-HBcAg humoral IgG responses following oral or intravenous immunisation of B10 mice, although responses were higher in BRD969 immunised animals. IgG subclass analysis revealed a predominantly IgG2a response in these animals. BRD974, but not BRD969, induced anti-E7 humoral IgG responses. Anti-HBcAg (BRD969 and BRD974) and anti-E7 (BRD974) IgA responses were detected in the intestines of orally immunised mice. Anti-Salmonella but not anti-HBcAg or anti-E7 T helper cell responses were detected in mice immunised with BRD509, BRD969 and BRD974. Thus Salmonella vaccine strains can be used to efficiently deliver HBcAg and E7 epitopes to the mucosal and systemic immune systems.

A vaccine conjugate of 'ISCAR' immunocarrier and peptide epitopes of the E7 cervical cancer-associated protein of human papillomavirus type 16 elicits specific Th1- and Th2-type responses in immunized mice in the absence of oil-based adjuvants.

TraT protein, known as ISCAR (= Immunostimulatory Carrier), is one of a family of integral membrane proteins (Imps) of Escherichia coli representing powerful carrier molecules which when injected into experimental animals generate substantial antibody and T proliferative responses to molecules conjugated to it. We extend these findings to show that ISCAR functions to stimulate Th1- and Th2-type responses, including specific cytotoxic T cells and tumour protection. We report here that by conjugating to ISCAR a 19mer peptide containing linear B epitopes, a T helper (Th) epitope, and a H-2b-restricted T cytotoxic (CTL) epitope of E7 protein of human papillomavirus type 16 (HPV16), and immunizing C57B1/6 (H-2b) mice, we elicited (i) specific IgG2a and IgG1 antibodies; (ii) IL-2 and IL-4 production by specifically recalled lymph node cells in vitro; (iii) cytotoxic T lymphocytes which specifically killed both E7 peptide-pulsed, and whole E7 gene-transfected tumour target cells; and (iv) in vivo protection against an E7 gene-transfected tumour cell inoculum. These findings have implications for the design of vaccines to stimulate immune responses to endogenously processed target antigens (e.g. tumour-associated antigens) without the unwanted side effects of oil-based adjuvants. In addition they support the case for a E7-targeted therapeutic vaccine for HPV-associated human cervical cancer.

Immunological responses in human papillomavirus 16 E6/E7-transgenic mice to E7 protein correlate with the presence of skin disease.

The human papillomavirus (HPV) oncogenes, E6 and E7, are believed to contribute to the development of cervical cancers in women infected with certain HPV genotypes, most notably HPV-16 and HPV-18. Given their expression in tumor tissue, E6 and E7 have been implicated as potential tumor-specific antigens. We have examined an HPV-16 E6- and E7-transgenic mouse lineage for immune responses to these viral oncoproteins. Mice in this lineage express the HPV-16 E6 and E7 genes in their skin and eyes, and on aging, these mice frequently develop squamous cell carcinomas and lenticular tumors. Young transgenic mice, which had measurable E7 protein in the eye but not in the skin, were immunologically naive to E7 protein. They mounted an immune response to E7 on immunization comparable to that of nontransgenic controls, suggesting a lack of immune tolerance to this protein. Older line 19 mice, which are susceptible to skin disease associated with transcription of the E6 and E7 open reading frames, had measurable E7 protein in their skin. These older transgenic mice spontaneously developed antibody responses to endogenous E7 protein, particularly in association with skin disease. Also detected in older mice were delayed-type hypersensitivity responses to E7. These finding parallel the humoral immune response to E7 protein in patients with HPV-associated cervical cancer and suggest that line 19 mice may provide a model for studying the immunobiology of HPV-associated cancers.

T-helper epitopes of the E7 transforming protein of cervical cancer associated human papillomavirus type 18 (HPV18).

The presence of T-helper epitopes within the E7 transforming protein of human papillomavirus type 18 (HPV18) was sought using a series of overlapping synthetic 15-20 mer peptides spanning the entire 105 amino acid sequence of this protein. Two H-2k restricted T-helper epitopes were defined, comprising 44VNHQHLPARRA55 and 81DDLRAFQQLF90 as the minimal T proliferative epitopes. Peptides containing these epitopes were able to provide cognate help to B epitopes from HPV18E7 protein for production of antibody to this protein in vivo in CBA/CaH mice. No H-2b or H-2d restricted epitopes were demonstrable, and in H-2d mice this was associated with poor antibody response to the E7 protein. There is no "promiscuous" T-helper epitope in HPV18 E7 comparable to the 49DRAHYNI55 sequence in HPV16 E7, and restricted T-helper epitope availability may be a determinant of poor immune responses to this protein after natural infection.