[Show abstract][Hide abstract] ABSTRACT: Persistent infection with the high-risk Human Papillomavirus type 16 (HPV 16) is
the causative event for the development of cervical cancer and other malignant
tumors of the anogenital tract and of the head and neck. Despite many attempts to
develop therapeutic vaccines no candidate has entered late clinical trials. An
interesting approach is a DNA based vaccine encompassing the nucleotide
sequence of the E6 and E7 viral oncoproteins. Because both proteins are
consistently expressed in HPV infected cells they represent excellent targets for
immune therapy. Here we report the development of 8 DNA vaccine candidates
consisting of differently rearranged HPV-16 E6 and E7 sequences within one
molecule providing all naturally occurring epitopes but supposedly lacking
transforming activity. The HPV sequences were fused to the J-domain and the
SV40 enhancer in order to increase immune responses. We demonstrate that one
out of the 8 vaccine candidates induces very strong cellular E6- and E7- specific
cellular immune responses in mice and, as shown in regression experiments,
efficiently controls growth of HPV 16 positive syngeneic tumors. This data
demonstrates the potential of this vaccine candidate to control persistent HPV 16
infection that may lead to malignant disease. It also suggests that different
sequence rearrangements influence the immunogenecity by an as yet unknown
PLoS ONE 11/2014; 9(11):e113461. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Infection with different species of cutaneous human papillomaviruses (cHPV) of genus alpha (cαHPVs) and associated skin disease are highly prevalent in solid organ transplant recipients (OTR), documenting the importance of the immunological control of HPV infection.Objectives
To investigate the natural course of cαHPV-specific cellular and humoral immune responses during systemic long-term immunosuppression.Methods
Integrating bead-based multiplex serology and flow cytometry we analyzed natural cαHPV-specific antibodies and TH cell responses against the major capsid protein L1 of HPV types 2, 27, 57 (species 4) and 3, 10 and 77 (species 2) in sera and blood of OTR before and after initiation of iatrogenic immunosuppression and in comparison to immunocompetent individuals (IC).ResultsAmong OTR we observed an overall 42% decrease in humoral L1-specific immune responses during the course of iatrogenic immunosuppression, comparing median values 30d before and 30d after initiation of immunosuppressive therapy (p < 0.05). This difference disappeared after long-term (>1 year) immunosuppression. The predominant cellular L1-specific immune response was of type TH1 (CD4+CD40L+IL-2+IFN-γ+). Consistent with the detected L1-specific antibody titres, L1-specific TH1 responses were unchanged in long-term immunosuppressed OTR compared to IC. Notably, cαHPV-L1-specific IL-2+/CD40L+CD4+ or IFN-γ+/CD40L+ CD4+ TH cell responses against any of the cαHPV-L1 types were significantly higher in OTR with clinically apparent common warts.Conclusion
The systemic humoral immune response against cαHPV may reflect the individual degree of iatrogenic immunosuppression indicating a higher susceptibility for cαHPV infection among OTR during the early phase after organ transplantation. Humoral cαHPV-specific immune responses may show a reconstitution to pre-transplantation levels despite continuous potent immunosuppression.
[Show abstract][Hide abstract] ABSTRACT: Eight HPV types (HPV26, 53, 66, 67, 68, 70, 73 and 82) that are phylogenetically closely related to 12 WHO-defined high-risk (HR-)HPV have been rarely but consistently identified as single HPV infections in about 3% of cervical cancer (CxCa) tissues. Due to lack of biological data, these types are referred to as probable/possible (p)HR-HPV. To analyze their biological activity in direct comparison to HR-HPV types, we selected 55 formalin-fixed paraffin-embedded (FFPE) CxCa tissues harboring single pHR-HPV infections (2-13 cases per type) and 266 tissues harboring single HR-HPV (7-40 cases per type) from a worldwide, retrospective, cross-sectional study. Single HPV infection was verified by two genotyping methods. Presence of type-specific spliced E6*I mRNA transcripts and expression of cellular proteins indicative of HPV transformation were assessed in all cases. In 55 CxCa tissues with pHR-HPV, E6*I mRNA expression was 100%, high p16INK4a, 98%; low pRb, 96%; low CyD1, 93% and low p53, 84%. Compared to HPV16 tissues as a reference, individual frequencies of these five markers did not differ significantly, either for any of the eight pHR-HPV and the 11 other HR-types individually, or for the groups of pHR- and HR-types without HPV16. We conclude that the eight pHR-HPV types, when present as a single infection in CxCa, are biologically active and affect the same cellular pathways as any of the fully-recognized carcinogenic HR-HPV types. Therefore we have provided molecular evidence of carcinogenicity for types currently classified as probably/possibly carcinogenic. Although this evidence is crucial for HPV type carcinogenicity classification, per se it is not sufficient for inclusion of these HPV types into population-wide primary and secondary prevention programs. Such decisions have to include careful estimation of effectiveness and cost-benefit analyses.
The Journal of Pathology 07/2014; · 7.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The genus beta human papillomavirus 8 (HPV8) is involved in the development of cutaneous squamous cell carcinomas (SCCs) in individuals with epidermodysplasia verruciformis. Immunosuppressed transplant recipients are prone to harbor particularly high betapapillomavirus DNA loads, which may contribute to their highly increased risk of SCC. Tumor induction in HPV8 transgenic mice correlates with increased expression of viral oncogenes E6 and E2. In an attempt to prevent skin tumor development, we evaluated an HPV8-E6-DNA vaccine, which was able to stimulate a detectable HPV8-E6-specific cell-mediated immune response in 8/15 immunized mice. When skin of HPV8 transgenic mice was grafted onto non-transgenic littermates, the grafted HPV8 transgenic tissue was not rejected and papillomas started to grow within 14 days all over the transplant of 9/9 non-vaccinated and 7/15 not successfully vaccinated mice. In contrast, no papillomas developed in 6/8 successfully vaccinated mice. In the other two of these eight mice, a large ulcerative lesion developed within the initial papilloma growth or papilloma development was highly delayed. As the vaccine completely or partially prevented papilloma development without rejecting the transplanted HPV8 positive skin, the immune system appears to attack only keratinocytes with increased levels of E6 protein, which would give rise to papillomas.
Medical Microbiology and Immunology 01/2014; · 3.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Helicobacter pylori infection that is usually acquired in childhood and lasts for lifetime is mostly asymptomatic but associated with severe gastrointestinal disease including cancer. During chronic infection, the gastric mucosa is histologically changing. This forces H. pylori to permanent adaptation in its gastric habitat by expression of different proteins which might be reflected in distinctive antibody patterns.
To characterize dynamics of the immune response to H. pylori we analysed 1797 sera of a cross-sectional study representative for the German population (age range 1-82 years) with multiplex serology, a fluorescent bead-based antibody binding assay that allows simultaneous and quantitative detection of antibodies. Fifteen recombinant, affinity-purified H. pylori proteins (UreA, GroEL, Catalase, NapA, CagA, CagM, Cagδ, HP0231, VacA, HpaA, Cad, HyuA, Omp, HcpC and HP0305) were used as antigens.
H. pylori seroprevalence (positivity for at least three antigens) was 48% and increased with age from 12% in children <15 years to 69% in females and 90% in males >65 years. Prevalences were highest (>83%) for Omp, VacA and GroEL. For 11 proteins, seroprevalence was higher in males than females (P < 0.05) from age 55 onwards. For all antigens, the median prevalence increase per age decade was stronger in males (8.4%, range 3.8-12.9%) than females (6.1%, range 3.4-10.8%). However, among seropositives the median number of antigens recognized increased from children <15 years to individuals >65 years stronger in females (P = 0.02). Antibody reactivities to GroEL, HyuA, CagM, Catalase, NapA and UreA also increased stronger in females (average 1.7-fold/decade, SD 0.5) than in males (1.5-fold/decade, SD 0.4).
H. pylori antibody response accumulates qualitatively and quantitatively with age. This may reflect a lifelong stimulation of the immune response by chronically active infection.
[Show abstract][Hide abstract] ABSTRACT: Many findings support a possible involvement of a subgroup of human papillomaviruses (HPVs), called cutaneous beta HPV types, in the development of non-melanoma skin cancer (NMSC). The skin of transgenic (Tg) mice expressing viral oncoproteins E6 and E7 from different cutaneous beta HPV types, including HPV38, showed an increased susceptibility to UV-induced and/or chemically induced skin carcinogenesis compared with wild-type animals. In this study, we show that beta HPV38 E6 and E7 oncoproteins act as promoter and progression factors in multi-stage skin carcinogenesis, strongly cooperating with the initiator and DNA damage agent 7,12-dimethylbenz[a]anthracene (DMBA). In contrast, exposure of HPV38 E6/E7 Tg mice to the promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) did not significantly result in the development of skin lesions. These findings further support the role of beta HPV types in skin carcinogenesis, providing additional insight into their precise contribution to the multi-step process.
Journal of General Virology 12/2012; · 3.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Judging the carcinogenicity of human papillomavirus (HPV) types rarely found in cervical cancer (CxCa) is hindered by lack of studies of their biological activity in cancer tissues. To asses transcriptional activity of HPV types, we have developed ultra-short amplimer, splice-site specific, E6*I mRNA RT-PCR assays for 12 high-risk (HR)-HPV (IARC Group 1) and eight probable/possible high-risk (pHR)-HPV types (IARC Group 2A/B carcinogens). Previously unreported E6*I splice sites of the six pHR-HPV types 26, 53, 67, 70, 73 and 82 were identified by cloning and sequencing. We analyzed 97 formalin-fixed paraffin-embedded (FFPE) Mongolian CxCa biopsies for presence of HPV DNA by two sensitive genotyping assays, for E6*I transcripts of all HR-/pHR-HPV types identified and for expression of HPV surrogate markers p16(INK4a) , pRb and p53. E6*I of at least one HR-/pHR-HPV was expressed in 94 (98%) of cancer tissues including seven with pHR-HPV types 26, 66, 70 or 82 as single transcribed types. Fifty-eight of E6*I mRNA transcribing cases were analyzable by immunohistochemistry and displayed p16(INK4a) overexpression in 57 (98%), pRb downregulation in 56 (97%) and p53 downregulation in 36 (62%) tissues. The newly developed E6*I mRNA RT-PCR assays appeared to be highly sensitive method to analyze HPV transcription in FFPE materials. Our finding of viral oncogene transcription of pHR-HPV types 26, 66, 70 and 82 in cervical tumors, in the absence of any other transcriptionally active HR-type and with p16(INK4a) overexpression and pRb downregulation, may support a reassessment of the carcinogenicity classification of these pHR-HPV types.
International Journal of Cancer 04/2012; · 6.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cervical cancer is the second most common cancer in women worldwide. Persistent high-risk human papillomavirus (HPV) infection has been identified as the causative event for the development of this type of cancer. Recombinant adeno-associated viruses (rAAVs) are currently being developed and evaluated as vaccine vector. In previous work, we demonstrated that rAAVs administered intranasally in mice induced high titers and long-lasting neutralizing antibodies against HPV type 16 (HPV16). To extend this approach to a more human-related species, we immunized rhesus macaques (Macaca mulatta) with AAVs expressing an HPV16 L1 protein using rAAV5 and 9 vectors in an intranasal prophylactic setting. An rAAV5-L1 vector followed by a boost with rAAV9-L1 induced higher titers of L1-specific serum antibodies than a single rAAV5-L1 immunization. L1-specific antibodies elicited by AAV9 vector neutralized HPV16 pseudovirions and persisted for at least 7 months post immunization. Interestingly, nasal application of rAAV9 was immunogenic even in the presence of high AAV9 antibody titers, allowing reimmunization with the same serotype without prevention of the transgene expression. Two of six animals did not respond to AAV-mediated intranasal vaccination, although they were not tolerant, as both developed antibodies after intramuscular vaccination with HPV16 virus-like particles. These data clearly show the efficacy of an intranasal immunization using rAAV9-L1 vectors without the need of an adjuvant. We conclude from our results that rAAV9 vector is a promising candidate for a noninvasive nasal vaccination strategy.
Human gene therapy 03/2012; 23(7):733-41. · 4.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The human papillomavirus (HPV) minor capsid protein L2 is a promising candidate for a broadly protective HPV vaccine yet the titers obtained in most experimental systems are rather low. Here we examine the potential of empty AAV2 particles (AAVLPs), assembled from VP3 alone, for display of L2 epitopes to enhance their immunogenicity. Insertion of a neutralizing epitope (amino acids 17-36) from L2 of HPV16 and HPV31 into VP3 at positions 587 and 453, respectively, permitted assembly into empty AAV particles (AAVLP(HPV16/31L2)). Intramuscularly vaccination of mice and rabbits with AAVLP(HPV16/31L2)s in montanide adjuvant, induced high titers of HPV16 L2 antibodies as measured by ELISA. Sera obtained from animals vaccinated with the AAVLP(HPV16/31L2)s neutralized infections with several HPV types in a pseudovirion infection assay. Lyophilized AAVLP(HPV16/31L2) particles retained their immunogenicity upon reconstitution. Interestingly, vaccination of animals that were pre-immunized with AAV2--simulating the high prevalence of AAV2 antibodies in the population--even increased cross neutralization against HPV31, 45 and 58 types. Finally, passive transfer of rabbit antisera directed against AAVLP(HPV16/31L2)s protected naïve mice from vaginal challenge with HPV16 pseudovirions. In conclusion, AAVLP(HPV16/31L2) particles have the potential as a broadly protective vaccine candidate regardless of prior exposure to AAV.
PLoS ONE 01/2012; 7(6):e39741. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The 27th International Papillomavirus Conference and Clinical Workshop, held in Berlin (17-22 September 2011), brought together more than 2000 scientists, clinicians and public health experts who shared new findings in the knowledge of the HPV and the prevention and treatment of HPV-related disease. In this second of three reports of the conference, the applied clinical science sessions are summarized, which focused on immunology, new HPV tests, benign HPV infections, noncervical HPV-related disease, primary and secondary prevention of cervical cancer by HPV-based screening and prophylactic HPV vaccination and treatment of HPV-induced disease. The clinical workshops discussed possible alternative schedules of prophylactic HPV vaccination, prevention of anal cancer and anal precancer, validation of HPV genotyping assays, establishment of standards and laboratory proficiency in testing for HPV DNA and anti-HPV antibodies through the WHO LabNet, and currently heavily debated questions on the role of colpo
[Show abstract][Hide abstract] ABSTRACT: The cutaneous beta human papillomavirus (beta HPV) types appear to be involved in skin carcinogenesis. However, only a few beta HPVs have been investigated so far. Here, we compared the properties of E6 and E7 oncoproteins from six uncharacterized beta HPVs (14, 22, 23, 24, 36, 49). Only HPV49 E6 and E7 immortalized primary human keratinocytes and efficiently deregulated the p53 and pRb pathways. Furthermore, HPV49 E6, similarly to E6 from the oncogenic HPV16, promoted p53 degradation.
Journal of Virology 12/2011; 86(4):2366-70. · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The currently licensed human papillomavirus (HPV) vaccines are safe and highly effective at preventing HPV infection for a select number of papillomavirus types, thus decreasing the incidence of precursors to cervical cancer. It is expected that vaccination will also ultimately reduce the incidence of this cancer. The licensed HPV vaccines are, however, type restricted and expensive, and also require refrigeration, multiple doses and intramuscular injection. Second-generation vaccines are currently being developed to address these shortcomings. New expression systems, viral and bacterial vectors for HPV L1 capsid protein delivery, and use of the HPV L2 capsid protein will hopefully aid in decreasing cost and increasing ease of use and breadth of protection. These second-generation vaccines could also allow affordable immunization of women in developing countries, where the incidence of cervical cancer is high.
[Show abstract][Hide abstract] ABSTRACT: Cutaneous beta human papillomavirus (HPV) types appear to be involved in the development of non-melanoma skin cancer (NMSC); however, it is not entirely clear whether they play a direct role. We have previously shown that E6 and E7 oncoproteins from the beta HPV type 38 display transforming activities in several experimental models. To evaluate the possible contribution of HPV38 in a proliferative tissue compartment during carcinogenesis, we generated a new transgenic mouse model (Tg) where HPV38 E6 and E7 are expressed in the undifferentiated basal layer of epithelia under the control of the Keratin 14 (K14) promoter. Viral oncogene expression led to increased cellular proliferation in the epidermis of the Tg animals in comparison to the wild-type littermates. Although no spontaneous formation of tumours was observed during the lifespan of the K14 HPV38 E6/E7-Tg mice, they were highly susceptible to 7,12-dimethylbenz(a)anthracene (DMBA)/12-0-tetradecanoylphorbol-13-acetate (TPA) two-stage chemical carcinogenesis. In addition, when animals were exposed to ultraviolet light (UV) irradiation, we observed that accumulation of p21(WAF1) and cell-cycle arrest were significantly alleviated in the skin of Tg mice as compared to wild-type controls. Most importantly, chronic UV irradiation of Tg mice induced the development of actinic keratosis-like lesions, which are considered in humans as precursors of squamous cell carcinomas (SCC), and subsequently of SCC in a significant proportion of the animals. In contrast, wild-type animals subjected to identical treatments did not develop any type of skin lesions. Thus, the oncoproteins E6 and E7 from beta HPV38 significantly contribute to SCC development in the skin rendering keratinocytes more susceptible to UV-induced carcinogenesis.
[Show abstract][Hide abstract] ABSTRACT: Infections with high-risk human papillomaviruses (HPV), mainly HPV type 16, can cause malignant transformation of the human cervical epithelium and cervical cancer (CxCa). Very little is known about the quantitative expression of HPV16 transcripts in cervical lesions of different grades. We have analysed the viral transcriptome in 80 HPV16 DNA positive cervical smears including lesions of different cytological grades, using nucleic acid sequence-based amplification (NASBA)-Luminex hybridisation assays quantifying spliced and unspliced HPV16 transcripts. Based on the quantitative analysis of single transcripts, highly significant changes in transcript levels were observed between different grades of cervical lesions. In conclusion, quantitative expression changes of HPV16 transcript markers may be involved in tumour progression. This study provides a basis for selection of candidate RNA markers for diagnostics of HPV16-related disease.
Molecular and Cellular Probes 06/2011; 25(5-6):260-5. · 1.87 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: ΔNp73α, a dominant-negative inhibitor of p53 and p73, exhibits antiapoptotic and transforming activity in in vitro models and is often found to be upregulated in human cancers. The mechanisms involved in the regulation of ΔNp73α protein levels in normal and cancer cells are poorly characterized. Here, we show that that IκB kinase beta (IKKβ) increases ΔNp73α protein stability independently of its ability to activate NF-κB. IKKβ associates with and phosphorylates ΔNp73α at serine 422 (S422), leading to its accumulation in the nucleus, where it binds and represses several p53-regulated genes. S422A mutation in ΔNp73α abolished IKKβ-mediated stabilization and inhibition of p53-regulated gene expression. Inhibition of IKKβ activity by chemical inhibitors, overexpression of dominant-negative mutants, or gene silencing by siRNA also resulted in ΔNp73α destabilization, which under these conditions was rapidly translocated into the cytoplasm and degraded by a calpain-mediated mechanism. We also present evidence for the IKKβ and ΔNp73α cross talk in cancer-derived cell lines and primary cancers. Our data unveil a new mechanism involved in the regulation of the p73 and p53 network.
Molecular and Cellular Biology 06/2011; 31(11):2210-26. · 5.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The prophylactic HPV vaccines consist of virus-like particles (VLP) of HPV 16 and 18 produced by recombinant DNA technology. These viruses induce about 70% of the worldwide occurring cases of cervical cancer as well as a substantial fraction of malignant tumors of the vulva, penis, perianum and oropharynx. One of the vaccines contains in addition VLPs of HPV 6 and 11 for prevention of genital warts (condylomata acuminata). In clinical trials with up to 6.4 years of follow-up the vaccines demonstrated an efficacy of almost 100% against infection by HPV 16 and 18 and the high-grade intraepithelial lesions (CIN, VIN, VaIN) they induce. Most likely due to cross-neutralizing antibodies one of the vaccines also protects significantly against lesions that are induced by genetically related non-vaccine types. Prevention of cases of cervical cancer will only be realized about 10 years after introduction of the vaccine in populations-based trials. First success of a vaccination campaign was already appreciated in Australia by reduction of the incidence of genital warts. Vaccinating males is a reasonable means to reduce the risk of infection in women but also provides to them a direct benefit through the recently documented efficacy of the quadrivalent vaccine against genital warts.
[Show abstract][Hide abstract] ABSTRACT: NK cell recognition of tumor cells is mediated by a delicate balance of signals received by MHC class I-binding inhibitory NK cell receptors and activating NK cell receptors, which mainly bind to virus-, stress- or tumor-induced ligands. In addition, adhesion molecules such as the intercellular adhesion molecule-1 (ICAM-1) and its receptors, the lymphocyte function-associated antigen-1 (LFA-1) and Mac-1, are crucial for immune synapse formation and NK cell-mediated killing. In this study, we show that expression of the adhesion molecule ICAM-1 was rapidly induced by E6 and -E7 oncoproteins of HPV16, -18, -5 and -8, but not of HPV38 and -6 in primary human keratinocytes after retroviral transduction. ICAM-1 was upregulated in E6E7-expressing keratinocytes both at mRNA and protein levels. The observed ICAM-1 upregulation in HPV16-E6E7-expressing keratinocytes was partially dependent on activation of the NF-κB pathway. Importantly, the upregulated ICAM-1 expression in HPV16-E6E7-expressing keratinocytes led to enhanced conjugate formation with NK cells. We previously showed that HPV16-positive cervical carcinomas frequently express low levels of inhibitory NK cell ligands and high levels of activating NK cell ligands. Moreover, levels of the adhesion molecule ICAM-1 are enhanced by HPV16-E6/E7. Therefore, strategies that aim at harnessing NK cells might be beneficial for the treatment of cervical carcinoma.
International Journal of Cancer 03/2011; 128(5):1104-13. · 6.20 Impact Factor