做TCT、HPV、阴道镜的顺序？如果TCT正常是否还要做HPV...
做TCT、HPV、阴道镜的顺序？如果TC...
病情描述（发病时间、主要症状、症状变化等）：做TCT、HPV、阴道镜的顺序？如果TCT正常是否还要做HPV或阴道镜？如果TCT和HPV都正常，是否还要做阴道镜？曾经治疗情况和效果：一直外阴瘙痒，3个月前做过TCT和HPV，显示有中度炎症，其它诊断正常，只是开了些灌洗的药。但外阴瘙痒一直时断时续，也就隔三差五去医院检查，上药、灌洗、吃药。今天医生说做个阴道镜和活体检验。想得到怎样的帮助：如果TCT和HPV都正常，是否还要做阴道镜？
医院出诊医生
擅长：小儿内科
擅长：外科
共4条医生回复
因不能面诊，医生的建议及药品推荐仅供参考
职称：住院医师
专长：心脑血管疾病、皮肤病尖锐湿疣、牛皮癣、荨麻疹和其他...
&&已帮助用户：255
病例分析:如果炎症没有改善则应做下检查查找病因，以便对症治疗。意见建议:建议中药治疗。
职称：医师
专长：滴虫阴道炎,功能失调性子宫出血病,盆腔积液卵巢囊肿
&&已帮助用户：1414
问题分析：你好，TCT、HPV检查结果都正常，基本上排除宫颈的恶性病变，阴道镜检查宫颈疾病，有特异性，准确度相对来说比较高。意见建议：建议到有条件的公立医院，进行阴道镜检查，必要时进行病理检查。
职称：主治医师
专长：功血,子宫息肉,卵巢早衰,盆腔炎性肿块,阴道念珠菌...
&&已帮助用户：33648
问题分析：针对你提出的问题，外阴部瘙痒到医院检查，医生给你治疗宫颈TCT和HPV检查，检查结果是宫颈有中度炎症。意见建议：然后给予治疗，但是外阴部仍然有时出现瘙痒，这种情况医生说做个阴道镜和活体检验，如果你目前宫颈糜烂不严重，一般是不需要做的。
职称：主治医师
专长：盆腔炎,子宫肌瘤,多囊卵巢综合征
&&已帮助用户：7252
问题分析：您好，一般排除宫颈的问题的话，TCT和HPV检查正常的话，是不需要做阴道镜的检查的，如果是TCT和HPV的检查超过半年或者一年以上的，需要复查TCT和HPV，TCT和HPV没有问题的话，可以先不要做阴道镜的检查。意见建议：但是你外阴瘙痒一直治疗不好的话，需要考虑其它的问题，目前医生叫你做阴道镜和活体检查，要考虑你外阴的问题，而TCT和HPV的检查是排除宫颈的问题，这个是不一样的，建议可以做一下阴道镜的检查，同进最好活检一下比较好。祝健康！
问我也是这个问题。HPV阳性，医生开了6盒，我记得他说每...
职称：医师
专长：胃、十二指肠溃疡,面神经炎,低血压
&&已帮助用户：245428
指导意见：您好，Hpv阳性是指对人乳头瘤病毒的判断标准，HPV阳性代表经检测发现了感染HPV病毒。HPV是一种DNA病毒，人类是HPV唯一的宿主。HPV进入机体皮肤粘膜后，主要潜伏于表皮内基底细胞间，一旦时机成熟它就会致病。目前为止，科学家们已经发现了它有60多个亚型，不同的亚型导致不同的疾病。患有hpv对自身的危害极大，如果发现患有hpv应及时到医院进行检查、治疗。
问高危HPV16.18,怀孕了可以要孩子吗
职称：主治医师
专长：功能失调性子宫出血病,细菌性阴道炎,经前期综合症
&&已帮助用户：15901
问题分析：你好！hpv-人乳头瘤病毒，有好多分型，其中hpv16，hpv18是引起是宫颈癌的高危致病型，hpv16、hpv18携带说明有宫颈癌高风险存在，但不会通过胎盘影响胎儿的发育，故不影响怀孕。意见建议：建议你备孕以前，如果阴道、宫颈有炎症存在，一定要先治愈后，再怀孕要宝宝。
问hpv阳性，但是tct正常，为何还需要做阴道镜检查？老公...
职称：医师
专长：妇产科及妇幼保健
&&已帮助用户：200
问题分析：2013年最新子宫颈癌筛查指南中指出，HPV阴性而TCT正常的话，可以有两种选择，一是12个月时复查TCT和HPV,二是进行HPV分型检测，如为16或16、18型，则考虑阴道镜检查，如阴性，则12月时复查TCT和HPV.意见建议：所以，你可以根据这两种情况做出选择哦！但是HPV必须要治疗呢，男方不用检查什么
问镜检和活检查出是慢性宫颈炎伴高级上皮内病变CIN2级医...
职称：医生会员
专长：妇产科、
&&已帮助用户：82573
指导意见：建议坚持宫颈炎的治疗，注意局部卫生，定期做宫颈液基细胞学检查，动态观察宫颈的变化，做到预防宫颈癌的发生。
问尖锐湿疣9个月没复发HPV阴性还要抽血作HPV抗体化验吗
职称：医师
&&已帮助用户：29019
问题分析：根据你的描述来看的话，病毒检测没有的话，可以不用进行抗体以及抽血化验了的，目前需要做的就是增强抵抗力的。意见建议：暂时可以不用进行抽血检查的，平时多休息，避免过度劳累，适当运动，增加营养，增强抵抗力，减少复发的几率的。
问宫颈hpv阳性能治好吗？
专长：外科其它、前列腺、男性不育
&&已帮助用户：218754
考虑如果感染的是低危型的HPV，可能会导致宫颈癌前病变，如果尖锐湿疣这一类病变的可能性比较大，那么导致癌的可能性就会小一些。治愈的几率就会大一些。
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Save publications, articles and searchesGet email alertsGet all the benefits mentioned below!医生没有换手套前面是一个高威宫颈癌感染者我该怎么办我4个月前刚检查完HPVTC一切都好怎么办呀_百度拇指医生
&&&普通咨询
?医生没有换手套前面是一个高威宫颈癌感染者我该怎么办我4个月前刚检查完HPVTC一切都好怎么办呀
女35岁|科室：妇科综合
邢台县医院
你说的这个情况，一般进行妇科检查的时候都是一人一用一灭菌，只要是正规的医院都是不会有什么问题，放心好了，再说这种检查一般也不会出现感染，放心吧！进行正常的妇科体检，一定要到正规的公立性的医院
拇指医生提醒您：医生建议仅供参考。
向医生提问
完善患者资料：*性别：
如果宫腔积液比较少，应该考虑是属于一种生理性的症状，应该注意观察，定期进行复查。
你这个症状是需要考虑和同房的时候局部的粘膜损伤有关系的，这个一般是不考虑什么宫颈...
病情分析：
宫颈癌术后放化疗后近期需要没过3个月复查一次腹部彩超。
指导意见：
病情分析： 你好，宫颈癌的期别是根据癌灶的侵润的深度和有无远处器官的转移来判定的
你好，这种情况不会那么巧的，艾滋病一般是多是有血液，性交传播的，你的情况可以注意...
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* 百度拇指医生解答内容由公立医院医生提供，不代表百度立场。* 由于网上问答无法全面了解具体情况，回答仅供参考，如有必要建议您及时当面咨询医生
向医生提问HPV DNA Vaccines and Methods of Use Thereof
United States Patent Application
Human papillomavirus (HPV) infection is the etiological factor for cervical cancer. Provided are HPV vaccines that generate a humoral immune response to prevent new infection, as well as cell-mediated immunotherapy to eliminate established infection or HPV-related disease. HPV vaccines include nucleic acid sequences encoding HPV16 early proteins E6 and E7. Additional nucleic acid sequences in the vaccines include sequences encoding calreticulin and/or the HPV16 late protein L2. Methods using these vaccines are provided that result in therapeutic effects.
Inventors:
Wu, Tzyy-choou (Stevenson, MD, US)
Hung, Chien-fu (Timonium, MD, US)
Roden, Richard (Severna Park, MD, US)
Application Number:
Publication Date:
10/23/2008
Filing Date:
03/17/2008
Export Citation:
Johns Hopkins University (Baltimore, MD, US)
Primary Class:
Other Classes:
424/204.1,
International Classes:
A61K39/00; A61P37/00; C07H21/00
View Patent Images:
&&&&&&PDF help
Related US Applications:
August, 2008Lombardo et al.May, 2004Robert et al.October, 2008HasbrouckJuly, 2003Mellott et al.October, 2002Chung et al.August, 2007Andreasen et al.November, 2009Goto et al.October, 2009Hurst et al.August, 2003Krugner-higby et al.September, 2007SoginJuly, 2007Palu et al.
Other References:
Cheng et al. Tumor-specific immunity and antiangiogenesis generated by a DNA vaccine encoding calreticulin linked to a tumor antigen. J Clin Invest. ):669-78.
Primary Examiner:
ZOU, NIANXIANG
Attorney, Agent or Firm:
Foley Hoag, LLP (w/JHV) (World Trade Center West, 155 Seaport Blvd, Boston, MA, , US)
1. A nucleic acid composition, comprising: (a) a first nucleic acid comprising a first sequence encoding an E6 or E7 protein of a human papillomavirus (HPV), wherein linked to the first sequence, directly or via a linker, is a second sequence that encodes an HPV late protein L2; and (b) a second nucleic acid encoding a calreticulin, wherein the first nucleic acid and the second nucleic acid are operably linked either directly or via a linker.
2. A nucleic acid composition, comprising: (a) a first sequence encoding a human papillomavirus (HPV) E6 protein or an immunogenically act (b) a second sequence encoding a HPV E7 protein or an immunogenically act (c) a third sequence encoding an HPV late protein L2 or an immunogenically act and (d) a fourth sequence encoding a calreticulin or an immunogenically active fragment thereof.
3. The composition of claim 2, wherein the HPV is HPV-16, and wherein the calreticulin is human calreticulin.
4. A DNA vaccine composition comprising a plasmid vector comprising the nucleic acid composition of claim 2 and an immunologically acceptable excipient or carrier.
5. A particle suitable for introduction into a cell or an animal, to which particle is bound the nucleic acid composition of claim 2.
6. The particle of claim 5, comprising a gold particle.
7. A method of inducing or enhancing an antigen-specific immune response in a mammalian subject, comprising administering to the subject an effective amount of the composition of claim 2, thereby inducing or enhancing the antigen specific immune response.
8. A method of inducing or enhancing an antigen-specific immune response in a mammalian subject, comprising administering to the subject an effective amount of the particle of claim 5, thereby inducing or enhancing the antigen specific immune response.
9. The method of claim 8, wherein the antigen specific immune response is mediated at least in part by CD8+ cytotoxic T lymphocytes (CTL).
10. The method of claim 8, wherein the antigen specific immune response is mediated at least in part by CD8- cytotoxic T lymphocytes (CTL).
11. The method of claim 8, wherein the mammalian subject is a human.
12. The method of claim 8, wherein the particle is administered intradermally by particle bombardment.
13. The method of claim 8, wherein the mammalian subject is a human having a tumor, and wherein the particle is administered intratumorally or peritumorally.
14. The method of claim 8, wherein the immune response is (i) specific for HPV E6 or E7 protein or immunogenically active fragment thereof, and (ii) greater in magnitude than an immune response induced by a DNA that encodes HPV E6, E7 and L2 without a DNA encoding the calreticulin or fragment thereof.
15. The composition of claim 2, wherein the first sequence encodes a HPV E6 protein comprising an amino acid sequence selected from the group consisting of LSRHFMHQKRTAMFQDPQERPRKILPQ(SEQ ID NO: 13)and AMFQDPQERPRKLPQLCTELQTTIHDIILEC.(SEQ ID NO: 14)
16. The composition of claim 2, wherein the second sequence encodes a HPV E7 protein comprising an amino acid sequence selected from the group consisting of PTLHEYMLDLQPETTDLYCYEQ,(SEQ ID NO: 15) HEYMLDLQPET(SEQ ID NO: 16) TLHEYMLDLQPETTD,(SEQ ID NO: 17) EYMLDLQPETTDLY,(SEQ ID NO: 18) DEIDGPAGQAEPDRAHY(SEQ ID NO: 19)and GPAGQAEPDRAHYNI.(SEQ ID NO: 20)
17. The composition of claim 2, wherein the third sequence encodes a HPV L2 protein comprising an amino acid sequence selected from the group consisting of (SEQ ID NO: 21)TGVPIDPAVPDSSIVPLLES, (SEQ ID NO: 22)GAEIEIAEVHPPPVYEGPE, (SEQ ID NO: 23)VTIGDIEEPPILEVVPETHPTand (SEQ ID NO: 24)SRMKRASATQLYKTCKQAGTCPPDIISKVEGKTIADQILQYGSMGVFFGG LGIGTGSGTGGRTGYIPLGTRPPTATDTLA.
18. The composition of claim 2, wherein the fourth sequence encodes a calreticulin protein comprising the amino acid sequence (SEQ ID NO: 26)MLLSVPLLLGLLGLAVAEPAVYFKEQFLDGDGWTSRWIESKHKSDFGKFV LSSGKFYGDE.
19. A nucleic acid composition comprising SEQ ID NO: 1.
20. A nucleic acid composition encoding an amino acid sequence comprising SEQ ID NO: 2.
Description:
CROSS-REFERENCE TO RELATED APPLICATIONSThis application claims the benefit of U.S. Provisional Application No. 60/918,278, filed Mar. 15, 2007, the contents of which are specifically incorporated by reference herein.GOVERNMENT INTERESTThis invention was made using funds from grants from the U.S. National Institutes of Health, including grants CA098252 and CA. The U.S. government therefore retains certain rights in the invention.FIELD OF THE INVENTIONThe present invention in the fields of molecular biology, immunology and medicine relates to nucleic acid vaccines for the prevention and treatment of diseases such as cancer. Specifically, treatment and prevention of cancers involving human papillomavirus or papilloma virus (HPV) infections are provided by vaccines that include nucleic acid sequences encoding HPV early proteins E6 and E7, the HPV late protein L2, and calreticulin.DESCRIPTION OF THE BACKGROUND ARTCervical cancer is the 2nd leading cause of cancer deaths in women worldwide. The primary factor in the development of cervical cancer is infection by the human papillomavirus. HPV is one of the most common sexually transmitted diseases in the world. It is now known that cervical cancer is a consequence of persistent infection with high-risk type HPV. HPV infection is a necessary factor for the development and maintenance of cervical cancer and thus, effective vaccination against HPV represents an opportunity to control cervical cancer.Cytotoxic T lymphocytes (CTL) are critical effectors of anti-viral and anti-tumor responses (reviewed in Chen, C H et al., J Biomed Sci. 5: 231-252, 1998; Pardoll, D M. Nat Med. 4: 525-531, 1998; Wang, R F et al., Immunol Rev. 170: 85-100, 1999). Activated CTL are effector cells that mediate antitumor immunity by direct lysis of their target tumor cells or virus-infected cells and by releasing cytokines that orchestrate immune and inflammatory responses that interfere with tumor growth or metastasis, or viral spread. Depletion of CD8+ CTL leads to the loss of anti-tumor effects of several cancer vaccines (Lin, K-Y et al., Canc Res 56: 21-26, 1996; Chen, C-H et al., Canc Res. 60: 00). Therefore, the enhancement of antigen presentation through the MHC class I pathway to CD8+ T cells has been a primary focus of cancer immunotherapy.Naked DNA vaccines have emerged as attractive approaches for vaccine development (reviewed in Hoffman, S L et al., Ann N Y Acad Sci 772: 88-94, 1995; Robinson, H L. Vaccine 15: 785-787, 1997; Donnelly, J J et al., Annu Rev Immunol 15: 617-648, 1997; Klinman, D M et al., Immunity 11: 123-129, 1999; Restifo, N P et al., Gene Ther 7: 89-92, 2000; Gurunathan, S et al., Annu Rev Immunol 18: 927-974, 2000). DNA vaccines generate long-term cell-mediated immunity (reviewed in Gurunathan, S et al., Curr Opin Immunol 12: 442-447, 2000) and generate CD8+ T cell responses in vaccinated humans (Wang, R et al. Science 282: 476-480, 1998). However, one limitation of these vaccines is their lack of potency, since the DNA vaccine vectors generally do not have the intrinsic ability to be amplified and to spread in vivo, as do some replicating viral vaccine vectors. Furthermore, some tumor antigens such as the E7 and E6 proteins of human papillomavirus-16 (“HPV-16”) are viewed as weak immunogens. Therefore, there is a need in the art for strategies to enhance DNA vaccine potency, particularly for more effective cancer and viral immunotherapy.Intradermal administration of DNA vaccines via gene gun can efficiently deliver genes of interest into professional antigen presenting cells (APCs) in vivo (Condon C et al., Nat Med, 2: 96). The skin contains numerous bone marrow-derived APCs (called Langerhans cells) that are able to move through the lymphatic system from the site of injection to draining lymph nodes (LNs), where they can prime antigen-specific T cells (Porgador A et al., J Exp Med 188: , 1998). APCs exist in other sites, particularly in lymphatic tissue as dendritic cells (DC). Gene gun immunization therefore provides the opportunity to provide HPV vaccine strategies that involve direct delivery of nucleic acids (e.g., DNA or RNA) to APCs.SUMMARY OF THE INVENTIONThis invention includes in one aspect a nucleic acid composition that contains a first nucleic acid including a first sequence encoding an E6 or E7 protein of a human papillomavirus (HPV), linked to a second sequence that encodes an HPV late protein L2, and a second nucleic acid encoding a calreticulin. The linkage between nucleic acid sequences can occur directly or via a linker.In another aspect, the invention provides a nucleic acid composition that contains a first sequence encoding a human papillomavirus (HPV) E6 protein or an immunogenically active fragment thereof, a second sequence encoding a HPV E7 protein or an immunogenically active fragment thereof, a third sequence encoding an HPV late protein L2 or an immunogenically active fragment thereof, and a fourth sequence encoding a calreticulin or an immunogenically active fragment thereof. Preferably, the HPV is HPV-16. In certain embodiments, the calreticulin is human calreticulin. The nucleic acid composition may be contained in a plasmid vector, such as an expression vector, and an immunologically acceptable excipient or carrier. In certain embodiments the invention provides a nucleic acid composition bound to a particle suitable for introduction into a cell or an animal. For example, this particle is a gold particle.The invention also provides a method of inducing or enhancing an antigen-specific immune response in a mammalian subject by administering to the subject an effective amount of a nucleic acid composition as described herein, thereby inducing or enhancing the antigen specific immune response.The invention further provides a method of inducing or enhancing an antigen-specific immune response in a mammalian subject by administering to the subject an effective amount of a nucleic acid composition bound to a particle, thereby inducing or enhancing the antigen specific immune response. In certain embodiments, the antigen-specific immune response is mediated at least in part by CD8+ cytotoxic T lymphocytes (CTL). In other embodiments, the antigen specific immune response is mediated at least in part by CD8- cytotoxic T lymphocytes. The mammalian subject is, for example, a human, such as a human having a tumor, and the nucleic acid composition or a particle containing the nucleic acid composition is administered intratumorally or peritumorally. The particle is capable of being administered intradermally by particle bombardment. The induced or enhanced immune response is preferably specific for HPV E6 or E7 protein or an immunogenically active fragment thereof. In other embodiments, the induced or enhanced immune response is, greater in magnitude than an immune response induced by a DNA that encodes HPV E6, E7 and L2 without a DNA encoding the calreticulin or fragment thereof.The nucleic acid compositions of the present invention include nucleic acids encoding modified proteins, such as protein fragments. In certain embodiments, a HPV E6 protein contains the sequences LSRHFMHQKRTAMFQDPQERPRKLPQ or AMFQDPQERPRKLPQLCTELQTTIHDIILEC. In other embodiments, the HPV E7 protein contains the sequences PTLHEYMLDLQPETTDLYCYEQ, HEYMLDLQPET, TLHEYMLDLQPETTD, EYMLDLQPETTDLY, DEIDGPAGQAEPDRAHY or GPAGQAEPDRAHYNI. In still other embodiments, the HPV L2 protein includes the sequences TGVPIDPAVPDSSIVPLLES, GAEIEIAEVHPPPVYEGPE, VTIGDIEEPPILEVVPETHPT, SRMKRASATQLYKTCKQAGTCPPDIISKVEGKTIAD QILQYGSMGVFFGGLGIGTGSGTGGRTGYIPLGTRPPTATDTLA, or MKRASATQLYKTCKQAGTCPPDIISKVEGKTIADQILQYGSMGVFFGGLGIGTGSGTGGRT GYIPLGTRPPTATDTLAPVRPPLTVDP. In additional embodiments, the calreticulin protein contains the amino acid sequence MLLSVPLLLGLLGLAVAEPAVYFKEQFLDGDG WTSRWIESKHKSDFGKFVLSSGKFYGDE.The present invention also provides a nucleic acid composition containing SEQ ID NO: 1, and a nucleic acid composition encoding an amino acid sequence that contains SEQ ID NO: 2.BRIEF DESCRIPTION OF THE DRAWINGSFIG. 1 demonstrates the experimental characterization of expression of E6, E7 and L2 in DNA transfected cells. BHK 21 cells were transfected with 2 μg of the various DNA constructs, pNGVL4a, pNGVL4a-L2, pNGVL4a-hCRT, pNGVL4a-hCRTL2, pNGVL4a-hCRTE6E7 or pNGVL4a-hCRTE6E7L2 as described herein. FIG. 1 shows a photograph of Western blot analysis performed using 50 μg of protein from the cell lysates 24 hours after transfection. Protein expression was determined using mouse anti-HPV 16 E6 (upper panel), E7 (middle panel) or rabbit anti-HPV L2 (lower panel) polyclonal antibody. Western blot analysis showing the expression of HPV 16 E6, E7 and L2 proteins in BHK 21 cells transfected with each of the six DNA constructs. The arrows indicate the molecular weights. Expression of E6 and E7 proteins was observed in the 4a-hCRTE6E7 (lane 5) and the 4a-hCRTE6E7L2 (lane 6) constructs. Expression of L2 protein was observed in the 4a-L2 (lane 2), 4a-hCRTL2 (lane 4) and the 4a-hCRTE6E7L2 (lane 6) constructs.FIG. 2 demonstrates the characterization of IFNγ-secreting E6-specific CD8+ T cell precursors in mice vaccinated with recombinant DNA vaccines. One set of C57BL/6 mice (3 per group) was immunized intradermally via gene gun with 2 μg of DNA per mouse of the various recombinant DNA vaccines, four times with one-week intervals. Another set of C57BL/6 mice (3 per group) was immunized intramuscularly with 50 μg per mouse of the various recombinant DNA vaccines four times with one-week intervals. The splenocytes from vaccinated mice were incubated with H-2Kb-restricted E6 CTL peptide (aa 50-57) overnight. Determination of the E6-specific CD8+ T cells was performed by intracellular IFN-γ staining followed by flow cytometry analysis. FIG. 2A shows a series of graphs of representative flow cytometry data showing the number of E6-specific IFNγ+ CD8+ T cells in the mice vaccinated either intradermally via gene gun (left panel) or intramuscularly (right panel) with the various DNA vaccines. FIG. 2B provides a bar graph showing the number of E6-specific IFNγ+ CD8+ T cells from mice immunized intradermally with the various DNA vaccines via gene gun with (shaded bars) or without (empty bars) stimulation with the E6 peptide. The data are shown as mean ±s.d. (p&0.05). FIG. 2C provides a bar graph showing the number of E6-specific IFNγ+ CD8+ T cells from mice immunized intramuscularly with the various DNA vaccines with (shaded bars) or without (empty bars) stimulation with the E6 peptide. The data are shown as mean ±s.d. (p&0.05).FIG. 3 demonstrates the characterization of IFNγ-secreting E7-specific CD8+ T cell precursors in mice vaccinated with recombinant DNA vaccines. One set of C57BL/6 mice (3 per group) was immunized intradermally via gene gun with 2 μg of the various recombinant DNA vaccines four times with one-week intervals. Another set of C57BL/6 mice (3 per group) was immunized intramuscularly with 50 μg of the various recombinant DNA vaccines four times with one-week intervals. The splenocytes from vaccinated mice were incubated with H-2Db-restricted E7 CTL peptide (aa 49-57) overnight. Determination of the E7-specific CD8+ T cells was performed by intracellular IFN-γ staining followed by flow cytometry analysis. FIG. 3A is a series of images providing representative flow cytometry data showing the number of E7-specific IFNγ+ CD8+ T cells in the mice vaccinated either intradermally via gene gun (left panel) or intramuscularly (right panel) with the various DNA vaccines. FIG. 3B is a bar graph showing the number of E7-specific IFNγ+ CD8+ T cells from mice immunized intradermally with the various DNA vaccines via gene gun with (shaded bars) or without (empty bars) stimulation with the E7 peptide. The data are shown as mean ±s.d. (p&0.05). FIG. 3C is a bar graph showing the number of E7-specific IFNγ+ CD8+ T cells from mice immunized intramuscularly with the various DNA vaccines with (shaded bars) or without (empty bars) stimulation with the E7 peptide. The data are shown as mean ±s.d. (p&0.05).FIG. 4 demonstrates in vivo tumor protection and treatment. FIG. 4A is a graph showing an in vivo tumor protection experiment as described herein. C57BL/6 mice (5 per group) were immunized intradermally with the various DNA vaccines via gene gun twice with a one-week interval. Seven days after the last immunization, the mice were challenged subcutaneously with 5×104 cells/mouse of TC-1 tumor cells. Tumor growth was monitored by visual inspection and palpation twice a week and examined by Kaplan-Meier analysis. FIG. 4B is a bar graph depicting the quantification of the number of pulmonary nodules in mice treated with the various DNA vaccines. C57BL/6 mice (5 per group) were challenged with 1×104/mouse of TC-1 cells via the tail vein. Three and ten days later, the mice were treated intradermally with the various recombinant DNA vaccines via gene gun. Mice were sacrificed 28 days after tumor challenge and the numbers of pulmonary tumor nodules were quantified and compared. The data are shown as mean ±SE. (p&0.01).FIG. 5 provides the characterization of antibody levels against full length L2 protein by ELISA. One group of C57BL/6 mice (3 per group) was immunized intradermally via gene gun with 2 μg of the various recombinant DNA vaccines four times with one-week intervals. Another group of C57BL/6 mice (3 per group) was immunized intramuscularly with 50 μg of the various recombinant DNA vaccines four times with one-week intervals. ELISA analysis was performed to determine the L2-specific antibody responses in vaccinated mice. FIG. 5A is a bar graph depicting the L2-specific antibody responses in mice vaccinated intradermally with the various DNA vaccines via gene gun. FIG. 5B is a bar graph depicting the L2-specific antibody responses in mice vaccinated intramuscularly with the various DNA vaccines. The results from 1:100, 1:500 and 1:1000 dilutions are represented, showing mean absorbance (OD 450 nm)±s.d. The data collected from all of the above experiments are from one representative experiment of two performed. Serum from a rabbit vaccinated with L2 was used as standard control.FIG. 6 shows the results of neutralization assays using HPV-16 pseudovirions as demonstrated by graphical representations of the neutralization activity in mice vaccinated with the various DNA vaccines. FIG. 6A shows vaccination using gene gun delivery. One set of C57BL/6 mice (3 per group) was immunized intradermally via gene gun with 2 μg of the various recombinant DNA vaccines four times with one-week intervals. FIG. 6B shows vaccination using intramuscular injection. Another set of C57BL/6 mice (3 per group) was immunized intramuscularly with 50 μg of the various recombinant DNA vaccines four times with one-week intervals. Neutralizing assays were performed using HPV-16 pseudovirion to determine the L2-specific neutralizing antibody responses in vaccinated mice. RG1 is the 17-36aa anti-peptide serum that is used as a positive control antibody that generates a neutralizing response. B6I is the non-neutralizing negative control antibody.DETAILED DESCRIPTIONCervical cancer is one of the most common cancers in women worldwide. Persistent infection with human papillomavirus (HPV) is considered to be the etiological factor for cervical cancer. Provided herein are effective vaccines against HPV infections that lead to the control of cervical cancer. Beneficially, HPV vaccines generate both humoral immune response to prevent new infections as well as cell-mediated immunity to eliminate established infection or HPV-related disease. Disclosed herein are preventive and therapeutic HPV DNA vaccines using human calreticulin (CRT), HPV16 early proteins E6 and E7, and the HPV late protein L2. The DNA vaccines described herein generate effective CTL and antibody responses by delivering antigens to APCs that stimulate CD4+ and CD8+ T cells. Compared to live viral or bacterial vectors, naked DNA plasmid vaccines are safe and can be easily administered. Furthermore, DNA vaccines are easy to prepare on a large scale with high purity and high stability and can be engineered to express antigenic peptides or proteins. Additionally, DNA has the unique ability to be maintained long term in an episomal form. This enables prolonged expression of antigens and enhancement of immunologic memory. Using DNA vaccines to express proteins bypasses MHC restriction and thus maintains higher CTL responses than current protein vaccines. DNA vaccines can also be repeatedly applied to the same patient safely and effectively, unlike live vector vaccines. These features make DNA vaccines a useful approach for HPV vaccine development. While DNA has considerable advantages, one major limitation is its limited potency. This is due to the fact that DNA vaccines, unlike viral vectors, lack the intrinsic ability to amplify in transfected cells.Calreticulin (CRT) is an abundant 46 kDa Ca2+-binding protein which is located in the endoplasmic reticulum (ER). CRT is considered to be related to the family of heat shock proteins (HSPs). This protein has been shown to associate with peptides delivered into the ER by transporters associated with antigen processing (TAP-1 and TAP-2) and with MHC class I-β2 microglobulin molecules to aid in antigen presentation. CRT has been previously employed to create effective DNA vaccines using HPV-16 E6, E7 or SARS-Co-V as target antigens. The inventors have shown that DNA vaccines encoding calreticulin (CRT) linked to the target antigen generate the high levels of antigen-specific CD8+ T-cell responses as well as significant antigen-specific humoral immunity. Thus, CRT is used in a HPV DNA vaccine development in order to generate strong T cell specific responses as well as humoral responses against the HPV antigens, E6, E7 and L2. The employment of E6, E7 and L2 protein of HPV16 has been previously explored in protein-based vaccines to generate a chimeric L2E7E6 fusion protein (also called TA-CIN). Vaccination of healthy volunteers with TA-CIN induced serum antibody that neutralizes across papillomavirus species.In the current invention, an HPV DNA vaccine is provided that encodes calreticulin linked to HPV16 early proteins, E6 and E7, and the late protein L2 (hCRTE6E7L2). It is demonstrated that vaccination with hCRTE6E7L2 DNA vaccine induces a potent E6/E7-specific CD8+ T cell immune response and results in a significant therapeutic effect against E6/E7-expressing tumor cells. Furthermore, vaccination with hCRTE6E7L2 generates significant L2-specific neutralizing antibody responses against HPV-16 pseudovirion infection. Thus, the hCRTE6E7L2 DNA vaccines generate potent preventive and therapeutic effects in vaccinated mice. However, the present invention is not limited to the exemplified antigens. Rather, one of skill in the art will appreciate that the same results are expected for any antigen (and epitopes thereof) for which a T cell-mediated response is desired. The response so generated will be effective in providing protective or therapeutic immunity, or both, directed to an organism or disease in which the epitope or antigenic determinant is involved—for example as a cell surface antigen of a pathogenic cell or an envelope or other antigen of a pathogenic virus, or a bacterial antigen, or an antigen expressed as or as part of a pathogenic molecule.Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art of this invention. As used herein, the following terms have the meanings ascribed to them unless specified otherwise.The term “antigen” or “immunogen” as used herein refers to a compound or composition or cell comprising a peptide, polypeptide or protein which is “antigenic” or “immunogenic” when administered in an appropriate amount (an “immunogenically effective amount”), i.e., capable of inducing, eliciting, augmenting or boosting a cellular and/or humoral immune response and of being recognized by the products of that response (T cells, antibodies). A nucleic acid such as DNA that encodes an immunogen and is used as a vaccine is referred to as a “DNA immunogen,” as the encoded polypeptide is expressed in vivo after administration of the DNA. An immunogen may be effective when given alone or in combination, or linked to, or fused to, another substance (which can be administered at one time or over several intervals). An immunogenic composition can comprise an antigenic peptide/polypeptide of at least about 5, or about 10 or about 15, or about 20 amino acids, etc. Smaller antigens may require presence of a “carrier” polypeptide e.g., as a fusion protein, aggregate, conjugate or mixture, preferably linked (chemically or otherwise) to the antigen to be immunogenic. The immunogen can be recombinantly expressed from a vaccine vector, which can be naked DNA which comprises the polypeptide immunogen's coding sequence operably linked to a promoter, e.g., an expression vector or cassette as described herein. The immunogen includes one or more antigenic determinants or epitopes which may vary in size from about 3 to about 15 or more amino acids.The term “epitope” as used herein refers to an antigenic determinant or antigenic site that interacts with an antibody or a T cell receptor (TCR), e.g., MHC class I-binding peptides used in the methods of the invention. The specific conformational or stereochemical “domain” to which an antibody or a TCR bind is an “antigenic determinant” or “epitope.” TCRs bind to peptide epitopes which are physically associated with a third molecule, a major histocompatibility complex (MHC) class I or class II protein.The term “recombinant” refers to (1) a nucleic acid or polynucleotide synthesized or otherwise manipulated in vitro or ex vivo, (2) methods of using recombinant DNA technology to produce gene products in cells or other biological systems, or (3) a polypeptide encoded by a recombinant nucleic acid. For example, the hCRTE6E7L2-encoding nucleic acid or polypeptide can be recombinant. “Recombinant means” includes ligation of nucleic acids having various coding regions or domains or promoter sequences from different sources into a single unit in the form of an expression cassette or vector for expression of the coding sequences in the vectors resulting in production of the encoded polypeptide. In one embodiment, the isolated or recombinant nucleic acid molecule is operatively linked to a promoter, such as, e.g., a constitutive, an inducible or a tissue-specific promoter. The promoter can be expressed in any cell, including cells of the immune system, including, e.g., antigen presenting cells (APCs), e.g., in a constitutive, an inducible or a tissue-specific manner. In alternative embodiments, the APCs are DCs, keratinocytes, astrocytes, monocytes, macrophages, B lymphocytes, a microglial cell, or activated endothelial cells, and the like. A “linker” is a moiety such as a nucleic acid or amino acid sequence that operably connects a first nucleic acid sequence with a second nucleic acid sequence, or connects a first amino acid sequence with a second amino acid sequence. Linkers may be cleavable by endonucleases or peptidases. Linkers may contain additional functionality, such as a label or attachment site. A nucleic acid linker may be from 1 to about 120 n an amino acid linker may be from 1 to about 60 amino acid residues in length.Vectors, Antigen, and IPP Nucleic Acids and PolypeptidesPlasmid SequencesPlasmids used herein include the pET28a and pNGVL4a vectors as described in Example 1. Any nucleic acid expression vector can be employed in the present invention. pNGVL4a, a preferred plasmid backbone for the present invention was originally derived from the pNGVL3 vector, which has been approved for human vaccine trials. The pNGVL4a vector includes two immunostimulatory sequences (tandem repeats of CpG dinucleotides) in the noncoding region. Whereas any other plasmid DNA that can transform either APCs, preferably DC's or other cells which, via cross-priming, transfer the antigenic moiety to DCs, is useful in the present invention, pNGFVLA4a is preferred because of the fact that it has already been approved for human therapeutic use.Antigen Polypeptide SequencesThe present invention includes a combined DNA vaccine composition that includes multiple DNA sequences encoding antigenic polypeptides, which are termed “DNA immunogens” herein. For example, a DNA vaccine includes a DNA sequence encoding an E6 or an E7 immunogen, a DNA sequence encoding L2, and a DNA sequence encoding a calreticulin. In another example, the DNA vaccine encodes calreticulin, E6, E7, and L2. In any DNA vaccine containing multiple DNA sequences, the relative order of the DNA sequences (and thus the order of the expressed polypeptides) can be altered by one of skill in the art so long as the immunogenicity of each polypeptide is not eliminated.DNA Encoding HPV E7The E7 nucleic acid sequence and amino acid sequence from HPV-16 are shown below (see Accession Number NC—001526) (SEQ ID NO: 3)atg cat gga gat aca cct aca ttg cat gaa tat atg tta gat ttg caa cca gag aca act60Met His Gly Asp Thr Pro Thr Leu His Glu Tyr Met Leu Asp Leu Gln Pro Glu Thr Thr20 gat ctc tac tgt tat gag caa tta aat gac agc tca gag gag gag gat gaa ata gat ggt120Asp Leu Tyr Cys Tyr Glu Gln Leu Asn Asp Ser Ser Glu Glu Glu Asp Glu Ile Asp Gly40 cca gct gga caa gca gaa ccg gac aga gcc cat tac aat att gta acc ttt tgt tgc aag180Pro Ala Gly Gln Ala Glu Pro Asp Arg Ala His Tyr Asn Ile Val Thr Phe Cys Cys Lys60 tgt gac tct acg ctt cgg ttg tgc gta caa agc aca cac gta gac att cgt act ttg gaa240Cys Asp Ser Thr Leu Arg Leu Cys Val Gln Ser Thr His Val Asp Ile Arg Thr Leu Glu80 gac ctg tta atg ggc aca cta gga att gtg tgc ccc atc tgt tct cag gat aag ctt297Asp Leu Leu Met Gly Thr Leu Gly Ile Val Cys Pro Ile Cys Ser Gln Asp Lys Leu99
In single letter code, the wild type E7 amino acid sequence is (SEQ ID NO:4)MHGDTPTLHE YMLDLQPETT DLYCYEQLND SSEEEDEIDG99 PAGQAEPDRA HYNIVTFCCK CDSTLRLCVQ STHVDIRTLE DLLMGTLGIV CPICSQDKL In another embodiment (See GenBank Accession No. AF125673, nucleotides 562-858 and the E7 amino acid sequence) the C-terminal four amino acids QDKL (and their codons) above are replaced with the three amino acids QKP (and the codons cag aaa cca) yielding a protein of 98 residues.When an oncoprotein or an epitope thereof is the immunizing moiety, it is preferable to reduce the tumorigenic risk of the vaccine itself. Because of the potential oncogenicity of the HPV E7 protein, the E7 protein is preferably used in a “detoxified” form.For example, to reduce oncogenic potential of E7 in a construct of this invention, one or more of the following positions of E7 is mutated:Preferrednt PositionOriginalMutantcodon(in SEQ IDAmino acid (inresidueresiduemutationNO: 3)SEQ ID NO: 4)CysGly (or Ala)TGT→GGT7024GluGly (or Ala)GAG→GGG7726(or GCG)CysGly (or Ala)TGC→GGC27191 The preferred E7 (detox) mutant sequence has the following two mutations: a TGT→GGT mutation resulting in a Cys→Gly substitution at position 24 of SEQ ID NO:4 a and GAG→GGG mutation resulting in a Glu→Gly substitution at position 26 of SEQ ID NO:4. This mutated amino acid sequence is shown below with the replacement residues underscored.(SEQ ID NO:5)MHGDTPTLHE YMLDLQPETT DLYGYEGLND SSEEEDEIDG97 PAGQAEPDRA HYNIVTFCCK CDSTLRLCVQ STHVDIRTLE DLLMGTLGIV CPICSQKP These substitutions completely eliminate the capacity of the E7 to binding capacity to Rb, and thereby nullify its transforming activity.Any nucleotide sequence that encodes encoding the above E7 or E7 (detox) polypeptide, or an antigenic fragment or epitope thereof, can be used in the present compositions and methods.DNA Encoding HPV E6The wild type E6 nucleotide (SEQ ID NO:6) and amino acid (SEQ ID NO:7) sequences are shown below (see GenBank accession #'s K02718 and NC—001526)):atg cac caa aag aga act gca atg ttt cag gac cca cag gag cga ccc aga aag tta cca60Met His Gln Lys Arg Thr Ala Met Phe Gln Asp Pro Gln Glu Arg Pro Arg Lys Leu Pro20 cag tta tgc aca gag ctg caa aca act ata cat gat ata ata tta gaa tgt gtg tac tgc120Gln Leu Cys Thr Glu Leu Gln Thr Thr Ile His Asp Ile Ile Leu Glu Cys Val Tyr Cys40 aag caa cag tta ctg cga cgt gag gta tat gac ttt gct ttt cgg gat tta tgc ata gta180Lys Gln Gln Leu Leu Arg Arg Glu Val Tyr Asp Phe Ala Phe Arg Asp Leu Cys Ile Val60 tat aga gat ggg aat cca tat gct gta tgt gat aaa tgt tta aag ttt tat tct aaa att240Tyr Arg Asp Gly Asn Pro Tyr Ala Val Cys Asp Lys Cys Leu Lys Phe Tyr Ser Lys Ile80 agt gag tat aga cat tat tgt tat agt ttg tat gga aca aca tta gaa cag caa tac aac300Ser Glu Tyr Arg His Tyr Cys Tyr Ser Leu Tyr Gly Thr Thr Leu Glu Gln Gln Tyr Asn100 aaa ccg ttg tgt gat ttg tta att agg tgt att aac tgt caa aag cca ctg tgt cct gaa360Lys Pro Leu Cys Asp Leu Leu Ile Arg Cys Ile Asn Cys Gln Lys Pro Leu Cys Pro Glu120 gaa aag caa aga cat ctg gac aaa aag caa aga ttc cat aat ata agg ggt cgg tgg acc420Glu Lys Gln Arg His Leu Asp Lys Lys Gln Arg Phe His Asn Ile Arg Gly Arg Trp Thr140 ggt cga tgt atg tct tgt tgc aga tca tca aga aca cgt aga gaa acc cag ctg taa474Gly Arg Cys Met Ser Cys Cys Arg Ser Ser Arg Thr Arg Arg Glu Thr Gln Leu stop158 The wild type HPV E6 amino acid sequence (see GenBank Accession Number NC—001526) (SEQ ID NO:7) is shown below. This sequence has 158 amino acids.MHQKRTAMFQ DPQERPRKLP QLCTELQTTI HDIILECVYC158 KQQLLRREVY DFAFRDLCIV YRDGNPYAVC DKCLKFYSKI SEYRHYCYSL YGTTLEQQYN KPLCDLLIRC INCQKPLCPE EKQRHLDKKQ RFHNIRGRWT GRCMSCCRSS RTRRETQL E6 proteins from cervical cancer-associated HPV types such as HPV-16 induce proteolysis of the p53 tumor suppressor protein through interaction with E6-AP. Human mammary epithelial cells (MECs) immortalized by E6 display low levels of p53. HPV-16 E6 as well as other cancer-related papillomavirus E6 proteins also binds the cellular protein E6BP (ERC-55). As with E7, it is preferred to used a non-oncogenic mutated form of E6, referred to as “E6 (detox).” Several different E6 mutations and publications describing them are discussed below.The preferred amino acid residues to be mutated are underscored in the E6 amino acid sequence above. Some studies of E6 mutants are based upon a shorter E6 protein of 151 nucleic acids, wherein the N-terminal residue was considered to be the Met at position 8 in SEQ ID NO: 7 above. That shorter version of E6 is shown below as SEQ ID NO:8.MFQDPQERPR KLPQLCTELQ TTIHDIILEC VYCKQQLLRR EVYDFAFRDL CIVYRDGNPY AVCDKCLKFY SKISEYRHYC YSLYGTTLEQ QYNKPLCDLL IRCINCQKPL CPEEKQRHLD KKQRFHNIRG RWTGRCMSCC RSSRTRRETQ L To reduce oncogenic potential of E6 in a construct of this invention, one or more of the following positions of E6 is mutated:OriginalMutantaa position inaa position inresidueresidueSEQ ID NO: 7SEQ ID NO: 8CysGly (or Ala)7063CysGly (or Ala)113106IleThr135128 Nguyen M et al., J Virol. 6:02, described a mutant of HPV-16 E6 deficient in binding α-helix partners, which displays reduced oncogenic potential in vivo. This mutant, that involves a replacement of Ile with Thr as position 128 (of SEQ ID NO:8), may be used in accordance with the present invention to make an E6 DNA vaccine that has a lower risk of being oncogenic. This E6(I128T) mutant is defective in its ability to bind at least a subset of α-helix partners, including E6AP, the ubiquitin ligase that mediates E6-dependent degradation of the p53 protein.Cassetti M C et al., Vaccine 22:520-52, 2004, examined the effects of mutations of four or five amino acid positions in E6 and E7 to inactivate their oncogenic potential. The following mutations were examined: E6-C63G and E6 C106 G (positions based on SEQ ID NO:8); E7-C24 G, E7-E26G, and E7 C91G (positions based on SEQ ID NO:4). Venezuelan equine encephalitis virus replicon particle (VRP) vaccines encoding mutant or wild type E6 and E7 proteins elicited comparable CTL responses and generated comparable antitumor responses in several HPV16 E6(+)E7(+) tumor challenge models: protection from either C3 or TC-1 tumor challenge was observed in 100% of vaccinated mice. Eradication of C3 tumors was observed in approximately 90% of the mice. The predicted inactivation of E6 and E7 oncogenic potential was confirmed by demonstrating normal levels of both p53 and Rb proteins in human mammary epithelial cells infected with VRPs expressing mutant E6 and E7 genes.The HPV16 E6 protein contains two zinc fingers important for st one cysteine (C) amino acid position in each pair of C-X-X-C (where X is any amino acid) zinc finger motifs are preferably was mutated at E6 positions 63 and 106 (based on SEQ ID NO:8). Mutants are created, for example, using the Quick Change Site-Directed Mutagenesis Kit (Stratagene, La Jolla, Calif.). HPV16 E6 containing a single point mutation in the codon for Cys106 in SEQ ID NO:8 (=Cys 113 in SEQ ID NO:7). Cys106 neither binds nor facilitates degradation of p53 and is incapable of immortalizing human mammary epithelial cells (MEC), a phenotype dependent upon p53 degradation. A single amino acid substitution at position Cys63 of SEQ ID NO:8 (=Cys70 in SEQ ID NO:7) destroys several HPV16 E6 functions: p53 degradation, E6TP-1 degradation, activation of telomerase, and, consequently, immortalization of primary epithelial cells.Any nucleotide sequence that encodes this E6 polypeptide, or preferably, one of the mutants thereof discussed herein, or an antigenic or immunogenic fragment or epitope thereof, can be used in the present invention. Other mutations can be tested and used in accordance with the methods described herein including those described in Cassetti et al., supra. These mutations can be produced from any appropriate starting sequences by mutation of the coding DNA.The present invention also includes the use of a tandem E6-E7 vaccine, using one or more of the mutations described herein to render the oncoproteins inactive with respect to their oncogenic potential in vivo. VRP vaccines (described in Cassetti et al., supra) include fused E6 and E7 genes in one open reading frame which are mutated at four or five amino acid positions. Thus, the present constructs may include one or more epitopes of E6 and E7, which may be arranged in their native order or shuffled in any way that permits the expressed protein to bear the E6 and E7 antigenic epitopes in an immunogenic form. DNA encoding amino acid spacers between E6 and E7 or between individual epitopes of these proteins may be introduced into the vector, provided again, that the spacers permit the expression or presentation of the epitopes in an immunogenic manner after they have been expressed by transduced host cells.DNA Encoding HPV L2Wild type L2 nucleotide (SEQ ID NO:9) and amino acid (SEQ ID NO:10) sequences are shown below:Wild type L2 nucleotide sequence obtained from HPV type 16 genomic DNA (see GenBank Accession No. K02718) includes the following sequence as SEQ ID NO: 9:atgcga cacaaacgtt ctgcaaaacg cacaaaacgt gcatcggcta cccaacttta taaaacatgc aaacaggcag gtacatgtcc acctgacatt atacctaagg ttgaaggcaa aactattgct gaacaaatat tacaatatgg aagtatgggt gtattttttg gtgggttagg aattggaaca gggtcgggta caggcggacg cactgggtat attccattgg gaacaaggcc tcccacagct acagatacac ttgctcctgt aagaccccct ttaacagtag atcctgtggg cccttctgat ccttctatag tttctttagt ggaagaaact agttttattg atgctggtgc accaacatct gtaccttcca ttcccccaga tgtatcagga tttagtatta ctacttcaac tgataccaca cctgctatat tagatattaa taatactgtt actactgtta ctacacataa taatcccact ttcactgacc catctgtatt gcagcctcca acacctgcag aaactggagg gcattttaca ctttcatcat ccactattag tacacataat tatgaagaaa ttcctatgga tacatttatt gttagcacaa accctaacac agtaactagt agcacaccca taccagggtc tcgcccagtg gcacgcctag gattatatag tcgcacaaca caacaggtta aagttgtaga ccctgctttt gtaaccactc ccactaaact tattacatat gataatcctg catatgaagg tatagatgtg gataatacat tatatttttc tagtaatgat aatagtatta atatagctcc agatcctgac tttttggata tagttgcttt acataggcca gcattaacct ctaggcgtac tggcattagg tacagtagaa ttggtaataa acaaacacta cgtactcgta gtggaaaatc tataggtgct aaggtacatt attattatga tttaagtact attgatcctg cagaagaaat agaattacaa actataacac cttctacata tactaccact tcacatgcag cctcacctac ttctattaat aatggattat atgatattta tgcagatgac tttattacag atacttctac aaccccggta ccatctgtac cctctacatc tttatcaggt tatattcctg caaatacaac aattcctttt ggtggtgcat acaatattcc tttagtatca ggtcctgata tacccattaa tataactgac caagctcctt cattaattcc tatagttcca gggtctccac aatatacaat tattgctgat gcaggtgact tttatttaca tcctagttat tacatgttac gaaaacgacg taaacgttta ccatattttt tttcagatgt ctctttggct gcctag Wild type L2 amino acid sequence (see GenBank Accession Numbers AAD33258) includes the following sequence as SEQ ID NO: 10: 1 mrhkrsakrt krasatqlyk tckqagtcpp diipkvegkt iadqilqygs mgvffgglgi
61 gtgsgtggrt gyiplgtrpp tatdtlapvr ppltvdpvgp sdpsivslve etsfidagap 121 tsvpsippdv sgfsittstd ttpaildinn tvttvtthnn ptftdpsvlq pptpaetggh 181 ftlssstist hnyeeipmdt fivstnpntv tsstpipgsr pvarlglysr ttqqvkvvdp 241 afittptkli tydnpayegi dvdntlyfss ndnsiniapd pdfldivalh rpaltsrrtg 301 irysrignkq tlrtrsgksi gakvhyyydf stidsaeeie lqtitpstyt ttshaalpts 361 innglydiya ddfitdtstt pvpsvpstsl sgyipantti pfggaynipl vsgpdipini 421 tdqapslipi vpgspqytii adagdfylhp syymlrkrrk rlpyffsdvs laa The present inventors and their colleagues have described HPV L2 nucleic acids and polypeptides, and fragments thereof. See e.g., PCT Publication No. WO, and U.S. Pat. No. 6,599,739. Further, synthetic or recombinant L2 nucleic acids are provided herein. See, e.g., GenBank Accession No. AJ313180. Variant L1 and L2 genes are provided. See, e.g., GenBank Accession No. U37217.DNA Encoding Calreticulin (CRT)The present inventors and their colleagues have described the use of calreticulin in DNA vaccines. See Cheng W F et al., Vaccine. 23:05. As discussed herein and in the references, DNA vaccines encoding CRT linked either to E6 or to E7 generate significant antitumor effects against E6- and E7-expressing tumors, respectively. Moreover, simultaneous vaccination with both CRT/E6 and CRT/E7 DNA vaccines generated significant E6- and E7-specific T-cell immune responses and significantly better therapeutic antitumor effects against E6- and E7-expressing tumors than vaccination with either CRT/E6 DNA or CRT/E7 DNA alone.The three domains of CRT also produce E7-specific antitumor immunity and antiangiogenic effects (Cheng W F et al., Vaccine. 23:05). DNA vaccines encoding each of N, P, and C domains of CRT linked to E7 antigen produced significant stimulation of E7-specific CD8+ T cell precursors and antitumor effects against E7-expressing tumors. The N domain of CRT also showed antiangiogenic properties that might have contributed to the antitumor effect. Thus, the present invention includes DNA immunogens expressing the N, P, or C domain of CRT, or a combination thereof.“Calreticulin” or “CRT” describes the well-characterized ~46 kDa resident protein of the ER lumen that has lectin activity and participates in the folding and assembly of nascent glycoproteins. CRT acts as a “chaperone” polypeptide and a member of the MHC class I transporter TAP CRT associates with TAP1 and TAP2 transporters, tapasin, MHC Class I heavy chain polypeptide and β2 microglobulin to function in the loading of peptide epitopes onto nascent MHC class I molecules (Jorgensen, Eur. J. Biochem. 267:02). The term “calreticulin” or “CRT” refers to polypeptides and nucleic acids molecules having substantial identity (defined herein) to the exemplary CRT sequences as described herein. A CRT polypeptide is a polypeptides comprising a sequence identical to or substantially identical to the amino acid sequence of CRT. An exemplary nucleotide and amino acid sequence for a CRT used in the present compositions and methods are presented herein. The terms “calreticulin” or “CRT” encompass native proteins as well as recombinantly produced modified proteins that induce an immune response, including a CTL response. CRT encompasses homologues and allelic variants of CRT, including variants of native proteins constructed by in vitro techniques, and proteins isolated from natural sources. The CRT polypeptides of the invention, and sequences encoding them, also include fusion proteins including non-CRT sequences, particularly MHC class I- and also further including other domains, e.g., epitope tags, enzyme cleavage recognition sequences, signal sequences, secretion signals and the like.The term “endoplasmic reticulum chaperone polypeptide” as used herein means any polypeptide having substantially the same ER chaperone function as the exemplary chaperone proteins CRT, tapasin, ER60 or calnexin. Thus, the term includes all functional fragments or variants or mimics thereof. A polypeptide or peptide can be routinely screened for its activity as an ER chaperone using assays known in the art, such as that set forth in Example 1 of U.S. patent application Ser. No. 11/773,162, filed Jul. 3, 2007. While the invention is not limited by any particular mechanism of action, in vivo chaperones promote the correct folding and oligomerization of many glycoproteins in the ER, including the assembly of the MHC class I heterotrimeric molecule (heavy (H) chain, β2m, and peptide). They also retain incompletely assembled MHC class I heterotrimeric complexes in the ER (Hauri FEBS Lett. 476:32-37, 2000).The sequences of CRT, including human CRT, are well known in the art (McCauliffe, J. Clin. Invest. 86:332-5, 1990; Burns, Nature 367:476-80, 1994; Coppolino, Int. J. Biochem. Cell Biol 30:553-8, 2000). The nucleic acid sequence appears as GenBank Accession No. NM 004343 and is SEQ ID NO:11. 1 gtccgtactg cagagccgct gccggagggt cgttttaaag ggccgcgttg ccgccccctc
61 ggcccgccat gctgctatcc gtgccgctgc tgctcggcct cctcggcctg gccgtcgccg
121 agcccgccgt ctacttcaag gagcagtttc tggacggaga cgggtggact tcccgctgga
181 tcgaatccaa acacaagtca gattttggca aattcgttct cagttccggc aagttctacg
241 gtgacgagga gaaagataaa ggtttgcaga caagccagga tgcacgcttt tatgctctgt
301 cggccagttt cgagcctttc agcaacaaag gccagacgct ggtggtgcag ttcacggtga
361 aacatgagca gaacatcgac tgtgggggcg gctatgtgaa gctgtttcct aatagtttgg
421 accagacaga catgcacgga gactcagaat acaacatcat gtttggtccc gacatctgtg
481 gccctggcac caagaaggtt catgtcatct tcaactacaa gggcaagaac gtgctgatca
541 acaaggacat ccgttgcaag gatgatgagt ttacacacct gtacacactg attgtgcggc
601 cagacaacac ctatgaggtg aagattgaca acagccaggt ggagtccggc tccttggaag
661 acgattggga cttcctgcca cccaagaaga taaaggatcc tgatgcttca aaaccggaag
721 actgggatga gcgggccaag atcgatgatc ccacagactc caagcctgag gactgggaca
781 agcccgagca tatccctgac cctgatgcta agaagcccga ggactgggat gaagagatgg
841 acggagagtg ggaaccccca gtgattcaga accctgagta caagggtgag tggaagcccc
901 ggcagatcga caacccagat tacaagggca cttggatcca cccagaaatt gacaaccccg
961 agtattctcc cgatcccagt atctatgcct atgataactt tggcgtgctg ggcctggacc 1021 tctggcaggt caagtctggc accatctttg acaacttcct catcaccaac gatgaggcat 1081 acgctgagga gtttggcaac gagacgtggg gcgtaacaaa ggcagcagag aaacaaatga 1141 aggacaaaca ggacgaggag cagaggctta aggaggagga agaagacaag aaacgcaaag 1201 aggaggagga ggcagaggac aaggaggatg atgaggacaa agatgaggat gaggaggatg 1261 aggaggacaa ggaggaagat gaggaggaag atgtccccgg ccaggccaag gacgagctgt 1321 agagaggcct gcctccaggg ctggactgag gcctgagcgc tcctgccgca gagcttgccg 1381 cgccaaataa tgtctctgtg agactcgaga actttcattt ttttccaggc tggttcggat 1441 ttggggtgga ttttggtttt gttcccctcc tccactctcc cccaccccct ccccgccctt 1501 tttttttttt tttttaaact ggtattttat cctttgattc tccttcagcc ctcacccctg 1561 gttctcatct ttcttgatca acatcttttc ttgcctctgt gccccttctc tcatctctta 1621 gctcccctcc aacctggggg gcagtggtgt ggagaagcca caggcctgag atttcatctg 1681 ctctccttcc tggagcccag aggagggcag cagaaggggg tggtgtctcc aaccccccag 1741 cactgaggaa gaacggggct cttctcattt cacccctccc tttctcccct gcccccagga 1801 ctgggccact tctgggtggg gcagtgggtc ccagattggc tcacactgag aatgtaagaa 1861 ctacaaacaa aatttctatt aaattaaatt ttgtgtctc1899
Human CRT protein (GenBank Accession No. NM 004343), is shown below as SEQ ID NO: 12:
1 MLLSVPLLLG LLGLAVAEPA VYFKEQFLDG DGWTSRWIES KHKSDFGKFV LSSGKFYGDE
61 EKDKGLQTSQ DARFYALSAS FEPFSNKGQT LVVQFTVKHE QNIDCGGGYV KLFPNSLDQT 121 DMHGDSEYNI MFGPDICGPG TKKVHVIFNY KGKNVLINKD IRCKDDEFTH LYTLIVRPDN 181 TYEVKIDNSQ VESGSLEDDW DFLPPKKIKD PDASKPEDWD ERAKIDDPTD SKPEDWDKPE 241 HIPDPDAKKP EDWDEEMDGE WEPPVIQNPE YKGEWKPRQI DNPDYKGTWI HPEIDNPEYS 301 PDPSIYAYDN FGVLGLDLWQ VKSGTIFDNF LITNDEAYAE EFGNETWGVT KAAEKQMKDK 361 QDEEQRLKEE EEDKKRKEEE EAEDKEDDED KDEDEEDEED KEEDEEEDVP GQAKDEL417 Nucleic Acids Encoding Calreticulin, E6 E7 and L2Shown below as SEQ ID NO: 1 is a nucleic acid sequence of the invention that contains nucleic acid sequences encoding calreticulin (nucleotides 1-1251), E6 (), E7 () and L2 (). Additionally, SEQ ID NO: 1 contains two linker sequences (nucleotides
and ). | 10 | 20 | 30 | 40 | 50 | 60 | 70 | 80 1 atgctgctat ccgtgccgct gctgctcggc ctcctcggcc tggccgtcgc cgagcctgcc gtctacttca aggagcagtt80
81 tctggacggg gacgggtgga cttcccgctg gatcgaatcc aaacacaagt cagattttgg caaattcgtt ctcagttccg160
161 gcaagttcta cggtgacgag gagaaagata aaggtttgca gacaagccag gatgcacgct tttatgctct gtcggccagt240
241 ttcgagcctt tcagcaacaa aggccagacg ctggtggtgc agttcacggt gaaacatgag cagaacatcg actgtggggg320
321 cggctatgtg aagctgtttc ctaatagttt ggaccagaca gacatgcacg gagactcaga atacaacatc atgtttggtc400
401 ccgacatctg tggccctggc accaagaagg ttcatgtcat cttcaactac aagggcaaga acgtgctgat caacaaggac480
481 atccgttgca aggatgatga gtttacacac ctgtacacac tgattgtgcg gccagacaac acctatgagg tgaagattga560
561 caacagccag gtggagtccg gctccttgga agacgattgg gacttcctgc cacccaagaa gataaaggat cctgatgctt640
641 caaaaccgga agactgggat gagcgggcca agatcgatga tcccacagac tccaagcctg aggactggga caagcccgag720
721 catatccctg accctgatgc taagaagccc gaggactggg atgaagagat ggacggagag tgggaacccc cagtgattca800
801 gaaccctgag tacaagggtg agtggaagcc ccggcagatc gacaacccag attacaaggg cacttggatc cacccagaaa880
881 ttgacaaccc cgagtattct cccgatccca gtatctatgc ctatgataac tttggcgtgc tgggcctgga cctctggcag960
961 gtcaagtctg gcaccatctt tgacaacttc ctcatcacca acgatgaggc atacgctgag gagtttggca acgagacgtg1040 1041 gggcgtaaca aaggcagcag agaaacaaat gaaggacaaa caggacgagg agcagaggct taaggaggag gaagaagaca1120 1121 agaaacgcaa agaggaggag gaggcagagg acaaggagga tgatgaggac aaagatgagg atgaggagga tgaggaggac1200 1201 aaggaggaag atgaggagga agatgtcccc ggccaggcca aggacgagct gGAATTCATG CACCAGAAGA GAACCGCCAT1280 1281 GTTCCAGGAC CCCCAGGAGA GACCCAGGAA GCTGCCCCAG CTGTGCACCG AGCTGCAGAC CACCATCCAC GACATCATCC1360 1361 TGGAGTGCGT GTACTGCAAG CAGCAGCTGC TGAGGAGAGA GGTGTACGAC TTCGCCTTCC GGGACCTGTG CATCGTGTAC1440 1441 AGAGACGGCA ACCCCTACGC CGTGGGCGAC AAGTGCCTGA AGTTCTACAG CAAGATCAGC GAGTACAGAC ACTACTGCTA1520 1521 CAGCCTGTAC GGCACCACCC TGGAGCAGCA GTACAACAAG CCCCTGTGCG ACCTGCTGAT CCGGTGCATC AACGGCCAGA1600 1601 AGCCCCTGTG CCCCGAGGAG AAGCAGCGGC ACCTGGACAA GAAGCAGAGA TTCCACAACA TCAGGGGCCG GTGGACCGGC1680 1681 AGatgcatga gctgctgcag gagcagccgg accagacggg agacccagct gatgcacggc gacaccccca ccctgcacga1760 1761 gtacatgctg gacctgcagc cagagaccac cgacctgtac ggctacggcc agctgaacga cagcagcgag gaggaggacg1840 1841 agatcgacgg ccccgccggc caggccgagc ccgaccgggc ccactacaac atcgtgacct tctgctgcaa gtgcgacagc1920 1921 accctgaggc tgtgcgtgca gagcacccac gtggacatca gaaccctgga ggacctgctg atgggcaccc tgggcatcgt2000 2001 gtgccccatc tgcagccaga agcccTCTAG AATGAAGAGG GCCAGCGCCA CCCAGCTGTA CAAGACCTGC AAGCAGGCCG2080 2081 GCACCTGCCC CCCCGACATC ATCTCCAAGG TGGAGGGCAA GACCATCGCC GACCAGATCC TGCAGTACGG CAGCATGGGC2160 2161 GTGTTCTTCG GCGGCCTGGG CATCGGCACC GGCAGCGGCA CCGGCGGCAG GACCGGCTAC ATCCCCCTGG GCACCAGGCC2240 2241 CCCCACCGCC ACCGACACCC TGGCCCCCGT GAGGCCCCCC CTGACCGTGG ACCCCGTGGG CCCCAGCGAC CCCAGCATCG2320 2321 TGAGCCTGGT GGAGGAGACC AGCTTCATCG ACGCCGGCGC CCCCACCAGC GTGCCCAGCA TCCCCCCCGA CGTGAGCGGC2400 2401 TTCAGCATCA CCACCAGCAC CGACACCACC CCCGCCATCC TGGACATCAA CAACACCGTG ACCACCGTGA CCACCCACAA2480 2481 CAACCCCACC TTCACCGACC CCAGCGTGCT GCAGCCCCCC ACCCCCGCCG AGACCGGCGG CCACTTCACC CTGAGCAGCA2560 2561 GCACCATCAG CACCCACAAC TACGAGGAGA TCCCCATGGA CACC2604 | 10 | 20 | 30 | 40 | 50 | 60 | 70 | 80 Polypeptide Antigens Contains Calreticulin, E6, E7 and L2Shown below as SEQ ID NO: 2 is an amino acid sequence of the invention that contains nucleic acid sequences encoding calreticulin (amino acid residues 1-417), E6 (420-577), E7 (578-675) and L2 (678-868). Additionally, SEQ ID NO: 2 contains two amino acid linker peptides (amino acid residues 418-419 and 676-677). 10 | 20 | 30 | 40 | 50 | 60 | 70 | 80 1 mllsvplllg llglavaepa vyfkeqfldg dgwtsrwies khksdfgkfv lssgkfygde ekdkglqtsq darfyalsas80
81 fepfsnkgqt lvvqftvkhe qnidcgggyv klfpnsldqt dmhgdseyni mfgpdicgpg tkkvhvifny kgknvlinkd160 161 irckddefth lytlivrpdn tyevkidnsq vesgsleddw dflppkkikd pdaskpedwd erakiddptd skpedwdkpe240 241 hipdpdakkp edwdeemdge weppviqnpe ykgewkprqi dnpdykgtwi hpeidnpeys pdpsiyaydn fgvlgldlwq320 321 vksgtifdnf litndeayae efgnetwgvt kaaekqmkdk qdeeqrlkee eedkkrkeee eaedkedded kdedeedeed400 401 keedeeedvp gqakdelefM HQKRTAMFQD PQERPRKLPQ LCTELQTTIH DIILECVYCK QQLLRREVYD FAFRDLCIVY480 481 RDGNPYAVGD KCLKFYSKIS EYRHYCYSLY GTTLEQQYNK PLCDLLIRCI NGQKPLCPEE KQRHLDKKQR FHNIRGRWTG560 561 RCMSCCRSSR TRRETQLmhg dtptlheyml dlqpettdly gygqlndsse eedeidgpag qaepdrahyn ivtfcckcds640 641 tlrlcvqsth vdirtledll mgtlgivcpi csqkpSRMKR ASATQLYKTC KQAGTCPPDI ISKVEGKTIA DQILTYGSMG720 721 VFFGGLGIGT GSGTGGRTGY IPLGTRPPTA TDTLAPVRPP LTVDPVGPSD PSIVSLVEET SFIDAGAPTS VPSIPPDVSG800 801 FSITTSTDTT PAILDINNTV TTVTTHNNPT FTDPSVLQPP TPAETGGHFT LSSSTISTHN YEEEIPMDT868 | 10 | 20 | 30 | 40 | 50 | 60 | 70 | 80
Other Antigens Associated with Pathogens A major use for the present invention is as a therapeutic vaccine for cancer and for major chronic viral infections that cause morbidity and mortality worldwide. Such vaccines are designed to eliminate infected cells, which requires a T cell response. The vaccines of the present invention are designed to meet these needs.Preferred antigens are epitopes of pathogenic microorganisms against which the host is defended by effector T cells responses, including CTL and delayed type hypersensitivity. Thus, the types of antigens included in the vaccine compositions of this invention are any of those associated with such pathogens (in addition to tumor-specific antigens).The two most common cancers worldwide, hepatoma and cervical cancer, are associated with viral infection. Hepatitis B virus (HBV) (Beasley, R. P. et al., Lancet 2: (1981) has been implicated as etiologic agent of hepatomas. 80-90% of cervical cancers express the E6 and E7 antigens (discussed above and exemplified herein) from one of four “high risk” human papillomavirus types: HPV-16, HPV-18, HPV-31 and HPV-45 (Gissmann, L. et al., Ciba Found Symp. 120:190-207, 1986; Beaudenon, S., et al. Nature 321:246-9, 1986). As described herein, the HPV E6 and E7 antigens are the most promising targets for virus associated cancers in immunocompetent individuals because of their ubiquitous expression in cervical cancer. In addition to their importance as targets for therapeutic cancer vaccines, virus associated tumor antigens are also ideal candidates for prophylactic vaccines. Indeed, introduction of prophylactic HBV vaccines in Asia have decreased the incidence of hepatoma (Chang, M H et al. New Engl. J. Med. 336,
(1997), representing a great impact on cancer prevention.Additional viruses associated with chronic human viral infections are hepatitis C Virus (HCV), human immunodeficiency virus (HIV-1 and HIV-2), herpesviruses such as Epstein Barr Virus (EBV), cytomegalovirus (CMV) and HSV-1 and HSV-2, and influenza virus. Useful antigens include HBV surface antigen or HBV ppUL83 or pp 89 of CMV; antigens of gp120, gp41 or p24 proteins of HIV-1; ICP27, gD2, gB of HSV; or influenza hemagglutinin or nucleoprotein (Anthony, L S et al., Vaccine -83). Other antigens associated with pathogens that can be utilized as described herein are antigens of various parasites, includes malaria, preferably malaria peptide based on repeats of NANP.In addition to its applicability to human cancer and infectious diseases, the present invention is also intended for use in treating animal diseases in the veterinary medicine context. Thus, the approaches described herein may be readily applied by one skilled in the art to treatment of veterinary infections including equine herpesviruses, bovine viruses such as bovine viral diarrhea virus (for example, the E2 antigen), bovine herpesviruses, Marek's disease virus in chi animal retroviral and lentiviral diseases (e.g., feline leukemia, feline immunodeficiency, simian immunodeficiency viruses, etc.); pse and the like.As for tumor antigens, any tumor-associated or tumor-specific antigen that can be recognized by T cells, preferably by CTL, can be used. These include, without limitation, mutant p53, HER2/neu or a peptide thereof, or any of a number of melanoma-associated antigens such as MAGE-1, MAGE-3, MART-1/Melan-A, tyrosinase, gp75, gp100, BAGE, GAGE-1, GAGE-2, GnT-V, and p15 (see, for example, U.S. Pat. No. 6,187,306).General Recombinant DNA MethodsBasic texts disclosing general methods of molecular biology, all of which are incorporated by reference, include: Sambrook, J et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press, Cold Spring H}