子宫内的DNA疫苗接种：诱导新生儿保护性免疫的新方法

以下为《子宫内的DNA疫苗接种：诱导新生儿保护性免疫的新方法》的无排版文字预览，完整内容请下载

Vaccine 22 (2004) 1717–1727 DNA vaccination in utero: a new approach to induce protective immunity in the newborn Volker Gerdts∗, Cemaine Tsang, Philip J. Griebel, Lorne A. Babiuk Vaccine and Infectious Disease Organization, University of Saskatchewan, 120 Veterinary Road, Saskatoon, Sask., Canada S7N 5E3 Abstract Infectious diseases are the primary cause of neonatal morbidity and mortality in people, resulting in millions of deaths every year. Infection of the newborn with some of the pathogens involved, such as herpes simplex virus (HSV), human immunode?ciency virus (HIV), hepatitis B virus (HBV), human cytomegalovirus (HCMV) or group B Streptococcus sp. (GBS), usually occurs at the end of pregnancy, during birth or by breast feeding. Therefore, active immunization of the fetus might represent an effective approach to reduce the high risk of neonatal diseases. We recently showed that DNA immunization in utero within the third trimester of gestation induced strong humoral and cell-mediated immune responses in immunized fetal lambs. Here, we demonstrate that fetal immunization was safe and did not affect fetal gestation, neonatal viability, or signi?cantly alter blood leukocyte populations. In utero immunization resulted in the induction of protective mucosal immunity and immune memory in the newborn lamb. Furthermore, there was no evidence that in utero DNA immunization induced immune tolerance. Our results also indicate that the uptake and expression of the plasmid DNA already occurred within the epithelium of the oral cavity. This correlates with our previous ?ndings that local immune responses were found exclusively in the retropharyngeal lymph nodes draining the oral cavity. © 2004 Elsevier Ltd. All rights reserved. Keywords: DNA vaccination; Fetal immunization; In utero; Newborn; Herpesvirus; Hepatitis B; Mucosal immunity 1. Introduction Early life infections with pathogens such as herpes simplex virus (HSV), human immunode?ciency virus (HIV), hepatitis B virus (HBV), group B Streptococcus sp. (GBS), Haemophilus sp. or Chlamydia sp. are responsible for signi?cant morbidity and mortality in large numbers of human infants every year (reviewed in [1,2]). Disease transmission from mother to infant frequently occurs shortly before, during or after birth by early rupture of the amniotic membranes or direct contact with infectious secretions during labor. Infections can also result from non-sterile delivery techniques, by breast feeding or during the ?rst days of life in a perinatal nursery. To reduce the risk of infection, improved neonatal care together with caesarian sections, antibiotic treatments, and maternal antiviral therapy are currently being used to prevent vertical disease transmission. However, there is a signi?cant need for alternative therapeutic approaches to prevent neonatal infection. Vaccination has provided a very cost-effective approach to prevent infectious diseases. However, the induction of ∗ Corresponding author. Tel.: +1-***; fax: +1-***. E-mail address: gerdts@sask.usask.ca (V. Gerdts). 0264-410X/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2003.05.004 tolerance or a state of non-responsiveness was previously thought to preclude vaccination as an effective therapy in the fetus or newborn [3]. We recently demonstrated that DNA immunization in utero induced strong immunity in fetal lambs when delivered into the oral cavity into the amniotic ?uid at days 120–125 of gestation [4,5] (gestation period of sheep is 145 days). Strong humoral and cell-mediated immune responses were found in all immunized fetuses following a single administration of 500 $.g plasmid DNA encoding the truncated version of glycoprotein gD of bovine herpesvirus-1 (BHV-1). Additionally, antigen (Ag)-speci?c antibody-secreting cells were found in the retropharyngeal lymph node draining the oral cavity. Thus, we showed for the ?rst time that in utero immunization with plasmid DNA induces strong immunity in the fetus, which could have a signi?cant impact on survival and quality of life for the large number of infants infected during or shortly after birth [4,5]. However, since manipulations in utero could result in abortions or fetal misdevelopment, we addressed the safety of in utero DNA vaccination by analyzing fetal development, neonatal viability and development of the neonatal immune system. Fetal lambs represent a valuable model for analyzing in utero vaccination because of a number of physiological 1718 V. Gerdts et al. / Vaccine 22 (2004) 1717–1727 similarities between the ovine and human fetal development and the ontogeny of their immune systems. Using fetal lambs we con?rmed observations in higher primates where fetal immunization with a recombinant protein vaccine induced active immunity in the newborn [6]. Three in utero immunizations with a hepatitis B (HB) vaccine induced detectable immune responses in 75% (5/8) of newborn baboons [6]. Thus, these observations verify that the last term fetus is fully immunocompetent and that in utero immunization may represent a useful approach to prevent vertical disease transmission in diseased pregnant women. Vertical disease transmission occurs primarily through mucosal surfaces and we therefore analyzed the induction of mucosal immunity and immune memory in the newborn by in utero immunization which would signi?cantly enhance disease protection in the neonate. To identify the sites of uptake and expression of the plasmid DNA we used luciferase encoding plasmid DNA for in utero delivery and analyzed local expression within the oral cavity and other mucosal sites. Neonatal immune memory was addressed by analyzing immune responses to either a secondary DNA vaccination or a viral infection within the ?rst 10 days after birth. Moreover, immune protection against a respiratory viral infection was assessed in an experimental challenge with infectious BHV-1. To compare the ef?cacy of fetal DNA immunization with a licensed protein vaccine, we analyzed the induction of protective immunity against HBV, an important pathogen of newborn infants (reviewed in [7,8]) following fetal immunization with either a HBV surface antigen (HBsAg) protein or DNA vaccine. ligated into the vector to create pMASIA-luc. Plasmids were puri?ed using the Qiagen endotoxin removing Giga-Kit (Qiagen, Mississauga, Ont., Canada). DNA concentration was estimated after agarose gel electrophoresis by comparison with known standards and by spectrophotometry. For immunization, DNA was dissolved in sterile pyrogen-free PBS, pH 7.3 (Sigma–Aldrich, Oakville, Ont., Canada). 2.2. Animals Suffolk sheep were obtained from the Department of Animal and Poultry Science (University of Saskatchewan, Saskatoon, Sask., Canada) and were cared for in accordance with Guidelines of the Canadian Council for Animal Care (CCAC). The timed mating of ewes and con?rmation of pregnancy was performed as previously described and pregnant ewes were seronegative for gD-speci?c antibodies to ensure there was no passive transfer of gD-speci?c antibody to newborn lambs. The newborn lambs were housed individually with their mothers for 3 days after birth and then housed as a group until weaned at 7 weeks of age. For the BHV-1 challenge experiment, lambs were separated from their mothers 7 days after birth and housed in an isolation facility. Lambs were fed fresh cow milk (Department of Animal and Poultry Science, University of Saskatchewan) throughout the challenge experiment. 2.3. Fetal immunization 2. Methods 2.1. Cells, viruses, plasmids Bovine viral diarrhea virus-free Madin Darby bovine kidney (MDBK) cells were cultured in minimum essential medium (MEM; Gibco-BRL, Burlington, Ont., Canada) supplemented with 5% fetal bovine serum (FBS; Gibco-BRL). BHV-1 strain 108, is a virulent ?eld isolate [9] and was propagated in MDBK cells. Plasmid pSLIA-tgD [10] encoding the truncated version of glycoprotein D (tgD) of BHV- 内容过长，仅展示头部和尾部部分文字预览，全文请查看图片预览。 Hengartner H, Zinkernagel RM. Protective long-term antibody memory by antigen-driven and T-help dependent differentiation of long-lived memory B cells to short-lived plasma cells independent of secondary lymphoid organs. Proc Natl Acad Sci USA 2000;97:13263. [42] Greenwood PL, Bell AW, Hermanson JW, Slepetis RM. An ultrasound-guided procedure to administer a label of DNA synthesis in fetal sheep. Reprod Fertil Dev 1999;11:303. [43] Krieg AM. From A to Z on CpG. Trends Immunol 2002;23(2):64–5. [文章尾部最后500字内容到此结束,中间部分内容请查看底下的图片预览]请点击下方选择您需要的文档下载。

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