Oral DNA immunization in the second trimester fetal lamb and secondary immune responses in the neonate

We previously demonstrated that oral DNA vaccination of the third trimester fetus may be an effective strategy to prevent vertical disease transmission despite an immature immune system and the possibility of developing tolerance. The present investigation examined oral DNA vaccine delivery and immunogenicity in the second trimester fetal lamb (50-100 days of gestation (dg)). Histological examination revealed nucleated cells in the superficial layers of the oral mucosa. Therefore, luciferase-encoding DNA plasmid was injected into the oral cavity of 65-70 dg fetuses to monitor plasmid expression. Luciferase activity was detected in the oral mucosa of all fetuses but the level of luciferase activity varied among individual fetuses. Luciferase activity was also detected within the tonsils and lymph nodes draining the oral cavity. Oral DNA immunization between 55 and 83 dg with a truncated glycoprotein D (tgD)-encoding plasmid induced germinal centres in the draining lymph nodes and detectable tgD-specific serum antibody titers and/or IFNgamma-secreting cell responses in 16 of 24 (67%) fetuses. The tgD-specific antibody and IFNgamma responses persisted until birth in some fetuses but the magnitude of antibody titers in second trimester fetuses was low relative to antibody titers induced following oral DNA immunization in the third trimester. Lambs immunized during the second trimester of gestation responded to neonatal DNA immunization and anamnestic responses were detected in some lambs immunized as early as 67-72 dg. These observations confirmed that oral DNA immunization of the early second trimester fetus induced antigen-specific immune responses with no evidence of tolerance induction.     

In utero DNA immunisation. Immunity over tolerance in fetal life

The function and plasticity of the developing immune system during embryonic life has been central to immunological thinking for half a century. A classical view is that antigen encountered during fetal life induces a state of acquired immunological tolerance. However, the ability to develop T cell immune responses during the perinatal period would be of great importance against intracellular pathogens. Recent experiments have challenged this notion and shown that neonatal tolerance can be circumvented by extrinsic immunological manipulations. Here, we used DNA immunization targeted at B lymphocytes to induce a CD4 T cell response that could be measured 2 weeks after birth. We conclude that T cell immunity can be programmed in utero by manipulating the parameters of the immune response in the fetal environment. Furthermore, our data suggest that under appropriate conditions the fetal immune system can be programmed to immunity.     

Induction of immunological memory in baboons primed with DNA vaccine as neonates

DNA immunization is a potential vaccination strategy for neonates and infants. We tested the ability of a prototype DNA vaccine against influenza virus to prime lasting immunity when administered to newborn non-human primates. Neonatal DNA vaccination triggered virus-specific and neutralizing antibodies of titers and persistence depending on the vaccine dose. Subsequent exposure to influenza virus, revealed significantly increased recall responses in the baboons vaccinated with DNA during the neonatal stage. The humoral and cellular responses were enhanced in the baboons primed with DNA vaccine as neonates. Thus, neonatal DNA vaccination of non-human primates triggered immune memory that persisted beyond infancy.     

Immunogenicity of viral vector,prime-boost SIV vaccine regimens in infant rhesus macaques: Attenuated vesicular stomatitis virus (VSV) and modified vaccinia Ankara (MVA) recombinant SIV vaccines compared to live-attenuated SIV

In a previously developed infant macaque model mimicking HIV infection by breast-feeding, we demonstrated that intramuscular immunization with recombinant poxvirus vaccines expressing simian immunodeficiency virus (SIV) structural proteins provided partial protection against infection following oral inoculation with virulent SIV. In an attempt to further increase systemic but also local antiviral immune responses at the site of viral entry, we tested the immunogenicity of different orally administered, replicating vaccines. One group of newborn macaques received an oral prime immunization with a recombinant vesicular stomatitis virus expressing SIVmac239 gag, pol and env (VSV-SIVgpe), followed 2 weeks later by an intramuscular boost immunization with MVA-SIV. Another group received two immunizations with live-attenuated SIVmac1A11, administered each time both orally and intravenously. Control animals received mock immunizations or non-SIV VSV and MVA control vectors. Analysis of SIV-specific immune responses in blood and lymphoid tissues at 4 weeks of age demonstrated that both vaccine regimens induced systemic antibody responses and both systemic and local cell-mediated immune responses. The safety and immunogenicity of the VSV-SIVgpe + MVA-SIV immunization regimen described in this report provide the scientific incentive to explore the efficacy of this vaccine regimen against virulent SIV exposure in the infant macaque model.     

Oral administration of low doses of plant-based HBsAg induced antigen-specific IgAs and IgGs in mice,without increasing levels of regulatory T cells

Plant-based oral vaccines run the risk of activating regulatory T cells (Tregs) and suppressing the antigen-specific immune response via oral tolerance. Mice humanized for two HLA alleles (HLA-A2.1 and HLA-DR1) were used to measure changes in Tregs and antigen-specific immune responses induced by the oral administration of tobacco (Nicotiana tabacum), expressing the hepatitis B surface antigen (HBsAg). Antigen-specific CD8+ T cell activation was not detected, but the plant-based oral immunization, without adjuvant, resulted in humoral responses comparable to those obtained by adjuvanted DNA immunization. Treg titers did not increase with DNA immunization. In contrast, with plant immunization, Tregs increased linearly to reach a plateau at high antigen doses. The highest humoral IgA and IgG responses correlated with the lowest plant antigen dose (0.5 ng), while for DNA immunization the best antibody responses were obtained at higher antigen doses. These experiments suggest that plant-based oral vaccines could be adjusted to minimize tolerance, while still inducing an immune response. Oral tolerance and adjuvant engineering in plants are discussed.     

Effects of monobutyl and di(n-butyl) phthalate in vitro on steroidogenesis and Leydig cell aggregation in fetal testis explants from the rat: comparison with effects in vivo in the fetal rat and neonatal marmoset and in vitro in the human

BACKGROUND: Certain phthalates can impair Leydig cell distribution and steroidogenesis in the fetal rat in utero, but it is unknown whether similar effects might occur in the human. OBJECTIVES: Our aim in this study was to investigate the effects of di(n-butyl) phthalate (DBP), or its metabolite monobutyl phthalate (MBP), on testosterone production and Leydig cell aggregation (LCA) in fetal testis explants from the rat and human, and to compare the results with in vivo findings for DBP-exposed rats. We also wanted to determine if DBP/MBP affects testosterone production in vivo in the neonatal male marmoset. METHODS: Fetal testis explants obtained from the rat [gestation day (GD)19.5] and from the human (15-19 weeks of gestation) were cultured for 24-48 hr with or without human chorionic gonadotropin (hCG) or 22R-hydroxycholesterol (22R-OH), and with or without DBP/MBP. Pregnant rats and neonatal male marmosets were dosed with 500 mg/kg/day DBP or MBP. RESULTS: Exposure of rats in utero to DBP (500 mg/kg/day) for 48 hr before GD21.5 induced major suppression of intratesticular testosterone levels and cytochrome P450 side chain cleavage enzyme (P450scc) expression; this short-term treatment induced LCA, but was less marked than longer term (GD13.5-20.5) DBP treatment. In vitro, MBP (10(-3) M) did not affect basal or 22R-OH-stimulated testosterone production by fetal rat testis explants but slightly attenuated hCG-stimulated steroidogenesis; MBP induced minor LCA in vitro. None of these parameters were affected in human fetal testis explants cultured with 10(-3) M MBP for up to 48 hr. Because the in vivo effects of DBP/MBP were not reproduced in vitro in the rat, the absence of MBP effects in vitro on fetal human testes is inconclusive. In newborn (Day 2-7) marmosets, administration of a single dose of 500 mg/kg MBP significantly (p = 0.019) suppressed blood testosterone levels 5 hr later. Similar treatment of newborn co-twin male marmosets for 14 days resulted in increased Leydig cell volume per testis (p = 0.011), compared with co-twin controls; this is consistent with MBP-induced inhibition of steroidogenesis followed by compensatory Leydig cell hyperplasia/hypertrophy. CONCLUSIONS: These findings suggest that MBP/DBP suppresses steroidogenesis by fetal-type Leydig cells in primates as in rodents, but this cannot be studied in vitro.     

Experimental Rhodococcus equi and equine infectious anemia virus DNA vaccination in adult and neonatal horses: effect of IL-12, dose, and route

Improving the ability of DNA-based vaccines to induce potent Type1/Th1 responses against intracellular pathogens in large outbred species is essential. Rhodoccocus equi and equine infectious anemia virus (EIAV) are two naturally occurring equine pathogens that also serve as important large animal models of neonatal immunity and lentiviral immune control. Neonates present a unique challenge for immunization due to their diminished immunologic capabilities and apparent Th2 bias. In an effort to augment R. equi- and EIAV-specific Th1 responses induced by DNA vaccination, we hypothesized that a dual promoter plasmid encoding recombinant equine IL-12 (rEqIL-12) would function as a molecular adjuvant. In adult horses, DNA vaccines induced R. equi- and EIAV-specific antibody and lymphoproliferative responses, and EIAV-specific CTL and tetramer-positive CD8+ T lymphocytes. These responses were not enhanced by the rEqIL-12 plasmid. In neonatal foals, DNA immunization induced EIAV-specific antibody and lymphoproliferative responses, but not CTL. The R. equi vapA vaccine was poorly immunogenic in foals even when co-administered with the IL-12 plasmid. It was concluded that DNA immunization was capable of inducing Th1 responses in horses; dose and route were significant variables, but rEqIL-12 was not an effective molecular adjuvant. Additional work is needed to optimize DNA vaccine-induced Th1 responses in horses, especially in neonates.     

Immunization with virus-like particles of enterovirus 71 elicits potent immune responses and protects mice against lethal challenge

Enterovirus 71 (EV71) is an etiologic agent responsible for seasonal epidemics of hand-foot-and-mouth disease and causes outbreaks with significant mortality among young children. To develop the vaccine, we have produced and purified the EV71 virus-like particle (VLP) that resembles the authentic virus in appearance, capsid structure and protein composition. In this study, we further evaluated the potential of VLP as a vaccine by comparing the humoral and cellular immune responses elicited by the purified VLP, denatured VLP and heat-inactivated EV71 virus. After immunization of BALB/c mice, EV71 VLP induced potent and long-lasting humoral immune responses as evidenced by the high total IgG titer and neutralization titer. The splenocytes collected from the VLP-immunized mice exhibited significant cell proliferation and produced high levels of IFN-gamma, IL-2 and IL-4 after stimulation, indicating the induction of Th1 and Th2 immune responses by VLP immunization. More importantly, the VLP immunization of mother mice conferred protection (survival rate up to 89%) to neonatal mice against the lethal (1000 LD(50)) viral challenge. Compared with the VLP immunization, immunization with denatured VLP and heat-inactivated EV71 elicited lower neutralization titers and conferred less effective protection to newborn mice, although they induced comparable levels of total IgG and cellular immune responses. These data collectively indicate the importance of the preservation of VLP structure and implicate the potential of VLP as a vaccine to prevent EV71 infection.     

Influence of maternal immunity on antibody and T-cell response in mice

The prevalence of maternal antibodies (Abs) and an immature neonate immune system, which is inclined to a T-helper 2 (Th2) response, are factors that counteract active immunization in early life. In a mouse model, the maternal influence on an active immunization of 2-day-old offspring with Sendai virus (SV) envelope proteins was explored. Maternal immunizations were conducted with the same SV antigen preparation as used for offspring immunization, presented in three different formulations, namely micelles of SV (SV-MIC), Al(OH)(3)-adjuvanted SV-MIC (SV-aluMIC) for Th2 and immune stimulation complex (iscom)-adjuvanted SV (SV-ISC) for Th1. An inversely correlation was found between the immunoglobulin G2a (IgG2a) Abs of the mothers and the interleukin 5 (IL-5) levels of the offspring. Although a maternally derived immunity induced by SV-aluMIC suppressed both B- and T-cell responses of the newborn to SV-ISC immunization, significant priming effects of the neonatal immunization on IgG2a Abs and IFN-gamma levels were recorded after reimmunization at adult age.     

Comparative analysis of the immunogenicity of SARS-CoV nucleocapsid DNA vaccine administrated with different routes in mouse model

The development of strategies to augment the immunogenicity of DNA vaccines is critical for improving their clinical utility. One such strategy involves using the different immune routes with DNA vaccines. In the present study, the immunogenicity of SARS-CoV nucleocapsid DNA vaccine, induced by using the current routine vaccination routes (intramuscularly, by electroporation, or orally using live-attenuated Salmonella typhimurium), was compared in mouse model. The comparison between the three vaccination routes indicated that immunization intramuscularly induced a moderate T cell response and antibody response. Mice administrated by electroporation induced the highest antibody response among the three immunization groups and a mid-level of cellular response. In contrast, the orally DNA vaccine evoked vigorous T cell response and a weak antibody production. These results indicated that the distinct types of immune responses were generated by the different routes of DNA immunization. In addition, our results also show that the delivery of DNA vaccines by electroporation and orally using live-attenuated Salmonella in vivo is an effective method to increase the immune responses. Further studies could be carried out using a combination strategy of both oral and electroporation immunizations to stimulate higher cellular and humoral immune responses.     

Early life humoral response of ducks to DNA immunization against hepadnavirus large envelope protein

DNA vaccination may represent an interesting strategy for early life immunization. However, in some cases, this approach has been shown to induce a tolerance rather than immunity. We have compared the efficiency of neonatal DNA or protein immunization against hepadnavirus envelope protein using the duck hepatitis B virus (DHBV) model. Three-day-old ducklings were immunized with either a plasmid encoding the DHBV pre-S/S large envelope protein (L), or a recombinant preS protein, followed by sequential DNA or protein boosts at weeks 4 and 15. Our results showed that genetic immunization of duck neonates induced specific humoral response to DHBV L protein. Interestingly, an enhanced antibody response was elicited when animals received DNA priming-DNA boosting as compared to DNA priming-protein boosting.     

Local and systemic antibody responses and immunological memory in humans after immunization with cholera B subunit by different routes

A single oral or intramuscular immunization with purified cholera B subunit induced an intestinal secretory immunoglobulin A (IgA) antitoxin response in, respectively, 10 out of 11 and 9 out of 12 Bangladeshi volunteers. The IgA titre rise in intestinal lavage fluid was similar by either route of immunization, but the duration of the response was usually longer after the oral dose. A second immunization by either route, given 25 days after the first, and a third dose (oral only), given 15 months later, resulted in intestinal immune responses which did not differ in magnitude from that induced by the initial immunization but were observed significantly earlier, usually by day 3. Both the first oral and intramuscular immunizations induced significant antitoxin titre rises, mainly IgG, in the serum in most vaccinees but the magnitude of the response was considerably higher after the intramuscular dose. Significant IgA antitoxin titre rises in saliva and breast milk were seen after both oral and intramuscular immunization.     

乙型肝炎病毒携带者妊娠对新生儿的影响探讨

目的 :探讨乙型肝炎病毒慢性携带者妊娠对新生儿的影响。方法 :检测 66例 HBs Ag阳性产妇静脉血、脐血、乳汁中 HBV DNA含量并对其分娩及新生儿指标进行分析。结果 :脐血 HBV DNA阳性率为 59.1% ;产妇血 HBVDNA含量与脐血 HBV DNA含量的相关系数为 0 .3 72 (P<0 .0 5) ,呈正相关 ;产妇血、脐血 HBV DNA含量与新生儿体重无关 ,有新生儿窒息与无新生儿窒息者比较 ,产妇血、脐血 HBV DNA含量均无显著性差异。结论 :乙型肝炎病毒慢性携带者妊娠可造成新生儿乙型肝炎病毒感染 ,孕妇分娩前的感染状况不能反映其对胎儿发育、胎盘功能的影响     

Anti-major histocompatibility complex antibody responses in macaques via intradermal DNA immunizations

In simian immunodeficiency virus (SIV) models, immunization of macaques with uninfected human cells or human major histocompatibility complex (MHC) proteins can induce xenogeneic immune responses which can protect the animals from subsequent SIV challenges. These studies suggest that the induction of anti-MHC immune responses can be a viable vaccine strategy against human immunodeficiency virus type 1 (HIV-1). We have previously shown in mouse studies that DNA immunization with class I and class II MHC-encoding plasmids can elicit both xenogeneic and allogeneic antibody responses against conformationally intact MHC molecules (Vaccine 17 (1999) 2479-92). Here we take these observations one step closer to human applications and report that intradermal needle immunizations of non-human primates with plasmid DNA encoding human MHC alleles can safely elicit xenogeneic anti-MHC antibody responses. Moreover, injecting macaques with DNA encoding a specific macaque allogeneic MHC induced anti-allogeneic MHC antibodies production. These studies show that DNA immunization with MHC-encoding vectors can indeed be used to induce specific anti-human xenogeneic, as well as anti-macaque allogeneic MHC immunity in non-human primates. This strategy could thus be used to mobilize anti-MHC antibody response which may be useful as part of an anti-HIV-1 vaccination approach.     

Induction of immune responses in newborn lambs following enteric immunization with a human adenovirus vaccine vector

We investigated the antigen-specific mucosal and systemic immune responses of newborn lambs following enteric immunization, targeting jejunal Peyer's patches with a human adenovirus vector that expressed the glycoprotein D (gD) of bovine herpesvirus-1. Both humoral and cell-mediated gD-specific mucosal immune responses were detected in newborn lambs (1-4 days old) after a single immunization and these responses were qualitatively and quantitatively similar to those detected in 5-6-week-old lambs. Passively transferred gD-specific maternal antibody did not significantly alter either mucosal or systemic gD-specific immune responses. Furthermore, enteric immunization of newborn lambs primed mucosal immune responses in the lungs. These observations confirmed that gut-associated lymphoid tissue of a newborn ruminant is immune competent and that enteric immunization may be an effective approach for the induction of both mucosal and systemic immune responses in the neonate.     

Perinatal Toxicology: Its Recognition and Fundamentals

Perinatal toxicology is the study of aberrant or toxic responses to environmental agents when exposure occurs from conception through the neonatal period. The current increased interest in perinatal toxicology reflects the concern of both society and the individual in the resultant deficits induced by exposure to agents in the workplace, home. environment and by therapeutic intervention during early development. Not only is differentiation during organogenesis (the first eight weeks in human gestation) a highly susceptible period to the induction of malformations, but the fetal/neonatal developmental phases are just as sensitive for certain developmental deficits. Long-term postnatal evaluations are in most instances critical for the documentation of fetal/neonatal functional defects, which include carcinoogenesis, behavioral impairment, endocrine and immune dysfunction, Ethylnitrosource, estrogens (diethylstilbestrol), and cadmium are discussed in relationship to specificity of their fetal effects, long-term follow-up, and possible mechanisms of toxic action.     

Comparison of mucosal and systemic humoral immune responses after transcutaneous and oral immunization strategies

In order to compare the ability of transcutaneous and oral immunization strategies to induce mucosal and systemic immune responses, we inoculated mice transcutaneously with cholera toxin (CT) or the non-toxic B subunit of cholera toxin (CtxB), or orally with Peru2(pETR1), an attenuated vaccine strain of Vibrio cholerae expressing CtxB. In addition, we also evaluated dual immunization regimens (oral inoculation with transcutaneous boosting, and transcutaneous immunization with oral boosting) in an attempt to optimize induction of both mucosal and systemic immune responses. We found that transcutaneous immunization with purified CtxB or CT induces much more prominent systemic IgG anti-CtxB responses than does oral inoculation with a vaccine vector strain of V. cholerae expressing CtxB. In comparison, anti-CtxB IgA in serum, stool and bile were comparable in mice either transcutaneously or orally immunized. Overall, the most prominent systemic and mucosal anti-CtxB responses occurred in mice that were orally primed with Peru2(pETR1) and transcutaneously boosted with CT. Our results suggest that combination oral and transcutaneous immunization strategies may most prominently induce both mucosal and systemic humoral responses.     

Induction of HIV-specific functional immune responses by a multiclade HIV-1 DNA vaccine candidate in healthy Ugandans

A phase I randomized, double blind, placebo-controlled trial to assess the immunogenicity of a multiclade HIV-1 DNA plasmid vaccine was conducted in 31 HIV-1-negative Ugandans. Following immunization with DNA at 0, 1, and 2 months, the frequency of HIV-specific immune responses was assessed up to 10 months using a standard chromium release assay (CRA), lymphoproliferative assay (LPA), and antibody dependent cell-mediated cytotoxicity assay (ADCC). Seven of 15 (47%) vaccinees demonstrated CTL activity using the CRA to HIV-1 Env B with responses observed 1 month following the second vaccination and as late as 7 months following complete immunization. Additionally, lymphoproliferative reponses were observed in 14/15 vaccinees against p24. No CTL or LPA responses were observed at baseline or in the placebo group. ADCC activity was minimally induced by DNA vaccination. This study demonstrates that immunization with DNA alone induces CTL and lymphoproliferative responses in a population that will participate in a phase IIb study evaluating HIV-1 DNA priming followed by boosting with a replication-defective recombinant adenovirus vector.     

Intramuscular delivery of a cholera DNA vaccine primes both systemic and mucosal protective antibody responses against cholera

Cholera is a potentially lethal diarrhea disease caused by the gram-negative bacterium Vibrio cholerae. The need for an effective cholera vaccine is clearly indicated but the challenges of eliciting both systemic and mucosal immune responses remains a significant challenge. In the current report, we discovered that a DNA vaccine expressing a protective cholera antigen, cholera toxin B subunit (CTB), delivered parenterally can elicit both systemic and mucosal anti-CTB antibody responses in mice. The priming effect by DNA immunization was demonstrated by higher mucosal antibody responses following one boost with the inactivated cholera vaccine (KWC-B) delivered orally when compared to the twice oral administration of KWC-B alone. This finding indicates that DNA vaccines delivered parenterally are effective in eliciting mucosal protective immune responses—a unique advantage for DNA vaccination that has not yet been well realized and should bring value to the development of novel vaccination approaches against mucosally transmitted diseases.     

Gene-based neonatal immune priming potentiates a mucosal adenoviral vaccine encoding mycobacterial Ag85B

Tuberculosis remains a major public health hazard worldwide, with neonates and young infants potentially more susceptible to infection than adults. BCG, the only vaccine currently available, provides some protection against tuberculous meningitis in children but variable efficacy in adults, and is not safe to use in immune compromised individuals. A safe and effective vaccine that could be given early in life, and that could also potentiate subsequent booster immunization, would represent a significant advance. To test this proposition, we have generated gene-based vaccine vectors expressing Ag85B from Mycobacterium tuberculosis (Mtb) and designed experiments to test their immunogenicity and protective efficacy particularly when given in heterologous prime-boost combination, with the initial DNA vaccine component given soon after birth. Intradermal delivery of DNA vaccines elicited Th1-based immune responses against Ag85B in neonatal mice but did not protect them from subsequent aerosol challenge with virulent Mtb H37Rv. Recombinant adenovirus vectors encoding Ag85B, given via the intranasal route at six weeks of age, generated moderate immune responses and were poorly protective. However, neonatal DNA priming following by mucosal boosting with recombinant adenovirus generated strong immune responses, as evidenced by strong Ag85B-specific CD4+ and CD8+ T cell responses, both in the lung-associated lymph nodes and the spleen, by the quality of these responding cells (assessed by their capacity to secrete multiple antimicrobial factors), and by improved protection, as indicated by reduced bacterial burden in the lungs following pulmonary TB challenge. These results suggest that neonatal immunization with gene-based vaccines may create a favorable immunological environment that potentiates the pulmonary mucosal boosting effects of a subsequent heterologous vector vaccine encoding the same antigen. Our data indicate that immunization early in life with mycobacterial antigens in an appropriate vaccine setting can prime for protective immunity against Mtb.