Association of gamma interferon and interleukin-17 production in intestinal CD4+ T cells with protection against rotavirus shedding in mice intranasally immunized with VP6 and the adjuvant LT(R192G)

Mucosal immunization of mice with chimeric, Escherichia coli-expressed VP6, the protein that comprises the intermediate capsid layer of the rotavirus particle, together with attenuated E. coli heat-labile toxin LT(R192G) as an adjuvant, reduces fecal shedding of rotavirus antigen by >95% after murine rotavirus challenge, and the only lymphocytes required for protection are CD4+ T cells. Because these cells produce cytokines with antiviral properties, the cytokines whose expression is upregulated in intestinal memory CD4+ T cells immediately after rotavirus challenge of VP6/LT(R192G)-immunized mice may be directly or indirectly responsible for the rapid suppression of rotavirus shedding. This study was designed to identify which cytokines are significantly upregulated in intestinal effector sites and secondary lymphoid tissues of intranasally immunized BALB/c mice after challenge with murine rotavirus strain EDIM. Initially, this was done by using microarray analysis to quantify mRNAs for 96 murine common cytokines. With this procedure, the synthesis of mRNAs for gamma interferon (IFN-gamma) and interleukin-17 (IL-17) was found to be temporarily upregulated in intestinal lymphoid cells of VP6/LT(R192G)-immunized mice at 12 h after rotavirus challenge. These cytokines were also produced in CD4+ T cells obtained from intestinal sites specific to VP6/LT(R192G)-immunized mice after in vitro exposure to VP6 as determined by intracellular cytokine staining and secretion of cytokines. Although genetically modified mice that lack receptors for either IFN-gamma or IL-17 remained protected after immunization, these results provide suggestive evidence that these cytokines may play direct or indirect roles in protection against rotavirus after mucosal immunization of mice with VP6/LT(R192G).     

Mice develop effective but delayed protective immune responses when immunized as neonates either intranasally with nonliving VP6/LT(R192G) or orally with live rhesus rotavirus vaccine candidates

Rotavirus vaccines are delivered early in life, when the immune system is immature. To determine the effects of immaturity on responses to candidate vaccines, neonatal (7 days old) and adult mice were immunized with single doses of either Escherichia coli-expressed rotavirus VP6 protein and the adjuvant LT(R192G) or live rhesus rotavirus (RRV), and protection against fecal rotavirus shedding following challenge with the murine rotavirus strain EDIM was determined. Neonatal mice immunized intranasally with VP6/LT(R192G) were unprotected at 10 days postimmunization (dpi) and had no detectable rotavirus B-cell (antibody) or CD4(+) CD8(+) T-cell (rotavirus-inducible, Th1 [gamma interferon and interleukin-2 {IL-2}]-, Th2 [IL-5 and IL-4]-, or ThIL-17 [IL-17]-producing spleen cells) responses. However, by 28 and 42 dpi, these mice were significantly (P >or= 0.003) protected and contained memory rotavirus-specific T cells but produced no rotavirus antibody. In contrast, adult mice were nearly fully protected by 10 dpi and contained both rotavirus immunoglobulin G and memory T cells. Neonates immunized orally with RRV were also less protected (P=0.01) than adult mice by 10 dpi and produced correspondingly less rotavirus antibody. Both groups contained few rotavirus-specific memory T cells. Protection levels by 28 dpi for neonates or adults were equal, as were rotavirus antibody levels. This report introduces a neonatal mouse model for active protection studies with rotavirus vaccines. It indicates that, with time, neonatal mice develop full protection after intranasal immunization with VP6/LT(R192G) or oral immunization with a live heterologous rotavirus and supports reports that protection depends on CD4(+) T cells or antibody, respectively.     

Functional mapping of protective domains and epitopes in the rotavirus VP6 protein

The purpose of this study was to determine which regions of the VP6 protein of the murine rotavirus strain EDIM are able to elicit protection against rotavirus shedding in the adult mouse model following intranasal (i.n.) immunization with fragments of VP6 and a subsequent oral EDIM challenge. In the initial experiment, the first (fragment AB), middle (BC), or last (CD) part of VP6 that was genetically fused to maltose-binding protein (MBP) and expressed in Escherichia coli was examined. Mice (BALB/c) immunized with two 9-microg doses of each of the chimeras and 10 microg of the mucosal adjuvant LT(R192G) were found to be protected against EDIM shedding (80, 92, and nearly 100% reduction, respectively; P 相似文献   

Intranasal administration of an Escherichia coli-expressed codon-optimized rotavirus VP6 protein induces protection in mice

We are developing rotavirus vaccines based on the VP6 protein of the human G1P[8] [corrected] [J. Virol. 73 (1999) 7574] CJN strain of rotavirus. One prototype candidate consisting of MBP::VP6::His6, a chimeric protein of maltose-binding protein, VP6 and hexahistidine, was expressed mainly as truncated polypeptides in Escherichia coli BL21(DE3) cells. A possible reason for this extensive truncation is the high frequencies of rare bacterial codons within the rotavirus VP6 gene. Expression of truncated recombinant VP6 was found to be reduced, and expression of complete VP6 protein was simultaneously increased, when the protein was expressed in Rosetta(DE3)pLacI E. coli cells that contain increased amounts of transfer RNAs for a selection of rare codons. The same observation was made when a synthetic codon-optimized CJN-VP6 gene was expressed in E. coli BL21 or Rosetta cells. To increase protein recovery, recombinant E. coli cells were treated with 8M urea. Denatured, full-length MBP::VP6::His6 protein was then purified and used for intranasal vaccination of BALB/c mice (2 doses administered with E. coli heat-labile toxin LT(R192G) as adjuvant). Following oral challenge with the G3P[16] [corrected] [J. Virol. 76 (2002) 560] EDIM strain of murine rotavirus, protection levels against fecal rotavirus shedding were comparable (P>0.05) between groups of mice immunized with denatured codon-optimized or native (not codon-optimized) immunogen with values ranging from 87 to 99%. These protection levels were also comparable to those found after immunization with non-denatured CJN VP6. Thus, expression of complete rotavirus VP6 protein was greatly enhanced by codon optimization, and the protection elicited was not affected by denaturation of recombinant VP6.     

CD4 T Cells Are the Only Lymphocytes Needed To Protect Mice against Rotavirus Shedding after Intranasal Immunization with a Chimeric VP6 Protein and the Adjuvant LT(R192G)

Intranasal immunization of mice with a chimeric VP6 protein and the mucosal adjuvant Escherichia coli heat labile toxin LT(R192G) induces nearly complete protection against murine rotavirus (strain EDIM [epizootic diarrhea of infant mice virus]) shedding for at least 1 year. The aim of this study was to identify the protective lymphocytes elicited by this new vaccine candidate. Immunization of mouse strains lacking one or more lymphocyte populations revealed that protection was dependent on alphabeta T cells but mice lacking gammadelta T cells and B cells remained fully protected. Furthermore, depletion of CD8 T cells in immunized B-cell-deficient mice before challenge resulted in no loss of protection, while depletion of CD4 T cells caused complete loss of protection. Therefore, alphabeta CD4 T cells appeared to be the only lymphocytes required for protection. As confirmation, purified splenic T cells from immunized mice were intraperitoneally injected into Rag-2 mice chronically infected with EDIM. Transfer of 2 x 10(6) CD8 T cells had no effect on shedding, while transfer of 2 x 10(5) CD4 T cells fully resolved shedding in 7 days. Interestingly, transfer of naive splenic CD4 T cells also resolved shedding but more time and cells were required. Together, these results establish CD4 T cells as effectors of protection against rotavirus after intranasal immunization of mice with VP6 and LT(R192G).     

Antibody-Dependent and -Independent Protection following Intranasal Immunization of Mice with Rotavirus Particles

The ability to elicit protective immune responses after intranasal immunization with rotavirus particles, either with or without the attenuated Escherichia coli heat-labile enterotoxin LT(R192G) as an adjuvant, was examined in the adult mouse model. BALB/c mice were administered one or two inoculations of psoralen/UV-inactivated, triple-layered (tl) or double-layered (dl) purified rotavirus particles. Four weeks after immunization, mice were challenged with the murine rotavirus strain EDIM, and the shedding of rotavirus antigen was quantified. Rotaviruses used for immunization included EDIM and heterotypic simian (RRV), bovine (WC3), and human (89-12) strains. tl EDIM stimulated both systemic and intestinal rotavirus antibody responses and complete protection with as little as one 1-microgram dose. Inclusion of LT(R192G) (10 micrograms) significantly increased rotavirus antibody responses and reduced antigen concentrations needed for full protection. Both dl EDIM and heterotypic dl and tl particles stimulated protection, but they did so less than tl EDIM at comparable concentrations, either with or without LT(R192G). When B-cell-deficient microMt mice were immunized with tl EDIM particles, protection was reduced to levels similar to those induced with dl EDIM and heterotypic particles in BALB/c mice. However, dl EDIM particles induced similar levels of protection in both mouse strains. The protection stimulated by tl or dl EDIM particles was not diminished by CD8 cell depletion prior to immunization in either strain of mice. These results indicate that tl EDIM induced immunity at least partially through responses to its outer capsid proteins, presumably by stimulation of serotype-specific neutralizing antibody. In contrast, the other particles stimulated protection primarily by an antibody-independent mechanism. Finally, depletion of CD8 cells had no effect on protection by either mechanism.     

Rotavirus virus-like particles administered mucosally induce protective immunity.

We have evaluated the immunogenicity and protective efficacy of rotavirus subunit vaccines administered by mucosal routes. Virus-like particles (VLPs) produced by self-assembly of individual rotavirus structural proteins coexpressed by baculovirus recombinants in insect cells were the subunit vaccine tested. We first compared the immunogenicities and protective efficacies of VLPs containing VP2 and VP6 (2/6-VLPs) and G3 2/6/7-VLPs mixed with cholera toxin and administered by oral and intranasal routes in the adult mouse model of rotavirus infection. VLPs administered orally induced serum antibody and intestinal immunoglobulin A (IgA) and IgG. The highest oral dose (100 microg) of VLPs induced protection from rotavirus challenge (> or = 50% reduction in virus shedding) in 50% of the mice. VLPs administered intranasally induced higher serum and intestinal antibody responses than VLPs administered orally. All mice receiving VLPs intranasally were protected from challenge; no virus was shed after challenge. Since there was no difference in immunogenicity or protective efficacy between 2/6- and 2/6/7-VLPs, protection was achieved without inclusion of the neutralization antigens VP7 and VP4. We also tested the immunogenicities and protective efficacies of 2/6-VLPs administered intranasally without the addition of cholera toxin. 2/6-VLPs administered intranasally without cholera toxin induced lower serum and intestinal antibody titers than 2/6-VLPs administered with cholera toxin. The highest dose (100 microg) of 2/6-VLPs administered intranasally without cholera toxin resulted in a mean reduction in shedding of 38%. When cholera toxin was added, higher levels of protection were achieved with 10-fold less immunogen. VLPs administered mucosally offer a promising, safe, nonreplicating vaccine for rotavirus.     

Rotavirus 2/6 Viruslike Particles Administered Intranasally with Cholera Toxin, Escherichia coli Heat-Labile Toxin (LT), and LT-R192G Induce Protection from Rotavirus Challenge

We have shown that rotavirus 2/6 viruslike particles composed of proteins VP2 and VP6 (2/6-VLPs) administered to mice intranasally with cholera toxin (CT) induced protection from rotavirus challenge, as measured by virus shedding. Since it is unclear if CT will be approved for human use, we evaluated the adjuvanticity of Escherichia coli heat-labile toxin (LT) and LT-R192G. Mice were inoculated intranasally with 10 μg of 2/6-VLPs combined with CT, LT, or LT-R192G. All three adjuvants induced equivalent geometric mean titers of rotavirus-specific serum antibody and intestinal immunoglobulin G (IgG). Mice inoculated with 2/6-VLPs with LT produced significantly higher titers of intestinal IgA than mice given CT as the adjuvant. All mice inoculated with 2/6-VLPs mixed with LT and LT-R192G were totally protected (100%) from rotavirus challenge, while mice inoculated with 2/6-VLPs mixed with CT showed a mean 91% protection from challenge. The availability of a safe, effective mucosal adjuvant such as LT-R192G will increase the practicality of administering recombinant vaccines mucosally.     

Immunization with baculovirus-expressed recombinant rotavirus proteins VP1, VP4, VP6, and VP7 induces CD8+ T lymphocytes that mediate clearance of chronic rotavirus infection in SCID mice.

Clearance of chronic murine rotavirus infection in SCID mice can be demonstrated by adoptive transfer of immune CD8+ T lymphocytes from histocompatible donor mice immunized with a murine homotypic rotavirus (T. Dharakul, L. Rott, and H.B. Greenberg, J. Virol 64:4375-4382, 1990). The present study focuses on the protein specificity and heterotypic nature of cell-mediated clearance of chronic murine rotavirus infection in SCID mice. Heterotypic cell-mediated clearance was demonstrated in SCID mice infected with EDIM (murine) rotavirus after adoptive transfer of CD8+ T lymphocytes from BALB/c mice that were immunized with a variety of heterologous (nonmurine) rotaviruses including Wa (human, serotype 1), SA11 and RRV (simian, serotype 3), and NCDV and RF (bovine, serotype 6). This finding indicates the serotypic independence of T-cell-mediated rotavirus clearance. To further identify the rotavirus proteins that are capable of generating CD8+ T cells that mediate virus clearance, donor mice were immunized with SF-9 cells infected with a baculovirus recombinant expressing one of the following rotavirus proteins: VP1, VP2, NS53 (from RF), VP4, VP7, NS35 (from RRV), VP6, and NS28 (from SA11). SCID mice stopped shedding rotavirus after receiving CD8+ T cells from mice immunized with VP1, VP4, VP6, and VP7 but not with VP2, NS53, NS35, NS28, or wild-type baculovirus. These results suggest that heterotypic cell-mediated clearance of rotavirus in SCID mice is mediated by three of the major rotavirus structural proteins and by a putative polymerase protein.     

Serum-neutralizing antibody to VP4 and VP7 proteins in infants following vaccination with WC3 bovine rotavirus.

Serum specimens from infants 2 to 12 months old vaccinated with the WC3 bovine rotavirus were analyzed to determine the relative concentrations of neutralizing antibody to the VP4 and VP7 proteins of the vaccine virus. To do this, reassortant rotaviruses that contained the WC3 genome segment for only one of these two neutralization proteins were made. The segment for the other neutralization protein in these reassortants was from heterotypic rotaviruses that were serotypically distinct from WC3. Sera were examined from 31 infants who had no evidence of a previous rotavirus infection and the highest postvaccination WC3-neutralizing antibody titers (i.e., 160 to 600) of the 103 subjects administered the vaccine. A reassortant (3/17) that contained both neutralization proteins from the heterotypic rotaviruses, i.e., EDIM (EW strain of mouse rotavirus) VP7 and rhesus rotavirus VP4, was not neutralized by these sera (geometric mean titer [GMT], less than 20). A reassortant (E19) that contained EDIM VP7 and WC3 VP4 was also very poorly neutralized by these antisera (GMT = 20). In contrast, antibody titers to a reassortant (R20) that contained WC3 VP7 and rhesus rotavirus VP4 were higher than those against WC3 (GMTs of 458 and 313, respectively). Thus, VP7 appeared to be the dominant immunogen for production of neutralizing antibody after intestinal infection of previously uninfected infants vaccinated with WC3 bovine rotavirus.     

Immunodominance of the VP4 neutralization protein of rotavirus in protective natural infections of young children.

Natural infection by very similar strains of rotavirus during the 1988-1989 rotavirus season in Cincinnati, Ohio, provided complete protection of young children against subsequent rotavirus illnesses for a period of at least 2 years. Using this limited strain variability, we characterized the association between the titers of antibody to either the VP4 or the VP7 neutralization protein and protection against subsequent rotavirus disease. This was done by using reassortants that contained only one of the two rotavirus neutralization proteins of 89-12, a culture-adapted isolate representative of the protective rotavirus strains. The other neutralization protein in these reassortants was derived from a heterologous rotavirus (WC3 or EDIM) to which the infected subjects made little or no neutralizing antibody (titers, < or = 20). The geometric mean titer (GMT) of antibody to 89-12 in convalescent-phase sera from the 21 subjects analyzed was 2,323. The GMT of antibody to a reassortant (strain WC-4) that contained the VP7 protein of 89-12 and VP4 of WC3 was 387. In contrast, the GMT of antibody to a reassortant (strain EDIM-7) that contained the VP4 protein of 89-12 and the VP7 protein of EDIM was 1,078. Thus, the major neutralization response was directed against VP4 rather than VP7, a finding that has important implications for development of appropriate rotavirus vaccines.     

表达轮状病毒G2和G3型vp7基因重组腺病毒的免疫效果

为探索以非复制型腺病毒为表达载体的多价轮状病毒(Rotavirus,RV)基因工程疫苗的可行性,在前期工作的基础上,对表达我国G2和G3型RV流行毒株vp7基因的重组腺病毒的免疫效果进行了研究。分别用表达G2和G3型vp7基因的重组腺病毒rvAdG2VP7、rvAdG3VP7经滴鼻和灌胃两种途径免疫Balb/c小鼠,对免疫后小鼠的血清抗体、黏膜抗体和相关的细胞因子水平进行了检测和比较。结果表明,用表达G2和G3型vp7基因的重组腺病毒经滴鼻和灌胃两种途径免疫小鼠后,均可诱导机体产生较强的RV特异性免疫反应,包括体液免疫、细胞免疫和黏膜免疫,并能产生中和抗体。但免疫反应以Th2类为主,Th1类反应也占有相当的比例。本研究为新型RV基因工程疫苗的深入研究奠定了基础。     

Heterotypic passive protection induced by synthetic peptides corresponding to VP7 and VP4 of bovine rotavirus.

We have evaluated the potential of two peptides derived from highly conserved regions of rotavirus outer capsid proteins (VP7 and VP4) to act as a rotavirus vaccine. The capacity of peptides coupled to rotavirus VP6 spherical particles to provide passive protection in a murine model was compared with the protection induced by peptide-keyhole limpet hemocyanin (KLH) conjugates. Female mice were immunized a total of three times before and during pregnancy. Suckling mouse pups were challenged at 7 days of age with either homologous or heterologous rotavirus serotypes. The efficacy of vaccination was determined by analyzing the clinical symptoms and measuring xylose adsorption in the intestine. In this model the VP4 peptide-VP6 conjugate provided protection equal to that obtained using bovine rotavirus (BRV) as the immunogen. The VP7 peptide-VP6 conjugate provided slightly less protection than the VP4 peptide-VP6 conjugate. A mixture of the VP4 peptide-VP6 and VP7 peptide-VP6 conjugates provided better heterologous protection than immunization with BRV. In contrast, KLH-conjugated peptides provided only partial protection. The significance of a synthetic-peptide-based rotavirus vaccine in the prevention of rotavirus infections is discussed.     

Relative concentrations of serum neutralizing antibody to VP3 and VP7 proteins in adults infected with a human rotavirus.

Two outer capsid rotavirus proteins, VP3 and VP7, have been found to elicit neutralizing-antibody production, but the immunogenicity of these proteins during human rotavirus infection has not been determined. The relative amounts of serum neutralizing antibody against the VP3 and VP7 proteins of the CJN strain of human rotavirus were, therefore, determined in adult subjects before and after infection with this virus. Reassortant strains of rotavirus that contained the CJN gene segment for only one of these two neutralization proteins were isolated and used for this study. The geometric mean titer of serum neutralizing antibody to a reassortant virus (CJN-M) that contained VP7 of CJN and VP3 of another human rotavirus was 12.7 times less than that of antibody to CJN before infection and 20.3 times less after infection. This indicated that most neutralizing antibody was against the VP3 rather than the VP7 protein of CJN. This result was confirmed with other reassortants between CJN and animal rotavirus strains (EDIM and rhesus rotavirus). These findings suggest that VP3 is the primary immunogen that stimulates neutralizing antibody during at least some rotavirus infections of humans.     

Immunization with baculovirus-expressed VP4 protein passively protects against simian and murine rotavirus challenge.

A baculovirus-expressed VP4 protein derived from the simian rhesus rotavirus (RRV) was used to parenterally immunize murine dams. VP4-immunized dams developed high levels of neutralizing antibodies against RRV and low levels of cross-reactive neutralizing antibodies against human strains Wa, ST3, and S2 and animal strains SA-11, NCDV, and Eb. Newborn mice suckled on VP4-immunized dams were protected against a virulent challenge dose of the simian strain RRV and against murine rotavirus Eb. The cross-reactive nature of the serum-neutralizing response generated by VP4 immunization and the protective efficacy of the immunization suggest that recombinant-expressed VP4 proteins should be considered as viable vaccine candidates.     

Identification of VP7 epitopes associated with protection against human rotavirus illness or shedding in volunteers.

Sera from 17 of 18 adult volunteers challenged with a virulent serotype 1 rotavirus strain (D) were examined for prechallenge antibody levels against several well-defined rotavirus VP7 and VP4 neutralization epitopes by a competitive epitope-blocking immunoassay (EBA) in order to determine whether correlates of resistance to diarrheal illness could be identified. The presence of prechallenge serum antibody at a titer of greater than or equal to 1:20 that blocked the binding of a serotype 1 VP7-specific monoclonal antibody (designated 2C9) that maps to amino acid residue 94 in antigenic site A on the serotype 1 VP7 was significantly associated with resistance to illness or shedding (P less than 0.001) or illness and shedding (P less than 0.01) following challenge with the serotype 1 virus. In addition, an EBA antibody titer of greater than or equal to 1:20 in prechallenge serum against a serotype 3 VP7-specific epitope (defined by monoclonal antibody 954/159) that maps to amino acid 94 on the serotype 3 VP7 was also significantly associated with resistance to illness or shedding (P = 0.02), with a trend for protection against illness and shedding. A trend was also noted between the presence of EBA antibody against a cross-reactive VP4 epitope common to many human rotavirus strains, including the challenge virus, or a rhesus monkey rotavirus strain-specific VP4 antigenic site, and resistance to illness or shedding. These data confirm that the presence of serum antibody correlates with resistance to rotavirus illness or shedding but, in addition, demonstrate the association of antibody to a specific epitope with resistance to illness or shedding. These data also suggest that antigenic site A on the rotavirus VP7, composed of amino acids 87 to 96, may be involved in the formation of a major protective epitope. Further study of the role of this epitope in the development of homotypic and heterotypic immunity to rotaviruses following natural or vaccine-induced infection may be important in the development of strategies for control of rotavirus diarrheal disease.     

Infection immunity of piglets to either VP3 or VP7 outer capsid protein confers resistance to challenge with a virulent rotavirus bearing the corresponding antigen.

A single-gene substitution reassortant 11-1 was generated from two porcine rotaviruses, OSU (serotype 5) and Gottfried (serotype 4). This reassortant derived 10 genes, including gene 4 encoding VP3, from the OSU strain and only gene 9, encoding a major neutralization glycoprotein (VP7), from the Gottfried strain and was thus designated VP3:5; VP7:4. Oral administration of this reassortant to colostrum-deprived gnotobiotic newborn pigs induced a high level of neutralizing antibodies not only to Gottfried VP7 but also to OSU VP3, thus demonstrating that VP3 is as potent an immunogen as VP7 in inducing neutralizing antibodies during experimental oral infection. Gnotobiotic piglets infected previously with the reassortant were completely resistant to oral challenge with the virulent Gottfried strain (VP3:4; VP7:4), as indicated by failure of symptoms to develop and lack of virus shedding. Similarly, prior infection with the reassortant induced almost complete protection against diarrhea and significant restriction of virus replication after oral challenge with the virulent OSU strain (VP3:5; VP7:5). Thus, it appears that (i) the immune system of the piglet responds equally well to two rotavirus outer capsid proteins, VP3 and VP7, during primary enteric rotavirus infection; (ii) antibody to VP3 and antibody to VP7 are each associated with resistance to diarrhea; and (iii) infection with a reassortant rotavirus bearing VP3 and VP7 neutralization antigens derived from two viruses of different serotype induces immunity to both parental viruses. The relevance of these findings to the development of effective reassortant rotavirus vaccines is discussed.     

沙眼衣原体分泌性蛋白Pgp3滴鼻免疫增强小鼠对衣原体生殖道感染的保护作用

【目的】探讨不同免疫途径沙眼衣原体(Chlamydia trachomatis,Ct)分泌性蛋白Pgp3的免疫保护效果,分析其可能的保护机制,以确定Pgp3蛋白疫苗在Ct疫苗研制中的应用价值。【方法】分泌性蛋白Pgp3经滴鼻或肌注途径免疫雌性Balb/c小鼠,免疫60 d后,阴道接种鼠沙眼衣原体(Chlamydia muridarum,Cm)建立生殖道感染动物模型,在该模型中评价Pgp3蛋白疫苗抗Cm感染的保护效果,并探讨其机制。【结果】滴鼻或肌注免疫后,小鼠血清及生殖道中检测到了特异性抗体;小鼠脾淋巴细胞产生IFN-γ、IL-17及IL-5水平均明显高于对照组,且滴鼻免疫组IFN-γ水平升高较肌注组更显著(P<0.05);Pgp3蛋白滴鼻免疫组小鼠经Cm生殖道感染后,阴道带菌时间明显缩短,输卵管病理改变轻而肌注免疫组其保护作用不明显。【结论】Pgp3蛋白经滴鼻免疫可有效诱导小鼠产生明显的抗Cm保护效应。其可能的免疫保护机制与诱导Th1型为主的细胞免疫应答及高效价的特异性抗体有关,提示Pgp3蛋白疫苗具有潜在的疫苗研究与开发价值。     

Evidence that resolution of rotavirus infection in mice is due to both CD4 and CD8 cell-dependent activities.

The effector functions responsible for resolution of shedding in mice orally inoculated with the murine rotavirus strain EDIM were identified in B-cell-deficient and normal BALB/c mice after monoclonal antibody (MAb) depletion of CD4 and CD8 cells. When depleted of CD8 cells, B-cell-deficient muMt mice resolved their infections more slowly than nondepleted animals, but CD4 cell depletion caused chronic, high-level shedding. This finding indicated that CD4 cell-dependent immunological effectors other than, or in addition to, CD8 cells played roles in rotavirus resolution in muMt mice in the absence of antibody. The roles of CD4 and CD8 cells in resolution of rotavirus shedding were further characterized in immunologically normal BALB/c mice. Depletion of CD4 cells before EDIM inoculation resulted in rapid resolution of most shedding, but chronic, low-level shedding continued for weeks. When the CD4 cell-depleted BALB/c mice were subsequently depleted of CD8 cells, shedding levels increased significantly (P < 0.001), indicating that CD8 cells were responsible for the rapid but incomplete suppression of rotavirus shedding. Further experimentation revealed that little rotavirus antibody was made in CD4 cell-depleted BALB/c mice, and only after CD4 cells were repopulated did antibody production increase and virus shedding fully resolve. Thus, resolution of rotavirus shedding in both muMt and BALB/c mice was associated with CD4 and CD8 cell effector activities.     

Relative Importance of Rotavirus-Specific Effector and Memory B Cells in Protection against Challenge

Adult BALB/c mice were orally inoculated with murine (strain EDIM), simian (strain RRV), or bovine (strain WC3) rotavirus. Six or 16 weeks after inoculation, mice were challenged with EDIM. At the time of challenge and in the days immediately following challenge, production of rotavirus-specific immunoglobulin A (IgA), IgG, and IgM by small intestinal lamina propria lymphocytes (LPL) was determined by fragment culture, and quantities of virus-specific antibodies at the intestinal mucosal surface were determined by intestinal lavage. Mice immunized with EDIM were completely protected against EDIM challenge both 6 and 16 weeks after immunization. Protection was associated with production of high levels of IgA by LPL and detection of virus-specific IgA at the intestinal mucosal surface. In addition, animals immunized and later challenged with EDIM did not develop a boost in antibody responses, suggesting that they were also not reinfected. We also found that in mice immunized with nonmurine rotaviruses, (i) quantities of virus-specific IgA generated following challenge were greater 16 weeks than 6 weeks after immunization, (ii) immunization enhanced the magnitude but did not hasten the onset of production of high quantities of virus-specific IgA by LPL after challenge, and (iii) immunization induced partial protection against challenge; however, protection was not associated with either production of virus-specific antibodies by LPL or detection of virus-specific antibodies at the intestinal mucosal surface.