Lactobacilli and Bifidobacteria Promote Immune Homeostasis by Modulating Innate Immune Responses to Human Rotavirus in Neonatal Gnotobiotic Pigs

The effects of co-colonization with Lactobacillus rhamnosus GG (LGG) and Bifidobacterium lactis Bb12 (Bb12) on 3-dose vaccination with attenuated HRV and challenge with virulent human rotavirus (VirHRV) were assessed in 4 groups of gnotobiotic (Gn) pigs: Pro+Vac (probiotic-colonized/vaccinated), Vac (vaccinated), Pro (probiotic-colonized, non-vaccinated) and Control (non-colonized, non-vaccinated). Subsets of pigs were euthanized pre- [post-challenge day (PCD) 0] and post (PCD7)-VirHRV challenge to assess diarrhea, fecal HRV shedding and dendritic cell/innate immune responses. Post-challenge, Pro+Vac and Vac groups were completely protected from diarrhea; protection rates against HRV shedding were 100% and 83%, respectively. Diarrhea and HRV shedding were reduced in Pro compared to Control pigs following VirHRV challenge. Diarrhea scores and virus shedding were significantly higher in Controls, compared to all other groups, coincident with significantly higher serum interferon-alpha levels post-challenge. LGG+Bb12 colonization ±vaccine promoted immunomaturation as reflected by increased frequencies of CD4, SWC3a, CD11R1, MHCII expressing mononuclear cells (MNCs) and conventional dendritic cells in intestinal tissues and blood post-challenge. Colonization decreased frequencies of toll-like receptors (TLR) 2 and TLR4 expressing MNCs from vaccinated pigs (Pro+Vac) pre-challenge and increased frequencies of TLR3 expressing MNCs from Pro pigs post-challenge, suggesting that probiotics likely exert anti-inflammatory (TLR2 and 4 down-regulation) and antiviral (TLR3 up-regulation by HRV dsRNA) actions via TLR signaling. Probiotic colonization alone (Pro) increased frequencies of intestinal and systemic apoptotic MNCs pre-challenge, thereby regulating immune hyperreactivity and tolerance. However, these frequencies were decreased in intestinal and systemic tissues post-challenge, moderating HRV-induced apoptosis. Additionally, post-challenge, Pro+Vac and Pro groups had significantly decreased MNC proliferation, suggesting that probiotics control excessive lymphoproliferative reactions upon VirHRV challenge. We conclude that in the neonatal Gn pig disease model, selected probiotics contribute to immunomaturation, regulate immune homeostasis and modulate vaccine and virulent HRV effects, thereby moderating HRV diarrhea.     

IgY Antibodies Protect against Human Rotavirus Induced Diarrhea in the Neonatal Gnotobiotic Piglet Disease Model

Group A Rotaviruses are the most common cause of severe, dehydrating diarrhea in children worldwide. The aim of the present work was to evaluate protection against rotavirus (RV) diarrhea conferred by the prophylactic administration of specific IgY antibodies (Ab) to gnotobiotic piglets experimentally inoculated with virulent Wa G1P[8] human rotavirus (HRV). Chicken egg yolk IgY Ab generated from Wa HRV hyperimmunized hens specifically recognized (ELISA) and neutralized Wa HRV in vitro. Supplementation of the RV Ab free cow milk diet with Wa HRV-specific egg yolk IgY Ab at a final ELISA Ab titer of 4096 (virus neutralization –VN- titer = 256) for 9 days conferred full protection against Wa HRV associated diarrhea and significantly reduced virus shedding. This protection was dose-dependent. The oral administration of semi-purified passive IgY Abs from chickens did not affect the isotype profile of the pig Ab secreting cell (ASC) responses to Wa HRV infection, but it was associated with significantly fewer numbers of HRV–specific IgA ASC in the duodenum. We further analyzed the pigś immune responses to the passive IgY treatment. The oral administration of IgY Abs induced IgG Ab responses to chicken IgY in serum and local IgA and IgG Ab responses to IgY in the intestinal contents of neonatal piglets in a dose dependent manner. To our knowledge, this is the first study to show that IgY Abs administered orally as a milk supplement passively protect neonatal pigs against an enteric viral pathogen (HRV). Piglets are an animal model with a gastrointestinal physiology and an immune system that closely mimic human infants. This strategy can be scaled-up to inexpensively produce large amounts of polyclonal IgY Abs from egg yolks to be applied as a preventive and therapeutic passive Ab treatment to control RV diarrhea.     

Effects of Maternal Antibodies on Protection and Development of Antibody Responses to Human Rotavirus in Gnotobiotic Pigs

Although maternal antibodies can protect against infectious disease in infancy, they can also suppress active immune responses. The effects of circulating maternal antibodies, with and without colostrum and milk antibodies, on passive protection and active immunity to human rotavirus (HRV) were examined in gnotobiotic pigs. Pigs received intraperitoneal injections of high-titer serum (immune pigs [groups 1 and 2]) from immunized sows, low-titer serum from naturally infected sows (control pigs [groups 3 and 4]), or no serum (group 5). Immune or control colostrum and milk were added to the diet of groups 2 and 4, respectively. After inoculation (3 to 5 days of age) and challenge (postinoculation day [PID] 21) with virulent HRV, the effects of maternal antibodies on protection (from diarrhea and virus shedding), and on active antibody responses (measured by quantitation of antibody-secreting cells [ASC] in intestinal and systemic lymphoid tissues by ELISPOT) were evaluated. Groups 1 and 2 had significantly less diarrhea and virus shedding after inoculation but higher rates of diarrhea and virus shedding after challenge than did groups 3 and 5. Group 1 and 2 pigs had significantly fewer immunoglobulin A (IgA) ASC in intestinal tissues at PID 21 and at postchallenge day (PCD) 7 compared to group 5. Significantly fewer IgG ASC were present in the intestines of group 2 pigs at PID 21 and PCD 7 compared to group 5. There was a trend towards fewer ASC in intestinal tissues of group 2 than group 1, from PID 21 on, with significantly fewer IgA ASC at PCD 7. IgG ASC in the duodenum and mesenteric lymph nodes of group 3 and 4 pigs were significantly fewer than in group 5 at PCD 7. These decreases in ASC emphasize the role of passive antibodies in impairing induction of ASC rather than in merely suppressing the function of differentiated B cells. To be successful, vaccines intended for populations with high titers of maternal antibodies (infants in developing countries) may require higher titers of virus, multiple doses, or improved delivery systems, such as the use of microencapsulation or immune stimulating complexes, to overcome the suppressive effects of maternal antibodies.     

Viremia and nasal and rectal shedding of rotavirus in gnotobiotic pigs inoculated with Wa human rotavirus

Respiratory symptoms with rotavirus shedding in nasopharyngeal secretions have been reported in children with and without gastrointestinal symptoms (Zheng et al., 1991, J. Med. Virol. 34:29-37). To investigate if attenuated and virulent human rotavirus (HRV) strains cause upper respiratory tract infections or viremia in gnotobiotic pigs, we inoculated them with attenuated or virulent HRV intranasally, intravenously, or orally or via feeding tube (gavage) and assayed virus shedding. After oral or intranasal inoculation with attenuated HRV, the pigs remained asymptomatic, but 79 to 95% shed virus nasally and 5 to 17% shed virus rectally. After inoculation by gavage, no pigs shed virus nasally or rectally, but all pigs seroconverted with antibodies to HRV. No viremia was detected through postinoculation day 10. Controls inoculated intranasally with nonreplicating rotavirus-like particles or mock inoculated did not shed virus. In contrast, 100% of pigs inoculated with virulent HRV (oral, intranasal, or gavage) developed diarrhea, shed virus nasally and rectally, and had viremia. The infectivity of sera from the viremic virulent HRV-inoculated pigs was confirmed by inoculating gnotobiotic pigs orally with pooled HRV-positive serum. Serum-inoculated pigs developed diarrhea and fecal and nasal virus shedding and seroconverted with serum and intestinal HRV antibodies. Pigs inoculated intravenously with serum or intestinal contents from the viremic virulent HRV-inoculated pigs developed diarrhea, virus shedding, and viremia, similar to the orally inoculated pigs. This study provides new evidence that virulent HRV causes transient viremia and upper respiratory tract infection in addition to gastrointestinal infection in gnotobiotic pigs, confirming previous reports of rotavirus antigenemia (Blutt et al., Lancet 362:1445-1449, 2003). Our data also suggest that intestinal infection might be initiated from the basolateral side of the epithelial cells via viremia. Additionally, virus shedding patterns indicate a different pathogenesis for attenuated versus virulent HRV.     

Cytokine responses in gnotobiotic pigs after infection with virulent or attenuated human rotavirus

To understand the role of cytokines during rotavirus infection, we assessed the kinetics of tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6) (proinflammatory), IL-12 (Th1 inducer), gamma interferon (IFN-gamma) (Th1), IL-4 and IL-10 (Th2), and transforming growth factor beta (Th3) cytokine responses by enzyme-linked immunosorbent assay in serum and intestinal contents of neonatal gnotobiotic pigs and IL-12, IFN-gamma, IL-4, and IL-10 cytokine-secreting cell (CSC) responses of mononuclear cells from ileum, spleen, and blood by ELISPOT. Pigs received the virulent Wa P1A[8]G1 strain of human rotavirus (HRV) (VirHRV), attenuated Wa HRV (AttHRV), or mock (controls). The TNF-alpha levels peaked earlier and remained elevated in serum of the VirHRV group but peaked later in the AttHRV group. In serum, IL-6 was significantly elevated at postinoculation day (PID) 1 in the VirHRV group and at PID 3 in both HRV groups. The IL-12 was detected in serum of all pigs including controls with significantly elevated peaks in both HRV-infected groups, indicating a role for IL-12 in the induction of immune responses to rotavirus infection. Only low and transient IFN-gamma responses occurred in serum and intestinal contents of the AttHRV-infected pigs, compared to significantly higher and prolonged IFN-gamma responses in the VirHRV-infected pigs. This observation coincides with the diarrhea and viremia induced by VirHRV. The number of IFN-gamma-secreting cells was significantly higher in the ileum of the VirHRV group than in that of the controls. The number of IL-4 CSCs was significantly higher in ileum of both HRV groups than in that of the controls. Significantly higher levels of IL-10 in the serum occurred early in the VirHRV group, compared to lower levels in the AttHRV group. However, the number of IL-10 CSCs was significantly higher later in ileum and spleen of the AttHRV than in the VirHRV group, suggesting a delayed initiation of a Th2 response induced by AttHRV. A significantly higher percentage of pigs had IFN-gamma and IL-10 responses in serum after VirHRV infection than after AttHRV infection or in controls. These data indicate a balanced Th1/Th2 response during rotavirus infection, with higher cytokine levels early after infection with VirHRV compared to that with AttHRV. Mapping the kinetics and patterns of cytokine responses after rotavirus infection has important implications for induction of protective immunity by HRV vaccines. Higher protection rates may be associated with more balanced Th1- and Th2-type responses, but induction of higher earlier IFN-gamma (Th1) and proinflammatory cytokines triggered by VirHRV may also play an important role in the higher intestinal immunoglobulin A responses and protection rates induced by VirHRV.     

Probiotic Lactobacillus rhamnosus GG Enhanced Th1 Cellular Immunity but Did Not Affect Antibody Responses in a Human Gut Microbiota Transplanted Neonatal Gnotobiotic Pig Model

This study aims to establish a human gut microbiota (HGM) transplanted gnotobiotic (Gn) pig model of human rotavirus (HRV) infection and diarrhea, and to verify the dose-effects of probiotics on HRV vaccine-induced immune responses. Our previous studies using the Gn pig model found that probiotics dose-dependently regulated both T cell and B cell immune responses induced by rotavirus vaccines. We generated the HGM transplanted neonatal Gn pigs through daily feeding of neonatal human fecal suspension to germ-free pigs for 3 days starting at 12 hours after birth. We found that attenuated HRV (AttHRV) vaccination conferred similar overall protection against rotavirus diarrhea and virus shedding in Gn pigs and HGM transplanted Gn pigs. HGM promoted the development of the neonatal immune system, as evidenced by the significantly enhanced IFN-γ producing T cell responses and reduction of regulatory T cells and their cytokine production in the AttHRV-vaccinated pigs. The higher dose Lactobacillus rhamnosus GG (LGG) feeding (14 doses, up to 10⁹ colony-forming-unit [CFU]/dose) effectively increased the LGG counts in the HGM Gn pig intestinal contents and significantly enhanced HRV-specific IFN-γ producing T cell responses to the AttHRV vaccine. Lower dose LGG (9 doses, up to 10⁶ CFU/dose) was ineffective. Neither doses of LGG significantly improved the protection rate, HRV-specific IgA and IgG antibody titers in serum, or IgA antibody titers in intestinal contents compared to the AttHRV vaccine alone, suggesting that an even higher dose of LGG is needed to overcome the influence of the microbiota to achieve the immunostimulatory effect in the HGM pigs. This study demonstrated that HGM Gn pig is an applicable animal model for studying immune responses to rotavirus vaccines and can be used for studying interventions (i.e., probiotics and prebiotics) that may enhance the immunogenicity and protective efficacy of vaccines through improving the gut microbiota.     

Protective immunity and antibody-secreting cell responses elicited by combined oral attenuated Wa human rotavirus and intranasal Wa 2/6-VLPs with mutant Escherichia coli heat-labile toxin in gnotobiotic pigs

Two combined rotavirus vaccination regimens were evaluated in a gnotobiotic pig model of rotavirus infection and disease and were compared to previously tested rotavirus vaccination regimens. The first (AttHRV/VLP2x) involved oral inoculation with one dose of attenuated (Att) Wa human rotavirus (HRV), followed by two intranasal (i.n.) doses of a rotavirus-like particle (2/6-VLPs) vaccine derived from Wa (VP6) and bovine RF (VP2) rotavirus strains. The 2/6-VLPs were coadministered with a mutant Escherichia coli heat-labile toxin, LT-R192G (mLT) adjuvant. For the second regimen (VLP2x/AttHRV), two i.n. doses of 2/6-VLPs+mLT were given, followed by one oral dose of attenuated Wa HRV. To compare the protective efficacy and immune responses induced by the combined vaccine regimens with individual rotavirus vaccine regimens, we included in the experiments the following vaccine groups: one oral dose of attenuated Wa HRV (AttHRV1x and Mock2x/AttHRV, respectively), three oral doses of attenuated Wa HRV (AttHRV3x), three i.n. doses of 2/6-VLPs plus mLT (VLP3x), three i.n. doses of purified double-layered inactivated Wa HRV plus mLT (InactHRV3x), mLT alone, and mock-inoculated pigs. The isotype, magnitude, and tissue distribution of antibody-secreting cells (ASCs) in the intestinal and systemic lymphoid tissues were evaluated using an enzyme-linked immunospot assay. The AttHRV/VLP2x regimen stimulated the highest mean numbers of intestinal immunoglobulin A (IgA) ASCs prechallenge among all vaccine groups. This regimen induced partial protection against virus shedding (58%) and diarrhea (44%) upon challenge of pigs with virulent Wa HRV. The reverse VLP2x/AttHRV regimen was less efficacious than the AttHRV/VLP2x regimen in inducing IgA ASC responses and protection against diarrhea (25% protection rate) but was more efficacious than VLP3x or InactHRV3x (no protection). In conclusion, the AttHRV/VLP2x vaccination regimen stimulated the strongest B-cell responses in the intestinal mucosal immune system at challenge and conferred a moderately high protection rate against rotavirus disease, indicating that priming of the mucosal inductive site at the portal of natural infection with a replicating vaccine, followed by boosting with a nonreplicating vaccine at a second mucosal inductive site, may be a highly effective approach to stimulate the mucosal immune system and induce protective immunity against various mucosal pathogens.     

Systematic and intestinal antibody-secreting cell responses and correlates of protective immunity to human rotavirus in a gnotobiotic pig model of disease.

Neonatal gnotobiotic pigs orally inoculated with virulent (intestinal-suspension) Wa strain human rotavirus (which mimics human natural infection) developed diarrhea, and most pigs which recovered (87% protection rate) were immune to disease upon homologous virulent virus challenge at postinoculation day (PID) 21. Pigs inoculated with cell culture-attenuated Wa rotavirus (which mimics live oral vaccines) developed subclinical infections and seroconverted but were only partially protected against challenge (33% protection rate). Isotype-specific antibody-secreting cells (ASC were enumerated at selected PID in intestinal (duodenal and ileal lamina propria and mesenteric lymph node [MLN]) and systemic (spleen and blood) lymphoid tissues by using enzyme-linked immunospot assays. At challenge (PID 21), the numbers of virus-specific immunoglobulin A (IgA) ASC, but not IgG ASC, in intestines and blood were significantly greater in virulent-Wa rotavirus-inoculated pigs than in attenuated-Wa rotavirus-inoculated pigs and were correlated (correlation coefficients: for duodenum and ileum, 0.9; for MLN, 0.8; for blood, 0.6) with the degree of protection induced. After challenge, the numbers of IgA and IgG virus-specific ASC and serum-neutralizing antibodies increased significantly in the attenuated-Wa rotavirus-inoculated pigs but not in the virulent-Wa rotavirus-inoculated pigs (except in the spleen and except for IgA ASC in the duodenum). The transient appearance of IgA ASC in the blood mirrored the IgA ASC responses in the gut, albeit at a lower level, suggesting that IgA ASC in the blood of humans could serve as an indicator for IgA ASC responses in the intestine after rotavirus infection. To our knowledge, this is the first report to study and identify intestinal IgA ASC as a correlate of protective active immunity in an animal model of human-rotavirus-induced disease.     

Diarrheal response of gnotobiotic pigs after fetal infection and neonatal challenge with homologous and heterologous human rotavirus strains.

Pigs exposed in utero to human rotavirus (HRV) strain Wa serotype 1 from 15 to 36 days prior to birth responded immunologically by modifying their clinical response to neonatal oral challenge with a pathogenic dose of homologous Wa or heterologous M serotype 3 HRV. In these cases, diarrhea was prevented in 12 of 14 pigs and greatly reduced in the other two. However, fecal virus shedding was not significantly modified, since it was detected in 12 of 14 pigs. These results suggest the existence of a closer antigenic relationship between these two different HRV serotypes which may only be expressed in an in vivo test system. Exposure of fetal pigs to HRV DS-1 serotype 2 failed to cause infection or to induce any protection when pigs were challenged at birth with HRV Wa. This model for cross-protection studies in gnotobiotic piglets offers good possibilities for the evaluation of potential HRV vaccine candidates, for the in vivo study of antigenic similarities between rotavirus serotypes, and for the understanding of protective immune responses against diarrhea and virus shedding.     

Antibody-Secreting Cell Responses and Protective Immunity Assessed in Gnotobiotic Pigs Inoculated Orally or Intramuscularly with Inactivated Human Rotavirus

Newborn gnotobiotic pigs were inoculated twice perorally (p.o.) (group 1) or intramuscularly (i.m.) (group 2) or three times i.m. (group 3) with inactivated Wa strain human rotavirus and challenged with virulent Wa human rotavirus 20 to 24 days later. To assess correlates of protection, antibody-secreting cells (ASC) were enumerated in intestinal and systemic lymphoid tissues from pigs in each group at selected postinoculation days (PID) or postchallenge days. Few virus-specific ASC were detected in any tissues of group 1 pigs prior to challenge. By comparison, groups 2 and 3 had significantly greater numbers of virus-specific immunoglobulin M (IgM) ASC in intestinal and splenic tissues at PID 8 and significantly greater numbers of virus-specific IgG ASC and IgG memory B cells in spleen and blood at challenge. However, as for group 1, few virus-specific IgA ASC or IgA memory B cells were detected in any tissues of group 2 and 3 pigs. Neither p.o. nor i.m. inoculation conferred significant protection against virulent Wa rotavirus challenge (0 to 6% protection rate), and all groups showed significant anamnestic virus-specific IgG and IgA ASC responses. Hence, high numbers of IgG ASC or memory IgG ASC in the systemic lymphoid tissues at the time of challenge did not correlate with protection. Further, our findings suggest that inactivated Wa human rotavirus administered either p.o. or parenterally is significantly less effective in inducing intestinal IgA ASC responses and conferring protective immunity than live Wa human rotavirus inoculated orally, as reported earlier (L. Yuan, L. A. Ward, B. I. Rosen, T. L. To, and L. J. Saif, J. Virol. 70:3075–3083, 1996). Thus, more efficient mucosal delivery systems and rotavirus vaccination strategies are needed to induce intestinal IgA ASC responses, identified previously as a correlate of protective immunity to rotavirus.     

轮状病毒灭活疫苗和减毒活疫苗序贯免疫的体液免疫应答效果

目的:评价采用轮状病毒灭活疫苗进行初始免疫,减毒活疫苗进行加强免疫的序贯免疫方案的体液免疫应答效果。方法:将实验小鼠随机分为4组(口服疫苗组、序贯疫苗组、口服对照组及序贯对照组),按相应方案免疫后,ELISA检测血清轮状病毒特异性IgG和IgA、肠道轮状病毒特异性IgA;微量中和实验检测血清病毒特异性中和抗体;同时采用ELISA分析口服活疫苗后病毒排出情况。结果:与对照组相比,序贯疫苗组小鼠产生的轮状病毒特异性血清IgG、IgA、中和抗体及肠道IgA水平显著升高。与口服疫苗组相比,序贯疫苗组的免疫方案诱发的轮状病毒特异性血清IgG、IgA、中和抗体水平显著升高,肠道IgA水平两组间没有显著差异。同时,与口服疫苗组相比,序贯疫苗组中轮状病毒灭活疫苗进行的初始免疫未影响第一次口服活疫苗后病毒的排出量和排出时间,但序贯疫苗组第二次口服活疫苗后病毒的排出量迅速减少,排毒时间快速缩短,与口服疫苗组第三次服苗后病毒的排出量和排出时间相似。结论: 轮状病毒灭活疫苗和减毒活疫苗序贯免疫可有效诱发小鼠全身和黏膜局部的体液免疫应答,该方案将有可能成为轮状病毒疫苗临床应用的候选方案。     

人类轮状病毒基因DNA免疫的初步研究

本文研究人类轮状病毒基因ＤＮＡ免疫及应用。通过构建重组质粒ｐＣＩ／ｖｐ７,ｐＣＩ／ｖｐ４及ｐＣＩ／ｖｐ６,以肌注法导入ＢＡＬＢ／ｃ小鼠肌肉组织,其中ｐＣＩ／ｖｐ７及ｐＣＩ／ｖｐ４能　有效引起机体免疫应答,而且对轮状病毒Ｗａ株有一定中和效应。从本次试验的结果来看　,人类轮状病毒ＤＮＡ疫苗能作为预防病毒性小儿腹泻的一种手段。     

Transgenic Tobacco Expressing a Modified VP6 Gene Protects Mice Against Rotavirus Infection

Elevated expression of the rotavirus VP6 antigen in transgenic plants is a critical factor in the development of a safe and effective rotavirus vaccine. Using codon optimization, a gene that encodes the inner capsid protein VP6 of the human group A rotavirus was synthesized （sVP6）. The VP6 and sVp6 genes were transformed into tobacco （Nicotiana tabacum L.） plants using Agrobacterium tumefaciens. The expression level of the sVP6 gene in transgenic plants was 3.8-34-fold higher than that of controls containing the non-modified VP6 gene, accounting for up to 0.34% of the total soluble protein （TSP）. Then, BALB/ c female mice that had been gavaged weekly with 10 mg TSP containing 34 p.g VP6 protein, in which VP6-specific serum IgG and mucosal IgA antibodies were investigated. The severity and duration of diarrhea caused by simian rotavirus SA-11 challenge were reduced significantly in passively immunized pups, which indicates that anti-VP6 antibodies generated in orally immunized female mice can be passed onto pups and provide heterotypic protection. An edible vaccine based on the VP6 of human rotavirus group A could provide a means to protect children and young animals from severe acute diarrhea.     

Expression of Human Rotavirus Chimeric Fusion Proteins from Replicating but Non Disseminating Adenovectors and Elicitation of Rotavirus-Specific Immune Responses in Mice

The aim of this study was to evaluate the usefulness of replicating but non disseminating adenovirus vectors (AdVs) as vaccine vector using human rotavirus (HRV) as a model pathogen. HRV VP7, VP4, or VP4Δ (N-terminal 336 amino acids of VP4) structural proteins as well as the VP4Δ::VP7 chimeric fusion protein were expressed in mammalian cells when delivered with the AdVs. A preliminary experiment demonstrated that VP4Δ was able to induce a HRV-specific IgG response in BALB/c mice inoculated intramuscularly with AdVs expressing the rotaviral protein. Moreover, an AdV-prime/plasmid DNA-boost regimen of vectors resulted in VP4Δ-specific antibody (Ab) titers ~4 times higher than those obtained from mice immunized with AdVs alone. Subsequently, the various HRV protein-encoding AdVs were compared using the AdV-prime/plasmid DNA-boost regimen. Higher IgG and IgA responses to HRV were obtained when VP4Δ::VP7 fusion protein was used as an immunogen as compared to VP7 or VP4 alone or to a mix of both proteins delivered independently by AdVs. A synergetic effect in terms of Ab was obtained with VP4Δ::VP7. In conclusion, this study demonstrated for the first time the suitability of using replicating but non disseminating AdVs as vaccine vector and the VP4Δ::VP7 fusion protein as an immunogen for vaccination against HRV.     

轮状病毒疫苗研究进展及其转基因植物疫苗的开发前景

轮状病毒是目前婴幼儿秋冬季腹泻的最主要病原物,作者通过轮状病毒减毒株疫苗,亚单位疫苗和DNA疫苗研究进展的综合分析,指出了减毒株疫苗在实际应用中存在的弊端,论述了开发新型轮状病毒疫苗,特别是转基因植物疫苗的必要性和可行性。     

Entrapment of H1N1 Influenza Virus Derived Conserved Peptides in PLGA Nanoparticles Enhances T Cell Response and Vaccine Efficacy in Pigs

Pigs are believed to be one of the important sources of emerging human and swine influenza viruses (SwIV). Influenza virus conserved peptides have the potential to elicit cross-protective immune response, but without the help of potent adjuvant and delivery system they are poorly immunogenic. Biodegradable polylactic-co-glycolic acid (PLGA) nanoparticle (PLGA-NP) based vaccine delivery system enhances cross-presentation of antigens by the professional antigen presenting cells. In this study, Norovirus P particle containing SwIV M2e (extracellular domain of the matrix protein 2) chimera and highly conserved two each of H1N1 peptides of pandemic 2009 and classical human influenza viruses were entrapped in PLGA-NPs. Influenza antibody-free pigs were vaccinated with PLGA-NPs peptides cocktail vaccine twice with or without an adjuvant, Mycobacterium vaccae whole cell lysate, intranasally as mist. Vaccinated pigs were challenged with a virulent heterologous zoonotic SwIV H1N1, and one week later euthanized and the lung samples were analyzed for the specific immune response and viral load. Clinically, pigs vaccinated with PLGA-NP peptides vaccine had no fever and flu symptoms, and the replicating challenged SwIV was undetectable in the bronchoalveolar lavage fluid. Immunologically, PLGA-NP peptides vaccination (without adjuvant) significantly increased the frequency of antigen-specific IFNγ secreting CD4 and CD8 T cells response in the lung lymphocytes, despite not boosting the antibody response both at pre- and post-challenge. In summary, our data indicated that nanoparticle-mediated delivery of conserved H1N1 influenza peptides induced the virus specific T cell response in the lungs and reduced the challenged heterologous virus load in the airways of pigs.     

Detection and Typing of Human Rotavirus in Reference to Repeated Acute Gastroenteritis in Infants

Stool specimens from infants who visited a clinic because of acute gastroenteritis were tested for the presence of human rotavirus. Among the samples obtained were specimens taken from seven patients who had visited the clinic at two different times. In six of these seven children, human rotavirus (HRV) was detected in only one of the specimens taken (i.e. during only one of the two visits). One patient was shown to have excreted HRV twice; in both cases the HRV was serotyped to be type 1. The present results indicate that the symptomatic reinfection of HRV was not a widely occurring phenomenon in the group of infants tested.     

Rotavirus vaccine administered parenterally induces protective immunity.

We performed experiments to determine whether parenteral immunization with SA11 rotavirus can induce active protective immunity in a rabbit model of rotavirus infection. After one or two intramuscular injections of 1 ml of live or formalin-inactivated SA11 virus, we evaluated the mucosal and serologic immune response and protection from challenge with a high dose of live, virulent rabbit (Ala) rotavirus. Inactivated SA11 virus preparations, evaluated by enzyme-linked immunosorbent assay (ELISA) with a panel of VP4- and VP7-specific neutralizing and nonneutralizing monoclonal antibodies, did not show a loss of epitopes from the inactivation procedure compared with live virus. Administration of two doses of vaccine, one at zero days postvaccination (DPV) and a booster shot at 49 DPV, followed by challenge at 71 DPV with 3.5 x 10(5) PFU of Ala virus resulted in protection from challenge. None of the two-dose virus-vaccinated rabbits shed virus after challenge, while virus shedding was detected in all control rabbits (P = 0.001, Fisher's exact two-tailed test). Differences in total serum immunoglobulin (Ig) antirotavirus ELISA titers (P < 0.05, Wilcoxon's rank sum test) were observed between groups vaccinated with virus in aluminum phosphate or Freund's adjuvant but not between groups vaccinated with live or inactivated virus in either adjuvant. All rabbits given two doses of vaccine had detectable antirotavirus intestinal antibody of the IgG, but not IgA, isotype. After challenge, fourfold or greater increases in intestinal IgG antibody responses were observed in three rabbits, whereas all controls and all but one virus-vaccinated rabbit had an intestinal IgA antibody response. In contrast, vaccination of rabbits with one dose of SA11 followed by challenge at 21 DPV did not protect from challenge; no difference in the mean number of days of virus shedding between any of the vaccinated groups and controls was observed. A serologic, but not a mucosal, antibody response was observed after the one-dose vaccination regimen. Differences in serologic antibody titers were not observed between any of the one-dose virus-vaccinated groups. These data indicate that parenteral vaccination with two, but not one, doses of rotavirus in either Freund's adjuvant or aluminum phosphate can induce active protection from challenge.(ABSTRACT TRUNCATED AT 400 WORDS)     

Evaluation of the efficacy of a low-passage bovine rotavirus (strain WC3) vaccine in children in Central Africa

The safety and efficacy of a WC3 rotavirus vaccine was evaluated in a double-blind placebo-controlled trial involving 472 children in Bangui (Central African Republic). Each child received two doses of either placebo (235 children) or vaccine (237 children) at a 1-month interval, the first dose being given at 3 months of age. During the follow-up survey 9 months after the first dose, 117 rotavirus diarrhoeas were observed, 59 in the placebo group, 58 in the vaccinated group. The only positive effect of the vaccine was a significantly higher proportion of mild rotavirus diarrhoeal episodes in the vaccinated group than in the placebo group. Of the children in the vaccinated group, 60% had a positive immune response to WC3 rotavirus when tested by plaque reduction seroneutralization.     

In Vitro and in Vivo Evaluation of the Efficacy of Bovine Colostrum against Human Rotavirus Infection

We found that skimmed and concentrated bovine late colostrum (SCBLC) obtained from normal cows at 6–7 d after parturition exhibited high potency in inhibiting replication of human rotavirus (HRV) in vitro. Furthermore, prophylactic oral administration of SCBLC once before inoculation of HRV prevented the development of diarrhea in suckling mice in vivo. SCBLC from normal cows might be useful in the prevention of HRV-induced severe gastroenteritis in immunocompromised hosts.