Assembly of cholera toxin B subunit full-length rotavirus NSP4 fusion protein oligomers in transgenic potato

A CTB-NSP4(175) fusion gene encoding the entire 175-aa murine rotavirus NSP4 enterotoxin protein was transferred into Solanum tuberosum cells by Agrobacterium tumefaciens-mediated transformation. The CTB-NSP4(175) enterotoxin fusion gene was detected in the genomic DNA of transformed leaves by PCR DNA amplification. Synthesis and assembly of the full-length CTB-NSP4(175) fusion protein into oligomeric structures of pentamer size was detected in transformed tuber extracts by immunoblot analysis. The binding of CTB-NSP4(175 )fusion protein pentamers to intestinal epithelial cell membrane receptors was quantified by G(M1)-ganglioside enzyme-linked immunosorbent assay (G(M1)-ELISA). The ELISA results showed that CTB-NSP4(175) fusion protein was 0.006-0.026% of the total soluble tuber protein. The synthesis of CTB-NSP4(175) monomers and their assembly into biologically active oligomers in transformed potato tubers demonstrates the feasibility of using edible plants for the synthesis of enterocyte-targeted full-length rotavirus enterotoxin antigens that retain all of their pathogenic epitopes for initiation of a maximum mucosal immune response.     

A plant-based multicomponent vaccine protects mice from enteric diseases

Cholera toxin (CT) B and A2 subunit complementary DNAs (cDNAs) were fused to a rotavirus enterotoxin and enterotoxigenic Escherichia coli fimbrial antigen genes and transferred into potato. Immunoblot and enzyme-linked immunosorbent assay (ELISA) results indicated that the fusion antigens were synthesized in transformed tuber tissues and assembled into cholera holotoxin-like structures that retained enterocyte-binding affinity. Orally immunized mice generated detectable levels of serum and intestinal antibodies against the pathogen antigens. Elevated levels of interleukin 2 (IL2) and interferon gamma (INFgamma) detected in immunogen-challenged spleen cells from the immunized mice indicated the presence of a strong Th1 immune response to the three plant-synthesized antigens. This result was supported by flow cytometry analysis of immunized mouse spleen cells that showed a significant increase in CD4+ lymphocyte numbers. Diarrhea symptoms were reduced in severity and duration in passively immunized mouse neonates following rotavirus challenge. The results suggest that food plants can function as vaccines for simultaneous protection against infectious virus and bacterial diseases.     

Oral immunisation of mice with transgenic rice calli expressing cholera toxin B subunit fused to consensus dengue cEDIII antigen induces antibodies to all four dengue serotypes

Dengue virus (DENV) infection is an emerging global health threat. DENV consists of four distinct serotypes, necessitating a tetravalent vaccine. In this study, expression of consensus envelope protein domain III (cEDIII) fused to cholera toxin B subunit (CTB) in transgenic rice calli was improved using the luminal binding protein BiP at the N-terminus and the SEKDEL signal sequences at the C-terminus, targeting the recombinant protein to endoplasmic reticulum (ER). We found that the fusion protein showed higher levels of expression when compared to the fusion proteins using rice amylase 3D (RAmy3D) or CTB native signal sequence only. The CTB-cEDIII fusion protein was evaluated as an oral dengue vaccine candidate in mice. Serotype specific systemic IgG antibodies and specific IgA response in feces were detected and furthermore, T cell proliferation and high frequency antibody-secreting B cells were detected in the spleen. These results suggest the possible use of plant-based dengue tetravalent vaccine targeted to the mucosal immune system for induction of systemic and mucosal immune responses to DENV infection.     

Protection against murine intestinal amoebiasis induced by oral immunization with the 29 kDa antigen of Entamoeba histolytica and cholera toxin

Entamoeba histolytica antigens recognized by salivary IgA from infected patients include the 29 kDa antigen (Eh29), an alkyl hydroperoxide reductase. Here, we investigate the potential of recombinant Eh29 and an Eh29-cholera toxin subunit B (CTxB) fusion protein to confer protection against intestinal amoebiasis after oral immunization. The purified Eh29-CTxB fusion retained the critical ability to bind ganglioside GM₁, as determined by ELISA. Oral immunization of C3H/HeJ mice with Eh29 administered in combination with a subclinical dose of whole cholera toxin, but not as an Eh29-CTxB fusion, induced elevated levels of intestinal IgA and serum IgG anti-Eh29 antibodies that inhibited trophozoites adherence to MDCK cell monolayers. The 80% of immunized mice seen to develop IgA and IgG immune responses showed no evidence of infection in tissue sections harvested following intracecal challenge with virulent E. histolytica trophozoites. These results suggest that Eh29 is capable of inducing protective anti-amoebic immune responses in mice following oral immunization and could be used in the development of oral vaccines against amoebiasis.     

流感新型黏膜疫苗的研究

目的评价PorA、PorB和Class4对流感裂解疫苗的免疫增强作用,从中挑选出最有效的流感黏膜佐剂,为发展流感黏膜疫苗提供理论基础。方法流感三价裂解抗原按比例与PorA、PorB和Class4非共价结合,滴鼻免疫Balb／c小鼠3次,采取间接ELISA检测血清特异性IgG抗体及抗体亚型,检测鼻咽、肺、小肠和阴道冲洗液中IgA效价,采用血凝抑制试验检测血清中HAI效价。结果PorB重组蛋白佐剂组较无佐剂的流感裂解抗原组在提高小鼠早期免疫应答的同时诱导较强的系统免疫应答和黏膜免疫应答;PorA组也有黏膜佐剂的功能,但和无佐剂的流感裂解抗原组相比,差异无统计学意义。结论在蛋白体的三分子中,以PorB为佐剂的流感黏膜疫苗不仅提高了抗原的系统免疫应答,而且诱导了较强的小鼠呼吸道、生殖道的局部黏膜免疫应答,为流感黏膜疫苗的研制奠定了理论基础。     

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.     

禽IL-2与传染性法氏囊VP2融合蛋白免疫学特性

为研究禽细胞因子IL-2与IBDV主要保护性抗原VP2基因融合蛋白的免疫学特性,将重组的rVP2-IL-2融合蛋白免疫鸡,通过IBDV-VP2 ELISA抗体效价、抗体亚型(IgG1和IgG2a)、淋巴细胞增殖、INF-γ和IL-4细胞因子的分泌水平、中和抗体以及动物攻毒试验检测评价其对鸡体免疫水平的影响。抗体滴度测定和淋巴细胞增殖试验结果显示,rVP2-IL-2融合蛋白免疫鸡体的体液和细胞免疫应答水平均明显高于单独的VP2蛋白免疫组。抗体亚型测定结果显示,rVP2-IL-2融合蛋白免疫组鸡体能产生一个平衡的IgG1和IgG2a抗体反应。细胞因子ELISA试验结果表明rVP2-IL-2融合蛋白能有效平衡Th1(γ-IFN)和Th2(IL-4)类型的细胞免疫反应。动物攻毒试验rVp2-IL-2融合蛋白免疫组鸡体获得了85%的保护率,表明构建的rVP2-IL-2融合蛋白对IBDV的攻击具有较好的免疫保护作用。本研究为进一步研制IBD高效的基因工程疫苗奠定了基础。     

Synthesis and assembly of a cholera toxin B subunit-rotavirus VP7 fusion protein in transgenic potato

A gene encoding VP7, the outer capsid protein of simian rotavirus SA11, was fused to the carboxyl terminus of the cholera toxin B subunit gene. A plant expression vector containing the fusion gene under control of the mannopine synthase P₂ promoter was introduced into Solanum tuberosum cells by Agrobacterium tumefaciens-mediated transformation. The CTB::VP7 fusion gene was detected in the genomic DNA of transformed potato leaf cells by polymerase chain reaction (PCR) amplification methods. Immunoblot analysis of transformed potato tuber tissue extracts showed that synthesis and assembly of the CTB::VP7 fusion protein into oligomers of pentameric size occurred in the transformed plant cells. The binding of CTB::VP7 fusion protein pentamers to sialo-sugar containing GM1 ganglioside receptors on the intestinal epithelial cell membrane was quantified by enzyme-linked immunosorbent assay (ELISA). The ELISA results showed that the CTB::VP7 fusion protein made up approx 0.01% of the total soluble tuber protein. Synthesis and assembly of CTB::VP7 monomers into biologically active pentamers in transformed potato tubers demonstrates the feasibility of using edible plants as a mucosal vaccine for the production and delivery system for rotavirus capsid protein antigens.     

A组轮状病毒VP6与霍乱毒素B亚基融合蛋白在大肠杆菌中的表达及生物活性分析

利用霍乱毒素B亚基 (CholeratoxinBsubunit,CTB)的免疫载体作用 ,将轮状病毒相关抗原引入口服免疫体系 ,可激起有效的粘膜免疫反应 ,这里报道了CTB基因与A组轮状病毒地方株T114VP6全基因的融合 ,并在大肠杆菌BL21(DE3 )中进行了融合蛋白的表达。在IPTG诱导下得到分子量为 5 6kD的融合蛋白 ,表达量占菌体蛋白的15 %。分别用抗CT的抗体和抗A组轮状病毒的高价免疫血清进行WesternBlot检测 ,结果证明融合蛋白CTB VP6保留了天然霍乱毒素B亚基及轮状病毒VP6的抗原性。G_M1-ELISA检测表明 ,复性后的融合蛋白具有与神经节苷脂GM1 结合的能力。     

Immunogenicity of a Cholera Toxin B Subunit <Emphasis Type="Italic">Porphyromonas gingivalis</Emphasis> Fimbrial Antigen Fusion Protein Expressed in <Emphasis Type="Italic">E. coli</Emphasis>

The gram-negative anaerobic oral bacterium Porphyromonas gingivalis initiates periodontal disease through fimbrial attachment to saliva-coated oral surfaces. To study the effects of immunomodulation on enhancement of subunit vaccination, the expression in E. coli and immunogenicity of P. gingivalis fimbrial protein (FimA) linked to the C-terminus of the cholera toxin B subunit (CTB) were investigated. Complementary DNAs encoding the P. gingivalis 381 fimbrillin protein sequence FimA1 (amino acid residues 1-200) and FimA2 (amino acid residues 201-337) were cloned into an E. coli expression vector downstream of a cDNA fragment encoding the immunostimulatory CTB. CTB-FimA1 and CTB-FimA2 fusion proteins synthesized in E. coli BL21 (DE3) cells were purified under denaturing conditions by Ni2+-NTA affinity column chromatography. Renaturation of the CTB-FimA1 and CTB-FimA2 fusion proteins, permitted identification of CTB-FimA pentamers and restored CTB binding activity to GM1-ganglioside to provide a biologically active CTB-FimA fusion protein. Mice orally inoculated with purified CTB-FimA1 or CTB-FimA2 fusion proteins generated measurable FimA1 and FimA2 IgG antibody titers, while no serum fimbrial IgG antibodies were detected when mice were inoculated with FimA1 or FimA2 proteins alone. Immunoblot analysis confirmed that sera from mice immunized with CTB linked to FimA1 or FimA2 contained antibodies specific for P. gingivalis fimbrial proteins. In addition, mice immunized with FimA2 or CTB-FimA2 generated measurable intestinal IgA titers indicating the presence of fimbrial antibody class switching. Further, mice orally immunized with CTB-FimA1 generated higher IgA antibody titers than mice inoculated with FimA1 alone. The experimental data show that the immunostimulatory molecule CTB enhances B cell-mediated immunity against linked P. gingivalis FimA fusion proteins, in comparison to immunization with FimA protein alone. Thus, linkage of CTB to P. gingivalis fimbrial antigens can increase subunit vaccine immunogenicity to provide enhanced protection against periodontal disease.     

表达轮状病毒nsp4基因重组腺病毒的构建与免疫评价

目的：应用非复制腺病毒表达系统构建表达人轮状病毒非结构蛋白4（NSP4）的重组腺病毒,初步评价其免疫保护效果。方法：构建含野生轮状病毒NSP4基因的穿梭质粒pshuttle-NSP4,与腺病毒骨架质粒pAdeasy经同源重组后在Ad-293细胞中包装获得pAd-NSP4重组腺病毒颗粒。电镜、RT-PCR、免疫荧光等方法鉴定病毒特征及在体外细胞中的表达。肌肉注射及滴鼻方式免疫小鼠,检测小鼠血清抗体效价及其中和保护效果。结果：获得了滴度为10^8.25CCID50/ml的重组腺病毒pAd-NSP4,免疫荧光检测到特异性目的蛋白的表达。二次免疫后肌肉注射和滴鼻小鼠的ELISA血清平均效价分别为1：320 和1：1436.8;中和抗体效价1：45.3和1：71.8。结论：表达轮状病毒NSP4蛋白的非复制型重组腺病毒颗粒具有良好的免疫原性。滴鼻途径比肌肉注射可更加有效地诱导小鼠的免疫应答。     

4种禽流感病毒M2e多肽的串联表达和小鼠口服免疫效果

为研制广谱性禽流感口服疫苗,将4种禽流感病毒特异的基质蛋白2胞外区(matrix protein 2 ectodomain,M2e)多肽与黏膜免疫佐剂CTA1-DD串联,原核表达并纯化CTA1DD-AVI4M2e融合蛋白。以200 μg融合蛋白CTA1DD-AVI4M2e口服免疫BALB/c小鼠,结果显示实验组肠液IgA抗体效价和血清IgG抗体效价比对照组显著升高,血清中特异性IgG抗体效价达约360倍。分割的3段肠样中：第1段浸出液IgA抗体效价约为360倍,第2段约为120倍,第3段约为 9 720 倍。结果表明,本研究制备的CTA1DD-AVI4M2e具有显著口服免疫效果,为后续研究提供了基础。     

Synthesis of an HIV-1 Tat transduction domain-rotavirus enterotoxin fusion protein in transgenic potato

A DNA fragment encoding a 12-amino acid (aa) HIV-1 Tat transduction peptide fused to a 90-aa murine rotavirus NSP4 enterotoxin protein (Tat-NSP4₉₀) was transferred to Solanum tuberosum by Agrobacterium tumefaciens-mediated transformation. The fusion gene was detected in the genomic DNA of transformed plant leaf tissues by PCR DNA amplification. The Tat-NSP4₉₀ fusion protein was identified in transformed tuber extracts by immunoblot analysis using anti-NSP4₉₀ and anti-Tat as the primary antibodies. Enzyme-linked immunosorbent assay results showed that the Tat-NSP4₉₀ fusion protein made up to 0.0015% of the total soluble tuber protein. The synthesis of Tat-NSP4₉₀ fusion protein in transformed potato tuber tissues demonstrates the feasibility of plant cell delivery of the HIV-1 Tat transduction domain as a carrier for non-specific targeting of fused antigens to the mucosal immune system.Abbreviations APC Antigen-presenting cells - BA Benzyladenine - BSA Bovine serum albumin - CT Cholera toxin - CTB Cholera toxin B subunit - CTL Cytotoxic T lymphocytes - 2,4-D 2,4-Dichlorophenoxyacetic acid - ELISA Enzyme-linked immunosorbent assay - HIV-1 Human immunodeficiency virus type 1 - MHC Major histocompatibility complex - IAA Indole-3-acetic acid - NAA -Naphthaleneacetic acid - NPT II Neomycin phosphotransferase II - NSP4 Rotavirus enterotoxin non-structural protein - PBS Phosphate-buffered saline - PBST Phosphate-buffered saline containing 0.05% Tween-20 - PTD Protein transduction domain Communicated by W.A. Parrott     

Protective mucosal immunity in aging is associated with functional CD4+ T cells in nasopharyngeal-associated lymphoreticular tissue

Our previous studies showed that mucosal immunity was impaired in 1-year-old mice that had been orally immunized with OVA and native cholera toxin (nCT) as mucosal adjuvant. In this study, we queried whether similar immune dysregulation was also present in mucosal compartments of mice immunized by the nasal route. Both 1-year-old and young adult mice were immunized weekly with three nasal doses of OVA and nCT or with a nontoxic chimeric enterotoxin (mutant cholera toxin-A E112K/B subunit of native labile toxin) from Brevibacillus choshinensis. Elevated levels of OVA-specific IgG Abs in plasma and secretory IgA Abs in mucosal secretions (nasal washes, saliva, and fecal extracts) were noted in both young adult and 1-year-old mice given nCT or chimeric enterotoxin as mucosal adjuvants. Significant levels of OVA-specific CD4(+) T cell proliferative and OVA-induced Th1- and Th2-type cytokine responses were noted in cervical lymph nodes and spleen of 1-year-old mice. In this regard, CD4(+), CD45RB(+) T cells were detected in greater numbers in the nasopharyngeal-associated lymphoreticular tissues of 1-year-old mice than of young adult mice, but the same did not hold true for Peyer's patches or spleen. One-year-old mice given nasal tetanus toxoid plus the chimeric toxin as adjuvant were protected from lethal challenge with tetanus toxin. This result reinforced our findings that age-associated immune alterations occur first in gut-associated lymphoreticular tissues, and thus nasal delivery of vaccines for nasopharyngeal-associated lymphoreticular tissue-based mucosal immunity offers an attractive possibility to protect the elderly.     

Protective mucosal immunity to ocular herpes simplex virus type 1 infection in mice by using Escherichia coli heat-labile enterotoxin B subunit as an adjuvant

The potential of nontoxic recombinant B subunits of cholera toxin (rCtxB) and its close relative Escherichia coli heat-labile enterotoxin (rEtxB) to act as mucosal adjuvants for intranasal immunization with herpes simplex virus type 1 (HSV-1) glycoproteins was assessed. Doses of 10 microg of rEtxB or above with 10 microg of HSV-1 glycoproteins elicited high serum and mucosal anti-HSV-1 titers comparable with that obtained using CtxB (10 microg) with a trace (0.5 microg) of whole toxin (Ctx-CtxB). By contrast, doses of rCtxB up to 100 microg elicited only meager anti-HSV-1 responses. As for Ctx-CtxB, rEtxB resulted in a Th2-biased immune response with high immunoglobulin G1 (IgG1)/IgG2a antibody ratios and production of interleukin 4 (IL-4) and IL-10 as well as gamma interferon by proliferating T cells. The protective efficacy of the immune response induced using rEtxB as an adjuvant was assessed following ocular challenge of immunized and mock-immunized mice. Epithelial disease was observed in both groups, but the immunized mice recovered by day 6 whereas mock-immunized mice developed more severe corneal disease leading to stromal keratitis. In addition, a significant reduction in the incidence of lid disease and zosteriform spread was observed in immunized animals and there was no encephalitis compared with 95% encephalitis in mock-immunized mice. The potential of such mucosal adjuvants for use in human vaccines against pathogens such as HSV-1 is discussed.     

Intranasal immunization with recombinant toxin-coregulated pilus and cholera toxin B subunit protects rabbits against Vibrio cholerae O1 challenge

Intranasal immunization, a noninvasive method of vaccination, has been found to be effective in inducing systemic and mucosal immune responses. The present study was aimed at investigating the efficacy of intranasal immunization in inducing mucosal immunity in experimental cholera by subunit recombinant protein vaccines from Vibrio cholerae O1. The structural genes encoding toxin-coregulated pilus A (TcpA) and B subunit of cholera toxin (CtxB) from V. cholerae O1 were cloned and expressed in Escherichia coli . Rabbits were immunized intranasally with purified TcpA and CtxB alone or a mixture of TcpA and CtxB. Immunization with TcpA and CtxB alone conferred, respectively, 41.1% and 70.5% protection against V. cholerae challenge, whereas immunization with a mixture of both antigens conferred complete (100%) protection, as assayed in the rabbit ileal loop model. Serum titers of immunoglobulin G (IgG) antibodies to TcpA and CtxB, and anti-TcpA- and anti-CtxB-specific sIgA in intestinal lavage of vaccinated animals were found to be significantly elevated compared with unimmunized controls. Vibriocidal antibodies were detected at remarkable levels in rabbits receiving TcpA antigen and their titers correlated with protection. Thus, mucosal codelivery of pertinent cholera toxoids provides enhanced protection against experimental cholera.     

Protective immunity to rotavirus shedding in the absence of interleukin-6: Th1 cells and immunoglobulin A develop normally

We investigated whether interleukin-6 (IL-6) was required for the development of immunoglobulin A (IgA)- and T-helper 1 (Th1)-associated protective immune responses to rotavirus by using adult IL-6-deficient mice [BALB/c and (C57BL/6 x O1a)F(2) backgrounds]. Naive IL-6(-) mice had normal frequencies of IgA plasma cells in the gastrointestinal tract. Consistent with this, total levels of IgA in fecal extracts, saliva, and sera were unaltered. In specific response to oral infection with rhesus rotavirus, IL-6(-) and IL-6(+) mice exhibited efficient Th1-type gamma interferon responses in Peyer's patches with high levels of serum IgG2a and intestinal IgA. Although there was an increase in Th2-type IL-4 in CD4(+) T cells from IL-6(-) mice following restimulation with rotavirus antigen in the presence of irradiated antigen-presenting cells, unfractionated Peyer's patch cells failed to produce a significant increase in IL-4. Moreover, virus-specific IgG1 in serum was not significantly increased in IL-6(-) mice in comparison with IL-6(+) mice. Following oral inoculation with murine rotavirus, IL-6(-) and IL-6(+) mice mediated clearance of rotavirus and mounted a strong IgA response. When IL-6(-) and IL-6(+) mice [(C57BL/6 x O1a)F(2) background] were orally inoculated with rhesus rotavirus and later challenged with murine rotavirus, all of the mice maintained high levels of IgA in feces and were protected against reinfection. Thus, IL-6 failed to provide unique functions in the development of IgA-secreting B cells and in the establishment of Th1-associated protective immunity against rotavirus infection in adult mice.     

Mucosal immune responses are related to reduction of bacterial colonization in the stomach after therapeutic Helicobacter pylori immunization in mice

The aim of this study was to investigate the capacity of oral and parenteral therapeutic immunization to reduce the bacterial colonization in the stomach after experimental Helicobacter pylori infection, and to evaluate whether any specific immune responses are related to such reduction. C57BL/6 mice were infected with H. pylori and thereafter immunized with H. pylori lysate either orally together with cholera toxin or intraperitoneally (i.p.) together with alum using immunization protocols that previously have provided prophylactic protection. The effect of the immunizations on H. pylori infection was determined by quantitative culture of H. pylori from the mouse stomach. Mucosal and systemic antibody responses were analyzed by ELISA in saponin extracted gastric tissue and serum, respectively, and mucosal CD4+ T cell responses by an antigen specific proliferation assay. Supernatants from the proliferating CD4+ T cells were analyzed for Th1 and Th2 cytokines. The oral, but not the parenteral therapeutic immunization induced significant decrease in H. pylori colonization compared to control infected mice. The oral immunization resulted in markedly elevated levels of serum IgG+M as well as gastric IgA antibodies against H. pylori antigen and also increased H. pylori specific mucosal CD4+ T cell proliferation with a Th1 cytokine profile. Although the parenteral immunization induced dramatic increases in H. pylori specific serum antibody titers, no increases in mucosal antibody or cellular immune responses were observed after the i.p. immunization compared to control infected mice. These findings suggest that H. pylori specific mucosal immune responses with a Th1 profile may provide therapeutic protection against H. pylori.     

Flagellin in Fusion with Human Rotavirus Structural Proteins Exerts an Adjuvant Effect When Delivered with Replicating but Non-Disseminating Adenovectors Through the Intrarectal Route

Human rotavirus (HRV) is the worldwide leading cause of gastroenteritis in young children. Two live attenuated HRV vaccines have been approved since 2006. However, these live vaccines still have potential risks including reversion of virulence. Adenoviruses are suitable vectors for mucosal administration of subunit vaccines. In addition to the adjuvant effect of certain adenovirus components, the use of an adjuvant like flagellin is also another means to increase the immune response to the immunogen. The aim of this study was to determine whether flagellin in fusion with HRV structural proteins stimulates the innate immune response and enhances the HRV-specific immune response when delivered through the intrarectal route with replicating but non-disseminating adenovector (R-AdV). Salmonella typhimurium flagellin B (FljB) in fusion with HRV VP4Δ::VP7 protein induced IL-1β production in J774A.1 macrophages exposed to the R-AdV. Intrarectal administration of R-AdVs expressing either VP4Δ::VP7 or VP4Δ::VP7::FljB in BALB/c mice resulted in HRV-specific mixed Th1/Th2 immune responses. The HRV-specific antibody response elicited with the use of R-AdV expressing VP4Δ::VP7::FljB was higher than that with R-AdV expressing VP4Δ::VP7. The results also show that the replication capability of R-AdVs contributed to enhance the HRV-specific immune response as compared with that obtained with non-replicative AdVs. This work lays the foundation for using the R-AdV system and FljB-adjuvanted formulation to elicit a mucosal immune response specific to HRV.     

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.