Oral immunization of mice with <Emphasis Type="Italic">Lactococcus lactis</Emphasis> expressing the rotavirus VP8* protein

The efficacy of recombinant Lactococcus lactis as a delivery vehicle for a rotavirus antigen was evaluated in a mouse model. The rotavirus VP8* protein was expressed intracellularly and extracellularly in L. lactis wild type and in an alr mutant deficient in alanine racemase activity, necessary for the synthesis of the cell-wall component d-alanine. When the mucosal immune response was evaluated by measuring VP8*-specific IgA antibody in faeces, wild-type L. lactis triggered a low IgA synthesis only when the secreting strain was used. In contrast, VP8*-specific IgA was detected in faeces of both groups of mice orally given the alr mutant expressing extracellular VP8* and intracellular VP8*, which reached levels similar to that obtained with the wild type secreting strain. However, oral administration of the recombinant strains did not induce serum IgG or IgA responses. L. lactis cell-wall mutants may therefore provide certain advantages when low-antigenic proteins are expressed intracellularly. However, the low immune response obtained by using this antigen-bacterial host combination prompts to the use of new strains and vaccination protocols in order to develop acceptable rotavirus immunization levels.     

Ability of Lactococcus lactis to export viral capsid antigens: a crucial step for development of live vaccines

The food grade bacterium Lactococcus lactis is a potential vehicle for protein delivery in the gastrointestinal tract. As a model, we constructed lactococcal strains producing antigens of infectious bursal disease virus (IBDV). IBDV infects chickens and causes depletion of B-lymphoid cells in the bursa of Fabricius and subsequent immunosuppression, morbidity, or acute mortality. The two major IBDV antigens, i.e., VP2 and VP3, that form the viral capsid were expressed and targeted to the cytoplasm, the cell wall, or the extracellular compartment of L. lactis. Whereas VP3 was successfully targeted to the three compartments by the use of relevant expression and export vectors, VP2 was recalcitrant to export, thus confirming the difficulty of translocating naturally nonsecreted proteins across the bacterial membrane. This defect could be partly overcome by fusing VP2 to a naturally secreted protein (the staphylococcal nuclease Nuc) that carried VP2 through the membrane. Lactococcal strains producing Nuc-VP2 and VP3 in various bacterial compartments were administered orally to chickens. The chickens did not develop any detectable immune response against VP2 and VP3 but did exhibit an immune response against Nuc when Nuc-VP2 was anchored to the cell wall of lactococci.     

乳酸乳球菌食品级诱导表达系统的构建及异源蛋白的表达

以α-aga基因为食品级选择标记构建了乳酸乳球菌食品级高效诱导细胞内和细胞壁锚定表达系统,并用这一表达系统表达了铜绿假单胞菌融合外膜蛋白基因OprF/H。首先以pRAF800和pNZ8048构建了含有α-aga、PnisA-MCS-TpepN和θ复制子的乳酸乳球菌食品级细胞内诱导表达载体pRNA48,再以pRNA48和pVE5524为出发载体构建了含有α-aga、PnisA-SPUsp45-nucA-CWAM6-t1t2和θ复制子的乳酸乳球菌细胞壁锚定诱导表达载体pRNV48。然后以食品级载体pRNA48和pRNV48为基础,构建了不含抗生素抗性选择标记的铜绿假单胞菌融合外膜蛋白基因的表达质粒pRNA48-OprF/H和pRNV48-OprF/H。利用nisin进行重组乳酸乳球菌菌株的诱导表达,通过SDS-PAGE和Western blot分析,检测到表达蛋白分别占细胞内可溶蛋白的9.6%和细胞壁锚定蛋白的9.8%,表达产物具有免疫原性,可与含OprF/H的乳球菌以及铜绿假单胞菌发生特异性的凝集反应。     

Oral vaccination of mice against Helicobacter pylori with recombinant Lactococcus lactis expressing urease subunit B

To determine whether a protective immune response could be elicited by oral delivery of a recombinant live bacterial vaccine, Helicobacter pylori urease subunit B (UreB) was expressed for extracellular expression in food-grade bacterium Lactococcus lactis . The UreB-producing strains were then administered orally to mice, and the immune response to UreB was examined. Orally vaccinated mice produced a significant UreB-specific serum immunoglobulin G (IgG) response. Specific anti-UreB IgA responses could be detected in the feces of mice immunized with the secreting lactococcal strain. Mice vaccinated orally were significantly protected against gastric Helicobacter infection following a challenge with H. pylori strain SS1. In conclusion, mucosal vaccination with L. lactis expressing UreB produced serum IgG and UreB-specific fecal IgA, and prevented gastric infection with H. pylori .     

Peroral immunization of microencapsulated human VP8 in combination with cholera toxin induces intestinal antibody responses

To develop an orally delivered subunit vaccine for rotavirus infection, a trypsin cleavage product of VP4, recombinant VP8*, was expressed in Escherichia coli. The recombinant VP8* (rVP8*), purified by affinity chromatography, was reactive against human rotavirus positive serum in Western-blot analysis. To further evaluate the immunogenicity of the oral-delivered rVP8*, it was encapsulated with alginate-microsphere and administered in combination with cholera toxin (CT) as a mucosal adjuvant perorally into mice. The ELISPOT assay showed that the number of rVP8*-specific IgG1 antibody secreting cells increased about 3-fold and about 2-fold in spleen and Peyer's patch, respectively as compared to non-immune mice. In addition, the number of rVP8*-specific IgA antibody secreting cells increased about 2-fold in Peyer's patch. Finally, rVP8*-specific IgA antibody response was significantly enhanced in the intestinal fluids from the mice immunized perorally with encapsulated rVP8* and CT. Taken together, these results indicate that rVP8* possessed proper immunogenicity and it would be potentially useful as a subunit vaccine against rotavirus-associated disease through peroral immunization.     

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.     

Mice immunization with live lactococci displaying a surface anchored HPV-16 E7 oncoprotein

E7 oncoprotein of human papillomavirus-16 (HPV-16) is constitutively produced in cervical cancer (CxCa) and is a good candidate for the design of therapeutic vaccines. In this work, the nisin-controlled expression system was used to display the E7 protein at the cell surface of the food-grade Gram-positive bacterium Lactococcus lactis. An efficient cell wall anchoring of E7 was obtained. Intranasal administration of these recombinant lactococci in mice induced an HPV-16 E7-specific immune response. This is the first report of E7 cell wall anchoring in L. lactis and represents one more step towards the use of live food-grade bacteria to fight against CxCa.     

Bovine rotavirus nonstructural protein 4 produced by Lactococcus lactis is antigenic and immunogenic

Rotavirus nonstructural protein 4 (NSP4) can induce diarrhea in mice. To get insight into the biological effects of NSP4, production of large quantities of this protein is necessary. We first tried to produce the protein in Escherichia coli, but the nsp4 gene proved to be unstable. The capacity of the generally regarded as safe organism Lactococcus lactis to produce NSP4 either intra- or extracellularly was then investigated by using the nisin-controlled expression system. Production of recombinant NSP4 (rNSP4) was observed in L. lactis for both locations. In spite of a very low secretion efficiency, the highest level of production was obtained with the fusion between a lactococcal signal peptide and rNSP4. Cultures of the rNSP4-secreting strain were injected into rabbits, and a specific immune response was elicited. The anti-rNSP4 antibodies produced in these rabbits recognized NSP4 in MA104 cells infected by rotavirus. We showed that L. lactis is able to produce antigenic and immunogenic rNSP4 and thus is a good organism for producing viral antigens.     

Llama-derived single-chain antibody fragments directed to rotavirus VP6 protein possess broad neutralizing activity in vitro and confer protection against diarrhea in mice

Group A rotavirus is one of the most common causes of severe diarrhea in human infants and newborn animals. Rotavirus virions are triple-layered particles. The outer capsid proteins VP4 and VP7 are highly variable and represent the major neutralizing antigens. The inner capsid protein VP6 is conserved among group A rotaviruses, is highly immunogenic, and is the target antigen of most immunodiagnosis tests. Llama-derived single-chain antibody fragments (VHH) are the smallest molecules with antigen-binding capacity and can therefore be expected to have properties different from conventional antibodies. In this study a library containing the VHH genes of a llama immunized with recombinant inner capsid protein VP6 was generated. Binders directed to VP6, in its native conformation within the viral particle, were selected and characterized. Four selected VHH directed to conformational epitopes of VP6 recognized all human and animal rotavirus strains tested and could be engineered for their use in immunodiagnostic tests for group A rotavirus detection. Three of the four VHH neutralized rotavirus in vivo independently of the strain serotype. Furthermore, this result was confirmed by in vivo partial protection against rotavirus challenge in a neonatal mouse model. The present study demonstrates for the first time a broad neutralization activity of VP6 specific VHH in vitro and in vivo. Neutralizing VHH directed to VP6 promise to become an essential tool for the prevention and treatment of rotavirus diarrhea.     

用重组腺病毒表达A组轮状病毒主要中和抗原VP7可获得良好的免疫学效果

为探索利用重组腺病毒表达轮状病毒的结构抗原以制备轮状病毒基因工程疫苗的可行性,构建了一株可表达A组轮状病毒主要中和抗原VP7的重组腺病毒AdEasyCVP7.AdEasyCVP7感染293细胞后,RT－PCR证明VP7基因有转,Western blotting试验可检测到VP7的表达。随后,用AdEasyCVP7通过灌胃和滴鼻两种不同途径免疫小鼠,并对免疫后小鼠的血清抗体和粘膜抗体进行了比较。初次免疫后,两组小鼠均有应答,但血清抗体滴度及阳转率不同。再次免疫后,滴鼻组小鼠显示出明显的加强效果。对肺灌洗液中的sIgA及肺、肠粘膜组织匀浆中的IgA进行检测发现滴鼻组的免疫学效果明显优于灌胃组。对血清中和抗体的检测表明,初次和再次免疫后,两组小鼠血清中均有中和抗体产生。该研究为轮状病毒基因工程疫苗的免疫方案、免疫途径及免疫保护作用等的进一步研究奠定了基础。     

Receptor binding domain of Escherichia coli F18 fimbrial adhesin FedF can be both efficiently secreted and surface displayed in a functional form in Lactococcus lactis

Adherence of F18 fimbrial Escherichia coli to porcine intestinal epithelial cells is mediated by the adhesin (FedF) of F18 fimbriae. In a previous study, we demonstrated the specificity of the amino acid residues between 60 and 109 as the receptor binding domain of FedF. In this study, different expression, secretion, and anchoring systems for the receptor binding domain of the FedF adhesin in Lactococcus lactis were evaluated. Two partially overlapping receptor binding domains (42 and 62 amino acid residues) were expressed as fusions with L. lactis subsp. cremoris protein PrtP for evaluation of secretion efficiency. To evaluate the cell surface display of these FedF-PrtP fusions, they were further combined with different lengths of PrtP spacers fused with either the L. lactis AcmA anchor or the PrtP cell wall binding domain. An HtrA-defective L. lactis NZ9000 mutant was constructed to determine its effect on the level of secreted or anchored fusion proteins. Recombinant L. lactis clones secreting the receptor binding domain of F18 fimbriae as a fusion with the H domains of L. lactis protein PrtP were first constructed by using two different signal peptides. FedF-PrtP fusions, directed by the signal sequence of L. brevis SlpA, were throughout found to be secreted at significantly higher quantities than corresponding fusions with the signal peptide of L. lactis Usp45. In the surface display systems tested, the L. lactis AcmA anchor performed significantly better, particularly in the L. lactis NZ9000DeltahtrA strain, compared to the L. lactis PrtP anchor region. Of the cell surface display constructs with the AcmA anchor, only those with the longest PrtP spacer regions resulted in efficient binding of recombinant L. lactis cells to porcine intestinal epithelial cells. These results confirmed that it is possible to efficiently produce the receptor binding domain of the F18 adhesin in a functionally active form in L. lactis.     

Nucleotide sequence of VP4 and VP7 genes of human rotaviruses with subgroup I specificity and long RNA pattern: implication for new G serotype specificity.

We sequenced the genes coding for the two neutralization proteins, VP4 and VP7, of human rotavirus strains L26 and L27 with subgroup I specificity but the long RNA pattern. The deduced VP7 amino acid sequence of strains L26 and L27 showed a low homology (73.6 to 81.9%) to those of rotavirus strains of the established serotypes. This finding, together with the previous serological characterizations, suggests that the VP7 (G) serotype of the L26 and L27 strains is distinct from those of strains of the previously established serotypes. In contrast, the VP4 sequences of the L26 and L27 strains were quite similar to those of virulent serotype 2 strains (DS-1, S2, and RV-5).     

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。     

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.     

A novel mucosal vaccine based on live Lactococci expressing E7 antigen and IL-12 induces systemic and mucosal immune responses and protects mice against human papillomavirus type 16-induced tumors

Current strategies to prevent or treat human papillomavirus type 16 (HPV-16) infection are promising, but remain costly. More economical but efficient vaccines are thus needed. In this study, we evaluated the protective effects of mucosally coadministered live Lactococcus lactis strains expressing cell wall-anchored E7 Ag and a secreted form of IL-12 to treat HPV-16-induced tumors in a murine model. When challenged with lethal levels of tumor cell line TC-1 expressing E7, immunized mice showed full prevention of TC-1-induced tumors, even after a second challenge, suggesting that this prophylactic immunization can provide long-lasting immunity. Therapeutic immunization with L. lactis recombinant strains, i.e., 7 days after TC-1 injection, induced regression of palpable tumors in treated mice. The antitumor effects of vaccination occurred through a CTL response, which is CD4+ and CD8+ dependent. Furthermore, immunized mice developed an E7-specific mucosal immune response. These preclinical results suggest the feasibility of the low-cost mucosal vaccination and/or immunotherapy strategies against HPV-related cervical cancer in humans.     

Comparison of the local immune response against Giardia lamblia cyst wall protein 2 induced by recombinant Lactococcus lactis and Streptococcus gordonii

Lactococcus lactis and Streptococcus gordonii are lactic acid bacteria (LAB) currently being advocated for use as live antigen delivery vehicles to mucosal sites. Since both vehicles differ in their capability to persist within the small intestine and in their mode of antigen delivery, we sought to compare them to determine which one was superior. In this study, we compared the efficacy of recombinant L. lactis and S. gordonii to stimulate intestinal immune responses against Giardia lamblia cyst wall protein-2 in BALB/c mice. Oral administration of either vector significantly increased the number of CD4(+) T helper and B-cells in the mesenteric lymph nodes (MLN) and Peyer's patches (PP) of immunized animals. Delivery of recombinant CWP2 (rCWP2) by L. lactis stimulated a balanced IFN-gamma/IL-4 response (MLN and PP cells) and a CWP2-specific intestinal IgA antibody response. Alternatively, delivery of rCWP2 by S. gordonii stimulated a higher frequency of IFN-gamma secreting MLN and PP cells, as well as doubling the amount of CWP2-specific intestinal IgA. In challenge studies, L. lactis and S. gordonii reduced cyst output by 71 and 90%, respectively. When compared to each other, S. gordonii-immunized animals shed 65% fewer cysts than their L. lactis-immunized counterparts. Based on these findings, we concluded that S. gordonii was superior to L. lactis as an intestinal vaccine delivery vehicle.     

Immune response to rotavirus VP4 expressed in an attenuated strain of Shigella flexneri

An attenuated strain of Shigella flexneri was utilised to express viral protein (VP) 4 of rotavirus and the immunogenicity of the recombinant constructs was studied in BALB/c mice. VP4 was expressed as a fusion with maltose binding protein (MBP) in both the cytoplasm and periplasm, with a much higher level of expression occurring in the former. While all constructs induced a Shigella-specific response in mice, only the construct expressing MBP-VP4 in the cytoplasm of Shigella stimulated an immune response specific to rotavirus. This study demonstrates that Shigella can be used to deliver rotavirus antigens and induces an immune response directed towards both rotavirus and Shigella.     

Yeast expression of the VP8* fragment of the rotavirus spike protein and its use as immunogen in mice

The VP8* fragment from the rotavirus spike protein was expressed as a fusion protein with two different cell wall proteins of Saccharomyces cerevisiae, Icwp (Ssr1p) and Pir4, to achieve cell wall targeting or secretion to the growth medium of the fusion proteins. Two different host strains were used for the expression of the fusion proteins, a standard S. cerevisiae strain and a mnn9 glycosylation deficient strain, the later to reduce hyper-glycosylation. The Icwp-VP8* fusion could only be detected in the growth medium, indicating that the presence of the VP8* moiety interferes with the anchorage of Icwp to the cell wall. In the case of the Pir4-VP8* fusion proteins, we achieved cell wall targeting or secretion depending on how the gene fusion had been performed. In all cases, the fusion proteins expressed in the mnn9 strain showed a reduced level of glycosylation. Mice were inoculated intraperitoneally either with Pir4-VP8* or Icwp-VP8* fusion proteins purified from the growth medium of mnn9 strains expressing them or with whole cells of an mnn9 strain expressing a Pir4-VP8 fusion protein on its cell walls. Hundred percent of mice inoculated with the Pir4-VP8* fusion protein and 25% of those inoculated with the Icwp-VP8* fusion protein showed high titers of anti-VP8* antibodies. No specific immune response was detected in those mice inoculated with whole cells. Finally, susceptibility to rotavirus infection of the offspring of immunized dams was determined and protection was found in a percentage of approximately 60% with respect to the control group.     

Ability of Lactococcus lactis To Export Viral Capsid Antigens: a Crucial Step for Development of Live Vaccines

Thefood grade bacterium Lactococcus lactis is a potential vehicle for protein delivery in the gastrointestinal tract. As a model, we constructed lactococcal strains producing antigens of infectious bursal disease virus (IBDV). IBDV infects chickens and causes depletion of B-lymphoid cells in the bursa of Fabricius and subsequent immunosuppression, morbidity, or acute mortality. The two major IBDV antigens, i.e., VP2 and VP3, that form the viral capsid were expressed and targeted to the cytoplasm, the cell wall, or the extracellular compartment of L. lactis. Whereas VP3 was successfully targeted to the three compartments by the use of relevant expression and export vectors, VP2 was recalcitrant to export, thus confirming the difficulty of translocating naturally nonsecreted proteins across the bacterial membrane. This defect could be partly overcome by fusing VP2 to a naturally secreted protein (the staphylococcal nuclease Nuc) that carried VP2 through the membrane. Lactococcal strains producing Nuc-VP2 and VP3 in various bacterial compartments were administered orally to chickens. The chickens did not develop any detectable immune response against VP2 and VP3 but did exhibit an immune response against Nuc when Nuc-VP2 was anchored to the cell wall of lactococci.