A Combined YopB and LcrV Subunit Vaccine Elicits Protective Immunity against Yersinia Infection in Adult and Infant Mice.

Yersinia enterocolitica causes a severe enteric infection in infants and young children. There is no vaccine approved for use in humans. We investigated the immunogenicity and protective capacity of Yersinia YopB, a conserved type III secretion system protein, alone or combined with LcrV in adult mice immunized intranasally. YopB or LcrV (5 µg) administered with the Escherichia coli double mutant heat-labile toxin (dmLT) adjuvant afforded modest (10-30%) protection against lethal Y. enterocolitica oral infection. The combination of YopB and LcrV (5 µg each) dramatically improved vaccine efficacy (70-80%). Additionally, it afforded complete protection against Y. pestis pulmonary infection. Immunization with YopB/LcrV+dmLT resulted in Ag-specific serum IgG, systemic and mucosal Ab-secreting cells, as well as IFN-γ, TNF-α, IL-2, IL-6, IL-17A, and KC production by spleen cells. Serum Abs elicited by YopB/LcrV+dmLT had enhanced bactericidal and opsonophagocytic killing activity. After Y. enterocolitica challenge, YopB/LcrV+dmLT-vaccinated mice exhibited intact intestinal tissue, active germinal centers in mesenteric lymph nodes, IgG+ and IgA+ plasmablasts in the lamina propria, and Abs in intestinal fluid. On the contrary, complete tissue destruction and abscesses were seen in placebo recipients that succumbed to infection. Mice immunized as infants with YopB+dmLT or LcrV+dmLT achieved 60% protection against lethal Y. enterocolitica infection, and vaccine efficacy increased to 90-100% when they received YopB/LcrV+dmLT. YopB+dmLT also afforded substantial (60%) protection when administered intradermally to infant mice. YopB/LcrV+dmLT is a promising subunit vaccine candidate with the potential to elicit broad protection against Yersinia spp.

A Novel PspA Protein Vaccine Intranasal Delivered by Bacterium-Like Particles Provides Broad Protection Against Pneumococcal Pneumonia in Mice.

BACKGROUND: Streptococcus pneumoniae is a major pathogen accounting for a large number of pneumococcal disease in worldwide. Due to the mucosal immune pathway induces both systemic and mucosal immune responses, the potential strategy to prevent pneumococcal disease may be to develop a mucosal vaccine. METHOD: In this study, we developed an intranasal pneumococcal protein vaccine based on a bacterium-like particle (BLP) delivery system. PspA is expressed and exposed on the surface of all pneumococcal strains, which confers the potential to induce immune responses to protect against pneumococcal infection. We fused one of the pneumococcal surface proteins (PspA, family2 clade4) with the protein anchor (PA) protein in order to display PspA on the surface of BLPs. RESULT: The current results showed that intranasal immunization with BLPs/PspA-PA efficiently induced both PspA-specific IgG in the serum and PspA-specific IgA in mucosal washes. And intranasal immunization of BLPs/PspA-PA could provide complete protection in a mouse challenge model with pneumococci of different two clades of both homologous and heterologous PspA families. DISCUSSION AND CONCLUSION: Thus, targeted delivery of multiple bacterial antigens via BLPs may prevent pneumococcal disease by inducing both systemic and mucosal immune responses.

Biophysical Characterization of the Type III Secretion System Translocator Proteins and the Translocator Proteins Attached to Bacterium-Like Particles.

Diarrhea caused by Shigella, Salmonella, and Yersinia is an important public health problem, but development of safe and effective vaccines against such diseases is challenging. A new antigen delivery platform called bacterium-like particles (BLPs) was explored as a means for delivering protective antigens from the type III secretion systems (T3SS) of these pathogens. BLPs are peptidoglycan skeletons derived from Lactococcus lactis that are safe for newborns and can carry multiple antigens. Hydrophobic T3SS translocator proteins were fused to a peptidoglycan anchor (PA) for BLP attachment. The proteins and protein-BLP complexes associated with BLPs were characterized and the resulting data used to create three-index empirical phase diagrams (EPDs). On the basis of these EPDs, IpaB (Shigella) and SipB (Salmonella) behave distinctly from YopB (Yersinia) under different environmental stresses. Adding the PA domain appears to enhance the stability of both the PA and translocator proteins, which was confirmed using differential scanning calorimetry, and although the particles dominated the spectroscopic signals in the protein-loaded BLPs, structural changes in the proteins were still detected. The protein-BLPs were most stable near neutral pH, but these proteins' hydrophobicity made them sensitive to environmental stresses.

Shigella IpaB and IpaD displayed on L. lactis bacterium-like particles induce protective immunity in adult and infant mice.

Shigella spp. are among the enteric pathogens with the highest attributable incidence of moderate-to-severe diarrhea in children under 5 years of age living in endemic areas. There are no vaccines available to prevent this disease. In this work, we investigated a new Shigella vaccine concept consisting of nonliving, self-adjuvanted, Lactococcus lactis bacterium-like particles (BLP) displaying Shigella invasion plasmid antigen (Ipa) B and IpaD and examined its immunogenicity and protective efficacy in adult and newborn/infant mice immunized via the nasal route. Unique advantages of this approach include the potential for broad protection due to the highly conserved structure of the Ipas and the safety and practicality of a probiotic-based mucosal/adjuvant delivery platform. Immunization of adult mice with BLP-IpaB and BLP-IpaD (BLP-IpaB/D) induced high levels of Ipa-specific serum IgG and stool IgA in a dose-dependent manner. Immune responses and protection were enhanced by BLP delivery. Vaccine-induced serum antibodies exhibited opsonophagocytic and cytotoxic neutralizing activity, and IpaB/D IgG titers correlated with increased survival post-challenge. Ipa-specific antibody secreting cells were detected in nasal tissue and lungs, as well as IgG in bronchoalveolar lavage. Bone marrow cells produced IpaB/D-specific antibodies and contributed to protection after adoptive transfer. The BLP-IpaB/D vaccine conferred 90% and 80% protection against S. flexneri and S. sonnei, respectively. Mice immunized with BLP-IpaB/D as newborns also developed IpaB and IpaD serum antibodies; 90% were protected against S. flexneri and 44% against S. sonnei. The BLP-IpaB/D vaccine is a promising candidate for safe, practical and potentially effective immunization of children against shigellosis.

Biophysical characterization of the type III secretion tip proteins and the tip proteins attached to bacterium-like particles.

Bacterium-like particles (BLPs), derived from Lactococcus lactis, offer a self-adjuvanting delivery vehicle for subunit protein vaccines. Proteins can be specifically loaded onto the BLPs via a peptidoglycan anchoring (PA) domain. In this study, the tip proteins IpaD, SipD, and LcrV belonging to type III secretion systems of Shigella flexneri, Salmonella enterica, and Yersinia enterocolitica, respectively, were fused to the PA and loaded onto the BLPs. Herein, we biophysically characterized these nine samples and condensed the spectroscopic results into three-index empirical phase diagrams (EPDs). The EPDs show distinctions between the IpaD/SipD and LcrV subfamilies of tip proteins, based on their physical stability, even upon addition of the PA. Upon attachment to the BLPs, the BLPs become defining moiety in the spectroscopic measurements, leaving the tip proteins to have a subtle yet modulating effect on the structural integrity of the tip proteins-BLPs binding. In summary, this work provides a comprehensive view of physical stability of the tip proteins and tip protein-BLPs and serves as a baseline for screening of excipients to increase the stability of the tip protein-BLPs for future vaccine formulation.

An expression system for the efficient incorporation of an expanded set of tryptophan analogues.

Biosynthetic incorporation of tryptophan (Trp) analogues in recombinant proteins using an E. coli Trp auxotroph expression host is limited to analogues modified with a small substituent like a fluoro atom or a hydroxyl or amine group. We report here the efficient incorporation (>89 %) of chloro- and bromo atoms containing Trp analogues in alloproteins at high expression levels using a Lactococcus lactis Trp auxotroph strain. This result was only obtained after coexpression of the enzyme tryptophanyl-tRNA synthetase (TrpRS) of L. lactis, an enzyme believed to show a more relaxed substrate specificity than TrpRS from E. coli. Chloro- and bromo-Trps are attractive intrinsic phosphorescence probes as these Trp analogues are much less sensitive for quenchers in the medium, like oxygen, than Trp. Coexpression of TrpRS was also essential for the biosynthetic incorporation (94 %) of the Trp analogue 5,6 difluoroTrp. This makes our expression system ideally suited to generate a set of methyl- and fluoro-substituted Trp analogue-containing alloproteins in high yield for investigating the involvement of the Trp residue in cation-pi or pi-pi interactions. Taken together, the presented Trp auxotroph expression system features the most relaxed specificity for Trp analogue structures reported to date and gives a high alloprotein yield.

Monitoring lysin motif-ligand interactions via tryptophan analog fluorescence spectroscopy.

The lysin motif (LysM) is a peptidoglycan binding protein domain found in a wide range of prokaryotes and eukaryotes. Various techniques have been used to study the LysM-ligand interaction, but a sensitive spectroscopic method to directly monitor this interaction has not been reported. Here a tryptophan analog fluorescence spectroscopy approach is presented to monitor the LysM-ligand interaction using the LysM of the N-acetylglucosaminidase enzyme of Lactococcus lactis. A three-dimensional model of this LysM protein was built based on available structural information of a homolog. This model allowed choosing the amino acid positions to be labeled with a Trp analog. Four functional single-Trp LysM mutants and one double-Trp LysM mutant were constructed and biosynthetically labeled with 7-azatryptophan or 5-hydroxytryptophan. These Trp analogs feature red-shifted absorption spectra, enabling the monitoring of the LysM-ligand interaction in media with a Trp background. The emission intensities of four of the five LysM constructs were found to change markedly on exposure to either L. lactis bacterium-like particles or peptidoglycan as ligands. The method reported here is suitable to monitor LysM-ligand interactions at (sub)micromolar LysM concentrations and can be used for the detection of low levels of peptidoglycan or microbes in solutions.

Bacterium-like particles supplemented with inactivated influenza antigen induce cross-protective influenza-specific antibody responses through intranasal administration.

Administration of influenza vaccines through the intranasal (IN) route forms an attractive alternative to conventional intramuscular (IM) injection. It is not only a better accepted form of vaccine administration but it also has the potential to induce, in addition to systemic antibodies, local protective antibodies, i.e. S-IgA. Most commercially available vaccines however are inactivated non-replicating vaccines and have a low immunogenicity when administered intranasally. Local administration of these vaccines would therefore need an adjuvant to boost systemic and local antibody responses. Here we explored the use of a safe adjuvant system, i.e. bacterium-like particles (BLPs) derived from the food-grade bacterium in Lactococcus lactis, in the induction of protective antibody responses after intranasal immunization of mice. Supplementation of H1N1 split vaccine with BLPs significantly increased levels of serum influenza-specific IgG and hemagglutination-inhibiting antibodies: this was dependent on the dose of admixed BLPs and number of immunizations. Admixing BLPs further boosted local influenza-specific S-IgA antibody levels at lung and nasal mucosal sites, but also at distant mucosal sites such as the vaginal mucosal tissue. Mice immunized IN with BLP-adjuvanted vaccine and IM with non-adjuvanted vaccine were protected against weight loss upon homologous infection with H1N1 A/PR/8/34. Full protection against weight loss upon heterologous challenge with H1N1 A/PR/8/34 was seen in mice immunized IN with BLP-adjuvanted H1N1 A/New Caledonia-derived split virus vaccine, but not in those receiving the split virus vaccine IM. Mice immunized IN with BLP-adjuvanted vaccine had significantly lower lung viral titers upon homologous and heterologous challenge when compared to titers detected in mice immunized by IM injection of non-adjuvanted vaccine. Thus, adjuvantation of IN-administered influenza vaccines with BLPs effectively enhances systemic and local antibody responses leading to a superior protection against homologous and heterologous influenza infection compared to conventional IM immunization.

Bacterium-like particles as multi-epitope delivery platform for Plasmodium berghei circumsporozoite protein induce complete protection against malaria in mice.

BACKGROUND: Virus-like particles have been regularly used as an antigen delivery system for a number of Plasmodium peptides or proteins. The present study reports the immunogenicity and protective efficacy of bacterium-like particles (BLPs) generated from Lactococcus lactis and loaded with Plasmodium berghei circumsporozoite protein (PbCSP) peptides. METHODS: A panel of BLP-PbCSP formulations differing in composition and quantity of B-cell, CD4+ and CD8+ T-cell epitopes of PbCSP were tested in BALB/c mice. RESULTS: BLP-PbCSP1 induced specific humoral responses but no IFN-γ ELISPOT response, protecting 30-40% of the immunized mice. BLP-PbCSP2, with reduced length of the non-immunogenic part of the T-cell-epitopes construct, increased induction of IFN-γ responses as well as protection up to 60-70%. Compared to controls, lower parasitaemia was observed in unprotected mice immunized with BLP-PbCSP1 or 2, suggestive for partial immunity. Finally, further increase of the number of B-cell epitopes and codon optimization (BLP-PbCSP4) induced the highest anti-CSP antibody levels and number of IFN-γ spots, resulting in sterile immunity in 100% of the immunized mice. CONCLUSION: Presentation of Plasmodium-derived antigens using BLPs as a delivery system induced complete protection in a murine malaria model. Eventually, BLPs have the potential to be used as a novel versatile delivery platform in malaria vaccine development.

Intranasal delivery of influenza subunit vaccine formulated with GEM particles as an adjuvant.

Nasal administration of influenza vaccine has the potential to facilitate influenza control and prevention. However, when administered intranasally (i.n.), commercially available inactivated vaccines only generate systemic and mucosal immune responses if strong adjuvants are used, which are often associated with safety problems. We describe the successful use of a safe adjuvant Gram-positive enhancer matrix (GEM) particles derived from the food-grade bacterium Lactococcus lactis for i.n. vaccination with subunit influenza vaccine in mice. It is shown that simple admixing of the vaccine with the GEM particles results in a strongly enhanced immune response. Already after one booster, the i.n. delivered GEM subunit vaccine resulted in hemagglutination inhibition titers in serum at a level equal to the conventional intramuscular (i.m.) route. Moreover, i.n. immunization with GEM subunit vaccine elicited superior mucosal and Th1 skewed immune responses compared to those induced by i.m. and i.n. administered subunit vaccine alone. In conclusion, GEM particles act as a potent adjuvant for i.n. influenza immunization.

Lactococcus lactis as expression host for the biosynthetic incorporation of tryptophan analogues into recombinant proteins.

Incorporation of Trp (tryptophan) analogues into a protein may facilitate its structural analysis by spectroscopic techniques. Development of a biological system for the biosynthetic incorpor-ation of such analogues into proteins is of considerable importance. The Gram-negative Escherichia coli is the only prokaryotic expression host regularly used for the incorporation of Trp analogues into recombinant proteins. Here, we present the use of the versatile Gram-positive expression host Lactococcus lactis for the incorporation of Trp analogues. The availability of a tightly regulated expression system for this organism, the potential to secrete modified proteins into the growth medium and the construction of the trp-synthetase deletion strain PA1002 of L. lactis rendered this organism potentially an efficient tool for the incorporation of Trp analogues into recombinant proteins. The Trp analogues 7-azatryptophan, 5-fluorotryptophan and 5-hydroxytryptophan were incorporated with efficiencies of >97, >97 and 89% respectively. Interestingly, 5-methylTrp (5-methyltryptophan) could be incorporated with 92% efficiency. Successful biosynthetical incorporation of 5-methylTrp into recombinant proteins has not been reported previously.

Development of lactococcal GEM-based pneumococcal vaccines.

We report the development of a novel protein-based nasal vaccine against Streptococcus pneumoniae, in which three pneumococcal proteins were displayed on the surface of a non-recombinant, killed Lactococcus lactis-derived delivery system, called Gram-positive Enhancer Matrix (GEM). The GEM particles induced the production of the proinflammatory cytokine tumour necrosis factor-alpha (TNF-alpha) by macrophages as well as the maturation of dendritic cells. The pneumococcal proteins IgA1 protease (IgA1p), putative proteinase maturation protein A (PpmA) and streptococcal lipoprotein A (SlrA) were anchored in trans to the surface of the GEM particles after recombinant production of the antigens in L. lactis as hybrids with a lactococcal cell wall binding domain, named Protein Anchor domain (PA). Intranasal immunisation with the SlrA-IgA1p or trivalent vaccine combinations without additional adjuvants showed significant protection against fatal pneumococcal pneumonia in mice. The GEM-based trivalent vaccine is a potential pneumococcal vaccine candidate that is expected to be easy to administer, safe and affordable to produce.

Lactococcus lactis GEM particles displaying pneumococcal antigens induce local and systemic immune responses following intranasal immunization.

The present work reports the use of non-living non-recombinant bacteria as a delivery system for mucosal vaccination. Antigens are bound to the cell-wall of pretreated Lactococcus lactis, designated as Gram-positive enhancer matrix (GEM), by means of a peptidoglycan binding domain. The influence of the GEM particles on the antigen-specific serum antibody response was studied. Following nasal immunization with the GEM-based vaccines, antibody responses were induced at systemic and local levels. Furthermore, different GEM-based vaccines could be used consecutively in the same mice without adverse effects or loss of activity. Taken together, the results evidence the adjuvant properties of the GEM particles and indicate that GEM-based vaccines can be used repeatedly and are particularly suitable for nasal immunization purposes.

Novel surface display system for proteins on non-genetically modified gram-positive bacteria.

A novel display system is described that allows highly efficient immobilization of heterologous proteins on bacterial surfaces in applications for which the use of genetically modified bacteria is less desirable. This system is based on nonliving and non-genetically modified gram-positive bacterial cells, designated gram-positive enhancer matrix (GEM) particles, which are used as substrates to bind externally added heterologous proteins by means of a high-affinity binding domain. This binding domain, the protein anchor (PA), was derived from the Lactococcus lactis peptidoglycan hydrolase AcmA. GEM particles were typically prepared from the innocuous bacterium L. lactis, and various parameters for the optimal preparation of GEM particles and binding of PA fusion proteins were determined. The versatility and flexibility of the display and delivery technology were demonstrated by investigating enzyme immobilization and nasal vaccine applications.

Mucosal vaccine delivery of antigens tightly bound to an adjuvant particle made from food-grade bacteria.

Mucosal immunization with subunit vaccines requires new types of antigen delivery vehicles and adjuvants for optimal immune responses. We have developed a non-living and non-genetically modified gram-positive bacterial delivery particle (GEM) that has built-in adjuvant activity and a high loading capacity for externally added heterologous antigens that are fused to a high affinity binding domain. This binding domain, the protein anchor (PA), is derived from the Lactococcus lactis AcmA cell-wall hydrolase, and contains three repeats of a LysM-type cell-wall binding motif. Antigens are produced as antigen-PA fusions by recombinant expression systems that secrete the hybrid proteins into the culture growth medium. GEM particles are then used as affinity beads to isolate the antigen-PA fusions from the complex growth media in a one step procedure after removal of the recombinant producer cells. This procedure is also highly suitable for making multivalent vaccines. The resulting vaccines are stable at room temperature, lack recombinant DNA, and mimic pathogens by their bacterial size, surface display of antigens and adjuvant activity of the bacterial components in the GEM particles. The GEM-based vaccines do not require additional adjuvant for eliciting high levels of specific antibodies in mucosal and systemic compartments.

Type I signal peptidases of Gram-positive bacteria.

Proteins that are exported from the cytoplasm to the periplasm and outer membrane of Gram-negative bacteria, or the cell wall and growth medium of Gram-positive bacteria, are generally synthesized as precursors with a cleavable signal peptide. During or shortly after pre-protein translocation across the cytoplasmic membrane, the signal peptide is removed by signal peptidases. Importantly, pre-protein processing by signal peptidases is essential for bacterial growth and viability. This review is focused on the signal peptidases of Gram-positive bacteria, Bacillus and Streptomyces species in particular. Evolutionary concepts, current knowledge of the catalytic mechanism, substrate specificity requirements and structural aspects are addressed. As major insights in signal peptidase function and structure have been obtained from studies on the signal peptidase LepB of Escherichia coli, similarities and differences between this enzyme and known Gram-positive signal peptidases are highlighted. Notably, while the incentive for previous research on Gram-positive signal peptidases was largely based on their role in the biotechnologically important process of protein secretion, present-day interest in these essential enzymes is primarily derived from the idea that they may serve as targets for novel anti-microbials.

Detergent-independent in vitro activity of a truncated Bacillus signal peptidase.

The Gram-positive eubacterium Bacillus subtilis contains five chromosomally encoded type I signal peptidases (SPases) for the processing of secretory pre-proteins. Even though four of these SPases, denoted SipS, SipT, SipU and SipV, are homologous to the unique SPase I of Escherichia coli, they are structurally different from that enzyme, being almost half the size and containing one membrane anchor instead of two. To investigate whether the unique membrane anchor of Bacillus SPases is required for in vitro activity, soluble forms of SipS of B. subtilis, SipS of Bacillus amyloliquefaciens and SipC of the thermophile Bacillus caldolyticus were constructed. Of these three proteins, only a hexa-histidine-tagged soluble form of SipS of B. amyloliquefaciens could be isolated in significant quantities. This protein displayed optimal activity at pH 10, which is remarkable considering the fact that the catalytic domain of SPases is located in an acidic environment at the outer surface of the membrane of living cells. Strikingly, in contrast to what has been previously reported for the soluble form of the E. coli SPase, soluble SipS was active in the absence of added detergents. This observation can be explained by the fact that a highly hydrophobic surface domain of the E. coli SPase, implicated in detergent-binding, is absent from SipS.