Intrinsic Adjuvant Properties Research Articles

Scavenger receptor-mediated dendritic cell internalization and activation by Polyguanine-conjugated antigen (P4234)

Abstract Targeting specific antigens to antigen presenting cells for efficient antigen processing and immune activation is an essential design consideration for vaccine development. Our group has developed polyguanine (Poly(dG))-conjugated antigens with intrinsic antigen targeting and adjuvant properties. These antigen conjugates can potentially increase vaccine efficiency by transforming soluble antigens into aggregated particulates that are targeted to the scavenger receptor in dendritic cells (DCs), resulting in efficient internalization, activation, and cross-presentation. We have successfully conjugated Poly(dG) residues to the model Ag ovalbumin (OVA) and found that Poly(dG)-OVA can induce robust antigen-specific helper, cytotoxic, memory T cell, and antibody responses. In this study, we measure DC internalization and demonstrate that Poly(dG)-OVA is internalized several times more efficiently than OVA alone. This internalization is specifically inhibited by scavenger receptor ligands. Treatment with Poly(dG)-OVA also resulted in upregulation of CCR7 expression and secretion of MCP-1, TNF-α, and IL-6 by DCs in vitro. These results suggest that coupling Poly(dG) to antigens enables efficient DC targeting and activation through the scavenger receptor. This vaccine design can be applied to viral or tumor protein or peptide antigens for the production of more efficient antiviral and antitumor vaccines without the need for additional adjuvants.

Evaluation of an Intranasal Virosomal Vaccine against Respiratory Syncytial Virus in Mice: Effect of TLR2 and NOD2 Ligands on Induction of Systemic and Mucosal Immune Responses

IntroductionRSV infection remains a serious threat to newborns and the elderly. Currently, there is no vaccine available to prevent RSV infection. A mucosal RSV vaccine would be attractive as it could induce mucosal as well as systemic antibodies, capable of protecting both the upper and lower respiratory tract. Previously, we reported on a virosomal RSV vaccine for intramuscular injection with intrinsic adjuvant properties mediated by an incorporated lipophilic Toll-like receptor 2 (TLR2) ligand. However, it has not been investigated whether this virosomal RSV vaccine candidate would be suitable for use in mucosal immunization strategies and if additional incorporation of other innate receptor ligands, like NOD2-ligand, could further enhance the immunogenicity and protective efficacy of the vaccine.ObjectiveTo explore if intranasal (IN) immunization with a virosomal RSV vaccine, supplemented with TLR2 and/or NOD2-ligands, is an effective strategy to induce RSV-specific immunity.MethodsWe produced RSV-virosomes carrying TLR2 (Pam3CSK4) and/or NOD2 (L18-MDP) ligands. We tested the immunopotentiating properties of these virosomes in vitro, using TLR2- and/or NOD2-ligand-responsive murine and human cell lines, and in vivo by assessing induction of protective antibody and cellular responses upon IN immunization of BALB/c mice.ResultsIncorporation of Pam3CSK4 and/or L18-MDP potentiates the capacity of virosomes to activate (antigen-presenting) cells in vitro, as demonstrated by NF-κB induction. In vivo, incorporation of Pam3CSK4 in virosomes boosted serum IgG antibody responses and mucosal antibody responses after IN immunization. While L18-MDP alone was ineffective, incorporation of L18-MDP in Pam3CSK4-carrying virosomes further boosted mucosal antibody responses. Finally, IN immunization with adjuvanted virosomes, particularly Pam3CSK4/L18-MDP-adjuvanted-virosomes, protected mice against infection with RSV, without priming for enhanced disease.ConclusionMucosal immunization with RSV-virosomes, supplemented with incorporated TLR2- and/or NOD2-ligands, represents a promising approach to induce effective and safe RSV-specific immunity.

Specific humoral immune response to the Thomsen-Friedenreich tumor antigen (CD176) in mice after vaccination with the commensal bacterium Bacteroides ovatus D-6.

The tumor-specific Thomsen-Friedenreich antigen (TFα, CD176) is an attractive target for a cancer vaccine, especially as TF-directed antibodies play an important role in cancer immunosurveillance. However, synthetic TF vaccines have not overcome the low intrinsic immunogenicity of TF. Since natural TF-directed antibodies present in human sera are generated in response to microbes found in the gastrointestinal tract, microbial TF structures are obviously more immunogenic than synthetic TF. We recently isolated a new strain (D-6) of the human gut bacterium Bacteroides ovatus, which carries the true TFα antigen. Here, we present experimental data on the immunogenicity of this strain. Mice immunized with B. ovatus D-6 in the absence of adjuvants developed specific anti-TFα IgM and IgG antibodies which also bound to human cancer cells carrying TFα. Our data suggest that B. ovatus D-6 presents a unique TFα-specific immunogenicity based on a combination of several inherent properties including: expression of the true TFα antigen, clustering and accessible presentation of TFα as repetitive side chains on a capsular polysaccharide, and intrinsic adjuvant properties. Therefore, B. ovatus strain D-6 is an almost perfect candidate for the development of the first adjuvant-free TFα-specific anti-tumor vaccine.

Recombinant vaccines and the development of new vaccine strategies

Vaccines were initially developed on an empirical basis, relying mostly on attenuation or inactivation of pathogens. Advances in immunology, molecular biology, biochemistry, genomics, and proteomics have added new perspectives to the vaccinology field. The use of recombinant proteins allows the targeting of immune responses focused against few protective antigens. There are a variety of expression systems with different advantages, allowing the production of large quantities of proteins depending on the required characteristics. Live recombinant bacteria or viral vectors effectively stimulate the immune system as in natural infections and have intrinsic adjuvant properties. DNA vaccines, which consist of non-replicating plasmids, can induce strong long-term cellular immune responses. Prime-boost strategies combine different antigen delivery systems to broaden the immune response. In general, all of these strategies have shown advantages and disadvantages, and their use will depend on the knowledge of the mechanisms of infection of the target pathogen and of the immune response required for protection. In this review, we discuss some of the major breakthroughs that have been achieved using recombinant vaccine technologies, as well as new approaches and strategies for vaccine development, including potential shortcomings and risks.

Tailoring the immune response by targeting C-type lectin receptors on alveolar macrophages using “pathogen-like” amphiphilic polyanhydride nanoparticles

C-type lectin receptors (CLRs) offer unique advantages for tailoring immune responses. Engagement of CLRs regulates antigen presenting cell (APC) activation and promotes delivery of antigens to specific intracellular compartments inside APCs for efficient processing and presentation. In these studies, we have designed an approach for targeted antigen delivery by decorating the surface of polyanhydride nanoparticles with specific carbohydrates to provide pathogen-like properties. Two conserved carbohydrate structures often found on the surface of respiratory pathogens, galactose and di-mannose, were used to functionalize the surface of polyanhydride nanoparticles and target CLRs on alveolar macrophages (AMϕ), a principle respiratory tract APC. Co-culture of functionalized nanoparticles with AMϕ significantly increased cell surface expression of MHC I and II, CD86, CD40 and the CLR CIRE over non-functionalized nanoparticles. Di-mannose and galactose functionalization also enhanced the expression of the macrophage mannose receptor (MMR) and the macrophage galactose lectin, respectively. This enhanced AMϕ activation phenotype was found to be dependent upon nanoparticle internalization. Functionalization also promoted increased AMϕ production of the pro-inflammatory cytokines IL-1β, IL-6 and TNF-α. Additional studies demonstrated the requirement of the MMR for the enhanced cellular uptake and activation provided by the di-mannose functionalized nanoparticles. Together, these data indicate that targeted engagement of MMR and other CLRs is a viable strategy for enhancing the intrinsic adjuvant properties of nanovaccine adjuvants and promoting robust pulmonary immunity.

High cell density fed-batch fermentation for the production of recombinant E. coli K-12 ghost vaccine against streptococcal disease

The bacterial ghost system is a novel vaccine delivery method which provides versatile carrier functions for foreign antigens with excellent natural intrinsic adjuvant properties. In this study, ghost bacteria of E. coli K-12/pHCE-InaN-GAPDH-ghost 27 SDM were created for mass production of a Streptococcus iniae ghost vaccine. The optimal fed-batch process for high cell density culture of E. coli K-12/pHCE-InaN-GAPDH-ghost 27 SDM was developed using the nutrient feeding strategy with Riesenberg defined medium. Fermentation was conducted in four phases as follow: (1) initial batch phase, (2) fed-batch phase for high cell density culture, (3) thermal induction phase for the formation of ghost by the expression of lysis gene E, and (4) high temperature holding phase to increase ghost formation efficiency. The maximum ghost bacteria vaccine (GBV) was obtained from the fed-batch fermentation of 34.9 g dry cell weight (dcw)/L. The expression of antigen glyceraldehyde 3-phosphate dehydrogenase (GAPDH) on the ghost cell with a high temperature holding phase was confirmed with outer-membrane protein fractionation using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Results indicate no damage to the expressed antigen on the ghost cell surfaces even after the temperature was increased to 47°C for high efficiency ghost cell formation. Efficacy of the GBV was evaluated by the challenge test in which vaccinated Olive flounder were infected with live S. iniae. The E. coli K-12 host strain, E. coli K-12/pHCE vector control, and formalin-killed cell (FKC) -treated vaccine groups showed 100, 100, and 65% cumulative mortality, respectively. The GBV-treated groups showed 50% cumulative mortality with increased survival ratios. Hence, the immunoprotective efficacy of GBV against S. iniae was better than that of the FKC vaccine. Therefore, the GBV is proposed as an effective vaccine in aquaculture for the prevention of streptococcal disease.

Iron-Regulated Lysis of Recombinant Escherichia coli in Host Releases Protective Antigen and Confers Biological Containment

The use of a recombinant bacterial vector vaccine is an attractive vaccination strategy to induce an immune response to a carried protective antigen. The superiorities of live bacterial vectors include mimicry of a natural infection, intrinsic adjuvant properties, and the potential for administration by mucosal routes. Escherichia coli is a simple and efficient vector system for production of exogenous proteins. In addition, many strains are nonpathogenic and avirulent, making it a good candidate for use in recombinant vaccine design. In this study, we screened 23 different iron-regulated promoters in an E. coli BL21(DE3) vector and found one, P(viuB), with characteristics suitable for our use. We fused P(viuB) with lysis gene E, establishing an in vivo inducible lysis circuit. The resulting in vivo lysis circuit was introduced into a strain also carrying an IPTG (isopropyl-β-d-thiogalactopyranoside)-inducible P(T7)-controlled protein synthesis circuit, forming a novel E. coli-based protein delivery system. The recombinant E. coli produced a large amount of antigen in vitro and could deliver the antigen into zebrafish after vaccination via injection. The strain subsequently lysed in response to the iron-limiting signal in vivo, implementing antigen release and biological containment. The gapA gene, encoding the protective antigen GAPDH (glyceraldehyde-3-phosphate dehydrogenase) from the fish pathogen Aeromonas hydrophila LSA34, was introduced into the E. coli-based protein delivery system, and the resultant recombinant vector vaccine was evaluated in turbot (Scophtalmus maximus). Over 80% of the vaccinated fish survived challenge with A. hydrophila LSA34, suggesting that the E. coli-based antigen delivery system has great potential in bacterial vector vaccine applications.

Probiotics: multifarious oral vaccine against infectious traumas

Microorganisms have been used for a long time in food and alcoholic fermentation. In the last few years they have undergone scientific scrutiny of their preventative and therapeutic aspects. This has led to the discovery of a new term, probiotics. Lactic acid bacteria (LAB) are microbial communities normally present in the intestine of most animals. They play an important role in humans and other animals, and act as immunomodulators. They are helpful in the treatment and prevention of disease as well as improving the digestion and absorption of nutrients. Probiotic microorganisms include the LAB Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus plantarum and Lactobacillus rhamnosus. Use of these live bacteria to elicit an immune response or to carry a vaccine component is a new invention in vaccine development. The advantage of live bacterial vaccines is that they mimic natural infection, have intrinsic adjuvant properties and can be given orally. Components of pathogenic and nonpathogenic food-related microorganisms are currently being evaluated as candidates for oral vaccines.

P19-02. High protection of female macaques from repeated intravaginal challenges with SHIV-162P3 upon mucosal vaccination with Gp41 subunits-virosomes

Methods We have developed a mucosal vaccine candidate based on two complementary conserved gp41 subunit antigensa trimeric recombinant gp41 deleted in known immunodominant regions (rGp41) and the 35 amino acid peptide P1, this later adopting the 3D conformation of the gp41 MPER and target of HIV-neutralizing IgA in highly exposed but persistently seronegative individualsfor focusing the immune response on relevant neutralizing epitopes. Gp41 subunit antigens are grafted on virosomes, a market-approved vaccine carrier with intrinsic adjuvant properties. Female Macaca mulata were immunized 4 times with both rGp41and P1 peptide-virosomes, using either the sole intra-muscular or the combined intra-muscular/intra nasal routes. Five weeks post-vaccination, animals were challenged 13 times intra-vaginally with low dose of SHIV162p3 at a biweekly frequency. Results Up to 13 weeks post-challenge, 5/5 animals vaccinated by the combined intra muscular/intra nasal routes were fully protected against SHIV162p3 (undetectable viremia, antiHIV IgG seronegativity), as compared to the 6/6 infected control group, or the 5/6 infected animals vaccinated by only the intra muscular route. Protection was correlated to gp41-specific IgA in vaginal secretions with in vitro neutralizing activities against transcytosis and CD4+ cell infection.

A novel technology for the production of a heterologous lipoprotein immunogen in high yield has implications for the field of vaccine design

We have developed a novel platform technology that can express high levels of recombinant lipoproteins with intrinsic adjuvant properties. In this study, Ag473 (a lipoprotein from Neisseria meningitidis) can be produced in high yields using Escherichia coli strain C43 (DE3). After testing a non-lipoimmunogen (E3, from dengue virus) fused with different lipid signal peptides from other lipoproteins as well as Ag473 fragments of different lengths, we identified that the fusion sequence has to contain at least the N-terminal 40 residues, D1, of Ag473 to achieve high expression levels of the recombinant lipo-immunogen (rlipo-D1E3). The rlipo-D1E3 was found to elicit stronger anti-E3 and virus neutralizing antibody responses in animal studies than those from rE3 alone or rE3 formulated with alum adjuvant. These results have successfully demonstrated the merit of lipo-immunogens for novel vaccine development.

The Effects of Heat-Killed Wild-Type Lactobacillus casei Shirota on Allergic Immune Responses in an Allergy Mouse Model

Background: Probiotics are used as a management strategy for allergic diseases, but their effects on allergic responses in sensitized allergic individuals remain unclear. This study explored the immunomodulatory effects of probiotics on allergen-specific allergic reactions in an allergy mouse model. Methods: C57BL/6 mice were presensitized by epicutaneous patching with recombinant Der p 2, and were subsequently administered orally with either heat-killed wild-type Lactobacillus casei or NaHCO₃ buffer for 5 weeks (n = 6 per group). All mice then received 2 subcutaneous immunizations of Der p 2 to mimic allergen immunotherapy, followed by aerosol challenge with Der p 2 a week later. Results: The Der p 2-sensitized mice fed L. casei showed significantly lower Der p 2-specific IgE and IgG1 after subcutaneous immunizations and airway challenge with Der p 2 compared to the control, unfed group. Splenic T cells of mice fed L. casei showed suppression of Th-2 (IL-5, IL-13, IL-10 and IL-4) and proinflammatory (TNF-α, IFN-γ) cytokines. Following airway allergen challenge, the mice fed L. casei showed histological evidence of attenuation of lung inflammation, as well as reductions in the total cell count and Th2 and proinflammatory cytokines in bronchoalveolar lavage fluid, compared to controls. Taken together, these results suggest that the oral administration of heat-killed L. casei could effectively downregulate the pre-existing Th-2 allergic responses and pulmonary inflammatory responses upon subcutaneous and airway allergen challenge. Conclusions:L. casei has intrinsic adjuvant and immunomodulatory properties that could potentially be exploited for secondary prevention or treatment of allergic respiratory diseases.

New malaria vaccine candidates based on the Plasmodium vivax Merozoite Surface Protein-1 and the TLR-5 agonist Salmonella Typhimurium FliC flagellin

The present study evaluated the immunogenicity of new malaria vaccine formulations based on the 19kDa C-terminal fragment of Plasmodium vivax Merozoite Surface Protein-1 (MSP119) and the Salmonella enterica serovar Typhimurium flagellin (FliC), a Toll-like receptor 5 (TLR5) agonist. FliC was used as an adjuvant either admixed or genetically linked to the P. vivax MSP119 and administered to C57BL/6 mice via parenteral (s.c.) or mucosal (i.n.) routes. The recombinant fusion protein preserved MSP119 epitopes recognized by sera collected from P. vivax infected humans and TLR5 agonist activity. Mice parenterally immunized with recombinant P. vivax MSP119 in the presence of FliC, either admixed or genetically linked, elicited strong and long-lasting MSP119-specific systemic antibody responses with a prevailing IgG1 subclass response. Incorporation of another TLR agonist, CpG ODN 1826, resulted in a more balanced response, as evaluated by the IgG1/IgG2c ratio, and higher cell-mediated immune response measured by interferon-γ secretion. Finally, we show that MSP119-specific antibodies recognized the native protein expressed on the surface of P. vivax parasites harvested from infected humans. The present report proposes a new class of malaria vaccine formulation based on the use of malarial antigens and the innate immunity agonist FliC. It contains intrinsic adjuvant properties and enhanced ability to induce specific humoral and cellular immune responses when administered alone or in combination with other adjuvants.

Translating innate response into long‐lasting antibody response by the intrinsic antigen‐adjuvant properties of papaya mosaic virus

Identifying the properties of a molecule involved in the efficient activation of the innate and adaptive immune responses that lead to long-lasting immunity is crucial for vaccine and adjuvant development. Here we show that the papaya mosaic virus (PapMV) is recognized by the immune system as a pathogen-associated molecular pattern (PAMP) and as an antigen in mice (Pamptigen). A single immunization of PapMV without added adjuvant efficiently induced both cellular and specific long-lasting antibody responses. PapMV also efficiently activated innate immune responses, as shown by the induction of lipid raft aggregation, secretion of pro-inflammatory cytokines, up-regulation of co-stimulatory molecules on dendritic cells and macrophages, and long-lasting adjuvant effects upon the specific antibody responses to model antigens. PapMV mixed with Salmonella enterica serovar Typhi (S. typhi) outer membrane protein C increased its protective capacity against challenge with S. typhi, revealing the intrinsic adjuvant properties of PapMV in the induction of immunity. Antigen-presenting cells loaded with PapMV efficiently induced antibody responses in vivo, which may link the innate and adaptive responses observed. PapMV recognition as a Pamptigen might be translated into long-lasting antibody responses and protection observed. These properties could be used in the development of new vaccine platforms.

Bacterial ghosts as adjuvant particles

The development of more advanced and effective vaccines is of great interest in modern medicine. These new-generation vaccines, based on recombinant proteins or DNA, are often less reactogenic and immunogenic than traditional vaccines. Thus, there is an urgent need for the development of new and improved adjuvants. Besides many other immunostimulatory components, the bacterial ghost (BG) system is currently under investigation as a potent vaccine delivery system with intrinsic adjuvant properties. BGs are nonliving cell envelope preparations from Gram-negative cells, devoid of cytoplasmic contents, while their cellular morphology and native surface antigenic structures remain preserved. Owing to the particulate nature of BGs and the fact that they contain many well known immune-stimulating compounds, BGs have the potential to enhance immune responses against ghost-delivered target antigens.

Live bacterial vaccines – a review and identification of potential hazards

The use of live bacteria to induce an immune response to itself or to a carried vaccine component is an attractive vaccine strategy. Advantages of live bacterial vaccines include their mimicry of a natural infection, intrinsic adjuvant properties and their possibility to be administered orally. Derivatives of pathogenic and non-pathogenic food related bacteria are currently being evaluated as live vaccines. However, pathogenic bacteria demands for attenuation to weaken its virulence. The use of bacteria as vaccine delivery vehicles implies construction of recombinant strains that contain the gene cassette encoding the antigen. With the increased knowledge of mucosal immunity and the availability of genetic tools for heterologous gene expression the concept of live vaccine vehicles gains renewed interest. However, administration of live bacterial vaccines poses some risks. In addition, vaccination using recombinant bacteria results in the release of live recombinant organisms into nature. This places these vaccines in the debate on application of genetically modified organisms. In this review we give an overview of live bacterial vaccines on the market and describe the development of new live vaccines with a focus on attenuated bacteria and food-related lactic acid bacteria. Furthermore, we outline the safety concerns and identify the hazards associated with live bacterial vaccines and try to give some suggestions of what to consider during their development.

Immobilization of plasmid DNA in bacterial ghosts

The development of novel delivery vehicles is crucial for the improvement of DNA vaccine efficiency. In this report, we describe a new platform technology, which is based on the immobilization of plasmid DNA in the cytoplasmic membrane of a bacterial carrier. This technology retains plasmid DNA ( Self– Immobilizing Plasmid, pSIP) in the host envelope complex due to a specific protein/DNA interaction during and after protein E-mediated lysis. The resulting bacterial ghosts (empty bacterial envelopes) loaded with pDNA were analyzed in detail by real time PCR assays. We could verify that pSIP plasmids were retained in the pellets of lysed Escherichia coli cultures indicating that they are efficiently anchored in the inner membrane of bacterial ghosts. In contrast, a high percentage of control plasmids that lack essential features of the self-immobilization system were expelled in the culture broth during the lysis process. We believe that the combination of this plasmid immobilization procedure and the protein E-mediated lysis technology represents an efficient in vivo technique for the production of non-living DNA carrier vehicles. In conclusion, we present a “self-loading”, non-living bacterial DNA delivery vector for vaccination endowed with intrinsic adjuvant properties of the Gram-negative bacterial cell envelope.

Induction of T-cell immunity to oligosaccharide antigens immobilized on crystalline bacterial surface layers (S-layers)

Immunization of Balb/c mice with conjugates of oligosaccharide haptens and crystalline bacterial surface-layer proteins (S-layers) primed the mice for a strong, hapten-specific, delayed-type hypersensitivity (DTH) response. Conjugates of haptens with bovine serum albumin produced only weak DTH responses but, when mixed with aluminium hydroxide, elicited DTH responses comparable to those against S-layer conjugates. Surface-layer conjugates also elicited strong anti-hapten DTH responses when administered by an oral/nasal route. Apparently, the natural assembly of S-layer proteins into large, two-dimensional arrays endows them with intrinsic adjuvant properties.

Meningococcal lipopolysaccharide (LPS)-derived oligosaccharide-protein conjugates evoke outer membrane protein- but not LPS-specific bactericidal antibodies in mice: influence of adjuvants.

Meningococcal lipopolysaccharide (LPS)-derived oligosaccharides (OS) were coupled to tetanus toxoid (TT) and purified P1.7,16 outer membrane proteins (OMP). The immunogenicities of the conjugates with and without the addition of the adjuvant Quil A or the nonionic block polymer L121 were studied in mice. Immunotype L2 and L3,7,9 OS-TT conjugates induced immunoglobulin G (IgG) responses that were strongly augmented by Quil A and L121. These adjuvants not only enhanced the amount of IgG evoked but also shifted the IgG subclass distribution from mainly IgG1 toward the complement-activating subclasses IgG2a and IgG2b. The antibodies induced were directed against the OS part of meningococcal LPS. They were not bactericidal for group B meningococci. Both the L3,7,9 OS-P1.7,16 OMP conjugate and purified P1.7,16 OMP evoked a strong IgG response against the P1.7,16 OMP but not against the L3,7,9 LPS. These anti-OMP IgG responses were comparable to the IgG OMP-specific responses induced by the H44/76 or HIII-5 outer membrane vesicles but still did not lyse group B meningococcal strains. The IgG response evoked with OS-OMP or purified OMP consisted mainly of the IgG1 subclass, whereas the H44/76 or HIII-5 outer membrane vesicles induced high amounts of bactericidal IgG2a and IgG2b antibodies next to the IgG1 antibodies. The addition of the adjuvant Quil A or L121 to OS-OMP or OMP resulted in the induction of high levels of bactericidal anti-P1.7,16-specific OMP antibodies, as reflected by the presence of substantial amounts of IgG2a and IgG2b antibodies. These results indicate that (i) mouse anti-LPS antibodies evoked by LPS-derived OS-protein conjugates are not bactericidal for group B meningococci, (ii) extensive purification of P1.7,16 OMP can lead to the loss of the intrinsic adjuvant properties of outer membrane vesicle preparations, and (iii) the addition of suitable adjuvants restores the ability of these purified P1.7,16 OMP to induce bactericidal antibodies.