Recent Advances in Design and Synthesis of Self-Adjuvanting Lipopeptide Vaccines

Synthetic lipopeptide vaccines are being increasingly investigated mainly because of the advantages they offer over traditional vaccines, including safety of use in humans, high specificity in eliciting immune responses, greater purity and large scale/cost-effective production capacity. Moreover, a number of lipopeptide vaccines designed to possess self-adjuvanting properties have been developed and tested in vitro and in vivo. Producing high levels of serum-specific antibodies against incorporated peptide epitopes, they are showing their potential as effective vaccine candidates without the need for a co-administered adjuvant and/or carrier protein, often associated with undesirable effects in humans. This review presents recent insights on lipopeptide vaccine research and development, particularly on (1) the influence of the orientation of peptide epitopes and lipids on immune responses, (2) the use of carbohydrates for vaccine targeting, adjuvanting or as peptide epitope carriers, and (3) synthetic approaches to highly pure, multi-epitopic vaccine molecules using native chemical ligation techniques. Incorporation of different types of antigens within the same lipopeptide construct could provide a lipopeptide vaccine candidate suitable for safe and effective mucosal administration, which is a comfortable way of drug delivery.     

Effect of adjuvants on responses to skin immunization by microneedles coated with influenza subunit vaccine

Recent studies have demonstrated the effectiveness of vaccine delivery to the skin by vaccine-coated microneedles; however there is little information on the effects of adjuvants using this approach for vaccination. Here we investigate the use of TLR ligands as adjuvants with skin-based delivery of influenza subunit vaccine. BALB/c mice received 1 μg of monovalent H1N1 subunit vaccine alone or with 1 μg of imiquimod or poly(I:C) individually or in combination via coated microneedle patches inserted into the skin. Poly(I:C) adjuvanted subunit influenza vaccine induced similar antigen-specific immune responses compared to vaccine alone when delivered to the skin by microneedles. However, imiquimod-adjuvanted vaccine elicited higher levels of serum IgG2a antibodies and increased hemagglutination inhibition titers compared to vaccine alone, suggesting enhanced induction of functional antibodies. In addition, imiquimod-adjuvanted vaccine induced a robust IFN-γ cellular response. These responses correlated with improved protection compared to influenza subunit vaccine alone, as well as reduced viral replication and production of pro-inflammatory cytokines in the lungs. The finding that microneedle delivery of imiquimod with influenza subunit vaccine induces improved immune responses compared to vaccine alone supports the use of TLR7 ligands as adjuvants for skin-based influenza vaccines.     

Application of chitosan microspheres for nasal delivery of vaccines

Nasal vaccines offer several benefits, such as highly vascular mucous membranes, low enzymatic degradation compared to oral vaccines, and greater acceptability to patients. Nasal vaccines, however, have to overcome several limitations, including mucociliary clearance and the inefficient uptake of soluble antigens. Therefore, nasal vaccines require potent adjuvants and delivery systems to enhance their immunogenicity and to protect their antigens. Chitosan is a cheap, biocompatible, biodegradable, mucoadhesive, and nontoxic natural polymer. Chitosan microspheres have been investigated to determine whether they allow the controlled release of drugs and vaccines. They have figured in various studies on the vaccine delivery system through the nasal route. Several researchers have developed modified chitosan microspheres through their concomitant use with adjuvants or immunomodulators for an additive and a synergistic effect, and through the mannosylation of chitosan for receptor-mediated targeting antigen-presenting cells. The results of the recent researches on chitosan microspheres used as a nasal vaccine delivery system are discussed in this review.     

Adjuvants--essential components of new generation vaccines

Adjuvants are essential components of vaccines that augment an immunological reaction of organism. New vaccines based on recombinant proteins and DNA, are more save than traditional vaccines but they are less immunogenic. Therefore, there is an urgent need for the development of new, improved vaccine adjuvants. There are two classes of adjuvants: vaccine delivery systems (e.g. emulsions, microparticles, immune-stimulating complexes ISCOMs, liposomes) and immunostimulatory adjuvants (e.g. lipopolysaccharide, monophosphoryl lipid A, CpG DNA, or muramylpeptides). The discovery of more potent and safer adjuvants may allow to development better prophylactic and therapeutic vaccines against chronic infectious (e.g., HSV, HIV, HCV, HBV, HPV, or Helicobacter pylori) and noninfectious diseases as multiple sclerosis, insulin-dependent diabetes, rheumatoid arthritis, allergy and tumors (e.g., melanoma, breast, or colon cancer).     

The partially degraded lipopolysaccharide of Burkholderia cepacia and ornithine-containing lipids derived from some Gram-negative bacteria are useful complex lipid adjuvants

The partially degraded lipopolysaccharide of Burkholderia cepacia (LPSdegr) and the ornithine-containing lipids were purified from some bacteria. The substances were developed as complex lipid adjuvants, because they have weak toxicity and are able to activate the immune systems of the living body. After various toxoid antigens such as pertussis toxoid, diphtheria toxoid and tetanus toxoid were mixed with the complex lipid adjuvants, the mixtures were administered to mice subcutaneously. Antitoxoid IgG antibody titers in the serum were measured several times over 3 months. The efficacy of the LPSdegr as adjuvant was almost as high as that of the ornithine-containing lipids, and it was almost equal to that of the aluminum hydroxide adjuvant (Alum), which is generally used as a vaccine adjuvant.     

Mucosal adjuvants and delivery systems for protein-, DNA- and RNA-based vaccines

Almost all vaccinations today are delivered through parenteral routes. Mucosal vaccination offers several benefits over parenteral routes of vaccination, including ease of administration, the possibility of self-administration, elimination of the chance of injection with infected needles, and induction of mucosal as well as systemic immunity. However, mucosal vaccines have to overcome several formidable barriers in the form of significant dilution and dispersion; competition with a myriad of various live replicating bacteria, viruses, inert food and dust particles; enzymatic degradation; and low pH before reaching the target immune cells. It has long been known that vaccination through mucosal membranes requires potent adjuvants to enhance immunogenicity, as well as delivery systems to decrease the rate of dilution and degradation and to target the vaccine to the site of immune function. This review is a summary of current approaches to mucosal vaccination, and it primarily focuses on adjuvants as immunopotentiators and vaccine delivery systems for mucosal vaccines based on protein, DNA or RNA. In this context, we define adjuvants as protein or oligonucleotides with immunopotentiating properties co-administered with pathogen-derived antigens, and vaccine delivery systems as chemical formulations that are more inert and have less immunomodulatory effects than adjuvants, and that protect and deliver the vaccine through the site of administration. Although vaccines can be quite diverse in their composition, including inactivated virus, virus-like particles and inactivated bacteria (which are inert), protein-like vaccines, and non-replicating viral vectors such as poxvirus and adenovirus (which can serve as DNA delivery systems), this review will focus primarily on recombinant protein antigens, plasmid DNA, and alphavirus-based replicon RNA vaccines and delivery systems. This review is not an exhaustive list of all available protein, DNA and RNA vaccines, with related adjuvants and delivery systems, but rather is an attempt to highlight many of the currently available approaches in immunopotentiation of mucosal vaccines.     

畜禽疫苗佐剂研究进展

畜禽用疫苗主要是亚单位疫苗、减毒或灭活细菌/病毒疫苗,为了使疫苗对动物能达到长效持久的保护,必须使用有效的佐剂.根据不同的作用方式,目前可将佐剂主要分为2大类:疫苗传递系统的佐剂、免疫刺激作用佐剂.本探讨了近年来畜禽用疫苗佐剂的功能及其发展趋势,概述了其应用研究的进展.     

Recent advances in veterinary vaccine adjuvants

Next generation veterinary vaccines are going to mainly comprise of either subunit or inactivated bacteria/viruses. These vaccines would require optimal adjuvants and delivery systems to accord long-term protection from infectious diseases in animals. There is an urgent need for the development of new and improved veterinary and human vaccine adjuvants. Adjuvants can be broadly divided into two classes, based on their principal mechanisms of action: vaccine delivery systems and 'immunostimulatory adjuvants'. Vaccine delivery systems are generally particulate e.g. emulsions, microparticles, ISCOMS and liposomes, and mainly function to target associated antigens into antigen presenting cells (APC). In contrast, immunostimulatory adjuvants are predominantly derived from pathogens and often represent pathogen associated molecular patterns, e.g. LPS, MPL and CpG DNA, which activate cells of the innate immune system. Recent progress in innate immunity is beginning to yield insight into the initiation of immune responses and the ways in which immunostimulatory adjuvants might enhance this process in animals and humans alike.     

Recent developments in adjuvants for vaccines against infectious diseases

New generation vaccines, particularly those based on recombinant proteins and DNA, are likely to be less reactogenic than traditional vaccines, but are also less immunogenic. Therefore, there is an urgent need for the development of new and improved vaccine adjuvants. Adjuvants can be broadly separated into two classes, based on their principal mechanisms of action; vaccine delivery systems and 'immunostimulatory adjuvants'. Vaccine delivery systems are generally particulate e.g. emulsions, microparticles, iscoms and liposomes, and mainly function to target associated antigens into antigen presenting cells (APC). In contrast, immunostimulatory adjuvants are predominantly derived from pathogens and often represent pathogen associated molecular patterns (PAMP) e.g. LPS, MPL, CpG DNA, which activate cells of the innate immune system. Once activated, cells of innate immunity drive and focus the acquired immune response. In some studies, delivery systems and immunostimulatory agents have been combined to prepare adjuvant delivery systems, which are designed for more effective delivery of the immunostimulatory adjuvant into APC. Recent progress in innate immunity is beginning to yield insight into the initiation of immune responses and the ways in which immunostimulatory adjuvants may enhance this process. However, a rational approach to the development of new and more effective vaccine adjuvants will require much further work to better define the mechanisms of action of existing adjuvants. The discovery of more potent adjuvants may allow the development of vaccines against infectious agents such as HIV which do not naturally elicit protective immunity. New adjuvants may also allow vaccines to be delivered mucosally.     

Assembly of single-walled carbon nanohorn supported liposome particles

Nanoparticle-supported liposomes can be a promising platform for drug delivery, vaccine development, and biomedical imaging. Single-walled carbon nanohorns are a relatively new carbon nanomaterial, and they could be used as carriers of drug and imaging reagents. Assembling lipids around carbon nanohorns would confer this nanomaterial much broader applications such as vaccine development and targeted drug delivery by embedding a target protein or immunogenic protein into the lipid bilayer structure. Here, we show the assembly of functionalized single-walled carbon nanohorns (-CH(2)-CH(2)-COOH(x), ~100 nm) with positively charged lipids through a freeze and thaw cycle. The assembled complex particles can be readily separated from individual nanohorns or liposomes under specific centrifugation conditions. The results from transmission electronic microscopy, flow cytometry through nitrobenzoxadiazole labeled lipids, and zeta potential analysis clearly show that the nanohorns are encapsulated by liposomes with a median size of ca. 120 nm.     

粘膜免疫佐剂研究进展

粘膜免疫佐剂在新型疫苗的设计中具有重要作用。常用的粘膜免疫佐剂主要包括细菌性物质、细胞因子以及抗原递送系统。本文综述了这些佐剂的研究进展,以期为新型疫苗研究提供参考。     

Conjugation,characterization and toxicity of lipophosphoglycan-polyacrylic acid conjugate for vaccination against leishmaniasis

Research on the conjugates of synthetic polyelectrolytes with antigenic molecules, such as proteins, peptides, or carbohydrates, is an attractive area due to their highly immunogenic character in comparison to classical adjuvants. For example, polyacrylic acid (PAA) is a weak polyelectrolyte and has been used in several biomedical applications such as immunological studies, drug delivery, and enzyme immobilization. However, to our knowledge, there are no studies that document immune-stimulant properties of PAA in Leishmania infection. Therefore, we aimed to develop a potential vaccine candidate against leishmaniasis by covalently conjugating PAA with an immunologically vital molecule of lipophosphoglycan (LPG) found in Leishmania parasites. In the study, LPG and PAA were conjugated by a multi-step procedure, and final products were analyzed with GPC and MALDI-TOF MS techniques. In cytotoxicity experiments, LPG-PAA conjugates did not indicate toxic effects on L929 and J774 murine macrophage cells. We assume that LPG-PAA conjugate can be a potential vaccine candidate, and will be immunologically characterized in further studies to prove its potential.     

Vaccine instability in the cold chain: Mechanisms,analysis and formulation strategies

Instability of vaccines often emerges as a key challenge during clinical development (lab to clinic) as well as commercial distribution (factory to patient). To yield stable, efficacious vaccine dosage forms for human use, successful formulation strategies must address a combination of interrelated topics including stabilization of antigens, selection of appropriate adjuvants, and development of stability-indicating analytical methods. This review covers key concepts in understanding the causes and mechanisms of vaccine instability including (1) the complex and delicate nature of antigen structures (e.g., viruses, proteins, carbohydrates, protein-carbohydrate conjugates, etc.), (2) use of adjuvants to further enhance immune responses, (3) development of physicochemical and biological assays to assess vaccine integrity and potency, and (4) stabilization strategies to protect vaccine antigens and adjuvants (and their interactions) during storage. Despite these challenges, vaccines can usually be sufficiently stabilized for use as medicines through a combination of formulation approaches combined with maintenance of an efficient cold chain (manufacturing, distribution, storage and administration). Several illustrative case studies are described regarding mechanisms of vaccine instability along with formulation approaches for stabilization within the vaccine cold chain. These include live, attenuated (measles, polio) and inactivated (influenza, polio) viral vaccines as well as recombinant protein (hepatitis B) vaccines.     

Archaeosomes as adjuvants for combination vaccines

The present study evaluated the potential of archaesomes, prepared from the total polar lipids extracted from Methanobrevibacter smithii, as adjuvants for combination (multivalent) vaccines. Groups of Balb/c mice were immunized subcutaneously at day 0 and 21 with one of the following vaccines: trivalent vaccine formulated by the simultaneous co-encapsulation of bovine serum albumine (BSA), ovalbumin (OVA) and hen egg lysozyme (HEL) into archaeosomes (CEC vaccine); an univalent archaeosome vaccine (UVE vaccine) containing either BSA, OVA or HEL; or an admixture vaccine (AMC vaccine) consisting of the three UVE vaccines. Serum specific antibody (IgG + M) responses were determined at day 32, 112 and 203, and specific IgG1 and IgG2a responses were determined at day 112. Mice immunized with the CEC of AMC vaccine developed strong and sustained specific antibody responses to all three antigens at a magnitude similar to those seen in control mice immunized with UVE vaccines. Moreover, the serum BSA-, OVA-, and HEL-specific IgG1 and IgG2a levels in the CEC and AMC immunized mice were overall comparable to those of the UVE immunized control mice. Boosting CEC and AMC vaccinated mice with antigens alone at day 203 elicited strong antibody memory responses, comparable to those in the UVE vaccinated groups. These results show that archaeosomes could be used as adjuvants in developing combination vaccines.     

Beyond empiricism: informing vaccine development through innate immunity research

Although a great public heath success, vaccines provide suboptimal protection in some patient populations and are not available to protect against many infectious diseases. Insights from innate immunity research have led to a better understanding of how existing vaccines work and have informed vaccine development. New adjuvants and delivery systems are being designed based upon their capacity to stimulate innate immune sensors and target antigens to dendritic cells, the cells responsible for initiating adaptive immune responses. Incorporating these adjuvants and delivery systems in vaccines can beneficially alter the quantitative and qualitative nature of the adaptive immune response, resulting in enhanced protection.     

The choice of adjuvants in Mycoplasma vaccines

The use of adjuvants in vaccine production is an important aspect of potent vaccines. This investigation was concerned with finding the most efficient adjuvants for use in Mycoplasma vaccines produced in Nigeria. Four different vaccines were produced from the Gladysdale strain of Mycoplasma mycoides subspecies mycoides. They differed depending on the type of adjuvants used. Each vaccine was used to vaccinate eight cattle using a dose of 1 ml. Two other groups of eight cattle were used as controls. One of the two groups received 1 ml dose of inactivated Gladysdale vaccine without adjuvant while the second group received 1 ml dose of saline. The number of cattle that had the peak complement fixing (CF) antibody titres of 1/80 in each group of cattle was four for vaccine containing aluminium hydroxide gel, eight for vaccine containing liquid paraffin, one for vaccine containing sodium alginate and one for vaccine without adjuvant. Seven cattle from the group vaccinated with vaccine containing Freund's incomplete adjuvant had peak CF antibody titres of 1/80 or higher. The two groups vaccinated with vaccine containing liquid paraffin and Freund's incomplete adjuvant survived challenge at 6 months post vaccination. Freund's incomplete adjuvant and liquid paraffin containing 10% Arlacel A are the most efficient adjuvants.     

Dendritic cell‐targeting DNA‐based nasal adjuvants for protective mucosal immunity to Streptococcus pneumoniae

To develop safe vaccines for inducing mucosal immunity to major pulmonary bacterial infections, appropriate vaccine antigens (Ags), delivery systems and nontoxic molecular adjuvants must be considered. Such vaccine constructs can induce Ag‐specific immune responses that protect against mucosal infections. In particular, it has been shown that simply mixing the adjuvant with the bacterial Ag is a relatively easy means of constructing adjuvant‐based mucosal vaccine preparations; the resulting vaccines can elicit protective immunity. DNA‐based nasal adjuvants targeting mucosal DCs have been studied in order to induce Ag‐specific mucosal and systemic immune responses that provide essential protection against microbial pathogens that invade mucosal surfaces. In this review, initially a plasmid encoding the cDNA of Flt3 ligand (pFL), a molecule that is a growth factor for DCs, as an effective adjuvant for mucosal immunity to pneumococcal infections, is introduced. Next, the potential of adding unmethylated CpG oligodeoxynucleotide and pFL together with a pneumococcal Ag to induce protection from pneumococcal infections is discussed. Pneumococcal surface protein A has been used as vaccine for restoring mucosal immunity in older persons. Further, our nasal pFL adjuvant system with phosphorylcholine‐keyhole limpet hemocyanin (PC‐KLH) has also been used in pneumococcal vaccine development to induce complete protection from nasal carriage by Streptococcus pneumoniae . Finally, the possibility that anti‐PC antibodies induced by nasal delivery of pFL plus PC‐KLH may play a protective role in prevention of atherogenesis and thus block subsequent development of cardiovascular disease is discussed.

     

用于疫苗的免疫增强剂和递送系统

疫苗是抵抗传染病的最重要的预防性手段。在开发有效疫苗的过程中,人们面临的挑战一方面是要鉴定最为相关的免疫原及有效的免疫接种程序,另一方面则是免疫原用不同的能够影响抗体应答亲和性成熟和细胞介导的免疫应答的佐剂配成制剂。因此,有必要开发既能激发天然免疫应答,又能激发适应性免疫应答的有效佐剂和递送系统。主要阐述了不同免疫增强剂以及可利用于新一代疫苗的递送系统的研制进展情况。     

No phospholipid monolayer-sugar interactions

Studies by a number of workers using the Langmuir film balance have shown that when carbohydrates, such as sucrose or glycerol, are dissolved in a subphase on which a phospholipid is spread, film expansion occurs (Cadenhead & Demchak, 1969; Cadenhead & Bean, 1972; Maggio et al., 1976; Maggio & Lucy, 1978). Recently such effects have been observed again, particularly with the carbohydrates galactose and trehalose (Johnston et al., 1984). The origin of these film expansions was uncertain, and various suggestions have been made to explain them. One idea was that they might be due to interactions which these carbohydrates have with the water molecules close to the polar head groups of the lipids. Recent studies in our two laboratories, described here, show that the magnitude of the expansion effects is variable and that in general they arise from surfactant impurities in the sugars. These impurities are observed in carbohydrates which are reputedly of high grade; the amount of impurity present can vary from batch to batch, and sometimes they can be difficult to remove. Film balance techniques or subphase preparation can mask the detection of minor impurities. The presence of surfactant impurities in reputedly pure carbohydrates needs to be considered in other biochemical and biophysical studies of lipids and cell membranes.     

Inulin-derived adjuvants efficiently promote both Th1 and Th2 immune responses

There has been a recent resurgence of interest into new and improved vaccine adjuvants. This interest has been stimulated by the need for new vaccines to combat problematic pathogens such as SARS and HIV, and to counter potential bioterrorist attacks. A major bottleneck in vaccine development is the low immunogenicity of purified subunit or recombinant proteins, creating the need for safe human adjuvants with high potency. A major problem in the search for the ideal adjuvant is that adjuvants that promote cell-mediated (Th1) immunity (e.g. Freund's complete adjuvant) generally have unacceptable local or systemic toxicity that precludes their use in human vaccines. There is a need for a safe, non-toxic adjuvant that is able to stimulate both cell-mediated and humoral immunity. Inulin-derived adjuvants that principally stimulate the innate immune system through their ability to activate the alternative complement pathway have proven ability to induce both cellular and humoral immunity. With their excellent tolerability, long shelf-life, low cost and easy manufacture, they offer great potential for use in a broad range of prophylactic and therapeutic vaccines. Based on successful animal studies in a broad range of species, human trials are about to get underway to validate the use of inulin-based adjuvants in prophylactic vaccines against hepatitis B, malaria and other pathogens. If such trials are successful, then it is possible that inulin-derived adjuvants will one day replace alum as the adjuvant of choice in most human prophylactic vaccines.