Biotechnology approaches to produce potent,self-adjuvanting antigen-adjuvant fusion protein subunit vaccines

Traditional vaccination approaches (e.g. live attenuated or killed microorganisms) are among the most effective means to prevent the spread of infectious diseases. These approaches, nevertheless, have failed to yield successful vaccines against many important pathogens. To overcome this problem, methods have been developed to identify microbial components, against which protective immune responses can be elicited. Subunit antigens identified by these approaches enable the production of defined vaccines, with improved safety profiles. However, they are generally poorly immunogenic, necessitating their administration with potent immunostimulatory adjuvants. Since few safe and effective adjuvants are currently used in vaccines approved for human use, with those available displaying poor potency, or an inability to stimulate the types of immune responses required for vaccines against specific diseases (e.g. cytotoxic lymphocytes (CTLs) to treat cancers), the development of new vaccines will be aided by the availability of characterized platforms of new adjuvants, improving our capacity to rationally select adjuvants for different applications. One such approach, involves the addition of microbial components (pathogen-associated molecular patterns; PAMPs), that can stimulate strong immune responses, into subunit vaccine formulations. The conjugation of PAMPs to subunit antigens provides a means to greatly increase vaccine potency, by targeting immunostimulation and antigen to the same antigen presenting cell. Thus, methods that enable the efficient, and inexpensive production of antigen-adjuvant fusions represent an exciting mean to improve immunity towards subunit antigens. Herein we review four protein-based adjuvants (flagellin, bacterial lipoproteins, the extra domain A of fibronectin (EDA), and heat shock proteins (Hsps)), which can be genetically fused to antigens to enable recombinant production of antigen-adjuvant fusion proteins, with a focus on their mechanisms of action, structural or sequence requirements for activity, sequence modifications to enhance their activity or simplify production, adverse effects, and examples of vaccines in preclinical or human clinical trials.     

Boosting the immune response: the use of iNKT cell ligands as vaccine adjuvants

Natural killer T (NKT) cells comprise a small, but important T cell subset and are thought to bridge the innate and adaptive immune responses. The discovery of NKT cells and extensive research on their activating ligands have paved the way for modulation of these potent immunoregulatory cells in order to improve the outcome of various clinical conditions. Efforts to modulate NKT cell effector functions have ranged from therapy for influenza to antitumor immunotherapy. These approaches have also led to the use of NKTcell agonists such as ??-Galactosylceramide (??-GalCer) and its analogs as vaccine adjuvants, an approach that is aimed at boosting specific B and Tcell responses to a vaccine candidate by concomitant activation of NKT cells. In this review we will provide a comprehensive overview of the efforts made in using ??-GalCer and its analogs as vaccine adjuvants. The diverse array of vaccination strategies used, as well as the role of NKTcell activating adjuvants will be discussed, with focus on vaccines against malaria, HIV, influenza and tumor vaccines. Collectively, these studies demonstrate the efficacy of NKT cell-specific agonists as adjuvants and suggest that these compounds warrant serious consideration during the development of vaccination strategies.     

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.     

Recombinant viruses as tools to induce protective cellular immunity against infectious diseases.

Infections by intracellular pathogens such as viruses, some bacteria and many parasites, are cleared in most cases after activation of specific T cellular immune responses that recognize foreign antigens and eliminate infected cells. Vaccines against those infectious organisms have been traditionally developed by administration of whole live attenuated or inactivated microorganisms. Nowadays, research is focused on the development of subunit vaccines, containing the most immunogenic antigens from the particular pathogen. However, when purified subunit vaccines are administered using traditional immunization protocols, the levels of cellular immunity induced are mostly low and not capable of eliciting complete protection against diseases caused by intracellular microbes. In this review, we present a promising alternative to those traditional protocols, which is the use of recombinant viruses encoding subunit vaccines as immunization tools. Recombinant viruses have several interesting features that make them extremely efficient at inducing immune responses mediated by T-lymphocytes. This cellular immunity has recently been demonstrated to be of key importance for protection against malaria and AIDS, both of which are major targets of the World Health Organization for vaccine development. Thus, this review will focus in particular on the development of new vaccination protocols against these diseases.     

Synergism/complementarity of recombinant adenoviral vectors and other vaccination platforms during induction of protective immunity against malaria

The lack of immunogenicity of most malaria antigens and the complex immune responses required for achieving protective immunity against this infectious disease have traditionally hampered the development of an efficient human malaria vaccine. The current boom in development of recombinant viral vectors and their use in prime-boost protocols that result in enhanced immune outcomes have increased the number of malaria vaccine candidates that access pre-clinical and clinical trials. In the frontline, adenoviruses and poxviruses seem to be giving the best immunization results in experimental animals and their mutual combination, or their combination with recombinant proteins (formulated in adjuvants and given in sequence or being given as protein/virus admixtures), has been shown to reach unprecedented levels of anti-malaria immunity that predictably will be somehow reproduced in the human setting. However, all this optimism was previously seen in the malaria vaccine development field without many real applicable results to date. We describe here the current state-of-the-art in the field of recombinant adenovirus research for malaria vaccine development, in particular referring to their use in combination with other immunogens in heterologous prime-boost protocols, while trying to simultaneously show our contributions and point of view on this subject.     

Prospects for the use of sulfated polysaccharides from brown seaweeds as vaccine adjuvants

The data from the Russian and foreign literature on the effects of brown seaweed-derived sulfated polysaccharides (fucoidans) used as potential vaccine adjuvants to enhance the anti-infection and anti-tumor immune response are discussed in the present review. Due to their low toxicity, high biocompatibility, safety, and good tolerability by macroorganisms, as well as their mechanisms of immunomodulatory activity, fucoidans can be considered as promising adjuvants to administer in the composition of prophylactic and therapeutic vaccines. Fucoidans are agonists to receptors of innate immunity and are potent inducers of the cellular and humoral immune response, which is an important factor to be taken into account in the development of vaccines against various pathogens, including viruses, as well as anti-tumor vaccines. The results of numerous studies in which sulfated polysaccharides were tested as components of experimental vaccines, as presented in this review, show that these substances can be used as safe and effective adjuvants.     

Malaria vaccine development: current status

The development of an effective malaria vaccine represents one of the most important approaches that would provide a cost-effective intervention for addition to currently available malaria control strategies. Here, Howard Engers and Tore Godal review recent advances. Over the past decade there has been considerable progress in the understanding of immune mechanisms involved in conferring protection to malaria and in the identification of vaccine candidate antigens and their genes. Several new vaccines have entered Phase I/II trials recently, new adjuvants have been developed for human use and new approaches, such as DNA vaccines and structural modification of antigens to circumvent some of the strategies the parasite uses to avoid the immune response, are being applied. Thus, from the TDR perspective, global malaria vaccine development is entering a crucial period with unprecedented opportunities.     

DNA vaccine against malaria: a long way to go

Vaccination is the attempt to mimic certain aspects of an infection for the purpose of causing an immune response that will protect the individual from that infection. Malaria, a disease responsible for immense human suffering, is caused by infection with Plasmodium spp. parasites, which have a very complex life cycle--antigenically unique stages infect different tissues of the body. It is a parasitic disease for which no successful vaccine has been developed so far, despite considerable efforts to develop a subunit vaccine that offers protective immunity. Due to the spread of drug-resistant malaria, efforts to develop an effective vaccine have become increasingly critical. DNA vaccination provides a stable and long-lived source of protein vaccine capable of inducing both antibody- and cell-mediated immune responses to a wide variety of antigens. Injected DNA enters the cells of the host and makes the protein, which triggers the immune response. According to present needs, the flexibility of DNA vaccine technology permits the combination of multiple antigens from both the preerythrocytic and erythrocytic stages of malaria parasite. DNA vaccines with genes coding for different antigenic parts of malaria proteins have been created and presently some of these are undergoing field trials. The results from these trials will help to determine the likelihood of success of this technology in humans. This review presents an update of the studies carried out in malaria using DNA vaccine approach, the challenges, and the future prospects.     

The requirement for potent adjuvants to enhance the immunogenicity and protective efficacy of protein vaccines can be overcome by prior immunization with a recombinant adenovirus

A central goal in vaccinology is the induction of high and sustained Ab responses. Protein-in-adjuvant formulations are commonly used to achieve such responses. However, their clinical development can be limited by the reactogenicity of some of the most potent preclinical adjuvants and the cost and complexity of licensing new adjuvants for human use. Also, few adjuvants induce strong cellular immunity, which is important for protection against many diseases, such as malaria. We compared classical adjuvants such as aluminum hydroxide to new preclinical adjuvants and adjuvants in clinical development, such as Abisco 100, CoVaccine HT, Montanide ISA720, and stable emulsion-glucopyranosyl lipid A, for their ability to induce high and sustained Ab responses and T cell responses. These adjuvants induced a broad range of Ab responses when used in a three-shot protein-in-adjuvant regimen using the model Ag OVA and leading blood-stage malaria vaccine candidate Ags. Surprisingly, this range of Ab immunogenicity was greatly reduced when a protein-in-adjuvant vaccine was used to boost Ab responses primed by a human adenovirus serotype 5 vaccine recombinant for the same Ag. This human adenovirus serotype 5-protein regimen also induced a more cytophilic Ab response and demonstrated improved efficacy of merozoite surface protein-1 protein vaccines against a Plasmodium yoelii blood-stage challenge. This indicates that the differential immunogenicity of protein vaccine adjuvants may be largely overcome by prior immunization with recombinant adenovirus, especially for adjuvants that are traditionally considered poorly immunogenic in the context of subunit vaccination and may circumvent the need for more potent chemical adjuvants.     

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.     

DNA Vaccine Against Malaria: A Long Way To Go

Vaccination is the attempt to mimic certain aspects of an infection for the purpose of causing an immune response that will protect the individual from that infection. Malaria, a disease responsible for immense human suffering, is caused by infection with Plasmodium spp. parasites, which have a very complex life cycle — antigenically unique stages infect different tissues of the body. It is a parasitic disease for which no successful vaccine has been developed so far, despite considerable efforts to develop a subunit vaccine that offers protective immunity. Due to the spread of drug-resistant malaria, efforts to develop an effective vaccine have become increasingly critical. DNA vaccination provides a stable and long-lived source of protein vaccine capable of inducing both antibody- and cell-mediated immune responses to a wide variety of antigens. Injected DNA enters the cells of the host and makes the protein, which triggers the immune response. According to present needs, the flexibility of DNA vaccine technology permits the combination of multiple antigens from both the preerythrocytic and erythrocytic stages of malaria parasite. DNA vaccines with genes coding for different antigenic parts of malaria proteins have been created and presently some of these are undergoing field trials. The results from these trials will help to determine the likelihood of success of this technology in humans. This review presents an update of the studies carried out in malaria using DNA vaccine approach, the challenges, and the future prospects.     

Saponin-adjuvanted particulate vaccines for clinical use

Saponins are well recognised as potent immune stimulators, but their applicability as vaccine adjuvants have been limited due to associated toxicity. Formulation of saponin adjuvant with cholesterol and phospholipid produces the particulate ISCOMATRIX adjuvant, and when antigen is also contained within the particle, an ISCOM vaccine is produced. These particulate vaccines retain the adjuvant activity of the saponin component but without toxicity. Saponin-adjuvanted particulate vaccines have significant potential as a novel strategy in vaccine development. This review discusses (i) recent methodologies which have attempted to increase the flexibility and applicability of this technology by modifying either the vaccine composition or the mode of formulation; (ii) recent evaluations of these technologies for inducing protection against infectious diseases and as cancer immunotherapeutics.     

猪瘟DNA疫苗研究进展

自1990年DNA疫苗问世以来,已有许多研究者构建了不同类型的DNA疫苗。这些载体能诱发机体产生不同程度的特异性体液免疫和(或)细胞免疫。研究者们也在猪瘟DNA疫苗研究方面做出了很多努力并取得了一定的成果。以下从猪瘟DNA疫苗的构建和评价、佐剂在猪瘟DNA疫苗中的应用、猪瘟DNA疫苗与其他疫苗的联合应用以及目前猪瘟DNA疫苗存在的问题和解决途径等方面做了比较全面的阐述。     

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.     

Immune response against Mycobacterium tuberculosis: implications for vaccine development

Tuberculosis remains a major health problem globally. Although this threat would best be controlled by a combination of chemotherapy and vaccination, satisfactory vaccines are not available yet. Rational design of a novel vaccine generation against tuberculosis has become possible on the basis of recent achievements in molecular genetics of the pathogen and immunology of the host. Currently, two different strategies are pursued. First, the subunit vaccine approach attempting to induce efficacious immunity by unique antigens in defined adjuvants. Second, the whole bacterial vaccine approach relying on multiple antigens and built-in adjuvanticity. Time will tell which type of vaccine is best suited for eradication of tuberculosis.     

Enhancing blood-stage malaria subunit vaccine immunogenicity in rhesus macaques by combining adenovirus, poxvirus, and protein-in-adjuvant vaccines

Protein-in-adjuvant formulations and viral-vectored vaccines encoding blood-stage malaria Ags have shown efficacy in rodent malaria models and in vitro assays against Plasmodium falciparum. Abs and CD4(+) T cell responses are associated with protective efficacy against blood-stage malaria, whereas CD8(+) T cells against some classical blood-stage Ags can also have a protective effect against liver-stage parasites. No subunit vaccine strategy alone has generated demonstrable high-level efficacy against blood-stage infection in clinical trials. The induction of high-level Ab responses, as well as potent T and B cell effector and memory populations, is likely to be essential to achieve immediate and sustained protective efficacy in humans. This study describes in detail the immunogenicity of vaccines against P. falciparum apical membrane Ag 1 in rhesus macaques (Macaca mulatta), including the chimpanzee adenovirus 63 (AdCh63), the poxvirus modified vaccinia virus Ankara (MVA), and protein vaccines formulated in Alhydrogel or CoVaccine HT adjuvants. AdCh63-MVA heterologous prime-boost immunization induces strong and long-lasting multifunctional CD8(+) and CD4(+) T cell responses that exhibit a central memory-like phenotype. Three-shot (AdCh63-MVA-protein) or two-shot (AdCh63-protein) regimens induce memory B cells and high-titer functional IgG responses that inhibit the growth of two divergent strains of P. falciparum in vitro. Prior immunization with adenoviral vectors of alternative human or simian serotype does not affect the immunogenicity of the AdCh63 apical membrane Ag 1 vaccine. These data encourage the further clinical development and coadministration of protein and viral vector vaccine platforms in an attempt to induce broad cellular and humoral immune responses against blood-stage malaria Ags in humans.     

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.     

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.     

Advances in vaccine adjuvants.

Currently, aluminum salts and MF59 are the only vaccine adjuvants approved for human use. With the development of new-generation vaccines (including recombinant subunit and mucosal vaccines) that are less immunogenic, the search for more potent vaccine adjuvants has intensified. Of the novel compounds recently evaluated in human trials, immunostimulatory molecules such as the lipopolysaccharide derived MPL and the saponin derivative QS21 appear most promising, although doubts have been raised as to their safety in humans. Preclinical work with particulate adjuvants, such as the MF59 microemulsion and lipid-particle immune-stimulating complexes (Iscoms), suggest that these molecules are also potent elicitors of humoral and cellular immune responses. In addition, preclinical data on CpG oligonucleotides appear to be encouraging, particularly with respect to their ability to selectively manipulate immune responses. While all these adjuvants show promise, further work is needed to better define the mechanisms of adjuvant action. Ultimately, the development of more potent adjuvants may allow vaccines to be used as therapeutic, rather than prophylactic, agents.     

Pathogen-associated molecular patterns on biomaterials: a paradigm for engineering new vaccines

Vaccine development has progressed significantly and has moved from whole microorganisms to subunit vaccines that contain only their antigenic proteins. Subunit vaccines are often less immunogenic than whole pathogens; therefore, adjuvants must amplify the immune response, ideally establishing both innate and adaptive immunity. Incorporation of antigens into biomaterials, such as liposomes and polymers, can achieve a desired vaccine response. The physical properties of these platforms can be easily manipulated, thus allowing for controlled delivery of immunostimulatory factors and presentation of pathogen-associated molecular patterns (PAMPs) that are targeted to specific immune cells. Targeting antigen to immune cells via PAMP-modified biomaterials is a new strategy to control the subsequent development of immunity and, in turn, effective vaccination. Here, we review the recent advances in both immunology and biomaterial engineering that have brought particulate-based vaccines to reality.