Fasciola gigantica vaccine construct: an in silico approach towards identification and design of a multi-epitope subunit vaccine using calcium binding EF-hand proteins.

Continuous attempts have been made to pinpoint candidate vaccine molecules and evaluate their effectiveness in order to commercialise such vaccines for the treatment of tropical fascioliasis in livestock. The pathophysiology of fascioliasis can be related to liver damage brought on by immature flukes that migrate and feed, as well as immunological reactions to chemicals produced by the parasites and alarm signals brought on by tissue damage. Future research should, in our opinion, concentrate on the biology of invasive parasites and the resulting immune responses, particularly in the early stages of infection. The goal of the current study was to use the calcium-binding proteins from F. gigantica to create a multi-epitope subunit vaccine. The adjuvant, B-cell epitopes, CTL epitopes, and HTL epitopes that make up the vaccine construct are all connected by certain linkers. The antigenicity, allergenicity, and physiochemical properties of the vaccine construct were examined. The vaccine construct was docked with toll-like receptor 2, and simulations of the molecular dynamics of the complex's stability, interaction, and dynamics were run. After performing in silico cloning and immunosimulation, it was discovered that the construct was suitable for further investigation. New vaccination technologies and adjuvant development are advancing our food safety procedures since vaccines are seen as safe and are accepted by the user community. This research is also applicable to the F. hepatica system.

A Global Review on Short Peptides: Frontiers and Perspectives.

Peptides are fragments of proteins that carry out biological functions. They act as signaling entities via all domains of life and interfere with protein-protein interactions, which are indispensable in bio-processes. Short peptides include fundamental molecular information for a prelude to the symphony of life. They have aroused considerable interest due to their unique features and great promise in innovative bio-therapies. This work focusing on the current state-of-the-art short peptide-based therapeutical developments is the first global review written by researchers from all continents, as a celebration of 100 years of peptide therapeutics since the commencement of insulin therapy in the 1920s. Peptide "drugs" initially played only the role of hormone analogs to balance disorders. Nowadays, they achieve numerous biomedical tasks, can cross membranes, or reach intracellular targets. The role of peptides in bio-processes can hardly be mimicked by other chemical substances. The article is divided into independent sections, which are related to either the progress in short peptide-based theranostics or the problems posing challenge to bio-medicine. In particular, the SWOT analysis of short peptides, their relevance in therapies of diverse diseases, improvements in (bio)synthesis platforms, advanced nano-supramolecular technologies, aptamers, altered peptide ligands and in silico methodologies to overcome peptide limitations, modern smart bio-functional materials, vaccines, and drug/gene-targeted delivery systems are discussed.

Exploring the possible use of saponin adjuvants in COVID-19 vaccine.

There is an urgent need for a safe, efficacious, and cost-effective vaccine for the coronavirus disease 2019 (COVID-19) pandemic caused by novel coronavirus strain, severe acute respiratory syndrome-2 (SARS-CoV-2). The protective immunity of certain types of vaccines can be enhanced by the addition of adjuvants. Many diverse classes of compounds have been identified as adjuvants, including mineral salts, microbial products, emulsions, saponins, cytokines, polymers, microparticles, and liposomes. Several saponins have been shown to stimulate both the Th1-type immune response and the production of cytotoxic T lymphocytes against endogenous antigens, making them very useful for subunit vaccines, especially those for intracellular pathogens. In this review, we discuss the structural characteristics, mechanisms of action, structure-activity relationship of saponins, biological activities, and use of saponins in various viral vaccines and their applicability to a SARS-CoV-2 vaccine.

Poly(amino acids) as a potent self-adjuvanting delivery system for peptide-based nanovaccines.

To be optimally effective, peptide-based vaccines need to be administered with adjuvants. Many currently available adjuvants are toxic, not biodegradable; they invariably invoke adverse reactions, including allergic responses and excessive inflammation. A nontoxic, biodegradable, biocompatible, self-adjuvanting vaccine delivery system is urgently needed. Herein, we report a potent vaccine delivery system fulfilling the above requirements. A peptide antigen was coupled with poly-hydrophobic amino acid sequences serving as self-adjuvanting moieties using solid-phase synthesis, to produce fully defined single molecular entities. Under aqueous conditions, these molecules self-assembled into distinct nanoparticles and chain-like aggregates. Following subcutaneous immunization in mice, these particles successfully induced opsonic epitope-specific antibodies without the need of external adjuvant. Mice immunized with entities bearing 15 leucine residues were able to clear bacterial load from target organs without triggering the release of soluble inflammatory mediators. Thus, we have developed a well-defined and effective self-adjuvanting delivery system for peptide antigens.

Preclinical development of peptide vaccination combined with oncolytic MG1-E6E7 for HPV-associated cancer.

Human papilloma virus (HPV)-associated cancer is a significant global health burden and despite the presence of viral transforming antigens within neoplastic cells, therapeutic vaccinations are ineffective for advanced disease. HPV positive TC1 cells are susceptible to viral oncolysis by MG1-E6E7, a custom designed oncolytic Maraba virus. Epitope mapping of mice vaccinated with MG1-E6E7 enabled the rational design of synthetic long peptide (SLP) vaccines against HPV16 and HPV18 antigens. SLPs were able to induce specific CD8+ immune responses and the magnitude of these responses significantly increased when boosted by MG1-E6E7. Logically designed vaccination induced multi-functional CD8+ T cells and provided complete sterilising immunity of mice challenged with TC1 cells. In mice bearing large HPV-positive tumours, SLP vaccination combined with MG1-E6E7 was able to clear tumours in 60% of mice and these mice were completely protected against a long term aggressive re-challenge with the TC1 tumour model. Combining conventional SLPs with the multi-functional oncolytic MG1-E6E7 represents a promising approach against advanced HPV positive neoplasia.

Type III hypersensitivity reactions to a B cell epitope antigen are abrogated using a depot forming vaccine platform.

Peptide antigens are combined with an adjuvant in order to increase immunogenicity in vivo. The immunogenicity and safety of a RSV vaccine formulated in a novel oil-based platform, DepoVax™ (DPX), was compared to an alum formulation. A peptide B cell epitope derived from RSV small hydrophobic ectodomain (SHe) served as the antigen. Both vaccines induced SHe-specific antibodies after immunization of mice. A single dose of the DPX-based formulation resulted in anti-SHe titres for up to 20 weeks. Boosting with Alum-SHe, but not with DPX-SHe, led to unexpected clinical signs such as decreased activity, cyanosis and drop in body temperature in mice but not in rabbits. The severity of adverse reactions correlated with magnitude of SHe-specific IgG immune responses and decreased complement component 3 plasma levels, indicating a type III hypersensitivity reaction. By RP-HPLC analysis, we found that only 8-20% of the antigen was found to be adsorbed to alum in vitro, indicating that this antigen is likely released systemically upon injection in vivo. Clinical signs were not observed in rabbits, indicating the response correlates with peptide dose relative to size of animal. These results suggest that peptide antigens targeted to produce B cell mediated response may result in increased incidence of type III hypersensitivity reactions when delivered in non-depot forming vaccines. The DPX formulation induced strong antibody titres to the antigen without causing adverse events, likely due to the strength of the depot in vivo, and demonstrates the potential safety and immunogenicity of this platform for B cell peptide antigens.

Exploring Leishmania secretory proteins to design B and T cell multi-epitope subunit vaccine using immunoinformatics approach.

Visceral leishmaniasis (VL) is a fatal form of leishmaniasis which affects 70 countries, worldwide. Increasing drug resistance, HIV co-infection, and poor health system require operative vaccination strategy to control the VL transmission dynamics. Therefore, a holistic approach is needed to generate T and B memory cells to mediate long-term immunity against VL infection. Consequently, immunoinformatics approach was applied to design Leishmania secretory protein based multi-epitope subunit vaccine construct consisting of B and T cell epitopes. Further, the physiochemical characterization was performed to check the aliphatic index, theoretical PI, molecular weight, and thermostable nature of vaccine construct. The allergenicity and antigenicity were also predicted to ensure the safety and immunogenic behavior of final vaccine construct. Moreover, homology modeling, followed by molecular docking and molecular dynamics simulation study was also performed to evaluate the binding affinity and stability of receptor (TLR-4) and ligand (vaccine protein) complex. This study warrants the experimental validation to ensure the immunogenicity and safety profile of presented vaccine construct which may be further helpful to control VL infection.

In silico analyses of heat shock protein 60 and calreticulin to designing a novel vaccine shifting immune response toward T helper 2 in atherosclerosis.

Recent experiments demonstrated that atherosclerosis is a Th1 dominant autoimmune condition, whereas Th2 cells are rarely detected within the atherosclerotic lesions. Several studies have indicated that Th2 type cytokines could be effective in the reduction and stabilization of atherosclerotic plaque. Therefore, the modulation of the adaptive immune response by shifting immune responses toward Th2 cells by a novel vaccine could represent a promising approach to prevent from progression and thromboembolic events in coronary artery disease. In the present study, an in silico approach was applied to design a novel multi-epitope vaccine to elicit a desirable immune response against atherosclerosis. Six novel IL-4 inducing epitopes were selected from HSP60 and calreticulin proteins. To enhance epitope presentation, IL-4 inducing epitopes were linked together by AAY and HEYGAEALERAG linkers. In addition, helper epitopes selected from Tetanus toxin fragment C (TTFrC) were applied to induce CD4+ helper T lymphocytes (HTLs) responses. Moreover, cholera toxin B (CTB) was employed as an adjuvant. A multi-epitope construct was designed based on predicted epitopes which was 320 residues in length. Then, the physico-chemical properties, secondary and tertiary structures, stability, intrinsic protein disorder, solubility and allergenicity of this chimeric protein were analyzed using bioinformatics tools and servers. Based on bioinformatics analysis, a soluble, and non-allergic protein with 35.405kDa molecular weight was designed. Expasy ProtParam classified this chimeric protein as a stable protein. In addition, predicted epitopes in the chimeric vaccine indicated strong potential to induce B-cell mediated immune response and shift immune responses toward protective Th2 immune response. Various in silico analyses indicate that this vaccine is a qualified candidate for improvement of atherosclerosis by inducing immune responses toward T helper 2.

Immunoinformatics analysis and in silico designing of a novel multi-epitope peptide vaccine against Staphylococcus aureus.

Staphylococcus aureus is a pathogen that causes a variety of infections in humans. Methicillin-resistant S. aureus, which is an antibiotic-resistant form, is responsible for nosocomial staphylococcal infections, whose frequency is increasing in healthy people. Thereby, the development of novel techniques is required to overcome this bacterial infection. In this context, the use of vaccines to control infections is an appropriate alternative. In this study, immunoinformatics analysis is used on three antigenic determinants as vaccine candidates, and a novel multi-epitope vaccine is designed to induce cellular, humoral, and innate immune responses against S. aureus. Alpha-enolase, clumping factor A, and iron surface determinant B were selected as the protective antigens; and phenol-soluble modulin alpha 4was applied as the adjuvant. Epitopes identification was done for each antigen using various immunoinformatics servers. Moreover, the tertiary structure of our protein vaccine was predicted and validated. Subsequently, the best-modeled protein structure was used for the refinement process. There fined model was then applied for docking studies with Toll-like receptor 2 (TLR2).In the next step, molecular dynamics (MD) simulation was used to evaluate the stability of vaccine molecule and TLR2-vaccine complex. The high ranked epitopes were selected from the mentioned antigens. The selected epitopes and the adjuvant were fused together by proper linkers. Then, the modeled protein structure was selected and validated. Validation results indicated that the initial model needs refinement. After a refinement process, the final model was generated. Finally, the best-docked model of vaccine and TLR2 complex was selected. In this research, we attempted to design an efficient subunit vaccine, which could stimulate humoral and cellular immune responses. Therefore, we expect that our designed vaccine could defeat antibiotic-resistant staphylococcal infections.

Novel lipopeptides of ESAT-6 induce strong protective immunity against Mycobacterium tuberculosis: Routes of immunization and TLR agonists critically impact vaccine's efficacy.

Mycobacterium tuberculosis (Mtb), the bacterial cause of tuberculosis, is a leading infectious agent worldwide. The development of a new vaccine against Mtb is essential to control global spread of tuberculosis, since the current vaccine BCG is not very effective and antibiotic resistance is a serious, burgeoning problem. ESAT-6 is a secreted protein of Mtb, which is absent in BCG but has been implicated in inducing protective immunity against Mtb. Peptide based subunit vaccines are attractive due to their safety and high specificity in eliciting immune responses, but small synthetic peptides are usually not very immunogenic. We have designed a novel subunit vaccine for Mtb by using simple lipid (palmitic acid) modified derivatives of peptides from ESAT-6 protein corresponding to dominant human T cell epitopes and examined their ability to stimulate protective immunity against Mtb by intranasal and subcutaneous immunization in mice. We also investigated how individual TLR agonists as adjuvants (PolyI:C, MPL and GDQ) contribute to enhancing the induced immune responses and resulting protective efficacy of our vaccine. We observed that single C-terminal palmitoyl-lysine modified lipopeptides derived from ESAT-6 induce significant cellular immune responses on their own upon mucosal and subcutaneous immunizations. Intriguingly, a combination of immunogenic lipopeptides of ESAT-6 antigen exhibited local (pulmonary) and systemic immune responses along with efficient protective efficacy when administered intranasally or subcutaneously. Surprisingly, combination of ESAT-6 derived lipopeptides with a TLR-4 agonist (MPL) enhanced protection, whereas TLR-3 (Poly I:C) and TLR-7/8 agonists (gardiquimod, GDQ) led to reduced protection associated with specific local and systemic immune modulation. Our studies demonstrate the potential of ESAT-6 derived lipopeptides as a promising vaccine candidate against Mtb, and emphasize that selection of adjuvant is critical for the success of vaccines. These findings demonstrate the promise of synthetic lipopeptides as the basis of a subunit vaccine for TB.

Advances in strategies and methodologies in cancer immunotherapy.

Since the invention of Coley's toxin by William Coley in early 1900s, the path for cancer immunotherapy has been a convoluted one. Although still not considered standard of care, with the FDA approval of trastuzumab, Provenge and ipilimumab, the medical and scientific community has started to embrace the possibility that immunotherapy could be a new hope for cancer patients with otherwise untreatable metastatic diseases. This review aims to summarize the development of some major strategies in cancer immunotherapy, from the earliest peptide vaccine and transfer of tumor specific antibodies/T cells to the more recent dendritic cell (DC) vaccines, whole cell tumor vaccines, and checkpoint blockade therapy. Discussion of some major milestones and obstacles in the shaping of the field and the future perspectives is included. Photoimmunotherapy is also reviewed as an example of emerging new therapies combining phototherapy and immunotherapy.

A Targeted Multiple Antigenic Peptide Vaccine Augments the Immune Response to Self TGF-ß1 and Suppresses Ongoing Hepatic Fibrosis.

Transforming growth factor (TGF)-ß1 expression is induced upon liver injury, and plays a critical role in hepatic fibrosis. Antibodies against TGF-ß1 represent a novel approach in the treatment of hepatic fibrosis. However, TGF-ß1 is not a suitable antigen due to immunological tolerance. In the current study, we synthesized a multiple antigenic peptide (MAP) vaccine against the dominant B-cell epitope of TGF-ß1. The immunogenicity and potential therapeutic effects of this vaccine were examined using a rat model of hepatic fibrosis. Dominant B-cell epitopes of TGF-ß1 were identified using bioinformatic program. An MAP vaccine corresponding to the 90-98 amino acid domain of TGF-ß1 and containing four dendritic arms was synthesized using a 9-fluorenylmethoxycarbonyl solid phase method. Hepatic fibrosis which was induced in male Sprague-Dawley rats received a high-fat diet and ethanol (1.8 g/kg). Starting from the third week, rats were exposed to 40 % carbon tetrachloride (CCl4; 150 µl/100 g body weight twice weekly, initially 200 µl/100 g) treatment for a duration of 8 weeks. Rats received the MAP vaccine (100 µg) or Freund's adjuvant at weeks 1, 3, 5. A group of rats receiving the fibrosis-inducing regimen alone and a group of healthy rats (receiving an olive oil vehicle alone) were included as controls. At the conclusion of the experiment, serum titre of TGF-ß1 antibody was measured using ELISA and a standard liver functional test panel was examined. The extent of hepatic fibrosis was determined by measuring hydroxyproline content in the liver as well as hematoxylin-eosin (HE) and van Gieson (VG) staining. The expression of TGF-ß1 and alpha-smooth muscle actin (α-SMA) was examined using immunohistochemistry, and presented as positive staining cells. The MAP purity was >90 % upon reverse phase high-performance liquid chromatography, with apparent molecular weight at 4.77 kDa. Serum TGF-ß1 antibody titre was 1:256. The fibrosis-inducing treatment produced significant liver damage, as reflected by increases in liver functional test, HE and VG staining. The MAP vaccine attenuated such damage, as reflected by decreased alanine aminotransferase, aspartate aminotransferase, total bilirubin, and hepatic hydroxyproline (116.78 ± 23.76 vs. 282.71 ± 136.94 IU/L; 319.78 ± 82.48 vs. 495.29 ± 137.13 IU/L; 2.02 ± 0.27 vs. 4.01 ± 0.52 µmol/L; 263.67 ± 41.18 vs. 439.14 ± 43.29 µg/g vs. in model rats, respectively; p < 0.01), as well as fibrosis extent by HE and VG staining. The MAP vaccine reduced TGF-ß1 and α-SMA expression in rats (0.325 ± 0.059 vs. 0.507 ± 0.044 IOD/area; 0.318 ± 0.058 vs. 0.489 ± 0.029 IOD/area vs. model rats, respectively; p < 0.05). The TGF-ß1 MAP vaccine could generate sufficient antibody that suppresses the development of hepatic fibrosis.

Structure and immunogenicity of a peptide vaccine, including the complete HIV-1 gp41 2F5 epitope: implications for antibody recognition mechanism and immunogen design.

The membrane-proximal external region (MPER) of gp41 harbors the epitope recognized by the broadly neutralizing anti-HIV 2F5 antibody, a research focus in HIV-1 vaccine development. In this work, we analyze the structure and immunogenic properties of MPERp, a peptide vaccine that includes the following: (i) the complete sequence protected from proteolysis by the 2F5 paratope; (ii) downstream residues postulated to establish weak contacts with the CDR-H3 loop of the antibody, which are believed to be crucial for neutralization; and (iii) an aromatic rich anchor to the membrane interface. MPERp structures solved in dodecylphosphocholine micelles and 25% 1,1,1,3,3,3-hexafluoro-2-propanol (v/v) confirmed folding of the complete 2F5 epitope within continuous kinked helices. Infrared spectroscopy (IR) measurements demonstrated the retention of main helical conformations in immunogenic formulations based on alum, Freund's adjuvant, or two different types of liposomes. Binding to membrane-inserted MPERp, IR, molecular dynamics simulations, and characterization of the immune responses further suggested that packed helical bundles partially inserted into the lipid bilayer, rather than monomeric helices adsorbed to the membrane interface, could encompass effective MPER peptide vaccines. Together, our data constitute a proof-of-concept to support MPER-based peptides in combination with liposomes as stand-alone immunogens and suggest new approaches for structure-aided MPER vaccine development.

Structurally designed attenuated subunit vaccines for S. aureus LukS-PV and LukF-PV confer protection in a mouse bacteremia model.

Previous efforts towards S. aureus vaccine development have largely focused on cell surface antigens to induce opsonophagocytic killing aimed at providing sterile immunity, a concept successfully applied to other Gram-positive pathogens such as Streptococcus pneumoniae. However, these approaches have largely failed, possibly in part due to the remarkable diversity of the staphylococcal virulence factors such as secreted immunosuppressive and tissue destructive toxins. S. aureus produces several pore-forming toxins including the single subunit alpha hemolysin as well as bicomponent leukotoxins such as Panton-Valentine leukocidin (PVL), gamma hemolysins (Hlg), and LukED. Here we report the generation of highly attenuated mutants of PVL subunits LukS-PV and LukF-PV that were rationally designed, based on an octameric structural model of the toxin, to be deficient in oligomerization. The attenuated subunit vaccines were highly immunogenic and showed significant protection in a mouse model of S. aureus USA300 sepsis. Protection against sepsis was also demonstrated by passive transfer of rabbit immunoglobulin raised against LukS-PV. Antibodies to LukS-PV inhibited the homologous oligomerization of LukS-PV with LukF-PV as well heterologous oligomerization with HlgB. Importantly, immune sera from mice vaccinated with the LukS mutant not only inhibited the PMN lytic activity produced by the PVL-positive USA300 but also blocked PMN lysis induced by supernatants of PVL-negative strains suggesting a broad protective activity towards other bicomponent toxins. These findings strongly support the novel concept of an anti-virulence, toxin-based vaccine intended for prevention of clinical S. aureus invasive disease, rather than achieving sterile immunity. Such a multivalent vaccine may include attenuated leukotoxins, alpha hemolysin, and superantigens.

Advances in peptide-based human papillomavirus therapeutic vaccines.

Cervical cancer is the second leading cause of cancer in women worldwide. Human papillomavirus (HPV) is responsible for all cases of cervical cancer. Commercial prophylactic HPV vaccines are now available, but unfortunately these vaccines have no therapeutic effect against established HPV infections. In order to accelerate the control of cervical cancer and treat established HPV infections, it is necessary to develop therapeutic vaccines to eradicate HPV by generating cell-mediated immunity against HPV infected cells. Two HPV-encoded early proteins, the E6 and E7 oncoproteins, are the preferred targets because they are consistently expressed in virtually all cervical cancer cells and are necessary for the induction and maintenance of HPV-associated disease. A variety of vaccine strategies have been employed targeting immune responses to these proteins. Peptide-based vaccines are a promising strategy for the development of therapeutic HPV vaccines because of their safety, stability, and ease of production. This review summarizes the prospects of peptide-based vaccines for the treatment of established HPV infections. We address the challenges that scientists currently face for developing peptide-based vaccines and explore feasible strategies for improving the potency of the induced immune response with the aim of treating established HPV infections.

Erbium:YAG laser enhances transdermal peptide delivery and skin vaccination.

Transdermal delivery of peptides and the related vaccines is attractive for treatment and prevention of diseases. However, this delivery method is limited by the low permeability of the stratum corneum (SC). The objective of this study was to enhance and control skin permeation of peptides and related vaccines using an erbium:yttrium-aluminum-garnet (Er:YAG) laser. The amount of peptide transported through nude mouse skin was measured using a Franz diffusion apparatus. The SC layer was partly ablated by an Er:YAG laser, resulting in a greater enhancement effect on peptide delivery. A thinned and discontinuous SC layer was observed after laser exposure. The flux of peptides across skin treated with a laser at various fluences was 3-140-fold higher than that across intact skin. The molecular size, lipophilicity, and sequence of the peptides were found to play important roles in modulating the enhancement of activity. The result of confocal laser scanning microscopy (CLSM) indicated a significant increase in the skin deposition of peptides into laser-treated skin. In an in vivo study, mouse skin was treated with the laser followed by skin vaccination with a lysozyme antigen. It was demonstrated for the first time that laser treatment with no adjuvant or penetration enhancer significantly enhanced the production of antibodies in the serum by 3-fold. To reveal the mechanisms underlying these changes, a proteomic technique combined with mass spectrometry was used. This is, to our knowledge, the first report of a laser being used to immunize intact animals.

Nanodecoy system: a novel approach to design hepatitis B vaccine for immunopotentiation.

The progress toward subunit vaccines has been limited by their poor immunogenicity and limited stability. To enhance the immune response, subunit vaccines universally require improved adjuvants and delivery vehicles. In the present study, we propose the use of ceramic core based nanodecoy systems for effective immunization, which seems to exhibit a broad range of surface properties. Nanodecoy systems were prepared by self-assembling of hydroxyapatite core and cellobiose and finally the hepatitis B surface antigen (HBsAg) was adsorbed over the preformed nanodecoy systems. HBsAg loaded nanodecoy systems were characterized for size, shape and antigen loading efficiency. The effect of processing steps on the stability and integrity of HBsAg was assessed by in vitro antigenicity and SDS-PAGE experiments. Nanodecoy preparations were nanometric in size range and almost spherical in shape. SDS-PAGE studies confirmed the integrity of HBsAg protein in the formulation. Vaccine efficacy was determined in female Balb/c mice and results indicated that specific anti-HBsAg antibody titers in mice receiving nanodecoy system were more efficient than the conventional adjuvant alum followed by subcutaneous immunization. Studies also indicated that nanodecoy formulations could elicit combined Th1 and Th2 immune response. It is inferred that nanodecoy systems are a class of novel carriers and hold potential as an alternative adjuvant in vaccine technology.

Formulation of botulinum neurotoxin heavy chain fragments for vaccine development: mechanisms of adsorption to an aluminum-containing adjuvant.

Heavy chain fragments of botulinum neurotoxin serotypes A and B are being developed as a bivalent vaccine for botulism. To potentiate the immune response, an aluminum containing adjuvant will be formulated with the two antigens. The adsorption mechanisms of each antigen to aluminum phosphate and aluminum hydroxide adjuvants were studied. The adsorption of the serotype A antigen to each adjuvant, and the serotype B antigen to aluminum phosphate adjuvant, is dependent on electrostatic attractive forces. The serotype A antigen is basic, and pretreatment with phosphate anions is required for favorable adsorption conditions to aluminum hydroxide adjuvant. In contrast, the serotype B antigen displays a high affinity to aluminum hydroxide adjuvant even when the two species possess the same charge. It is proposed that the serotype B antigen is adsorbed to aluminum hydroxide adjuvant by a ligand exchange mechanism.

General strategies for efficient adjuvant incorporation of recombinant subunit immunogens.

We have previously reported strategies for Escherichia coli production of recombinant immunogens fused to hydrophobic peptides or lipid tags to improve their capacity to be incorporated into an adjuvant formulation, e.g., immunostimulating complexes (iscoms). Recently, we also explored the strong interaction between biotin and streptavidin to achieve iscom association of recombinant immunogens. Plasmodium falciparum,Toxoplasma gondii and Neospora caninum antigens have served as model immunogens in the different studies. Generated fusion proteins have been found to be successfully incorporated into iscoms and high-titer antigen-specific antibody responses have been obtained upon immunization of mice. We believe that the different concepts presented, utilizing either hydrophobic peptide or lipid tags, or the recently explored biotin-streptavidin principle, offer convenient methods to achieve efficient adjuvant incorporation of recombinant immunogens.