Continuous antigen delivery from controlled release implants induces significant and anamnestic immune responses.

Two continuous delivery injectable silicone implants were tested to determine if they were capable of delivering vaccines in a single shot. The Type A implant delivers antigen in vitro over a 1-month-period and the Type B over several months. Vaccination studies in sheep were designed to compare the responses induced by the Type A and B implants, Alzet mini-osmotic pumps and conventional antigen delivery. A model antigen, avidin, was used along with IL-1beta or alum as adjuvants. Sheep were immunised with various formulations and the titre and isotype of the antigen specific antibodies monitored. The Type B implant induced antibody (Ab) titres of greater magnitude and duration than soluble vaccines or the Type A implant with adjuvant, but only if IL-1beta was included in the formulation. Both implants induced antibodies of IgG1 and IgG2 isotype. A memory response to soluble antigen challenge was induced by the Type B+IL-1beta implant, which was predominantly of an IgG1 isotype.     

The application of biodegradable collagen minipellets as vaccine delivery vehicles in mice and sheep.

Collagen minipellets are injectable delivery vehicles that release antigen and adjuvant over several days in a first-order release profile. In vaccination experiments in mice, secondary antibody responses induced by minipellets formulated with avidin and IL-1beta as adjuvant were equivalent to those induced by a conventional immunization with avidin in alum. When no adjuvant was used, anti-avidin responses induced by minipellets were 10-20-fold higher than those induced by injection of avidin in saline. In sheep, conventional vaccination with avidin in alum induced antibody responses initially exceeding that induced by minipellets formulated with avidin and IL-1beta, while following a secondary vaccination, the minipellet antibody response was equal to or greater than the alum-adjuvanted control groups. Increasing levels of IL-1beta adjuvant resulted in enhanced persistence of the antibody response. When clostridial vaccine antigens were incorporated into the minipellets, total antibody responses induced in sheep were equivalent to those induced by vaccination with the clostridial antigens in alum. Neutralizing antibody titres exceeded those induced by conventional vaccination. No adverse site reactions were observed at the implant site, with immunohistological study showing that the cellular infiltrate was dominated by a transient influx of neutrophils. This is a typical response to delivery of bioactive IL-1beta. The minipellets were completely degraded within 35 days of implantation.     

Influence of antigenic forms and adjuvants on the IgG subclass antibody response to Aujeszky's disease virus in mice.

The influence of antigenic forms of Aujeszky's disease virus (ADV) and adjuvant types on the production of IgG subclass antibodies in mice was investigated. Particulate antigen, inactivated ADV, alone induced IgG1 and lower IgG2a antibody production, while the antigen adsorbed onto aluminum phosphate gel (alum) enhanced IgG1 antibody production but suppressed IgG2a antibody production as well as solubilized ADV antigen adsorbed onto alum. QS21 saponin purified from Quillaja saponaria promoted the production of IgG1 and IgG2a antibodies in a large extent against the both particulate and soluble antigens, while this saponin has strong hemolytic activity. Lablaboside F saponin isolated from Dolichos lablab without hemolytic activity, also induced the production of large IgG1 and little IgG2a antibody against both antigens. Oil-based adjuvant, ISA70 of water-in-oil type and ISA25 of oil-in-water type, increased IgG1 and IgG2a antibodies against the both soluble and particulate antigens, whereas a combination of ISA25 and soluble antigen reduced IgG2a antibody response. These results indicate that IgG1 antibody production was not suppressed by a combination of antigenic form and adjuvant type, however, IgG2a antibody production was influenced.     

Evaluation of novel aggregate structures as adjuvants: composition, toxicity studies and humoral responses

Adjuvants are compounds that, when combined with an antigen, potentiate an immune response in an immunized species. There are numerous pathogens for which there are no protective vaccines and since alum is the only adjuvant licensed for use in humans, there is a clear need for more effective adjuvant preparations. In this study we describe the immunopotentiating properties of three novel molecular aggregate formulations based on tomatine (RAM1), a glycosylamide lipid (RAM2) and a fifth generation dendrimeric polymer (RAM3) respectively. These formulations were evaluated for their ability to augment antigen-specific antibody responses when administered with a soluble protein antigen. All three adjuvants were shown to be nontoxic to mice and elicited antigen-specific antibody responses. Of the three formulations, RAM1 was found to induce the highest titers of antibody; these were substantially higher than those induced by reference control adjuvants. RAM1 elicited antibodies of the IgG1 and IgG2a subclasses indicating, indirectly, that this adjuvant can stimulate Th2 and Th1 type immunity.     

Adjuvantation of epidermal powder immunization

The skin is an immunologically active site and an attractive vaccination route. All current vaccines, however, are administered either orally, intramuscularly, or subcutaneously. We previously reported that epidermal powder immunization (EPI) with an extremely small dose of powdered influenza vaccine induces protective immunity in mice. In this study, we report that commonly used adjuvants can be used in EPI to further enhance the immune responses to an antigen.The IgG antibody response to diphtheria toxoid (DT) following EPI was augmented by 25- and 250-fold, when 1 microg DT was co-delivered with aluminum phosphate (alum) and a synthetic oligonucleotide containing CpG DNA motifs (CpG DNA), respectively. These antibodies had toxin-neutralization activity and were long lasting. Furthermore, EPI using an adjuvant selectively activated different subsets of T helper cells and gave either a Th1 or a Th2 type of immune response. Similar to needle injection into deeper tissues, EPI with alum adsorbed DT promoted a predominantly IgG1 subclass antibody response and elevated level of IL-4 secreting cells. These are indicative of Th2-type immunity. In contrast, co-delivery of CpG DNA adjuvant via EPI led to Th-1 type of response as characterized by the increased production of IgG2a antibodies and IFN-gamma secreting cells. This study indicated that EPI using appropriate adjuvants can produce an augmented antibody response and desirable cellular immune responses. EPI is a promising immunization method that may be used to administer a broad range of vaccines including vaccines with adjuvants.     

PyNTTTTGT prototype oligonucleotide IMT504 is a potent adjuvant for the recombinant hepatitis B vaccine that enhances the Th1 response

PyNTTTTGT oligodeoxinucleotides (ODNs) cause activation, proliferation and immunoglobulin secretion on B cells, and the expression of co-stimulatory molecules on plasmacytoid dendritic cells of primates. It has now been discovered that these ODNs are also active on rat cells. This fact allowed us to investigate the adjuvant properties of PyNTTTTGT ODNs in a human Hepatitis B vaccine using this animal model. A very significant increment, as compared with the antigen alone, was observed in the antibody production induced by vaccination with the recombinant Hepatitis B surface antigen adjuvated with the PyNTTTTGT prototype IMT504 ODN. Analysis of the IgG subclass distribution in the sera of vaccinated animals indicated that, although an increase was observed in the titer of all the IgG subclasses, the increase on the Th1-associated IgG2b subclass was clearly more pronounced. Remarkably, this effect on the IgG2b titer was observed even if alum, a Th2 promoting adjuvant, was present together with IMT504 in the vaccine formulation. The increase in the Th1 response induced by IMT504 was also suggested by in vitro gamma interferon secretion assays. Monkeys of the species Cebus apella immunized with the recombinant Hepatitis B surface antigen plus alum and IMT504 also showed titers of antibodies against the antigen several times superior to the titers observed in control animals immunized with the antigen plus alum without ODN. Since rat and monkey cells are significantly less immunostimulated "in vitro" by PyNTTTTGT ODNs than human cells, the present results reasonably predict a very good performance of these ODNs as adjuvants in human vaccination.     

RLJ-NE-299A: a new plant based vaccine adjuvant

Alum has been in use since long as an adjuvant for vaccines. However, its use as a vaccine adjuvant offers limitation in supporting cell mediated response. Therefore, a new plant based product RLJ-NE-299A from Picrorhiza kurroa reported for its immunostimulatory activity, has been explored for its potential as an alternative adjuvant. In order to compare the adjuvant activity with alum, antigen-specific immune responses were evaluated following immunization with a formulation containing hepatitis B surface antigen (HBsAg) adjuvanted with RLJ-NE-299A and alum in mice. The adjuvant RLJ-NE-299A up-regulated remarkably the expression of Th1 cytokines IL-2, IL-12, IFN-gamma, TNF alpha and Th2 cytokine IL-4 in lymph node cell cultures after 2 weeks of primary immunization with HBsAg. Further, the levels of both immunoglobulins IgG2a (Th1) and IgG1 (Th2) subtypes increased profoundly in blood sera of mice immunized with HBsAg/RLJ-NE-299A. The results indicated that RLJ-NE-299A has strong potential to increase both cell mediated and humoral immune responses and is capable of sustaining the total antigen-specific antibody response. Besides, the RLJ-NE-299A provides a signal to gear up both CD4 helper cells (Th1 and Th2) and CD8 cells populations, which may have important implications for vaccination against hepatitis B virus. Variable doses of RLJ-NE-299A (0.312-40 microg) containing vaccine antigen (HBsAg) were well tolerated with optimum T cell response at 2.5 microg/ml. Not only this, the adjuvant was also able to induce cellular immune responses to HBsAg as evidenced by Th1 and Th2 cytokines upregulation, which enabled mice to overcome the unresponsiveness to antigen HBsAg encountered with alum-adjuvanted vaccine in otherwise non-responding mice population. The study presents evidence that the HPLC standardized fraction RLJ-NE-299A, is an adjuvant of choice over alum in improving and maintaining the improved immune status against HBsAg, and may also prove useful adjuvant candidate with other vaccine antigens, too.     

Poly[di(sodium carboxylatoethylphenoxy)phosphazene] (PCEP) is a potent enhancer of mixed Th1/Th2 immune responses in mice immunized with influenza virus antigens

We investigated the ability of a novel polyphosphazene polyelectrolyte, poly[di(sodium carboxylatoethylphenoxy)phosphazene] (PCEP) to enhance antigen-specific immune responses. BALB/c mice were immunized once subcutaneously with either bovine serum albumin (BSA) or influenza virus X:31 antigen alone, or in combination with PCEP, or either of the adjuvants poly[di(sodium carboxylatophenoxy)phosphazene] (PCPP) and alum. Both PCEP and PCPP significantly enhanced serum antigen-specific total IgG, IgG1 and IgG2a antibody titers, and these responses were highest in PCEP-immunized mice. Alum induced only a modest enhancement of antibody responses. Reducing the dose of X:31 antigen by 25-fold had no effect on antibody responses in mice immunized with PCPP and PCEP, but resulted in reduced titers in those immunized with alum. Analysis of X:31 antigen-specific cytokines revealed that alum and PCPP were associated with a predominantly IL-4 response. In contrast, PCEP was associated with production of both IFNgamma and IL-4. We conclude that PCEP is a potent enhancer of antigen-specific Th1 and Th2 immune responses and is a promising adjuvant for vaccine applications.     

Evaluation of recombinant Onchocerca volvulus activation associated protein-1 (ASP-1) as a potent Th1-biased adjuvant with a panel of protein or peptide-based antigens and commercial inactivated vaccines

Alum, the only adjuvant approved for clinical applications, can induce strong humoral (Th2) but weak cellular (Th1) immune responses. It is necessary to develop safe and effective adjuvants capable of inducing both humoral and cellular immune responses. We previously showed that activation-associated protein-1 (ASP-1) derived from Onchocerca volvulus has potent adjuvant activity. In this study, we have further evaluated the adjuvanticity of recombinant ASP-1 using a panel of recombinant proteins or synthetic peptide-based antigens, including ovalbumin (OVA), synthetic HIV peptide (HIV-p), recombinant HIV gp41 (rgp41) and HBV HBsAg, as well as three commercially available inactivated vaccines against haemorrhagic fever with renal syndrome (HFRS), Influenza and Rabies. Our results indicate that ASP-1 induced significantly higher IgG1 (Th2-associated) and IgG2a (Th1-associated) responses than alum adjuvant against OVA antigen, HIV-p, and rgp41. Consistently, it induced similar level of IgG1 responses as alum but higher level of IgG2a and IFN-gamma-producing T cell responses than alum adjuvant against HBsAg. Further, ASP-1 improved both IgG1 and IgG2a responses to three commercial inactivated vaccines when used separately or in combination. In conclusion, the recombinant ASP-1, unlike alum adjuvant, is able to induce both Th1 and Th2-associated humoral responses and Th1 cellular responses, suggesting that it can be further developed as a promising adjuvant for subunit-based and inactivated vaccines.     

Systemic immune responses in sheep, induced by a novel nano-bead adjuvant

Although a number of adjuvants are currently approved for use in veterinary species, only alum has been widely used in humans. While it induces strong antibody responses, cell mediated responses are often low and inflammatory reactions at the site of injection are common. We investigated the immunological properties of a novel nano-bead adjuvant in a sheep large-animal model. In contrast to alum, antigen covalently coupled to nano-beads induced substantial cell mediated responses along with moderate humoral responses. No adverse reactions were seen at the site of immunisation in the sheep. Thus, nano-bead adjuvants in veterinary species may be useful for the induction of immunity to viral pathogens, where a cell mediated response is required. These findings also highlight the potential usefulness of nano-bead vaccines for intracellular pathogens in humans.     

CpG DNA induces stronger immune responses with less toxicity than other adjuvants

The ability to augment protective immune responses with minimal side effects is quintessential for a good adjuvant. This study has compared various adjuvants that are used in animal research (Freund's complete and incomplete adjuvants, Titermax Gold), are licensed for human use (alum), or are in clinical testing for humans (monophosphoryl lipid, CpG DNA), for their ability to augment humoral responses to a model antigen (hepatitis B surface antigen) and for the degree of damage they caused in the injected muscle. According to the data, the adjuvant combination CpG DNA+alum had the greatest potential to augment immune responses with minimal side effects at the injection site. Evaluation of antibody isotypes indicated Th2 responses (no IgG2a) with all adjuvants except monophosphoryl lipid and CpG DNA, which gave mixed Th1/Th2 responses (IgG1 and IgG2a). Strong Th1 responses (predominantly IgG2a) were obtained with combinations of CpG DNA with other adjuvants.     

Comparison of the immunological memory after DNA vaccination and protein vaccination against anthrax in sheep

Currently available live spore vaccines against anthrax in animals have many drawbacks, one of which is their presumed inability to induce a long lasting immunity. In the present study we compared the immunological memory after a protein vaccination with DNA vaccinations in sheep. The antigen used was the protective antigen (PA83) of Bacillus anthracis. Sheep were vaccinated three times with either PA83 plus alhydrogel, or with one of four different plasmid DNA formulations, which all encoded either the full-length PA83 or its domain 4. Two pDNA formulations included Vaxfectin adjuvant, the other two were injected in PBS without adjuvant. Initially, the antibody titres of protein vaccinated sheep were significantly higher than the titres of pDNA vaccinated sheep. After 5 months, however, the antibody titres of protein vaccinated sheep had dropped remarkably, while the titres of all four pDNA vaccinated groups were either stable or had increased. Humoral responses of sheep immunised with pDNA formulated with Vaxfectin adjuvant were higher than the responses of the corresponding groups that received pDNA in PBS only.     

Subcutaneous inoculation of a whole virus particle vaccine prepared from a non-pathogenic virus library induces protective immunity against H7N7 highly pathogenic avian influenza virus in cynomolgus macaques

Development of H7N7 highly pathogenic avian influenza virus (HPAIV) vaccines is an urgent issue since human cases of infection with this subtype virus have been reported and most humans have no immunity against H7N7 viruses. We made an H7N7 vaccine combining components from an influenza virus library of non-pathogenic type A influenza viruses. Antibody and T cell recall responses specific against the vaccine strain were elicited by subcutaneous inoculation with the whole virus particle vaccine with or without alum as an adjuvant in cynomolgus macaques. No significant difference was observed in magnitude of antibody responses between vaccination with alum and vaccination without alum, though vaccination with alum induced longer recall responses of CD8⁺ T cells than did vaccination without alum. After challenge with a subtype of H7N7 HPAIV, the virus was detected in nasal swabs of unvaccinated macaques for 8 days but only for 1 day in the animals vaccinated either with or without alum, although the macaques vaccinated with alum showed elevated body temperature more briefly after infection. These findings demonstrated that this H7N7 HPAIV strain is pathogenic to macaques and that the vaccine conferred protective immunity to macaques against H7N7 HPAIV infection.     

Cationic lipid DC-Chol induces an improved and balanced immunity able to overcome the unresponsiveness to the hepatitis B vaccine.

Th1 and Th2 immune responses against antigens can be modulated by the use of adjuvants. Since antibody isotypes (IgG1 and IgG2a) and cytokines induced may reflect the Th differentiation taking place during the immune response, the humoral and cellular immune responses induced in mice against hepatitis B virus surface antigen (HBsAg) were examined when the antigen was either adsorbed to aluminum hydroxyde or administered with a new adjuvant the cationic lipid 3beta-[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol (DC-Chol). The use of DC-Chol increased antibody responses in responding BALB/c mice, induced more consistent IgG1 and IgG2a antibody responses in OF1 mice and overcame the nonresponse to HBsAg in B10.M mice. Furthermore, DC-Chol was able to induce cellular immune responses to HBsAg. The DC-Chol induced a balanced Th1/Th2 response, which enabled mice to overcome the inherited unresponsiveness to HBsAg encountered with aluminum-adjuvanted vaccine. Thus, the DC-Chol provides a signal to switch on both Th1 and Th2 responses, which may have important implications for vaccination against hepatitis B virus, as well as for enhancing weak immunogenicity of other recombinant purified antigens in a nonresponder population.     

Development of novel fusogenic vesosomes for transcutaneous immunization

Transcutaneous immunization (TCI) is a novel vaccination strategy based on the application of antigen together with an adjuvant onto hydrated bare skin. This simple and non-invasive immunization procedure elicits systemic and cell mediated immune responses and therefore, it provides a viable and cost-effective strategy for disease prevention. In the present study, novel fusogenic vesicular carrier constructs, i.e. vesosomes were developed and evaluated for topical delivery of vaccines using tetanus toxoid (TTx) as a model antigen. Prepared vesosomes were characterized for size, shape, entrapment efficiency and zeta potential. The prepared novel systems were examined for in process antigen stability and long-term storage stability studies. In vitro skin permeation and fluorescence microscopy study were also preformed for prepared novel vesicular systems for the evaluation of skin penetration efficiency. The immune stimulating activity of these vesicles was studied by measuring the serum anti-tetanus toxoid IgG titer and isotype ratio IgG2a/IgG1 following topical immunization in three different protocols and results were compared with the alum adsorbed tetanus toxoid given intramuscularly and topically administered plain tetanus toxoid solution, plain liposomes and cationic fusogenic liposomes. Serum IgG titers after three consecutive topical administrations were significantly better (*P < 0.05) than single administration of TTx antigen with vesosomal systems, suggesting an effective stimulation of serum immune response. Furthermore, notable serum anti-TTx antibody titers also occurred in animals primed with alum adsorbed TTx and subsequently boosted with topical administration of novel vesosomal systems. In each immunization studies, the vesosomal systems could elicit combined Th1 and Th2 immune responses following topical administration. These results suggest that the investigated vesosomal systems can be effective as topical delivery of vaccines.     

Cattle immune responses to tetanus toxoid elicited by recombinant S. typhimurium vaccines or tetanus toxoid in alum or Freund's adjuvant

Cattle were immunised orally, nasally or subcutaneously with either S. typhimurium 4/74 aroA(-) aroD(-) or S. typhimurium 4/74 htrA-based live vaccines expressing Fragment C (TetC) of tetanus toxin from plasmid pTetnir15. Oral inoculation with S. typhimurium 4/74 aroA(-) aroD(-)- (pTetnir15) elicited mucosal anti-TetC IgA but no measurable systemic humoral responses to TetC. Subcutaneous inoculation with the same strain elicited both mucosal IgA and systemic anti-TetC IgG1 responses. Nasal inoculation did not elicit any detectable anti-TetC responses. Oral delivery of S. typhimurium htrA(-) proved fatal in inoculated animals. None of the animals inoculated with either mutant S. typhimurium developed detectable T cell proliferative responses to the guest antigen. Cattle were also inoculated with tetanus toxoid adsorbed in alum or emulsified in Freund's complete adjuvant. Animals inoculated subcutaneously with Ttox emulsified in FCA developed systemic IgG1 and IgG2 antibody, while animals inoculated with Ttox adsorbed in alum developed systemic IgG1 but little IgG2 to Ttox. Both of these groups of animals developed measurable TetC-specific proliferative T cell responses that were associated with the production of IFNgamma.     

Whole recombinant Hansenula polymorpha expressing hepatitis B virus surface antigen (yeast-HBsAg) induces potent HBsAg-specific Th1 and Th2 immune responses

Recent studies have suggested that yeast cell wall components possess adjuvant activities. In the present study, heat-killed whole recombinant Hansenula polymorpha yeast expressing hepatitis B surface antigen (yeast-HBsAg) was generated, and the immune responses elicited by yeast-HBsAg were investigated in mice. The studies showed that yeast-HBsAg as well as yeast greatly promotes the accumulation of immune cells in mouse spleen and contributes to the maturation of dendritic cells (DCs). Yeast-HBsAg not only induces significantly higher antibody responses (including IgG, IgG1 and IgG2a), but also increases the IgG2a/IgG1 ratio, while alum combined with HBsAg (HBsAg + alum) only enhances antibody responses, but not the IgG2a/IgG1 ratio compared to HBsAg alone. Analysis of HBsAg-specific cytokines revealed that yeast-HBsAg is associated with production of both IFN-γ and IL-4, but neither IFN-γ nor IL-4 was detected in the HBsAg + alum-immunized group. Moreover, yeast-HBsAg induces potent HBsAg-specific lymphocyte proliferation and Cytotoxic T lymphocyte (CTL) responses. In conclusion, yeast-HBsAg enhances both HBsAg-specific Th1 and Th2 immune responses, while alum only enhances Th2 immune responses, suggesting that yeast-HBsAg may be an ideal candidate for an effective vaccine for the control of chronic hepatitis B virus (HBV) infection.     

A new vaccine adjuvant (BOS 2000) a potent enhancer mixed Th1/Th2 immune responses in mice immunized with HBsAg

Adjuvants in vaccines are immune stimulants that play an important role in the induction of effective and appropriate immune responses to vaccine component. In search of a potent vaccine adjuvant, the water-soluble biopolymeric fraction BOS 2000 from Boswellia serrata was evaluated for desired activity. We investigated the ability of BOS 2000 to enhance HBsAg specific immune responses. The effect was determined in the form of protective anti-HBsAg titers, neutralizing antibodies (IgG1 and IgG2a), spleen cell lymphocyte proliferation by using MTT assay, Th1 (IFN-gamma and TNF-alpha) and Th2 (IL-4) cytokines as well as T-lymphocyte subsets (CD4/CD8) and intracellular cytokines (IFN-gamma/IL-4), these responses were highest in BOS 2000 immunized mice. Alum induced only a modest enhancement of antibody responses. Reducing the dose of adjuvant by 18.1-fold in comparison to alum, total IgG and its subtypes (IgG1 and IgG2a) antibodies titer in serum was significantly enhanced. Analysis of HBsAg specific cytokines revealed that alum was associated with a predominantly IL-4 response. In contrast, BOS 2000 was associated with production of both IFN-gamma and IL-4. We conclude that BOS 2000 is a potent enhancer of antigen-specific Th1 and Th2 immune responses in comparison to alum with Th2 limitation and is a promising adjuvant for vaccine applications.     

A novel adjuvant for vaccine development in the aged

A conformationally-biased, response-selective agonist of human C5a(65-74) (EP67) activated antigen presenting cells (APC) from aged C57Bl/6 mice in vitro and the generation of antigen (Ag)-specific antibody (Ab) responses in aged mice in vivo. EP67, induced the release of the pro-inflammatory cytokines IL-6, TNFα, and INFγ from splenic APCs obtained from both aged and young mice. Both aged and young mice produced high Ag-specific IgG Ab titers when immunized with EP67-containing vaccines to ovalbumin (OVA-EP67) and to a protein (rPrp1) from the cell wall of Coccidioides (rPrp1-EP67). Immunization with EP67-containing vaccines resulted in higher IgG titers in both young and aged mice compared to mice immunized with OVA adsorbed to alum (OVA/alum) and Prp1 admixed with CpG (rPrp1 +CpG). Aged and young mice immunized with the EP67-containing vaccines generated higher titers of IgG1 and IgG2b relative to their aged-matched counterparts immunized with OVA/alum or Prp1 +CpG. These results indicate that EP67 induces humoral immunity in aged mice not obtainable with alum and CpG. These results support the use of EP67 as a potential vaccine adjuvant suited to the elderly.