Effect of different adjuvant formulations on the antibody response of horses to porcine zona pellucida proteins.

Immunization with porcine zona pellucida (PZP) proteins is being used successfully to induce infertility in wildlife including horses. However, widespread adoption of this method to control the growth of horse populations requires further refinement in order to induce long-term infertility, reduce the frequency and severity of injection site reactions, and make the vaccines easier to administer. The next generation of PZP-based vaccines will likely be a controlled-release formulation with different adjuvants from the Freund's adjuvants used in existing vaccines. We evaluated the response of equine peripheral blood mononuclear cells to a cationic nanoparticle adjuvant, Nano-11, alone and with the TLR agonists poly(I:C) and CpG ODN as a screen to develop an adjuvant system suitable for immunization of horses. The secretion of IL-1ß, TNF and CXCL10 were used as readouts. The combination of poly(I:C) with Nano-11 significantly increased the secretion of IL-1ß and TNF in comparison with Nano-11 only, with little effect of further addition of CpG ODN. The efficacy of the Nano-11/poly(I:C) adjuvant to enhance the immune response to native PZP proteins was determined in horses. Horses were immunized twice with the licensed Zonastat-H vaccine or PZP with Nano-11/poly(I:C) emulsified in silicone oil. A third group received PZP with the saponin adjuvant QA-21 emulsified in silicone oil. The horse sera collected monthly after the injections had increased anti-PZP IgG antibodies with the strongest response observed with Zonastat-H. We conclude that Nano-11/poly(I:C) is a potential candidate for the development of a controlled release formulation of a next generation PZP-based immunocontraception.

Effect of medium-chain fatty acids on growth, health, and immune response of dairy calves.

It is necessary for the dairy industry to reduce calf morbidity and mortality, and the reliance on antibiotics to treat sick calves, to address the growing concern regarding antibiotic resistant bacteria. The primary objective of this study was to evaluate the effect that feeding dairy calves medium-chain fatty acids (MCFA) has on growth performance and health, and the secondary objective was to evaluate the effect of MCFA on energy status around weaning and the adaptive immune response following a vaccine challenge. Thirty-three Holstein bull calves (5 ± 1.6 d of age) were randomly assigned to 1 of 2 treatments. Control (CON) calves were fed milk replacer with no C8:0 or C10:0 oil added and MCFA calves were fed milk replacer with 0.5% of a combination of C8:0 or C10:0 oil added. Body weight and average daily gain were measured weekly. Feed efficiency (gain/feed) and the change in body condition score, hip width, hip height, heart girth, and paunch girth were calculated for the duration of the study. Fecal scores were recorded daily and all medical treatments were documented for the duration of the trial. On d 42, 49, and 56 of the study, a serum sample was collected from each calf and used to measure nonesterified fatty acids, ß-hydroxybutyric acid, insulin, and glucose concentrations to evaluate energy status around weaning. A subset of 11 calves per treatment were enrolled in a vaccine challenge. At 21 ± 1.9 d of age (mean ± standard deviation) calves were vaccinated intramuscularly with 1 mL of endotoxin-free ovalbumin (OVA) mixed with aluminum hydroxide adjuvant. At 42 d of age (±1.9 d), blood samples were collected and used to analyze OVA-specific IgG1 and IgG2, and calves were vaccinated a second time. At 56 d of age (±1.9 d), blood samples were collected to analyze IgG1 and IgG2 as well as IFN-γ and IL-4 secreted from peripheral blood mononuclear cells (PBMC) treated with OVA or phytohemagglutinin. Data were analyzed as a completely randomized design with repeated measures when applicable. A tendency for greater daily fecal score was observed for MCFA calves compared with CON. At d 42 of the study, nonesterified fatty acid concentrations were greater in CON calves compared with MCFA. At 42 and 56 d of age, anti-OVA IgG1 concentrations for CON and MCFA calves were greater than prevaccination samples. This study suggests that feeding MCFA to calves affects the energy status of calves around weaning and vaccinating dairy calves with ovalbumin combined with an aluminum hydroxide adjuvant is an effective way to evaluate the adaptive immune responses.

Preclinical safety study of a recombinant Streptococcus pyogenes vaccine formulated with aluminum adjuvant.

A recombinant vaccine composed of a fusion protein formulated with aluminum hydroxide adjuvant is under development for protection against diseases caused by Streptococcus pyogenes. The safety and local reactogenicity of the vaccine was assessed by a comprehensive series of clinical, pathologic and immunologic tests in preclinical experiments. Outbred mice received three intramuscular injections of 1/5th of the human dose (0.1 ml) and rabbits received two injections of the full human dose. Control groups received adjuvant or protein antigen. The vaccine did not cause clinical evidence of systemic toxicity in mice or rabbits. There was a transient increase of peripheral blood neutrophils after the third vaccination of mice. In addition, the concentration of acute phase proteins serum amyloid A and haptoglobin was significantly increased 1 day after injection of the vaccine in mice. There was mild transient swelling and erythema of the injection site in both mice and rabbits. Treatment-related pathology was limited to inflammation at the injection site and accumulation of adjuvant-containing macrophages in the draining lymph nodes. In conclusion, the absence of clinical toxicity in two animal species suggest that the vaccine is safe for use in a phase I human clinical trial. Copyright © 2016 John Wiley & Sons, Ltd.

Challenges in pre-clinical testing of anti-cancer drugs in cell culture and in animal models.

Experiments with cultures of human tumor cell lines, xenografts of human tumors into immunodeficient mice, and mouse models of human cancer are important tools in the development and testing of anti-cancer drugs. Tumors are complex structures composed of genetically and phenotypically heterogeneous cancer cells that interact in a reciprocal manner with the stromal microenvironment and the immune system. Modeling the complexity of human cancers in cell culture and in mouse models for preclinical testing is a challenge that has not yet been met although tremendous advances have been made. A combined approach of cell culture and mouse models of human cancer is most likely to predict the efficacy of novel anti-cancer treatments in human clinical trials.

Effect of the strength of adsorption of HIV 1 SF162dV2gp140 to aluminum-containing adjuvants on the immune response.

The importance of the strength of antigen adsorption by aluminum-containing adjuvants on immunopotentiation was studied using HIV 1 SF162dV2gp140 (gp140), a potential HIV/AIDS antigen. The strengths of adsorption by aluminum hydroxide (AH) adjuvant and aluminum phosphate adjuvant, as measured by the Langmuir adsorptive coefficient, were 1900 and 400 mL/mg, respectively. The strength of adsorption by AH was modified by pretreatment of AH with two different concentrations of potassium dihydrogen phosphate to produce phosphate-treated aluminum hydroxide adjuvants having adsorptive coefficients of 1200 and 800 mL/mg. The four adjuvants were used to prepare vaccines containing either 1 or 10 µg of gp140 per dose. Antibody studies in mice revealed that the presence of an adjuvant increased the immune response in comparison with a solution of gp140 when the dose was 1 µg. Furthermore, the immune response was inversely related to the adsorptive coefficient. In contrast, no significant difference in immunopotentiation was observed between treatments in the presence or absence of an adjuvant when the dose of gp140 was 10 µg. Analysis of the binding of gp140 to CD4 and anti-gp140 monoclonal antibodies by surface plasmon resonance suggests that tight binding induced structural changes in the antigen.

Mechanism of immunopotentiation by aluminum-containing adjuvants elucidated by the relationship between antigen retention at the inoculation site and the immune response.

The relationship between depot formation and immunopotentiation was studied by comparing the retention of antigen at the inoculation site with antibody production in rats. A model (111)In-labeled alpha casein (IDCAS) antigen was formulated into four vaccines: IDCAS adsorbed onto either aluminum hydroxide adjuvant (AH) or aluminum phosphate adjuvant (AP); non-adsorbed IDCAS with phosphate-treated AP (PTAP); and IDCAS solution. Gamma scintigraphy showed the order of retention following subcutaneous administration to be: AH adsorbed>AP adsorbed>non-adsorbed with PTAP=solution. The antibody titers followed the order: non-adsorbed with PTAP=AP adsorbed>AH adsorbed>>solution. The presence of an aluminum-containing adjuvant was essential for immunopotentiation, but retention of the antigen at the inoculation site was not required.

Effect of the strength of adsorption of hepatitis B surface antigen to aluminum hydroxide adjuvant on the immune response.

Hepatitis B surface antigen (HBsAg) is known to adsorb to aluminum hydroxide adjuvant (AH) by ligand exchange between its accessible phosphate groups and surface hydroxyl groups of the adjuvant. To study the effect of the binding strength, five vaccines were prepared with AH or four samples of AH that were modified by pretreatment with different concentrations of potassium dihydrogen phosphate. The adsorptive coefficients ranged from 3660 to 250mL/mg based on the Langmuir adsorption isotherm and degrees of elution ranged from 1 to 31% when the vaccines were exposed to interstitial fluid in vitro. When tested in mice the four vaccines containing phosphate-treated AH (PTAH) induced significantly greater antibody responses than the vaccine containing AH, which had the highest adsorptive coefficient and the smallest degree of elution of HBsAg. The results indicated that antibody production is reduced when the antigen is adsorbed too strongly. Thus, the strength of adsorption of the antigen to an aluminum-containing adjuvant can affect the immunogenicity of the vaccine and should be optimized during vaccine formulation.

Relationship between physical and chemical properties of aluminum-containing adjuvants and immunopotentiation.

Aluminum-containing adjuvants are an important component of many vaccines because they safely potentiate the immune response. The structure and properties of aluminum hydroxide adjuvant, aluminum phosphate adjuvant and alum-precipitated adjuvants are presented in this review. The major antigen adsorption mechanisms, electrostatic attraction and ligand exchange, are related to the adjuvant structure. The manner by which aluminum-containing adjuvants potentiate the immune response is related to the structure, properties of the adjuvant and adsorption mechanism. Immunopotentiation occurs through the following sequential steps: inflammation and recruitment of antigen-presenting cells, retention of antigen at the injection site, uptake of antigen, dendritic cell maturation, T-cell activation and T-cell differentiation.

Activation of dendritic cells and induction of CD4(+) T cell differentiation by aluminum-containing adjuvants.

Aluminum-containing adjuvants are widely used in licensed human and veterinary vaccines. However, the mechanism by which these adjuvants enhance the immune response and predominantly stimulate a T(H)2 humoral immune response is not well understood. In this study, the effects of aluminum hydroxide and aluminum phosphate adjuvants on antigen presentation, expression of costimulatory molecules and cytokines by mouse dendritic cells (DCs) and the ability of DCs to induce T helper cell differentiation were investigated. Dendritic cells pulsed with ovalbumin (OVA) adsorbed to aluminum-containing adjuvants activated antigen-specific T cells more effectively than DCs pulsed with OVA alone. Aluminum hydroxide adjuvant had a significantly stronger effect than aluminum phosphate adjuvant. Both aluminum-containing adjuvants significantly increased the expression of CD86 on DCs but only aluminum hydroxide adjuvant also induced moderate expression of CD80. Aluminum-containing adjuvants stimulated the release of IL-1beta and IL-18 from DCs via caspase-1 activation. DCs incubated with LPS and OVA induced T(H)1 differentiation of naïve CD4(+) T cells. In contrast, DCs incubated with aluminum/OVA activated CD4(+) T cells to secrete IL-4 and IL-5 as well as IFN-gamma. Addition of neutralizing anti-IL-1beta antibodies decreased IL-5 production and addition of anti-IL-18 antibodies decreased both IL-4 and IL-5 production. Inhibition of IL-1beta and IL-18 secretion by DCs via inhibition of caspase-1 also led to a marked decrease of IL-4 and IL-5 by CD4(+) T cells. These results indicate that aluminum-containing adjuvants activate DCs and influence their ability to direct T(H)1 and T(H)2 responses through the secretion of IL-1beta and IL-18.

Potentiation of the immune response to non-adsorbed antigens by aluminum-containing adjuvants.

The degree of antigen adsorption by aluminum-containing adjuvants is considered an important characteristic of vaccines that is related to immunopotentiation by the adjuvant. This study examined immunopotentiation by aluminum phosphate adjuvant in three model vaccines in which the antigen was not adsorbed in the vaccine formulation nor when mixed in vitro with interstitial fluid. In the first model vaccine, aluminum phosphate adjuvant was pre-treated with 0.5 M KH2PO4 to minimize the adsorption of dephosphorylated alpha casein. The second model vaccine was composed of aluminum phosphate adjuvant and ovalbumin that was dephosphorylated by treatment with potato acid phosphatase. The third model vaccine consisted of aluminum phosphate adjuvant and lysozyme (LYS). In order to prevent adsorption of lysozyme, the aluminum phosphate adjuvant was pre-treated with fibrinogen, a protein present in interstitial fluid that binds strongly to aluminum phosphate adjuvant. Immunopotentiation was evaluated by measuring antibody production in mice. It was found that all three model vaccines induced antibody titers that were statistically higher than induced by a solution of antigen without adjuvant and similar to vaccines in which the antigens were adsorbed by aluminum phosphate adjuvant. Confocal microscopy experiments suggested that the antigens used in these experiments, even though not adsorbed to the aluminum phosphate adjuvant, were trapped in void spaces within the adjuvant aggregates, resulting in uptake of antigen by dendritic cells.

Role of aluminum-containing adjuvants in antigen internalization by dendritic cells in vitro.

An important step in the induction of an immune response to vaccines is the internalization of antigens by antigen presenting cells, such as dendritic cells (DCs). Many current vaccines are formulated with antigens adsorbed to an aluminum-containing adjuvant. Following injection of the vaccine the antigens may either elute or stay adsorbed to the adjuvant surface. Antigens, which elute from the adjuvant surface, are internalized by dendritic cells through macropinocytosis while those that remain adsorbed are internalized with the adjuvant particle by phagocytosis. The relative efficiency of these two routes of internalization was studied. Alpha casein (AC) labeled with a green fluorescent dye was selected as the model antigen. In order to model vaccine antigens that elute from aluminum-containing adjuvants following administration, dendritic cells were incubated with a solution of fluorochrome-labeled alpha casein. To model vaccine antigens that do not elute from aluminum-containing adjuvants following administration, dendritic cells were exposed to fluorochrome-labeled alpha casein adsorbed to aluminum hydroxide adjuvant (AH). Alpha casein has eight phosphate groups and adsorbs to aluminum hydroxide adjuvant through ligand exchange. Alpha casein does not elute from aluminum hydroxide adjuvant upon exposure to cell culture media. The uptake of antigen by dendritic cells was determined at 0.5, 1, 2 and 3h by confocal microscopy and flow cytometry. Dendritic cells internalized both alpha casein in solution and alpha casein adsorbed to aluminum hydroxide adjuvant. However, the mean fluorescence intensity of dendritic cells incubated with adsorbed alpha casein was four times greater than dendritic cells incubated with alpha casein in solution. In addition, the internalization of alpha casein was enhanced when the mean aggregate diameter of the adjuvant in the cell culture media was reduced from 17 microm to 3 microm. It was concluded that antigen internalization by dendritic cells was enhanced when the antigen remained adsorbed to the aluminum-containing adjuvant following administration and the aggregate size of the adjuvant was smaller than dendritic cells which are approximately 10 microm in diameter.

Mechanism of adsorption of hepatitis B surface antigen by aluminum hydroxide adjuvant.

Hepatitis B surface antigen (HBsAg) differs from many antigens because of its associated lipid bilayer that is largely composed of phospholipids. In general, phosphate groups adsorb strongly to hydroxylated mineral surfaces by ligand exchange. The purpose of this study was to investigate the mechanism of adsorption of hepatitis B surface antigen to aluminum hydroxide adjuvant with emphasis on the role of phospholipids in this adsorption. The adsorption of HBsAg by aluminum hydroxide adjuvant exhibits a high affinity adsorption isotherm. The Langmuir equation was used to calculate the adsorptive capacity (1.7 microg/microg Al), which is the amount of HBsAg adsorbed at monolayer coverage and the adsorptive coefficient (6.0 ml/microg), which is a measure of the strength of the adsorption force. The relatively high value of the adsorptive coefficient indicates that adsorption is due to a strong attractive force. Ligand exchange between a phosphate of the antigen and a surface hydroxyl of the adjuvant provides the strongest adsorption mechanism. The adsorption capacity of HBsAg was not affected by increased ionic strength indicating that electrostatic attraction is not the predominant adsorption force. Adsorption was also not affected by the addition of ethylene glycol indicating that hydrophobic interactions were not the predominant adsorption force. The strength of the adsorption force was indicated by the resistance of HBsAg to elution when exposed to interstitial fluid. Less than 5% of the HBsAg adsorbed to aluminum hydroxide adjuvant in a model vaccine was eluted during a 12 h in vitro exposure to interstitial fluid at 37 degrees C. Less than 1% of the adsorbed HBsAg in two commercial vaccines was eluted by in vitro exposure to interstitial fluid for 48 h at 37 degrees C. Thus, it was concluded that adsorption of HBsAg by aluminum hydroxide adjuvant is predominantly due to ligand exchange between the phospholipids in HBsAg and surface hydroxyls in aluminum hydroxide adjuvant.

Effect of the degree of phosphate substitution in aluminum hydroxide adjuvant on the adsorption of phosphorylated proteins.

Aluminum hydroxide adjuvant was pretreated with six concentrations of potassium dihydrogen phosphate to produce a series of adjuvants with various degrees of phosphate substitution for surface hydroxyl. The adsorption of three phosphorylated proteins (alpha casein, dephosphorylated alpha casein, and ovalbumin) by the phosphate-treated aluminum hydroxide adjuvants was studied. The phosphorylated proteins were adsorbed by ligand exchange of phosphate for hydroxyl even when an electrostatic repulsive force was present. However, the extent (adsorptive capacity) and strength (adsorptive coefficient) of adsorption was inversely related to the degree of phosphate substitution of the aluminum hydroxide adjuvant. Exposure of vaccines containing aluminum hydroxide adjuvant and phosphorylated antigens to phosphate ion in the formulation or during manufacture should be minimized to produce maximum adsorption of the antigen.

Relationship between the degree of antigen adsorption to aluminum hydroxide adjuvant in interstitial fluid and antibody production.

The effect of the degree of adsorption after exposure to interstitial fluid on the immune response in mice to model vaccines containing ovalbumin, alpha casein or dephosphorylated alpha casein adsorbed to aluminum hydroxide adjuvant was studied. Ovalbumin and dephosphorylated alpha casein were adsorbed in the vaccine but were completely eluted when exposed to interstitial fluid for 4 h. The presence of aluminum hydroxide adjuvant in the vaccine produced immunopotentiation compared to a solution of the protein even though the protein desorbed rapidly upon subcutaneous administration. In contrast, alpha casein was completely adsorbed to aluminum hydroxide adjuvant in both the vaccine and upon exposure to interstitial fluid. Immunopotentiation by aluminum hydroxide adjuvant was also observed in this model vaccine compared to a solution of alpha casein. The results indicated that antigen presenting cells can take up desorbed antigen from interstitial fluid as well as antigen adsorbed to aluminum-containing adjuvants.

Immunization of rabbits against a bacterial pathogen with an alginate microparticle vaccine.

Pasteurella multocida is an important bacterial pathogen of domestic rabbits. To evaluate the ability of a thiocyanate extract (PTE) of P. multocida to stimulate an immune response and protect against infection with P. multocida, rabbits were immunized subcutaneously or intranasally on Days 7, 21 and 35. Cholera toxin, a potent mucosal adjuvant, was included in one treatment group. Rabbits immunized subcutaneously (SC) or intranasally (IN) had significant increases in serum anti-PTE IgG but not IgA. In contrast, only rabbits immunized IN with PTE developed significant titers of nasal lavage anti-PTE IgA and cholera toxin significantly enhanced this response. In a second study rabbits were immunized via the drinking water with PTE incorporated into alginate microparticles on Days 7, 14 and 21. Mild increases in serum IgG were noted in rabbits immunized with PTE in microparticles, with or without cholera toxin, and this increase was significant (P