Group A Streptococcus Vaccine Targeting the Erythrogenic Toxins SpeA and SpeB Is Safe and Immunogenic in Rabbits and Does Not Induce Antibodies Associated with Autoimmunity.

Group A streptococcus (GAS) is a global pathogen associated with significant morbidity and mortality for which there is currently no licensed vaccine. Vaccine development has been slow, mostly due to safety concerns regarding streptococcal antigens associated with autoimmunity and related complications. For a GAS vaccine to be safe, it must be ensured that the antigens used in the vaccine do not elicit an antibody response that can cross-react with host tissues. In this study, we evaluated the safety of our GAS vaccine candidate called VaxiStrep in New Zealand White rabbits. VaxiStrep is a recombinant fusion protein comprised of streptococcal pyrogenic exotoxin A (SpeA) and exotoxin B (SpeB), also known as erythrogenic toxins, adsorbed to an aluminum adjuvant. The vaccine elicited a robust immune response against the two toxins in the rabbits without any adverse events or toxicity. No signs of autoimmune pathology were detected in the rabbits' brains, hearts, and kidneys via immunohistochemistry, and serum antibodies did not cross-react with cardiac or neuronal tissue proteins associated with rheumatic heart disease or Sydenham chorea (SC). This study further confirms that VaxiStrep does not elicit autoantibodies and is safe to be tested in a first-in-human trial.

An alternative approach to combination vaccines: intradermal administration of isolated components for control of anthrax, botulism, plague and staphylococcal toxic shock.

BACKGROUND: Combination vaccines reduce the total number of injections required for each component administered separately and generally provide the same level of disease protection. Yet, physical, chemical, and biological interactions between vaccine components are often detrimental to vaccine safety or efficacy. METHODS: As a possible alternative to combination vaccines, we used specially designed microneedles to inject rhesus macaques with four separate recombinant protein vaccines for anthrax, botulism, plague and staphylococcal toxic shock next to each other just below the surface of the skin, thus avoiding potentially incompatible vaccine mixtures. RESULTS: The intradermally-administered vaccines retained potent antibody responses and were well- tolerated by rhesus macaques. Based on tracking of the adjuvant, the vaccines were transported from the dermis to draining lymph nodes by antigen-presenting cells. Vaccinated primates were completely protected from an otherwise lethal aerosol challenge by Bacillus anthracis spores, botulinum neurotoxin A, or staphylococcal enterotoxin B. CONCLUSION: Our results demonstrated that the physical separation of vaccines both in the syringe and at the site of administration did not adversely affect the biological activity of each component.The vaccination method we describe may be scalable to include a greater number of antigens, while avoiding the physical and chemical incompatibilities encountered by combining multiple vaccines together in one product.

A rational, systematic approach for the development of vaccine formulations.

With the continuous emergence of new infectious diseases and new strains of current diseases, such as the novel H1N1 influenza in 2009, in combination with expanding competition in the vaccine marketplace, the pressure to develop vaccine formulations right the first time is increasing. As vaccines are complex, costly, and have high risk associated with their development, it is necessary to maximize the potential for development of a successful formulation quickly. To accomplish this goal, the historical empirical approach to formulation development needs to be updated with a rational, systematic approach allowing for more rapid development of safe, efficacious, and stable vaccine formulations. The main components to this approach are biophysical characterization of the antigen, evaluation of stabilizers, investigation of antigen interactions with adjuvants, evaluation of product contact materials, and monitoring stability both in real time and under accelerated conditions. An overview of investigations performed for each of these components of formulation development is discussed. The information gained in these studies is valuable in forming the base of knowledge for the design of a robust formulation. With the use of continually advancing technology in combination with maintaining a rational, systematic approach to formulation development, there is a great increase in the probability of successfully developing a safe, effective, and stable vaccine formulation.

Synthetic Toll-like receptor 4 agonist enhances vaccine efficacy in an experimental model of toxic shock syndrome.

The development of new protein subunit vaccines has stimulated the search for improved adjuvants to replace traditional aluminum-containing products. We investigated the adjuvant effects of a synthetic Toll-like receptor 4 (TLR4) agonist on vaccine efficacy in an experimental model of toxic shock syndrome. The TLR4 agonist E6020 has a simplified structure consisting of a hexa-acylated acyclic backbone. The vaccine examined is a recombinantly attenuated form of staphylococcal enterotoxin B (STEBVax). Using cells stably transfected with TLRs, E6020 transduced signals only through TLR4, suggesting monospecificity, while Escherichia coli 055:B5 lipopolysaccharide activated both the TLR2/6 heterodimer and TLR4. Coadministration of E6020 with STEBVax, by the intramuscular or intranasal route, induced significant levels of immunoglobulin G (IgG) in BALB/c mice. Further, increased IgG production resulted from the combination of E6020 with aluminum hydroxide adjuvant (AH). The antibody response to the vaccine coadministered with E6020 was a mixed Th1/Th2 response, as opposed to the Th2-biased response obtained with AH. Mice vaccinated with STEBVax coadministered with AH, TLR4 agonists, or a combination of both adjuvants were protected from toxic shock. Our data demonstrate the effectiveness of the synthetic TLR4 agonist E6020 as an alternative adjuvant for protein subunit vaccines that may also be used in combination with traditional aluminum-containing adjuvants.

Relationship of adsorption mechanism of antigens by aluminum-containing adjuvants to in vitro elution in interstitial fluid.

The objective of this research was to determine how the mechanism by which antigens adsorb to aluminum-containing adjuvants affects the elution upon exposure to interstitial fluid. Antigens (alpha lactalbumin, bovine serum albumin, lysozyme and myoglobin) that adsorb to aluminum-containing adjuvants principally by electrostatic attraction were found to elute readily in vitro when exposed to interstitial fluid. Phosphorylated antigens (alpha casein, hepatitis B surface antigen and phosphorylated bovine serum albumin) that adsorb to aluminum-containing adjuvants principally by ligand exchange exhibit little if any elution during 12-24 h in vitro exposure to interstitial fluid. Dephosphorylated alpha casein, which contains less than two phosphate groups, was less strongly adsorbed by ligand exchange in comparison to alpha casein, which contains eight phosphate groups. Dephosphorylated alpha casein was completely eluted when exposed to interstitial fluid. The results of this study lead to the generalization that antigens that adsorb to aluminum-containing adjuvants by electrostatic attraction are more likely to elute upon intramuscular or subcutaneous administration than antigens that adsorb by ligand exchange.

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.

Effect of phosphorylation of ovalbumin on adsorption by aluminum-containing adjuvants and elution upon exposure to interstitial fluid.

The phosphate content of commercial ovalbumin was increased from 1.8 to 3.2 mol PO(4)/mol ovalbumin by conjugation of phosphoserine and reduced to 1.2 or 0.14 mol PO(4)/mol ovalbumin by treatment with potato acid phosphatase. The four ovalbumin samples were completely adsorbed by aluminum hydroxide adjuvant due to electrostatic attraction of the negatively charged ovalbumin and the positively charged aluminum hydroxide adjuvant as well as by ligand exchange of phosphate groups with surface hydroxyl groups. Elution from aluminum hydroxide adjuvant upon exposure to interstitial fluid was inversely related to the degree of phosphorylation of the ovalbumin. The ovalbumin sample containing 3.2 mol PO(4)/mol ovalbumin did not elute while the ovalbumin sample containing 0.14 mol PO(4)/mol ovalbumin eluted completely from aluminum hydroxide adjuvant during exposure to interstitial fluid for 30 min. Adsorption of the four ovalbumin samples by aluminum phosphate adjuvant was directly related to the degree of phosphorylation of ovalbumin. Adsorption was due to ligand exchange as an electrostatic repulsive force operated between the negatively charged ovalbumin samples and the negatively charged aluminum phosphate adjuvant. The potential for ligand exchange decreased as the phosphorylation of ovalbumin decreased. Elution upon exposure to interstitial fluid was inversely related to the degree of phosphorylation and was more extensive than observed for aluminum hydroxide adjuvant. Adsorption of ovalbumin by aluminum-containing adjuvants and elution upon exposure to interstitial fluid can be controlled by the degree of phosphorylation of both ovalbumin and the aluminum-containing adjuvant.

Distribution of adsorbed antigen in mono-valent and combination vaccines.

The distribution of alpha-casein, bovine serum albumin (BSA), myoglobin and recombinant protective antigen (rPA) in mono-valent and combination vaccines containing aluminum hydroxide adjuvant was studied by fluorescence microscopy and flow cytometry. Green and red fluorescent probes were conjugated to the antigens. Adsorption isotherms of the fluorescently labeled proteins to aluminum hydroxide adjuvant demonstrated that incorporation of the fluorescent probe did not significantly affect the adsorption. In mono-valent vaccine systems, antigen adsorption occurred within one minute and uniform surface coverage of the adjuvant aggregates was observed within 1h. Content uniformity was achieved through a cycle of de-aggregation and re-aggregation of the aluminum hydroxide adjuvant aggregates caused by mixing. For combination vaccines, two antigens were adsorbed separately to the aluminum hydroxide adjuvant prior to combination. Following combination, cycles of de-aggregation and re-aggregation occurred due to mixing, which led to uniform distribution of both antigens. The results of this study indicate that content uniformity should not be an issue during the production of mono-valent or combination vaccines as long as adequate mixing procedures are followed.

Effect of thermal treatment during the preparation of aluminum hydroxide adjuvant on the protein adsorption capacity during aging.

Six aluminum hydroxide adjuvants, poorly crystalline aluminum oxyhydroxide (AlOOH) were prepared using different thermal treatments of amorphous aluminum hydroxide (Al(OH)3) in an effort to increase the protein adsorption capacity. All of the adjuvants initially exhibited a higher protein adsorption capacity. However, the protein adsorption capacity decreased during aging at room temperature. X-ray and differential centrifugal sedimentation analysis revealed that complete dehydration of amorphous aluminum hydroxide to aluminum oxyhydroxide is required to produce a stable adjuvant. Any residual amorphous aluminum hydroxide will spontaneously transform to crystalline aluminum hydroxide during aging at room temperature. Since crystalline aluminum hydroxide has a small surface area, the protein adsorption capacity of adjuvants containing amorphous aluminum hydroxide decreased by 30-40% when stored for 6 months at room temperature.