Considerable Differences in Vaccine Immunogenicities and Efficacies Related to the Diluent Used for Aluminum Hydroxide Adjuvant

Abstract

We are developing an anticandidal vaccine using the recombinant N terminus of Als3p (rAls3p-N). We report that although more rAls3p-N was bound by aluminum hydroxide diluted in saline than by aluminum hydroxide diluted in phosphate-buffered saline (PBS), its immunogenicity and efficacy were superior in PBS. Thus, protein binding, by itself, may not predict the efficacy of some vaccines with aluminum adjuvants.Hundreds of millions of doses of aluminum adjuvants have been administered to humans over the past century (5,11,12). One standard formulation is Alhydrogel, a gelatinous matrix of aluminum hydroxide, which minimizes lot-to-lot variability and enables facile adsorption of proteins (5, 12). As noted by Hem et al., aluminum hydroxide in its dehydrogenated, crystalline form is chemically aluminum oxyhydroxide [AlO(OH)] (7), but in its aqueous phase, it acquires an additional water molecule to become aluminum trihydroxide [Al(OH) 3 ].We have developed a vaccine using the recombinant N terminus of the candidal adhesin Als3p (rAls3p-N), which has been shown to protect mice against otherwise-lethal disseminated candidiasis (8,9,15,16). The vaccine is also effective in combination with Alhydrogel diluted in phosphate-buffered saline (PBS) (8). Vaccine efficacy requires an adjuvant, as the vaccine does not significantly improve survival if no adjuvant is utilized. To determine the optimal diluent for the continued development of the rAls3p-N vaccine, we assessed its relative binding to Al(OH) 3 , its immunogenicity, and its efficacy in the presence of PBS versus in the presence of saline. rAls3p-N (amino acids 17 to 432 of Als3p) was produced in Saccharomyces cerevisiae and purified by Ni-nitrilotriacetic acid matrix affinity purification as previously described (15). The isoelectric point of several batches of rAls3p-N was determined to be 7.1 using a Ready Gel IEF pH 5-to-8 gel (Bio-Rad) per the manufacturer's instructions (data not shown).To quantify protein binding to Al(OH) 3 , rAls3p-N was diluted to 0.2 mg/ml in saline (pH 6.5, 0.9% sodium chloride) or PBS (pH 7.40, 0.9% sodium chloride, 0.1% sodium-potassium phosphate), with or without 0.13 mg/ml of Alhydrogel (Brenntag Biosector, Frederikssund, Denmark). The solutions were vortexed, incubated for 30 min at room temperature, and centrifuged at 13,000 rpm for 10 min on a tabletop microcentrifuge. The protein concentration in the supernatant (i.e., unbound) was calculated by UV absorption as follows: (concentration without Alhydrogel -concentration with Alhydrogel)/(concentration without Alhydrogel). Protein binding was found to be slightly but significantly higher in the presence of saline than in the presence of PBS (median

Immunological Reactivity of Blood from Healthy Humans to the rAls3p‐N Vaccine Protein

“…Indeed, we found high IgG antibody titers and cellular production of both IFN- γ and IL-17 in response to rAls3p-N in healthy donors, confirming that humans have preexisting memory lymphocytes specific for the protein. The magnitude of cytokine responses detected were similar to those found to be produced by immune lymphocytes from vaccinated mice that were protected against infection [3, 7]. …”

Increasing the Potency of an Alhydrogel-Formulated Anthrax Vaccine by Minimizing Antigen-Adjuvant Interactions

“…Consequently, the ability of phosphate to modulate the adsorption coefficient of an acidic protein antigen and aluminum oxyhydroxide adjuvants might appear to be irrelevant. However, enhanced immunogenicity was seen when a recombinant Candida antigen (rAls3p-N) was diluted with phosphate-buffered saline versus saline alone (44). Similarly, phosphate modulation of aluminum oxyhydroxide in both hepatitis B surface antigen and HIV-1 (SF162dV2gp140) subunit vaccines demonstrated enhancement of immunogenicity (8,9).…”

Immunological surrogate marker of rAls3p-N vaccine-induced protection againstStaphylococcus aureus: Table 1

“…We analyzed IgG 2a titers as a subset of IgG titers because interferon‐γ (IFN‐γ) induces class switching to IgG 2a (Snapper et al , 1988; Stevens et al , 1988), and hence IgG 2a antibodies are produced in the setting of type 1 immunity (Spellberg & Edwards, 2001). Although Al(OH) 3 is not typically considered a type 1‐polarizing adjuvant, we have previously reported that when administered with rAls3p‐N, Al(OH) 3 adjuvant resulted in strong priming of type 1 inflammatory cytokines in reactive murine splenocytes (Lin et al , 2008). Furthermore, a recent study reported that a major mechanism by which aluminum adjuvants enhance immune responses was by direct agonism of an intracellular pattern recognition receptor, Nalp3 (Eisenbarth et al , 2008).…”