It is intuitively attractive that adsorption should have effects on protein structure. Although aluminum-containing adjuvants have been in use in vaccine formulations for nearly a century and it has been assumed during much of that period that adsorption of antigens to those adjuvants is important, investigations into the direct effects of antigen adsorption on antigen conformation and stability have only recently begun. The work of Hem and colleagues demonstrated that the environment of the protein adsorbed to a mineral salt adjuvant can be significantly different from the bulk environment in which the protein stability is normally studied.31
The pH difference, which could be several units, suggests that caution should be taken when applying solution studies at bulk pH to predict the stability or conformation of the adsorbed antigen. Finally, it is also valid to assume that there may be differences in polarity and charge density on the adjuvant surface, which have implications for protein binding.31,32
Recent studies report contradicting evidence regarding the extent of structural perturbation following adsorption. In one of the earliest such studies published, Tleugabulova et al used SDS-PAGE, denaturing size exclusion chromatography (SEC) and microscopy to investigate the structural perturbations of HBsAg following adsorption.33
Under reducing conditions it was observed that, following adsorption, only half of the HBsAg was recoverable. It was further reported that increasing the time that the antigen was exposed to the reducing conditions (SDS sample buffer with dithiothreitol and 2-mercaptoethanol at 100°C, boiling) only increased the amount of the degradation peaks in the antigen exposed to adjuvant. They stated that this may suggest that the “structure of VLPs may be compromised by the adsorption on adjuvant.”33
It is important to note however that both the chromatography and the SDS-PAGE were performed on antigen that had been intentionally reduced and denatured. Further evidence for structural modifications upon adsorption was detected via microscopy techniques.
Later works investigating the potential structural perturbation upon adsorption using more direct, spectroscopic techniques not requiring antigen reducing or boiling prior to analysis have mostly focused on model antigens. In a study of three model vaccines using lysozyme (adsorbed to aluminum phosphate), ovalbumin (adsorbed to aluminum hydroxide) and bovine serum albumin, BSA, (adsorbed to aluminum hydroxide) potential structural perturbations upon adsorption and as a consequence of thermal treatment were assessed via fluorescence spectroscopy, attenuated total reflectance (ATR) FTIR spectroscopy, and differential scanning calorimetry.34
At relatively low temperatures (10–25°C), the spectral techniques suggested structural changes when the proteins were adsorbed. In another study, Zheng et al used ATR-FTIR to compare the secondary structure of soluble and aluminum hydroxide adjuvant adsorbed BSA and β-lactoglobulin in dried films and also concluded that the structures of the model antigens are altered following adsorption.35
Moreover, Zheng et al, found that the structural perturbations were dependent on the amount of protein adsorbed, with lower amounts of adsorbed protein yielding greater spectral alterations following adsorption.
Not all studies investigating the effect of adsorption on antigen structure detected changes. Dong et al did not detect adsorption-induced changes in the secondary structure of six model protein antigens, including ovalbumin.36
Unlike the other studies to detect secondary structural changes, Dong et al used transmission FTIR, which avoids potential artifacts associated with ATR-FTIR. The differences in the findings with ovalbumin might also be attributable to the differences in the buffer, Dong et al used phosphate buffers, which as stated above is known to attenuate the binding interaction between ovalbumin and aluminum hydroxide adjuvant. Agopian et al used ATR-FTIR and concluded that the secondary structure of their antigen of interest, gp41, was not altered following adsorption onto aluminum hydroxide adjuvant.37
They measured the spectra in 5 mM sodium formate buffer pH 5.2 or acetonitrile /water/detergent mixtures, and found α-helix and β-sheet contents similar, though not identical, to that of the soluble protein in 50 mM sodium formate buffer or ACN/water/detergent mixtures. They stated that 50 mM sodium formate or acetonitrile/water/no detergent prevent adsorption. Moreover, adjuvant dissolution was observed in 50 mM sodium formate buffer. This would suggest that the antigen is not tightly bound, but the strength of the binding interaction is not discussed. Nevertheless, both studies indicate that the secondary structures of antigens are not significantly altered under conditions that do not maximize the binding interaction. Collectively, studies investigating adsorption-induced structural perturbations of antigens suggest that all factors affecting antigen adsorption – including the antigen, buffer, and physiochemical properties of the aluminum-containing adjuvant – will dictate to what extent the antigen is perturbed. Antigen structures must be monitored in the formulation of interest, as extrapolations based on other formulations may prove unreliable.
Potential effects of adsorption and structural perturbations on the inherent thermal stability of antigens in the presence of adjuvant have been investigated as well. In the Jones et al. study using lysozyme, ovalbumin and BSA, marked changes in thermal behavior, including a decreased unfolding temperature and alterations of the number of thermally-induced transitions, were observed when these model antigens were bound to adjuvants.34
Similar studies by others revealed that several potential vaccine antigens, including malaria,38
and botulinum toxin39
experience similar thermal destabilization when adsorbed to adjuvants when monitored by DSC, fluorescence, and second derivative UV spectroscopy. These studies suggest that it is inappropriate to use solution state stability data alone to predict the stability of the antigen in the presence of an aluminum-containing adjuvant. Instead, ensuring optimization of the vaccine requires that the antigen stability be studied in the presence of the adjuvant.
The thermal stability of cytochrome c, α-chymotrypsinogen A and ovalbumin as a function of adsorption onto aluminum hydroxide was determined by Bai and Dong using transmission FTIR spectroscopy.40
They determined that the changes in the secondary structures of the proteins as a function of temperature were, at most, marginally affected by adsorption. Although they observed little to no differences in the relative intensity of the predominant second derivative peaks for the proteins in the adsorbed and solution states, they did determine that the strength of intermolecular β-sheet aggregations formed as a function of heating the samples was weaker in the adsorbed samples than the solution state proteins. Finally, ovalbumin exhibited the biggest spectral differences as a function of adsorption when heated, with the adsorbed sample having a band for random coil that was absent in the solution state. It was stated that this band represented an incomplete transition of the adsorbed protein from an unfolded state to aggregate.
In contradiction to the work by Jones et al, Zheng et al. suggest that the secondary structures of BSA and BLG in the adsorbed state had increased thermal stability in comparison to the solution state proteins.41
ATR-FTIR data monitoring the amide I and amide II regions of the antigens showed that BSA in solution lost α-helix structure and gained intermolecular β-sheet structure at high temperature, but no significant spectral changes were observed for BSA adsorbed to aluminum. Similar results were obtained for BLG although it was stated that “the thermal effect on stability of the adsorbed BSA and BLG is not the same.”41
Although the authors conclude that adsorption stabilizes the structures of the proteins, it is important to keep in mind that the spectra of each protein in the adsorbed and solution states prior to heating were not alike. Thus, the “stabilized state” of the adjuvant formulation was already perturbed from that of the solution state. It was not the conformational state of the protein observed in the adjuvant-free formulation. Zheng et al. suggested that there were multiple reasons for the differences of this work in comparison to Jones et al including measurement temperature (Jones et al measured samples at elevated temperatures and Zheng et al measured previously heated samples at room temperature). They also point out differences in sample handling: Jones et al analyzed a slurry following centrifugation while Zheng et al. analyzed evaporated films. Therefore, although it would be advantageous to compare these studies it may be impossible.34,35,41
While the immediate effects of binding to adjuvant are somewhat ambiguous or small, longer-term studies indicate greater effects of structural perturbation. Studies on a potential trivalent botulinum toxin vaccine showed greater changes in pH, DSC profile, fluorescence maximum, and 2D-UV peak position for antigen bound to adjuvant over 9 weeks, at 4°C and 37°C, than controls stored in similar conditions without adjuvant.39
In addition, adsorbed antigens also showed a sharply decreased ability to desorb from the adjuvant. Similarly treated botulinum toxin vaccines were also examined by MALDI-TOF mass spectrometry after protease digestion in an attempt to localize sites of chemically modified residues (through oxidation or deamidation) for each of the proteins involved.32
It appears that binding to adjuvants causes chemical modifications to appear sooner, within two weeks of storage rather than 9 weeks, though in some cases it also decreases the number of chemical modifications present.32
These differences could indicate that the minor structural perturbations detected in most studies exposed different sections of the protein to conditions under which they are reactive, including the increased microenvironment pH surrounding adjuvants.31
There is some evidence that addition of excipients can improve stability of adjuvant-bound proteins, as will be discussed later. A significant consequence of chemical and physical alterations is the inability to adequately desorb proteins from the adjuvant, which as alluded to early in this review may negatively impact the immunogenicity of the vaccine:17,25,29
although it must be noted that this has not been investigated.32,39