Alhydrogel® (aluminum hydroxide) is a widely used adjuvant in the US. Regulatory authorities require that vaccines be tested to determine the antigen content in the final vaccine product. The level of formulated antigen is currently determined in our laboratory by the o-Phthalaldehyde (OPA) fluorescent protein assay, and antigen identity and integrity are determined by Western blot and SDS-PAGE. However, OPA assay is non-specific and only limited to detection of total protein content, and it is often not sensitive enough to detect antigens in low dose formulations. Furthermore, antigens used in identity and integrity tests must be extracted from vaccines using an extraction procedure which is time-consuming and may not completely recover antigens for analysis or may alter the structures of antigens during extraction. The present study developed a Direct Alum Formulation Immunoassay (DAFIA) which was designed to directly (without antigen extraction), accurately, and sensitively determine the antigen content, identity and integrity on alum. The AMA1-C1/Alhydrogel formulation was used as a model vaccine in assay development and validation. The results showed that the DAFIA is highly antigen-specific, accurate (87-100%), sensitive (0.16 µg/ml), reproducible, and simple with a linear detection range of 0.16-10 µg/ml. These results demonstrate that DAFIA is an excellent assay to determine antigen content, identity and integrity of antigens bound to alum and may be used in routine vaccine quality control for testing antigens in Alhydrogel-based vaccines.
Efficient antigen extraction from vaccines formulated on aluminum hydroxide gels is a critical step for the evaluation of the quality of vaccines following formulation. It has been shown in our laboratory that the efficiency of antigen extraction from vaccines formulated on Alhydrogel decreased significantly with increased storage time. To increase antigen extraction efficiency, the present study determined the effect of surfactants on antigen recovery from vaccine formulations. The Plasmodium falciparum apical membrane antigen 1 (AMA1) formulated on Alhydrogel and stored at 2-8 °C for three years was used as a model in this study. The AMA1 on Alhydrogel was extracted in the presence or absence of 30 mM sodium dodecyl sulfate (SDS) or 20 mM cetylpyridinium chloride in the extraction buffer (0.60 M citrate, 0.55 M phosphate, pH 8.5) using our standard antigen extraction protocols. Extracted AMA1 antigen was analyzed by 4-20% Trisglycine SDS-PAGE followed by silver staining or western blotting. The results showed that inclusion of SDS or cetylpyridinium chloride in extraction buffer increased the antigen recovery dramatically and can be used for efficient characterization of Alhydrogel vaccines.
Pre-erythrocytic vaccines prevent malaria by targeting parasites in the clinically silent sporozoite and liver stages and preventing progression to the virulent blood stages. The leading pre-erythrocytic vaccine RTS,S/AS01E (Mosquirix®) entered implementation programs in 2019 and targets the major sporozoite surface antigen called circumsporozoite protein or CSP. However, in phase III clinical trials, RTS,S conferred partial protection with limited durability, indicating a need to improve CSP-based vaccination. Previously, we identified highly expressed liver stage proteins that could potentially be used in combination with CSP and are referred to as pre-erythrocytic vaccine antigens (PEVA). Here, we developed heterologous prime-boost CSP vaccination models to confer partial sterilizing immunity against Plasmodium yoelii (Py)(protein prime/adenovirus 5 (Ad5) boost) and P. berghei (Pb) (DNA prime/Ad5 boost) in mice. When combined as individual antigens with PyCSP, 3 of 8 PyPEVA significantly enhanced sterile protection against sporozoite challenge, compared to PyCSP alone. Similar results were obtained when 3 PbPEVA and PbCSP were combined in a single vaccine regimen. In general, PyCSP antibody responses were similar after CSP alone versus CSP+PEVA vaccinations. Both Py and Pb CSP+PEVA combination vaccines induced robust CD8 + T cell responses including signature IFN-γ increases. In the Pb model system, IFN-γ responses were significantly higher in hepatic than splenic CD8 + T cells. The addition of novel antigens may enhance the degree and duration of sterile protective immunity conferred by a human vaccine such as RTS,S.
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