Abstract
BackgroundThe RV144 clinical trial showed for the first time that vaccination could provide modest but significant protection from HIV-1 infection. To understand the protective response, and to improve upon the vaccine's efficacy, it is important to define the structure of the immunogens used in the prime/boost regimen. Here we examined the heterogeneity in net charge, attributable to glycoform variation, of the gp120 immunogens contained in the AIDSVAX B/E vaccine.Methodology/Principal FindingsIsoelectric focusing and glycosidase digestion were used to assess variation in net charge of the gp120s contained in the AIDSVAX B/E vaccine used in the RV144 trial. We observed 16 variants of MN-rgp120 and 24 variants of A244-rgp120. Glycoform variation in gp120 produced in Chinese hamster ovary cells was compared to glycoform variation in gp120 produced in the 293F human embryonic kidney cell line, often used for neutralization assays. We found that gp120 variants produced in CHO cells were distinctly more acidic than gp120 variants produced in 293 cells. The effect of glycoform heterogeneity on antigenicity was assessed using monoclonal antibodies. The broadly neutralizing PG9 MAb bound to A244-rgp120, but not to MN-rgp120, whether produced in CHO or in 293. However, PG9 was able to bind with high affinity to MN-rgp120 and A244-rgp120 produced in 293 cells deficient in N-acetylglucosaminyltransferase I.Conclusions/SignificanceMN- and A244-rgp120 used in the RV144 trial exhibited extensive heterogeneity in net charge due to variation in sialic acid-containing glycoforms. These differences were cell line-dependent, affected the antigenicity of recombinant envelope proteins, and may affect assays used to measure neutralization. These studies, together with recent reports documenting broadly neutralizing antibodies directed against carbohydrate epitopes of gp120, suggest that glycoform variation is a key variable to be considered in the production and evaluation of subunit vaccines designed to prevent HIV infection.
Highlights
Interest in HIV-1 envelope-based subunit vaccines has been rekindled by the results from the recent RV144 phase 3 clinical trial, involving more than 16,000 Thai volunteers [1]
A prime-boost immunization regimen involving priming with a recombinant canarypox virus encoding the gp120, gag, and protease genes from HIV-1 [2] followed by booster immunizations with the bivalent gp120 subunit vaccine, AIDSVAX B/E [3,4], provided modest (31%) but statistically significant protection from HIV infection
We evaluated the binding of the monoclonal antibodies (MAbs) and CD4-IgG to A244-rgp120 produced in Chinese hamster ovary (CHO) and 293 cells
Summary
Interest in HIV-1 envelope-based subunit vaccines has been rekindled by the results from the recent RV144 phase 3 clinical trial, involving more than 16,000 Thai volunteers [1]. This study was notable since it was the first demonstration that vaccination can protect humans from HIV-1 infection Based on these results, further studies with new vaccine antigens are planned to verify the results, optimize the immunization regimen, and collect additional information regarding correlates of protection [5]. Further studies with new vaccine antigens are planned to verify the results, optimize the immunization regimen, and collect additional information regarding correlates of protection [5] In this regard, it is important to characterize the vaccine immunogens that elicited the protective response in the RV144 trial and to know the extent to which newly-manufactured gp120 subunit vaccines replicate their structure. We examined the heterogeneity in net charge, attributable to glycoform variation, of the gp120 immunogens contained in the AIDSVAX B/E vaccine
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