Background
Certain Env features, such as low cell surface expression and conformational lability, have hampered mRNA vaccine development for HIV. We previously found that ablation of Env endocytosis motifs and incorporation of a truncated HIV/SIV chimeric cytoplasmic tail (SIV.CT) increased surface expression on virions and cells transduced with vaccinia-vectored immunogens. Here, we incorporated this SIV.CT modification into mRNA immunogens and measured its impact on cell surface expression and immunogenicity.
Methods
mRNAs encoding prefusion-stabilized versions of Subtype A, AE, C, and D HIV-1 Envs, including germline targeting Envs RC1 and 11MUTB, were produced. Cell surface protein expression from mRNA was assessed following transfection of 293F cells and staining with conformationally-dependent antibodies (e.g., PGDM1400, 17b) using flow cytometry. To test immunogenicity, BALB/c mice were immunized intramuscularly at weeks 0 and 4 with 1 ug of mRNA-lipid nanoparticles encoding SIV.CT or gp160 variants of RC1 or 11MUTB. Serum drawn at weeks 2, 3, and 6 was evaluated using autologous antigen-specific ELISAs and neutralization assays.
Results
Properly folded, prefusion RC1 and 11MUTB trimers were expressed on the surface of mRNA-transfected cells as demonstrated by high PGDM1400 (V2-apex) signal and low 1393A (V2i) and 17b (CD4i) signals. Notably, DH270.6 (V3) and PGDM1400 signals were ~1 log higher for Envs containing the SIV.CT modification as compared to gp160 Envs. Application of our stabilization + SIV.CT approach to 8 additional HIV-1 strains yielded similarly high cell surface expression of properly folded trimers. Sera from RC1- and 11MUTB-vaccinated mice drawn 2 weeks post-prime demonstrated autologous binding antibodies in all animals, with SIV.CT-immunized animals showing consistently higher titers than gp160-immunized ones (RC1 SIV.CT- vs. gp160-primed geomean AUC: 59.2 vs 33.4; 11MUTB-primed: 67.7 vs 23.9). Further, SIV.CT variants elicited 10-28-fold greater neutralizing titers 3 weeks post-prime (geomean reciprocal ID50 RC1-primed animals: 2,155 vs. 219; 11MUTB-primed: 7,246 vs. 251) that remained 4.2-11.7-fold higher following homologous boost at week 6.
Conclusions
Engineering of the mRNA-encoded Env CT increases cell surface expression and, in combination with trimer stabilization, confers enhanced immunogenicity in mice. This strategy may be applied to generate mRNA vaccines from genetically diverse Env strains for preventative and therapeutic vaccine applications.
Table or Figure
