Structure-Based Design of Head-Only Fusion Glycoprotein Immunogens for Respiratory Syncytial Virus

Boyington, Jeffrey C.; Joyce, M. Gordon; Sastry, Mallika; Stewart-Jones, Guillaume B. E.; Chen, Man; Kong, Wing-Pui; Ngwuta, Joan O.; Thomas, Paul V.; Tsybovsky, Yaroslav; Yang, Yongping; Zhang, Baoshan; Chen, Lei; Druz, Aliaksandr; Georgiev, Ivelin S.; Ko, Kiyoon; Zhou, Tongqing; Mascola, John R.; Graham, Barney S.; Kwong, Peter D.

Neutralization titers were measured by a fluorescence plate reader neutralization assay as described previously [15, 16] . Briefly, 2.4 x 10 4 HEp-2 cells were seeded in 384-well black optical bottom plates (Nunc 384-well plates, Thermo Fisher Scientific, MA). Sera samples were assayed in four-fold dilutions from 1:10 to 1:40960, mixed with an equal volume of recombinant mKate-RSV expressing prototypic F genes from subtype A (strain A2) and the Katushka fluorescent protein, incubated at 37°C for one hour. Next, 50 μl diluted sample and virus were added to HEp-2 cells in 384-well assay plate, and incubated at 37°C for 22-26 hours. After incubation, fluorescence intensity was measured in a fluorescence plate reader at an excitation of 588 nm and an emission of 635 nm (SpectraMax Paradigm, Molecular Devices, CA). The IC 50 for each sample was calculated by curve fitting and non-linear regression using GraphPad Prism (GraphPad Software Inc., CA). Sera neutralization titers elicited from the immunization prime followed by the first boost were measured once for each mouse. Neutralization titers elicited from the second boost were measured four times and averaged for each mouse, except for sera elicited from the final i-447 boost and from the 3×DS-Cav1 immunization, which were measured three times for each mouse before averaging.