equal sized groups, with varying dose and route of vaccine administration. This is the 88 immunogen being employed in the Moderna vaccine entering phase 3 clinical trials 89
(mRNA-1273) 18 . Immunizations were performed by the intradermal (ID) and 90 intramuscular (IM) routes with 10 and 30 µg doses. Preimmune and serum 4 weeks 91
after the second immunization were analyzed for neutralization of pseudoviruses with 92 the D614 and G614 sequences. Preimmune sera from all mice scored negative. At 4 93 weeks after the second immunization, a relative increase in NAb titer for G614 over 94 D614 was observed for each animal (Figures 1a and S1a, Table S1 ); analyses were 95 done both as a single group and separately by dose and route. Across all routes and 96 doses (N=40), the geometric mean for the G614:D614 ratio was 5.16-fold (p < 0.001) for 97 ID 50 and for 4.44 for ID 80 (p<0.001). Similar patterns were seen across the 10 µg and 30 98 µg doses and for both IM and ID routes (Table 1) . Table 1 shows the geometric mean 99
for the ratio of G614:D614 NAb titers across route and doses, which ranged from, 3.87 100 to 6.49 ID 50 and 4.29-4.57 ID 80 with p<0.001 for all comparisons. 108 the second immunization were tested for neutralization against pseudoviruses with the D614 109 All rights reserved. No reuse allowed without permission.
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Eleven rhesus macaques were immunized with the nucleoside-modified mRNA-125 LNP vaccine platform using 2 different immunogens by the IM route (50 µg). The first 126 encoded the Wuhan index strain sequence 1 cell surface Spike protein with a mutated 127 furin cleavage site (furin mut). The second encoded the RBD domain as a secreted 128 monomer. Sera obtained at baseline and 4 weeks after the second immunization were 129 assessed for neutralization of the D614 and G614 variants (Figure 1b and c, Figure S1b  130 and c, Table S1 ). Baseline (pre-immune) sera were all negative. Post-immunization 131 sera exhibiting greater neutralization of the G614 variant virus for both immunogens. For 132 the cell surface Spike immunogen with a mutated furin site (N=5), the geometric mean 133
for the G614: D614 ID 50 NAb titer ratio was 5.37-fold (p = 0.001) and for ID 80 was 2.89-134 fold (p < 0.001) (Figure 1b ). For the soluble RBD (N=6), the G614:D614 ID 50 NAb titer 135 ratio had a geometric mean of 6.45-fold (p < 0.001) and for ID 80 was 3.22-fold (p< 136 0.001) (Figure 1c ). The observation that the G variant was more sensitive to antibodies 137 induced by both a D614 containing cell surface Spike trimer and an RBD secreted 138 monomer suggests that the G614 mutation increases RBD mediated neutralization.
Preliminary results from a phase 1/2 clinical trial using the nucleoside-modified 141 mRNA-LNP vaccine platform that delivered a secreted RBD trimer was recently 142
published and demonstrated potent ELISA binding and neutralization in all subjects at 143 all tested doses, 10, 30, and 100 µg 19 . Virus neutralizing titers after the second 144 immunization of 10 and 30 μg were 1.8-fold and 2.8-fold greater, respectively, than a 145 convalescent serum panel from SARS-CoV-2 infected patients 19 , although the 146 convalescent patients had a wide range and were older than vaccinated subjects.
Sera obtained preimmunization and 7 days after the second immunization with 149 50 μg (N=3), 30 μg (N=1), and 10 μg (N=1) of human subjects from a phase 1 trial 150 performed in Germany using the same vaccine were analyzed for neutralization of the 151 D614 and G614 Spike variant pseudoviruses. Similar to the data in murine and NHP 152 All rights reserved. No reuse allowed without permission.
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The copyright holder for this preprint this version posted July 24, 2020. . immunizations, the G614 pseudovirus was more potently neutralized compared to the 153 D614 pseudovirus by the same serum samples (Figures 1d and S1d, Table S1 ). The 154
geometric mean of the G614:D614 ID 50 NAb titer ratio was 4.22-fold (p = 0.014) and for 155 ID 80 was 3.1-fold (p = 0.017). No neutralizing activity was detected in the corresponding 156 preimmune sera.
The observation that sera from macaques and humans immunized with RBD-159 only immunogens also enhanced neutralization of the G614 pseudovirus suggested that 160 the elicited response was RBD-directed, and the neutralization enhancement was due 161
to a structural change in the expressed Spike. To evaluate this possibility, we used 162 negative stain electron microscopy (EM) and single particle reconstruction coupled with 163 3D classification to determine the structure and variability of the two variants in furin- 179 All rights reserved. No reuse allowed without permission.
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The copyright holder for this preprint this version posted July 24, 2020. 
Soon after the G614 mutation in Spike appeared early in the pandemic it rapidly 185
replaced the D614 variant in many countries 12 . This mutation is associated with 186 increased infectivity 12,13 when tested in in vitro model systems. Over 100 vaccines using 187
various platforms and immunogens are being developed to combat COVID-19 and end 188 the devastating financial, societal, and health burdens. Currently, 23 vaccines are in 189 clinical testing, some of which will soon enter phase 3 trials. Most SARS-CoV-2 190 vaccines were originally designed using the D614 variant of the Spike protein, which 191
was present in the first sequence of SARS-CoV-2 from Wuhan 1 . The most critical 192 finding that will ease the concern for most current vaccines in clinical trials is the finding 193
that the SARS- from the S1 region of one protomer to the S2 region of an adjacent protomer. This 202 interaction would bracket the furin and S2 cleavage sites. Potentially, it could reduce 203
shedding of S1 from viral-membrane-bound S2 and the introduction of G614 would 204 increase S1 release. Our structural data demonstrates that, in the context of the 205 stabilized ectodomain, this mutation leads to an increase in the percent of 1 RBD region 206
per trimer being in the up position, which is necessary for binding to ACE2 and infection 207 of target cells (Figure 2) . A recent publication demonstrated a similar effect of the G614 208 mutation to increase the number of RBDs in the up position 21 . Using an alternative 209 structural analysis method, extensive microsecond timescale atomistic molecular 210 dynamics simulations, reveal that in the G-form, the inter-protomer interactions in the 211
Spike trimer become more symmetric compared to the D-form. This equalization of 212
inter-protomer energetics results in a higher population of one-up Spike conformations, 213
leading to increased encounter between RBD and ACE2 receptor and greater exposure 214 of RBD domain for neutralization (Gnana, personal communication to be replaced with 215 Biorxiv).
Potential drawbacks to our studies include: 1) while we studied 4 different 218
variations of the Spike immunogen, we only used a single type of vaccine, the 219 nucleoside-modified mRNA-LNP platform (reviewed in 15, 22 (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted July 24, 2020. studies were performed in the context of a furin cleavage-deficient Spike ectodomain. 232
While this soluble ectodomain has been shown to be a good mimic of the native Spike, 233 and the shift in the proportion of RBD "up" conformation between the D614 and G614 234
forms suggest an allosteric effect of the D614G mutation on the RBD conformations, the 235 structures of the native Spike may have some differences from what we observe in the 236
context of the ectodomain.
We demonstrate that vaccinated mice, NHPs, and humans using the nucleoside-239 modified mRNA-LNP vaccine platform encoding 4 different SARS-CoV-2 Spike 240
immunogens generate antibody responses that not only recognize the G614 mutation 241 that has taken over the pandemic in many countries and has demonstrated increased 242 infectivity 13 12 , they have stronger titers of neutralization to this virus variant. The 243 mechanism appears to be that the mutation increases the up formation of the RBD in 244
the Spike trimer, increasing the exposure of neutralization epitopes. Twenty-three 245
vaccines are currently in clinical trial testing. Most of the immunogens used were either 246 derived from the initial D614 virus or contain this mutation in the Spike. The observation 247
that the G614 variant that has replaced the original D614 mutation in the SARS-CoV-2 248
Spike throughout much of the world is not an escape mutation and, in fact, is better 249 neutralized by sera from mice, NHPs, and humans immunized with immunogens 250 containing or derived from the D614 viral Spike alleviates a major concern in the 251 development of an effective SARS CoV-2 vaccine.
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The copyright holder for this preprint this version posted July 24, 2020. Lipid nanoparticles (LNPs) used in this study contain ionizable lipids proprietary 280
to Acuitas /DSPC/Cholesterol/PEG-Lipid 26 . Encoding mRNA was encapsulated in LNPs 281 using a self-assembly process in which an aqueous solution of mRNA at 4.0 pH was 282 rapidly mixed with a solution containing the aforementioned lipids premixed at mol/ mol 283 percent ratio of 50:10:38.5:1.5 and dissolved in ethanol. mRNA was encapsulated into 284
LNPs at a nucleic acid to total lipid ratio of ~0.05 (wt/wt) and aliquoted at a nucleic acid 285 concentration of ~1 mg/ml. All LNPs were characterized post-production at Acuitas 286 pharmaceutical (Vancouver, BC, Canada) for their size, surface charge using a Malvern 287
Zetasizer (Zetasizer Nano DS, Malvern, UK), encapsulation efficiency, and shipped on 288 dry ice and stored at -80°C until use.
Administration of test articles (immunization) and blood collection 291 292
8-12 week old Balb/c mice were immunized with either 10 µg of mRNA-LNPs via 293 the intramuscular (IM) route of administration using a 29G X1/2'' Insulin syringe. Mice 294 received a booster injection on day 28 (4 weeks). Blood was collected at day 0, 28 and 295 56 through the retro-orbital route using non heparinized micro hematocrit capillary tubes 296
(ThermoFisher Scientific,Waltham, MA, USA). Serum was separated by centrifugation 297
(10 000 g, 5 min) using a non-refrigerated Eppendorf 5424 centrifuge (Eppendorf, 298
Enfield, CT, USA), heat-inactivated (56 o C) for 30 minutes, and stored at -20 o C until 299
analysis. 300 301
Administration of mRNA/LNPs to rhesus macaques 302 303
Fifty micrograms of either mRNA/LNPs encoding an unstabilized 304 transmembrane (TM) Spike protein or a monomeric soluble RBD were administered 305 intramuscularly in two sites in the left and right quadriceps on weeks 0, and 4. 306
Monkeys immunized 50 µ g IM; 500 µ L total (250 µ L in R + L quad). Immunizations at 307 wk 0, 4. Animals were anesthetized with ketamine prior to blood draws from the femoral 308
vein. Serum samples were analyzed in 5 macaques immunized with mRNA/LNPs 309 encoding unstabilized TM Spike and in 6 animals immunized with mRNA/LNPs 310 encoding soluble RBD. All studies were performed at Bioqual, Inc, Rockville, MD 311
following IACUC approval.
Clinical trial samples 314 315
Serum from subjects enrolled in a phase 1/2 clinical trial of a nucleoside-modified 316 mRNA-LNP vaccine encoding trimeric SARS-CoV-2 RBDs (BNT162b1) were obtained 317 (NCT04380701). Five subjects that received 2 immunizations at a 3-week interval with 318 10, 30, or 50 μg of mRNA were used. All subjects were considered a single group, as 319 similar serologic data was obtained for each dose and the comparison and calculation 320 of statistical significance was performed within each sample. Serum was obtained prior 321
to the first immunization and 7 days after the second immunization. immediately added to all wells (10,000 cells in 100 µL of growth medium per well). SARS-CoV-2 ectodomain constructs 3 were produced and purified as follows. 365
Genes encoding residues 1−1208 of the SARS-CoV-2 S (GenBank: MN908947) with a 366 "GSAS" substitution at the furin cleavage site (residues 682-685), with and without 367 proline substitutions of residue K986 and V987 (S-GSAS/PP or S-GSAS), a C-terminal 368 T4 fibritin trimerization motif, an HRV3C protease cleavage site, a TwinStrepTag and an 369 8XHisTag were synthesized and cloned into the mammalian expression vector pαH. 370
The S-GSAS template was used to include the D614G mutation (S-GSAS(D614G)).
Plasmids were transiently transfected into FreeStyle-293F cells using Turbo293 372 (SpeedBiosystems). Protein was purified on the sixth day post-transfection from filtered 373 supernatant using StrepTactin resin (IBA), followed by size-exclusion chromatography 374 (SEC) purification using a Superose 6 10/300 GL column (GE healthcare) with 375 equilibrated in 2mM Tris, pH 8.0, 200 mM NaCl, 0.02% sodium azide buffer.
Negative-stain electron microscopy 378 379
Samples of S-GSAS and S-GSAS (D614G) ectodomain constructs were diluted 380
to 100 µg/ml with room-temperature buffer containing 20 mM HEPES pH 7.4, 150 mM 381
NaCl, 5% glycerol and 7.5 mM glutaraldehyde, and incubated 5 min; then 382 glutaraldehyde was quenched for 5 min by addition of 1M Tris stock to a final 383 concentration of 75 mM. A 5-µl drop of sample was applied to a glow-discharged, 384
carbon-coated grid for 10-15 s, blotted, stained with 2% uranyl formate, blotted and air-385
dried. Images were obtained with a Philips EM420 electron microscope at 120 kV, 386
82,000× magnification, and a 4.02 Å pixel size. The RELION program 28 was used for 387 particle picking, 3D classification, and 3D refinements. samples assayed from mice, nonhuman primates and a phase 1/2 clinical trial. 404 All rights reserved. No reuse allowed without permission.
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The copyright holder for this preprint this version posted July 24, 2020. trimer. Orange, pseudovuirus bearing D614 Spike; Blue, pseudovirus bearing G614 414
Spike.
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The copyright holder for this preprint this version posted July 24, 2020. . https://doi.org/10.1101/2020.07.22.20159905 doi: medRxiv preprint