key: cord-0737956-ya91dloq
authors: Ullah, Irfan; Prévost, Jérémie; Ladinsky, Mark S; Stone, Helen; Lu, Maolin; Anand, Sai Priya; Beaudoin-Bussières, Guillaume; Benlarbi, Mehdi; Ding, Shilei; Gasser, Romain; Fink, Corby; Chen, Yaozong; Tauzin, Alexandra; Goyette, Guillaume; Bourassa, Catherine; Medjahed, Halima; Mack, Matthias; Chung, Kunho; Wilen, Craig B; Dekaban, Gregory A.; Dikeakos, Jimmy D.; Bruce, Emily A.; Kaufmann, Daniel E; Stamatatos, Leonidas; McGuire, Andrew T.; Richard, Jonathan; Pazgier, Marzena; Bjorkman, Pamela J.; Mothes, Walther; Finzi, Andrés; Kumar, Priti; Uchil, Pradeep D.
title: Live imaging of SARS-CoV-2 infection in mice reveals neutralizing antibodies require Fc function for optimal efficacy
date: 2021-03-22
journal: bioRxiv
DOI: 10.1101/2021.03.22.436337
sha: 14087a1bc9f9225cee1905543d5df7edad9eb82c
doc_id: 737956
cord_uid: ya91dloq

Neutralizing antibodies (NAbs) are effective in treating COVID-19 but the mechanism of immune protection is not fully understood. Here, we applied live bioluminescence imaging (BLI) to monitor the real-time effects of NAb treatment in prophylaxis and therapy of K18-hACE2 mice intranasally infected with SARS-CoV-2-nanoluciferase. We visualized sequential spread of virus from the nasal cavity to the lungs followed by systemic spread to various organs including the brain, culminating in death. Highly potent NAbs from a COVID-19 convalescent subject prevented, and also effectively resolved, established infection when administered within three days of infection. In addition to direct neutralization, in vivo efficacy required Fc effector functions of NAbs, with contributions from monocytes, neutrophils and natural killer cells, to dampen inflammatory responses and limit immunopathology. Thus, our study highlights the requirement of both Fab and Fc effector functions for an optimal in vivo efficacy afforded by NAbs against SARS-CoV-2.

when systemic spread occurred. The first signs of infection in the lungs were observed at 1 dpi. 137

The nLuc signal then steadily increased in the lungs until 3 dpi and plateaued thereafter. We 138 detected nLuc signals in the cLNs and brain region (imaging in ventral position) at 4 dpi. There 139 was a steep rise in nLuc activity in the brain from 4 to 6 dpi indicating neuroinvasion and robust 140 virus replication ( Figure 1B , C, Video S1). This was accompanied by widespread replication of 141 the virus in the gut and genital tract with concomitant loss in body weight. By 6 dpi, the infected 142 K18-hACE2 mice lost 20% of their initial body weight, became moribund and succumbed to the 143 infection ( Figure 1D, E) . In contrast, as expected, B6 mice did not experience any weight loss 144 and survived the virus challenge. 145

To visualize the extent of viral spread with enhanced sensitivity and resolution, we imaged 146 individual organs after necropsy ( Figure 1B, F) . nLuc signal was absent in B6 mice while most 147 organs analyzed from K18-hACE2 mice showed nLuc activity with maximum signal detected in 148 the brain followed by the lung and nasal cavity ( Figure 1F ). These observations mirrored viral 149 loads [Focus Forming Units (FFUs) and nLuc activity] in the brain, lung and nasal cavity ( Figure  150 1G, H). Real-time PCR analyses to detect N gene mRNA as well as histological analyses of 151 organs confirmed widespread infection ( Figure S1A, B) . 152

Reporter-expressing viruses often purge foreign genes, particularly in vivo, due to fitness 153 and immune pressure (Falzarano et al., 2014; Ventura et al., 2019) . To estimate the stability of 154 nLuc reporter, we compared the copy numbers of SARS-CoV-2 nucleocapsid (N) to nLuc in the 155 viral RNA by real-time PCR analyses of input virions and virions isolated from sera of mice at 6 156 dpi. The ratio of copy numbers between the two samples sets did not change significantly ( Figure  157 1I) indicating that the reporter was stable throughout the experimental timeline. Thus, nLuc activity 158 was an excellent surrogate to follow virus replication in vivo. 159 SARS-CoV-2 infection triggers an imbalanced immune response and a cytokine storm 160 that contributes significantly to pathogenesis (Del Valle et al., 2020) . We compared the mRNA 161 levels of inflammatory cytokines IL6, CCL2, CXCL10 and IFNγ in the lungs and brains of mice 162 after necropsy at 6 dpi. Indeed, most cytokines mRNAs were significantly upregulated in both 163 organs of infected K18-hACE2 mice compared to B6 ( Figure 1J, K) . Overall, cytokine mRNAs 164 were higher in the brain compared to lungs with CXCL10 mRNA copy numbers reaching ~1000 165 fold higher in K18-hACE2 than in B6 mice corroborating extensive infection ( Figure 1J, K) . 166 We next used BLI to illuminate areas of infected regions within lungs, brain, and testis for 167 We recently reported and characterized plasma from a COVID-19 convalescent subject 178 (S006) with potent neutralizing activity and high levels of SARS-CoV-1 cross-reactive Abs (Lu et 179 al., 2020) . We probed the B cell receptor (BCR) repertoire from this donor to isolate broad and 180 potent NAbs. Using a recombinant SARS-CoV-2 S ectodomain (S2P) as a bait to identify antigen-181 specific B cells, we collected and screened a library of S-targeted BCR clones and identified two 182 most potent NAb candidates: CV3-1 and CV3-25. We first characterized their epitope specificity 183 using ELISA, cell-surface staining, virus capture assay and surface plasmon resonance (SPR) 184 (Ding et al., 2020; Prevost et al., 2020) . Both NAbs recognized SARS-CoV-2 S efficiently with a 185 low-nanomolar affinity, as a stabilized ectodomain (S-6P) or when displayed on cells and virions 186 ( Figure 3A-E) . While CV3-1 bound the SARS-CoV-2 RBD, CV3-25 targeted the S2 subunit 187

CV3-1 NAb alone completely protected K18-hACE2 mice against SARS-CoV-2-induced mortality. 240

We therefore explored if CV3-1 could also cure infected mice. Mice infected with SARS-CoV-2-241 nLuc were administered CV3-1 at 1, 3, and 4 dpi after confirming SARS-CoV-2 infection was 242 established in the lungs of all mice ( Figure 5A ). Temporal imaging and quantification of nLuc 243 signal revealed that CV3-1, when administered at 1 and 3 dpi, controlled virus spread and 244 successfully prevented neuroinvasion (Figure 5B-D, G, H) . This was corroborated by no weight 245 loss and/or recuperation of body weight, undetectable viral loads as well as near-baseline levels 246 of inflammatory cytokines in tissues ( Figure 5E -K). CV3-1 therapy at 4 dpi, however, could neither 247 control virus spread nor neuroinvasion resulting in death of 75% of the mice in this cohort ( Figure  248 5B-F) with loss in body weight, high levels of inflammatory cytokines and tissue viral loads, similar 249 to that in the control cohort ( Figure 5E-K) . Thus, the therapeutic window of maximal efficacy for 250 CV3-1 treatment extends for up to 3 days from the initiation of SARS-CoV-2 infection to 251 successfully prevent lethality. 252

Highly potent antibodies can effectively neutralize free viruses and may also mediate Fc-255 recruitment of immune cells to eliminate infected cells. We therefore explored a role for Fc-256 mediated effector functions in protection in vivo. We generated Leucine to Alanine (L234A/L235A, 257 LALA) mutant versions of both NAbs to impair interaction with Fc receptors (Saunders, 2019) . 258

Our in vitro assays confirmed that, while ADCC and ADCP activities were compromised, LALA 259 mutations had no impact on S binding and neutralizing capacities of both NAbs ( Figure S5) . 260

Biodistribution analyses of AF647-conjugated CV3-1 and CV3-25 LALA NAbs, 24h after i.p. 261 administration indicated penetration into most tissues ( Figure S5 ). 262

We next tested the impact of LALA mutations on the prophylactic efficacy of CV3-1 and 263 CV3-25 ( Figure S6A ). Longitudinal non-invasive BLI and terminal imaging analyses after 264 necropsy, body weight changes, survival and viral load estimations revealed that LALA mutations 265 had indeed compromised the protective efficacy of both antibodies ( Figure S6A-I) . SARS-CoV-2 266 replicated better, invaded the brain and induce body weight loss in cohorts treated with LALA 267

NAbs compared to the corresponding wild-type NAbs ( Figure S6D-E) . Histology at 6 dpi revealed 268 that both LALA NAbs had penetrated the brain tissue during the course of infection and bound 269 the surface of infected neurons ( Figure S5G, H) . While CV3-1-treated animals had no detectable 270 viral loads at 6 dpi, CV3-1 LALA pretreated mice had higher tissue viral loads indicating 271 compromised protective efficacy ( Figure S6I) . Similarly, while tissue viral loads in CV3-25 treated 272 mice were reduced by a log, those in CV3-25 LALA treated mice were comparable to that in 273 control cohorts. Moreover, the delayed mortality and 25% protective efficacy offered by CV3-25 274 was abrogated and the ability of CV3-1 to provide 100% protection from SARS-CoV-2-induced 275 mortality was reduced to 62.5% with the corresponding LALA mutants ( Figure S6F ). Additionally, 276

there was an overall increase in the signature inflammatory cytokine profile in mice pre-treated 277 with LALA NAbs (Figure S6J, K) . 278

The requirement for Fc effector function during CV3-1 prophylaxis was surprising as we 279 did not detected infection in CV3-1 treated mice both by non-invasive and post-necropsy tissue 280 imaging at 6 dpi ( Figure 4) . However, examination of tissues at 3 dpi did reveal weak nLuc signals 281 in the nasal cavity and lungs despite absence of signal by non-invasive imaging ( Figure S5J-M) . 282 PCR analyses also confirmed the presence SARS-CoV-2 N RNA in these tissues at 3 dpi ( Figure  283   S5N ). The data indicated that some of the incoming virions did not encounter CV3-1 and managed 284 to establish infection during prophylaxis and hence Fc effector functions were required to 285 eliminate them. 286

Our data thus implied that immune cell components would be critical during CV3-1 therapy 287 ( Figure 6A) . Indeed, while CV3-1 treatment at 3 dpi controlled infection, cohorts treated with CV3-288 1 LALA displayed rapidly spreading lung infection and fully succumbed by 6 dpi after an 289 accelerated loss in body weight ( Figure 6B-F) . High viral loads and cytokine levels in nose, lung, 290 and brain also reflected the failure of the LALA NAbs to treat pre-established viral infection ( Figure  291 6G). Notably, while the lung viral loads in CV3-1 LALA NAb-treated cohort were similar to that in 292 the control, inflammatory cytokine mRNA levels in lungs, CXCL10 in particular, were significantly 293 higher suggesting a crucial requirement for Fc-engagement in curbing a cytokine-storm like 294 phenotype ( Figure 6H-I) . 295 296

To identify the immune cell types engaged by NAbs, we initiated NK cell depletion (α-NK1.1) prior 298 to CV3-1 prophylaxis. Flow cytometric analyses confirmed NK cell depletion in α-NK1.1 treated 299 cohorts. (Figure S7J , K). Our BLI-guided analyses showed appearance of weak nLuc signals in 300 lungs of infected mice prophylactically treated with CV3-1 as well as αNK1.1 mAbs compared to 301 those in the control cohort treated with CV3-1 and with an isotype control ( Figure S7A-D) . In 302 addition, two of the mice that underwent NK cell depletion in the CV3-1 pretreated group 303 experienced a temporary but significant decrease in body weight before recovering ( Figure S7E) . 304

Nevertheless, all the mice receiving CV3-1 prophylaxis survived despite NK cell depletion ( Figure  305 S7F). We did observe a marginal increase in viral loads in target organs upon NK depletion in 306 mice under CV3-1 prophylaxis ( Figure S7G ). In addition, the ability of CV3-1 to suppress 307 inflammatory cytokines was significantly compromised upon NK cell-depletion ( Figure S7H , I-K). 308

Thus, while NK cells do contribute to in vivo efficacy of CV3-1, their requirement was not 309 significant enough to compromise protection offered by CV3-1 prophylaxis. 310

To investigate NK cell-requirement during CV3-1 therapy, we depleted them in SARS-311

CoV-2-nLuc infected mice where CV3-1 treatment was initiated at 3 dpi ( Figure 7A ). Our BLI-312 centric multiparametric analyses revealed that NK cell depletion partially compromised the 313 efficacy of CV3-1 therapy with 25% of the mice succumbing to SARS-CoV-2 infection compared 314

to CV3-1-treated controls where all the mice survived ( Figure 7B-F) . We next investigated if 315 Ly6G + neutrophils and Ly6C hi CD11b + classical monocytes accounted for additional Fc effector 316 activities by using the anti-Ly6G and anti-CCR2 depleting mAbs in mice under CV3-1 therapy 317 ( Figure 7A , B, S7L-O) (Mack et al., 2001) . Neutrophil or monocyte depletion under CV3-1 therapy 318 led to 75% and 80% of the mice failed to control SARS-CoV-2 spread and loss in body weight 319 resulting in death although neuroinvasion was overall weaker than isotype control cohorts ( Figure  320 Our data also establishes that neutralizing capacity alone is not enough to garner clinical 359 protection by NAbs. LALA variants of CV3-1 revealed a crucial role for Fc-mediated interactions 360 in augmenting in vivo protection for prophylaxis as well as therapy. The requirement for Fc effector 361 functions during CV3-1 prophylaxis, needed for eliminate infected cells originating from virions 362 that eluded neutralization, is noteworthy and contrasting to a recent report where they were 363 required only during NAb therapy (Winkler et al., 2021) . Introducing LALA mutation in CV3-1 364 completely compromised its ability to therapeutically cure and was in agreement with previous 365 observations (Winkler et al., 2021) . Surprisingly, we noticed a more severe loss in body weight in 366 mice that were therapeutically administered CV3-1 LALA variants and significantly higher 367 inflammatory responses (CCL2, CXCL10, IFNγ) in lungs than isotype-treated control animals. 368

These data suggest that the Fc region plays an additional protective role by limiting 369 immunopathology through dampening of inflammatory responses. A previously reported NAb 370 engaged only monocytes for in vivo activity (Winkler et al., 2021) . However, our studies revealed 371 that CV3-1 engaged Fc-interacting neutrophils, monocytes and NK cells for its in vivo efficacy. 372

Thus, in addition to its potent neutralizing activity, a superior engagement of innate immune 373 components contributed to the high in vivo potency of CV3-1 374 Experiments using low doses of NAbs indicated that CV3-1 did not enhance infection at 375

concentrations that protected only 50% of animals in the group. Thus, our data add to the growing 376 body of evidence that suggest the absence of an antibody-dependent enhancement (ADE) 377 mechanism with a protective rather than a pathogenic role for Fc effects during SARS-CoV (CD11b + Ly6G + ) and monocytes (CCR2 + Ly6 hi CD11b + ) in K18-hACE2 mice therapeutically treated 613 with CV3-1 NAb (i.p.,12.5 mg/kg body weight) at 3 dpi after challenge with SARS-CoV-2-nLuc. 614 αNK1.1 mAb (i.p., 20 mg/kg body weight), αLy6G mAb (i.p., 20 mg/kg body weight) and αCCR2 615 mAb (i.p., 2.5 mg/kg body weight) were used to deplete NK cells, neutrophils and monocytes 616 respectively every 48h starting at 1 dpi. Corresponding human (for CV3-1) and rat (for αNK1.1 617 and αLy6G mAb or αCCR2) monoclonal antibodies served as non-specific isotype controls (Iso). 618

The mice were followed by non-invasive BLI every 2 days from the start of infection. factor. Subsequently, these concentration values were multiplied with the homogenization volume 688 and divided by the total organ weight. 689 (E) Persistence and redistribution of neutralizing NAbs in SARS-CoV-2 infected mice. Images of 690 brain tissue from K18-hACE2 mice infected with SARS-CoV-2-nLuc at 6 dpi that were 691

prophylactically treated with CV3-1 or CV3-25 (12.5 mg/kg body weight), 24 h before infection. 692

Actin (green) was labelled using phalloidin, CV3-1 and CV3-25 (magenta) were detected using 693 anti-hIgG conjugated to Alexa Fluor 647 and infected cells (red) were identified using antibodies 694 to SARS-CoV-2 N. CV3-1 localizes to the endothelial walls of blood vessels and CV3-25 695 redistributes to decorate infected neurons in addition to endothelium (seen in UI mice; Figure S2 ). Leucine to Alanine (LALA) mutants (12.5 mg/kg body weight) delivered intraperitoneally (i.p.) 1 787 day before challenging K18-hACE2 mice with 1 x 10 5 FFU of SARS-CoV-2 nLuc. Human IgG1-788 treated (12.5 mg/kg body weight) mice were used as control (Iso). Mice were followed by non-789 invasive BLI every 2 days from the start of infection using IVIS Spectrum after retroorbital ), 293T-ACE2, CF2Th, TZM-bl and TZM-bl-ACE2 cells  919 were maintained at 37°C under 5% CO 2 in DMEM media, supplemented with 5% FBS and 100 920 All standard operating procedures and protocols for IVIS imaging of SARS-CoV-2 infected 983 animals under ABSL-3 conditions were approved by IACUC, IBSCYU and YARC. All the imaging 984 was carried out using IVIS Spectrum® (PerkinElmer) in XIC-3 animal isolation chamber 985 (PerkinElmer) that provided biological isolation of anesthetized mice or individual organs during 986 the imaging procedure. All mice were anesthetized via isoflurane inhalation (3 -5 % isoflurane, 987 oxygen flow rate of 1.5 L/min) prior and during BLI using the XGI-8 Gas Anesthesia System. Prior 988 to imaging, 100 µL of nanoluciferase substrate, furimazine (NanoGlo TM , Promega, Madison, WI) 989 diluted 1:40 in endotoxin-free PBS was retroorbitally administered to mice under anesthesia. The 990 mice were then placed into XIC-3 animal isolation chamber (PerkinElmer) pre-saturated with 991 isothesia and oxygen mix. The mice were imaged in both dorsal and ventral position at indicated 992 days post infection. The animals were then imaged again after euthanasia and necropsy by 993 spreading additional 200 µL of substrate on to exposed intact organs. Infected areas of interest 994 identified by carrying out whole-body imaging after necropsy were isolated, washed in PBS to 995 remove residual blood and placed onto a clear plastic plate. Additional droplets of furimazine in 996 PBS (1:40) were added to organs and soaked in substrate for 1-2 min before BLI. 997

Images were acquired and analyzed with the manufacturer's Living Image v4.7.3 in vivo 998 software package. Image acquisition exposures were set to auto, with imaging parameter 999 preferences set in order of exposure time, binning, and f/stop, respectively. Images were acquired 1000 with luminescent f/stop of 2, photographic f/stop of 8. Binning was set to medium. Comparative 1001 images were compiled and batch-processed using the image browser with collective luminescent 1002 scales. Photon flux was measured as luminescent radiance (p/sec/cm2/sr). During luminescent 1003 threshold selection for image display, luminescent signals were regarded as background when 1004 minimum threshold levels resulted in displayed radiance above non-tissue-containing or known 1005 uninfected regions. To determine the pattern of virus spread, the image sequences were acquired 1006 every day following administration of SARS-CoV-2 (i.n). Image sequences were assembled and 1007 converted to videos using Image J. 1008 1009

Mice were intraperitoneally (i.p) administered with 250 µg of unconjugated (12.5 mg/kg body 1011 weight), Alexa Fluor 647 or Alexa Fluor 594-labeled antibodies to non-infected or SARS-CoV-2 1012 infected hACE2 mice. 24 h later all organs (nose, trachea, lung, cervical lymph nodes, brain, liver, 1013 spleen, kidney, gut, testis and seminal vesicles) were isolated after necropsy and images were 1014 acquired with an IVIS Spectrum® (PerkinElmer) and fluorescence radiance intensities were 1015 analyzed with the manufacturer's Living Image v4.7.3 in vivo software package. Organs were cut 1016 into half and weighed. One half was fixed in 4 % PFA and processed for cryoimmunohistology. 1017

The other half was resuspended in serum-free RPMI and homogenized in a bead beater for 1018 determination of antibody levels using quantitative ELISA. 1019 1020

Recombinant SARS-CoV-2 RBD and S-6P proteins were used to quantify CV3-1 and CV3-25 1022 antibody levels, respectively, in mice organs. SARS-CoV-2 proteins (2.5 μg/ml), or bovine serum 1023 albumin (BSA) (2.5 μg/ml) as a negative control, were prepared in PBS and were adsorbed to pressure freezing machine (Leica Microsystems, Vienna Austria). The frozen samples were transferred under liquid nitrogen to cryotubes (Nunc) containing a frozen solution of 2.5 % osmium

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CoV-2 Spike in longitudinal convalescent plasma samples

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COVID-19 Vaccines: Should We Fear ADE? The 1554

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Structure-based design of prefusion-stabilized SARS-1583 CoV-2 spikes

The potential danger of suboptimal antibody responses in 1586 COVID-19

Animal and translational models of SARS-CoV-2 1590 infection and COVID-19

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The functions of SARS-CoV-2 neutralizing and infection-1619 enhancing antibodies in vitro and in mice and nonhuman primates. bioRxiv

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Fc effector functions for optimal therapeutic protection

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Indicated organs (nasal cavity, brain, lungs from infected or uninfected mice were collected, 1064 weighed, and homogenized in 1 mL of serum free RPMI media containing penicillin-streptomycin 1065 and homogenized in 2 mL tube containing 1.5 mm Zirconium beads with BeadBug 6 homogenizer 1066 (Benchmark Scientific, TEquipment Inc). Virus titers were measured using three highly correlative 1067 methods. Frist, the total RNA was extracted from homogenized tissues using RNeasy plus Mini 1068 kit (Qiagen Cat # 74136), reverse transcribed with iScript advanced cDNA kit (Bio-Rad Cat 1069 #1725036) followed by a SYBR Green Real-time PCR assay for determining copies of SARS-1070CoV-2 N gene RNA using primers SARS-CoV-2 N F: 5'-ATGCTGCAATCGTGCTACAA-3' and 1071 SARS-CoV-2 N R: 5'-GACTGCCGCCTCTGCTC-3'. 1072Second, serially diluted clarified tissue homogenates were used to infect Vero-E6 cell culture 1073 monolayer. The titers per gram of tissue were quantified using standard plaque forming assay 1074 described above. Third, we used nanoluciferase activity as a shorter surrogate for plaque assay. 1075Infected cells were washed with PBS and then lysed using 1X Passive lysis buffer. 

For evaluating the effect of NK cell depletion during CV3-1 prophylaxis, anti-NK1.1 (clone PK136; 1100 12.5 mg/kg body weight) or an isotype control mAb (BioXCell; clone C1.18.4; 12.5 mg/kg body 1101 weight) was administered to mice by i.p. injections every 2 days starting at 48 h before SARS-1102CoV-2-nLuc challenge till 8 dpi. The mice were bled after two days of antibody depletion, necropsy 1103 or at 10 dpi (surviving mice) for analyses. To evaluate the effect of NK cell and neutrophil depletion 1104 during CV3-1 therapy, anti-NK1.1 (clone PK136; 12.5 mg/kg body weight) or anti-Ly6G (clone: 1105 1A8; 12.5 mg/kg body weight) was administered to mice by i.p injection every two days starting 1106 at 1 dpi respectively. Rat IgG2a mAb (BioXCell; clone C1.18.4; 12.5 mg/kg body weight) was 1107 used as isotype control. The mice were sacrificed and bled at 10 dpi for analyses. For evaluating 1108 the effect of monocyte depletion on CV3-1 therapy, anti-CCR2 (clone MC-21; 2.5 mg/kg body 1109 weight) (Mack et al., 2001) or an isotype control mAb (BioXCell; clone LTF-2; 2.5 mg/kg body 1110 weight) was administered to mice by i.p injection every two days starting at 1 dpi. The mice were 1111 sacrificed and bled 2-3 days after antibody administration or at 10 dpi to ascertain depletion of 1112 desired population. test (two-tailed). To obtain statistical significance for survival curves, grouped data were 1444 compared by log-rank (Mantel-Cox) test. To obtain statistical significance for grouped data we 1445 employed 2-way ANOVA followed by Dunnett's or Tukey's multiple comparison tests. 1446 p values lower than 0.05 were considered statistically significant. P values were indicated as * , p 1447 < 0.05; * * , p < 0.01; * * * , p < 0.001; * * * * , p < 0.0001. 1448 1449 Schematics