Time elapsed between Zika and dengue virus type 2 infections alters the magnitude of antibody and T cell responses but not viremia in rhesus macaques

1 Department of Microbiology and Medical Zoology, University of Puerto Rico-Medical Sciences 9 Campus, San Juan, Puerto Rico, United States of America. 2 Unit of Comparative Medicine, 10 Caribbean Primate Research Center and Animal Resources Center, University of Puerto Rico11 Medical Sciences Campus, San Juan, Puerto Rico, United States of America. 3 Department of 12 Molecular Microbiology & Immunology, Saint Louis University School of Medicine, Saint Louis, 13 Missouri, United States of America. 4 Department of Biology, University of Puerto Rico-Río 14 Piedras Campus, San Juan, Puerto Rico, United States of America. 5 Texas Biomedical Research 15 Institute, San Antonio, Texas, United States of America. 6 Departments of Microbiology & 16 Immunology, University of North Carolina-Chapel Hill, North Carolina, United States of America. 17 7 Division of Vaccine Discovery, La Jolla Institute for Immunology, La Jolla, California, United 18 States of America. 8 Department of Internal Medicine, University of Puerto Rico-Medical Sciences 19 Campus, San Juan 00936, Puerto Rico, United States of America. # Current Address: Takeda 20 Vaccines Inc, Cambridge, Massachusetts, United States of America. 21 22

The clinical status was monitored to determine if the presence of ZIKV immunity affected 165 the clinical outcome of DENV infection. Vital signs such as weight (kg), and temperature (°C) were 166 monitored. Also, complete blood cell counts (CBC), and comprehensive metabolic panel (CMP) 167 were performed before (baseline: day 0) and after DENV infection at multiple timepoints (CBC: 0, 168 7, 15 dpi; CMP: 0, 7, 15, 30 dpi). Neither symptomatic manifestations nor significant differences 169 in weight or temperature were observed in any of the animals after DENV infection up to 90 dpi 170 (Supplementary Fig. 1a-b). Likewise, no significant differences between groups were detected in 171 were quantified by qRT-PCR at baseline, 1 to 10, and 15 dpi to determine if the presence of early-183 (ZIKVPR-2mo) or mid-convalescence (ZIKVPF-10mo) to ZIKV alters DENV RNAemia kinetics. 184 No significant differences between groups were observed in detected levels of DENV genome 185 copies per ml of serum overtime (Fig. 2a). We noted that in the ZIKVPF-10mo group 3 out of 4 186 animals were able to keep the RNAemia level below 10 3 genome copies the next day after DENV 187 infection. This group started an early clearance of the RNAemia at 7 dpi, with only 1 out of 4 188 animals having detectable levels by days 8 and 9 pi. For ZIKVPR-2mo and naïve animals, the 189 clearance of detectable RNAemia started at 8 dpi, in 4 out of 6 and 1 out of 4 of the animals, 190 respectively. Naïve animals had the most delayed clearance of RNAemia with at least half of the 191 animals with detectable levels of viral RNA until day 9 pi. RNAemia was completely resolved in 192 all animals by 10 dpi. In summary, ZIKVPF-10mo had 7.25, ZIKVPR-2mo 7.5, and naïve animals 193 8 mean days of detectable RNAemia after DENV infection (Fig. 2b). In addition, the area under 194 the curve (AUC) was calculated but no statistically significance differences were observed in the 195 RNAemia peak among groups ( Supplementary Fig. 2). However, the AUC trend to be lower in 196 both ZIKV-immune groups. In terms of the kinetics, a delay in the peak RNAemia set point was 197 observed in both ZIKV-immune groups (switch from day 2 to days 5 and 6) followed by higher, 198 but non-significant, levels compared to the naïve group, and a subsequent early RNAemia 199 clearance in both ZIKV-immune groups. Together these results show that, although no statistically 200 significant differences among groups were observed, previous immunity to ZIKV is not associated 201 with an increase in DENV RNAemia; even more, a mid-convalescence to ZIKV tended to develop 202 a shorter viremic period. Tukey test) and 10 pi (p=0.0009 vs ZIKVPR-2mo, Two-way Anova Tukey test). CXCL10 is a T 220 cell-activating chemokine and chemoattractant for many other immune cells. Also, this group 221 showed higher levels of perforin ( Fig. 3h) at day 10 (p=0.0024 vs Naïve and p=0.0190 vs ZIKVPR-222 2mo, Two-way Anova Tukey test) and 15 pi (p=0.0178 vs Naïve, Two-way Anova Tukey test). 223 Perforin is an effector cytolytic protein released by activated cytotoxic CD8+ T cells and natural 224 killer (NK) cells. No significant differences between groups were observed for other pro-225 inflammatory citokines such as monocyte chemoattractant protein 1 (MCP-1), macrophage 226 inflammatory protein 1-beta (MIP-1β) and IL-1 receptor antagonist (IL-1RA) (Fig. 3d-f). 227 Collectively, these results demonstrate that the presence of ZIKV immunity does not exacerbate 228 pro-inflammatory status after DENV infection while mid-convalescence immunity to ZIKV 229 stimulated levels of mediators mainly involved in the activation of cell-mediated immune response. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/621094 doi: bioRxiv preprint

DENV and ZIKV cross-reactive antibody response is boosted by ZIKV immunity and is 232 influenced by the time span of the previous ZIKV infection. An ELISA-based serological 233
profile was performed to determine the contribution of ZIKV immunity in the cross-reactive Ab 234 response before and after DENV infection. We assessed the levels of DENV IgM and IgG, and 235 cross-reactivity with ZIKV (IgM, IgG, NS1-IgG and EDIII-IgG) at multiple timepoints 236 ( Supplementary Fig. 3). Naïve cohort showed a significant higher peak of IgM (Supplementary 237 suggesting that although different pre-infecting ZIKV strains, the previous elicited IgG response 254 against both ZIKV strains is comparable. After DENV infection, an increase of ZIKV IgG was 255 shown and remain constantly high at 15, 30, 60 and 90 dpi in both ZIKV-immune groups 256 (p<0.0001 vs naïve for all timepoints, Two-way Anova Tukey test), suggesting that DENV has the 257 potential to stimulate ZIKV-binding Ab-producing plasmablasts. In addition, to elucidate the 258 composition of similar ZIKV IgG levels in ZIKV-immune groups, we measured ZIKV-specific NS1 259 IgG ( Supplementary Fig. 3e) and ZIKV-specific EDIII IgG ( Supplementary Fig. 3f) levels. Although 260 ZIKVPR-2mo showed significant differences compared to naïve at 30, 60 and 90 dpi (p<0.0001, 261 p=0.0001, p=0.0159; Two-way Anova Tukey test), we observed a significantly higher expansion 262 and long-lasting response of ZIKV NS1-specific Abs in the ZIKVPF-10mo group compared to the 263 ZIKVPR-2mo group at baseline, 60 and 90 dpi (p=0.0036, p=0.0071, p=0.0294; Two-way Anova 264 Tukey test) and also compared to naïve animals at all timepoints (p<0.0001, Two-way Anova 265 Tukey test). Moreover, higher magnitude of ZIKV-specific EDIII-IgG levels in the ZIKVPF-10mo 266 group than in the ZIKVPR-2mo group was observed compared to naïve at baseline (ZIKVPF-267 10mo only), 15, 30 and 60 (ZIKVPF-10mo vs Naïve: p=0.0092, p<0.0001, p<0.0001, p=0.0034; 268 ZIKVPR-2mo vs Naïve: p=0.0003, p=0.0014, p=0.0055; Two-way Anova Tukey test), suggesting 269 that a ZIKV mid-convalescence promotes an expansion of higher magnitude of ZIKV EDIII-IgG 270 Abs from ZIKV memory B cells (MBC). However, those higher cross-reacting levels decrease 271 overtime as expected. In summary, a boost of DENV and ZIKV Abs is triggered by the presence 272 of ZIKV immunity and the expansion of specific-and cross-reactive Abs is higher on magnitude 273 and durability when a mid-convalescence immunity to ZIKV is present. response is known to define to a great extent the infection outcome 12,48 . Accordingly, we tested 279 the neutralization capacity of NAbs in serum from ZIKV-immune and naïve animals before and 280 after DENV infection, to determine whether an early-or mid-convalescence to ZIKV affected the 281 NAb response. Plaque Reduction Neutralization Test (PRNT) was performed to elucidate the NAb 282 titers of all groups against all DENV serotypes and both ZIKV pre-infecting strains. Before 283 infection with DENV the naïve groups had no detectable NAb levels (<1:20 PRNT60 titers) against 284 all DENV serotypes, while ZIKV-immune groups showed low cross-NAb titers against DENV-2 285 and DENV-4 (Fig. 4a). These cross-reactive levels were higher in the ZIKVPF-10mo group than 286 in the ZIKVPR-2mo group for both viruses. The peak of high NAb titers occurred at 30 days after 287 DENV infection for all groups (ZIKVPF-10mo>ZIKVPR-2mo>Naïve) against all DENV serotypes 288 (DENV-2>DENV-4>DENV-3>DENV-1) (Fig. 4b). The ZIKVPF-10mo group neutralized all DENV 289 serotypes with significant higher potency than naïve animals (p<0.0001, p=0.0337, p<0.0001, 290 p<0.0001 for DENV1-4; Two-way Anova Tukey test) and the ZIKVPR-2mo group, except for 291 DENV-2, that both ZIKV-immune groups have comparable neutralization magnitude at 30 dpi 292 (p=0.0002, p=0.7636, p=0.0016, p=0.0004 for DENV1-4; Two-way Anova Tukey test). However, 293 the neutralization kinetics by sigmoidal response curves suggest higher percent of neutralization 294 against DENV-2 overtime in the group with mid-convalescence to ZIKV ( Supplementary Fig. 4). 295 On the other hand, the ZIKVPR-2mo group showed significantly higher potency of the NAb 296 response only against DENV-1 compared to naive animals (p=0.0146; Two-way Anova Tukey 297 test) (Fig. 4b). 298 . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/621094 doi: bioRxiv preprint In addition, we tested whether the NAb titers that peak at 30 dpi for all groups remain 299 constant over time (up to 90 dpi) against all DENV serotypes ( Fig. 4c-f). In general, the 300 neutralizing response of the ZIKVPF-10mo maintained higher NAb titers up to 90 dpi compared 301 to ZIKVPR-2mo and naïve groups. Significant differences between ZIKVPF-10mo and ZIKVPR-302 2mo groups were observed against DENV-1,-3 and -4 at day 30 pi (p=0.0002, p=0.0016, 303 p=0.0004; Two-way Anova Tukey test) and at day 60 pi against DENV-2 and DENV-3 (p=0.0179, 304 p=0.0047; Two-way Anova Tukey test). The neutralizing Ab response of the ZIKVPF-10mo group 305 was even more significantly higher compared to the naïve group at day 15 (only performed for 306 the infecting serotype to monitor early neutralizing activity), day 30, 60 and 90 pi against DENV-307 2 (p=0.0022, p=0.0337, p=0.0146, p=0.0337; Two-way Anova Tukey test); at day 30 pi against 308 DENV-1 (p<0.0001, Two-way Anova Tukey test); at day 30 and 60 pi against DENV-3 (p<0.0001, 309 Two-way Anova Tukey test); and at day 30 pi against DENV-4 (p<0.0001, Two-way Anova Tukey 310 test). In contrast, the ZIKVPR-2mo group showed a neutralizing Ab response with a magnitude 311 and long-lasting levels comparable to the naïve group, except at day 15 and 30 pi against DENV-312 2 and DENV-1, respectively (p=0.0067, p=0.0146; Two-way Anova Tukey test). The neutralizing 313 response was long-lasting in the ZIKVPF-10mo group compared to other groups as supported by 314 the data from days 30 and 60 p.i. At day 90 pi, although no significant differences were observed 315 between all groups, the ZIKVPF-10mo group showed a consistent trend to maintain higher NAb 316 titers against all DENV serotypes indicating a higher and long-lasting breadth of cross-317 neutralization within DENV serocomplex. 318 Collectively, these results demonstrate that a mid-convalescence to ZIKV provokes a 319 boost of the magnitude and durability of the neutralizing response against all DENV serotypes 320 more effectively than in animals with an early-convalescence to ZIKV, but also higher compared 321 to a de novo DENV-specific NAb response of the naïve animals. 322 323 ZIKV cross-neutralizing antibody response is strain-independent and higher in magnitude 324 and durability in the presence of mid-convalescence to ZIKV. Previous exposure to ZIKV 325 strains in ZIKV-immune groups developed high levels of cross-reactive, non-neutralizing, and 326 neutralizing Abs before DENV infection (baseline). To determine if this memory Ab response is 327 strain-specific and if the difference in convalescence period to ZIKV alters the efficacy and 328 modulation after DENV infection, we assessed the NAb levels in ZIKV-immune (ZIKVPF-10mo 329 and ZIKVPR-2mo) and ZIKV-naïve serum with both pre-infecting contemporary Asian-lineage 330 H/PF/2013 and PRVABC59 ZIKV strains at multiple timepoints after DENV infection. At baseline, 331 both ZIKV-immune groups showed high NAb titers against H/PF/2013 strain, which suggest that 332 . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/621094 doi: bioRxiv preprint irrespective of pre-exposure to different ZIKV strains and different convalescent periods the Ab 333 response remains similarly effective (Fig. 5a). As early as day 15 after DENV infection, a potent 334 boost of NAb titers in both ZIKV-immune groups was developed. However, elevated NAb titers 335 were significantly higher in the ZIKVPF-10mo group compared to the ZIKVPR-2mo and naïve 336 groups at day 15 pi (p=0.0005, p<0.0001; Two-way Anova Tukey test) and day 30 pi (p=0.0067, 337 p=0.0012; Two-way Anova Tukey test). As expected, this elevated ZIKV cross-reactive NAb 338 levels decreased gradually overtime after 15 dpi in both ZIKV-immune groups. Nevertheless, the 339 ZIKVPF-10mo group retained higher NAb titers until 90 dpi while the titers of the ZIKVPR-2mo 340 group returned to baseline levels. Of note, the NAb titers of the naïve group were considered as 341 negative in all timepoints and failed to neutralize ZIKV throughout DENV infection even at 342 concentrated levels of the antibodies (Fig. 5a). These results are confirmed by the behavior of 343 neutralization kinetics by sigmoidal response curves where the ZIKVPF-10mo group retained 344 elevated magnitude of ZIKV neutralization overtime ( Supplementary Fig. 5). 345 To determine if the immune memory induced by different ZIKV strains play a role in the 346 modulation of the cross-NAb response triggered by a subsequent DENV infection, NAb titers were 347 measured against both ZIKV strains before and 30 days after DENV infection. The ZIKVPF-10mo 348 group showed significant higher NAb titers against both ZIKV strains compared to the ZIKVPR-349 2mo group before DENV infection (p=0.0093, p=0.0141; Two-way Anova Tukey test) (Fig. 5b). 350 Subsequently, DENV infection promote an equally 8-fold increase of NAb titers against both 351 strains in the ZIKVPF-10mo group, significantly higher than the 4-fold increase in the ZIKVPR-352 2mo group (p=0.0025, p=0.0011; Two-way Anova Tukey test) (Fig. 5c). To rule out that difference 353 in fitness between both ZIKV strains would bias the magnitude of the NAbs after DENV infection 354 we compared in parallel the NAb titers at 30 and 60 days after ZIKV infection (day 60 correspond 355 to the baseline of the ZIKVPR-2mo group). No significant differences were observed between 356 ZIKV-immune groups in the NAb titers induced by both strains at the same timepoints after ZIKV 357 infection ( Supplementary Fig. 6). Altogether, these results demonstrate that DENV infection 358 results in a significant increase in the magnitude and durability of the cross-neutralizing Ab 359 response against ZIKV in animals with a mid-convalescent period from ZIKV infection. The elicited 360 changes in neutralization capacity were likely driven more by the longevity of the immune memory 361 maturation and the associated memory recall of the ZIKV immunity than by a strict dependency 362 of the specific pre-exposed ZIKV strain. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/621094 doi: bioRxiv preprint early activation and proliferation of multiple immune cell subsets and how these parameters are 367 affected by the presence of pre-existing immunity to ZIKV on a subsequent DENV infection 368 ( Supplementary Fig. 7, 8, and 9 for gating strategy; Supplementary Table 2 for Ab panel). As part 369 of the innate immune response, the frequency of dendritic cells (DCs) and natural killer (NK) cells 370 subpopulations were measured. Plasmacytoid DCs (pDCs: Lin -HLA-DR + CD123 + ) are known to 371 respond to viral infection by production of IFN-α, while myeloid DCs (mDCs: Lin -HLA-DR + CD11c + ) 372 interacts with T cells. The frequency of pDCs was not significantly altered by DENV infection in 373 any group compared to baseline levels ( Supplementary Fig. 10a). At day 2 pi we detected a 374 significant increase of mDCs in the ZIKVPF-10mo group (p=0.0082; Two-way Anova Dunnett test) 375 ( Supplementary Fig. 10b). Furthermore, we determined the frequency of NK subpopulations 376 including: NKCD8, NKCD56, NKp30 and NKp46 ( Supplementary Fig. 11). In general, no 377 differences were detected between baseline and after DENV infection in all groups for all NK 378 subpopulations and receptor expression with the exception of the ZIKVPR-2mo group that 379 showed a significant increases in the following subpopulations: NKG2A + NKp30 and 380 NKp30 + NKp46 + at 7 dpi (p=0.0495, p=0.0006; Two-way Anova Dunnett test) and NKp46 + NKp30 + 381 at 7 and 10 dpi (p=0.0005, p=0.0001; Two-way Anova Dunnett test) ( Supplementary Fig. 11j, o, 382 s). 383 We next investigated cell subsets that are part of the bi-phasic (humoral/cellular) adaptive 384 immune response such as B (CD20+CD3-) and T (CD3+CD20-) cells. No differences were detected 385 in total B cells between groups following DENV infection compared to baseline levels (Supplementary 386 Fig. 12a), but ZIKV-immune groups had elevated levels of activated B cells (CD20+CD3-CD69+) 387 since baseline and a trend to increase these levels more than the naïve group overtime 388 ( Supplementary Fig. 12b). We detected a significant decrease of proliferating B cells (CD20+CD3-389 Ki67+) in naïve animals at 7 and 10 dpi (p=0.0031, p=0.0345; Two-way Anova Dunnett test), while 390 ZIKV-immune groups retained their proliferating levels ( Supplementary Fig. 12c). Interestingly, the 391 ZIKVPF-10mo group showed a significant increase of B cells that were proliferating and activated 392 simultaneously (CD20+CD3-CD69+Ki67+) as early as in day 1 pi (p=0.0240; Two-way Anova Dunnett 393 test) and maintained higher levels up to 10 dpi ( Supplementary Fig. 12d). Together, these 394 phenotyping results of B cells are consistent with the early and boosted production of binding and 395 neutralizing Abs in the ZIKVPF-10mo group compared to naïve animals. The frequency of total T cells 396 (CD3 + CD20 -) and CD4 + /CD8 + T cells subsets, was comparable at all timepoints before and after 397 DENV infection in all groups of animals ( Supplementary Fig. 13a-c). 398 Previous studies have demonstrated that DENV and ZIKV specific CD4 + and CD8 + T cells 399 are enriched in certain memory subsets 24,49 . Thus, we measured whether the early activation of 400 . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/621094 doi: bioRxiv preprint T cell subpopulations, such as effector memory (CD3 + CD4 + CD28 -CD95 + ) and central memory 401 (CD3 + CD4 + CD28 + CD95 + ) T cells (T-EM and T-CM), within each T cell compartment was 402 modulated following DENV infection in presence or absence of convalescence to ZIKV (Fig. 6,  403 and Supplementary Fig. 7 for gating strategy). The ZIKVPF-10mo group showed significant higher 404 frequency of activated CD4 + and CD8 + T-EM (CD3 + CD4 + CD28 -CD95 + CD69 + and 405 CD3 + CD8 + CD28 -CD95 + CD69 + ) following DENV infection compared to basal levels (CD4 + T-EM 406 at 7 and 10 dpi: p=0.0001, p=0.0072; CD8 + T-EM at 2 and 7 dpi: p=0.0291, p=0.0001; Two-way 407 Anova Dunnett test) (Fig. 6a, d). Interestingly, the ZIKVPR-2mo group showed a very limited 408 frequency and activation of the CD4 + and CD8 + T-EM compared to the ZIKVPF-10mo and naïve 409 groups. However, this group with an early convalescent period to ZIKV, contrary to the other two 410 groups, showed a very limited but significant activation of CD8 + T-CM 411 (CD3 + CD8 + CD28 + CD95 + CD69 + ) at day 7 and 10 pi (p=0.0007, p=0.0147; Two-way Anova 412 Dunnett test) (Fig. 6e). In contrast, naïve animals did not show any significant activation of these 413 memory cell subsets after DENV infection. Collectively, these results suggest that following DENV 414 infection: (i) animals with a mid-convalescence ZIKV immunity have a more dynamic B cell 415 response and are able to rapidly produce more activated effector memory T cells from both T cell  Table 3 for 429 Ab panel) was performed to quantify the production of effector immune markers such as the 430 cytotoxic marker CD107a, IFN-γ, and TNF-α by CD4 + and CD8 + T cells at baseline, 30, 60, and 431 90 days after DENV infection (Fig. 7). 432 To assess the ZIKV-primed specific-or cross-reactive effector T cell response we studied 433 the response against ZIKV or DENV stimuli before DENV infection. In general, before DENV 434 . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/621094 doi: bioRxiv preprint infection, we found that the ZIKV-primed effector T cell response was higher in CD8 + (Fig. 7m, q, 435 u) than in CD4 + (Fig 7a, (Fig. 7b). 459 The ZIKVPF-10mo group showed a remarkable significant increase of the IFN-γ-460 producing CD4 + T cells against DENV E protein since 60 dpi and is maintained up to 90 dpi 461 compared to other groups (ZIKVPF-10mo vs ZIKVPR-2mo at 60 and 90 dpi: p<0.0001, p=0.0024; 462 ZIKVPF-10mo vs Naïve at 60 and 90 dpi: p<0.0001, p=0.0037; Two-way Anova Tukey test) (Fig.  463 7g, h), and was the only group with significant increase in the IFN-γ producing CD8 + T cell 464 compartment at 60 dpi (ZIKVPF-10mo vs ZIKVPR-2mo: p=0.0253; Two-way Anova Tukey test) 465 ( Fig. 7s). On the other hand, the ZIKVPR-2mo group exhibited a significant increase of IFN-γ 466 producing CD4 + T cells earlier than other groups at 30 dpi (ZIKVPR-2mo vs ZIKVPF-10mo: 467 p<0.0001; ZIKVPR-2mo vs Naïve: Two-way Anova Tukey test) (Fig. 7f). Interestingly, the naïve 468 . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/621094 doi: bioRxiv preprint group showed an increase of cross-reactive TNF-α producing CD4 + T cells against ZIKV NS 469 proteins 30 days after DENV infection (Naïve vs ZIKVPR-2mo: p=0.0359; Two-way Anova Tukey 470 test) (Fig. 7j). The ZIKVPF-10mo group developed a significant effector T cell response by TNF-471 α producing CD4 + T cells against DENV and ZIKV E proteins at 60 days after DENV infection 472 (ZIKVPF-10mo vs ZIKVPR-2mo against DENV/ZIKV E protein: p=0.0163, p=0.0172; Two-way 473 Anova Tukey test) (Fig. 7k). Although all groups showed a boosted TNF-α effector response in 474 the CD8 + T cell compartment up to 90 days after DENV infection, no significant differences 475 between groups were observed. 476 Collectively, these results after DENV infection suggest that a mid-convalescence to ZIKV 477 translate in a more complete functional T cell response characterized by: (i) a cytotoxic CD107a + 478 phenotype directed to DENV E protein for both T cell compartments comparable to the DENV-479 specific de novo response of the naïve group, (ii) developed CD107a, IFN-γ and TNF-α producing 480 CD8 + T cell effector response that cross-react efficiently with DENV E protein since baseline and 481 is boosted after DENV infection, (iii) and promoted the higher T cell effector response against 482 ZIKV NS proteins. An early-convalescence to ZIKV results in (iv) a very limited cytotoxic activity 483

(limited expression of CD107a marker) which is in line with a very limited activation of the T-EM, 484
and with failed capability to react efficiently against E or NS proteins. The ZIKV-naïve group 485 response was characterized by: (v) production of a DENV-specific de novo functional T cell 486 response with similar magnitude between both T cell compartments, (vi) capable to cross-react 487 against ZIKV E and NS proteins, (vii) and able to mount a DENV-specific cytotoxic CD107a + 488 phenotype. 489 490

Discussion 491
We found that previous infection to ZIKV modulates the immune response against 492 subsequent DENV infection without an enhancement of DENV viremia nor pro-inflammatory 493 status, and that this modulation is influenced by the longevity of ZIKV convalescence-more after 494 longer ZIKV pre-exposure. The aftermath of the recent ZIKV epidemic has been related to a 495 remarkable decrease in DENV cases in Brazil 28 , and also in most of Latin American and 496 Caribbean countries (http://www.paho.org/data/index.php/es/temas/ indicadores-497 dengue/dengue-nacional/9-dengue-pais-ano.html?start=2) 25 . Yet, little is known about the role of 498 previous ZIKV immunity in the outcome of a subsequent DENV infection in human populations, 499 and if ZIKV immunity is supporting this epidemiological phenomenon observed post-ZIKV 500 epidemic 28 . To evaluate the hypothesis of a potential ZIKV-DENV cross-protection in humans 501 characterizing the immunological history of prospective cohorts 47 will be necessary, but human 502 . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/621094 doi: bioRxiv preprint samples for this purpose are scarce yet. Because of this, NHPs are key to provide knowledge 503 and anticipate different immunological scenarios when DENV epidemics re-emerge in human 504 populations with previous immunity to ZIKV. 505 Animals with pre-existing ZIKV immunity do not show an enhancement of DENV-induced 506 RNAemia, regardless of the period of convalescence from previous ZIKV infection (10 or 2 507 months) and different pre-infecting ZIKV strains. Previous ZIKV immunity is associated with a 508 trend of less RNAemia days during subsequent DENV infection. This effect is more evident in 509 animals with a ZIKV convalescence period of 10 months. Previous work reported that a period of 510 early-convalescence (56 days) to ZIKV (PRVABC59 strain) in rhesus macaques was associated 511 with a significant increase of DENV-2 RNAemia at day 5 after DENV infection and a pro-512 inflammatory cytokine profile. However, very similar to our results, it was noteworthy a delay at 513 early timepoints and an early clearance in late timepoints of the DENV-2 RNAemia in ZIKV-514 immune macaques in comparison to the naïve ones 38 . The lack of significant DENV RNAemia 515 enhancement found in the group with the early-convalescence period in our work, compared to 516 previous results 38 , may be attributable to the different sample types collected (plasma vs serum), 517 or different DENV-2 strains used for the challenge [New Guinea/1944 strain vs 518 Thailand/16681/1964 strain, from Asian II and Asian I Genotype, respectively]. This fact is of 519 relevance because it suggests that the effect of previous ZIKV immunity on a subsequent DENV 520 infection may differ between DENV serotypes or even within genotypes. Another possible 521 explanation is the genetic heterogeneity of rhesus macaques used in these two studies as they 522 are derived from different breeders. The importance of selecting genetic well-characterized 523 macaques have been discussed previously 50 . 524 Due to limited availability of ZIKV-immune cohorts we used animals infected with two 525 different ZIKV strains for our subsequent challenge with DENV-2. However, extensive revision of 526 the literature up to date reveals a broad consensus that these two contemporary ZIKV strains 527 behave very similar from an antigenic point of view 12,51-53 . Our results are confirmatory of those 528 results showing that both ZIKV strains were neutralized with same efficacy by serum within each 529 ZIKV-convalescent group, explained by the broadly neutralization activity against multiple ZIKV 530 strains irrespective of the infecting strain 52 . However, the magnitude of the neutralization of both 531 strains was statistically higher in animals exposed to DENV 10 months (mid-convalescence) after 532 ZIKV infection compared to the animals with a shorter ZIKV convalescence (2 months). These 533 results suggest that the differences in the neutralization profile between the two ZIKV-immune 534 groups are associated to the longevity of ZIKV convalescence which may be attributable to the 535 maturation of the cross-reactive immune memory elicited by the heterologous DENV infection and 536 . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/621094 doi: bioRxiv preprint no to the antigenic differences or the different replication capabilities in rhesus macaques of those 537 two pre-infecting ZIKV strains 17,54 . 538 The period of convalescence further had an impact in the maintenance of the 539 neutralization magnitude against ZIKV and DENV overtime. We observed a higher activation of 540 the memory immune response characterized by transiently higher peak levels of serum NAbs 541 against DENVs and ZIKV in ZIKVPF-10mo immune animals compared to ZIKVPR-2mo immune 542 animals challenged with DENV-2. However, unlike heterologous infections with different DENV 543 serotypes, by 90 days after DENV-2 infection, the naïve and ZIKV-immune animals had similar 544 levels of DENV-2 NAbs. Moreover, in the ZIKV-immune animals, the ZIKV NAbs returned to 545 steady-state levels similarly observed before DENV-2 challenge. Overall, these results 546 demonstrate that pre-existing ZIKV immunity leads to a transient increase in neutralizing Ab 547 responses in animals challenged with DENV-2 compared to naïve animals. This is in contrast with 548 previous findings were ZIKV-convalescent macaques show a lack of an early and delayed 549 anamnestic response overtime with limited induction of DENV NAbs compared to ZIKV-naïve 550 animals after DENV infection 39 . However, our results show the ability of DENV-2 to activate MBCs 551 stimulated by the previous ZIKV infection, but this activation is modest and short-lived compared 552 to the robust and sustained activation of MBCs on secondary DENV infections 11,31,55,56 . Is still 553 uncertain why the ZIKVPF-10mo animals have a slightly higher peak of Ab response compared 554 to the ZIKVPR-2mo animals. We speculate this may be caused by modification of MBCs overtime, 555 so that by 10 months the cells are able to better respond to antigen compared to cells at two 556 months. After ZIKV infection in human DENV-naïve subjects, the ZIKV/DENV cross-reactive MBC 557 response increased in magnitude (39% of total MBC proportion) after longer periods of ZIKV 558 convalescence (~8 months post-ZIKV infection) 57 , similar to the 10 months in the ZIKV mid-559 convalescent group that exhibited higher DENV cross-neutralization. Based upon studies of 560 human monoclonal Abs, plasmablasts response during secondary DENV infection is mainly of 561 MBC origin, resulting in a mature response characterized by cross-neutralizing Abs in vitro 58 . 562 However, there are very limited studies on how the affinity maturation develops during the initial 563 viral encounter and whether the affinity of MBCs is modified during a secondary heterologous 564 infection or as in this work, during a secondary DENV infection following a primary ZIKV infection. 565 These are seminal contributions to forecast and understand the cross-neutralization capacity of 566 further heterologous DENV epidemics in the context of previous ZIKV-DENV immunity. 567 Interestingly, ZIKV-convalescent animals showed some degree of cross-neutralization 568 against DENV-2 and DENV-4 before DENV infection. This is consistent with our previous results 569 showing that DENV-naïve ZIKV-infected animals also preferentially neutralized DENV-4 followed 570 . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/621094 doi: bioRxiv preprint by DENV-2 after ZIKV infection 17 . Longitudinal data of cross-neutralization of DENV serotypes in 571 DENV-naïve ZIKV-infected human subjects showed low cross-neutralization against all DENV 572 serotypes, but DENV-4 followed by DENV-2 were neutralized more efficiently up to 6 months after 573 ZIKV infection with comparable basal titers reported here 59 . There is no data yet that delineates 574 shared cross-neutralizing epitopes between ZIKV and DENV-2/-4, but it is known that DENV-4 575 genotypic diversity impact the capacity of its neutralization 60 . 576 One factor that plays a critical role in the induction of enhancement and disease severity 577 is the time elapsed between sequential heterologous DENV infections 10 . At this time, it is unknown 578 whether this factor plays a role when DENV accounts for a secondary infection following ZIKV. 579 Based on our previous works 17,54 , it is possible to argue that the sequence of DENV-ZIKV 580 infections induce a different immunological response-in terms of the neutralization magnitude, 581 cytokines profile and functionality of the cellular immune response-compared to the ZIKV-DENV 582 scenario shown here. However, in both scenarios, the role of the time interval between infections 583 seems to play a critical role in the quality and quantity of the immune response. 584 Early studies of T cells associate their contribution towards immunopathogenesis in DENV 585 secondary infections explained by the original antigenic sin 61 , but increasing evidence suggest 586 their protective role during primary and secondary DENV infections 62 . Recently, with the 587 introduction of ZIKV into The Americas, T cells from DENV immunity are being implicated in 588 mediating cross-protection against ZIKV 22-24 . We found that animals with a mid-convalescence to 589 ZIKV developed an early activation of CD4 + and CD8 + effector memory T cells after DENV 590 infection. This early activation has been observed for the opposite scenario in DENV-immune 591 ZIKV-infected patients 24 . Interestingly, the ZIKV early-convalescent group displays a modest 592 activation (T-CM>T-EM) early after DENV infection. Since this group was infected with ZIKV only 593 two months before DENV it is possible that after viral clearance and development of ZIKV-specific 594 T cell response, the T cell compartments were still under the contraction phase at the time of the 595 DENV challenge. Yellow fever virus (YFV) and vaccinia virus vaccinations in humans demonstrate 596 that T cell contraction start as early as approximately one-month post-vaccination and at least for 597 almost three months is still ongoing 63 . Also, a study shows that re-stimulation using alphavirus 598 replicons during T cell response contraction does not have significant impact modulating the pre-599 existing T cell response 64 . 600 The profile of ZIKV-specific CD8 + T cells in humans with convalescence to ZIKV is 601 characterized by the production of IFN-γ, and expression of activation and cytotoxic markers 65 . 602 Presence of sustained levels of IFN-γ prior and early after DENV challenge in vaccinees has been 603 associated with protection against viremia and/or severe disease 66,67 . We observed a similar 604 . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/621094 doi: bioRxiv preprint phenotype of the functional response of CD8 + T cells prior DENV infection in animals with longer 605 convalescence to ZIKV. Strikingly, this response recognizes more efficiently peptides from DENV 606 E protein than from ZIKV E protein. However, ZIKV-specific CD8 + T cells direct 57% of their 607 response against structural proteins, which may suggest these cells can recognize conserved 608 epitopes between ZIKV and DENV structural proteins. Cross-reactivity of T cells between 609 heterologous flavivirus infections is explained by selective immune recall of memory T cells that 610 recognize conserved epitopes between DENV and ZIKV 24 , which also has previously been 611 demonstrated during secondary heterotypic DENV infections 68,69 . In addition, an increased 612 cytotoxic profile as demonstrated by the higher frequency of CD107a-expressing CD4 + and CD8 + 613 T cells in the ZIKV mid-convalescent group correlates with the synchronously early activation of 614 CD4 + and CD8 + effector memory T cells and elevated levels of perforin release. 615 Higher proportion of IFN-γ and TNF-α producing T cells before a secondary heterologous 616 DENV infection has been associated to a subsequent subclinical outcome 70 . Herein, we observed 617 that the ZIKV mid-convalescent group had elevated levels of IFN-γ and TNF-α producing T cells 618 since baseline. In this group, DENV infection stimulated a higher frequency of these cells, but 619 remarkably, also increased highly cross-reactive IFN-γ-producing CD4 + T cells directed to DENV 620 E, and ZIKV E/NS proteins. A study showed that cross-reactive ZIKV-primed CD4 + T cells 621 T cells are also required to generate an effective humoral response against ZIKV 73 . Based on 627 this, the higher proportion of DENV-E-reactive IFN-γ-producing CD4 + T cells may play a role in 628 the induction of the robust Ab response in the ZIKV mid-convalescent group against ZIKV and all 629 DENV serotypes. On the other hand, we showed that naïve animals with DENV de novo response 630 did not cross-neutralized ZIKV at all, which state that although similar, antigenic differences are 631 sufficient to mount predominantly type-specific rather than cross-reactive responses during a 632 primary infection 51,57 . 633 A lack of ZIKV immunity promoted a more pro-inflammatory profile characterized by 634 significant elevated levels of IL-6 and MIG/CXCL9. IL-6 has been detected in high levels during 635 secondary DENV infections in children 74 , and the day patients suffer from shock (DSS) 75 or died 636 from DHF 76 . MIG/CXCL9 is known to be a risk factor for DENV severity involved in vascular 637 permeability 77 . Its detection varies between primary and secondary (higher levels) DENV 638 . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/621094 doi: bioRxiv preprint infections 78 , which may explain non-significant peaks within ZIKV-immune groups during a 639 secondary DENV infection. Interestingly, higher levels of IFN-α were observed in the ZIKV-naïve 640 animals. This antiviral cytokine is known to be actively produced during acute DENV infection in 641 vitro and in vivo 79 . Elevated levels have been correlated with severity in DHF patients, and to act 642 as a marker for elevated DENV replication 80,81 . On the other hand, the presence of a longer ZIKV 643 convalescence is associated with increased levels of CXCL10 and perforin. CXCL10 is an 644 immune mediator for T cells proliferation, recruitment of CD4 + and CD8 + activated T cells and IFN- and also naïve cohorts were available as well. After our laboratories prioritized ZIKV research 751 since 2016, DENV pre-exposed and naïve cohorts were infected with ZIKV and pre-exposed 752 animals became available for this study. All animals were housed within the Animal Resources 753 The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/621094 doi: bioRxiv preprint MA023, MA029, and MA062) as a control group. Cohort 1 and 3 were challenged on the same 775 day while cohort 2 was challenged 3 months later with the same stock of DENV-2. However, all 776 samples were frozen and analyzed together, except for the immunophenotyping analysis. 777 The ages of all animals are within the age range for young adults rhesus macaques 778 https://www.nc3rs.org.uk/macaques/macaques/life-history-and-diet/ (ZIKVPF-10mo: 6.8, 6.8, 779 5.8, and 5.9; ZIKVPR-2mo: 6.4, 6.5, 5.2, 4.3, 5.6, and 5.5; Naïve: 4.8, 6.6, 6.8, and 5.7). Prior to 780 DENV-2 challenge all animals were subjected to quarantine period. All cohorts were bled for 781 baseline and challenged subcutaneously (deltoid area) with 5x10 5 pfu/500 ul of DENV-2 New serotype (DENV-2) and ZIKV the NAbs were measured in early timepoints as well (7 and 15 dpi). 825 For the PRNT, serum samples were inactivated, diluted (2-fold), mixed with a constant inoculum 826 of virus (volume necessary to produce ~35 pfu/well) and then incubated for 1 hr at 37°C and 5% 827

CO2. After incubation, virus-serum mix dilutions were added to Vero-81 cells monolayer in flat 828
bottom 24-well plates seeded the day before for 1 hr at 37°C and 5% CO2, finally overlay medium 829 was added and incubated by several days (serotype dependent). Results were reported as 830 PRNT60 titers, NAb titer capable of reduce 60% or more of DENV serotypes or ZIKV strains pfu 831 compared with the mock (control of virus without serum). A PRNT60 1:20 titer was considered a 832 positive Neut titer, and <1:20 as a negative Neut titer. Non-neutralizing titers (<1:20) were 833 assigned with one-half of the limit of detection for graphs visualization. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/621094 doi: bioRxiv preprint respectively (Supplementary Table 4 for peptide sequences). The stimulation with peptides was 877 performed in presence of brefeldin A at 10 ug/ml -1 . After stimulation, the cells were stained for the 878 following markers: CD3, CD4, CD8, CD20 (excluded), CD107a (functional cytotoxicity). Levels of 879 IFN-γ and TNF-α also were measured in gated lymphocytes cell populations. Samples were 880 measured and data was collected on a LSRII (BD).    were assigned with one-half of the limit of detection for graphs visualization (1:10). Statistically significant differences between groups were calculated using Two-Way Anova adjusted for Tukey's multiple comparisons test including 4 and 6 families for heterologous serotypes and DENV-2, respectively, and 3 comparisons per family. Significant multiplicity adjusted p values (* ˂0.05, ** ˂0.01, *** ˂0.001, **** ˂0.0001) are shown. Blue and orange asterisks represent significant difference between the corresponded ZIKV immune groups and naive group, and gray asterisks indicate a significant difference between ZIKV immune groups.
. CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/621094 doi: bioRxiv preprint Figure 5 | ZIKV neutralization is more potent and durable in animals with midconvalescence to ZIKV and is independent of the pre-infecting ZIKV strain. (a) NAb titers against ZIKV H/PF/2013 were determined by PRNT60 at baseline, 7, 15, 30, 60 and 90 days after DENV infection. Comparison of NAb titers between pre-infecting ZIKV strains was performed (b) before and (c) after DENV infection. Symbols connected with full lines indicate mean levels of NAb titers detected per cohort over time: blue squares (ZIKVPF-10mo), orange squares (ZIKVPR-2mo) and black circles (Naïve). Error bars represent the standard error of the mean (SEM). PRNT60: NAb titer capable of reduce 60% or more of ZIKV strains plaque-forming units (pfu) compared with the mock (control of virus without serum). A PRNT60 1:20 titer was considered positive, and <1:20 as a negative Neut titer. Dotted line mark <1:20 for negative results. Nonneutralizing titers (<1:20) were assigned with one-half of the limit of detection for graphs visualization (1:10). Statistically significant differences between groups were calculated using Two-Way Anova adjusted for Tukey's multiple comparisons test including 6 and 2 families for panel a and b-c, respectively, and 3 comparisons per family. Significant multiplicity adjusted p values (* ˂0.05, ** ˂0.01, *** ˂0.001, **** ˂0.0001) are shown. Blue and orange asterisks represent significant difference between the corresponded ZIKV-immune groups and naive group, and gray asterisks indicate a significant difference between ZIKV-immune groups.

Figure 6 | Effector and central memory T cells within CD4 + and CD8 + T cell compartments are activated after DENV infection. Activation (CD69 + ) of effector memory (T-EM:
CD3 + CD4 + CD28 -CD95 + ) and central memory (T-CM: CD3 + CD4 + CD28 + CD95 + ) T cells within (ac) CD4 + and (d-f) CD8 + T cell compartments before and after DENV infection. Percent of cells were determined by immunophenotyping using flow cytometry ( Supplementary Fig. 7 for gating strategy). Blue, orange and black squares represent T-EM for ZIKVPF-10mo, ZIKVPR-2mo and Naïve, respectively. Gray squares represent T-CM for each group. Short black lines mark mean value for each group per timepoint. Cutted line divide % of T-EM and T-CM cells quantified before and after DENV infection. Statistically significant differences within groups were determined using Two-Way Anova adjusted for Dunnett's multiple comparisons test (comparison of each group response at each timepoint versus baseline of the same group) including 2 families, and 7 comparisons per family. Significant differences are reported as multiplicity adjusted p values (* ˂0.05, ** ˂0.01, *** ˂0.001, **** ˂0.0001). Asterisks represent significant difference between the corresponded timepoint and baseline within the same group.
Figure 7 | Longevity of ZIKV immunity shapes the functional response of CD4 + and CD8 + T cells. T cell functional effector response was determined by the quantification (%) of (a-d; m-p) CD107a-expressing and (e-h; q-t) IFN-γ or (i-l; u-x) TNF-α producing CD4 + and CD8 + T cells before (0) and 30, 60 and 90 days after DENV infection. Responses to several peptide pools that encode for DENV and ZIKV envelope (E) proteins or ZIKV non-structural (NS) protein were quantified. After antigenic stimulation intracellular cytokine staining was performed using flow cytometry analysis ( Supplementary Fig. 14 for gating strategy). Individual symbols represent each animal per antigenic stimulation over time: blue squares (ZIKVPF-10mo), orange squares (ZIKVPR-2mo) and black circles (Naïve). Short gray lines mark mean value for each group. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/621094 doi: bioRxiv preprint Temperature (°C) was monitored with an infrared device at baseline, 1-10, 15, 30, 60 and 90 dpi. Complete blood cell counts (CBC) parameters (thou/ul and/or % of total WBC) such as (c) white blood cells (WBC), (d) lymphocytes (LYM), (e) neutrophils (NEU), (f) monocytes (MON), and (g) platelets (PLT) were screened at baseline, 7, and 15 dpi. Comprehensive metabolic panel (CMP) was performed to assess levels (U/L) of (h) alkaline phosphatase (ALK PHOSPHATASE) and liver enzymes (i) aspartate transaminase (AST), and (j) alanine transaminase (ALT) at baseline, 7, 15 and 30 dpi. Normal range of AST and ALT are depicted for reference. (k) Age of rhesus macaques are depicted including the range of young adults for reference. Symbols represent mean level detected for each parameter per cohort per timepoint: blue squares (ZIKVPF-10mo), orange squares (ZIKVPR-2mo) and black circles (Naïve). Lines connect mean values detected over time. Error bars indicate the standard error of the mean (SEM) for each cohort per timepoint. Statistically significant differences between groups were determined using Two-Way Anova adjusted for Tukey's multiple comparisons test including 10, 15, 3, 4, and 3 families for panel a, b, c-g, h-j, and k, respectively, and 3 comparisons per family. For differences in ALT levels Two-Way Anova Dunnett's multiple comparisons test (comparison of each group response at each timepoint versus baseline of the same group) was performed including 3 families, and 3 comparisons per family due to divergence of non-specific levels between cohorts at baseline. Statistically differences are reported as multiplicity adjusted p values (* ˂0.05).
. CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/621094 doi: bioRxiv preprint

Supplementary Figure 2 | Previous ZIKV immunity modulates DENV RNAemia kinetics and is associated with a lower area under the curve.
The area under the curve (AUC) was calculated using log-transformed values of DENV-2 RNAemia in ZIKV-immune and naïve animals. The total area by group is depicted on the graph as light blue, light orange and gray for ZIKVPF-10mo, ZIKVPR-2mo, and Naïve, respectively. Lines mark the mean value of genome copies per group per timepoint. A value of 1 was assigned to all samples below the LOD in order to calculate the means. Error bars indicate the standard error of the mean (SEM) and dotted line mark the limit of detection for each individual ELISA. Results were read at OD 450, 405 or using ISR (Immune Status Ratio) following manufacturer's instructions. Statistically significant differences between groups were calculated using Two-Way Anova adjusted for Tukey's multiple comparisons test including 5, 6, 9, and 4 families, and 3 comparisons per family. Significant multiplicity adjusted p values (* ˂0.05, ** ˂0.01, *** ˂0.001, **** ˂0.0001) are shown. Blue and orange asterisks represent significant difference between the corresponded ZIKV immune groups and naive group, and gray asterisks indicate a significant difference between ZIKV immune groups. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/621094 doi: bioRxiv preprint group calculated by the transformation of PRNT60 Neut 2-fold titers into Log10 (1/serum dilution), and sigmoidal-dose response curves were generated. Each column of panels represent the % of DENV-2 neutralization for each group (ZIKVPF-10mo: blue squares/curves; ZIKVPR-2mo: orange squares/curves; Naïve: black circles/curves) and each row of panels represent a timepoint before and after DENV infection (baseline, 7, 15, 30, 60, 90 dpi).
. CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/621094 doi: bioRxiv preprint each animal per group calculated by the transformation of PRNT60 Neut 2-fold titers into Log10 (1/serum dilution), and sigmoidal-dose response curves were generated. Each column of panels represent the % of ZIKV neutralization for each group (ZIKVPF-10mo: blue squares/curves; ZIKVPR-2mo: orange squares/curves; Naïve: black circles/curves) and each row of panels represent a timepoint before and after DENV infection (baseline, 7, 15, 30, 60, 90 dpi).
. CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It  blue squares (ZIKVPF-10mo), orange squares (ZIKVPR-2mo) and black circles (Naïve). Short gray lines depict mean value for each group detected overtime. Cutted line divide % of DCs quantified before and after DENV infection. Statistically significant differences within groups were determined using Two-Way Anova Dunnett's multiple comparisons test (comparison of each group response at each timepoint versus baseline of the same group) including 3 families, and 5 comparisons per family. Significant differences are reported as multiplicity adjusted p values (* ˂0.05, ** ˂0.01, *** ˂0.001, **** ˂0.0001). Asterisks represent significant difference between the corresponded timepoint and baseline within the same group. . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/621094 doi: bioRxiv preprint