key: cord-0336380-elcg865t
authors: Izmirly, Abdullah M; Pelletier, Adam-Nicolas; Connors, Jennifer; Taramangalam, Bhavani; Alturki, Sawsan O.; Gordon, Emma A.; Alturki, Sana O.; Mell, Joshua C.; Swaminathan, Gokul; Karthik, Vivin; Kutzler, Michele A.; Kallas, Esper G.; Sekaly, Rafick-Pierre; Haddad, Elias K
title: Pre-vaccination Frequency of Circulatory Tfh is associated with Robust Immune Response to TV003 Dengue Vaccine
date: 2021-08-19
journal: bioRxiv
DOI: 10.1101/2021.08.19.456926
sha: 9f5c00ddc21cb98775e3185f80ebab995e77bf6d
doc_id: 336380
cord_uid: elcg865t

It has been estimated that more than 390 million people are infected with Dengue virus every year; around 96 millions of these infections result in clinical pathologies. To date, there is only one licensed viral vector-based Dengue virus vaccine CYD-TDV approved for use in dengue endemic areas. While initially approved for administration independent of serostatus, the current guidance only recommends the use of this vaccine for seropositive individuals. Therefore, there is a critical need for investigating the influence of Dengue virus serostatus and immunological mechanisms that influence vaccine outcome. Here, we provide comprehensive evaluation of sero-status and host immune factors that correlate with robust immune responses to a Dengue virus vector based tetravalent vaccine (TV003) in a Phase II clinical cohort of human participants. We observed that sero-positive individuals demonstrate a much stronger immune response to the TV003 vaccine. Our multi-layered immune profiling revealed that sero-positive subjects have increased baseline/pre-vaccination frequencies of circulating T follicular helper (cTfh) cells and the Tfh related chemokine CXCL13/BLC. Importantly, this baseline/pre-vaccination cTfh profile correlated with the vaccinees’ ability to launch neutralizing antibody response against all four sero-types of Dengue virus, an important endpoint for Dengue vaccine clinical trials. Overall, we provide novel insights into the favorable cTfh related immune status that persists in Dengue virus sero-positive individuals that correlate with their ability to mount robust vaccine specific immune responses. Such detailed interrogation of cTfh cell biology in the context of clinical vaccinology will help uncover mechanisms and targets for favorable immuno-modulatory agents. Author summary Dengue virus (DENV) is a worldwide threat that causes significant health and economic burden. Currently, there are several challenges in the development of a DENV vaccine including the existence of four different serotypes all; capable of causing disease and antibody dependent enhancement (ADE). For complete protection, a vaccine must be able to generate neutralizing antibodies against all 4 serotypes to avoid ADE. Currently, there is one licensed DENV vaccine, CYD-TDV (DENGVAXIATM). However, this vaccine is only efficacious in protecting against severe disease in DENV seropositive individuals therefore serostatus effect must be further studied for optimal vaccine design. A subset of CD4+ T cells called T-follicular helper (Tfh) cells have been well known to play a major role in aiding high affinity antibody production. Therefore, we chose to look at subsets of Tfh and the cytokines they produce in human blood that can serve as biomarkers for effective vaccine design. We found that DENV sero-positive participants had increased pre-vaccination frequencies of Tfh cells and higher levels of the Tfh related chemokine CXCL13/BLC that plays a role in directing antigen-specific responses. This pre-vaccination Tfh profile and CXCL13/BLC are then correlated positively with the vaccinees’ ability to produce neutralizing antibody against all four sero-types (breadth of the Response) of DENV, an important goal for all DENV vaccine trials.

To investigate the influence of Dengue virus serostatus on vaccine outcome, we have utilized 150 samples from Brazil that were obtained longitudinally during Live attenuated TV003 phase II 151 clinical trial. This cohort is a unique cohort because it could be utilized as a tool to characterize 152 the pre-vaccination basal microenvironment and how it differs between Dengue seropositive 153 and seronegative groups. We investigated both the cellular differences and the immune 154 signatures differences at baseline/pre-vaccination (Day 0) prior to TV003 vaccination in the two 155 groups. The vaccine response in this study was measured by two outcomes, which are the 156 neutralizing antibody titers as well as the breadth of the vaccine response (seroconversion). four serotypes compared to their Naïve counterparts, or individuals who received the placebo.

Interestingly, we observed large variability in the response amongst all vaccinated subjects 178 suggesting that pre-vaccination environment and conditions could play a role in the response 179 ( Fig 1A) . To further understand these variations, we used Principal Component Analysis (PCA) 180 that clusters samples in groups based on their similarity ( Fig 1B) . As expected, placebo controls 181 clustered to the left with minimal neutralizing antibody titers; whereas there is a clear distribution 182 difference between naïve vaccinated (seronegative at pre-vaccination) and Dengue immune 183 (seropositive at pre-vaccination) vaccinated subjects, with most of the higher response 184 observed in the Dengue immune vaccinated subjects. In addition, we observed minor 185 similarities between a) Dengue serotype 2 (DENV2) and serotype 3 (DENV3) specific 186 responses and b) Dengue serotype 1 (DENV1) and serotype 4 (DENV4) (Fig 1B) .

We further, demonstrate quantifiably that pre-existing dengue infection of any of the four 189 serotypes before vaccination was associated with significant increase in neutralizing antibodies Fig 1F) . This could be likely due to the fact that this cohort has much less exposure 199 to DENV4 due to the prevalence of of other serotypes circulating this particular geographical 200 locations. From this data, we can infer that immunization with this vaccine is capable of eliciting 201 robust immune responses in seropositive individuals against majority of the serotypes. Overall, 202 using unsupervised analysis, we observed qualitatively that seropositive (Immune) individuals 203 seem to elicit more vaccine induced neutralizing antibody titers as compared to their 204 seronegative (Naïve) counterparts (Fig 1 A, B) . Therefore, we next investigated the immune and 205 naïve neutralizing response AUCp quantitatively and found that pre-existing Dengue virus 206 seropositivity is associated with significant increases in neutralizing antibodies against DENV1, 207 DENV2, and DENV3 post-vaccination (AUCp) (Fig 1C-F) . is associated with significant increases in neutralizing antibody responses in a serotype-specific 219 manner for three of the four serotypes. Individuals who were seropositive for DENV1 before 220 vaccination, showed significantly higher levels of DENV1-specific nAbs (p<0.0001) and DENV3-221 specific nAbs (p=0.0436) post vaccination when compared to seronegative individuals (Fig 2A, 

2C). Interestingly, we did not observe any significant increase in DENV2 or DENV4-specific 223 nAbs in those subjects post vaccination (Fig 2B, 2D) . Additionally, individuals who were 224 seropositive for DENV3 before vaccination, showed significantly (p<0.0001) higher levels of 225 DENV3 specific nAb ( Fig 2G) and no difference in DENV-1,2,4-specific nAbs post vaccination.

Similarly, individuals who were seropositive for DENV4 before vaccination, showed significantly 227 (p=0.0061) higher levels of DENV4-specific nAbs ( Fig 2L) . However, we did not observe any 228 increase in DENV1, 2, 3-specific nAbs in those subjects (Fig 2 I-K) . This serotype-dependent 229 response was not seen in individuals who were previously immune to DENV2 (Fig S1) . We 230 suspect that this is due to the fact that there were fewer subjects in the seropositive cohort that 231 are seropositive only to DENV2. 

Together, this data suggests that this vaccine candidate triggers a robust serotype specific 238 immune response in which seropositive individuals generate significantly high neutralizing 239 antibody titers against the serotype that they were previously exposed to. 

and found no significant differences in any of the major T-cell subsets such as total CD4+ T 253 cells, total memory cells, total CD3+CD4-cells that differentiate Immune and Naïve subjects (S3 254 Fig) . However, we detected a significantly higher (p=0.0082) frequency of total circulatory Tfh 255 cells (cTfh; marked by CD3+CD4+CD45RA-CXCR5+ expression) in the subjects that belonged 256 to the Immune group compared to those in the Naïve group ( Fig 3A) . While not statistically 257 significant, it is intriguing to note that subtypes of cTfh cells were different in Immune vs Naïve 258 subjects. One of the subtypes of cTfh cells, cTfh1 (marked by expression of CXCR3 within the 259 cTfh population) seems to be trending higher in the Immune group, while cTfh2 (marked by 260 absence of CXCR3/CCR6 expression within the cTfh population) seems to be trending higher in 539 Neutralizing antibody titers 540 Neutralizing antibodies to DENV were determined by plaque-reduction neutralization titer 541 (PRNT) assays as previously described (53). Briefly, for all doners, an initial serum dilution of 542 1/5 was used for PRNT 50 assays. Seropositivity was defined by PRNT 50 cutoff titers (≥1/10) 543 before immunization and at any timepoint up to 91 days after vaccination (day 28, 56, or 91).

For DENV-naive participants, seroconversion was defined by PRNT 50 cutoff titers (≥1/10). For 545 participants previously exposed to DENV, seroconversion was defined as a four-fold or higher 546 increase in pre-existing neutralizing antibody titer after immunization (53). 

Whitney test, 95% confidence was calculated for (C-F). Before vaccination, seropositive 833 subjects with existing antibodies to any of the four Dengue virus serotypes due to prior infection 834 were referred to as ("Immune") , or seronegative subjects with no existing antibodies to any of 835 the four Dengue virus serotypes were referred to as ("Naïve"). prior infection before were referred to as ("Immune") or seronegative subjects with no existing 911 antibodies to any of the four Dengue virus serotypes were referred to as ("Naïve"). Paired non-912 parametric Wilcoxon test, 95% confidence was calculated for (C). 

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S2 Fig: T Cell Panel Gating strategy

Immune subject 970 example of total cTfh at pre-vaccination/baseline time point . (C) Naïve subject example of total 971 cTfh at pre-vaccination/baseline time point. Before vaccination, seropositive subjects with 972 existing antibodies to any of the four Dengue virus serotypes due to prior infection were referred 973 to as

No difference at pre-vaccination frequency of major T cell subsets was observed 977 between seropositive (Immune) and seronegative (Naïve) groups

B) Y axis is the total Treg frequency 980 CD3+ CD4+ CD45RA-CXCR5-Foxp3+. (C) Y axis is the total CD3+ CD4-frequency . Pre-981 vaccination/baseline subjects PBMCs and optimized T cell panel for ex-vivo staining was 982 utilized. 1 million cells per panel were stained. Unpaired non-parametric Mann Whitney test 95% 983 confidence was calculated for (A-C). (Naive N=44

Seropositive subjects with existing antibodies to any of the four Dengue virus serotypes due to 985 prior infection were referred to as

No difference at pre-vaccination frequency of major B cell subsets was observed 989 between seropositive (Immune) and seronegative (Naïve) groups

A) Y axis is the total B cell frequency: CD19+. (B) Y axis is the total switched B cell frequency: 992 CD19+CD10-IgM-IgD-. (C) Y axis is the activated memory B cell frequency

Pre-vaccination/baseline subjects PBMCs and optimized B cell panel for ex-994 vivo staining was utilized. 1 million cells per panel were stained. D) Heatmap of the 31 different 995 clusters expressing different phenotypes using the optimized B cell panel with markers are 996 shown in the x axis. Among them is cluster 23 that represents a B cell plasmablast cluster. The 997 table identifies cell type, immune group pre-vaccination/baseline difference, breadth and 998 neutralizing antibody titers (AUCp)

Before 1006 vaccination, seropositive subjects with existing antibodies to any of the four Dengue virus 1007 serotypes due to prior were referred to as *"Immune") or seronegative subjects with no existing 1008 antibodies to any of the four Dengue virus serotypes were referred to as

Spearman Correlation with 95% confidence intervals was used for correlations with neutralizing 1011 antibody titers