Impact of Th1 CD4 TFH skewing on Antibody Responses to an Impact of Th1 CD4 TFH skewing on Antibody Responses to an HIV-1 Vaccine in Rhesus Macaques HIV-1 Vaccine in Rhesus Macaques

41 Generating durable humoral immunity through vaccination depends upon effective interaction of follicular 42 helper T cells (T fh ) with germinal center (GC) B cells. T h 1 polarization of T fh cells is an important process 43 shaping the success of T fh -GC B cell interactions by influencing co-stimulatory and cytokine-dependent T fh help 44 to B cells. However, the question remains whether adjuvant-dependent modulation of T fh cells enhances HIV-1 45 vaccine-induced anti-Envelope (Env) antibody responses. We investigated whether an HIV-1 vaccine platform 46 designed to increase the number of T h 1-polarized T fh cells enhances the magnitude and quality of anti-Env 47 antibodies. Utilizing a novel interferon-induced protein (IP)-10-adjuvanted HIV-1 DNA prime, followed by an 48 MPLA+QS-21-adjuvanted Env protein boost in macaques (D IP-10 P ALFQ ), we observed higher anti-Env serum 49 IgG titers with greater cross-clade reactivity, specificity to V1V2, and effector functions when compared to 50 macaques primed with DNA lacking IP-10 and boosted with MPLA+alum-adjuvanted Env protein (DP ALFA ) The 51 D IP-10 P ALFQ vaccine regimen elicited higher anti-Env IgG1 and lower IgG4 antibodies in serum, showing for the 52 first time that adjuvants can dramatically impact the IgG subclass profile in macaques. The D IP-10 P ALFQ regimen 53 also increased vaginal and rectal IgA antibodies to a greater extent. Within lymph nodes, we observed 54 augmented GC B cell responses and promotion of T h 1 gene expression profiles in GC T fh cells. The frequency 55 of GC T fh cells correlated with both the magnitude and avidity of anti-Env serum IgG. Together, these data 56 suggest that adjuvant-induced stimulation of T h 1-T fh cells is an effective strategy for enhancing the magnitude 57 and quality of anti-Env antibody response. The results of the trial demonstrated that vaccination could prevent HIV transmission in humans and that longevity of anti-Env antibodies may be key to this protection. Efforts to improve upon the prime-boost vaccine regimen used in RV144 have indicated that booster immunizations increase serum anti-Env antibody titers but only transiently. Poor antibody durability hampers efforts to develop an effective HIV-1 vaccine. This study was designed to identify the specific elements involved in the immunological mechanism necessary to produce robust HIV-1 specific antibodies in rhesus macaques. By clearly defining immune-mediated pathways that improve the magnitude and functionality of the anti-HIV-1 antibody response, we will have the foundation necessary for rational development of an HIV-1 vaccine. Based on evidence that T h 1-polarized T fh cells correlate with higher antibody responses, we set out to investigate empirically whether an HIV-1 vaccine platform designed to increase the number of T h 1-polarized T fh cells would enhance the functional quality and magnitude of HIV-1 anti-Env antibodies. Utilizing a novel interferon-induced protein (IP)-10-adjuvanted HIV-1 DNA prime, followed by an MPLA+QS-21- 157 adjuvanted Env protein boost in macaques (D IP-10 P ALFQ ), we show increased HIV-1 anti-Env specific binding 158 antibody in serum and mucosal compartments compared to vaccination with DNA lacking IP-10 and an 159 MPLA+alum-adjuvanted Env protein boost (DP ALFA ). The D IP-10 P ALFQ vaccine regimen augmented GC B cell 160 responses and promoted T h 1 gene expression profiles in GC T fh cells. The number of GC T fh cells positively 161 correlated with both magnitude and avidity of anti-Env specific antibody responses. We report for the first time that adjuvants dramatically impact IgG antibody subclass profile in rhesus macaques . We made the striking 163 observation that while both vaccine regimens induced IgG1 antibodies to gp120, the DP ALFA regimen generated 164 much greater IgG4 responses. Together, these data show that by stimulating production of T h 1-T fh cells during 165 the prime and boost using an adjuvanted vaccine, we can enhance the magnitude and function of the anti-HIV- 166 1 -Env antibody response. primes. Based on robust induction of antibody The HVTN studies 070 and 080 employed the IL-12 DNA adjuvanted plasmid with the subtype B PENNVAX-B 490 (PV) DNA plasmid and showed 80% response rates after the third DNA vaccination in PV+IL-12 recipients 491 compared to a 44% response rate with the PV alone vaccine. A subsequent follow up study demonstrated 492 robust recall of binding anti-Env antibody titers with ADCC activity following an MVA boost in PV+IL-12 493 recipients (44, 45). Because IL-12 is a classic innate mediator of T h 1 responses, the data suggest that an 494 increase in T h 1 GC T fh cells may underlie the observed effects. Correspondingly, studies in rhesus macaques 495 with an ALVAC prime, ALVAC + gp120 protein boost using SIV immunogens showed higher SIV Env titers with 496 MF59 compared to aluminum adjuvanted protein boosts 2 weeks following the final immunization(46). While T fh 497 responses and memory antibody titers were not examined, a recent study in humans showed enhanced 498 binding antibody titers 26 weeks after booster immunization with a T h 1 GLA-SE-adjuvanted malaria antigen 499 relative to one formulated in aluminum(47). These studies in conjunction with our report provide support to the 500 immune potential of T h 1-T fh cells in fostering high magnitude antibody titers. In contrast, a study using a 501 homologous subtype C protein immunization reported induction of higher anti-Env antibody titers with 502 aluminum-hydroxide (Ahydrogel) relative to Addavax, an MF59 analog, in rabbits(48). Collectively, these data 503 indicate the importance of detailed studies to understand the context in which T h 1 responses are superior to 504 mixed T h 1+2 responses and how viral versus DNA vectors and subunit proteins influence this paradigm. Our findings raise the question of the mechanisms underlying the D IP-10 Pro ALFQ vaccine-mediated enhancement 506 in T fh responses. A few possibilities can be explored; IP-10 increases dendritic cell-T cell interactions, which 507 could have effector volumetric diluted PBS-washed, (1 effector (5 uL HiSeq lane. Quality 799 of data were verified using the Illumina SAV viewer; this included verifying low rates based on 800 alignments of the standard Illumina PhiX spike, and removal of PCR after alignments. Adapter 801 trimming, QC of sequencing data & demultiplexing was performed by the UC Bioinformatics Core. After 802 read filtering, reads were mapped to a reference genome using HISAT-aligner. On average 82.17% (~55-61% 803 uniquely mapped) reads were mapped, and the uniformity of the mapping each sample indicated comparability between samples. Prior to differential gene expression analysis, genes