CROI 2020 Abstract eBook

Abstract eBook

Poster Abstracts

285 METHOTREXATE BLOCKS PROLIFERATION NOT INFLAMMATION TO MODULATE IMMUNITY Michael L. Freeman 1 , Daniela Moisi 1 , Brian Clagett 1 , Leonard Calabrese 2 , Benigno Rodriguez 1 , Michael M. Lederman 1 1 Case Western Reserve University, Cleveland, OH, USA, 2 Cleveland Clinic, Cleveland, OH, USA Background: Inflammation associated with increased risk of comorbidities persists in people living with HIV (PLWH) on combination antiretroviral therapy (ART). Low-dose methotrexate (MTX) is an anti-inflammatory treatment for rheumatological disorders. A recent placebo-controlled trial (ACTG A5314) of low-dose MTX in PLWH found no changes in plasma inflammatory indices, but numbers of total and cycling (Ki-67+) CD4 and CD8 T cells decreased in the low-dose MTX arm. Methods: Effects of MTX (up to 100nM) on the release of IL-1ß, IL-6, IFNγ TNF, and IL-2 by PBMCs from PLWH (n=6) or HIV-uninfected controls (n=6) was measured by ELISA in culture supernatant following exposure to LPS, flagellin, antibodies to CD3 and CD28 (anti-CD3/CD28), or medium control. Effects of MTX on T cell proliferation (CellTrace Violet dilution), activation (CD69 and CD25 expression), and numbers from PLWH (n=6) or HIV-uninfected controls (n=11) were measured by flow cytometry following stimulation with anti-CD3/CD28, IL-2, IL-7, IL-15, or medium control. Results: At concentrations up to 100nM, MTX treatment did not inhibit IL-1ß and IL-6 release in response to LPS or flagellin; IFNγ, TNF, and IL-2 release in response to anti-CD3/CD28; or T cell activation (CD69 and CD25 expression) in response to stimulation with anti-CD3/CD28, IL-2, IL-7, or IL-15. T cell proliferative responses to anti-CD3/CD28 were inhibited by MTX (median IC 50 = 42.07nM), but IC 50 s were similar among cells from PLWH and controls (P = 0.099), with similar results seen for IL-2, IL-7, and IL-15. MTX treatment during anti-CD3/CD28 stimulation also resulted in a significant decrease in T cell numbers, suggesting activation-induced cell death. Addition of folinic acid (1μM) restored T cell proliferation to anti-CD3/CD28 stimulation, but did not rescue cell numbers. Conclusion: Our findings indicate that MTX at concentrations up to 100 nM does not inhibit expression of IFNγ, TNF, and IL-2 in response to T cell receptor (TCR) stimulation or IL-1ß and IL-6 after stimulation with the TLR4 and TLR5 agonists LPS and flagellin in vitro. Proliferation of CD4 and CD8 T cells in response to TCR and common gamma chain cytokine stimulation is profoundly reduced by MTX and is associated with cell death in vitro. Folinic acid could restore T cell proliferation, but did not fully rescue cell death. Our data are fully consistent with the in vivo effects of MTX in PLWH suggesting that the major effect of MTX on immune function is an inhibition of cellular proliferation. 286LB ENHANCED COMPLEMENT ACTIVITY DOES NOT IMPROVE PROTECTION IN SHIV-CHALLENGED MACAQUES David A. Spencer 1 , Benjamin Goldberg 2 , Jérémy Dufloo 3 , Timothée Bruel 3 , Olivier Schwartz 3 , Margaret Ackerman 2 , Ann J. Hessell 1 1 Oregon Health and Sciences University, Portland, OR, USA, 2 Dartmouth College, Hanover, NH, USA, 3 Institut Pasteur, Paris, France Background: Fc modified bNAbs are being developed for prophylactic and therapeutic treatment of HIV. Extended half-life and reduced immunogenicity modifications have proven effective, but attempts to improve bNAb efficacy by enhancing affinity for Fcg Receptors alone have not worked in SHIV-challenged macaques. In this model, ablating FcgR binding reduced protection, but the role of complement appeared limited. We hypothesized improving bNAb Fc-mediated complement activity and increasing affinity for FcgR would strengthen protection. Methods: We developed 10 Fc variant bNAbs with site mutations designed to increase CDC activity, C1q binding, and FcgR affinity and evaluated each for binding to FcgRs, C1q and infected cells plus functional CDC, ADCC and ADCP activity. MPER targeting 10E8v4 that weakly neutralizes SHIVSF162P3 (IC 50 30 mg/ml) and mediated Complement activity in vitro, but does not mediate ADCC was selected for macaque studies. Protection was evaluated with a single high dose intrarectal SHIVSF162P3 challenge 3 days after 5 mg/kg mAb infusion. Groups of 6 macaques received either unmodified 10E8v4, 10E8v4- LALA (Complement/FcgR dual knockout), 10E8v4-EFTAE, (>2-fold enhanced Complement deposition, viral lysis, and CDC, increased affinity for FcgRs with no ADCC or increased ADCP), or a control mAb. Blood draws monitored viremia, mAb kinetics, and neutralizing titers. Results: Unexpectedly, mean plasma viral loads (PVL) were elevated in the EFTAE group compared to unmodified 10E8v4 (P<0.0001) and LALA groups

(P=0.0070). Viremia was starkly increased in multiple lymphoid and gut tissues in the EFTAE group, over unmodified 10E8v4 (P<0.0001), LALA (P=0.0270), and control (P<0.0001) groups. EFTAE mutations led to lower serum concentrations and neutralizing titers at challenge and reduced serum half-life. Higher doses of 10 and 20 mg/kg EFTAE or unmodified mAb led to comparable PVL, suggesting neutralizing titers may mitigate effects of increased complement. Mechanistic studies show splenocytes treated with sub-neutralizing EFTAE increased infection over controls dependent on the presence of monocyte derived DCs. Conclusion: Our studies imply enhancing CDC in vitro may not predict in vivo function and supports evidence that increased affinity for FcgRs may not enhance protection. Implications of complement opsonization of HIV inhibiting effector cell function warrant further study. Importantly, consequences seen here of modulating complement in HIV infection may forewarn clinical safety and therapeutic trials with modified Fc bNAbs. 287 THE RV144 VACCINE PRIMED IgG4 AND V1V2-ADCP RESPONSES IN HIV BREAKTHROUGH INFECTIONS Thembi Mdluli 1 , Dominic Paquin-Proulx 1 , Bonnie Slike 1 , Gina Donofrio 1 , Sorachai Nitayaphan 2 , Punnee Pitisutthithum 3 , Supachai Rerks-Ngarm 4 , Margaret Ackerman 5 , Merlin L. Robb 1 , Nelson L. Michael 1 , Sandhya Vasan 1 , Michael A. Eller 1 , Shelly J. Krebs 1 , Morgane Rolland 1 , for the RV144 study group 1 US Military HIV Research Program, Silver Spring, MD, USA, 2 Armed Forces Research Institute of Medical Sciences in Bangkok, Bangkok, Thailand, 3 Mahidol University, Bangkok, Thailand, 4 Ministry of Public Health, Nonthaburi, Thailand, 5 Dartmouth College, Hanover, NH, USA Background: The RV144 vaccine efficacy trial showed a reduction in HIV infections that associated with stronger binding antibody (Ab) responses against Env variable loops V1V2. High levels of Ab-dependent cellular cytotoxicity (ADCC) together with low plasma levels of Env-specific IgA also correlated with decreased infection risk. Exact mechanisms of prevention remain unclear. Methods: To better understand the effect of vaccine priming, we performed a systems serology analysis in breakthrough infections in 42 vaccine and 63 placebo recipients, using plasma samples collected 6, 12 and 36 months after HIV diagnosis. We analyzed Ab binding features corresponding to immunoglobulin (Ig) subclasses and nine variant Fc gamma receptors (FcgR) (tested against 51 HIV antigens (Ag) for a total of 896 immune variables) together with functional Ab responses: ADCC, Ab-dependent cellular phagocytosis (ADCP), trogocytosis, NK cell activation measured by ICS and neutralization. Machine-learning analyses included clustering, CART, LASSO logistic model, and Partial Least Squares (sPLS). We filtered redundant immune features to reduce the number of variables to 221, 286 and 284 at 6, 12 and 36 months, respectively. Results: RV144 vaccination primed B cells responses post HIV-1 infection. Vaccinees were classified by a specific Fc binding profile with IgG4 responses as the strongest distinguishing feature dominating until 3 years after diagnosis. When effector functions were included, vaccinees were characterized by strong V1V2-specific Ab responses synergized with V1V2-specific ADCP responses, whereas placebo recipients had stronger IgG3 and gp120-specific responses. The development of neutralization breadth, which was linked to gp120/gp140 binding features, did not cluster with the vaccine group. Conclusion: Our results showed that the RV144 vaccine primed a specific IgG4 and V1V2-ADCP-dominated profile post-breakthrough infection while it did not prime broad neutralizing responses.These findings substantiate the importance of V2-specific binding Abs which were previously identified as a correlate of decreased risk of HIV infection in RV144 and show that vaccine-induced responses had consequences post HIV infection. By contrasting the immediate (post-vaccination, pre-infection) and long-term (post-infection) impact of vaccine priming, we can obtain a novel understanding of vaccine-elicited immunity, with characteristic features that can be harnessed to design more efficacious vaccine strategies. 288 PHASE I/IIA TRIAL OF HIV CLADE C DNA WITH MF59- OR AS01B- ADJUVANTED CLADE C PROTEIN Nigel Garrett 1 , Cynthia L. Monaco 2 , Philipp Mann 3 , Julia Hutter 4 , Allan C. deCamp 3 , One Dintwe 3 , Georgia Tomaras 5 , Megan Jones 3 , Giuseppe Pantaleo 6 , Song Ding 7 , Olivier Van Der Meeren 8 , Julie McElrath 3 , Larry Corey 3 , James G. Kublin 3 , for the HVTN 108 Study Team

Poster Abstracts

98

CROI 2020