We show that intensification of treatment with maraviroc in patients chronically infected with HIV-1 receiving successful long-term ART was not associated with improvements in HIV related morbidity, HIV reservoir, microbial translocation, immune activation or immune exhaustion in either gut or peripheral blood. The measurement of reservoir in both gut and blood longitudinally contributes to a paucity of data in the area

Cason, J

Davies, O

Fidler, S

Fox, J

Frater, John

Hopkins, Emily

Kaye, S

Klenerman, Paul

Mant, C

Meyerowitz, Jodi

Pace, Matt

Patel, K

Phalora, Prabhjeet

Ray, S

Robinson, Nicola

Sanderson, J

Tiraboschi, J

Wang, Y

AIDS Research and Human Retroviruses

English

Oxford University Research Archive

1  The effect of Maraviroc intensification on blood and gut reservoir   Juan Tiraboschi1, Sarah Fidler,3 Ranjababu Kulasagaram1, Matt Pace2, Prabhjeet Phalora2, Nicola Robinson, Yanzhong Wang5, Olubanke Davies1, Paul Klenerman2, John Cason5, Steve kaye3, Jeremy Sanderson1, Shuvra Ray1, Kemal Patel1 , John Frater,2 Julie Fox1,  1Harrison Wing, 2nd Floor Lambeth Wing, St. Thomas’ Hospital, London, SE1 7EH  1. Guys and St. Thomas' NHS Foundation Trust, London, United Kingdom 2. University of Oxford, Oxford, United Kingdom  3. Imperial College London, London, United Kingdom 4. Chelsea and Westminster Hospital NHS Foundation Trust, London, United Kingdom 5. Kings College London, London, UK   Journal:  AIDS RE HUM Retrovirus: - Short Communications, also subject to peer-review, are intended for the presentation of brief observations that do not warrant a full-length text, but also are not preliminary results. Include an abstract; do not use section headings in the body of the text; report methods, results, and discussion in a single section. Reference citations are identical in style to those of full-length papers, but appropriately abbreviated.  These submissions should not exceed 1,200 words with no more than 10 references and one table OR figure.  The word count does not include the abstract, acknowledgments, disclosure information, references, figure legends, or tables. - Letters to the Editor(s) are welcomed, but with a 500 word limit and no more than one (1) table OR figure, and with a maximum of four (4) references.  OR J virus eradication: http://viruseradication.com/page/author-instruction/   2  Background Despite the huge success of antiretroviral therapy (ART), increased morbidity compared with HIV uninfected individuals continues (Volberding Lancet 2010), particularly in those with a blunted CD4 response to ART (Dronda 2002; Goicoechea 2006 ; Kaufmann 2003). A cure for HIV remains elusive and this is in part due to an inaccessible long-lived pool of latently infected cell; the viral reservoir, which is focused in the gut-associated lymphoid tissue (Chun PNAS 1997).    Intensification of ART in individuals with suppressed plasma viremia has not consistently reduced viral reservoir or improved immune function (Puertas AIDS 2014; Ananworanich 2015; Gutierrez PloS One; Ghanid 2010; Hatano 2010). Results from the C-C chemokine receptor type 5 (CCR5)  entry inhibitor, maraviroc, have been less conclusive and suggested a CD4 count rise (Asmuth JAIDS 2010; Cuzin 2012), decline in immune activation (Funderburg 2010; Gutierrez PloS One ) and favorable tissue penetration (Walker Xenobiotic 2008:Fox 2016). The gut been investigated to a lesser extent (Lafeuillade 2014; Ananworanich 2015), with no consensus on microbial translocation (Abad-Fernández HIV Med 2015; Gutierrez PLOS One 2011; Hunt 2013), immune activation (Hunt 2013) and a novel finding that lymphocyte changes in blood merely reflect T cell redistribution from the gut (Hunt 2013).  We investigated the effect of maraviroc intensification on gut immune function and reservoir.   Methods Study Design and recruitment A prospective study whereby 10 individuals with chronic HIV infection with sustained virological suppression on ARV for >12 months and a CD4+ T cell count below 500 cells/mm,3 were intensified with a 24-week course of maraviroc. Blood samples were collected at week 0, 4, 12 and 24.. At baseline and week 24, individuals underwent flexible sigmoidoscopy, whereby eight-ten rectal biopsies were taken and fixed overnight in 10% normal buffered formalin at 4°C.  Laboratory Tests  Bacterial translocation Plasma bacterial 16s DNA was quantified by real time PCR. Plasma sCD14 was quantified using the QuantikineH Human sCD14 Immunoassay (R&D Systems, Minnesota, USA), according to manufacturer’s instructions. All samples were run in duplicate.  3  HIV Reservoir and residual Viraemia Low copy viral load was measured with an internally controlled ultrasensitive quantitative real-time RT-PCR (single copy assay) able to detect 3 copies/ml. Purified peripheral blood CD4 T cells were analyzed by qPCR for HIV-1 DNA (Total and Integrated) and cell-associated HIV-1 RNA unspliced transcripts (CA-RNA) as reported elsewhere (Williams 2014).  For preparation of the gut biopsies for HIV reservoir quantitation, a commercial kit was used (Qiagen, Cat. No. 56404), and the manufacturer’s protocol modified as follows to ensure maximum yield of DNA. Biopsy sections were removed from paraffin blocks using a scalpel and placed into 1.5ml micro centrifuge tubes. Samples were washed repeatedly with xylene and ethanol until the  precipitate had disappeared, then dried at room temperature.  Biopsy sections were incubated at 37°C with Buffer ATL and Proteinase K overnight, or until the sample had fully degraded, followed by 1 hour incubation at 90°C. Equal volumes of premixed Buffer AL and 100% ethanol were added before the samples were loaded into DNA extraction columns and spun by centrifugation at 8000 rpm for 1 min. Columns were washed with Buffer AW1 and Buffer AW2 and residual liquid removed by centrifugation before eluting DNA with 200μL RNase free water and spinning at 13,000 rpm for 1 minute.  The resulting DNA was analysed by qPCR using the same assays for peripheral blood CD4 T cells detailed above.  Immune activation and lymphocyte subsets  PBMC were stained with the anchor markers (CD3- VioBlue, CD4(VIT4)-VioGreen, CD8-APC) and a Live/Dead marker Near IR- APC-Cy7 plus either an activation panel (CD25(3G10)-PE, CD38-PE-Vio770, CD69-FITC, Anti-HLA-DR-PerCP) or an exhaustion panel (TIGIT-PE, TIM-3-FITC, LAG-3-PerCPeF710, PD1-PE-Cy7). Cells were run on a MACSQuant and analysed with FlowJo software v10 (Miltenyi Biotec).    Immunohistochemistry  Rectal biopsy sections were stained for CD4 (goat polyclonal, Novus Biologicals) and CD8 antibodies (rabbit polyclonal, Abcam). Images were analysed in ImageJ and the distribution of cells determined as described in Milano (2011).  Statistics For each outcome measure, the results at baseline and week 24 were compared using the paired T-test. Wilcoxon rank test was used to assess the impact of tropism on each parameter. 4   Results: Patient characteristics (table 1)  The median age was 46 years. All patients were male with subtype B virus and were receiving two nucleoside reverse transcriptase inhibitors (NRTI) combined with either a non-nucleoside reverse transcriptase inhibitor (NNRTIs) (n=5) or protease inhibitor (n=5).  The mean CD4 T cell count was 321 cells/uL (SD 112), CD4:CD8 0.51 (SD 0.27) and nadir CD4 117 cells/uL (SD 135).   All patients maintained an HIV viral load <50 copies/ml throughout the study. Maraviroc was well tolerated. One patient was lost to follow up at week 24.   Baseline to week 24:  - Clinical outcome / T-cell subsets composition in blood and gut Between baseline and week 24 in the blood, there was a non-significant increase of 48.22 CD4 T cells/uL (p=0.275) and 39.23 CD8 T cells/uL (p=0.672) whilst CD4:CD8 did not change. This was accompanied by a non-significant reduction in gut CD4 (-0.804 p= 0.087) and gut CD8 T cells (-5.59 p=0.052). Changes in blood CD4 and CD8 did not correlate with changes in these cells in the gut.  - Bacterial translocation Between baseline and week 24 there was no significant change over time in 16s DNA copies (change -0.6;p=0.797) or sCD14a (change +59.35  p=0.637).  - HIV reservoir Neither HIV RNA, HIV DNA (blood or gut) or cellular HIV RNA changed significantly between baseline and week 24 or between baseline and week 12.  However, gut HIV DNA levels (mean 101805.93 copies/million cells) were significantly higher than blood (3669.41 copies/million CD4 T cells') at baseline (p=0.017).    - T-cell activation and exhaustion The level of activation of CD4 + T cells and CD8+ T cells showed non- significant increases from baseline to week 24 in 6 parameters (including CD8+CD38+HLADR;p=0.677) with only %CD8+HLADR (+0.61 p=0.048) increasing significantly. These findings were the same when analysed from baseline to week 12 with only %CD8+ HLADR+ showing a significant change (+0.47; p= 0.001).  5  Eight out of 10 immune exhaustion markers increased from baseline to week 24 but only CD8% Tigit reached significance (+6.07; p=0.006). This was supported by analysis of week 0 to week 12 (% CD8 Tigit (mean increase +5.82; p=0.004).  Tropism: Six out 10 individuals had R5 virus. Tropism (R5 v X4 or X4/R5) did not correlate with changes in reservoir, immune activation , immune exhaustion or microbial translocation  (all P values > 0.05).   6  Discussion: In this pilot study, intensification of treatment with maraviroc in patients chronically infected with HIV-1 receiving successful long-term ART with a blunted CD4 immune response was not associated with any improvements in surrogate markers of HIV related morbidity (CD4 T cell count or CD4:CD8 ratio), microbial translocation, immune activation or immune exhaustion in both gut and peripheral blood. Furthermore, reservoir did not decline over time in either gut or blood. The results concur with the majority of research and reflect more resistant immune dysfunction than intensification with antiretroviral therapy can repair.  This may reflect the short duration of follow up or the fact that these individuals had no further capacity to improve their immune function and that individuals with a higher nadir CD4 count may show a different result.   The lack of impact of tropism on the effect of maraviroc on immunological function or reservoir size is novel but limited due to small sample size.  The large gut HIV reservoir supports interventions targeting this compartment. However the large intra-individual variation confirms that sample sizes may be large.     Overall, the study shows no role in maraviroc intensification to improve clinical outcome. The study is limited by the small sample size and lack of control group but the findings are in agreement with other similar studies in chronic infection. 7  References 1. Volberding PA, Deeks SG. Antiretroviral therapy and management of HIV infection. Lancet. 2010 Jul 3;376(9734):49-62 2. Dronda F, Moreno S, Moreno A, Casado JL, Perez-Elias MJ, Antela A. Long-term outcomes among antiretroviral-naive human immunodeficiency virus-infected patients with small increases in CD4+ cell counts after successful virologic suppression. Clin Infect Dis. 2002;35:1005–1009. 3. Goicoechea M, Smith DM, Liu L, May S, Tenorio AR, Ignacio CC, Landay A, Haubrich R. Determinants of CD4+ T cell recovery during suppressive antiretroviral therapy: association of immune activation, T cell maturation markers, and cellular HIV-1 DNA. J Infect Dis. 2006;194:29–37 4. Kaufmann GR, Perrin L, Pantaleo G, Opravil M, Furrer H, Telenti A, Hirschel B, Ledergerber B, Vernazza P, Bernasconi E, Rickenbach M, Egger M, Battegay M. CD4 T-lymphocyte recovery in individuals with advanced HIV-1 infection receiving potent antiretroviral therapy for 4 years: the swiss HIV cohort study. Arch Intern Med. 2003;163:2187–2195. 5. Chun TW, Fauci AS Latent reservoirs of HIV: obstacles to the eradication of virus. Proc Natl Acad Sci U S A 1999; 96:10958–61.  6. Puertas MC, Massanella M, Llibre JM, Ballestero M, Buzon MJ, Ouchi D, Esteve A, Boix J, Manzardo C, Miró JM, Gatell JM, Clotet B, Blanco J, Martinez-Picado J; MaraviBoost Collaborative Group. Intensification of a raltegravir-based regimen with maraviroc in early HIV-1 infection. AIDS. 2014 Jan 28;28(3):32 5-34 7. Ananworanich J, Chomont N, Fletcher JL, Pinyakorn S, Schuetz A, Sereti I, Rerknimitr R, Dewar R, Kroon E, Vandergeeten C, Trichavaroj R, Chomchey N, Chalermchai T, Michael NL, Kim JH, Phanuphak P, Phanuphak N. Markers of HIV reservoir size and immune activation after treatment in acute HIV infection with and without raltegravir and maraviroc intensification. J Virus Erad. 2015;1(2):116-122. Epub 2015  8. Gutiérrez C, Díaz L, Vallejo A, et al. Intensification of antiretroviral therapy with a CCR5 antagonist in patients with chronic HIV-1 infection: effect on T cells latently infected. PLoS ONE 2011;6(12):e27864 9. Gandhi RT, Bosch RJ, Aga E, Albrecht M, Demeter LM, et al. (2010) No evidence for decay of the latent reservoir in HIV-1-infected patients receiving intensive enfuvirtide-containing antiretroviral therapy. J Infect Dis 201: 293–6. 10. Hatano, H., et al. A randomized, controlled trial of raltegravir intensification in antiretroviral-treated, HIV-infected patients with a suboptimal CD4+ T cell response. The Journal of 8  infectious diseases 203, 960-968 (2011).  11. Asmuth DM, Goodrich J, Cooper DA, Haubrich R, Rajicic N, Hirschel B, et al. CD4+ T-cell restoration after 48 weeks in the maraviroc treatment-experienced trials MOTIVATE 1 and 2. J Acquir Immune Defic Syndr. 2010;54: 394–7.  12. Cuzin L, Trabelsi S, Delobel P, et al. ; ANRS 145 MARIMUNO Study Group. Maraviroc intensification of stable antiviral therapy in HIV-1-infected patients with poor immune restoration: MARIMUNO-ANRS 145 study. J Acquir Immune Defic Syndr 2012;61(5):557-564.  13. Funderburg N, Kalinowska M, Eason J, Goodrich J, Heera J, Mayer H, et al. Effects of maraviroc and efavirenz on markers of immune activation and inflammation and associations with CD4+ cell rises in HIV-infected patients. PLoS One. 2010;5: e13188  14. Walker DK, Bowers SJ, Mitchell RJ, Potchoiba MJ, Schroeder CM, Small HF. Preclinical assessment of the distribution of maraviroc to potential human immunodeficiency virus (HIV) sanctuary sites in the central nervous system (CNS) and gut-associated lymphoid tissue (GALT). Xenobiotica. 2008;38: 1330–9.  15. Fox J, Tiraboschi J, Herrera C, Else L, Dickinson L, Back D, Khoo S, Boffitto M. Pharmacokinetic/pharmacodynamic investigation of single dose oral maraviroc in the context of HIV-1 pre exposure prophylaxis (PrEP). J Acquir Immune Defic Syndr 2016 in press 16. Lafeuillade A, Assi A, Poggi C, Bresson-Cuquemelle C, Jullian E, Tamalet C. Failure of combined antiretroviral therapy intensification with maraviroc and raltegravir in chronically HIV-1 infected patients to reduce the viral reservoir: the IntensHIV randomized trial. AIDS Res Ther. 2014 Oct 7;11(1):33 17. Abad-Fernández M, Gutiérrez C, Madrid N, Hernández-Novoa B, Díaz L, Muñoz-Fernández MA, Moreno S, Vallejo A. Expression of gut-homing β7 receptor on T cells: surrogate marker for microbial translocation in suppressed HIV-1-infected patients? HIV Med. 2015 Jan;16(1):15-23. . 18. Hunt PW, Shulman NS, Hayes TL, et al. The immunologic effects of maraviroc intensification in treated HIV-infected individuals with incomplete CD4+ T-cell recovery: a randomized trial. Blood 2013;121(23):4635-4646. 19. Milano et al, 2011. An easy cell counting method for immunohistochemistry that does not use an image analysis program. Bologna –Histopathology, Oct;59(4):801-3.    9    Results Table 1: Demographic and baseline characteristics   Total N=10 Demographics  Age in years mean (SD 35 Gender, n (%)  Female 0 Male 10 Ethnicity n (%)     White 5    Black 3    Oriental 1    Mixed BC/white 1 CD4  CD4:CD8 ratio  HIV RNA <20 Nadir CD4 117 HBVs Ag  Yes 0 No 10 HCV IgG  Yes 0 No 10 ARV regime - PI based - NNRTI based - Other 5 5 0 CCR5 tropism -R5 - X4 - mixed  3 2 5  10    Table: Mean changes in parameters between week 0 and 24 Outcome variable Mean (SD) Week 0 (visit 2) N=10 Mean (SD)  Week 24 (visit 5)  N=9 Mean difference  (week 24 - week 0) N=9  P.value  Primary Outcome     plasma16s DNA  7.95(3.7)  7.69(6.73)  -0.6  0.7972  scd14s  1781.56(355.16)  1783.02(438.14)  59.35  0.6368       Secondary Outcomes     Peripheral Blood     CD4count  334.4(131.64)  407.56(154.63)  48.22  0.2751  CD4%  24.39(8.77)  24.39(7.02)  -1.29  0.3349  CD4:CD8 ratio  0.51(0.27)  0.54(0.21)  0  1       Reservoir     HIV DNA copy CD4 T cell  3669.41(1902.92)  3181.33(3484.3)  -343.76  0.5594  Cell HIV RNA copy per 18s RNA  2755.62(4400.75)  991.18(1130.88)  -1295.49  0.5279  Low copy plasma HIV RNA 16.1(14.96)  38.33(38.12)  21.78  0.1424       Immune Exhaustion     % CD8 lag3  0.91(1.08)  0.44(0.22)  -0.52  0.2669  % CD8pd1  0.27(0.23)  0.55(0.41)  0.27  0.1762  % CD8 tigit  47.23(13.79)  52.08(16.23)  6.07  0.0059  % CD8 tim3  0.1(0.09)  0.11(0.1)  0  0.9191  % CD4 lag3  0.24(0.21)  0.26(0.2)  0.08  0.3693  % CD4 pd1  1.01(1.08)  1.64(1.24)  0.98  0.0596  % CD4 tim3  0.32(0.35)  0.26(0.2)  -0.01  0.9074  % CD4 tigit  26.56(10.01)  25.3(10.36)  1.63  0.5215       Immune activation     % CD4CD25  1.73(0.89)  2.19(2.07)  0.58  0.4762  % CD4CD38  0.95(0.52)  1.15(0.94)  0.37  0.2302  % CD4CD69  1.4(1.15)  1.29(0.89)  0.21  0.3268  % CD4 HLA DR  4.74(2.5)  5.08(3.01)  1.32  0.163  11  % CD8CD38  45.67(27.47)  31.59(34.97)  -16.37  0.3669  % CD8CD25  2.04(1.3)  1.77(1.77)  -0.63  0.3275  % CD8CD69  5.7(4.34)  3.66(1.82)  -2.16  0.326  % CD8 HLA DR  0.82(0.59)  1.22(0.9)  0.61  0.0482  % CD8CD38 HLA DR  0.82(0.59)  1.23(0.84)  0.58  0.0326  Gut tissue     Reservoir     Gut HIV DNA copy  101805.93(117858.81)  51468.83(109191.98)  -44062.84  0.4738       Cell composition     Gut total cell number 93.9(22.33)  93.59(28.52)  1.26  0.9006  Gut CD4cell number 22.9(12.52)  16.41(9.98)  -8.04  0.0873  Gut 2 total cell number 95.17(11.9)  80.19(16.02)  -13.81  0.0199  Gut CD8 cell  number  11.5(5.79)  6.26(4.57)  -5.59  0.0522  Gut Proportion of cells CD4  0.25(0.16)  0.18(0.12)  -0.09  0.0644  Gut Proportion of cells CD8 0.12(0.05)  0.08(0.05)  -0.05  0.0898   Note: 1. Outcome variables are exact match of column C to AG in the compiled Oxford lab data spreadsheet. 2. 10 patients had week 0 (visit 2) data, but only 9 patients had week 24 (visit 5) data. Patient with study number 12 didn’t have week 24 (visit 5) data. 3. Mean changes are based on 9 patients with both visit 2 and 5 data. 4. P-values are calculated based on paired t-test on 9 patients. 5. SD: Standard deviation 12       

Lack of effect of Maraviroc intensification on blood and gut reservoir.

ORA - Oxford University Research Archive

Pace, M

Phalora, P

Robinson, N

Hopkins, E

Meyerowitz, J

Klenerman, P

Frater, J

Oxford University Research Archive (ORA)

https://ora.ox.ac.uk/objects/uuid:036fcb12-7c5e-4a07-8011-df627f837149/files/m6ebfbf9ee7000433c37609582c2de055

Lack of effect of Maraviroc intensification on blood and gut reservoir.

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