ANALYSIS OF HOST AND VIRAL FACTORS IN THE ELITE SUPPRESSION OF HIV-1 INFECTION. Robert W. Buckheit III

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1 ANALYSIS OF HOST AND VIRAL FACTORS IN THE ELITE SUPPRESSION OF HIV-1 INFECTION By Robert W. Buckheit III A dissertation submitted to Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, MD

2 ABSTRACT HIV-1 infection is typically characterized by robust viral replication and progressive CD4 + T cell depletion in the absence of antiretroviral therapy. Some unique individuals, termed elite suppressors (ES) have the remarkable capacity to restrict viral replication below the limit of detection of standard clinical testing (<50 HIV-1 RNA copies/ml of plasma). Interestingly, while ongoing replication occurs in ES, the number of latently infected cells has been shown to be reduced compared to chronic progressors. The mechanisms by which these individuals control viral replication are unknown, but a better understanding of both the viral and host factors that contribute to this disease course could aid in the development of a preventative HIV-1 vaccine. This thesis provides an analysis of both viral and host factors that contribute towards the control of HIV-1 infection. These results demonstrate that, in some cases, ES are infected with virus that has the capacity to cause progressive disease, indicating that ES possess a unique capacity to restrict viral replication independent of the fitness of the infective virus. Next, we focus our attention on the cytolytic CD8 + T cell response, using the most physiological CD8 + T cell suppression assay to date, which utilizes unstimulated CD8 + T cell effectors and CD4 + T cell targets. Using this assay, we examined the ability of individual activation and memory CD8 + T cell populations to suppress viral replication. We determined that the effector memory population was most effective at suppressing viral replication quickly, but phenotypic shifts in the CM population allowed for an increase in CD8 + T cell suppression overtime. Additionally, using a variation of this assay, designed to analyze cell elimination, we determined that ES CD8 + T cell can ii

3 eliminate non-productively infected cells, potentially allowing ES to prevent the seeding of the latent reservoir early during viral infection. Taken together, these data suggest that a potent CTL response is a key component in the control of viral replication that is mediated by HIV-1 infected elite suppressors. Further study is needed to unravel the complex interplay of the innate and acquired immune system, and whether the CTL response alone dictates control of HIV-1 infection. Readers/Advisors: Dr. Joel N. Blankson and Dr. Robert F. Siliciano iii

4 PREFACE I would like to thank the Cellular and Molecular Medicine Graduate program for allowing the opportunity to perform my graduate studies at Johns Hopkins. From the first visit to Baltimore, they have been extremely welcoming and supportive, and for that I am very grateful. Johns Hopkins was a wonderful place to complete my Ph.D., and allowed a unique blend of rigorous academic coursework in a clinical environment that was truly collaborative and collegiate. The potential to do translational, bench to bedside research was the notion that brought me to Hopkins initially, and I have had the unique experience of working an extremely challenging and unique clinical research that delved into the nature of HIV-1 infection and control. I would like to thank Drs. Andrea Cox, Chuck Drake and Stuart Ray as members of my Thesis Committee for their support and guidance, and for taking time out of their own busy schedules to help nurture my professional and personal development. Special thanks must be given to Dr. Stuart Ray for serving as my thesis committee chair. I am cognizant of the fact that I would not be where I was today without the help and support of many people throughout my life. I have been blessed to have grown up with many opportunities and an environment where learning was a pleasure, not a chore, and where I was encouraged to work hard to succeed. For this, I would like to thank my parents for their unwavering support throughout the years, and for encouraging me, at all times, to excel beyond my means. In addition, I would like to thank both of my sisters for pushing me to be a better brother and a better man. Without my family, I would not be the scientist, and more importantly, the person I am today. iv

5 I would also like to thank my mother-, father- and brother-in-law for their unconditional enthusiasm and support of my goals to get my Ph.D. I was consistently supported over my trek through graduate school, and I am forever grateful for their willingness and patience with my decision to come to graduate school. Many, if not all, of the furniture and kitchen supplies in my shoebox apartment in Fells Point were graciously gifted to me, in addition to the trove of new learning at how to be a homeowner. I could not ask for a better addition to my family and more respectable individuals to call my parents, brother and role models. I would also like to thank all of the people in my life that made science fun and exciting. From my elementary and high school science teachers, the college professors, and my Dad, a love of science was instilled at a young age, and nurtured over the years. No single instance made me want to be a scientist, but a love of learning and the development of an inquisitive nature was encouraged at all times, and cultured for my own development as a scientist. Additionally, I would be remiss to not mention my thanks and gratitude to my wife. For graciously, and generously accepting my decision to attend Hopkins in Baltimore instead of a closer school in Philadelphia, which was two hours closer to where she lived, for supporting my decision to continue my education, and for being my point of sanity and support when needed, I cannot thank her enough. To her, I would say that no words can ever express my thanks and love for all of the incredible things you do, and I cannot tell you how proud and happy I am to call you my wife. So, I simply say thank you, and know that you understand the depth of my caring and appreciation. v

6 Finally, I owe a debt of gratitude to Dr. Robert Siliciano and Dr. Joel Blankson. The Siliciano lab is a wonderful and supportive environment in which to complete a graduate thesis. I have had the pleasure of meeting, and collaborating with some of the smartest individuals that I have had the privilege to know, and I thank Dr. Siliciano for maintaining a friendly and collegiate environment in which to work. Personally, Bob has been an incredible role model for scientific development and proving you can be a great scientist without sacrificing your moral center. When I joined the lab, I was only Joel s second student, and I cannot say enough great things about my experience. I was quickly challenged with the pace and rigor of my scientific development, and have truly learned beyond the bounds of my own abilities. Joel has been consistently supportive, while providing drive and motivation to produce quality data. I have learned many things from Joel, but mainly I am in awe of Joel s work ethic, intelligence, drive, and ability to break down complex questions into manageable, answerable questions. Joel is a great role model for my career, a great physician-scientist, and I wish him nothing but the best in the future. Finally, for my own benefit, a mean to reminisce and potentially a learning or relearning opportunity for myself or others, these years in graduate school have been some of the most exciting times in my life. After graduating from college, I moved to Baltimore and had the opportunity to live as a city-slicker, albeit a medium sized city city-slicker, but nonetheless, my parallel parking skills are second to none. Additionally, during my time in graduate school I maintained a long distance relationship, started an Alumni Club in Baltimore, captained a championship IM soccer team, watched the Ravens win the Superbowl, watched (more surprisingly) the Orioles make the MLB vi

7 playoffs, successfully saved for an engagement ring, was engaged, married and had the opportunity to purchase a house. All the while I met some incredible people, and was able to share my path with many friends, new and old. I don t think there is another four and a half year period of my life more defining then my time at Hopkins. These years in school have defined my career path, and the events outside of school have defined my personal path. I hope, in the future, that I can remember some of the foundations upon which this path was built. From this, I hope to remember the gift and ability to multitask. I truly believe multitasking is the key to success. Additionally, per many conversations I have had with my wife, hard work and dedication are more important that brains, although brains surely help. But hard work defines your character and enables success. Without hard work, intelligence means nothing. Finally, remember balance. There will always be more work: that is the one thing that is inescapable. But remember the life experiences that occur along the way, and remember that life is about balance. It s a balance between friends and family, work and play, between stress and relaxation. Lose yourself in one, and you can forget about the others, for good or for ill. It s a complicated balancing act, but balance is the key to living a fulfilling life. So always remember your balance, and always remember the fixed points in your life that help you to establish your equilibrium, and hold them tight. Thanks to everyone who has made the dream of a Ph.D. possible. vii

8 TABLE OF CONTENTS List of Tables List of Figures x xi Introduction 1 Chapter 1: Host Factors Dictate Control of Viral Replication in two HIV-1 controller/chronic progressor transmission pairs 6 1.1: Background 6 1.2: Methods 8 1.3: Results : Discussion 30 Chapter 2: Inhibitory Potential of Subpopulations of CD8 + T cells in HIV-1 infected Elite suppressors : Background : Methods : Results : Discussion 59 Chapter 3: Primary CD8 + T cells from Elite Suppressors effectively kill non-productively HIV-1 infected resting and activated CD4 + T cells : Background : Methods : Results : Discussion 85 viii

9 References 88 Curriculum Vitae 102 ix

10 LIST OF TABLES Table 1: Transmission Pair Sequence Analysis 16 Table 2: Cellular Factors of Viral Control 20 x

11 LIST OF FIGURES Figure 1: Comparative, representative natural histories of infection for a typical progressor and an elite suppressors. 2 Figure 2: Comparison of Natural history of infection between transmission pairs 13 Figure 3: Analysis of viral sequences confirms transmission between patients. 17 Figure 4: Gag sequences from VC1 and CP1. 21 Figure 5: Gag and Nef Sequences from CP2 and ES Figure 6: Phylogenetic analysis and gag fitness in virus from ES38 and CP2. 25 Figure 7: Differential HIV-specific CD4 + T cell and CD8 + T cell cytokine response. 28 Figure 8: Strategies for calculation of the normalized percent inhibition and bulk CD8 suppression of viral replication by HD, CP, and ES. 41 Figure 9: Analysis of normalized percent inhibition for CD8 + T cell memory subpopulations on day 3 and 5 after infection. 46 Figure 10: Changes in the CM CD8 + T cell population by day five after infection. 49 Figure 11: Analysis of antigen-specific production of cytokines by CD8 + T cell memory populations. 51 xi

12 Figure 12: Correlation analysis for ES and CP memory populations. 54 Figure 13: The analysis of normalized percent inhibition for CD8 + T cell activation subsets on day 3 and 5 after infection. 57 Figure 14: Early Gag positivity in spinoculated cells is CD4 and co-receptor dependant. 73 Figure 15: Killing of non-productively infected CD4 + cells. 77 Figure 16: Killing of cells infected with inactivated virus. 79 Figure 17: Viral killing is constant over a range of inoculum sizes. 82 Figure 18: Equal killing of resting and activated CD4 + T cells 84. xii

13 INTRODUCTION Typically, Human Immunodeficiency Virus-1 (HIV-1) infection is characterized by very high viral loads in acute infection. During the chronic phase of infection, HIV-1 replication continues, concurrent with a progressive decline in CD4 + T cells counts. Without treatment, chronic progressors (CP) will progress to AIDS within 5-10 years (Figure 1). The patients who are placed on an effective antiretroviral therapy (ART) regimen, however, will experience a rebound in CD4 + T cell levels and a reduction in the HIV-1 plasma RNA level, usually to undetectable levels (<50 copies/ml). Long-term nonprogressors (LTNPs) are a subset of HIV-1 infected patients who maintain stable CD4 + T cells counts greater than 500 cells/µl for greater than 7 years, in the absence of ART. Once ultrasensitive assays to detect the HIV-1 plasma RNA levels were developed, it became clear that LTNPs were a phenotypically diverse population comprised of individual with varying HIV-1 plasma RNA levels. Elite Controllers or Suppressors (ES) and viremic controllers (VC) are distinct from LTNPs in that they are defined by the level of HIV-1 RNA plasma levels rather than their CD4 + T cell counts (Figure 1). ES are remarkable individuals maintain HIV-1 plasma RNA levels below the limit of detection of standard commercial assays (<50 copies/ml) in the absence of ART, and represent less than one percent of the total HIV-1 infected population [1]. VCs represent less than four percent of the total HIV-1 infected population, and maintain HIV- 1 plasma RNA to levels less than 2000 HIV-1 RNA copies/ml [2]. 1

14 Figure 1 Figure 1: Comparative, representative natural histories of infection for a typical progressor and an elite suppressors. HIV-1 infection in a progressive patient is characterized by robust viral replication during primary infection, and a reduction in CD4 + T cell counts. CD4 + T cell counts recover, and viral replication declines to a set point levels which persist for the duration of chronic infection. In the absence of therapy, progressive CD4 + T cell loss occurs. A patient is clinical defined as having AIDS once CD4 + T cell counts fall below 200 cells/µl, usually accompanied by an increasing viral load. In elite suppressors similarly have peak viremia in primary infection, but this has been shown to be blunted compared to progressive patients. In the absence of therapy viral load falls to undetectable limits (<50 copies/ml). No progressive CD4 + T cell loss or AIDS defining symptoms occur. Utilizing ultra sensitive viral load assays, low level viremia is still observed in many ESs. 2

15 The viral factors that are associated with elite control are poorly understood. Initial reports suggested that ES were infected with defective virus, including viruses that harbored large deletions or difficult to revert polymorphisms. Many of these studies looked at sequence analysis of proviral genes alone, however, and so the effect that these mutations or deletions had on the overall viral fitness was unclear [3-12]. Additionally, it was not clear whether the observed replication defects were due to infection with a defective virus, or whether the reduction in fitness was a result of a highly active immune response in patients that controlled viral replication. Using chimerical virus systems, individual HIV-1 viral genes were cloned from patients and the genes isolated from EC were seen to be less fit compared to those from CP [3, 8, 13]. Subsequently, replication competent virus was successfully isolated from ES, indicating that some ES were, in fact, infected with virus that replicated effectively in vitro [14-16]. Full genome sequence analysis also indicated that the replication-competent virus isolated from some patients contained no large scale deletions or gross mutations [14], and transmission pair studies have demonstrated that infection with genetically similar, replication competent viruses can result in drastically different clinical outcomes of infection [17, 18]. The data indicate that infection with defective virus is not the exclusive cause of elite control and that some ES are infected with virus that is able to cause pathogenic disease in vivo. This has been shown definitively in the macaque model of elite control [19] where some monkeys control fully pathogenic laboratory SIV isolates through CD8 + T cell responses [20, 21]. It is now generally accepted that host factors play a major role in elite control. The HLA-B*57 and B*27 alleles are overrepresented in ES [22-28], and these two alleles 3

16 along with a polymorphism in the promoter of HLA-C have been associated with slow progression in multiple GWAS studies [29-33, 33, 34]. HLA class I proteins are involved in presentation of peptides to CD8 + T cells, and this may explain why many studies have shown that HIV-specific CD8 + T cells from ES are more effective at controlling HIV replication in vitro than CD8 + T cells from patients with progressive disease [22, 35-38]. The maintenance of a polyfunctional HIV-1-specific CD8 + T cell response [39-41], as well as elevated proliferation and lytic granule loading have each been implicated in control of viral replication [22, 35, 36]. Additionally, unstimulated CD8 + T cell from ES have also been shown suppress viral replication more effectively compared to than CP CD8 + T cells [37, 38]. These data complement studies in the SIV model of elite control where depletion of CD8 + T cells in ES monkeys results in breakthrough viremia [20, 21]. The role of other host factors in elite control is more controversial. ES do not have elevated titers of neutralizing antibodies to autologous virus [42], but while one study suggested that these patients may have higher levels of antibody dependant cell mediated cytotoxicity (ADCC) than CP [43], a recent study found no differences in ADCC levels between ES and patients with progressive disease [44]. Other studies have shown that CD4 + T cells from many ES are fully susceptible to infection [15, 45, 46]. As a consequence of the normal physiology of CD4 + T cells, latency is established early in viral infection. These latently infected cells represent a major barrier to eradication in HIV-1 infected individuals using current strategies for treatment of infection [47]. A limiting dilution, co-culture assay that approximates the number of infectious, resting CD4 + T cells in a patient likely reflects the most accurate measure to quantify latently infected cells [48]. Using this assay, ES were observed to have a one 4

17 and a half log lower median infectious units per millions cells (IUPM) compared to CP [14]. ES have been shown to have significantly lower levels of integrated proviral DNA [49] compared to CPs, and a recent study that looked at four, unique ES with weakly reactive western blots, found that these patients had markedly lower levels of total and integrated proviral DNA compared to conventional ESs. These data suggests that the control of HIV-1 replication varies between ECs [50]. The reduction in the frequency of resting CD4 + T cells in ES may be a result of lower levels of HIV-1 RNA during the acute phase of infection [51, 52], or could be a result of a qualitatively superior CD8 + T cell response [22, 35-38], both of which could limit the seeding of the latent reservoir. Herein, we will analyze the role of host and viral factors that influence the control of HIV-1 infection mediated by ES. This analysis provides convincing evidence to suggest that host factors dictate the clinical outcome of infection in some, if not most, ES. Additionally, the nature of the CD8 + T cell response against HIV-1 infection was analyzed to determine which CD8 + T cell memory and activation subpopulation provided protection against infection. Finally, we suggest that a potent CD8 + T cell response is partially responsible for the reduced seeding of the latent reservoir early in infection. 5

18 Chapter 1: Host Factors Dictate Control of Viral Replication in two HIV-1 controller/chronic progressor transmission pairs 1.1 Background Defective or attenuated HIV-1 isolates were initially thought to be responsible for the LTNP phenotype. Deletions in viral genes such as nef have been shown to facilitate control of HIV-1 infection [3-11, 53, 54], and rare, stable polymorphisms in HIV-1 isolates from some ES have been documented [4]. It has been hypothesized that these defective isolates may explain the clinical status of these patients. However, it has become increasingly clear that viral factors alone cannot explain the variable nature of HIV-1 infection. Several studies have shown that replication-competent HIV-1 can be isolated from ES [14-16]. Full genome sequence analysis of replication-competent viral isolates from ES revealed no large deletions [14], and ongoing viral replication and evolution in ES have been documented in recent studies [55-57]. Taken together, these data suggest that in some cases, infection with attenuated virus cannot explain elite suppression. There is an increasing appreciation for the role played by the host immune response in this phenotype. The HLA-B*57 allele group is overrepresented in ES [22-28], and the HIV-1-specific CD8 + T cell response in these patients is generally superior to that seen in patients with progressive disease [22, 35-37, 37, 38, 38-41]. Data supporting a model of host control of viral replication comes from a previous study by our group examining an HIV-1 transmission pair. Virus was transmitted from an HLA-B*57 positive patient who developed AIDS to a recipient who was positive for both HLA-B*57 and HLA-B*27 group alleles and who subsequently 6

19 became an ES. The viruses infecting both individuals were shown to be closely related by phylogenetic analysis, but the virus isolated from the ES displayed a partial replication defect and mutations in gag were shown to contribute to this viral attenuation. It was hypothesized that selective pressure from HIV-specific CTLs caused evolution of the transmitted virus and maintained key escape mutations in HLA-B*27 and HLA-B*57 gag epitopes that resulted in viral attenuation [17]. However, it is possible that selective pressure from the chronic progressors CD8 + T cells resulted in mutations in HLA-B*57 epitopes early in his disease course. Some of these mutations, such as Gag T242N, have a significant fitness cost [58, 59], thus the virus may have been attenuated prior to transmission, which could have contributed to the elite control of viral replication. We currently present two additional cases of HIV-1 transmission from patients with progressive disease to patients who subsequently controlled viral replication. These cases differ from the previously reported case in that viruses isolated from all patients were shown to replicate equally well in vitro, suggesting that host factors can control replication of fully pathogenic HIV. 7

20 1.2 Methods Virus Isolation and Sequence Analysis Culture of replication-competent virus and full genome sequence analysis of proviral and plasma virus were performed as previously described [14]. Clonal nef sequences were amplified from resting CD4 + T cells as previously described [56]. Classical, maximum likelihood and Bayesian phylogenetic analysis were performed as described previously [55] Viral Fitness Assay Viral fitness was analyzed as described previously [14]. PBMCs from healthy donors were cultured for two days in the presence of IL-2 and PHA. CD4 + T cells were isolated (MACS, CD4 + T cell isolation Kit) and infected by spinoculation [60] (1200xg for 2 hours) with equal quantities (200ng/mL) of p24 from primary patient isolates, and with HIV-1BAL as a reference strain. Supernatant samples were taken over the course of 7 days. Viral replication was quantified using p24 ELISA (Perkin Elmer). To study the effect of mutations in HLA-B*57 epitopes on gag fitness, gag from patient viral isolates was amplified by PCR. The resulting amplicon was digested with BSSHII and SbfI restriction enzymes and ligated into a previously described pnl4-3- ΔEnv GFP NL43dE vector [61]. The pnl4-3-δenv GFP NL43dE vector with patientspecific gag was transformed into Stbl-3 cells (Invitrogen) to amplify, and co-transfected into 293T cells with an X4 Env plasmid to produce single round replication pseudovirus expressing patient specific gag. After a three day transfection, cellular debris were removed by centrifugation and filtration through Steriflip filters (Millipore). Virus was 8

21 collected by ultracentrifugation at 1,200 x g for two hours, at 25 o C. CD4 + T cells from healthy donor PBMCs were isolated by magnetic bead separation (Miltenyi), and were infected by spinoculation with NL4-3 (control), ES31-gag and CP2-gag pseudoviruses. Infection was analyzed after 3 days of infection by measuring green fluorescent protein (GFP) expression using a flow cytometer. The results were normalized by transfection efficiency Genetic Polymorphisms HLA-A and HLA-B locus allele identification was performed by direct sequencing of amplicons prepared from genomic DNA isolated from peripheral blood samples according to the instructions of the manufacturer (Abbott Molecular) with alignment of sequences to known alleles via software (Conexio). CCR5 from all patients was amplified from genomic DNA using gene specific primers. The presence or absence of the CCR5 Δ32 mutation was determined by relative size of the resulting PCR fragment. HLA-C single nucleotide polymorphism genotyping (rs ) was performed utilizing the Applied Biosystems 7300 real-time PCR System allelic discrimination assay, following the manufacturer s guidelines. Primers and probes were developed by Custom TaqMan SNP Genotyping assays (ABI). Determination of the KIR3DS1 and HLA-B Bw4-80Ile allele was performed using the Olerup SSP KIR Genotyping 12 Lot71E and KIR ligand genotyping Lot85E kits, following the manufacturer s guidelines Immunologic Assays Cytokine expression was determined by an intracellular cytokine staining assay. PBMCs were isolated, and 1x10 6 cells/well were cultured in a 48 well plate. One 9

22 microgram per millitliter anti-cd28 and anti-cd49d antibodies (BD Bioscinces) were added to each culture in addition to either HIV Gag peptides or HIV Nef peptides. For the analysis of CD8 + T cell cytokine production, patient-specific Gag or Nef peptides as determined by Elispot were used. For the analysis of CD4 + T cell cytokine production overlapping peptides spanning the entire amino acid sequence of B clade consensus Gag were used. PBMCs were cultured for 12 hours at 37 C in the presences of Golgi inhibitors (GolgiStop/GolgiPlug, BD Biosciences). Cells were washed twice, and then stained for surface antibodies (CD8, CD4, BD Biosciences). Cells were fixed and permeabilized using the CytoFix/CytoKit kits (BD Bio sciences) following manufacturers guidelines, and stained for intracellular antigens (Perforin, Diaclone Research; IL-2 and INF-gamma, BD Bioscicnes; TNF-α, BioLegend). Cells were analyzed by flow cytometry using a FACS Canto. For multicolor FACS analysis combinations of the following antibodies were used: anti-cd8 APC-H7, anti-cd4 FITC, anti-ifn-gamma Pe- Cy7, anti-tnf-α Pacific Blue, anti-perforin PE, and anti IL-2 APC. The Cytolytic T cell effect for VC1 compared to healthy donors, viremic patients and ES was determined by a CD8 suppression assay. PBMCs were isolated from patients, and CD8 + T cells were positively selected using Miltenyl magnetic beads (MACS, CD8 + T cell Isolation kit). CD8 + T cells were depleted of CD16 + cells (Invitrogen, Dynal Beads) to remove contaminating NK cells. CD4 + T cells were isolated by negative selection using Miltenyi magnetic beads. Purity of depletion was analyzed by flow cytometry. CD4 + T cells were infected by spinoculation at 1200xg for 2 hours with replication competent NL4-3 virus, in which GFP is engineered into nef. Autologous CD8 + T cells were added at a one to one ratio to CD4 + T cells, and viral 10

23 infection between cultures with and without CD8 + T cell addition was compared. FACS analysis was performed 5 days after infection. To compare the cytolytic T cell effect of VC1 and CP1, PBMCs were isolated and cultured for three days in stimulating media. CD4 + T cells were isolated after three days, and maintained in stimulating media for another four days. The CD4 + T cells were spinoculated at 1200xg for 2 hours with single round NL4-3 virus, in which env is replaced with GFP. Autologous CD8 + T cells, isolated directly ex vivo, were added at a one to one ratio to CD4 + T cells, and viral infection between cultures with and without CD8 + T cell addition. FACS analysis was performed five day after infection. Reactive CTL epitopes were defined by IFN-gamma Elispot. As previously described [17], whole blood was taken from each patient and PBMCs were isolated by Ficoll gradient centrifugation. PBMCs were aliquoted into each well of 96 well MultiScreen (Millipore) plates with conjugated IFN-gamma antibody. Cells were activated with overlapping peptides spanning the entire amino acid sequence of B clade consensus gag and nef. Alternatively, cells were activated with peptides representing conserved epitopes in HLA-B*57. PBMCs were cultured overnight, and subsequently analyzed. Quantification of spot forming units was performed independently and in a blinded fashion Nucleotide sequences accession numbers Full genome sequences from each patient have been submitted to GenBank and have been assigned accession numbers JN JN

24 1.3 Results Patient Characteristics The first transmission pair consists of a 40 year old male, chronic progressor (CP1), and a 37 year old female, viremic controller (VC1). Infection was first documented in CP1 in 1996, and antiretroviral therapy (ART) was initiated in The patient had a long history of poor adherence to multiple regimens, and his viral load fluctuated dramatically, as shown in Figure 2. CD4 + T cell counts progressively declined to the current level of 200 cells/µl. His partner, VC1, was also diagnosed with HIV-1 infection in She was originally started on ART for unknown reasons in Her baseline CD4 count was 741 cells/ul, and her baseline viral load was 1184 copies/ml. She was taken off treatment in 2000 due to poor adherence (Figure 2 A, B). Since that time, plasma HIV-1 RNA has been undetectable or very low and she has maintained an average CD4 + T cell count of 921 cells/µl. The second pair consists of a 35 year old male, chronic progressor (CP2), and a 21 year old female who became an ES (ES38). CP2 was found to be HIV-1 infected in June 2010, and has since maintained plasma HIV-1 RNA level of approximately 30,000 copies/ml. His CD4 + T cell count reached a nadir of 273 cells/ul before initiation of ART. ES38 was also diagnosed with HIV-1 infection in June, 2010 but has maintained a plasma HIV-1 RNA level of less than 50 copies/ml and an average CD4 + T cell count of 935 cells/ul (Figure 2 C, D). 12

25 Figure 2 Figure 2: Comparison of Natural history of infection between transmission pairs. CD4 + T cell counts (blue) and viral load (red) are observed over time for (A) CP1, (B) VC1, (C) CP2, and (D) ES38. Open squares denote viral loads below the limit of detection of the assay. Periods of antiretroviral therapy are indicated by shaded regions for CP1, CP2 and VC1. 13

26 1.3.2 Sequence Analysis Confirms Transmission To confirm transmission of virus between the relevant patients, virus was cultured from each patient s CD4 + T cells with a sensitive co-culture assay, as previously described [48]. Full length sequence analysis was performed, and the resulting genomes were compared to each other and to the consensus B clade sequence. No large deletions, premature stop codons, frame shifts or other gross defects were seen in isolates cultured from any of the patients. The numbers of synonymous nucleotide changes, as well as a number of amino acid differences, between transmission partners, are shown in Table 1. Phylogenetic analysis was performed, and full length env sequences from both transmission pairs were compared to other HIV-1 env sequences obtained from the Los Alamos database (Figure 3A). CP1 and VC1, and CP2 and ES38, were more similar to each other than to other B clade genes and they showed common ancestry, providing strong evidence of viral transmission between these patients Representative sequence alignments (Figure 3B) demonstrate that the sequences between the transmission pairs are closely related and that they have rare sequence polymorphisms unique to the transmission pairs. Comparative alignments of sequences from CP1 and VC revealed unique polymorphisms in tat at nucleotide positions 100 and 101, where aspartic acid (D) is followed by a histidine (H). The combination of these two amino acids at these positions occurs in only 5.3% of B clade isolates in the Los Alamos HIV sequence database (Figure 3B). This strongly suggests that the two patients are indeed a transmission pair. Similar, unique polymorphisims were seen in rev and vpu. Unique polymorphisms in tat also confirmed 14

27 transmission between CP2 and ES38: isolates from both patients had a combination of a phenylalanine (F) followed by a histidine (H) at positions 100 and 101, which is present in less than 1% of B clade isolates in the Los Alamos HIV-1 sequence database (Figure 3B). 15

28 Table 1 16

29 Figure 3 Figure 3: Analysis of viral sequences confirms transmission between patients. (A) Phylogenetic tree of env sequences from the CP1/VC1 and CP2/ES38 transmission pairs. (B) Tat amino acid sequences for CP1 and VC1 (top) and Tat amino acid sequences for CP2 and ES38 (bottom) are compared to consensus B clade sequence. Rare polymorphisms in Tat, shared within each transmission pair, are highlighted. (C) Fitness of viral isolates from transmission pairs. Error bars represent standard errors of the means from three independent experiments. On the left, growth kinetics of replication competent isolates from CP1 (blue) and VC1 (red) are compared to BaL (purple), and on the right growth kinetics of replication competent isolates from CP2 (orange) and ES38 (brown) are compared to Ba-L (purple). 17

30 1.3.3 No Significant Differences in Viral Fitness between Transmission Pairs Virus Isolates Previous studies have suggested that attenuation in infecting viruses could explain the clinical outcome of infection [3-11, 53, 54]. In addition, in a previously reported transmission pair there was clear viral attenuation in the isolate cultured from the ES, which could have contributed to elite suppression [17]. To determine if differences in viral fitness could explain the status of both transmission pairs, we isolated replicationcompetent virus from the latent reservoir of each patient. Multiple isolates were obtained from each patient, and viral replication was compared using healthy donor CD4 + T cells. For the first transmission pair, viral isolates from VC1 were as fit as isolates from CP1 (Figure 3C). The second transmission pair showed similar results, with no differences in viral fitness seen in isolates cultured from CP2 and ES38 (Figure 3C). All viral isolates replicated as efficiently as the HIV-1BAL reference strain The Role of Host Factors in the Control of Viral Replication Since no differences were seen in the virus between patients in each transmission pair, and since all viruses were as fit as the reference strain HIV-1BaL, we sought to determine whether cellular factors could be playing a predominant role in determining the clinical phenotype. Subjects who are heterozygous for the delta 32 mutation in CCR5 have slower progression of HIV-1 disease [13]. The CCR5 gene was analyzed by PCR analysis, and all patients were determined to have two wild type CCR5 alleles. HLA-typing was also performed on each patient. It is interesting to note that ES38 is HLA-B*57:03 positive. This is a protective allele group that is overrepresented in ES [22-28]. No known protective HLA alleles were present in VC1, CP1, or CP2 18

31 (Table 2). A single nucleotide polymorphism associated with the HLA-C promoter (rs ) has also been implicated in control of viral replication in a genome wide association study [29-33, 33, 34]. Of the four patients, none were homozygous for the protective allele (Table 2). Natural killer cells have been implicated in delaying the progression of HIV-1 disease. The interaction between the NK cell receptor KIR3DS1 and HLA-Bw4-80Ile motif has been shown to be protective [62]. We sought to determine if our patients possessed this protective combination. None of our patients were positive for the KIR3DS1 activating allele (Table 2), and only ES38 was positive for an HLA-B Bw4-80Ile motif. 19

32 Table 2 20

33 Figure 4 Figure 4: Gag sequences from VC1 and CP1. Patient-specific CTL epitopes were identified by IFN-gamma ELISPOT, and are highlighted for each patient. Numbering indicates amino acid position in Consensus B Clade Gag. 21

34 Figure 5 Figure 5: Gag and Nef Sequences from CP2 and ES38. Patient-specific CTL epitopes were identified by IFN-gamma ELISPOT, and are highlighted for each patient. HLA- B*57-restricted epitopes are indicated. Numbering indicates amino acid position in Consensus B Clade Gag or Nef. 22

35 1.3.5 CD8 + T cell response in Elite Suppression In order to determine how pressure from HIV-1-specific cytotoxic T lymphyocyte (CTL) responses might have led to differences in viral evolution in the patients, we performed IFN-gamma ELISPOT assays to determine which epitopes were targeted in Gag and Nef. The epitope DQ15 (Gag 219 to 233) was targeted by both CP1 and VC1, but mutations in this region were observed only in isolates from CP1 (Figure 4). Similarly, a mutation was present in NP15 (Gag 331 to 346), an epitope targeted by VC1, indicating potential virologic escape in this subject. CP2 and ES38 each targeted several epitopes in Gag (Figure 5). Interestingly, ES38 made responses to peptides that contained previously characterized HLA-B*57 restricted epitopes, and both ES38 and CP2 had mutations in HLA-B*57 restricted Gag and Nef epitopes, including L90V and H121N in Nef and I147L, G248A, I247V and E312D in Gag. Additionally, an S173T mutation that has been seen in HLA-B*57 individuals [63] was present adjacent to the immunodominant HLA-B*57-restricted KF11 epitope (gag ) in both patients. The presence of these mutations in both patients, including CP2 who is not positive for HLA-B*57 (Table 2), would suggest that either CP2 was originally infected with virus from an HLA- B*57 positive patient, or that ES38 transmitted virus to CP2 after these mutations developed. Phylogenetic analysis of clonal nef sequences (Figure 6A) amplified from resting CD4 + T cells from both patients was performed, but the results could not definitively distinguish between the two hypotheses. 23

36 It has been shown that some escape mutations in HLA-B*57 restricted epitopes in Gag have a negative effect on viral fitness [58, 59] and may contribute to elite control. ES38 did not possess the T242N mutation, which is known to revert when transmitted to HLA-B*57-negative individuals [64, 65]. Nonetheless, to determine whether other mutations in this gene had significant effects on fitness, we cloned the gag genes from CP2 and ES38 and compared infection of healthy donor CD4 + cells with these isolates to infection with NL4-3. There was no significant difference in the infectivity of virus expressing patient-derived Gag (Figure 6B, C). Thus the difference in the level of viral control in these patients cannot be attributed to differences in viral fitness. Additionally, the infection levels of both viruses was similar to that of the reference strain, further suggesting that ES38 was exerting control over a virus that replicates as well in vitro as does virus from a patient with progressive disease. Taken together, these data suggest that viral fitness is not the determining factor that explains differences in the clinical outcome seen in the patients in both of the reported transmission pairs. 24

37 Figure 6 Figure 6: Phylogenetic analysis and gag fitness in virus from ES38 and CP2. (A) Phylogentic tree of clonal nef sequences isolated from resting CD4 + T cells (circles) and replication competent virus isolates for reference. Other representative B clade isolates, consensus B clade nef, and the clade C outgroup are denoated. Fitness of patient-specific viral gag. (B) Representative FACS plots showing GFP expression indicates infection with single cycle, pseudotyped virus. (C) Percent infection with various volumes of pseudotyped virus containing gag from ES38 (red), CP2 (brown), and wild type NL4-3 (blue) was determined and repeated for three donors, producing similar results. Approximate levels of p24 in each infectious volume are shown, estimated and 25

38 normalized based on NL4-3. Error bars represent standard error of the means from three independent experiments. 26

39 ES38 was positive for the HLA-B*57 allele, which has been associated with elite control of HIV-1 replication in many studies. However, VC1 does not have any known protective HLA alleles, therefore we compared multiple aspects of her HIV-1-specific immune response to those seen in CP1. As shown in Figure 7A, stimulation of PBMCs with overlapping Gag peptides resulted in a substantially higher percentage of IFNgamma expressing HIV-specific CD4 + T cells that co-expressed IL-2 in VC1 compared to CP1, consistent with prior studies showing the preservation of IL-2 secretion in aviremic individuals [66-69]. CD8 + T cell responses have been intensely studied in ES. It has been shown that CTLs from ES are more likely to upregulate cytolytic granules containing perforin and granzyme B [22, 35, 36]. These cells are also more likely to coexpress IFN-gamma and TNF-α [39], which is a known correlate of cytotoxic activity [70]. IFN- -specific CD8 + T cells from VC1 fit this profile and coexpressed more perforin and TNF-α in response to stimulation with Gag and Nef peptides than did CD8 + T cells from CP1 (Figure 7B-D). In order to determine whether this phenotype was associated with actual inhibition of viral replication, a suppression assay was performed. Because CP1 was on HAART, CD4 + T cells from both patients were incubated for a week prior to infection with pseudotyped virus. Freshly isolated CD8 + T cells from CP1 had no effect on CD4 + T cell infection whereas CD8 + T cells from VC1 substantially reduced the percentage of infected cells (Figure 7E). In a traditional inhibition assay where there was no pre-incubation of CD4 + T cells prior to stimulation and infection, CD8 + T cells of VC1 were seen to be as effective as those from a cohort of HLA-B*57 ES in controlling replication-competent virus in vitro, whereas CD8 + T cells 27

40 from patients with progressive disease and uninfected individuals had low inhibitory activity (Figure 7F). 28

41 Figure 7 Figure 7: Differential HIV-specific CD4 + T cell and CD8 + T cell cytokine response. (A) Stimulation of patient PBMCs cells isolated directly ex vivo with overlapping Gag peptides. The percent of IFN-gamma expressing HIV-specific CD4 + T cells that coexpressed IL-2 is shown for VC1 and CP2. (B) Representative FACS plots showing the stimulation of PBMCs isolated from VC1 and CP1. The plots are gated on all CD8 + T cells, and the percentage of IFN-gamma positive cells that co-expressed TNF-α or perforin are indicated in the top right quadrant. Quantification of the percentage of IFN- 29

42 gamma expressing CD8 + T cells that co-expressed perforin, TNF-α, or both perforin and TNF-α after stimulation with patient-specific Gag (C) or Nef (D) peptides are shown for both VC1 and CP1. Comparative CD8 + T cell inhibition of viral replication. (E) CD4 + T cells from VC1 and CP1 were infected with GFP expressing recombinant NL4-3 virus. CD4 + T cells were cultured alone or with autologous CD8 + T cells at a 1:1 ratio. Percent infection was measure five days after infection by FACS analysis. (F) CD4 + T cells from 4 Healthy Donors (D1-4), an untreated Viremic patient, 6 ES, and VC1 were infected with GFP expressing replication-competent NL4-3 virus. CD4 + T cells were cultured alone or with autologous CD8 + T cells at a 1:1 ratio. Percent inhibition was quantified 5 days after infection. 30

43 1.4 Discussion Understanding the mechanisms of control of viral replication in VC and ES may lead to effective immunotherapy of HIV-1 infection. Determining the role of viral fitness versus host factors in these patients is critical because it bears directly on the fundamental mechanism of elite control of HIV-1 replication. Unfortunately, isolating replicationcompetent virus from ES has proven to be extremely challenging. As a result, many studies have focused on the analysis of proviral and/or plasma sequences from ES. Some studies have not found any deleterious mutations or large deletions [54, 71], whereas others have reported significant deletions [3-12] and/or reductions in fitness of recombinant viruses expressing viral genes amplified from ES [12, 53]. The results of these studies are limited by the fact that many proviral clones are not replicationcompetent and the viruses in the plasma of ES typically contain attenuating escape mutations not found in virus archived in resting CD4 + T cells [72]. Full genome sequence analysis has been performed for less than ten replication-competent isolates cultured from ES CD4 + T cells [4, 14, 17, 73], and there has been only one documented case of viral transmission from a CP to an ES described in the literature [17]. The two transmission pairs presented here are unique because they stand in contrast to the previously reported transmission pair: in this study, viruses isolated from both VC1 and ES38 were fully replication competent and as fit as viruses isolated from their partners with progressive HIV-1 disease. In the previous study, the ES was found to have an attenuated viral isolate. While we hypothesized that the diminished fitness in this patient was due to escape mutations that developed in response to strong selective 31

44 pressure from CTL, it is also possible that viral attenuation preceded transmission and contributed to elite suppression [17]. However, in this report, we have shown for the first time that replication-competent isolates obtained from patients who controlled viral replication had no evidence of attenuation and had the potential to cause progressive disease in vivo. This strongly suggests that in some cases elite suppression is not the result of infection with attenuated viruses. While ES38 is HLA-B*57:03 positive, no other known cellular factors could explain viral control in either of these transmission pairs. The HLA-C related SNP and the delta 32 CCR5 mutation that confer relative protection against HIV-1 infection, were not present in any of our patients, and all patients lacked the combination of KIR3DS1 and protective HLA-B alleles. CD8 + T cells have been extensively studied in control of HIV-1 infection, and strong evidence supports their key role in delaying disease progression [22, 23, 36, 38]. While we were unable to perform detailed HIV-specific CD8 + T cell analysis in ES38 due to a lack of cells, it is likely that HIV-specific HLA- B*57-restricted CTL played a role in elite suppression of viral replication. VC1 did not have any known protective HLA allele, but, unlike CP1, she had polyfunctional HIVspecific CD4 + T cells, and her HIV-specific CD8 + T cells expressed substantial levels of perforin and TNF-α upon activation and were as effective at inhibiting HIV-1 replication as CD8 + T cells from many HLA-B*57 positive ES. Thus, differences in CD8 + T cell function can probably explain the difference in clinical outcomes seen in these patients. These data support the hypothesis that ES are infected with virus that replicates as well as virus that causes progressive disease, and a combination of host immune factors, unique to ES, enables control of viral replication. While infection with an attenuated 32

45 virus can increase the chance of early and prolonged control of viral replication, this study and others supports a model in which host factors play a dominant role in determining the clinical outcome of infection. These results are consistent with the macaque model of elite control in which animals with protective MHC alleles are capable of controlling fully replication-competent laboratory strains of SIV (reviewed in [19]). Intriguingly, in two distinct elite monkey models, depletion of CD8 + T cells with monoclonal antibodies results in the transient loss of control of viral replication, [20, 21] implying that these cells play a major role in elite suppression. Importantly, these data suggest that ES have a unique capacity to restrict viral replication, independent of the fitness of the infecting virus. Our sequence analysis suggests that ES31 may have infected CP2 who then went on to develop a rapid decline in his CD4 + T cell count. Overall, the data presented here provides the strongest evidence to date that host factors rather than viral fitness determines clinical outcome. Thus, by identifying the contribution of individual host factors, the mechanism of elite suppression could form the basis for a therapeutic vaccine for HIV-1. 33

46 Chapter 2: Inhibitory Potential of Subpopulations of CD8 + T cells in HIV-1 infected Elite suppressors 2.1 Background The development of an effective vaccine against Human Immunodeficiency Virus Type-1 (HIV-1) is essential for the control of the HIV pandemic. ES provide a unique opportunity to better understand the mechanisms by which durable control is achieved. While the mechanism of are unclear, an improved understanding of the immune factors that enable this remarkable control can provide guidance for the development of an effective therapeutic vaccine for HIV-1 infection. Many studies have linked an effective cytolytic T lymphocyte response with control of HIV-1 replication. Studies in the macaque model of elite suppression have shown that depletion of CD8 + T cells with monoclonal antibodies results in a loss of viral control [20, 21]. The HLA-B*57 and HLA-B*5801 are overrepresented in ES [22-28], and amongst HLA-B*57 positive patients, the preferential targeting of conserved HLA- B*57-restricted epitopes has been associated with control of HIV-1 replication [23]. The targeting of conserved domains in Gag has been associated with escape mutations that may lead to viral attenuation, thus facilitating control of viral replication [58, 74]. Additionally, genome wide association studies have indicated that the HLA-B*57 and HLA-B*27 alleles are associated with viral control [29-33, 33, 34]. While some ES do not have protective HLA alleles or strong HIV-1-specific CD8 + T cell responses [25, 26, 37, 75], the HIV-1-specific CD8 + T cell response in many ES has also been shown to be qualitatively more effective than the response in CP [76]. 34

47 CD8 + T cells from ES maintain a polyfunctional response after stimulation with HIV-1 peptides [39-41], and there is significantly higher expression of granzyme B and perforin by HIV-1-specific CD8 + T cells from ES compared to CP [22, 35, 36]. In addition, CD8 + T cells from ES are much more effective at suppressing HIV-1 replication in autologous CD4 + T cells in vitro than CD8 + T cells from CP [17, 18, 22, 37, 38, 77]. The inhibitory potential of CD8 + T cells have recently been shown to be predictive of the rate of CD4 + T cell decline early in viral infection [78]. Current analysis of the CD8 + T cell response in HIV-1 infection has focused primarily on unfractionated populations of CD8 + T cells. However, an in vitro study showed that stimulation of CD8 + T cells with HIV-1 peptides for five days greatly improved the inhibitory potential of these cells [38] and a recent report suggested that a vaccine that elicits effector memory (EM) CD8 + T cell was able to induce early and durable control of viral replication in SIV-infected macaques [79]. Herein, we report a novel suppression assay in which unstimulated CD8 + T cells are isolated directly ex vivo, sorted by flow cytometry into subsets based on expression of memory or activation markers, and tested for ability to inhibit viral replication in autologous CD4 + T cells. The CD4 + T cells were infected directly ex vivo, and were maintained without exogenous cytokines to better recapitulate in vivo conditions. Thus, this variation of the CD8 + T cell suppression assay represents the most physiological analysis of the suppressive capacity of CD8 + T cells to date and the most detailed analysis of the relative inhibitory potential of different memory and activation subsets from ES. The results provide guidance for the development of an effective cell based vaccine against HIV-1 infection that can elicit immune responses and activation phenotypes those observed in ES. 35

48 2.2 Methods Patients All individuals provided written informed consent prior to participating in this study, and all studies were approved by the Johns Hopkins Institutional Review Board. All 8 ES maintained undetectable HIV-1 plasma RNA levels for the duration of study (<50 copies/ml) and are positive for the HLA-B*57 allele. The mean CD4 + T cell count for the ES used in this study was 927 cells/µl and the mean duration of infection was 14 years. The 8 CP had CD4 + T cell counts ranging from 223 to 788 (median 391) cells/µl and HIV-1 plasma RNA levels that ranged from 6,868 to 636,094 (median 38,648) copies/ml. None of the CP enrolled were currently on antiretroviral therapy. Seronegative, healthy donors (HD) were 8 healthy laboratory workers Isolation of CD4 + and CD8 + T Cells Peripheral blood mononuculear cells (PBMCs) were isolated from whole blood by Ficoll gradient separation. CD8 + T cells were then purified by positive selection from PBMCs using Human CD8 + Microbeads (Miltenyi) following the manufacturer s guidelines. CD8 + T cells were maintained in non-stimulating media (RPMI 1640 with 10% FBS without exogenous cytokines) on ice until cell sorting was performed. CD4 + T cells were then isolated from the CD8 + T cell depleted PBMCs using the Human CD4 + Isolation Kit II (Miltenyi) following the manufacturer s guidelines. CD4 + T cells were maintained in non-stimulating media on ice until infection. Healthy donor CD4 + and CD8 + T cells were also isolated to test for non-specific killing in the CD8 + suppression assay Fluorescence Activated Cell Sorting (FACS) 36

49 CD8 + T cells from each patient were sorted into memory or activation CD8 + subsets. For the memory subsets, CD8 + T cells were stained with anti-ccr7-pe (Biolegend) and anti-cd45ra-apc (Becton Dickinson) antibodies following the manufacturer s guidelines. The cells were then sorted using a FACS Aria (Becton Dickinson) or a MoFlo (Beckman Coulter) cell sorter into the following 4 populations: naïve (CCR7 + /CD45RA + ), central memory (CCR7 + /CD45RA - ), effector memory (CCR7 - /CD45RA - ), terminal effector (CCR7 - /CD45RA + ) [80, 81]. A representative FACS plot showing the memory cell sorting strategy is shown in Figure 2. CD8 + T cells were also sorted separately using anti-hla-dr-pe (Becton Dickinson) and anti-cd38-apc (Becton Dickinson) antibodies following the manufacturer s guidelines. The cells were then sorted into the following 4 populations: HLA-DR + /CD38 +, HLA-DR - /CD38 +, HLA- DR - /CD38 - and HLA-DR + /CD38 -. After sorting, all cells were re-suspending in nonstimulating media at a concentration of 1x10 6 cells/ml. Cells were kept on ice until use in the CD8 + suppression assay. An aliquot of bulk CD8 + T cells was taken after staining and prior to cell sorting for comparison in the CD8 + suppression assay. Cell purity after sorting was routinely observed to be greater than ninety five percent for each subset (data not shown). CD8 + T cells from healthy donors were also sorted for use as a negative control to test the specificity of suppression CD4 + T Cell Infection CD4 + T cells from each patient were infected ex vivo with a reporter virus by spinoculation at 1,200 x g for 2 hours at room temperature [60]. The virus used for infection has been routinely used by our lab group was a replication competent NL4-3 strain that was engineered to have GFP in the place of nef (NL43nGFP) [45]. CD4 + T 37

50 cells from all individuals were not stimulated prior to spinoculation and were subsequently cultured in non-stimulating media. An aliquot of uninfected CD4 + T cells was kept on ice to be used as a negative control. Infection of CD4 + T cells was performed concurrently with the sorting of CD8 + T cells. CD4 + T cells from healthy donors were also infected to be used as a negative control to test the specificity of suppression CD8 + T Cell Suppression Assay All cells were maintained in non-stimulating media for the duration of the experiment. All CD4 + T cells and CD8 + T cells were isolated, sorted, infected, and cultured on the same day. Sorted CD8 + T cells (effector cells) and infected CD4 + T cells (target cells) were co-cultured in a 96 well plate at varying effector to target ratios. The number of CD4 + T cells per well remained constant (100,000 cells per well) and the number of CD8 + T cells was varied. CD8 + T cells were serially diluted from a 1:1 effector to target ratio to a 1:128 effector to target ratio by two-fold dilutions. If the number of CD8 + T cells available after cell sorting was not sufficient for a 1:1 effector to target cell ratio, lower initial dilutions were used and two-fold dilutions were continued to a 1:128 effector to target ratio. CD8 + and CD4 + co-cultures were maintained in a final volume of 200 µl of non-stimulating media. Negative control wells with uninfected CD4 + T cells were present on each plate. Positive control wells with infected CD4 + target cells alone were also present on each plate. The percentage of infected cells in the positive controls ranged from 0.3 to 7.3 in CP versus 0.8 to 13.2 in ES and healthy donors, which is consist with our prior finding that CD4 + T cells from viremic patients were relatively resistant to infection in this assay [45, 46]. Data from the inhibition 38

51 assay was not used for the 4 CP where the infection rate of CD4 + T cells was less than 1.0% or where there was very poor cell viability. The 4 CP whose results in the inhibition assay were not used had higher plasma HIV-1 RNA levels than the 4 CP where superinfection of CD4 + T cells resulted in reasonable viability and infection rates (median of 297,882 vs. 29,841 copies/ml). The median percentage of infected CD4 + T cells in the absence of CD8 + T cells for these 4 CP was 3.6%, compared to a median of 2.9% for HD and 8.6% for ES. Each of the memory subsets (naïve, CM, EM, TE) and the activation subsets (DR + /38 +, DR - /38 +, DR - /38 -, DR + /38 - ) were tested individually using this suppression assay. The percent infection in each well was calculated on day 3 after infection (when GFP expression can first be reliably detected) and 2 days later for comparison. Cells were stained with anti-cd3-pacific Blue (Becton Dickinson) and anti-cd8-apch7 (Becton Dickinson) antibodies to distinguish target CD4 + T cells (CD3 + /CD8 - ) and effector CD8 + T cells (CD3 + /CD8 + ). For CM CD8 + T cells, cells were additionally stained with anti-ccr7-pe (Biolegend) and anti-cd45ra-apc (Becton Dickinson) antibodies to analyze the changes in the CM population over the course of infection. For cytometric analysis, lymphocytes were gated by forward scatter/side scatter, and CD3 + /CD8 - target cells were gated and analyzed for the expression of GFP (Figure 8A), which is indicative of infection. The normalized percent inhibition was calculated as follows: (Percent infection of CD4 + T cells alone wells Percent infection of a CD4 + and CD8 + co-culture well) / (Percent infection of CD4 + T cells alone) x 100. For example, in Figure 8, the normalized percent inhibition for the representative data would be calculated as ( )/(8.4)*100, resulting in a normalized percent inhibition of

52 percent. The normalized percent inhibition of each CD8 + T cell population at each effector to target ratio for each patient/healthy donor was calculated at day 3 and day 5. All cytometric analyses were performed using a FACS Canto II (Becton Dickinson) and analyzed using the FACS Diva software. A minimum of 100,000 events per sample were recorded Intracellular Cytokine Staining Cytokine production was measured by intracellular cytokine staining as previously described [17]. Briefly, bulk PBMCs from each patient were isolated directly ex vivo and stimulated overnight with overlapping Gag or Nef peptide mixtures that spanned the length of each protein at a concentration of 5 ug/ml. Prior to overnight incubation, cells were treated with a cocktail of anti-cd49d, anti-cd28, in addition to Golgi Plug and Golgi Stop (Becton Dickinson) as recommended by manufacturer. PBMCs treated in the same manner without HIV-1 peptides were analyzed to verify that the co-stimulatory factors alone did not result in stimulation. Cells were harvested after overnight incubation, and surface proteins were stained using anti-cd8-apch7, anti- CCR7-PE and anti-cd45ra-apc antibodies (Becton Dickinson). The Cytofix/Cytoperm kit (Becton Dickinson) was used to stain for intracellular cytokines according to the manufacturer s guidelines. Intracellular staining was performed using anti-ifn-gamma-pecy7 (Becton Dickinson) and anti-tnf-α-pacific Blue (Biolegend). Live lymphocytes were gated by forward and side scatter, then by CD8 expression, and then subdivided into the naïve, CM, EM and TE CD8 + T cell subsets by CCR7 versus CD45RA expression. Expression of IFN-gamma and TNF-α by each subset was then 40

53 analyzed using an FACS Canto II (Becton Dickinson). Data were analyzed using the FACSDiva software Statistical Analysis For the analysis of the significance of the difference between populations, the Mann-Whitney nonparametric T test was used. P values were calculated using SPSS software, and a P value of less than 0.05 was considered significant. Significant P values are indicated on each figure. For the correlation analysis, a Pearson s correlations analysis was used. Normalized percent inhibition is shown at E:T ratios of 1:4 since enough effectors were available at this ratio for the different subsets for all experiments. An E:T ratio of 1:32 was randomly selected for comparison. 41

54 Figure 8 Figure 8: Strategies for calculation of the normalized percent inhibition and bulk CD8 suppression of viral replication by HD, CP, and ES. (A) Representative data showing the strategy used to determine the normalized percent inhibition for each CD8 + T cell memory and activation subpopulation. Cells were stained with anti-cd3 and anti-cd8 antibodies to distinguish target CD4 + T cells (CD3 + /CD8 - ) and effector CD8 + T cells 42

55 (CD3 + /CD8 + ). Target cells were then gated, and the percent of cells that were GFP positive was calculated. Uninfected target CD4 + T cells were used as a negative control (Left panels). Infected CD4 + T cells cultured without CD8 + T cells were used as a positive infection control (center panels). CD8 + T cell subpopulations were cultured with infected CD4 + T cells at various E:T ratios to allow the analysis of normalized percent inhibition (right panels). (B) Bulk CD8 T cells from HD (n=8), CP (n=4) and ES (n=8) were used in a suppression assay with E:T ratios ranging from 1:1 to 1:128. HD (red line), CP (blue line) and ES (purple line) are shown, and the error bars represent the standard error of the mean. 43

56 2.3 Results CD8 + T cells from ES effectively inhibit viral replication We used a novel modification of a CD8 + T cell suppression assay where CD8 + T cells were isolated and assayed directly ex vivo. These unstimulated cells were cocultured at varying E:T ratios with autologous, unstimulated and freshly isolated target CD4 + T cells that had been infected with replication competent HIV-1 (NL4-3 ΔNef/GFP) by spinoculation. The E:T ratio ranged from 1:1 to 1:128. The percent infection for targets alone was compared to the percent infection of targets cells cultured with CD8 + T cells to calculate a normalized percent inhibition for each subpopulation at each E:T ratio (Figure 8A). CD8 + T cells from ES were markedly more effective than CD8 + T cells from CP and seronegative healthy donors at each E: T ratio (Figure 8 B). The effectiveness of CP CD8 + T cells shown here is likely biased by the fact that data from CP with higher plasma HIV-1 RNA levels could not be used because of very low levels of infection and/or very poor cell viability of target cells at day 3. We would expect that CD8 + T cells from these CP would be less effective at inhibiting viral replication The effector memory CD8 + T cells are the most effective subpopulation at suppressing viral replication To determine what memory population mediates the inhibition of viral replication in ES and CP, CD8 + T cells were isolated directly from 8 ES and 8 CP and stained using anti-ccr7 and anti-cd45ra antibodies. They were then sorted using a fluorescence activated cell sorter into previously defined [80, 81] subpopulations defined by these 44

57 markers: naive (N), central memory (CM), effector memory (EM), and terminal effector (TE) (Figure 9A). After 3 days of co-culture, a dose dependent relationship between the E:T ratio and the normalized percent inhibition was observed for each ES and CP population (Figure 9B) whereas CD8 + T cells from an uninfected, healthy donor had no inhibitory effect (data not shown). The inhibition mediated by CD8 + T cells from ES on day 3 was markedly higher when compared to CD8 + T cells from CP and an even greater level of inhibition mediated by ES CD8 + T cells was observed on day 5 (Figure 9B). The normalized percent inhibition values at E:T ratios of 1:4 and 1:32 were determined for each population. For ES, the bulk, EM, TE and CM subsets had a significantly higher percent inhibition than did naïve CD8 + T cells at a 1:4 E:T ratio. EM cells also had a significantly higher percent inhibition compared to bulk and CM CD8 + T cells at a 1:4 E:T ratio. Naïve CD8 + T cells had little inhibitory potential at either E:T ratio. At a 1:32 E:T ratio, EM CD8 + T cells caused significantly more inhibition of viral replication than did all other subpopulations and bulk CD8 + T cells, and TE CD8 + T cells inhibited viral replication significantly better than naïve CD8 + T cells. Interestingly, differences in the patterns of inhibition by T cell subsets were observed after five days of co-culture (Figure 9C). Naïve CD8 + T cells showed some activity in the five day suppression assay, but it was relatively low compared to all other subtypes and bulk CD8 + T cells at a 1:4 E:T ratio. The CM, TE and EM all showed similar levels of viral suppression, but only CM CD8 + T cells causes significantly higher inhibition relative to bulk CD8 + T cells at a 1:4 E:T ratio. At a 1:32 E:T ratio, the EM and CM subsets and bulk CD8 + T cells suppressed viral replication significantly better than 45

58 did naïve CD8 + T cells. The percent inhibition of TE CD8 + T cells was extremely variable at a 1:32 E:T ratio. The EM CD8 + T cells consistently exhibited high levels of inhibition of viral replication with a median percent inhibition of 69 percent at a 1:4 E:T ratio on day 3 after infection that increased to a median percent inhibition of 89 percent by day 5 after infection. While CM CD8 + T cells were observed to have a median percent inhibition of 48 percent at a 1:4 E:T ratio at day 3, the median percent inhibition increased to 94 percent by day 5 after infection. These data indicate potent suppression of viral replication mediated by ex vivo isolated CD8 + T cells from ES. Overall, EM CD8 + T had a consistent and potent inhibitory effect at both a 1:4 and a 1:32 E:T ratio that was maintained on both day 3 and day 5 after infection. CM CD8 + T cells, while initially producing lower inhibition than EM and TE cells, were observed to inhibit viral replication as effectively as EM CD8 + T cells by day 5 after infection. Naïve CD8 + T cells showed little to no inhibition of viral replication for the duration of the assay. At a 1:4 ratio, bulk CD8 T cells from CP (Figure 9D) were more effective at viral inhibition than any sorted population of cells at day 3 of infection and while all the memory subsets were more effective than naïve CD8 + T cells, there was no significant difference between EM, TE, and CM cell populations. Interestingly at a 1:32 E:T ratio, CM cells were the most effective at inhibiting viral replication. We were not able to analyze the effects of different subpopulations on viral replication at day 5 of infection because of very poor viability of the target cells at this time point, consistent with our prior observation that CD4 + T cells from CP die more quickly than cells from ES and HD after superinfection [45]. 46

59 Figure 9 Figure 9: Analysis of normalized percent inhibition for CD8 + T cell memory subpopulations on day 3 and 5 after infection. (A) CD8 + T cells were isolated by magnetic bead separation from PBMCs. Cells were stained with anti-ccr7-pe and anti- CD45RA-APC antibodies and sorted. Representative data indicating the sorting strategy for one ES patient are shown. All four of the memory subpopulations were collected, and CD8 + T cells were then used at various E:T ratios in the CD8 + T cell suppression assay. (B) The average, normalized percent inhibition plots for ES (n=8) and CP (n=4) at different E:T ratios on day 3 for CP and ES, and day 5 for ES. The normalized percent inhibition of bulk CD8 + T (blue diamonds), naïve CD8 + T cells (red squares), EM CD8 + T cells (orange triangles), CM CD8 + T cells (purple squares) and TE CD8 + T cells (light 47

60 blue circles) are shown. Open data points indicate a normalized percent inhibition of 0. Error bar represent the standard error of the mean. (C and D) Quantification and comparison of normalized percent inhibition for each subpopulation at a 1:4 and a 1:32 E:T ratio for the ES group (C, n=8) and the CP group (D, n=4). The normalized percent inhibition for each patient and each CD8 + T cell subpopulation is shown at a 1:4 E:T ratio (top panels) and a 1:32 E:T ratio (bottom panels) for day 3 (CP and ES) and day 5 (ES). Bulk (B), naïve (N), central memory (CM), terminal effector (TE), and effector memory (EM) CD8 + T cells were compared. Open circles indicate a normalized percent inhibition of 0. The median value of the normalized percent inhibition for each subpopulation is indicated. Only significant P-values are indicated. 48

61 2.3.3 A significant change in the phenotype of ES central memory CD8 + T cells occurs between days 3 and 5 of infection From day 3 to day 5 after infection, there was a dramatic increase in the percent inhibition by the ES CM CD8 + T cell subpopulation (Figures 9C). Therefore, in a subset of the ES, the expression of CCR7 and CD45RA by the CM CD8 + T cells was also analyzed on day 5 after infection to determine if there were changes in the phenotype of the cells in culture after the initial culturing of the pure, sorted CD8 + T cell memory populations (Figure 10). CM CD8 + T cells were the majority population in only one of the five ES that were analyzed. In a majority of patients, the majority cell population present was of the EM or TE phenotype. Thus, the increased suppressive ability after 5 days of infection for the sorted CM CD8 + T cell population may be a result of the differentiation of CM CD8 + T cells into effector CD8 + T cells. 49

62 Figure 10 Figure 10: Changes in the CM CD8 + T cell population by day five after infection. Sorted CD8 + T cells were co-cultured with infected CD4 + T cells on day 0. On day 5, CD8 + T cells were stained with anti-ccr7 and anti-cd45 RA antibodies and the expression of these markers was analyzed and quantified on day 5 after infection (n=5). The percentage of the CD8 + T cells expressing markers indicative of a TE (light blue bar), EM (orange bar), naïve (red bar) and CM (purple bar) phenotype are indicated for each ES analyzed. 50