Sep 24, 2015 - EVALUATE THE EFFECTS OF MARAVIROC ON PATIENT .... Maraviroc treatment in non-R5-HIV-1-infected patients results in the selection of ...
THE USE OF GENETIC SEQUENCING TECHNOLOGIES TO DETERMINE HIV-1 VIRAL TROPISM AND TO EVALUATE THE EFFECTS OF M ARAVIROC ON P ATIENT VIRAL POPULATIONS by
Rachel Ann McGovern B.Sc. (Hons.), Lakehead University, 2006
A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in
The Faculty of Graduate and Postdoctoral Studies (Experimental Medicine) UNIVERSITY OF BRITISH COLUMBIA (Vancouver) September 2015 ©Rachel Ann McGovern, 2015
Abstract HIV-1 infection is reliant on the ability of the virus to enter target cells characterized by the expression of either the CCR5 or CXCR4 co-receptor at the cell surface. It is now well established that the V3 loop of the HIV-1 envelope is the primary determinant of co-receptor use, and that genetic analysis of the V3 loop can be used to infer co-receptor use, or “tropism”. This became clinically relevant with the development of the entry inhibitor, maraviroc (MVC), an anti-HIV compound designed to inhibit HIV-1 cell entry exclusively at the CCR5 co-receptor. As such, the more pathogenic CXCR4-using variants are likely to thrive during MVC therapy. Anti-HIV treatment guidelines now strongly suggest a tropism test be performed when considering the clinical use of MVC. There are two primary objectives discussed in this thesis, 1) the validation of a populationbased sequencing tropism assay designed to predict virological response to MVC; 2) to apply a next generation sequencing tropism assay to investigate the selective pressures exerted by MVC on mixed tropic HIV-1 populations. The validation studies presented in this thesis demonstrate the reliability of a populationbased sequencing assay to infer virological response to MVC in two large, multinational cohorts. The results of these studies have promoted the worldwide expansion of this genotypic assay as a practical and affordable option for tropism inference. In addition, a more intensive investigation into the selective pressures exerted by MVC on mixed tropic HIV-1 populations demonstrated the reproducible outgrowth of preexisting variants most genetically characteristic of CXCR4-using virus. The results of these studies suggest MVC may be effective against a broader range of variants than previously thought. In general, the four studies described in this thesis demonstrate the clinical utility of genetic sequencing when considering the use of MVC.
ii
Preface The main body of the thesis features three peer-reviewed manuscripts published in the scientific literature, as well as a fourth manuscript not yet submitted for publication. The three published manuscripts are reprinted in this thesis with permission granted from the respective copyright holders. The candidate is primary author on all four manuscripts, responsible for composing the entirety of each. In addition to reporting the findings of each study to the scientific community, the candidate actively participated in the research described in these manuscripts, including study design, laboratory generation of data and data analysis. Co-authors at the candidate’s research laboratory include senior laboratory research assistants Theresa Mo and Winnie Dong; data analysts Chanson Brumme, Art Poon, Conan Woods and Xiaoyin Zhong as well as the candidate’s graduate supervisor, Dr. Richard Harrigan. There are a number of external collaborators associated with the studies from which samples used for the retrospective analyses presented in the thesis were taken. A version of chapter 2 has previously been published and reproduced here. Rachel A McGovern, Thielen A, Mo T, Dong W, Woods CK, Chapman D, Lewis M, James I, Heera J, Valdez H, Harrigan PR. (2010) Population-based V3 genotypic tropism assay: a retrospective analysis using screening samples from the A4001029 and MOTIVATE studies. AIDS 24:2517-2525. I am the primary author of this manuscript, as such the publisher of this journal does not require copyright permission to be granted for reproduction in a doctoral dissertation. ©Wolters Kluwer Health/Lippincott Williams & Wilkins. A version of chapter 3 has also been published in the scientific literature and reproduced here. Rachel A McGovern, Thielen A, Portsmouth S, Mo T, Dong W, Woods CK, Zhong X, Brumme CJ, Chapman D, Lewis M, James I, Heera J, Valdez H, Harrigan PR. (2012) Population-based sequencing of the V3 loop can predict the virological response to maraviroc in treatment-naïve patients of the MERIT trial. Journal of Acquired Immune Deficiency Syndromes 61(3):279-286. I am the
iii
primary author of this manuscript, as such the publisher of this journal does not require copyright permission to be granted for reproduction in a doctoral dissertation. ©Lippincott Williams & Wilkins. Lastly, chapter 4 has been published and reproduced here with permission under RightsLink license number: 3532690277492. Rachel A McGovern, Symons J, Poon AFY, Harrigan PR, van Lelyveld SFL, Hoepelman AIM, van Ham PM, Dong W, Wensing AMJ, Nijhuis M. (2013) Maraviroc treatment in non-R5-HIV-1-infected patients results in the selection of extreme CXCR4using variants with limited effect on the total viral setpoint. Journal of Antimicrobial Chemotherapy 68(9):2007-2014. ©Oxford University Press. Ethical approval was granted by the Providence Health Care–University of British Columbia Research Ethics Board for the studies presented in Chapters 2 and 3, H07-01901, as well as that presented in Chapter 5, H10-00565.
iv
Table of Contents Abstract.......................................................................................................................................... ii Preface.......................................................................................................................................... iii Table of Contents ........................................................................................................................ v List of Tables ............................................................................................................................ viii List of Figures .............................................................................................................................. ix List of Abbreviations ................................................................................................................. xi Acknowledgements.................................................................................................................. xiv Dedication ................................................................................................................................... xv Chapter One: A General Introduction and Thesis Objectives........................................... 1 1.1 The Human Immunodeficiency Virus .............................................................................. 1 1.1.1 The Identification of a Pandemic................................................................................... 1 1.1.2 HIV Types, Groups & Clades ........................................................................................ 2 1.1.3 Cross-Species Transmission ........................................................................................... 4 1.1.4 Transmission among Humans ....................................................................................... 4 1.2 HIV the Virus ......................................................................................................................... 5 1.2.1 The Genome & the Virion............................................................................................... 5 1.2.2 The Replication Cycle ..................................................................................................... 9 1.2.2.1 Cellular Entry & Reverse Transcription ..................................................................... 9 1.2.2.2 Integration & Replication.......................................................................................... 10 1.2.2.3 Budding & Maturation ............................................................................................. 12 1.2.3 HIV Disease Progression & Pathogenesis .................................................................. 14 1.3 HIV Entry .............................................................................................................................. 17 1.3.1 HIV Cell Interactions..................................................................................................... 17 1.3.2 HIV Receptors & Co-Receptors ................................................................................... 20 1.3.3 Viral Structures for Cell Entry ..................................................................................... 21 1.3.4 The Entry Mechanism ................................................................................................... 23 1.3.5 The Chemokine Co-Receptors & HIV Infection ........................................................ 25 1.3.5.1 Tropism Terminology ................................................................................................ 25 1.3.5.2 Tropism Switch with Disease Progression ................................................................ 26 1.4 The Clinical Management of HIV .................................................................................... 27 1.4.1 The Concept of Antiretroviral Therapy...................................................................... 27 1.4.2 The Antiretroviral Compounds ................................................................................... 29 1.4.3 The CCR5 Antagonists .................................................................................................. 31 1.4.3.1 Inhibition of HIV by Maraviroc ................................................................................ 32 1.4.3.2 The Clinical Trials of Maraviroc ............................................................................... 33 1.4.3.3 Maraviroc in the Presence of CXCR4-Using Virus.................................................. 35 v
1.4.4 Maraviroc in the Clinic ................................................................................................. 36 1.4.4.1 Treatment Guidelines ................................................................................................ 36 1.4.4.2 Testing for Tropism Prior to Initiating Maraviroc ................................................... 37 1.5 Sequencing ........................................................................................................................... 39 1.5.1 Understanding HIV through Genetics ....................................................................... 39 1.5.2 Sequencing Technologies ............................................................................................. 41 1.5.2.1 Concepts in Sequencing ............................................................................................ 41 1.5.2.2 Population-based Sequencing .................................................................................... 43 1.5.2.3 Next Generation “Deep” Sequencing ....................................................................... 46 1.5.3 From Sequence to Relevance........................................................................................ 52 1.5.4 Genotyping & Bioinformatics for Tropism Prediction ............................................. 53 1.6 Thesis Objectives & Organization ................................................................................... 56 1.6.1 Thesis Objectives ........................................................................................................... 56 1.6.2 Thesis Organization & Structure ................................................................................. 56 Chapter Two: Population-based V3 Genotypic Tropism Assay: a Retrospective Analysis Using Screening Samples from the A4001029 and MOTIVATE Studies .. 59 2.1 Introduction........................................................................................................................ 59 2.2 Methods ................................................................................................................................. 61 2.2.1 Study Population ........................................................................................................... 61 2.2.2 Sequencing...................................................................................................................... 62 2.2.3 Sequence Interpretation ................................................................................................ 63 2.2.4 Outcome Measures & Predictor Variables ................................................................ 64 2.2.5 Cutoff Point Optimization............................................................................................ 65 2.3 Results ................................................................................................................................... 66 2.4 Discussion ............................................................................................................................. 79 Chapter Three: Population-based Sequencing of the V3 Loop Can Predict the Virological Response to Maraviroc in Treatment-Naïve Patients of the MERIT Trial 83 3.1 Introduction .......................................................................................................................... 83 3.2 Methods ................................................................................................................................. 85 3.2.1 Study Population ........................................................................................................... 85 3.2.2 Sequencing...................................................................................................................... 86 3.2.3 Sequence Interpretation ................................................................................................ 87 3.2.4 Outcome Measures & Predictor Variables ................................................................. 87 3.3 Results ................................................................................................................................... 88 3.3.1 Subsequent Changes in Trofile Assay Results........................................................... 93 3.3.2 Comparison with ESTA ................................................................................................ 94 3.3.3 Assay Performance in Different HIV Clades ............................................................. 96 3.4 Discussion ............................................................................................................................. 97
vi
Chapter Four: Maraviroc Treatment in Non-R5-HIV-1-Infected Patients Results in the Selection of Extreme CXCR4-using Variants with Limited Effects on the Total Viral Setpoint ..................................................................................................................................... 101 4.1 Introduction ........................................................................................................................ 101 4.2 Methods ............................................................................................................................... 103 4.2.1 Study Population ......................................................................................................... 103 4.2.2 Laboratory Methods .................................................................................................... 104 4.2.3 Sequence Processing & Tropism Interpretation ...................................................... 105 4.2.4 Fitness Analysis ........................................................................................................... 106 4.3 Results ................................................................................................................................. 106 4.4 Discussion ........................................................................................................................... 113 Chapter Five: Next Generation “Deep” Sequencing to Evaluate Viral Tropism in HIV1 Patients Exposed to Short-term Maraviroc Add-on Therapy ....................................... 117 5.1 Introduction ........................................................................................................................ 117 5.2 Methods ............................................................................................................................... 119 5.2.1 Study Population ......................................................................................................... 119 5.2.2 Sample Preparation & “Deep” Sequencing ............................................................. 120 5.2.3 Sequence Processing ................................................................................................... 121 5.2.4 Bioinformatics Analyses ............................................................................................. 121 5.3 Results ................................................................................................................................. 123 5.4 Discussion ........................................................................................................................... 132 Chapter Six: General Discussion .......................................................................................... 138 6.1 Summary of Thesis............................................................................................................ 138 6.1.1 Validation of a Population-Based Genotypic Tropism Assay for Clinical Use .. 140 6.1.2 The Effects of Maraviroc on Non-R5 Virus Populations........................................ 143 6.2 Contributions to the Field of HIV .................................................................................. 145 6.3 Future Directions ............................................................................................................... 147 6.4 Closing Remarks................................................................................................................ 151 Bibliography ............................................................................................................................. 153 Appendix 1 ................................................................................................................................ 187 Chapter Three Supplementary Material ........................................................................... 187 Appendix 2 ................................................................................................................................ 196 Chapter Four Supplementary Material ............................................................................. 196 Appendix 3 ................................................................................................................................ 199 Chapter Five Supplementary Material .............................................................................. 199
vii
List of Tables Table 2.1. Percentage of patients with virological response to maraviroc at week eight. ...............69 Table 3.1. Baseline characteristics for participants of the MERIT trial. ..............................................90 Table 3.2. Results of rescreening the patient population of the MERIT trial for tropism. ...............91 Table 4.1. Changes in the most prevalent sequence following exposure to maraviroc in patients screened as having non-R5 virus. ...........................................................................................................109 Table 5.1. Baseline Characteristics Table (N=27). ................................................................................123 Table 5.2. The most prevalent sequence at Days 0 and 7 of maraviroc therapy from patients screened as having ≥2% non-R5 virus at any time point. ...................................................................128
viii
List of Figures Figure 1.1. The HIV classification system................................................................................................ 3 Figure 1.2. The HIV-1 genome. ................................................................................................................. 7 Figure 1.3. The HIV-1 virion. .................................................................................................................... 8 Figure 1.4. The HIV-1 replication cycle. .................................................................................................14 Figure 1.5. HIV-1 target cells and latency. .............................................................................................17 Figure 1.6. HIV-1 cell binding and membrane fusion. .........................................................................23 Figure 1.7. Conformational changes during HIV-1 cell entry. ............................................................25 Figure 1.8. The Sanger sequencing method. ..........................................................................................45 Figure 1.9. Next generation 454 “deep” sequencing. ............................................................................49 Figure 2.1. Virological response to maraviroc stratified by tropism test. ..........................................68 Figure 2.2. Virological response to maraviroc stratified by optimized background treatment. .....70 Figure 2.3. Virological response to maraviroc demonstrating concordance between Trofile and V3 genotype tropism assays. .....................................................................................................................72 Figure 2.4. Virological response to maraviroc in a subset of patients screened as having non-R5 virus and randomly selected patients screened as having R5 virus. ...................................................74 Figure 2.5. Frequency distribution of patient samples as determined by the assigned g2p score in accordance with therapeutic response categories. .................................................................................75 Figure 2.6. Population sequencing with optimized g2p cutoff points can predict the virological response to maraviroc. ...............................................................................................................................77 Figure 2.7. Population sequencing with optimized g2p cutoff points can effectively predict a) the probability of a tropism change and b) time to discontinuation in the study. ...................................78 Figure 3.1. Virological response to maraviroc in MERIT trial participants rescreened for tropism using a V3 genotypic assay. ......................................................................................................................92
ix
Figure 3.2. Time to change in tropism from R5 to non-R5 in MERIT trial participants rescreened for tropism using a V3 genotypic assay...................................................................................................94 Figure 3.3. Concordance between tropism results in MERIT trial participants rescreened for tropism using a V3 genotypic assay and ESTA. .....................................................................................96 Figure 4.1. Virological response to maraviroc in patients screened as having non-R5 virus. .......108 Figure 4.2. Virological response to maraviroc in R5 and non-R5 viral populations. .....................110 Figure 4.3. Changes in sequence prevalence by g2p FPR following maraviroc exposure.............111 Figure 4.4. Viral fitness in the presence of maraviroc as a function of g2p false positive rate. ....112 Figure 5.1. Virological response to short-term maraviroc exposure.................................................125 Figure 5.2. Changes in sequence frequency stratified by g2p FPR after short-term maraviroc therapy in patients found to have ≥2% non-R5 HIV at any time point. ............................................127 Figure 5.3. Viral fitness in the presence of maraviroc as a function of g2p FPR (FPR ≤ 20). .........129 Figure 5.4. Virological response to maraviroc demonstrating concordance between 454 “deep” sequencing and both ESTA and TROCAI. ............................................................................................131
x
List of Abbreviations 3TC – Lamivudine A – Adenine AIDS – Acquired Immune Deficiency Syndrome ART – Antiretroviral Therapy ATP – Adenosine Triphosphate AZT – Azidothymidine (Zidovudine) b.i.d. – twice-daily dosage bp – nucleotide base pair C – Cytosine CA – Capsid Protein CCR5 – C-C-Motif Receptor 5 CD4 – Cluster of Differentiation 4 CRF – Circulating Recombinant Form CXCR4 – C-X-C-Motif Receptor 4 CYP3A – Cytochrome P450, family 3, subfamily A D/M – Dual and/or Mixed Tropism DNA – Deoxyribonucleic Acid dNTP – Deoxynucleotide Triphosphate ddNTP – Dideoxynucleotide Triphosphate dTTP – Deoxythymidine Triphosphate ddTTP – Dideoxythymidine Triphosphate EFV – Efavirenz emPCR – emulsion PCR ESTA – Enhanced Sensitivity Trofile Assay FDA – United States of America Food and Drug Administration FPR – False Positive Rate G – Guanine g2p – geno2pheno bioinformatics algorithm GALT – Gut-Associated Lymphoid Tissue Gp41 – Envelope Glycoprotein 41 Gp120 – Envelope Glycoprotein 120
xi
Gp160 – Envelope Glycoprotein 160 GS-FLX – Genome Sequencer FLX (Roche/454 Life Sciences) GS Jr. – Genome Sequencer Junior (Roche/454 Life Sciences) GTR – General Time-Reversible (nucleotide substitution models) HAART – Highly Active Antiretroviral Therapy HIV – Human Immunodeficiency Virus HR – Heptad Repeat IN – Integrase INSTI – Integrase Strand Transfer Inhibitor IQR – Interquartile Range kb – kilobase pair LCB – Lower Confidence Bound LOCF – Last Observation Carried Forward LTR – Long Terminal Repeat MA – Matrix Protein MAAFT – Multiple Alignment using Fast Fourier Transform MCT – Maraviroc Clinical Test MERIT – Maraviroc versus Efavirenz in Treatment-Naïve Patients M=F – Missing Equals Failure MHC – Major Histocompatability Complex MID – Multiplex Identifier MIP – Macrophage Inflammatory Protein MOTIVATE – Maraviroc versus Optimized Therapy in Viremic Antiretroviral TreatmentExperienced Patients MT-2 – Human T-cell leukocyte cell line MSM – Men who have sex with Men MVC - Maraviroc NC – Nucleocapsid Protein Nef – Negative regulatory factor NNRTI – Non-Nucleoside Reverse Transcriptase Inhibitor NRTI – Nucleoside Reverse Transcriptase Inhibitor NSI – Non-Syncytium Inducing OBT – Optimized Background Therapy PBMC – Peripheral Blood Mononuclear Cell PBO – Placebo
xii
PCR – Polymerase Chain Reaction PI – Protease Inhibitor PIC – Pre-Integration Complex PPi – Pyrophosphate PR – Protease pr160 – Gag-Pol precursor protein 160 PSSM –Position-Specific Scoring Matrix PTP – Picotitre Plate pVL – Plasma Viral Load q.d. – once-daily dosage R5 – CCR5-Using HIV-1 RANTES – Regulated on Activation, Normal T-Cell Expressed and Secreted RAxML – Randomized Axelerated Maximum Likelihood Rev – Regulator of expression of virion proteins RNA – Ribonucleic Acid RNaseH – Ribonuclease H RT – Reverse Transcriptase RT-PCR – Reverse Transcription Polymerase Chain Reaction SDF – Stromal-Derived Factor SI – Syncytium-Inducing SIV – Simian Immunodeficiency Virus SP – Spacer Protein SVM – Support Vector Machine T – Thymine Tat – Transactivator of transcription tRNA – Transfer Ribonucleic Acid TROCAI – Tropism Coreceptor Assay Information UNAIDS – Joint United Nations Program on HIV/AIDS V3 – Third Hypervariable Loop Vif – Viral infectivity factor Vpr – Viral protein R Vpu – Viral protein U wSS – weighted Sensitivity Score X4 – CXCR4-Using HIV-1 ZDV - Zidovudine
xiii
Acknowledgements First and foremost, I would like to thank my PhD supervisor, Richard Harrigan. You are a truly patient, understanding and knowledgeable individual. I am honoured to have been a student under your tutelage in such a productive environment. I would also like to thank my Supervisory Committee, Thomas Kerr, Peter Phillips and Scott Tebbutt. To all members of my supervisory committee, your support, guidance and expertise have been invaluable. I am happy to have shared the graduate journey with my fellow PhD students Chanson Brumme, Guinevere Lee, Luke Swenson and those from other labs. Lunch dates, conference pals, commiserating companions, brainstorming comrades, thank you. The lab members, past and present, at the British Columbia Centre for Excellence in HIV/AIDS have been an incredibly supportive group. Every individual has helped to some capacity whether with lab work, study design, problem solving, data analysis, reviewing documents, moral support and even emotional support. I truly appreciate all they have done for me. I wish to thank study participants, coauthors and collaborators around the globe. To the study participants, as cliché as it sounds, we wouldn’t be here without you. Coauthors and collaborators, I am grateful for the opportunity to have met and worked with you. The pleasure was all mine. I would also like to recognize the dedicated teachers, professors and mentors of my youth, those who never ceased to encourage me in my academic pursuits and opened my eyes to what is and what could be. And of course, I would like to express my love and utmost appreciation to my primary support group, my four pillars. Mom, for your unconditional support, right-brain promotion and indisputable love; Grandma, for my inspiration, preschool education and appreciation of all puzzles; Rob, for your continuous encouragement, limitless support and for being my one true hero; and Charlie, for your everlasting respect, patience, partnership and for all of the adventures we have been on and have yet to embark upon. xiv
Dedication I dedicate this work to my four pillars.
xv
Chapter One: A General Introduction and Thesis Objectives 1.1 The Human Immunodeficiency Virus 1.1.1 The Identification of a Pandemic In 1981 young men were being diagnosed with and succumbing to opportunistic infections that were otherwise only observed in those with weakened immune systems. These young men happened to be homosexual, or men who have sex with men (MSM), living in New York and California. It became clear that these previously healthy men had failing immune systems and the search for a cause was initiated.1 Nearly two years later it was announced that a retrovirus, isolated from the tissues of a patient with lymphadenopathy, might be the cause of the newly termed Acquired Immunodeficiency Syndrome (AIDS).2 This retrovirus was later to become known as Human Immunodeficiency Virus (HIV) and confirmed as the infectious agent that leads to AIDS.3–5 Over thirty years later, in 2013 it was reported by UNAIDS that 35 million people worldwide were living with HIV.6 HIV is a pandemic.
The highest rates of HIV/AIDS-associated morbidity and mortality are in developing nations. UNAIDS estimates that 70% of global HIV infections are within Africa, and in 2013, Sub-Saharan Africa accounted for an estimated 74% of AIDS-related deaths worldwide.6 Though HIV is now considered manageable with antiretroviral therapy (ART), clinical management is dependent on available resources. With no curative strategies or working vaccines HIV continues to be a public health challenge. Global
1
initiatives are underway in attempts to lessen the burden and the spread of HIV, with a target of reducing transmission by 50% by the end of 2015.7 In the UNAIDS Gap Report released in 2013, rates of new infection had decreased by 13% over the past three years and AIDS-related deaths had decreased by 19%. In 2014, UNAIDS introduced the more ambitious 90-90-90 initiative.8 The 90-90-90 initiative focuses on the identification of HIVpositive individuals and ensuring they receive proper treatment. The initiative is defined by three goals to be achieved by 2020: 1) 90% of the HIV-positive population will know their HIV status; 2) 90% of the HIV-positive population will receive antiretroviral therapy; and 3) 90% of the HIV-positive individuals receiving antiretroviral therapy will attain viral suppression.8 With initiatives to expand HIV testing, care, treatment and prevention strategies, progress is being made.
1.1.2 HIV Types, Groups & Clades There are two primary types of HIV, HIV type 1, (HIV-1) and HIV type 2 (HIV-2). They are genetically distinct from each other and differ in many ways, including region of origin, species of origin, effects on disease progression and virulence. HIV-1 is the cause of the global pandemic. HIV-2 is concentrated in West Africa, and appears to be less virulent when compared to HIV-1.9,10 There are three primary groups of HIV-1 variants, and they are categorized as M (main), O (outlier) and N (non-M, non-O) based on genetic diversity and phylogeny. Group M viruses account for the vast majority of HIV infections, estimated to be responsible for 33 million (~94%) infections worldwide.11,12 More recently, the possibility of a fourth HIV-1 group has arisen. Currently, there are only two reported cases of HIV-1 demonstrating characteristics of a new group. For this reason, the new group is considered “putative” and has been designated group P.13–15 Variants within HIV2
1 group M are further subdivided into subtypes in order to account for genetic variation, these phylogenetic clusters are called clades.16,17 Genetic variation of HIV-1 within subtypes can be up to 17%, whereas genetic variation between subtypes can be up to 35%.16 According to the Los Alamos HIV sequence database, to date there are clades (A-D, F-H, J, K) with two sub subtypes in clades A (A1 and A2) and F (F1 and F2). In addition, there are over sixty-six circulating recombinant forms (CRFs) of HIV-1 group M viruses (Figure 1.1).18 Clades and CRFs tend to be geographically clustered, and in regions with multiple clades present may also be compartmentalized according to social and demographic characteristics.19–21 Clade C is the predominant infection in South Africa. It can also be found in India and China, accounting for approximately 50% of global HIV-1 infections.16 Clade B is the predominant HIV-1 subtype in the western countries of Europe, the Americas and Australia but only accounts for roughly 12% of the global infection.20,11
Figure 1.1. The HIV classification system. A diagram illustrating the four levels of classification (type, group, subtype and sub subtype) used to define HIV variants based on shared characteristics and genetic similarity. This image was created by the author.
3
1.1.3 Cross-Species Transmission The origins of HIV have been debated. It is now widely accepted that HIV is a human descendant of the Simian Immunodeficiency Virus (SIV)22 Using the endogenous effects of HIV as a molecular clock, it is believed that the first cross-species transmission of HIV-1 group M virus occurred at the beginning of the twentieth century, sometime between 1910 and 1930.23,24 Based on the analysis of tissue samples collected in Kinshasa, Democratic Republic of Congo, there is conclusive evidence of the presence of HIV-1 in Central Africa as early as 1959.24,25
It wasn’t until 1999 that the origin of HIV-1 was confirmed, when Gao et al. found HIV-1 virus to be phylogenetically similar to the form of SIV infecting chimpanzees, Pan troglodytes troglodytes.22,26 Keele et al. went further to differentiate between Pan troglodytes troglodytes communities inhabiting south-central and southeastern Cameroon, concluding that the two groups were the sources for group N and group M HIV-1, respectively.27 The initial route of transmission was likely via blood contact in the context of hunting chimpanzees as bushmeat.28–31
1.1.4 Transmission among Humans There are multiple routes of HIV transmission, all of which occur when bodily fluids containing virus such as the blood, or genital secretions of an infected individual come into contact with the blood, mucosal linings or damaged tissue of a new host. A recent literature review performed by Patel et al. ranked the risk of transmission for each route based on the incidence of new infections per 10 000 exposure events.32 The risk of
4
transmission from infected blood products is extremely high, followed by vertical motherto-child transmission. They also report a substantial risk when engaging in anal intercourse and when sharing needles for intravenous drug use, as well as risk associated with percutaneous needlesticks and penile-vaginal intercourse.32 It is important to note that there are various prevention strategies designed to reduce the risk of transmission for all routes, though many political, social and biological factors affect the extent to which those prevention strategies are implemented, accessed and beneficial.33–38
The rates of new infections associated with the different transmission routes vary between continents.6 Based on the geographical representation of HIV incidence among risk groups published in UNAIDS Gap Report 2013, the primary route of transmission in the Americas and Western Europe is homosexual intercourse, whereas the primary route in Africa is heterosexual intercourse. Transmission risks in Asia are greatly associated with sex work, and in Eastern Europe there are far more new infections associated with the intravenous drug use risk group. 6,39,40 Differences in the geographic distribution of transmission routes may be due to a number of societal factors including laws, policies, healthcare infrastructure, and the degree of social acceptance for such behaviours as homosexual intercourse, intravenous drug use and the treatment of women.
1.2 HIV the Virus 1.2.1 The Genome & the Virion HIV is a member of the Lentivirus genus within the Retroviridae family.2,41 The virion contains two single-stranded ribonucleic acid (RNA) molecules of nearly ten kilobases (kb)
5
in length.42,43 The relatively short HIV genome is organized into nine open reading frames. These reading frames overlap to some degree, allowing the virus to optimize the number of translated proteins (Figure 1.1).43–45 Characteristic of retroviruses, there are three large open reading frames referred to as gag, pol and env. The gag reading frame encodes for the Gag precursor protein, pr55, which is cleaved by the viral protein, protease (PR) during virion maturation to form the structural proteins: matrix protein (MA; p17), capsid protein (CA; p24), nucleocapsid protein (NC; p7) and three smaller peptides, spacer peptide 1 (SP1), spacer peptide 2 (SP2) and protein 6 (p6).44,46–48 Similarly, the env reading frame encodes the Env precursor protein (gp160), though this precursor is later cleaved by a host cellular protease to form the two elements of the envelope surface spike, glycoprotein 120 (gp120) and glycoprotein 41 (gp41).44,49,50 The pol reading frame overlaps with gag, creating a GagPol precursor protein (pr160).43 The enzymes reverse transcriptase (RT), ribonuclease H (RNaseH), integrase (IN) and protease are the products of viral proteolytic cleavage of the Gag-Pol precursor protein.44 In addition to the structural and enzymatic proteins, HIV also contains the genes for accessory proteins and regulatory proteins encoded for within the remaining six reading frames. The role of regulatory proteins such as regulator of viral expression (Rev) and transactivating protein (Tat) is to enhance viral gene expression whereas the accessory proteins, negative factor (Nef), viral infectivity factor (Vif), viral protein r (Vpr) and viral protein u (Vpu) all act to augment viral infectivity and pathogenicity.44,51,52 The entire genome is book-ended by the 5’ and 3’ long terminal repeats (LTRs), which are placed at either end of the proviral deoxyribonucleic acid (DNA) during reverse transcription. The LTR ends contain binding sites for cellular transcription factors
6
used in the integration and transcription of viral DNA, act as a promoter for viral gene expression and participate in the post-transcription polyadenylation.53–55
Figure 1.2. The HIV-1 genome. A linear schematic representation of the HIV-1 genome organized into the three reading frames. The gag, pol and env genes encode the structural, enzymatic and envelope proteins, respectively. This image was created by the author.
The mature infectious virion is spherical and has been estimated to be between 120 to 200 nm in diameter.56–58 The virion is composed of a lipid bilayer envelope, matrix, capsid, various viral proteins and two copies of single stranded RNA associated with nucleocapsid. The lipid portion of the viral envelope is of cellular origin, characterized by viral proteins at the surface often referred to as spikes. The spikes are trimeric glycoprotein complexes comprised of non-covalently linked surface gp120 protein and the transmembrane anchor gp41.59,60 The gp120-gp41 complex is necessary in the binding and fusion of the virion to the target cell.61–63 Matrix protein is associated with the inner surface of the viral envelope, and participates in the incorporation of gp120-gp41 spikes into the envelope of the new virion.64–66 Inside the mature virion is a conical capsid made of capsid protein, containing the RNA and viral proteins. The viral proteins found within the capsid include the enzymatic proteins protease, reverse transcriptase, RNaseH, integrase and the
7
structural nucleocapsid, which forms a nucleocapsid-RNA complex.57,67 The nucleocapsid has been described as coating the RNA and thought to be associated with RNA packaging, similar to the histones of eukaryotic DNA.68,69 In the micrograph and accompanying schematic presented in Figure 1.3, panels A) and B), one can clearly see the accumulation of gag proteins at the membrane of both the budding and immature viruses, on the left and right respectively. However, in the mature virion, pictured in the centre and featured in panel C), the formation of the capsid following proteolytic cleavage of the gag precursor protein can clearly be identified.70
Figure 1.3. The HIV-1 virion. Panel A is a micrograph image depicting three budding stages of HIV-1, from assembly to the mature virion. The micrograph is reproduced as a schematic in Panel B detailing the three stages, assembly of proteins at the cell membrane on the left, the released immature virion on the right and the mature virion in the centre. Panel C is a diagrammatic representation of the mature virion highlighting structural, enzymatic and envelope proteins. Panels A and B were taken from the open access article by Baumgärtel et al..70 The image in Panel C was created by the author.
8
1.2.2 The Replication Cycle 1.2.2.1 Cellular Entry & Reverse Transcription The first step in the HIV replication cycle is entry into the new host cell. The circulating infectious virion binds to a target cell, releasing its components into the cytoplasm. Target cells have two primary characteristics at their plasma membrane required for viral infection, the cluster of differentiation 4 (CD4) receptor and a chemokine co-receptor.71–75 There are two primary co-receptors used by HIV-1 to gain entry into the cell, chemokine (C-C motif) receptor 5 (CCR5) and chemokine (C-X-C motif) receptor 4 (CXCR4).73,74,76,77 In order to initiate the fusion between the virion and the cell, HIV-1 must first bind the CD4 receptor, which then exposes the viral binding site for either CCR5 or CXCR4.78,79 HIV binds to the cell surface receptors by way of the trimeric envelope glycoprotein spike, the gp120-gp41 complex.63 The conformational changes in the structure of the spike following the interactions of gp120 with both cell receptors pull the virion closer to the cell surface where gp41 can then initiate fusion of the virion with the cell membrane. As the virion and the cell membrane fuse, the envelope becomes incorporated into the membrane and the contents of the virion are released into the cellular cytoplasm.62,63
After the capsid is released from the virion into the cytoplasm it is “uncoated”.80 Capsid molecules are partially dissociated to form a reverse transcription complex. The details associated with the timing and location of when the capsid is uncoated have yet to be confirmed but uncoating has been shown to occur roughly one hour after viral fusion.80,81 Inside the reverse transcription complex, single stranded RNA is converted into
9
double stranded DNA through the process of reverse transcription, characteristic of retroviruses.53,82–84 First, the single-stranded viral RNA template is transcribed by the reverse transcriptase RNA-dependent DNA polymerase to synthesize a complementary DNA strand. The reverse transcriptase RNaseH then digests the viral RNA template leaving the newly synthesized DNA strand as the template from which the reverse transcriptase DNA-dependent DNA polymerase then generates a second complementary DNA strand. This second synthesis yields a double-stranded proviral DNA molecule.85 Reverse transcription can account for the high rate of diversity in HIV as it is a highly error prone process. Mutations arise as newly synthesized sequences are not checked for nucleotide accuracy by the 3’-5’ exonuclease “proof-reader” characteristic of more stable genomes.86,87 Unfortunately such alterations to the viral genome and genetic diversity make the design of treatments and vaccines a challenge.
1.2.2.2 Integration & Replication When the viral genome has been transcribed into a double-stranded DNA molecule, the reverse transcription complex dissociates and the complex is reassembled to form the pre-integration complex. It is largely accepted that the pre-integration complex contains viral DNA and the viral proteins reverse transcriptase, integrase, Vpr and matrix.88,89 The proviral DNA is ushered into the nucleus through the nuclear pore as part of the pre-integration complex.90 It is believed that Vpr helps to guide the pre-integration complex to the nuclear membrane but it is the nuclear localization signals found on matrix and integrase proteins that help guide and carry the pre-integration complex through the nuclear pore.91,92 Insertion of the newly synthesized proviral DNA recruits the host cell to begin replicating the viral genome. Integration of the viral genome into the host DNA is 10
characteristic of retroviruses. In most cases, the retrovirus requires a cell to be actively dividing such that the nuclear membrane is susceptible to nuclear entry and infection. HIV however, has the ability to infect resting or non-dividing cells as it enters the nucleus through the nuclear pore as part of the pre-integration complex.90,93
The integration process is catalyzed by integrase and can be divided into two stages.90 First the 3’ ends of the proviral DNA, near the LTR, are cleaved to produce two base pair overhanging “sticky ends”. This step is called 3’ processing and it prepares the proviral DNA for integration.94,95 Next, the host DNA is cleaved to produce a 5’ free end and a recessed 3’ end to which the viral DNA will be covalently linked. The host DNA polymerase then completes the covalent attachment of the 5’ end of the viral DNA to the 3’ end of the host DNA referred to as strand transfer and filling in the gaps.94–96
After the viral DNA has been integrated into the host genome, the cellular replication machinery transcribes the DNA into RNA. Replication begins in the “R” (repeat) region of the 5’ LTR by the host RNA polymerase II. Genomic regions for transcription regulation, particularly initiation, are found within the LTRs at both the 5’ and 3’ ends of the viral DNA.44,55 The viral transcripts are processed and modified in the same way as host RNA, including the addition of a string of adenosine nucleotides forming a “polyA” tale.44 The polyA tail aids the transcripts in exiting from the nucleus, as well as preventing their enzymatic degradation and promoting their translation in the cytoplasm.44,97 Spliced and unspliced RNA are transported out of the nucleus where the mRNA molecules are translated and folded into the viral proteins, where they await virion budding at the cellular membrane with the newly transcribed RNA genome.46,99,97
11
1.2.2.3 Budding & Maturation The Gag and Gag-Pol precursor proteins are translated late in the replication cycle. They move directly to the plasma membrane following translation and remain uncleaved until after budding. Gag is anchored at the plasma membrane by the N-terminal end of the matrix protein.67,100 The individual proteins within the polypeptide are organized radially at the cell membrane surface: matrix, capsid, SP2, nucleocapsid, SP1 and p6. The positions of the structural proteins in the polypeptide are relatively conserved within the mature virion (Figure 1.3). Late proteolytic cleavage of the Gag and Gag-Pol precursor proteins ensures budding does not occur prematurely.57,100,101
Prior to budding, additional components to be incorporated into the new virions assemble at the cell membrane, including the envelope proteins, enzymatic proteins, vpr and the genomic RNA. Host components tRNA and cyclophilin A are also collected at the membrane for packaging into the new virions.101 The former is incorporated for use in reverse transcription after the virion has entered a new host cell; the latter is believed to assist in the uncoating of the viral capsid.100,102 Prior to budding the matrix protein Nterminus is myristoylated, the myristoyl group playing a role in both transporting and attaching the matrix protein to the plasma membrane. Budding is triggered when the matrix N-terminal myristoyl group interacts with the plasma membrane.44,102 The budding process serves to encapsulate the collected viral components to form a new virus particle. The Gag polypeptides form a layered band along the inner surface of the immature virion, and the non-covalently linked gp120-gp41 complexes are incorporated to span the new viral envelope, forming the viral spikes that mark the surface of the virion.64,66,100,101 P6 interacts with a host trafficking system to complete budding, allowing the newly formed 12
virion to fully separate from the cell membrane.66,102 Even so, a further host restrictor called tetherin literally tethers the new virions to the surface of the cell. The protein Vpu overcomes tetherin, completing the budding process as the virion is fully released into the extracellular matrix.100,103 However, the newly released virus particle is in its immature form at this stage.
During the late stages of the HIV-1 replication cycle, protease is translated and it subsequently proteolytically cleaves itself from the Gag-Pol precursor protein. Following budding, protease is responsible for initiating the maturation of the immature virion by cleaving the radially organized Gag and Gag-Pol precursor proteins into their structural component proteins.57,100,102,104 After cleavage, the matrix remains associated with the envelope. The capsid proteins associate to form the capsid around the nucleocapsids, and the nucleocapsid proteins interact with the two copies of viral RNA to form the two nucleocapsid protein-RNA complexes housed within the capsid. The mature virion is now infectious (Figure 1.3c).57,101 A complete overview of the HIV-1 replication cycle is schematically represented in Figure 1.4.
13
Figure 1.4. The HIV-1 replication cycle. A schematic diagram illustrating the HIV replication cycle, which begins with the binding and fusion of the virion to the target membrane. Viral proteins and RNA are released into the cytoplasm where the RNA is reverse transcribed into DNA and carried into the cell nucleus via the preintegration complex (PIC). Once in the nucleus the DNA is incorporated into the host genome and replicated using the cellular mechanisms. The viral RNA is translated into proteins, which assemble at the cell surface alongside newly transcribed RNA. New virions encompassing two copies of viral RNA and viral proteins bud and are released from the cell surface into the cytoplasm. This figure has been adapted from Barré-Sinoussi et al., with the permission of Nature Publishing Group via RightsLink (License No. 3521620706908).105 ©2013
1.2.3 HIV Disease Progression & Pathogenesis The establishment and progression of an HIV infection can be subdivided into a number of phases characterized by the level of viremia and various immune factors. Approximately 10 days following an HIV transmission event viral RNA can be detected in the blood as the first sign of an established infection. This marks the beginning of the acute phase, which can last up to six months.106 During the acute phase increasing numbers of target cells become infected, facilitating the exponential growth of the infection and the
14
rapid spread of virus throughout the body.107 Virus can also be found in myeloid derived monocytes, as well as their macrophage and dendritic cell derivatives. Virus continues to replicate in the lymphoid tissues to which it has spread, most often reaching a peak in viral load within roughly three to four weeks of infection.107,108
The activated immune cells of the gut-associated lymphoid tissue (GALT) are primary targets in acute infection. This population of cells is severely depleted within the first three weeks of infection.106,109,110 At this point, flu-like symptoms including fever, lymphadenopathy, pharyngitis, skin rash, myalgia, arthralgia, fatigue and headache may emerge, indicating acute retroviral syndrome driven by the body’s natural immune response to viral infection.107,111 This symptomatic period of peak viral load is generally short-lived as the immune system begins to moderate viral replication. In so doing, the plasma viral load (pVL) decreases markedly and remains relatively stable at what is known as the viral “set point”.107,111 Though not fixed, the viral set point foreshadows the progression of infection. A high viral set point is most often associated with quicker progression to AIDS.112,113 Also at this time, HIV target cell populations recover incompletely, marking the end of the acute phase and the establishment of the chronic phase or the “clinical latency” period.114
Chronic infection is characterized by continuous viral replication and the infection of healthy CD4+ T-cells. Despite persistent infection in a number of tissues and reservoirs this period of active infection is relatively asymptomatic.115,116 Without treatment, the chronic phase can last up to ten years on average, though rates of progression vary between individuals.114,117 Current anti-HIV treatment can greatly reduce the rate of disease
15
progression during this phase, but it cannot eliminate latent viral reservoirs.118–120 As depicted in Figure 1.5, as early as acute infection, a portion of infected activated CD4+ Tcells will become resting cells, forming a latent cellular compartment able to evade the immune system for prolonged periods.118 However, the resting cells are capable of reactivating and feeding the infection by producing new virus and interacting with uninfected target cells.118,121 As well, activated, infected T-cells can be marked for apoptosis, leading to the progressive depletion of the CD4+ T-cell population.116 Control over viral replication starts to wane as CD4+ T-cell populations are depleted slowly over the course of the chronic phase.122,123 Nonspecific constitutional symptoms including fever, night sweats, weight loss, and non-threatening opportunistic infections such as oral candida begin to emerge, marking the end of the chronic phase.114
At this phase of infection, known as clinical AIDS, the immune system is severely impaired, and limited in its ability to fight infection. Pathogens that cannot overcome the immune system of a healthy host take advantage of this weakened immune state to cause opportunistic infection. On average, when left untreated an individual with AIDS will survive approximately three years, eventually falling to opportunistic infection(s) and or tumors. It was the identification of Pneumocystis pneumonia and Kaposi’s sarcoma in young homosexual men that lead to the discovery of AIDS over thirty years ago.1,124–127
16
Figure 1.5. HIV-1 target cells and latency. HIV-1 target cells and their role in latent infection. HIV-1 targets CD4+ immune cells, in particular activated CD4+ T-cells, which have the ability to become resting memory CD4+ cells, as well as resting memory CD4+ T-cells, as well as monocytes, macrophages and dendritic cells of the myeloid cell line. The latent reservoir is namely driven by the resting memory CD4+ T-cells, which can both subsist inactive and hidden from the immune system for prolonged periods as well as become reactivated to produce new virus, transmit virus or be marked for apoptosis. Though not depicted here, cells of the myeloid line are also capable of persisting as reservoirs. This figure has been adapted from Deeks et al. Nature Reviews.118 ©2012 Permission to reproduce this figure has been granted by Nature Publishing Group under RightsLink license No. 3521621023311.
1.3 HIV Entry 1.3.1 HIV Cell Interactions HIV interacts with a number of immune cells, but primarily targets CD4+ T-cells and macrophages. Following the transmission of HIV, the infectious virus particle is recognized as non-self by an antigen-presenting cell. Antigen-presenting cells include dendritic cells and tissue macrophages; they can be found throughout the body and are
17
integral in the innate immune defense mechanism.128,129,130 Antigen-presenting cells act as sentries to the immune system. Upon identifying as non-self, the antigen-presenting cell will engulf, digest and subsequently present an HIV epitope by displaying it on a major histocompatibility complex (MHC) found at its plasma membrane. The presentation of epitopes to immune cells stimulates the adaptive immune response, including the activation of CD4+ T-cells.131,130
The CD4+ T-cells are lymphocytes and part of the lymphatic system; a major component of the immune system. The lymphatic system is composed of various immune cells, lymph and lymphoid tissues like the thymus, GALT and lymph nodes. CD4+ T-cells, or T helper cells, congregate within the lymphoid tissues, particularly the secondary lymphoid tissues like the lymph nodes, where they encounter epitope-presenting cells. Here the CD4+ T-cells are activated and work to orchestrate the cellular immune response by releasing a variety of chemical messengers, or cytokines.132,133 Immune cells gather and interact in the lymphoid tissues exposing CD4+ cells to HIV thus hastening the spread of infection. It is the infection and accompanied immune deficiency caused by the depletion in CD4+ T-cell populations that are used to provide a clinical definition of AIDS. In 1990 it was shown that of all biological markers, CD4+ T cell count was the best predictor of HIV-1 disease progression.134
HIV targets tissue macrophages in addition to CD4+ T-cells. Marcophages play a large role in HIV infection, starting with the uptake of HIV in their role as an antigenpresenting cell type. After they have carried the virion to the lymphoid tissue, macrophages are capable of secreting chemokines as part of the immune response that
18
attract and activate CD4+ T-cells.135 Macrophages direct CD4+ T-cells to areas profuse with HIV thus providing a larger population of target cells for infection. In addition, macrophages have the ability to transfer HIV via cell-to-cell interactions allowing the virus to evade extracellular inhibitors, including those of the host immune response or antiretroviral therapies.136 It is believed that this process is quicker and more efficient than the cell entry of freely circulating HIV virions.137 Furthermore, they can serve as a longterm reservoir to the virus throughout the body, seemingly unaffected by the virusinduced cytopathic effects, safely storing the virus from the effects of the immune system and antiretroviral therapy in treated individuals.138–140
To a lesser extent other antigen-presenting cells like blood monocytes and dendritic cells have been found to become infected and harbour HIV. Blood monocytes are the precursor cell to both tissue macrophages and dendritic cells.141 They have been found to be somewhat resistant to HIV infection, however, this has more recently been shown to be a derivative of monocyte differentiation states.142 Though monocytes typically circulate for only a matter of days, they can act as a long-term reservoir of HIV as they do not fall victim to virus-induced cell death.143,144 Like macrophages, dendritic cells carry and present HIV to the CD4+ T-cells as part of the initial immune resonse.145 However, like monocytes they are less commonly infected with HIV. Depending on the sub-type of dendritic cell, infectious HIV can be retained at the dendritic cell surface from which it can be transferred to CD4+ T-cells.145–147
19
1.3.2 HIV Receptors & Co-Receptors Cell surface receptors mediate the signaling events used by the cell to monitor the extracellular environment. Receptors are integral membrane proteins, spanning the lipid membrane with extracellular, transmembrane and intracellular domains. When a receptor is engaged with a ligand at the cell surface it opens a route of communication triggering a cellular response.
The CD4 molecule is a glycoprotein receptor found on the surface of some immune cells, including the aforementioned macrophages, monocytes, dendritic cells and T helper cells. It is characterized by four immunoglobulin domains, which interact with the MHC II molecules found on antigen presenting cells, carrying antigen. When an antigen-carrying MHC-II complex is able to bind both a T-cell receptor and CD4, the CD4 receptor functions to augment the signals generated in this T-cell activation pathway. Signaling in this way stimulates the CD4+ T-cell to interact with other cells and release cytokines as an immune initiative. In the setting of HIV, CD4 is the primary receptor of the virus and is fundamental in viral entry.72,148
Though CD4 is the primary HIV receptor, the presence of a chemokine co-receptor at the surface of a target cell is necessary for viral entry. Though other chemokine receptors have been implicated in viral entry, it is now widely accepted that CXCR4 and CCR5 are the primary co-receptors used by HIV when entering a target cell in vivo.74,149–151 The chemokine receptors are members of the G protein-coupled family of receptors, characterized by seven transmembrane loops, of which four are extracellular.150 They convey extracellular messages to the cell by binding chemokines, small, proinflammatory
20
chemotactic cytokines, used in the activation and trafficking of leukocytes. The naturally occurring ligands for the CCR5 receptor are C-C chemokines RANTES (Regulated on Activation, Normal T-cell Expressed and Secreted), MIP-1α and MIP-1β (Macrophage Inflammatory Protein; two forms, 1α and 1β).74,152 The naturally occurring ligands for the CXCR4 receptor are C-X-C chemokines SDF-1α and SDF-1β (Stromal Cell-derived Factor; two forms, 1α and 1β).73 These naturally occurring CCR5 and CXCR4 chemokines inhibit HIV-1 replication, confirming the active role these co-receptors play in HIV infection.65,154,99 CXCR4 and CCR5 are expressed on leukocytes, though the level of expression differs between cell types. CCR5 is found to be more prevalent on macrophages and T-memory cells, whereas CXCR4 is found to be more prevalent on naïve T-cells. The two receptors are found on monocytes and dendritic cells relatively equally however the level of expression changes as these cell types mature.155,156
1.3.3 Viral Structures for Cell Entry HIV enters the target cell following the interaction of viral and cellular structures. The envelope-associated spike facilitates the binding, fusion and entry of the virion. The trimeric spike itself is composed of three copies of the heterodimer gp120 non-covalently linked to gp41.59,157 The gp120 trimer initiates contact between the virion and the target cell by binding to CD4 and a chemokine co-receptor, after which gp41 facilitates the fusion of the virion envelope and cell membrane.
Envelope gp120 is the extracellular portion of the virion spike. It is composed of five conserved regions (C1-C5) and five hypervariable regions (V1-V5).158 As the names suggest, the conserved regions have low genetic variability whereas a high level of genetic
21
diversity characterizes the variable regions. It is believed that this high level of variability in the envelope spike allows the virus to evade immune recognition. The core of gp120 is roughly heart-shaped with an inner and outer domain linked by a bridging sheet, the outer domain being more variable. As a glycoprotein, gp120 is characterized by a considerable amount of N-linked glycosylation at the outer domain.50,159 This glycosylation is thought to be a mechanism through which HIV can avoid host neutralizing antibodies.160,161
Despite the modest contributions of other gp120 regions, such as V1 and V2, the third hypervariable region (V3) has been confirmed as the primary envelope region for chemokine co-receptor interaction.162–164 Within the outer domain of the gp120 core, at the apex of the inverted heart-shaped protein, V3 extends slightly as a loop.78,165–167 The V3 loop is typically composed of approximately 35 amino acid residues. The loop structure is supported by the presence of a disulphide bridge formed between the single cysteine residues found at both the beginning and the end of the loop. V3 can be subdivided into three basic areas, a conserved base, a variable stem and a β-hairpin tip.167,168 The positions of amino acids within the variable stem are thought to recognize different regions of the coreceptor, and amino acid polymorphisms associated with co-receptor use have been identified in this region of the V3 loop.167,169 It is the variance in the amino acid sequence of the V3 loop that determines whether the virus will use the CCR5 and or CXCR4 coreceptor.
The gp41 trimer is the transmembrane portion of the gp120-gp41 complex, found at the base of gp120, in the shape of a mushroom.165,170 There are four primary domains of gp41. At the C-terminal are the transmembrane domain and the C-terminal heptad repeat
22
(HR2); at the N-terminal, the fusion peptide and N-terminal heptad repeat (HR1).171 The HR1 and HR2, each consisting of three α-helices, are linked by short, flexible peptides to form the gp41 ectodomain. The fusion peptide in the inactive state is found within a hydrophobic pocket of the gp120-gp41 complex. The transmembrane domain anchors the gp120-gp41 complex to the envelope, whereas the HR1, HR2 and fusion peptide, characterized by hydrophobic residues, facilitate membrane fusion between the virion and the target cell.44,172,173 The viral structures associated with each stage of the viral entry mechanism are illustrated in Figure 1.6.
Figure 1.6. HIV-1 cell binding and membrane fusion. A diagrammatic representation of the HIV-1 binding mechanism and subsequent fusion of the cell and viral membranes. HIV-1 binding first requires the binding of gp120 to the CD4 receptor, the interaction of which causes a conformational change allowing the V3 loop to contact one of the two chemokine co-receptors, either CCR5 or CXCR4, and illicit the fusion mechanism. Fusion occurs following co-receptor binding as the fusion peptide is inserted into the cell membrane and folds to pull the membranes together. This figure was adapted from Haqqani et al., Antiviral Research.174 ©2013 with permission under the RightsLink license No. 3521621226558.
1.3.4 The Entry Mechanism Upon encountering a target cell the HIV gp120 protein first binds to the surface CD4 receptor, putting in motion a complex cascade of events from a pre-fusion closed state to an intermediate open state leading to viral entry. The point of contact between CD4 and 23
gp120 is a recessed pocket found at the interface of the inner and outer domains at the surface of gp120.78,161 This binding interaction causes a conformational change in the gp120 protein whereby the trimer subunits rotate outwardly opening the gp120-gp41 complex, similar to the opening of a flower. This movement flexes the CD4 molecule and draws the cell membrane closer to the bound virus.165 It also causes V1/V2 to shift, exposing the coreceptor binding site associated with V3 and the bridging sheet.79,175,176 In the CD4-bound form the gp120 bridging sheet is stabilized and the V3 loop is oriented for co-receptor binding.70,165,117 When the V3 loop contacts the co-receptor, the V3 conserved base, variable stem and hairpin loop interact with the N-terminal, extracellular loops and transmembrane helices of the co-receptor binding pocket, respectively.177–179 This co-receptor activity shifts the CD4-bound virus nearer the cell membrane and conformational changes at the gp120gp41 interface expose gp41 to initiate fusion.180
Following HIV receptor binding, a second set of conformational changes in the gp120-gp41 complex causes the formation of a coiled coil composed of three parallel HR1 α-helices from each of the gp41 subunits of the envelope trimer. Formation of the coiled coil orients the fusion peptide toward the cell membrane. The fusion peptide extends through the extracellular space and into the target cell membrane, becoming a linear extension of the viral envelope and anchoring the virion to the target cell.50,172,181 Once the fusion peptide has become anchored in the cell membrane, the HR1 coiled coil folds back on itself interacting with the antiparallel HR2 helices. The HR2 helices fit into hydrophobic grooves formed between the HR1 helices to form a six-helical bundle and hairpin loop. The formation of the six-helical bundle draws the cell-embedded fusion peptide and the viral transmembrane domain near enough for the cell and viral membranes to fuse.62,180
24
Once fusion has occurred the virion is incorporated into the cell membrane, and the contents of the virion are released into the cytoplasm from which they can enter the replication cycle as described previously. Figure 1.7 demonstrates the conformational changes necessary for viral entry, occurring within the gp120-gp41 complex following HIV1 receptor binding.182
Figure 1.7. Conformational changes during HIV-1 cell entry. A model interpretation of the conformational changes occurring during the HIV-1 entry process. HIV-1 entry is mediated by the “envelope spike” composed of the trimeric gp120-gp41 complex. When bound to both the CD4 and chemokine co-receptor the spike is activated and the gp41 fusion peptide extends toward the cell membrane. The gp120 subunits rotate outward, allowing the gp41 subunit to reach the cell membrane, forming the pre-hairpin intermediate. Once anchored in the cell membrane the pre-hairpin intermediate folds on itself creating the six-helical bundle, drawing the cell and viral membranes together, allowing contact and fusion. This figure has been published by Tran et al., PLoS Pathogens.182 ©2012 (Open Access)
1.3.5 The Chemokine Co-Receptors & HIV Infection 1.3.5.1 Tropism Terminology HIV-1 is characterized by viral tropism, the co-receptor through which HIV enters the target cell. When virus enters the cell via the CCR5 or the CXCR4 chemokine coreceptor it is considered “CCR5-using” (R5) or “CXCR4-using” (X4), respectively.183 There are also instances of “dual/mixed” infection, where “dual” virus is capable of using both
25
co-receptors, and “mixed” characterizes a viral population that contains both R5 and X4 virus.184,185 Collectively, X4 and dual/mixed populations are often referred to as “non-R5”.
Historically, R5 virus has been referred to as macrophage-tropic, M-tropic, because R5 viral isolates are far more efficient at infecting primary macrophages when compared to T-cell lines. Conversely, X4 virus was originally designated as T cell line-tropic. It was called T-tropic because these viral isolates had greater infectivity in T-cell lines when compared to macrophages. Virus capable of infecting both macrophages and T cell lines were termed dual-tropic. These distinctions were also correlated with the formation of syncytia, which are large, irregularly shaped, multinucleated cells, in human T-cell leukocyte (MT-2) cell lines.186 T-tropic, or X4 virus was found to cause fusion of T-tropic infected cells, creating syncytia and thus termed syncytium inducing (SI). In contrast, Mtropic, or R5 virus was not found to be associated with the formation of syncytia and was termed non-syncytium inducing (NSI).183
1.3.5.2 Tropism Switch with Disease Progression It has long been accepted that HIV-1 can be dichotomized by R5 and X4 phenotypes, and that these phenotypes differ in many aspects, and are most clinically relevant in terms of the rate of disease progression.187 Both R5 and X4 variants are capable of being transmitted however R5 virus characterizes the vast majority of primary infections regardless of transmission route.188 Whether this observation is due to the preferential transmission of R5 virus, the prevalence of target cells, biased immune pressures limiting X4 virus, or a number of other hypothesized mechanisms, has yet to be determined.189,190 However, the extreme rarity of HIV infection in individuals homozygous for a deleterious
26
mutation in the CCR5 gene, causing deficient expression of the CCR5 co-receptor, supports the observation that R5 virus is seemingly transmitted and infection established more efficiently than X4.188,191–193 There are only handful of individuals with genetically defective CCR5 expression to be identified as HIV-1 positive, and in all cases the infection is characterized solely by X4 virus.194,195
As the majority of HIV infections are initially R5, generalizations made about infection and disease progression are based on the observations of R5 virus. However, roughly 50% of HIV-infected individuals will diverge from these generalizations and experience a tropism shift over the course of infection as increasing amounts of X4 virus emerge.196,197 This emergence of X4 virus is associated with the accelerated depletion of CD4+ T-cells and the progression to AIDS-defining illness.187,196,198–200 There is debate as to whether the emergence of X4 virus is a cause or a consequence of the immune system impairment experienced by HIV-1 positive individuals, as the mechanism driving this shift has yet to be determined. Regardless, it is widely accepted that X4 virus serves as a prognostic marker of accelerated disease progression and decline in patient health when left untreated.
1.4 The Clinical Management of HIV 1.4.1 The Concept of Antiretroviral Therapy In the decades since HIV was confirmed as the causative agent of AIDS, a wide range of antiretroviral compounds have been approved for use in ART. Though these compounds are not capable of curing HIV, they effectively interfere with the various stages
27
of the viral replication cycle, suppressing viral replication and limiting the amount of infectious virus in the blood. Interfering with the HIV replication cycle is key in the fight against HIV for three reasons: 1) a reduction in plasma viral load (pVL) greatly reduces the rate of immune depletion in the infected individual and gives rise to the associated benefits; 2) reduced rates of replication translate into reduced rates of HIV mutation and the emergence of new drug resistance polymorphisms; and 3) less infectious HIV in the blood reduces the risk of HIV transmission. For these reasons, identifying and treating HIV positive individuals has become the keystone of new initiatives to reduce rates of HIV and AIDS related-deaths worldwide.7,201
Initially, antiretroviral compounds were administered as monotherapies due to the limited number of compounds available. Despite the ongoing discovery of new antiretroviral compounds and classes, the development of drug resistance polymorphisms continued to be an issue in HIV treatment.202,203 In 1996 a new treatment strategy, highly active antiretroviral therapy (HAART), was introduced and accepted as the standard of care. HAART differed from the preexisting treatment methods by dictating the coadministration of at least three antiretroviral compounds.204–206 Typically HAART consists of a protease inhibitor or an non-nucleoside reverse transcriptase inhibitor (NNRTI) alongside two nucleoside reverse transcriptase inhibitors (NRTIs).207–209 As new compounds and new drug classes are approved for clinical use they are integrated into the HAART framework, providing the continued expansion of treatment options.
28
1.4.2 The Antiretroviral Compounds In 1987 the first anti-HIV compound was introduced, zidovudine (azidothymidine; ZDV or AZT), a nucleoside reverse transcriptase inhibitor.210–212 Zidovudine was discovered by screening pharmaceutical compound collections, and was originally designed as an anti-cancer drug in the 1960s.44 Unfortunately drug resistance polymorphisms negating the effects of zidovudine soon appeared within the treated HIV positive population.213,214 The pressure to discover and develop new antiretrovirals was great. Antiretroviral discovery quickly shifted to a target-based approach, with a number of potential targets within the HIV replication cycle including viral entry and the enzymatic proteins reverse transcriptase, integrase and protease.44 Following the introduction of zidovudine, a number of additional NRTIs were to follow including lamivudine (3TC) and emtricitabine. NRTIs are nucleoside analogues missing the 3’hydroxyl group on the deoxyribose moiety. Mimicking the nucleotide building blocks of DNA, NRTIs act as chain terminators during reverse transcription. When incorporated into the new DNA strand, the missing 3’-hydroxyl group interrupts the addition of the next sequential nucleotide. Unable to form the necessary bond between nucleotides, the extension of the new HIV DNA strand is terminated.211,85
The rapid emergence of NRTI resistance polymorphisms limited the efficacy of HIV treatment well into the 1990s. It wasn’t until 1995 that a new class of antiretroviral compounds was introduced, the protease inhibitors. Protease inhibitors act by blocking the active site of the protease enzyme thus preventing the proteolytic cleavage of the Gag-Pol precursor protein.215,216 A key step in the HIV replication cycle, cleavage of the Gag-Pol precursor protein is necessary for virion maturation and infectivity.104,217,218 It was later 29
discovered that protease inhibitors can affect the activity of the Cytochrome P450 (CYP3A) metabolic enzyme, which is known to quickly metabolize many antiretroviral compounds.219–221 Ritonavir has been shown to be most effective in inhibiting CYP3A, and it is now common clinical practice to use low-dose ritonavir to enhance the pharmacokinetics of a co-administered protease inhibitor, known as “boosting”.219,222–224
The non-nucleoside reverse transcriptase inhibitors debuted in 1996 with the introduction of nevirapine, and later efavirenz (EFV) in 1998.225 The NNRTIs inhibit reverse transcription, but unlike the NRTIs, they accomplish this by binding to the reverse transcriptase enzyme itself. Despite differences in shape and structure, all NNRTIs bind to a hydrophobic pocket that is created in the p66 subunit of reverse transcriptase. NNRTIs do not bind at the polymerase active site rather near the active site, allosterically locking the active site in an inactive state.226–228
The final HIV enzymatic protein, integrase, was targeted with the development of the Integrase Strand Transfer Inhibitors (INSTIs). As the name suggests, this class of ARV compounds inhibits the HIV replication cycle by blocking strand transfer during which the proviral DNA is incorporated into the chromosomal DNA.95 In 2007, raltegravir was the first integrase inhibitor to be approved for clinical use in United States.229–232 The current mechanism of integrase inhibition targets the Mg2+ molecule found at the active site of the integrase enzyme, competitively blocking the DNA strands from the active site.233–235
Inhibiting the entry of HIV has also become a target in anti-HIV approaches, not only preventing viral entry but the replication cycle. The entry inhibitors can be subdivided into fusion inhibitors and CCR5 antagonists, and together with the INSTIs are
30
the most recently approved anti-HIV drug classes. The fusion inhibitor enfuvirtide was approved for clinical use in 2003, and continues to be the only fusion inhibitor available. However, the administration of enfuvirtide requires an ill-favoured subcutaneous injection that has limited its use.236,237 Inhibition of fusion occurs at the gp41 envelope protein. By binding directly to the gp41 protein, enfuvirtide prevents the formation of the hairpin loop necessary for drawing the virus and cell membranes close for fusion and subsequent viral entry.238,239 In addition, viral entry can also be prevented by way of allosterically blocking the host CCR5 co-receptor, preventing HIV-co-receptor binding and thus fusion and entry. This is accomplished by the CCR5 antagonist compounds.
1.4.3 The CCR5 Antagonists It was discovered that a 32-base pair deletion in the gene encoding the CCR5 coreceptor when homozygous offered a natural resistance to HIV infection and partial resistance when heterozygous.191,193,240–243 Truncation of the gene leaves the co-receptor dysfunctional and not expressed on the cell surface thus preventing viral entry through the CCR5 co-receptor. As HIV co-receptor use was elucidated it became apparent that this mutation was exclusively effective against R5-virus.240,244 Inherited following classical mendelian genetics, this CCR5Δ32 mutation is found primarily in Caucasian populations particularly of Northern European decent, with an average allelic frequency of 10% in Europe.241,243–248 When it was shown that the CCR5Δ32 mutation had little effect on the health of individuals expressing it, an anti-HIV drug acting at the CCR5 co-receptor quickly became an objective, leading to the development of the CCR5 antagonist drug class.191,249
31
A number of different CCR5 antagonists have been investigated, including: aplaviroc, vicriviroc, cenicriviroc and maraviroc. The development of aplaviroc was discontinued following a series of Phase II clinical trials in which aplaviroc was outperformed by comparator compounds, and rates of hepatotoxicity were higher than expected.250–252 Vicriviroc was discontinued following the results of the Phase III clinical trials where the addition of vicriviroc to a strong optimized background therapy did not result in a significant increase in antiviral activity.253–255 Cenicriviroc is the most recent CCR5 antagonist to be explored, and is currently in the later stages of development following the promising results of the Phase IIb studies.256,257 Despite the clinical investigations of a number of CCR5 antagonists, maraviroc (MVC) is currently the only CCR5 antagonist approved for clinical use.
1.4.3.1 Inhibition of HIV by Maraviroc Of all the antiretroviral compounds in use, maraviroc is the first to target a host protein as opposed to a viral protein. Maraviroc is a small, non-peptidic molecule that is highly selective for the CCR5 cell receptor. Maraviroc does not alter the expression of CCR5 on the cell surface, nor does it seem to interrupt CCR5-related intracellular signaling.249 A number of studies agree that maraviroc works by allosteric inhibition.258–260 Instead of competitively blocking the active site where the gp120 would bind to the coreceptor, maraviroc binds elsewhere on the co-receptor leaving the active site vacant. However, this non-competitive binding induces conformational change at the active site, which prevents gp120 from recognizing and binding to the CCR5 co-receptor.
32
The N-terminal and the second extracellular loop of the CCR5 co-receptor are highly associated with CCR5 ligand binding, as well as HIV binding. 177,261–264 However, instead of engaging these regions of the co-receptor maraviroc likely binds in a pocket located deep within the transmembrane domain of the receptor, contorting the HIV-1 binding site.258,260 Some also believe that maraviroc serves to stabilize the co-receptor in an inactive state258,265 By non-competitively inhibiting gp120 at the CCR5 co-receptor, maraviroc prevents the final conformational changes associated with fusion and viral entry.
1.4.3.2 The Clinical Trials of Maraviroc In 2007 the United States Food and Drug Administration (FDA) approved MVC for clinical use in patients with treatment-experience following the successful Phase III clinical trials, MOTIVATE 1 and MOTIVATE 2 (Maraviroc versus Optimized Therapy in Viremic Antiretroviral Treatment-Experienced Patients). It was later shown to be effective and safe for treatment-naïve HIV-positive patients, as was demonstrated in the MERIT trial (Maraviroc versus Efavirenz in Treatment-Naïve Patients).
The MOTIVATE trials were multi-centre, randomized, double-blind, placebocontrolled studies run in parallel. MOTIVATE 1 was conducted in Canada and the United States, while MOTIVATE 2 was conducted in Australia, Europe and the United States. The studies were designed to test the efficacy and safety of maraviroc in treatment-experienced patients screened as having R5 virus compared to placebo when accompanied by an optimized background regimen.266 The optimized background regimen was designed for each patient based on safety precautions, the presence of drug resistance polymorphisms
33
and treatment history. The viral tropism was determined using the first clinically approved tropism testing method, the original phenotypic Trofile Assay by Monogram Biosciences. The primary end point was the mean change in plasma viral load after 48 weeks of maraviroc therapy.266,267 The results of both MOTIVATE 1 and 2 showed superior virological effects with similar safety profiles when maraviroc was compared to placebo in patients with R5 virus.266,268 Analyses of maraviroc for treatment-experienced patients conducted at 96 weeks also showed a preferential safety and efficacy profile when compared to placebo.269
As part of the maraviroc clinical trials, a study was conducted to determine the safety and efficacy of maraviroc in patients screened as having non-R5 virus by the original Trofile assay. This MOTIVATE sister study, A4001029, enrolled highly treatmentexperienced patients from Australia, Canada, Europe and the United States.270 Patients were randomized to receive either placebo or maraviroc with an optimized background regimen (OBT). The week 24 virological response revealed that maraviroc performed similarly when compared to placebo in patients with non-R5 virus, as would be expected given the mechanism of the drug.270
The MERIT trial was designed to evaluate treatment response to MVC when compared to efavirenz in treatment-naïve patients. The MERIT trial was a double-blind, double-dummy, multinational study enrolling only patients screened as having R5 virus by the original Trofile assay. Each patient was assigned to one of three treatment arms, either maraviroc once-daily (q.d.), twice-daily (b.i.d.) or efavirenz, with a co-formulated zidovudine-lamivudine backbone therapy.271 Maraviroc twice-daily was found to cause
34
both better CD4+ cell recovery and fewer adverse events. However, it was only following tropism reassessment using the improved Enhanced Sensitivity Trofile Assay (ESTA) and subsequent reanalysis that maraviroc taken twice-daily and efavirenz showed similar virological response.271–273 Rescreening using ESTA identified 15% of the enrolled study population as having dual/mixed or X4 virus partially or fully uninhibited by maraviroc, respectively. Reanalysis excluding these patients found maraviroc twice-daily to be noninferior to efavirenz at both of the co-primary endpoints, the number of patients with pVL 3.5); red branches represent non-R5 variants(non-R5≤3.5); grey branches represent variants existing at the other time point. The value at each tip indicates the number of times the sequence was identified.
201
Supplemental Figure 5.3a. Phylogenetic trees illustrating the change in non-R5 HIV-1 viral populations after short-term exposure to maraviroc. Phylogenetic trees based on maximum likelihood for patients experiencing a viral tropism switch, as determined by 454 “deep” sequencing, after eight days of MVC exposure. Patients 7 and 19 were screened as having non-R5 virus at baseline however after eight days of MVC exposure nonR5 variants were suppressed. Blue branches represent R5 variants (R5 FPR>3.5); red branches represent non-R5 variants(non-R5≤3.5); grey branches represent variants existing at the other time point. The value at each tip indicates the number of times the sequence was identified.
202
Supplementary Figure 5.3b. Phylogenetic trees illustrating the change in non-R5 HIV-1 viral populations after short-term exposure to maraviroc. Phylogenetic trees based on maximum likelihood for patients experiencing a viral tropism switch, as determined by 454 “deep” sequencing, after eight days of MVC exposure. Patients 13 and 17 were screened as having less than 2% non-R5 virus at baseline however after eight days of MVC exposure the non-R5 population expand to predominate. Blue branches represent R5 variants (R5 FPR>3.5); red branches represent non-R5 variants (non-R5≤3.5); grey branches represent variants existing at the other time point. The value at each tip indicates the number of times the sequence was identified.
203
