Association Among Ccr5 Genotypes, Ccr5 Expression, and In

Home , Amprenavir, CCR5, Didanosine, Nelfinavir, Zalcitabine

ASSOCIATION AMONG CCR5 GENOTYPES, CCR5 EXPRESSION, AND IN

VITRO HIV INFECTION

Bangan John

Submitted in partial fulfilment of the requirements

For the degree of Master of Science

Dissertation Advisor: Dr Peter A. Zimmerman

Department of Biology

CASE WESTERN RESERVE UNIVERSITY

May, 2013

CASE WESTERN RESERVE UNIVERSITY

SCHOOL OF GRADUATE STUDIES

We hereby approve the thesis/dissertation of

Bangan John ______

Master of Science candidate for the ______degree *.

Roy Ritzmann (signed)______(chair of the committee)

Peter Zimmerman ______

Christopher Cullis ______

Daniel Tisch ______

12/20/2012 (date) ______

*We also certify that written approval has been obtained for any proprietary material contained therein. Dedication:

I would like to dedicate this thesis to my family, the John’s. Table of Contents

List of Tables ...... ii List of Figures ...... iii Acknowledgements ...... iv Abbreviations ...... v Abstract ...... vi Chapter 1: Overview HIV infection, CCR5 chemokine receptors and CCR5 gene polymorphisms ...... 1 1.1 Introduction ...... 1 1.2 HIV-1: Infection and Tropism ...... 1 1.3 CCR5 receptor ...... 3 1.4 CCR5 Haplotype ...... 6 1.5 CXCR4 receptor...... 7 1.6 Antiretroviral therapy...... 8 1.7 Host genetics ...... 10 1.8 Cell Biology: Human T Lymphocytes ...... 11 Chapter 2: Rationale/Hypothesis and Method ...... 14 2.1 Aims: ...... 15 2.2 Methodology ...... 16 2.2.1 Participants and Sample ...... 16 2.2.2 DNA Extraction and Polymerase Chain Reaction ...... 16 2.2.3 Ligase Detection Reaction-Fluorescent Microsphere Assay ...... 17 2.2.4 Cells and Whole Blood Staining ...... 17 2.2.5 FACS-based HIV fusion/Productive Infection Assay ...... 18 2.2.5.1 Assay description [87] ...... 20 Chapter 3: Results and Discussion ...... 22 3.1 Genotyping: CCR5 genotypes from Human T lymphocytes ...... 22 3.2 Phenotyping: CCR5 expression on T lymphocytes in relations to CCR5 Genotypes ...... 25 3.3 CCR5 Genotype and Infectivity of Human T lymphocytes ...... 28 Chapter 4: Conclusion...... 31 4.1 Limitation and Future Directions ...... 33 Bibliography: ...... 34

List of Tables

Table 1. Six classes of ART and drugs ...... 10

Table 2. These are the channels and the antibodies that were used in the assays...... 21

Table 3. Number of individuals from various ethnicities...... 23

Table 4. CCR5 -2459 and Δ 32 allelic and genotypic frequencies ...... 24

List of Figures

Figure 1. General life cycle of HIV…………………………………………… 2

Figure 2. Chromosome 3 and location of the CCR5 gene…………………….. 3

Figure 3. Schematic overview of the viral strain, chemokine receptors and

their ligands…………………………………………………………. 4

Figure 4. Schematic overview of the viral strain, chemokine receptors and

their ligands…………………………………………………………. 8

Figure 5. Combination of Reporter virus system……………...... 22

Figure 6. CCR5 expression on TCM with CCR5 genotypes…………………. 26

Figure 7. CCR5 expression on TCM with CCR5 genotypes………………….. 27

Figure 8. In vitro HIV infectivity in TCM with CCR5 genotypes…………….. 28

iii

Acknowledgements

I would like to thank my advisor, Dr Peter Zimmerman, who provided guidance and support for me throughout my studies. Also, I would like to acknowledge my committee members for their patience and guidance: Dr. Peter Zimmerman, Dr.

Daniel Tisch, and Dr. Christopher Cullis. There are also people in the Zimmerman lab that I would like to thank, namely Dr. Rajeev Mehlotra – for his mentorship and advice, Melinda Blood, Dr. Scott Small, Akshaya Ramesh, Krufinta Bun, Tenisha

Phipps, Barne Willie, Kyle Logue, and Cara Halldin.

Abbreviations

AfA African American AIDS Acquired Immunodeficiency Disease Syndrome ART Antiretroviral Therapy AsA Asian American CA Caucasian American CCR5 CC Chemokine Receptor 5 CXC CXC Chemokine CXCR4 CXC Chemokine Receptor 4 DNA Deoxyribonucleic Acid FMA Fluorescent Microsphere Array G/A Combination of nucleotide G and A G/G Combination of nucleotide G and G HA Hispanic American HAART Highly Active Antiretroviral Therapy HIV Human Immunodeficiency Virus IND India LDR Ligase Detection Reaction NK Natural Killer Cells NNRTIs Non-Nucleoside Reverse Transcriptase Inhibitors ORF Open Reading Frame PCR Polymerase Chain Reaction PNG Papua New Guinea R5 R5 variant of HIV RANTES Regulated in Activation Normal T Expressed and Secreted protein SNPs Single Nucleotide Polymorphisms TCM Central Memory T cell TEM Effector Memory T cells TEMRA Effector Memory T cells with marker protein - CD45RA TM Memory T cells TN Naïve T cells TTM Transitional Memory T cells UHC IRB University Hospital of Cleveland Institutional Review Board US-FDA United States of America Food and Drug Administration

Association Among CCR5 Genotypes, CCR5 Expression, and In Vitro HIV Infection

Abstract

BANGAN JOHN

The single nucleotide polymorphisms (SNPs) in CCR5 (-2459 G>A [promoter], Δ32 deletion polymorphism [open reading frame, ORF]) influences the expression of

CCR5 chemokine receptor on human T lymphocytes. CCR5 is a major coreceptor for

HIV to bind to the T cell prior to fusion. Individuals have various frequencies of these polymorphisms, which influence their susceptible to HIV infection and disease progression. This thesis is focused on determining the association between these polymorphisms and CCR5 expression on T cells, as well as between these polymorphisms and in vitro HIV infection. The CCR5 -2459 genotype results that were obtained showed clear associations with CCR5 expression on the central memory T cell (TCM) subpopulation. That is, CCR5 -2459 G/G had lower levels of

CCR5 expression as compared to A/A and G/A on the TCM cells. While with the

CCR5 ORF genotypes, wildtype (Wt)/Δ32 had very low levels of CCR5 expression as compared to Wt/Wt. Furthermore, results obtained for CCR5 -2459 genotype and HIV infectivity of the TCM cells also clearly showed significant associations. These were observed when comparing CCR5 -2459 genotypes G/G and A/A (p=0.029), and also between G/A and G/G (p=0.049). That is, G/G had lower HIV infectivity of its TCM cells in comparison to those of both A/A and G/A genotypes. These findings, especially those of CCR5 -2459 G allele demonstrates its protective effect in the in vitro assay, thus, providing insights into expression and infection at the T cell subpopulation level.

Chapter 1: Overview HIV infection, CCR5 chemokine receptors and CCR5 gene polymorphisms

1.1 Introduction

Human immunodeficiency virus type (HIV) infection occurs when the virus gains entry into a host cell by first binding to the cell. For this to happen the host cell must have receptors expressed on its surface for the virus to bind to. These cell surface receptors include the primary receptor for HIV-1 infection, which is the cluster of differentiation 4 (CD4) and two specific chemokine coreceptors, CC chemokine receptor 5 (CCR5) and CXC chemokine receptor 4 (CXCR4) [1]. CCR5 and CXCR4 coreceptors are selectively targeted by different HIV-1 variants, R5, X4 or R5X4 respectively at different stages of the disease. There are polymorphisms in CCR5 and the ligand of CXCR4, which are significant in HIV infection and in the disease progression of acquired immune deficiency syndrome (AIDS). This review will focus on chemokine coreceptors and their ligands which make certain populations genetically more susceptible to HIV/AIDS than others.

1.2 HIV-1: Infection and Tropism

Receptor binding is the first stage of HIV-1 infection of target cells via the interaction of the viral glycoprotein 120 (gp120) with the CD4 molecule [2]. This causes a conformational change to the gp120, thus, exposing the chemokine coreceptors,

CCR5 and CXCR4, to the viral binding site [3].

The CD4 molecule, CCR5 and CXCR4 are expressed on the bilipid cellular membrane of specific cells thus making them susceptible targets to HIV-1 [2]. These are T-cells, dendritic cells and macrophages, which are well documented for their roles in the HIV-1 life cycle (Figure 1) and its importance in the human immune system. Nonetheless, there are other cells that co-express these receptors but do not have the capacity to support productive infection. T-cells are a type of white blood cell belonging to a sub-group of lymphocytes while macrophages are also a type of a white blood cell but are monocytes (phagocytes). T-cells compared to dendritic cells and macrophages have a much higher expression of CD4 molecules [4].

Figure 1. General life cycle of HIV[5]

As previously mentioned, different variants of HIV-1 are present at various stages of the disease progression to AIDS. The most predominant variant detectable when diagnosed with HIV-1 infection is the R5 variant. This variant is known as R5 tropic because it binds to the chemokine receptor, CCR5,. Likewise, the X4 variant is designated according to its chemokine receptor, CXCR4. However, the X4 variant is only detectable after the onset of AIDS. The R5X4 variant or the dual variant is

2 detectable during the transitional stage from HIV-1 infection to AIDS. As designated, the R5X4 variant is capable of causing infection via the CCR5 and CXCR4 chemokine coreceptors. It is important to note that at different stages of the disease progression certain variants are predominant. It means that other variants are present but only in very low numbers.

1.3 CCR5 receptor

CC chemokine receptor type 5 (CCR5) belongs to the seven-transmembrane G- protein coupled receptors (GPCRs) family and is encoded by the CCR5 gene, which is located on the short arm of chromosome 3, 3p21.31 (Figure 2) [6, 7]. CCR5 is expressed on cell surfaces and it functions as a beta (β)-chemokine receptor for the R5 variant [8]. However, polymorphisms in the CCR5 receptor have been shown to delay disease progression to AIDS or even resist HIV-1 infection. Developing resistance to

HIV infection meant that a person was genetically immune to the virus and can never get sick from it. Furthermore, the CCR5 receptors have three natural chemokine ligands known as chemokine (C-C motif) ligand 3 , CCL3 formerly known as macrophage inflammatory protein 1 alpha (MIP-1α), CCL4 (MIP-1β) and CCL5 formerly known regulated on activation normal T expressed and secreted protein

(RANTES) [6, 9]. These chemokine ligands compete with HIV-1 and have been associated with the control of HIV-1 infection (Figure 3) [10].

Figure 2. Chromosome 3 and location of the CCR5 gene

Figure 3. Schematic overview of the viral strain, chemokine receptors and their ligands[11].

There are eight polymorphisms on the CCR5 gene that have been identified. However, only a few of these polymorphisms are associated with susceptibility to HIV and

AIDS. These polymorphisms are named according to their location on the gene and their haplotype. These are CCR5: -2733A/G, -2554G/T, -2459G/A, -2135T/C, -

2132T/C, -2086A/G, -1835A/G and the open reading frame (ORF). In addition, another important receptor involved in HIV infection is the CCR2 ORF with polymorphism on position 64I.

The most well documented and known of these polymorphisms is the CCR5 delta 32 deletion (CCR5 -Δ32) [12-14]. It is in the ORF and is a 32 nucleotide deletion in the

CCR5 coding region. This polymorphism prevents or reduces the expression of CCR5 on the cell surface for R5 variants of HIV-1 in T-cells [13, 14]. This polymorphism

(Δ32) causes a frame shift and premature stop codon in the transmembrane, thus, the truncated protein product is not expressed on the cell surface [15-17]. For instance, individuals who are heterozygous for CCR5 -Δ32 have shown a delayed effect on

4 disease progression to AIDS, while, homozygous individuals are resistant to HIV-1 infection due to the lack of binding sites for R5 variants of HIV-1.

The CCR5 promoter polymorphism at position - 2459 (G/A) has been reported to be associated with different disease progression rates to AIDS [12, 18-20]. HIV-1 infected individuals with the CCR5 genotype -2459 G/G showed a slower disease progression than the -2459 A/A genotype [21]. Berger et al. suggested that the presence of the -2459 G/G may have decreased transcription and decreased the expression of CCR5 [15] while -2459 A/A might have the opposite effect.

Furthermore, studies have shown that the polymorphism CCR2 64I in combination with the ones mentioned above greatly diminish HIV-1 infectivity and or disease progression [22, 23].

CCL3 (MIP-1α), CCL4 (MIP-1β) and CCL5 (RANTES) are released by cells such as macrophages, effector T cells and natural killer (NK) cells at sites of inflammation

[24]. In response, both innate and adaptive immune cells, which express CCR5 are drawn to the site due to the presence of these chemokine ligands [25]. With HIV-1 infection these chemokines ligands will compete with R5 variants for the CCR5 receptors on the cells. Moreover, these chemokines have been demonstrated to bind effectively to CCR5 receptors and have suppressive activities in HIV-1 infection assays [26].

CCL3 (MIP-1α) is thought to down regulate HIV-1 infection by facilitating chemokine desensitization and lowering of CCR5 cell surface expression on CCR5 bearing cells [27]. It has been associated with decreased risk of HIV infection and

5 disease progression [9]. While CCL4 (MIP-1β) may have a similar effect to this receptor. Nevertheless, CCL5 (RANTES) chemokine ligand compared to CCL3

(MIP-1α) and CCL4 (MIP-1β) has been shown to be a better inhibitor of the R5 variant HIV-1 infection of the CD4+ T cells [28]. Studies performed to evaluate polymorphisms of RANTES, such as G4 and 28G in the promoter region have shown a protective effect against disease progression to AIDS [29-31]. That is, higher levels of RANTES have been shown to be associated with the regulation of HIV-1 infections [29].

1.4 CCR5 Haplotype

There are eight major polymorphisms in the CCR5 gene that determines the type of human haplotype (HH) an individual will have. These CCR5 SNPs previously mentioned, can be categorized into, older SNPs; -2733, -2554, -2459, -2135 and -

1835 as compared to the more recent SNPs; delta 32, -2132T and 2086G. The nine

HH generated from these SNPs are HHA, HHB, HHC, HHD, HHE, HHF1, HHF2,

HHG1 and HHG2 [32]. The most frequent of these CCR5 haplotype in the global population are HHA, HHC, HHE and HHF2 [33]. According to Mummidi et al [34] one of the mechanism underlying the effects of different CCR5 haplotypes might be differential transcriptional efficiency. In their study they were able to demonstrate that nucleotide substitutions in the cis-regulatory regions of CCR5 produced differences in transcriptional activity [34]. The HHA, an ancestral haplotype, had the lowest transcriptional activity, whereas the HHF haplotypes had the highest of the haplogroup-specific constructs tested [34]. Furthermore, results from another study on the analysis of association of CCR5 haplogroups and HIV-1 disease progression

6 suggests that HHA and HHF2 haplotypes are both associated with HIV-1 disease retardation in adults [32].

1.5 CXCR4 receptor

CXCR4, also known as fusin or CD184 is a member of the GPCR family. In humans,

CXCR4 is a protein that is encoded by the CXCR4 gene located on human chromosome 2 [7]. It acts as an α chemokine receptor and is the coreceptor for HIV-1

X4 variants [35]. CXCR4 functions as a chemokine receptor specifically for CXC chemokine ligand 12 (CXCL12), which is also called stromal derived factor-1 (SDF-

1), shown in figure 2.0. According to Martin et al (1998), there are no significant polymorphisms in CXCR4 [36]. However, its ligand, CXCL12, has a polymorphism that has been associated with a delay in disease progression.

Figure 4. Schematic overview of the viral strain, chemokine receptors and their ligands [8].

The CXCL12 is the only natural inhibitor for CXCR4 and has been reported to inhibit

X4 variant of HIV-1 infection due to a polymorphism in the 3’untranslated region

(3’UTR) of the CXCL12 gene. Recent findings show that CXCL12 is a ligand for the receptor CXCR7. The CXCL12 3’A polymorphism is a G>A transition at position 801 in the 3’UTR and is represented in the CXCL12 β transcript [37]. The association of 7

CXCL12 3’A with disease progression from HIV to AIDS is controversial. In some studies, the polymorphism has been associated with delayed disease progression in

HIV-1 infected individuals due to the upregulating of the transcription levels by the

CXCL12 gene [37-39]. However, other CXCL12 studies show no association between the polymorphism and HIV-1 disease progression [40-42]. Furthermore, there were no significant differences for patients who were either heterozygous or homozygous for the polymorphism from seroconversion to AIDS diagnosis and death [28]. The point is that various polymorphisms mentioned influence disease progression, likewise therapy, antiretroviral therapy (ART), administered to HIV positive individuals which will be briefly discussed next.

1.6 Antiretroviral therapy

To date, there no cure for HIV but there are treatments (ART) for HIV which reduce the viral load to undetectable levels and prolong life. The United States of American

Food and Drug Administration (US-FDA) has approved six distinct classes of drugs, of which there are 24 drugs available for treatment of HIV infection [43, 44]. These are the nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors (PIs), fusion inhibitors, CCR5 antagonists, and integrase inhibitors ART [44, 45].

The following is a general classification of these drugs and processes in the HIV life cycle that they interact with to reduce viral replication. Table 1 shows the various drugs that are classified under these six classes. The first class, NRTIs inhibits the

HIV life cycle by binding to the enzyme known as reverse transcriptase. This prevents

8 the viral ribonucleic acid [46] from converting into deoxyribonucleic acid (DNA) [43,

47]. The second class of drugs, NNRTIs, work by targeting reverse transcriptase structure and inhibiting the enzyme activity. Once the enzymatic activity for reverse transcription is interrupted, NNRTIs prevent the virus from reproducing, and NNRTIs are mostly used as first line treatment [43, 48]. The third class of drugs, PIs, inhibit the enzymatic activity, proteolysis, of the protease. By binding to the protease it interferes with the viral replication process, thus preventing proteins from being cleaved to assemble new HIV-1 virions [49, 50]. The fourth class, FIs, target viral entry by preventing fusion between virus and host cell. This drug is designed to interfere by blocking the viral envelope protein (gp120) and CD4 receptor of the host cell [51].

CCR5 antagonists are the fifth class of drugs that are specifically designed for entry inhibition. These small molecules bind to CCR5 receptors and prevent viral, R5 variant, entry into the host cell by competing for the available CCR5 [52]. The sixth class, integrase inhibitors, are designed to inhibit the action of integrase, a viral enzyme that inserts the viral genome into the DNA of the host cell [53]. Since integration is a vital step in retroviral replication, blocking it can halt further spread of the virus.

Table 1. Six classes of ART and drugs

US-FDA six classes of ART Names of drugs

Zidovudine, Didanosine, Zalcitabine, Nucleoside Reverse Transcriptase Stavudine, Lamivudine, Abacavir Sulfate, Inhibitors Emtricitabine,Tenofovir [44, 47] Non-Nucleoside Reverse Transcriptase Nevirapine, Efavirenz, Etravirine, Inhibitors Rilpivirine, Delavirdine [43, 44] Saquinavir, Ritonavir, Indinavir, Nelfinavir, Amprenavir, Darunavir, Protease Inhibitors Atazanavir, Fosamprenavir, Iopinavir, Tipranavir [44, 54, 55] Enfuvirtide (or T-20, monotherapy, only Fusion Inhibitors fusion inhibitor available) [56] CCR5 antagonist Aplaviroc, Vicriviroc, Maraviroc [52, 57] Raltegravir ( first integrase inhibitor Integrase Inhibitor approved by U.S. FDA, 2007), Elvitegravir [44, 53, 58]

1.7 Host genetics

Host genetics and highly active antiretroviral therapy (HAART, which is a combination of two or more ART regimens) responses are essential factors out of several that are used as markers to observe disease progression in AIDS patients.

Another important characteristic to consider is the rate of disease progression, which is highly variable between individuals and among ethnicities [59]. Host genetic predictors, such as CCR5 gene polymorphisms is likely to have associations with the responses of HAART [60], and since the mechanisms involved are not fully understood it would be beneficial to know their combined effect on disease progression is vital for a prolonged and healthy existence.

CCR5 -2459 G>A, a genotype previously described, has two alleles (-2459A/ -

2459G) which are race specific in response to treatment with the effect recessive in 10

European Americans and dominant in African Americans [61]. A study investigating the two main ethnicities in North America of European and African ancestries, found that HIV infected African Americans who had the allele -2459A while on HAART were more likely to progress faster to AIDS as compared to African Americans with -

2459 G and the European Americans [62]. Furthermore, CCR5 Δ32 polymorphism predominantly found in Caucasian populations is protective in both forms. Its homozygous form (Δ32/Δ32) is associated with resistance against HIV infection while the heterozygous (Δ32/wildtype) form is associated with slower rates of disease progression when compared to the others polymorphisms such as CXCL12-

3’A and CCR2-64I while on HAART [63, 64].

1.8 Cell Biology: Human T Lymphocytes

This is a review section on the host cell biology, human T cell population, in context to HIV interaction. As previously stated, the host – virus interaction is due to the CD4 molecule (glycoprotein) and the chemokine coreceptors, CCR5 and CXCR4.

However, the focus will be on the expression of the CCR5 coreceptor and other factors on the surface of the CD4+ T cells subset population which is a prerequisite for viral fusion and productive infection.

The T cells make up part of the adaptive immunity. The progenitor of all T cells are made in the bone marrow from haematopoietic stem cells which emigrate to and migrate through the thymus as thymocytes before becoming a naïve T cell, depending on the antigen it is presented with during the selection processes, it develops either into a CD4+ or CD8+ T cell subset before it is activated by an antigen. The human

CD4+ T cells is categorized as naïve T cell [65] and memory T cell (TM) based on expression of respective CD45RA and CD45RO isoforms [66]. These isoforms are generally expressed on T cells, preferentially memory and or activated cells and are

+ - - used as marker to discriminate TN (CD45RA CD45RO ) from TM (CD45RA

CD45RO+) [67]. Furthermore, the CD4+ T cell is required for immunity against viral infections such as HIV.

The TN is a cell population which frequently migrates to the lymph nodes (LN) to search for their antigen. According to Kaech et al. (2002), the migration capacity of

TN is partly due the homing receptors of the C-C chemokine, CCR7 and L – selectin,

CD62L, and obviously by CCR5 [68, 69]. Furthermore, based on several studies, TN population is resistant to HIV infection in vitro yet in some conditions HIV infection may occur [70-72]. However, TN in vitro and also in vivo do express the CCR5 chemokine coreceptor but with lower expression in comparison to TM, thus, R5 strain may still cause infection leading to integration, yet it is known that the efficiency of infection would be significantly lower in comparison to resting TM cells [71], although, the mechanism behind this is still unclear [73].

On the contrary, the TM population mostly migrate through the peripheral tissues [74] and is composed of subsets; central (TCM) [75] and effector memory with a marker protein CD45RA+CD45RO- (TEMRA). These TM subsets are distinguishable by the presence or absence of the CCR7 expression, with the concomitant of CD62L [66], and CCR5 coreceptor is preferentially expressed on all TM [76]. Moreover, TM is part of the T cell repertoire which have been activated by exposure due to antigen in the recent past [46, 77]. Furthermore, TM divides more rapidly, have adhesion

12 molecules (CD62L) that enable binding (CCR7 – also mediates migration of TM to inflamed tissues) and express the lower molecular weight isoforms of CD45RA-

CD45RO+.

The TCM and TEM are functional subsets of the TM and are distinct because of their polarity. TCM (CD45RO+CD45RA-, CCR7+) is non-polarized and expresses CCR7 and CD62L, migrates to the secondary lymphoid organs, on the contrary, TEM

(CD45RO+CD45RA-, CCR7-) is polarized, does not express CCR7 and migrates to the nonlymphoid tissues. In addition, TCM is one of several cell populations that have higher expression of CCR7 [66, 78] and lack potential effector function, while in comparison TEM has lower expression level of CCR7 and CD62L, and has effector functions [66, 79].

TEMRA (CD45RO-CD45RA+, CCR7-) is a small subset of CD4+ TEM, which has been found to greatly increase in some HIV infected individuals in relation to uninfected individuals [78]. Even with high levels of CCR5 expression TEMRA has shown to be resistant to the R5 HIV variant but remain susceptible to X4 HIV variant

[78].

Chapter 2: Rationale/Hypothesis and Method

HIV infection of T cells occurs via CD4 receptor and its co-receptors, CCR5 and

CXCR4. These receptors enable viral binding, fusion and the gradual destruction of the immune system resulting in AIDS. However, it has been reported that some groups of people are more susceptible to infection and experience a faster disease progression to AIDS than others [80]. The differences in the degree of susceptibility are attributed to host genetics, and it has been shown that DNA sequence polymorphisms in the genes that encode these receptors, mainly CCR5 co-receptor, accounts for these [18, 81, 82]. Essentially, SNP in the CCR5 gene have been associated with certain ethnicities and their predisposition to HIV infections and rapid disease progression.

There have been several CCR5 polymorphisms identified in the promoter and open reading frame (ORF) regions of the CCR5 gene which also determine the genotype.

Extensive studies on polymorphisms of the CCR5 gene, such as CCR5 Δ32 and CCR5

-2459 with variant G, respectively, have shown strong associations with resistance to

HIV and a slower disease progression to AIDS [83]. CCR5 Δ32 is only found in

Caucasians while CCR5 -2459G is found in all human ethnic groups [83, 84].

Ethnicities with a higher frequency of CCR5P1 (Haplotype: E, F1, F2, and G1) should have a higher expression of CCR5 receptors due to the -2459A variant as compared to

CCR5P2 – P5 (Haplotype: A, B, C and D) with the -2459G variant [18]. Our hypothesis is that if CCR5 genotypes are associated with CCR5 expression on T cells, then we expect to observe differences in HIV infection in vitro.

2.1 Aims:

1. To determine the association between CCR5 genotypes and CCR5 expression.

The following are hypotheses that were tested.

i) If CCR5 -2459G allele is present we expect to see lower expression of

CCR5, compared with if CCR5 - 2459A allele is present.

ii) If CCR5 Δ32 allele is present as heterozygote, we expect to see 50%

reduction in the expression of CCR5 as compared with CCR5 wild-type.

2. To determine the association between CCR5 genotype and HIV infection in

vitro. The following are hypotheses that were tested.

i. If CCR5 -2459G allele is presented we expect to observe a lower

infection in the in vitro assay as compared with if CCR5 -2459A allele

is present.

ii. If CCR5 Δ32 allele is present as heterozygote, we expect to observe a

50% reduction in infection in vitro as compared with CCR5 wild-type.

2.2 Methodology

2.2.1 Participants and Sample

Whole blood was collected using a vacutainer (9 ml) with sodium-heparin to prevent coagulation. There were 39 healthy individual samples collected in total, study participants were mainly North Americans, (n=33) – [Caucasians, CA, (n=25);

African, AfA, (n=3); Hispanic, HA, (n =1); Asia, AsA, (n=1) and unknown, (n=3)] and other minority groups [Indian, IND, (n=2) and Papua New Guinean, PNG, (n=4)].

However, eight of the 39 samples were duplicated bring the total to 47 samples. Prior to collecting venous blood, written informed consent was obtained from all study participants. Ethical clearance and approval for the study were given by the

University Hospital of Cleveland Institutional Review Board (UHC IRB) and assigned the protocol number: 08-03-33 (expiry date: 05/21/2013).

2.2.2 DNA Extraction and Polymerase Chain Reaction

Whole blood was collected from the respective participants mentioned above and then

DNA was extracted using the Qiagen QIAamp DNA Mini Kit (Qiagen Science,

Germantown, MD). Polymerase Chain Reaction (PCR) was performed on the DNA to amplify specific genes and which were later confirmed by the base pair of the bands on the gel electrophoresis. Upon confirmation of the result, the Ligase Detection

Reactions (LDR) assay and Fluorescent Microsphere Assay (FMA) were then performed.

2.2.3 Ligase Detection Reaction-Fluorescent Microsphere Assay

This three step genotyping assay has been described in detail by Mehlotra et al. and others [85, 86]. Step (1) involved the LDR assay, (2) followed by the FMA hybridisation, and then, (3) the streptavidin-R-phycoerythin (SA-PE) signal detection using the Bio-Plex suspension array system. This included a fluorescence reader and the Bio-Plex Manager analytical software (Bio-Rad Laboratories, Hercules,

California). Mehlotra et al. has described the basic principles and the necessary conditions used to perform the steps for this assay [85].

2.2.4 Cells and Whole Blood Staining

Phenotyping was performed on the healthy 39 participants’ samples; peripheral blood mononuclear cells (PBMC) were isolated from the whole blood by centrifugation over a Ficoll Histopaque cushion technique and the PBMC were stored at -80°C overnight then cryopreserved.

The cryopreserved PBMC were thawed at 37°C in a water bath and suspended in 10 ml of complete media [Roswell Park Memorial Institute medium (RPMI), 10% fetal bovine serum (FBS), 1% L-gutamine, 1% Pen strep, 1% HEPES]. This was centrifuged at 1600 rpm for 10 minutes (mins), the supernatant was discarded, then the PBMC were resuspended with RPMI medium and the centrifugation step was repeated before being mounted on a haemocytometer for counting under a microscope. The cells were added to Fluorescence-activated cell sorting (FACS) tubes and washed with 2 ml of phosphate buffer solution (PBS), microfuged for 2 mins, and resuspend in 1.5 ml of PBS. 1 µl of violet live/dead stain to each tube and incubated at

17 room temperature for 10 mins. Complete media (2 ml) was added to each tube and microfuged (2 mins) and the supernatant discarded. The cells were resuspended with wash buffer (2 ml), microfuged (2 mins), supernatant discarded, and the wash step was repeated twice. Cells were resuspended in 100 µl of wash buffer, and surface stained with correct amounts of antibody per tube, which were CD3 Qdot 655, DC4

AF700, mIgG2a PE, CD4RO PE, rIgG2a PE-Cy7, CCR7 PE-Cy7, mIgG2a APC,

CCR5 APC, VB violet and CD8 FITC, and incubated for 15 mins on ice. The wash step was then repeated twice and finally, the PBMC were resuspended in 200 µl of

1 % paraformaldehyde (PFA) and 200 µl of wash buffer.

2.2.5 FACS-based HIV fusion/Productive Infection Assay [87]

This assay was used for measuring the frequency and phenotype of the cells that are capable of supporting fusion during viral infection. Its samples were from aliquots of the PBMC that was previously described. It is done in two parts and takes several days (four) to complete.

On Day 1 cells are thawed in the cryotubes at 37°C, the cryotubes are sprayed with

70% ethanol, dried and then transferred to the hood. Media is added to the cryotubes and transferred into a clean 15 ml conical tube. Aliquots are taken for counting while the rest are centrifuged for eight minutes at 500g. This is followed by the setting up of the infection step. The cells are resuspended (1.0 x 106 cells/ml) in RPMI and aliquots of 200 µl (2 x 105 cells) per well into 96 well V bottom plates. One plate was assigned for fusion and another for productive infection assay. Cells were transferred from all experimental conditions and additional control wells. A final step for Day 1 is staining

18 with a fluorescent substrate [CCF2-AM; (per ml) 990 µl CO2-independent media, 8 µl solution B, 2 µl of CCF2-AM dye]. This required the addition of 25 µl of CO2- independent media to all the wells and centrifuged (1200g) for 10 mins and the supernatant aspirated. 100 µl of the CCF2-AM solution (light sensitive) is then added to resuspend each of the well, accept the control well, in a darkened hood. The control well was resuspended in 100 µl of CO2-independent media. The cells were then washed with 125 µl CO2-independent media and centrifuged (1200g) for 10 mins and the supernatant aspirated. 100 µl of probenecid solution (per ml: 900 µl CO2- independent media, 100 µl of PBS and 5 µl of probenecid stock) was added to resuspend all the wells before being wrapped in aluminium foil and incubated overnight at room temperature in the biosafety level hood.

On Day 2 to 4 is the staining of the cells with antibody. The cells are washed with 125

µl of PBS, centrifuged for 10 min (1200g) and the supernatant aspirated. 50 µl of memory panel antibody mix is added to resuspend each well except the control wells.

The control wells are resuspended in 50 µl of PBS/BSA. The wells are then incubated

(4°C) for 30 mins. Compensation control tubes are made by added 250 µl of PBS to each of the compensation control tubes. The cells were then washed with 175 µl of

PBS and centrifuged (1200g) for 10 mins and the supernatant aspirated. The cells were then resuspended in 125 µl of PBS/BSA with 1% of paraformaldehyde (PFA) and transferred to appropriately labelled U-bottom FACS tubes. The wells were washed again with another 125 µl of PBS/PFA (1%) and added to the U-bottom

FACS tubes (250 µl total). The tubes were wrapped in foil and stored at 4°C until ready for the flow cytometry.

2.2.5.1 Assay description [87]

This is brief description of a newly developed assay for fusion and productive infectivity of the T cells, thus, it only highlights the principle of the assays.

Binding of HIV-1 envelope glycoprotein gp120 to CD4 and subsequently to the

CCR5 or CXCR4 coreceptors induces conformational changes in gp120 [88, 89] that culminate with the insertion of the gp41 fusion peptide into the host cell membrane, formation of an energetically favourable six-helix bundle, and fusion between the viral and host cell membranes [90, 91]. Membrane fusion marks the end of the viral entry process and the beginning of a series of post-entry events that must occur successfully for progeny viruses to be produced, including uncoating, reverse transcription, integration, LTR-driven viral gene expression, protein production, and virion assembly and maturation.

We have developed novel flow cytometric panels (Table 2.) that precisely quantify the subsets of CD4+ T cells that are susceptible to HIV-mediated fusion (early viral life cycle event) and LTR-driven gene expression (late viral life cycle event) (Figure

5). Using this assay, we sought to address two questions with respect to CCR5 polymorphisms and HIV: (1) do CCR5 polymorphisms influence the CD4+ T cell subsets that can be infected by HIV, and (2) do CCR5 polymorphisms alter the susceptibility of CD4+ T cells to productive infection following infection? It is important to realize that these assays are performed on unstimulated CD4+ T cells in vitro, thereby eliminating confounding variables such as alteration of coreceptor expression levels due to stimulation.

Figure 5. Combination of Reporter virus system, a) HIV life cycle showing the sites that are being investigated using the assays for Fusion (CCF2) and Infectivity (EGFP), b) Fusion and productive infection of those sites, c) density of fusion and productive infection of T cell subpopulations.

Table 2. These are the channels and the antibodies that were used in the assays.

Channel Fusion Productive infection (CCF2 Cleavage) (EGFP) FITC CCF2- Cleaved EGFP PE CCR7 PE-TR CD45RO PE-Cy 5 CD38 PE-Cy 7 CD27 APC CD4 Alexa 700 CD1a ACP Cy 7 HLA-DR V440 CCF2-Cleaved V515 CCF2-uncleaved V575 Live/Dead BV605 CD14 BV650 CD3 BV705 CD8 CD4 CD3+CD4+CD8-CD14- Mono/Mø CD4+CD14+ LC Cd1a+HLA-DR+

Chapter 3: Results and Discussion

3.1 Genotyping: CCR5 genotypes from Human T lymphocytes

The CCR5 gene has been extensively studied after its role as a chemokine coreceptor for HIV binding was discovered. This led to the evaluation of several polymorphisms both in the CCR5 promoter and ORF regions via CCR5 genotyping, to establish if there were any relationships among the CCR5 SNPs and CCR5 expression. From these studies, CCR5 -2459 G>A (promoter) and CCR5 Δ32 (ORF) were shown to influence CCR5 expressions on T cell surfaces [18, 92], thus, affecting HIV infection and disease progression to AIDS [18]; however, the frequencies of CCR5 genotypes and haplotypes also vary among ethnicities. For instance, as previously stated CCR5

Δ32 SNPs is predominant among the Caucasian population, that is, approximately

10% are heterozygous while 1% are homozygous for the mutation [18, 93, 94], but is rarely found in other ethnicities. However, due to the uneven distribution of ethnicities among the 39 samples that we analyzed, a meaningful comparison among ethnicities is not possible for this study. Therefore, CCR5 genotyping for these samples provides data on the CCR5 SNPs, allowing the identification of allelic frequencies and use of the polymorphisms to make comparisons with CCR5 expression and infectivity of T cells subsets.

Table 3. Number of individuals from various ethnicities

Samples

(n=39)

Ethnicity Number, n, (%)

CA 25, (64%)

AfA 3, (8%)

AsA 1, (3%)

HA 1, (3%)

IND 2, (5%)

PNG 4, (10%)

Unknown 3, (8%)

All 39 samples were genotyped and grouped according to their ethnicities, which were self identified, as shown in Table 3. [For all of these samples, the Hardy-

Weinberg Equilibrium was applied to determine the relative frequencies of the CCR5 alleles and genotypes.] The allelic frequencies of CCR5 -2459G variant was 38% and the CCR5 -2459A variant was 62%, as shown in Table 4. As for the CCR5 ORF the allelic frequencies for the CCR5 Δ32 variant was 6% and the wild-type (Wt) was

94%. The genotypic frequencies for CCR5 -2459 SNPs for G/G was 23%, A/G was

30% and A/A was 46%. As for the CCR5 ORF the genotypic frequencies for Wt/Wt was 87% while Wt/ Δ32 was 13% for the CCR5 Δ32 SNPs.

Table 4. CCR5 -2459 and Δ 32 allelic and genotypic frequencies

Allelic Frequency

CCR5 -2459, n, (%) CCR5 Δ32, n, (%)

G=35, (38%) Wt=73, (94%)

A=62, (59%) Δ32=5, (6%)

Genotypic Frequency

CCR5 -2459 G>A, n, (%) CCR5 Δ32, n, (%)

G/G=9, (23%) Wt/Wt=34, (87%)

A/G=12, (30%) Wt/ Δ32=5, (13%)

A/A=18, (46%) Δ32/ Δ32=0, (0%)

There were five samples that were CCR5 Δ32 heterozygous (Wt/Δ32) and all were

Caucasian. Of these, only one was CCR5 -2459 homozygous (A/A) while the rest were heterozygous (A/G), [data not shown]. As mentioned, the CCR5 Δ32 SNP is dominant in the Caucasian population, as indicated in this study, while the CCR5 -

2459 SNPs are fairly distributed among all ethnicities for this data set.

3.2 Phenotyping: CCR5 expression on T lymphocytes in relations to CCR5

Genotypes

In this part of the experiment, staining of PBMCs was performed after genotyping to establish if there was any relationship between the CCR5 polymorphisms and the levels of CCR5 expression on T cell surface. There were nine antibodies, shown in the methods section, which were used to calculate the density of CCR5 expression for the

T cell subpopulations. The T cell subsets that were stained were TN and TM, also

TCM and TEM respectively, which are subtypes of TM. Initially, there were 12 samples stained, only eight samples had meaningful data from at least some of the four T cell subsets. However, from these, only five had meaningful data from the

TCM subtype.

Figure 6. The density of CCR5 receptors in percentage (%) on TCM cells for the CCR5 - 2459 genotypes, G/G and A/A.

CCR5 -2459 G/G genotype was compared with the pooled A/A genotypes. With limited samples available, there was still a trend that clearly show that the allele G, was associated with lower CCR5 expression on the T cell surface, while allele A was

25 associated with higher CCR5 expression, agreeing with other studies [95]. As previously stated, HIV preferentially infects TM cell subtypes [96], TEM and TCM because of their high levels of CCR5 expression [76], which in this study was significant for TCM. In Figure 6, TCM subtypes cells that had the CCR5 -2459 G/G homozygous genotype had lower levels of CCR5 expression than the A/A genotype in comparison. Thus, the CCR5 -2459G allele down regulates the expression of CCR5 in the TCM subtypes, which has been shown in other studies [95, 97, 98].

Figure 7. The density of CCR5 expression on TCM in percentage (%) for the CCR5 Wt/Wt and Wt/Δ32 with their CCR5 -2459 genotypes

Of those five samples that were stained for CCR5 and had data for TCM, two were

CCR5 Δ32 heterozygous; however, as previously mentioned, one had CCR5 -2459

A/G while the other was A/A. Figure 7, compares CCR5 Δ32 heterozygous samples with the wildtype, it does show a trend when considering CCR5 -2459 genotype as well, in context to the levels of CCR5 expression on TCM. When comparing the

26 trends of CCR5 Δ32 heterozygous for the sample with CCR5 -2459 heterozygous samples, the trend clearly indicates what has been documented, wildtype showing higher levels of CCR5 expression than CCR5 Δ32 heterozygous which has a 50% reduction of CCR5 expression on TCM [99] due to its polymorphism. Furthermore, there is a trend among the CCR5 ORF wildtype as well, where the CCR5 -2459 SNPs regulates CCR5 expression levels, as mentioned above.

The density of CCR5 expression on the TCM cells varied greatly among the two

CCR5 Δ32 heterozygous (Wt/Δ32) samples (Figure 7), which implicates a possible influence of CCR5 -2459 SNPs, as the sample with CCR5 -2459 G/A genotype had a value of 0.03% as compared to the A/A genotype which was 3.98%, similar observations have been reported in one study [99]. Furthermore, this demonstrates the influence that CCR5 -2459G allele has on the levels of CCR5 expression, by lowering it, in the presences or absence of CCR5 Δ32 SNPs, while CCR5 -2459A allele has the opposite effect.

3.3 CCR5 Genotype and Infectivity of Human T lymphocytes

Figure 8. The in vitro HIV infectivity in percentage (%) in TCM cells compared with CCR5 - 2459 genotype; G/G, G/A and G/G, and four CCR5 ORF Wt/32 genotypes

In this analysis, approximately 22 samples (some of which have been used in the analysis of Figures 6 and 7) were tested using in vitro assays. The infectivity of their

TCM cells were compared with the CCR5 -2459 genotypes, which were, G/G, A/G and A/A. Of these four samples were CCR5 Δ32 heterozygous (Wt/Δ32), in which three were A/G and one was A/A respectively. These are labeled on the graph as G/X and A/X, X indicating CCR5 Δ32 SNPs, in Figure 8. There were significant differences observed among the CCR5 -2459 genotypes in context to the infectivity of the TCM cells, between G/G and A/A with a p-value of 0.029 (unpaired t-test with

Welch’s correction), and also between G/A and A/A, with a p-value of 0.049. These show that the infection on the TCM cells of CCR5 -2459 G/G was significantly lower when compared to the CCR5 -2459 A/A SNPs. Similarly, the CCR5 -2459 G/A had

28 significantly lower infectivity than the CCR5 -2459 A/A SNPs with respect to their median values.

These in-vitro assay results support and confirm similar findings from other studies, where, CCR5 -2459 G/G genotype had a pronounced influence by down regulating the CCR5 expression due to the presences of two G alleles, thus, with lower CCR5 expressed implies less HIV binding which leads to a reduction in infectivity [98, 99].

On the contrary, the CCR5 -2459 A/A genotypes had more pronounced infection on its TCM subpopulation due to the presence of two A alleles, that is, an increase in the levels of CCR5 expressed on the TCM cells allowing more HIV to bind and increase infectivity [98].

An interesting observation was made between the SNPS on CCR5 -2459 and CCR5

Δ32, in relation to the levels of CCR5 infection on the TCM. From the four samples,

G/X and A/X, there was a large difference among the CCR5 Δ32 heterozygous samples. Intuitively, a heterozygous CCR5 Δ32 allele would mean very low infection or 50% reduction of infectivity for TCM cells; however, among these four, the G/X

SNPs had very low infections, which is expected for samples with the SNP, in comparison to A/X, which had considerably higher infection.

This study observed that CCR5 Δ32 heterozygous (Wt/Δ32) had a much lower infection compared to its homozygous wildtype (Wt/Wt). The possible mechanism behind this is due to their CCR5 -2459 SNPs, as discussed in the introduction. In other studies, the CCR5 -2459 G allele was shown to have a protective effect on disease progression to AIDS, that is, CCR5 -2459 G/G individuals had a significantly slower

29 time to disease progression compared to A/A which were much faster [21, 98].

However, this protective effect is seen in disease progression and not in protection against HIV infection.

Chapter 4: Conclusion

The human CCR5 gene is a significant component of the host genetics in relation to

HIV infection and disease progression to AIDS. As mentioned in this thesis, certain polymorphisms of the CCR5 gene are associated with the levels of CCR5 expression on PBMCs and other cells [100]. These polymorphisms have been identified, CCR5

Δ32 in the ORF and CCR5 -2459 G>A in the promoter regions [13, 18, 65]. The frequencies of these SNPs also vary among different ethnicities, thus, some groups of people are more susceptible to HIV infection and disease progression than others, as shown in epidemiological studies [99, 101]. Therefore, to better understand the relationship among these CCR5 SNPs, CCR5 expression and in vitro HIV infection, further investigations were carried out at the T cell subpopulation level.

For this study we looked at two CCR5 polymorphisms, specifically, the CCR5 -2459

G/A and the ORF Wt/Δ32 genotypes. Based on the levels of CCR5 expression we wanted to investigate the association with the CCR5 genotypes and the infectivity on the T cell subpopulation.

From the results obtained for the association between CCR5 genotypes and CCR5 expression, there was a significant trend showing the relationship between the CCR5 -

2459 SNPs with the levels of CCR5 expression on the TCM cells. That is, -2459 G/G genotype had lower levels of CCR5 expression than -2459 A/A due the presence of the G allele, that is, G allele down regulates CCR5 expression while A allele up regulates expression on TCM cells. Similarly, there was a clear trend showing the relationship with the CCR5 ORF, that is, Wt/Δ32 genotype had lower CCR5

31 expression as compared to Wt/Wt. Furthermore, we also observed a trend among the

CCR5 ORF wildtype with CCR5 -2459 SNPs, as mentioned previously mentioned.

As for the association between CCR5 genotypes and in vitro HIV infection, there were significant relationships demonstrated by the CCR5 -2459 genotypes with respect to the TCM cell subpopulation. That is -2459 G/G and A/A had pronounced influence on the level of infectivity of the TCM. That is, the G allele down regulated

CCR5 expression, thus, fewer CCR5 for HIV to bind to, while A allele had the opposite effect. As for the CCR5 ORF SNPs, heterozygous (Wt/Δ32) had showed a lower infectivity as compared to the homozygous (Wt/Wt), which had higher infectivity of its TCM cells. Furthermore, when comparing CCR5 ORF Wt/Δ32 and -

2459 SNPs in relation to infectivity of TCM cells, the trend showed the influence of -

2459G allele which had lower infectivity as compared to -2459A allele [99].

With the findings presented we were able to show the relationship between CCR5 genotype, CCR5 expression and the in vitro HIV infection of the T cells. That is, the association between CCR5 genotypes (-2459 G/A, Wt/Δ32) with the density of CCR5 expression and levels of infectivity on TCM cell subpopulation for these samples.

4.1 Limitation and Future Directions

The main challenge was the availability of samples. With that said, if we had more samples then we would have been able to do a more meaningful analysis on the CCR5 genotype frequencies, its relations with CCR5 expression and how this influences the in vitro HIV infection on T cell subpopulations. It would be interesting to investigate these relationships further with different ethnicities because of the difference in the distribution of CCR5 genotype frequencies. In addition, other interesting comparisons would be haplotype frequencies and the infectivity of the other T cells subpopulations as well.

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