HIV-1 phenotype and genetic variation in vertical transmission

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From the Microbiology and Tumorbiology Center, Karolinska Institutet, Stockholm, Sweden

HIV-1 PHENOTYPE AND GENETIC VARIATION IN VERTICAL TRANSMISSION

Peter Clevestig

Stockholm 2006

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All previously published papers were reproduced with permission from the publisher.

Published and printed by Karolinska University Press Box 200, SE-171 77 Stockholm, Sweden

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ABSTRACT

HIV-1 vertical transmission is increasing worldwide despite that successful strategies are available. Reduction of vertical transmission has been established by antiretroviral prophylaxis and elective Caesarian section, but this has been limited to the developed countries. In the developing world the lack of resources, the necessity of breastfeeding, social and ethical factors, have all contributed to high transmission rates. Thus, a better understanding of the basic mechanisms of vertical transmission of HIV-1 is important for future preventions in the developing world.

This thesis was based on blood samples from HIV-1 infected children and

mothers collected prospectively between March 1987 and September 1994, following a national program for pregnant women living in Sweden. In addition, sequence data from the HIV database in Los Alamos National Laboratory was analyzed to evaluate our results with data representative of globally circulating HIV-1.

HIV-1 isolates were subjected to co-receptor determination in U87 cell lines, expressing CD4 and chemokine receptors CCR5 or CXCR4, and additional receptors used by HIV-1. Co-receptor use was determined by syncythia induction together with p24 ELISA assays or env V3 directed PCR and by infection in GHOST(3) cell lines expressing these chemokine receptors. PCR was also used to confirm virus growth together with p24 ELISA assays. Samples were subjected to limiting dilution nested PCR, also directed towards env V3, to create single molecular clones for sequencing in conjunction with sequencing of the isolates. Phylogenetic analysis was used to obtain the correct genetic clade for each patient. It was also used to map the viral populations in two mother-child pairs and in another four subtype D infected women.

In the first study, we analyzed the co-receptor use of HIV-1 in 24 children over time, to relate clinical parameters with the emergence of X4 virus. In four children, the X4 phenotype emerged after immune deficiency was established, and was hence not the directly contributing cause of AIDS. In the second study, we wanted to examine the phenotypes in mother-child pairs. A statistically significant link was found between the presence of X4 in the mothers and the development of X4 in the children. To evaluate the nature of this link, we studied two mother-child pairs and related the maternal virus from during the pregnancies with the virus in the children. We could in detail describe the transmission of R5 from both mothers, followed by a transmission-independent evolution of X4 from the established R5 populations in the children. We preliminary observed a possible concordance between the V3 loop N-linked glycosylation motif and the R5 phenotype in these mother-child pairs, while a high V3 net charge was in accordance with X4 and dual-tropic viruses. We expanded our phylogenetic mapping of virus populations with four infected mothers of uninfected children. Here we could show a highly statistically significant association among R5 sequences to the N-linked glycosylation motif in the V3 loop of gp120. There was also an accumulation of X4 virus within this patient group in the context of subtype D. To examine the fidelity of the association between the V3 loop N-linked glycan and R5 viruses, we analyzed 176 individual sequences of group-O, -N and most group-M clades of HIV-, including recombinants. The glycosylation motif NNT was highly conserved among the M-group clades (99.2%). This strongly supported our conclusion of the importance of the N- linked glycosylation motif of the V3 loop for CCR5 binding, and hence possibly for transmission.

In summary, we have shown the population dynamics of HIV-1 in detail with regard to co-receptor use over time in HIV-1 infected mothers and children, and have identified a possible marker for transmissibility.

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“Science is not a search for answers; it is the continuous pursuit for more questions”

To my loving parents

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LIST OF PAPERS

This thesis is based on the following papers, referred to by their Roman numerals I. Casper C, Navér L, Clevestig P, Belfrage E, Leitner T, Albert J, Lindgren S,

Ottenblad C, Bohlin AB, Fenyö EM, Ehrnst A. Co-receptor change appears after immune deficiency is established in children infected with different HIV- 1 subtypes. AIDS Res Hum Retroviruses 2002;18:343-352.

II. Casper C, Clevestig P, Carlenor E, Leitner T, Anzén B, Lidman K, Belfrage E, Albert J, Bohlin AB, Navér L, Lindgren S, Fenyö EM, Ehrnst A. Link between the X4 phenotype in human immunodeficiency virus type 1-infected mothers and their children, despite the early presence of R5 in the child. J Infect Dis. 2002;186:914-921.

III. Clevestig P, Maljkovic I, Casper C, Carlenor E, Navér L, Lindgren S, Bohlin A-B, Fenyö EM, Leitner T, Ehrnst A. The X4 phenotype of HIV type 1 evolves from R5 in two children of mothers, carrying X4, and is not linked to transmission. AIDS Res Hum Retroviruses 2005;21:371-378.

IV. Clevestig P, Lindgren S, Leitner T, Ehrnst A. HIV-1 R5 and X4 populations and their gp120 V3 loop amino acid patterns in pregnant women, infected with HIV-1 of subtype D. Manuscript.

V. Clevestig P, Pramanik L, Leitner T, Ehrnst A. CCR5 use by HIV-1 is closely associated with the gp120 V3 loop N-linked glycosylation site. In press. J Gen Virol. 2006;87:March issue.

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LIST OF ABBREVIATIONS

aa amino acids

AIDS acquired immune deficiency syndrome AZT zidovudine

bp base pairs

C1-C5 constant regions 1 to 5 of the env gene

CCR5 / CCR5∆32 CC-chemokine receptor 5 / 32 base pair deletion of this gene

CD cluster of differentiation

CD4 cells helper/inducer T-lymphocytes CD8 cells suppressor/cytotoxic T-lymphocytes CDC centers for Disease Control and Prevention

CRF circulating recombinant form

CXCR4 CXC-chemokine receptor

DNA deoxyribonucleic acid

env envelope glycoprotein gene

ER endoplasmic reticulum

F84 Felsenstein, 1984 (nucleotide substitution model)

FCS fetal calf serum

gag group specific antigen gene gp glycoprotein gp41 env glycoprotein 41 kDa gp120 env glycoprotein 120 kDa gp160 env polyprotein (160 kDa)

HIV-1/2 human immunodeficiency virus type 1 or 2 HTLV human T-lymphotropic virus

Kb kilobase

LTR long terminal repeat

MHC major histacompatibility complex

MIP- α/β macrophage inflammatory protein-1 alpha/beta nm nanometer (one millionth of a meter)

NSI non-syncythia inducing

PBMC peripheral blood mononuclear cells

PCR polymerase chain reaction

pol polymerase gene

RANTES regulated upon activation, normal T-cell expressed or secreted

RNA ribonucleic acid

RT reverse transcriptase

SDF-1 α stromal derived factor 1 alpha

SI syncythia inducing

SIV simian immunodeficiency virus

SU subunit (gp120)

Taq Thermus aquaticus

TM transmembrane (gp41)

V1-V5 variable regions 1 to 5 of the env gene

V3 third variable region of env, contains the V3 loop

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IUPAC CODES

NUCLEOTIDES

Code Base

A Adenine C Cytosine G Guanine T (or U) Thymine (or Uracil)

R A or G

Y C or T

S G or C

W A or T

K G or T

M A or C

B C or G or T

D A or G or T

H A or C or T

V A or C or G

N Any base

. or - Gap

AMINO ACIDS

Code Abbreviation Amino acid

A Ala Alanine

C Cys Cystein

D Asp Aspartic acid

E Glu Glutamic acid

F Phe Phenylalanine

G Gly Glycine

H His Histidine

I Ile Isoleucine

K Lys Lysine

L Leu Leucine

N Asn Aspargine

P Pro Proline

Q Gln Glutamate

R Arg Arginine

S Ser Serine

T Thr Threonine

V Val Valine

W Trp Tryptophane

T Tyr Tyrosine

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INTRODUCTION

A BRIEF HISTORY OF HIV/AIDS

Twenty-five years have passed since the first cases of HIV infection in New York and Los Angeles were described in the form of Kaposi’s sarcoma, usually a benign form of cancer in the elderly, where it emerged in eight young homosexual men (Hymes et al., 1981; CDC, 1981a). At the same time, rare infections of

Pneumocystis carnii pneumonia (PCP) were also discovered among gay men in these cities (CDC, 1981b). This was noted by Sandra Ford, a drug technician working at the Centers for Disease Control and Prevention (CDC) in Atlanta, GA, while

investigating the unusual number of prescriptions for PCP treatments and was

published in a report later that year on the unusually high incidence of PCP and other severe opportunistic infections (CDC, 1981b). The following year, a report on gay men in Southern California suggested that the deterioration of the immune system seen among these patients was caused by a single, unidentified infectious agent and that it was sexually transmitted. By this time, the disease had been observed outside the gay community, in intravenous drug users, (Masur et al. 1981), haemophiliacs and in Haitian people (CDC, 1982a & 1982b). It was also discovered outside of America (du Bois et al., 1981; Vilaseca et al., 1982; Rozenbaum et al., 1982; Francioli et al., 1982; Serwadda et al., 1985) and the disease was later termed acquired

immunodeficiency syndrome (AIDS) (Gottleib et al., 1981; Marx, 1982; CDC, 1982c). Already the following year, a retrovirus was first isolated from a lymph node taken from a patient with lymphoadenopathy by Francoise Barré-Sinoussi in the laboratory of Jean-Claud Chermann in collaboration with the research group of Luc Monagnier at the Pasteur Institute in Paris (Barré-Sinoussi et al., 1983). The virus was hence named lymphoadenopathy-associated virus, and was suspected to be associated with AIDS. Soon to follow, Robert Gallo’s group in America confirmed that the virus was the causative agent of AIDS and was then named human T-cell lymphotropic virus type 3 (HTLV3) (Gallo et al., 1984; Levy et al., 1984). To avoid the confusion by the two names, the virus was agreed to be called human

immunodeficiency virus (Coffin et al., 1986), which we are all familiar with today.

THE HIV/AIDS PANDEMIC

Today, an estimated 40.3 million people are living with HIV/AIDS around the world, of which 2.3 million are children under the age of 15 (UNAIDS December 2005 report). Around 5 million new infections occurred and over 3 million people died from HIV/AIDS during. The majority of these infections occurred in Sub- Saharan Africa with 3.2 million people newly infected, followed by South and South- East Asia with 990.000 people, having the second highest prevalence compared to the 22.000 new infections in Europe and 43.000 in North America. The majority of the new infections (95%) occur in low to middle income countries due to poor social

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standards, lack of resources and social infrastructures to name a few contributing factors.

In Sub-Saharan Africa where the AIDS epidemic is the most severe, infection incidence in the demographic populations can be as high as 40% and does affects every single person in one way or the other. Social factors further contribute to the continued spread by widespread unprotected heterosexual intercourse, prostitution and stigmatism which invoke fear of disclosing to their sexual partners about their infection.

Another important factor of spread is mother-to-child transmission. The incidence of this transmission in regions of Africa have been very high, varying between 25 up to 40%, as HIV can be transmitted during pregnancy (in utero), during delivery (partus) and through breastfeeding (Dunn et al., 1992; Landesman et al., 1996). The term vertical transmission in this thesis refers to the transmission of HIV-1 from mother to child in utero and/or during delivery. The high transmission rates in association with breastfeeding impose a serious paradox for many Africans as breast milk provides infants with essential protection from more common and benign malaises which can turn out to be fatal. Also, breast milk supplements need clean water to be made, which is something not readily available in many parts of Sub-Saharan Africa.

In developed countries such as in Europe and North America, the incidence of vertical transmission has been reduced to less than 2%. This has been made possible by prophylactic treatment with antiretroviral drugs such as Zidovudine (AZT), given to the mother prior to and during delivery, and to the infant shortly after birth in conjunction with elective caesarean sections that decreases the exposure to infectious blood (Connor et al., 1994; Kind et al., 1998). Unfortunately, these highly effective regimes to minimize vertical transmission are not currently plausible in many developing countries. There is also little commercial interest in the development of low-cost effective drugs by pharmaceutical companies, especially when large

investments are required for research and development and where the drug later may be copied and developed by third party companies in the developing countries. This poses a serious dilemma that further delays the availability of otherwise cost effective and easily applicable regimens to reduce transmission rates in the regions most affected by this pandemic.

On a positive note, the non-nucleoside reverse transcriptase inhibitor nevirapine, (Viramune®), developed by Boehringer Ingelheim Roxanne Inc., is given at no cost by the company to pregnant mothers and children in these areas as a prophylactic drug during delivery and after birth. Nevirapine was discovered to be highly effective in reducing the growth of HIV-1 in vitro, but induced resistance quickly. Therefore, it had no clinical use in treating HIV-1 infected patients. When used for a short period of time, as when given to the mother during delivery and shortly after to the child, it is a highly effective drug in reducing vertical transmission rates (Guay et al., 1999).

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THE ORIGIN OF HIV

The origin of HIV is a topic of much controversy. However, it is widely accepted that the different types of HIV (HIV-1 and HIV-2) entered humans through zoonosis, the cross-species transmission pathway (Kanki et al., 1986; Hirsch et al., 1989; Gao et al., 1999; Hahn et al., 2000; Marx et al., 2004). Both HIV viruses are similar to

simian immunodeficiency viruses (SIV’s) commonly found in African monkeys.

HIV-2 has almost been confirmed to originate from SIV_SM, whose primate reservoir is the Sooty Mengaby monkeys from the fact that HIV-2 and SIV_SM share, an identical genome organization (Kanki et al., 1986; Hirsch et al., 1989; Huet et al., 1990). For HIV-1, the natural reservoir remains uncertain, but evidence points to HIV-1 being a descendant of SIV_CPZ found in Pan troglydes troglodyte, a subspecies of chimpanzee, as the genomic organization between these two viruses are almost identical (Gao et al., 1999; Corbet et al., 2000).

The first HIV-1 seropositive sample dates back to 1959, taken from an adult male who suffered AIDS-like symptoms and came from former Zaire, now the Democratic Republic of Congo (Nahmias et al., 1986; Clavel et al., 1986a; Zhu et al., 1998). This would suggest that HIV originally crossed the species barrier somewhere in Central Africa, perhaps in the 1940’s or early 1950’s (Zhu et al., 1998) or as early as 1930 based on advanced computer models (Korber, Jan 2000, 7^th Conference on

Retroviruses and Opportunistic Infections). HIV-2 infection was first identified in 1986 in West African patients with AIDS like symptoms, and remains today mostly endemic to this region of Africa (Clavel et al., 1986b; Arya et al., 1987).

HIV-1

HIV-1 is a lentivirus, belonging to the retroviridae genus of viruses that infects primates and other animals such as cats, horses and cattle. These viruses are called lentiviruses as they propagate slowly in their hosts, where symptoms of the disease usually develop after longer periods of time. HIV is found in two main forms, HIV-1 and HIV-2 which are very similar at a genomic level but differ in their epidemiology, transmission and pathogenicity. We will from here on focus on HIV-1.

Structure

HIV-1 is a spherical to conical enveloped virion with an average diameter of 100 nm (fig. 1). The envelope is a host derived lipid bi-layer with between 8 and 10 anchored viral trimeric structures containing gp120 (SU) and gp41 (TM) (Zhu et al., 2003). They are bound non-covalently together, forming a heterodimer, which together forms the trimere. Additional host specific proteins may also be present on the envelope surface, such as the major histocompatibility complex (MHC) (Arthur et al., 1995). The envelope encases a truncated cone-structured capsid (CA) through the association between a matrix protein (MA) shrink wrapping the envelope around the core. An additional protein, cellular cyclophillin-A, is associated with the matrix and capsid proteins and is essential for uncoating following entry in new cells (Braaten et al., 1996a), and is found in all but group-O HIV-1 viruses (Braaten et al., 1996b), The

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capsid core contains the viral genome in the form of two positive-strand RNA copies coated with nucleocapsid core proteins (NC) and two base-paired tRNA molecules that act as primers. The core also contains essential viral enzymes such as 10-15 copies of an RNA-dependant DNA polymerase known as reverse-transcriptase (RT), and integrase (IN) and protease (PR).

Figure 1. Illustration of the HIV-1 virion

Genome

The HIV-1 genome is a diploid structure consisting of two closely associated positive sense single-stranded RNA molecules, at around 9200 bases in length. This structural configuration provides the virus with an effective mechanism to repair damaged RNA through frequent recombination in addition to gaining genetic diversity.

This is an important aspect of retroviruses as they protect their genomes poorly from the harsh external environments (Coffin, 1979). A unique aspect of HIV is the association of two tRNA molecules to the genome copies involving complementary binding of 18 bases at the 3´ terminal of the tRNA molecules to a primer binding site (PB) on the HIV RNA genomes. The function of these tRNA molecules is to initiate reverse transcription through priming.

Figure 2. Genomic organization of HIV-1.

The genome of HIV-1 consists of three main genes, gag, pol and env (fig. 2).

The gag (group antigen) gene encodes the antigen precursor polyprotein which is proteolytically cleaved into the structural capsid proteins (p24), matrix proteins (p17),

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nucleocapsid proteins (p7/p9) and release proteins (p6). The pol (polymerase) gene encodes the viral reverse transcriptase (p66/p51) and integrase (p32) enzymes and also the protease enzyme (p10) together with gag. The env gene codes for the polyprotein gp160, which is proteolytically cleaved into the gp120 subunit (SU) and the

transmembrane subunit gp41 (TM) containing the fusion domain. Additional genes and proteins are shown in table 1.

Table 1. Additional proteins of HIV-1, and their functions (Adapted from Crandall, 1999).

Gene Product Function

vif p23 enhances infectivity of virions in some cells.

vpr p15 needed for entry into nucleus, arrests cell cycle.

vpu p16 downregulates CD4 expression, assists budding

tat p14 transactivator.

rev p19 helps in transporting unspliced mRNA, arrests cell cycle.

nef p27 downregulates CD4 and MHC-1, CD1b, is a cell activating

protein.

The HIV-1 genome also has two long terminal repeats (LTR) sequences which contain promoters, enhancers and other sequences that interact with host-cell

transcription factors needed for successful replication of the virus. Within the LTR are non-coding regions near each end of the genome. The first region is R, a short

repetitive sequence within the capping group (5´ end) and the poly-A (3´ end). The second region is the primer binding site (PBS) at the 5´ end which provides the primer with an initiation site for synthesis of the negative complementary DNA strand. The polypurine tract (PPT), a sequence repeat of purines (A’s and G’s) at the 3´end, provides the primer for the plus strand DNA. The U3 sequence, between the PPT and the R at the 3´ end, contains the control sites for binding the transcription factors.

Finally, the U5 region between R and the primer binding sequence (PBS) in the 5’ end provides a stabilizing leader-stem for primer directed initiation of transcription through an intermediary extended hairpin structure together with the PBS region (Bereens et al., 2000). During reverse transcription, the U3, R, and U5 sequences are copied at the end of the DNA forming the long terminal repeat (LTR), which enables the virus to

duplicate the correct end of the RNA and provides transcriptional control elements outside the actively transcribed region.

The Replication Cycle Binding and entry

HIV-1 primarily infects T-lymphocytes and cells of the macrophage lineage (including dendritic and glial cells) expressing CD4 through receptor-mediated membrane fusion (Dalgleish et al., 1984; Klatzmann et al., 1984; McDougal et al., 1986; Maddon et al., 1986; Habeshaw et al., 1989). This is a two step process as CD4 is only a partial entry receptor for HIV-1 (figure 3).

When the virus comes into contact with the host-cell, one or more pg120 subunits bind to the CD4 molecule with high affinity. This interaction allows the pg120 trimere

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to contort and alter the positions of its V1/V2 and V3 loops (Wyatt et al., 1995; Trkola et al., 1996; Wu et al., 1996), exposing conserved regions within the V3 loop (Rizzuto et al., 1998). This allows an additional interaction with either a C-C or C-X-C

chemokine receptor. The binding will lead to another conformational change that unveils the hydrophobic fusion domain of gp41 near its amino terminus (figure 3). The fusion domain will anchor into the hydrophobic compartment of the host cell

membrane and allows the formation of a prehairpin intermediate through interaction between the three N-terminal helices of gp41 (figure 3). The main hairpin structure is formed first when the C-terminal helices fold in anti-parallel around the N-terminal helix core, forming a 6-helix bundle (coiled-coil). This provides a mechanism for pulling the viral and host cell membranes closer together, allowing them to fuse and subsequently release the viral core into the host cell cytoplasm (figure 3) (Chan et al., 1997; Weissenhorn et al., 1997).

Figure 3. 1) Virus homes in on target cell. 2) Binding of the subunit glycoprotein (SU) to CD4 receptor.

3) Conformational change of the SU to expose conserved regions for binding to surface chemokine receptor. 4) Trans-membrane glycoprotein (TM) fusion domain penetrated target cell membrane. 5) Formation of the coiled-coil that drags both membranes closer together. 6) Membrane fusion and entry.

Reverse transcription and integration

After fusion of the membranes, the viral core together with NC, RT, and IN is released into the cytoplasm of the host cell. The core complex protein CA is associated to cyclophillin-A, a cellular chaperone protein, which destabilizes the core complex and leads to uncoating. Once the genome and accompanying enzymes are free, DNA

synthesis is initiated by RT together with the tRNA primer base-paired to the PBS. RT

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initiates transcription through adding nucleotides to the 3´end of the tRNA primer towards the end of the 5´LTR including the U5 and R regions. In conjunction with the creation of this DNA strand, RNase H degrades the copied RNA on the original

template strand, liberating the newly created DNA. This new DNA strand base-pairs to the R sequence of the opposite LTR at the 3énd, serving as a new primer for creating the whole genome minus DNA strand up to the PBS region of the 5énd. The original RNA template is degraded shortly after with the exception of the PPT sequence, which remains base-paired to the new minus strand DNA genome copy. This in turn acts as a primer for synthesis of a short plus stranded DNA segment up to the 3énd of the LTR, including a PBS sequence and the still attached tRNA primer. When completed, the minus strand DNA folds over into a circle and base-pairs its 3énd PBS sequence with the complementary PBS sequence of the shorter newly synthesized plus strand DNA strand. This allows the completion of the remaining plus-strand DNA, resulting in a complete double-stranded DNA genome including U3-R-U5 LTR sequences at both ends (figure 4).

The double stranded DNA forms a pre-integration complex (PIC) together with vpr, MA and IN. It includes a peptide signal that interacts with the cellular system for importing proteins into the nucleus. The PIC is then actively translocated into the nucleus and covalently combined into the host cell genome in random locations by IN, creating a provirus (Brown, 1997). This provirus is the basis for retroviral infection and is capable of producing progeny and establishing a persistent infection with constant production of new viral particles (figure 4). An important note is that integration is not required for production of new virus and that HIV-1 can be present episomally in the nucleus in asymptomatic individuals (Bukrinsky et al., 1991).

Transcription and translation

When integrated into the host cell chromosome, the provirus either remains latent or is transcribed when the cell is activated. Early viral genes are expressed by cellular RNA polymerase II, directed by a transcription initiation site at the U3-R boundary of the 3´LTR to produce full-length mRNA transcripts that are spliced via their poly(A) additions into sub-genomic mRNA and translated into tat, rev and nef. The tat protein enhances transcription through binding to the trans-activating region (TAR), a stem- loop structure within R in the LTR, where it recruits additional transcription factors that phosphorylate the cellular RNA polymerase II, enhancing its activity and enabling it to transcribe longer segment of mRNA. The rev protein assists in transporting spliced or unspliced mRNA into the cytoplasm for translation. It also directs expression of early genes to late genes when present at a peak concentration and acts as a negative

feedback regulator, keeping a balance between production of early and late genes. The nef protein down-regulates many different types of cell surface molecules with

important immune functions. One such molecule is cell surface CD4, which is internalized and degraded through the endo-lysosomal pathway (Aiken et al., 1994;

Piguet et al., 1999), as opposed to vpu, which promotes proteolysis of CD4 in the endoplasmic reticulum (ER). It also down-regulates antigen presenting MHC-I (Scheppler et al., 1989), and lipid presenting antigen CD1d (Chen et al., 2005). This

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down-regulation is likely a strategy developed by the virus to avoid detection by natural killer (NK) cells and other components of the immune system (Cohen et al., 1999). It also is an important protein for the formation of mature infectious particles (Lama, 2003), and enhances infectivity and viral propagation by super-induction of IL-2, leading to T-cell activation (Kinoshita et al., 1998; Manninen et al., 2000).

When the late genes are transcribed, unspliced or spliced transcripts are

translocated out into the cytoplasm and translated into structural proteins for the viral core capsid, and for RT, PR and IN by free polyribosomes in the cytoplasm, producing the gag and gag-pro-pol precursor polyprotein (Swanstrom & Willis, 1997). The env polyprotein (gp160) is translated into the ER by membrane bound polyribosomes.

Inside the ER, high mannose N-linked glycans are added and the polyprotein is

properly folded and transported into the Golgi apparatus for further processing. There it is cleaved into gp120 and gp41 and the N-linked glycans are trimmed and modified into complex and hybrid types with sialic-acid. The processed env proteins are then transported to the host-cell surface as complete membrane bound trimeres (figure 4).

Assembly and release

The gag and gag-pro-pol proteins assemble together near the membrane forming an immature capsid together with the viral RNA genome and cellular cyclophillin-A.

The env proteins, already present on the cell membrane surface, associate to the capsid in the budding process (figure 4). During budding, the particle gathers significant amounts of MHC and other membrane associated proteins.

Figure 4. Replication cycle of the HIV-1 virus.

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To avoid premature interactions between viral glycoproteins and CD4 during the replication cycle, HIV has developed three mechanisms preventing this; through vpu ,which removes newly synthesized CD4 from the ER, through nef removing both surface CD4 and MHC-1 (Garcia & Miller, 1992), and lastly though the env protein, which can directly block the CD4 molecule.

When the particle finally buds from the host cell membrane, it is still a premature virus particle. The pol portion of the gag-pol polyprotein is cleaved into PR, IN and RT and the gag polyprotein are subsequently cleaved into MA, CA and NC by PR, creating a mature virion ready to infect new cells.

BIOLOGICAL VARIABILITY

HIV-1 infects cells expressing the CD4 surface glycoprotein that ordinarily binds membrane-proximal domains of MHC class I and II molecules and interacts with the protein tyrosine kinase with its cytoplasmic domain. In addition to CD4, HIV-1 needs to bind to a secondary receptor (co-receptor) to enable fusion through gp41. The different types of cells identified expressing such receptors include monocytes, macrophages (Gartner et al., 1986; Koenig et al., 1986), microglia, dendritic cells (DC’s), Langerhans cells, thymocytes and the CD4+ T-cells (Klatzmann et al., 1984a).

The type of co-receptor used is a G-protein coupled receptor and its specific type is governed by the preference (tropism) of the infecting virus and can influence the strains infection capacity and may have implications for disease development.

Biological Classification

HIV-1 was first classified through its capacity to infect and replicate in established T-lymphoid and monocytoid cell lines (Åsjö et al., 1986). Viruses were divided either into rapid/high, which grew rapidly to high titers in PBMC and established a productive infection in T4-antigen-positive tumor cell-lines, or as slow/low, growing slowly with low titers and without the capacity to infect T4 cell-lines. A new classification was used based on the capacity of primary isolates to form syncythia in PBMC and MT-2 T-cell lines, naming the virus SI (syncythia inducing) or NSI (non-syncythia inducing) (Koot et al., 1993). A similar classification system was based on isolate capacity to replicate in primary macrophages or T-lymphocytes and monocytoid cells, named M-tropic or T- tropic, respectively (Schuitemaker et al., 1991). This provided a sharper distinction between the two phenotypes. These classifications provided a system for categorizing HIV-1 of different tropisms but also resulted in overlapping results (Björndal et al., 1997). Between 1995 and 1996, research on blocking HIV-1 from entry using RANTES and MIP-1α/β chemokines identified the chemokine receptors as the co- receptors used by HIV-1 to enter cells (Cocchi et al., 1995; Feng et al., 1996; Bleul et al., 1996; Oberlin et al., 1996). It was hence that the currently used co-receptor use (phenotype) classification system was adopted (Berger et al., 1998).

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Classification of HIV-1 by Co-Receptor Use

The current phenotype classification system has three main categories based on the three most common phenotypes (table 2). These include the R5 strains, using the CC- chemokine receptor CCR5 (CCL4), the X4 strains, using the CXC-chemokine receptor CXCR4 (CXCL12) and the dual-tropic (R5X4) phenotype that can use any of the two chemokine receptors (Björndal et al., 1997; Zhang & Moore, 1999). In addition to the mentioned receptors, HIV-1 has been found to use other chemokine and structurally related orphan receptors for entry into cells, but these remain to be studied in greater detail and are also more uncommon. Such receptors include CCR2d, CCR3, CCR9, CX3C and the orphan receptors BOB and BONZO (Moore et al., 1997).

Table 2. HIV-1 phenotypes, chemokine receptors, ligand's, and their relation to the earlier classifications.

Phenotype chemokine receptor natural ligand old classification

R5 CCR5 (CCL4) MIP-1α/β, RANTES NSI slow/low M-tropic

↕ ↕

R5X4 CCR5/CXCR4 SI rapid/high T-tropic

↕ X4 CXCR4 (CXCL12) SDF-1/vMIP-II* SI rapid/high T-tropic

↨ indicates overlaps between earlier classifications

*vMIP-II encoded by Kaposi’s sarcoma associated-herpes virus (HHV-8) and is not a naturally occurring ligand.

Chemokine Receptors

Both CCR5 and CXCR4 are chemokine receptors, belonging to a super-family of serpentine glycoprotein’s that signal through coupled heterotrimeric G-proteins and span through the membrane seven times. The receptors are classified by the position of their first cystein residues (Rollins, 1997) and are found in four classes, C (1), C-C (beta) and C-X-C (alpha) and C-X-C-3 which combined includes at least 50 members that vary in length between 68 and 120 aa. They have three intra- and extra cellular loops (ECL’s) held together by disulphide bonds, likely forming a compact barrel- shaped structure. The N-terminal of the polypeptide is located on the cell surface and is involved in the binding to the natural chemokine ligand. The C-terminal is intracellular and associated with the signal transducing heterotrimeric G-proteins (figure 5).

The chemokine receptors bind low-molecular-weight peptides (chemokines) that play an important part in the inflammatory response by inducing leukocytes to migrate into areas of infection or secondary lymphoid tissues by providing chemotaxis signals.

CD8+ T-cells are the major producers of these pro-inflamatory chemokines (Rollins 1997). The chemokine receptors can bind more than one natural ligand with the exception of the HIV-1 co-receptor CXCR4, which binds only one known natural ligand (stromal cell derived factor, SDF-1α). CXCR4 can also bind to the viral

macrophage inflammatory protein II (vMIP-II) that is encoded by the Kaposi’s sarcoma associated-herpes virus (Fernandez & Lolis, 2001). Both SDF-1α and vMIP-II can inhibit HIV-1 entry by blocking CXCR4.

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Figure 5. The general structure of the 7-transmembrane chemokine receptors. The extra cellular loops (ECL’s) are shown and the disulfide bonds between ECL1 and ECL2, N-terminus on the outside of the cell membrane and the C-terminus on the inside of the cell.

The chemokine receptors are found on a wide range of lymphoid cell types.

Specifically, CCR5 is expressed by T-lymphocytes, monocytes, macrophages and dendritic cells. CXCR4 is mainly expressed by myeloid cells, T-cells, B-cells, epithelial cells, endothelial cells and dendritic cells (Murdoch & Finn, 2000). On T-cells, CCR5 is suggested to be preferentially expressed by Th1 while CXCR4 is more highly expressed on Th2 cells and is up-regulated by IL-4 (Maggi et al., 1994; Meyaard et al., 1996; Bonecchi et al., 1998; Moonis et al., 2001).

HIV-1 infection and Co-Receptor Use

In patients with HIV-1 infections, the initial phenotype found is R5 and may remain R5 until the degradation of the immune system and the development of AIDS.

However, the X4 phenotype develops in about half of the individuals who progress to AIDS with CD4+ T-cell counts below 400 cells per µl blood (Koot et al., 1999) and has been associated with a more rapid disease progression (Koot et al., 1996; Connor et al., 1997; Scarlatti et al., 1997).

It appears that the R5 phenotype of HIV-1 has an advantage in transmission over X4, despite the fact that CXCR4 is found on both epithelial and endothelial cells (Murdoch & Finn 2000). A possible explanation for this could be that X4 viruses may have a higher CD4 dependency than R5 viruses and/or that macrophages have a general lower expression of CD4 (Kozak et al.,1997; Platt et al., 1998; Dimitrov et al., 1999;

Tokunaga et al., 2001). This is important as macrophages have been implicated as principal targets for the establishment of infection in new individuals by the macrophage tropic strains (R5) (Zhu et al., 1993; van’t Wout et al., 1994).

Additional support for the transmission advantage of R5 strains is though the CCR5∆32 allele, a 32 base deletion in their CCR5 gene producing a defected receptor molecule. Homozygous individuals carrying this allele deletion appear to be resistant

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from infection by HIV-1 (Liu et al., 1996; Samson et al., 1996). However, infection by X4 virus has been observed among a few of these individuals (Balotta et al., 1997;

Michael et al., 1998). Another possible factor is that the gp120 SU of R5 viruses have a higher frequency of N-linked glycosylation (Myers & Lenroot, 1992; Chabot et al., 2000; Pollakis et al., 2001; Polzer et al., 2001; Polzer et al., 2002), which provide a more efficient shielding from detection by the immune system (Miedema et al., 1990;

Schutten et al., 2001).

Other cells implicated as ports of entry for HIV-1 are the Langerhans and DC antigen-presenting cells. These cells are mainly found in peripheral tissues and transport captured antigens to the lymphoid tissues for presentation to T-cells. It is hypothesized that these antigen presenting cells could be infected by or carry the HIV-1 virus to the lymphoid tissues where it then can establish infection in the CD4+ T-cells.

Whether HIV-1 is carried by, or indeed infecting these cells, is a topic of controversy, but co-cultured DC’s and T-cells have shown to support HIV-1 infection (Pope, 1994;

Graelli-Piperno et al., 1996). The immature DC specific C-type lectin (DC-SIGN) has been shown to capture and present HIV-1 virus to T-cells without infection (Hladik et al., 1999; Geijtenbeek et al., 2000). However, this presentation of HIV-1 to CD4+ T- cells in the lymphoid tissues is not phenotype specific and is thus only considered as one possible mode of entry. However, immature DC’s have been observed to preferentially migrate towards HIV-1 R5 variants (Lin et al., 2000).

An additional influence on the initial establishment of infection by R5 virus could be that T-cells expressing CXCR4 generally are naïve or resting memory T-cells which when infected by X4 variants, can not provide the intracellular requirements needed for virus replication and production of progeny unless the T-cells are activated, and would give the R5 strains a replicative advantage (Zack et al., 1990; Zack et al., 1992). CCR5 is also transiently upregulated on mature CD3, CD4 and CD8 T-cells while CXCR4 is downregulated unless in naïve T-cells when migrating from the thymus. Also, the difference in co-receptor expression on Th1 and Th2 cells may be of great importance as they differ in their infectivity to R5 and X4 viruses (Moonis et al., 2001).

The nature of the development of X4 remains to be determined together with its association with a more rapid disease progression but has been suggested to evolve through the dual-tropic phenotype (van Rij et al., 2000). Very few mutations within the V3 region of gp120 are needed for a R5 strain to be able to use CXCR4 (Hwang et al., 1991; De Jong et al., 1992; Fouchier et al., 1992; Shioda et al., 1992; de Wolf et al., 1994; Shimizu et al., 1999; Verrier et al., 1999; Hu et al., 2000a; Hu et al., 2000b;

Briggs et al., 2000). An interesting observation is that the few changes needed for a co- receptor switch from CCR5 to CXCR4 would not be an obstacle for a multi-facetted virus such as HIV-1. It would instead suggest that the preferential establishment of R5 would depends on the lack of available or activated target cells for X4 viruses (Koning et al., 2003). A possible mechanism for the emergence of X4 is the immune activation in later stages of the disease which could result in the activation if infected and/or uninfected naïve T-cells, enabling X4 to finally proliferate (Hazenberg et al., 2000;

Davenport et al., 2002; De Boer et al., 2003).

Furthermore, the tat protein of HIV-1 has the capacity to upregulate the expression of C-X-C chemokine receptors in T-cells (Secchiero et al., 1999) and could contribute

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to the rapid disease progression seen in association with the emergence of X4 strains.

Also, the rapid replication rate of X4 strains seen in vitro could also be a contributing factor towards this phenomenon through a more rapid depletion of target cells. This does not suggest that X4 strains are more cytopathic than R5 strains, but suggests that this difference rather depends on the availability of target cells (i.e. a broader range of target cells) (Grivel & Margolis, 1999; Kwa et al., 2001).

It is well established through the use of phylogeny that both R5 and X4 virus populations continuously evolve in their infected hosts (either through random

misincorporations of nucleotides by RT or by random recombination events and other selection pressures). However, these random events drive the virus strains towards a broader use of co-receptors that can manifest itself in a change of co-receptor use for a subset or virus strains. Then the end result will depend on the availability of the specific target cells. Hence, X4 strains may readily evolve from R5 or dual-tropic strains but can not establish a subpopulation if target cells are not available, or they can co-evolve within their own ranges of target cells.

Glycoprotein 120 Interaction with the Co-receptor

The third variable region (V3) of the gp120 has been shown to be directly involved in the interaction with the co-receptor (Hwang et al., 1991; De Jong et al., 1992;

Fouchier et al., 1992; Shioda et al., 1992). The V3 region is an approximately 35 aa long sequence with a loop structure held together by two cystein residues forming a disulfide bond and is found to have a net positive charge of between +2 and +10 (at pH 7.0). The loop contains a conserved β-turn “crown” flanked by variable amino acids which ends with conserved “stems” of consecutive β-turns at the N-terminal region and a C-terminal helix (Rini et al., 1993; Catasti et al., 1995; Tugarinov et al., 2000). Single amino acids changes within the V3 loop have been shown to influence co-receptor use (Hwang et al., 1991; De Jong et al., 1992; Fouchier et al., 1992; Shioda et al., 1992; de Wolf et al., 1994; Shimizu et al., 1999; Verrier et al., 1999; Hu et al., 2000a & 2000b;

Briggs et al., 2000). These changes, involving substitutions with the positive charged amino acids arginine and lysine, have been associated with changes towards CXCR4 use, generating higher net charged for the entire V3 region (De Jong et al., 1992; de Wolf et al., 1994). In addition, loss of N-linked glycans in the V3 region has also been observed to confer CXCR4 use as well as increased sensitivity towards neutralization (Pollakis et al., 2001; Polzer et al., 2002; Polzer et al., 2001).

Mutations within the crown sequence affect co-receptor use (Shimizu et al., 1999;

Hu et al., 2000), as do changes in the flanking variable regions, likely through altering the stability and exposure of the crown (Chesebro et al., 1992; Catasti et al., 1995;

Tugarinov et al., 2000). In one study, soluble gp120 was shown to bind to soluble CCR5 N-terminal sulfopeptides by the stem region while binding to cell surface-bound CCR5 requires both the stem and the crown region (Cormier & Dragic, 2002).

The V1/V2 regions of gp120 are also of importance for co-receptor binding as they influence the accessibility of the V3 region (Cao et al., 1997; Wyatt et al., 1995). They occluded the crown region (Losman et al., 2001), which was made accessible by the internal structural rearrangement after binding to CD4 (Sullivan et al., 1998).

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Whatever the mechanism for co-receptor binding to either CCR5 or CXCR4, it is likely an interplay between multiple regions on both gp120 and the chemokine

receptor, with varying degrees of influence, which in turn makes it difficult to identify the true nature of this interaction.

GENETIC VARIABILITY

HIV-1 is a virus that displays a high degree of variation in its genome, similar to other RNA viruses. It is through the combination of a chronic infection and a

continuous production of new viral variants that HIV-1 poses such a challenge to combat with vaccines and antiretroviral drugs. The fact that the HIV-1 provirus is integrated into the host-cell chromosome and is subsequently hidden from the immune system provides another challenge.

The genetic variability seen in a population of HIV-1 particles, e.g. an infected patient, is the result of both positive and negative selection from both viral and host immune factors. The positive selection mechanisms depend on the fact that the RT enzyme lacks proof-reading activity. It has been estimated to produce 3.4 x 10^-5 misincorporations per nucleotide per replication cycle (Roberts et al., 1988; Preston et al., 1988; Mansky & Temin, 1995). In addition, antiretroviral drugs pushes selection further by selecting for virus strains resistant to the drug(s) in question. Negative selection occurs as virus with mutations in mainly pol and gag may become unable to produce infectious progeny. All these pressures lead to an accumulation of greatly diverse strains (Leitner at al., 1997). An additional contributing factor to the diversity of HIV-1 and other retroviruses is the close association of the two single stranded RNA copies in the capsid, which facilitates recombination events. This enables repair of the genome when breaks occur and exchange of generic material, which can also involve completely different strains during super infection. Another important factor is the high turnover rate of the virus in vivo, estimated to be up to 10¹⁰virus particles produced each day.

The consequence of all these factors is that each infected individual harbors a uniquely diverse virus population, referred to as intra-host variation and varies even greater between individuals, termed inter-host heterogeneity (Nowak, 1992a & 1992b).

Because many different HIV-1 variants may reside within a host, HIV-1 has been described as a quasispecies, consisting of genetically related but distinct sub-

populations which centre around one master-variant (Holland et al., 1992). Some authors, however, question weather HIV-1 is described by the quasispecies theory (Jenkins et al., 2001).

It has been suggested that in primary HIV-1 infection, a relatively homogeneous virus population resides in the beginning, only to diversify into a heterogeneous population over time. When the individuals progress into AIDS, the population reverts into homogeneity (figure 6) (Goodenow et al., 1989; McNearney et al., 1992; Wolfs et al., 1992; Shankarappa et al., 1999).

In vertical transmission, minor subsets of maternal variants have been shown to be transmitted (Wolinsky et al., 1992). However, recent studies have suggested that

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primary infection in children can occur with multiple variants (Nowak et al., 2002), only having the master variant detectable at HIV-1 diagnosis (Dickover et al., 2001). In addition to the general variability of HIV-1 within and between hosts, variation also occurs within different tissues, significantly within lymphoid organs and peripheral blood mononuclear cells, probably due to extreme immunological pressures and constant cell activation and/or replenishing. The rate of evolution can vary greatly depending on the development of the disease. This is seen in individuals who progress to AIDS more rapidly or have reached immune-suppression as they undergo lower levels of evolution, which could reflect a lack of selective pressures exerted from the immune system (Delwart et al., 1997).

Figure 6. a) A hypothetical model for the evolution of HIV-1 from the point of infection to the

development of AIDS, showing an increasing viral diversity, development of X4, followed by a leveling off and the revetment to a more homogeneity and stable viral population during AIDS. b) CD4+ cell counts and RNA levels. (Adapted from Shankarappa et al., 1999)

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The difference in variation within the HIV-1 genome is apparent for the function of the encoded proteins. The env gene is the most variable gene because the encoded surface proteins need a large variability to escape immune response. Within the gene are constant regions (C1-C5) interleaved with hypervariable regions (V1-V5). The pol gene varies the least, largely because the encoded enzymes must maintain their function to enable replication, while the gag gene varies intermediately.

HIV-1 Genetic Classification

Due to the highly variable nature of HIV-1, an extensive system has been developed to classify HIV-1 by its phylogenetic clustering patterns (Louwagie et al., 1993, Myers, 1993, Salminen et al., 1993). Initially, this classification was based on sequencing of env and/or gag genes only, but has later included whole genome

sequences as HIV-1 can contain parts from multiple clades. Today, new clades are still emerging and the list of genetic subtypes, sub-subtypes, and circulating recombinant forms of HIV-1 continues to grow.

Figure 7. Phylogenetic radial tree of HIV-1 genetic groups.

HIV-1 is genetically classified into three main lineages, called groups; the M (Major), O (Outer) and N (Non-M/Non-O) (De Leys et al., 1990; Chameau et al., 1994;

Korber et al., 1997; Simon et al., 1998). The M-group is the dominant group with a global geographic distribution and contains the subtypes, sub-subtypes, and circulating recombinant forms, while the O- and N-groups are endemic to countries in west equatorial Africa (also the region endemic to the P. t. troglodyte, the subspecies of chimpanzee believed to be the source of HIV-1).

Interestingly, the three groups are not each others closest phylogenetic relatives, but have instead closer phylogenetic relationships to subsets of SIVCPZ isolates (fig. 7), which suggests that the three groups of HIV-1 crossed species at separate points in time (Simon et al., 1998; Gao et al., 1999; Hahn et al., 2000).

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Figure 8. Phylogram showing the relationship between HIV-1 genetic groups and SIVcpz isolates (Adapted from Hahn et al., 2000).

Subtypes

The M-group contains 10 clades (fig.9) or subtypes. These are named with the letter codes A, B, C, D, F, G, H, J, K, and U, where U is a an actual denotation for unknown. This sequence belongs to the M-group, but does not yet have an assigned or new subtype association. There is also a subcategory of sub-subtypes for A and F where the sequences have an association to the main subtype group but is distinct through a sister-clade (Robertson et al., 1995a & 1995b).

Subtypes are classified after their close phylogenetic relationship to neighboring sequences of the associated clade (fig. 8). New subtypes are identified and classified after a certain set of criteria: 1) at least two have to be identified from two unrelated cases; 2) they should be similar enough to each other and different enough from other known clades, without having any tendency towards being a recombinant by sequence inconsistencies throughout their whole genomes; 3) the must not be directly

epidemiologically linked.

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Figure 9. Phylogenetic tree of the most common HIV-1 subtypes, sub-subtypes and circulating recombinant forms.

Circulating Recombinant Forms (CRF’s)

CRF’s are the result of recombination events in cells where two or more infecting viruses of different subtypes recombine into a new hybrid virus, named a circulating recombinant form (Carr et al., 1999). Around 16 CRF’s recombinants have been identified today (fig. 9), most notable the CRF01_AE, formerly known as subtype E (Louwagie et al., 1993). When the whole genome was analyzed, it was discovered to be an actual AE recombinant where the env gene was uniquely subtype E and pol/gag belonged to subtype A (Murphy et al., 1993; Carr et al., 1996; Gao et al., 1996). Some of these CRF’s have multiple subtype recombination sites and are termed _CPX for their complex mosaic forms (Srinivasan et al., 1989; Vidal et al., 2000).

Geographic Distribution

The geographic distribution of genetic clades in the world is an important

epidemiological tool for examining the distribution and continued spread of HIV/AIDS in the world (figure 10). Subtype C is the dominating HIV-1 subtype globally with the highest prevalence in North-Eastern and Southern African countries, India and China.

Subtype A together with CRF02_AG, the second most prevalent clades, are dominating in Eastern Africa, together with subtypes C and D (Osmanov et al., 2002). CRF02_AG

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appears to dominate in West and West Central Africa (Carr et al., 1999). Subtype B is the most commonly found subtype in developed countries as in Europe, North

America, and Australia. Therefore, it is the currently most studied clade of HIV-1. In Asia, the dominating clade is CRF01_AE (Ou et al., 1992; Gao et al., 1996; Carr et al., 1996). At the epicenter of the AIDS pandemic in sub-Saharan Africa, all known subtypes and CRF’s are present, including HIV-2. Many remain endemic to certain areas.

Today, most subtypes and CRF’s emerge into many European countries and in North America as more people from the African and Asian continents enter (Lukashov et al., 1999, Hamers et al., 1998, Alaeus et al., 1997).

Figure 10. Rough geographical distribution of HIV-1 subtypes.

Subtypes, Co-Receptor Use and Clinical Implications

Subtype specific patterns of co-receptor use have not been studied in detail, but the few studies performed have shown variable results. Studies with large sets of samples, where subtypes and co-receptor use were determined, have shown a tendency towards that subtype C less often uses CXCR4 and a tendency towards higher frequencies of CXCR4 use in subtypes D isolates (De Wolf et al., 1994; Tscherning et al., 1998;

Björndal et al., 1999; Tscherning-Casper et al., 2000).

Differences in mother-to-child transmission rates for subtypes have been reported.

In one such study, performed on a cohort in Western Kenya, a higher rate of

transmission in mothers infected with subtype D compared to subtype A was shown (Yang et al., 2003). In a more recent study with the same Tanzanian cohort, a higher in utero transmission rate or subtype C than for subtypes A, D and A-env or D-env recombinants was shown (Renjufi et al., 2004). An additional study on the Tanzanian cohort showed that viruses containing subtype D LTR’s were between 3.2 and 4.8 times less vertically transmissible than viruses containing subtype A or intersubtype recombinant LTR’s, respectively. Viruses with subtype C LTR’s appeared to be 6.1 times more transmissible than the viruses with the subtype D LTR’s (Blackard et al.,

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2001). Other studies have shown no significant differences in transmission rates between subtypes (Tapia et al., 2003). However, in one study, a tendency towards higher transmission rates for subtype D was observed (Eshleman et al., 2005).

One possible explanation for the higher vertical transmission rate for subtype C virus was recently suggested in a study on a Kenyan cohort, showing that women infected with subtype C virus were more prone to mucosal (vaginal) shedding of virus than women infected with subtypes A, C and D (John-Stewart et al., 2005). Other studies have suggested that viruses of different subtypes have preferences in their transmission pathways. One such study suggested that that subtypes C and CRF01_AE are better adapted to sexual transmission than subtype B (Mastro et al., 1997). A second study suggested that subtype B is associated with homosexual transmission while subtype C is associated with heterosexual transmission (van Harmelen et al., 1997).

This would be in agreement with the recent study on the higher vaginal shedding of subtype C virus (John-Stewart et al., 2005).

Clinical implications of infection by different subtypes have also been suggested.

In one such prospective study on female sex workers in Senegal, women infected with subtype A virus were 8 times less prone to develop AIDS than those women infected with HIV-1 of other subtypes (Kanki et al., 1999). Another study on 320 HIV-1 infected women in Nairobi, Kenya, showed that those women infected with subtype C virus were found to be at more advanced stages of immunosupression than the women infected with subtype A or D viruses (Neilson et al., 1999). The authors of this paper suggested that this difference was either due to subtype C being associated with a more rapid disease progression, or that subtype C represented an older and better established epidemic in this area.

Other studies have shown little clinical relevance for subtypes and for the

distribution of phenotypes (Alaeus, 2000) or when comparing subtypes with mother-to- child transmission rates (Morgado et al., 1998; Tapia et al., 2003). In a Tanzanian cohort, similar to the studies described previously, a lower mother-to-child transmission rate for subtype D than for subtypes A, and C, and CRF’s (Renjifo et al., 2001)

Whether subtypes truly have implications for the transmission/development of phenotypes, differences in transmission rates, and/or in the progression to AIDS, remains to be determined. However, the varying amounts of X4 virus observed among different subtypes and the association between X4 and a more rapid disease progression should not be ignored, as well as the other differences reported in the above mentioned studies. If and when the true implications of infection by different HIV-1 subtypes are identified, our current and future antiretroviral treatment regiments and vaccine strategies may have to be reevaluated and may also require tailoring.

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AIMS OF THESIS

The general aim of this thesis was to characterize the different subtypes in the Swedish cohort of HIV-1 infected mothers and their infected children, and to investigate the co- receptor use of the viruses.

In specific:

• To characterize the subtypes and co-receptor use of isolates from HIV-1 infected children and their relation to disease progression.

• To characterize the relation of phenotypes in mother-child pairs.

• To identify the origin of the X4 phenotype in vertically infected children, and whether it emerges through transmission or evolves from their own R5.

• To characterize pertinent amino acid differences between R5, R5X4, and X4 phenotypes.

• To determine if phenotype specific differences between R5 and X4, found in our cohort, applies to globally circulating HIV-1 of different genetic clades and phenotypes.

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MATERIALS & METHODS

PATIENTS AND SAMPLES

Papers I, II, III and IV were based on samples collected prospectively between March 1987 and September 1994 following a national program for pregnant women living in Sweden (Lindgren et al., 1993). These samples were all retrieved from the Department of Clinical Virology at Karolinska University Hospital Huddinge at one time point in 1997. All transferred samples have been listed. The lists have been signed by the then chief of the Department of clinical virology, Professor Anders Vahlne (co- signed by Dr Anders Sönnerborg), and the receipt by the then prefect of MTC,

Professor Ingemar Ernberg (co-signed by Professor Eva Maria Fenyö). Subsequently, they have been reported to a biobank register, as a sample collection. Originally they were received and handled at the Central Microbiological laboratory of the Stockholm County Council (SMCL) until June 1994, from which time they were received at Karolinska University Hospital Huddinge. The patients were HIV-1 infected pregnant women and children, most of whom were born to these mothers in Sweden. Samples were collected at birth, and at 6 months intervals, except during the first year when samples were collected also at 3 months, and if born after 1991, also at 6 weeks of age.

Cord blood was not used. After 18 months of age, samples were taken every following half or whole year.

Defibrinated whole blood was used for all virus culture from plasma and PBMC, as well as for HIV-1 detection by the polymerase chain reaction (PCR). The blood had been collected and separated by Ficoll and frozen as plasma supernatants and PBMC pellets. In the Swedish cohort, 220 distinct isolates were analyzed from 39 subjects in papers I to IV. Table 3 shows how they were included in each study, some of which had been described in the previous works. In the summary line, only he unique and non-overlaping numbers are shown. Paper V involved submitted and publicly available sequence data from the International HIV database in Los Alamos and included 176 HIV-1 infected individuals in whom the data included virus, classified according to phenotype and genotype (table 3). Thus, a total of 215 subjects were studies in papers I through V.

Table 3. Overview of the patients and samples included in the papers within this thesis and the total number of sequences and clones involved. A total of 220 isolates were characterized in papers I to IV.

Paper Subjects Isolates Sequences clones

PBMC Plasma

I 24 children 86 57 -

II 11 mother-child pairs 79 59 -

III 2 mother-child pairs 12 8 195

IV 5 mothers 17 12 290

V 176 individuals - - 176

In all 215 129 91 620

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METHODS

Virus Isolation (Papers I to IV)

Virus isolates were co-cultured with phytohemagglutinin stimulated PBMC from two healthy blood donors (Ehrnst et al., 1988). Virus stocks were obtained through passage through donor PBMC and monitored through gag p24 antigen ELISA (papers I, II and III) or through env V3 PCR (paper IV).

Determination of Co-receptor Use (Papers I to IV)

Co-receptor use was determined by inoculating U87 astroglioma cell lines expressing CD4 and chemokine receptors CCR5 or CXCR4 (paper IV) and other co- receptors CCR1, CCR2b, CCR3 (Deng et al., 1996; Deng et al., 1997; Berger et al., 1998). Additional co-receptor use determination was performed on GHOST(3) cells expressing CCR5, CXCR4 or orphan receptors BOB or BONZO (Deng et al., 1997) (papers I, II and III).

Sample Preparation for PCR (Papers I to IV)

Prior to PCR assays, infected PBMC or U87 cells were treated with a lysis buffer, containing Proteinase-K at different temperatures to first enable the proteinase-K to break down the cellular structures and expose the cell DNA and later to inactivate both virus and the active enzyme, rendering it safe for regular laboratory work and of good quality for PCR.

Nested PCR (Papers I to IV)

A nested PCR of the V3 region of gp120 was used as a base for subtyping of HIV- 1, to create molecular clones for DNA sequencing of isolates and clones, and lately for surveillance of HIV-1 infection in PBMC cultures and in co-receptor use

determinations.

Generation of DNA Clones (Papers III and IV)

To amplify integrated proviral DNA from one single cell, a limiting dilution technique based on the Poisson theory of random distribution was used (Brinchmann et al., 1991; Leitner et al., 1996a; Rodrigo et al., 1997). Quadruplet sets of dilutions for each sample were set up with an increasing four-fold dilution. The nested PCR

described above was used and an agarose gel electrophoresis conducted on all samples.

An average dilution was extrapolated from the resulting electrophoresis which would render one third of the reactions positive, likely representative of one provirus. Based on this dilution, a second round of nested PCR was performed on each sample with approximately 50 reactions. This give on average 20 positive clones, confirmed using agarose gel electrophoresis. As it was still difficult to accurately predict the outcome of positive reactions, we introduced a practice of using two sets of dilutions from which the best set was chosen for sequencing.

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Prior to sequencing, PCR samples were purified using nitrocellulose-membrane based filtration techniques to remove salts and other left over PCR components.

DNA Sequencing (Papers I to IV)

Sequencing PCR was performed on all positive PCR reactions from both isolate and clone samples, subjected to nested PCR. This cycle sequencing PCR was conducted using both inner primers JA168 and JA169 in separate tubes to provide two complementary sequences from each sample, enabling optimal base- calling (Leitner et al., 1996b). Samples were afterwards purified with

nitrocellulose-membrane based filtration techniques specific for sequencing reaction clean-ups.

Sequence Processing (Papers I to IV)

Raw sequence data was analyzed using base-calling software to determine an accurate consensus nucleotide sequence for each sample based on both

complementary sequences derived through each inner primer. This provided a tool for scrutinizing ambiguities and possible mismatches in the raw data

chromatograms and using correct IUPAC letter code abbreviation to identify true ambiguities. In general, multiple chromatogram peaks present on both

complementary strands with a height of at least 25% of the largest peak were accepted as ambiguities and given their appropriate IUPAC code. The criteria set up for accepting clone sequences for inclusion in our analysis were a maximum of one ambiguity. This was to ensure that each sequence we obtained was accurate in its nucleotide composition and when an ambiguity was found, present this sequence in its two configurations with designated suffixes a, and b. Sequences with multiple ambiguities were omitted as the original PCR amplification probably represented multiple pro-viruses and not a single molecular clone. The isolate sequences were allowed to contain multiple ambiguities. Prior to analysis, base-called sequenced were assembled with Se-Al sequence alignment software (Rambaut, 1995).

Hypermutation Analysis (Papers III)

Hypermutations were assessed among HIV-1 sequences in paper III using

Hypermut (Rose & Korber, 2000) with maternal sequence M1.2aD as master sequence (#GÆA>10, normal value 0-5). Sequences with abnormal GÆA mutations (Vartanian et al., 1991; Rose & Korber, 2000) were omitted in the analyses of the mother-to-child transmission to facilitate maximum resolution in our phylogenetic trees.

Phylogenetic Analysis (Papers III and IV)

An important tool in analyzing the data presented in this thesis has been the use of phylogeny. It is a tool for studying the evolutionary relatedness of species, or HIV-1 sequences in our cases, and provides a schematic overview of the ancestral

HIV-1 phenotype and genetic variation in vertical transmission

From the Microbiology and Tumorbiology Center, Karolinska Institutet, Stockholm, Sweden