Immunologic characteristics of healthy and HIV-1-infected Ethiopians - Chapter 1 General Introduction

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Immunologic characteristics of healthy and HIV-1-infected Ethiopians

Messele, T.

Publication date

2000

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Citation for published version (APA):

Messele, T. (2000). Immunologic characteristics of healthy and HIV-1-infected Ethiopians.

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HIV/AIDS S

Thee human immunodeficiency virus (HIV) which is the cause of acquired

immuno-defficiencyy syndrome (AIDS) was first isolated in 1983

"

. AIDS is the

ultimatee clinical stage of infection by HIV and is characterized by opportunistic

infectionss and specific malignant diseases in patients. HIV is an RNA virus and

belongss to the family retroviridae, subfamily lentiviridae*. HIV-1 and HIV-2 are

thee two known types of HIV with the former being distributed worldwide and the

latterr found primarily in West Africa

"

. HIV-2 was found to be closely related to

thee simian immunodeficiency virus (SIV)

. Although the degree of virulence

mayy be less for HIV-2, both HIV-1 and HIV-2 are associated with AIDS

'

.

Todayy most of the information on HIV has been obtained from studies on HIV-1.

Structurally,, HIV-1 is a spherical particle with a diameter of approximately

1000 nm. The outer membrane-protein of the virus covers the inner core, which

containss the viral RNA, along with several copies of the enzyme reverse

transcriptase

.. The envelope region of HIV-1 is highly variable. Based on the

variationn in the larger outer membrane protein, gp120, HIV-1 at present is

dividedd into 10 subtypes designated as subtypes A to J

. There is also a

heterologouss group of viruses designated as subtype O, which do not match

anyy of the subtypes described above

. The human CD4 molecule, which is a

55-kDD surface glycoprotein belonging to the immunoglobulin superfamilies,

servess as the primary receptor for the virus to enter susceptible cells

"

. CD4

iss expressed primarily on helper T lymphocytes and also on cells of the

monocyte/macrophagee lineage including dendritic cells, alveolar macrophages

inn the lung and Langerhans cells in the skin

"

. It is now known that the CD4

moleculee is necessary, but not sufficient for HIV entry into target cells.

Recently,, the chemokine receptors CCR5 and CXCR4 have been identified as

thee major co-receptors of HIV-1

"

. During infection, HIV first attaches to the

CD44 molecule and the chemokine receptors on the cell surface

. This step is

thenn followed by fusion of the viral and cellular membrane, resulting in the

penetrationn of the virus core. Uncoating is followed releasing the virus RNA

whichh is then reverse transcribed in to DNA by the viral reverse transcriptase.

Thee DNA provirus is then transported to the nucleus and is integrated to the

hostt cell genome with the help of virus-encoded enzyme, integrase

. The virus

dependss on host transcription and translation factors for its replication. The

synthesizedd virus genome and structural and regulatory proteins are

transportedd and assembled at the cell membrane and progeny viruses bud

fromm the cell membrane. A characteristic feature of HIV-1 infection is a highly

variablee period between infection and development of AIDS. Although at the

beginningg of the HIV epidemic it was thought that the viral load is low during the

asymptomaticc period, it is now known that a large number of viruses are

producedd per day throughout the infection period with continuous infection of

neww cells resulting in gradual CD4 depletion and ultimately development of

AIDS

. .

Thee global situation

Worldd wide, over 30 million people were infected with HIV by the

beginningg of 1998 and 11.7 million people had already lost their lives to the

disease.. HIV/ AIDS is among the top ten killers world wide

. Given the current

speedd of the spread of infection, it is expected that it may even move to be

amongg the top five killer diseases. Eighty nine percent of people with HiV live

inn sub-saharan Africa and the developing countries of Asia. In Africa, nearly 21

millionn men, women and children are infected and AIDS has become one of the

majorr causes of morbidity and mortality

.

Thee HIV problem in Ethiopia

Thee HIV/AIDS epidemic started relatively late in Ethiopia, the first

HIV-1-positivee sera being detected in 1984 and the first AIDS patients reported in

1986

.. However, since then the HIV-1/AIDS epidemic has spread to the entire

countryy to reach a prevalence ranging 7-20% in the 15-50 year age groups in

thee urban areas by 1998

. The circulating subtype is C , which has been

estimatedd to be the most prevalent (>48%) amongst HIV-1 -infected individuals

inn the world

.

HIVV infection and the immunological response

HIVV infection is characterized by dynamic and long-lasting interactions

betweenn the virus and the immune system. Both humorai and cellular immune

responsess are mounted to HIV infection.

Humorall immune response

Earlyy after infection, before seroconversion, there is high level viremia

andd as this decreases a substantial antibody response, including an early and

transientt IgM response and later a sustained IgG response specific for several

proteinss of the infecting virus is seen

. The antibodies are directed to all the

majorr antigens of the virus. As time goes on more broadly neutralizing

antibodiess are produced. However, these antibodies do not prevent HIV

diseasee progression and the generally accepted explanation for this

phenomenonn has been the occurrence of antibody-escape mutant viruses

partlyy because of the error-prone polymerase of the virus, which is thought to

generatee an average of one point mutation in each genome copy

and partly

Cell-mediatedd immune response

Cell-mediatedd immunity does play an important role in the immune

defensess against intracellular pathogens such as viruses. Cytotoxic T

lymphocytess (CTL) destroy infected cells after recognizing antigen presented by

majorr histocompatibility (MHC) class- molecule on the surface of such cells.

Highh frequencies of HIV-specific CTL have been detected in peripheral blood of

HIV-infectedd patients

. The activity of CTL has been found to decline as HIV

diseasee progresses

, and this has been also associated with impairment of

IL-22 production and a reduced clonogenic potential of CD8

T lymphocytes

.

AA correlation has been observed between generation of strong CTL response

andd persistence of an asymptomatic state in adults. Another study in infants

bornn to HIV-positive mothers showed that seroconversion of the infants is

associatedd with disappearance of HIV-specific CTL in peripheral blood

.

Specificc CTL responses, but not humoral immune responses are detected in

exposedd but uninfected individuals. Taken together, these observations

suggestt that CTL responses play a significant role in protection against infection

andd in the regulation of HIV disease. On the other hand, there are also a few

studiess which suggested a negative role of CTL on the immune system during

HIVV infection mediated by killing of infected cells and disruption of normal tissue

architecture

"

.. Furthermore a non-Iytic antiviral response which is active

againstt HIV-1, HIV-2 and SIV and which is not MHC-restricted was indicated in

CD8

T cells

. These cells are found at highest levels in asymptomatic

individualss and which decreases with disease progression

. The non-cytolytic

CD8

T cells anti-HlV response, termed CD8

T-cell anti-viral factor {CAF), was

initiallyy identified to be composed of p-chemokines

. However, subsequent

studiess have shown that the anti-viral response mediated by CAF can block

bothh SI and NSI viruses

"

and this effect cannot be suppressed by

antibodiess directed to p-chemokines suggesting that CAF's activity is not due

exclusivelyy to p-chemokines

"

.

Differentiatedd CD4 T-helper subsets, which are associated with distinct

typess of immune response, have been identified. These T-helper subsets

designatedd as T-helper 1(TH1) and T- helper 2 (TH2) were first described in

mouse

.. A third subset designated as T-helper 0 (THO) with both Th1 and Th2

propertiess was also described in humans. Tn1 cells produce cytokines such as

IFN-yy and IL-2 that increase cellular immune response, whereas Th2 cells

producee cytokines such as IL-4, IL-5, IL-6 and IL-10 that enhance antibody

production.. A change in the balance of production of especially IL-4 and IFN-y

hass been observed in several disease conditions

"

. There are also reports

suggestingg that a shift from Th1 to Th2 type of response contributes to the

immunee dysregulation observed in HIV infection and progression to AIDS is

dependentt on Th2 cytokine phenotype dominance

. In contrast, there is also

aa study arguing for the absence of an in-vivo Th1 to Th2 phenotype shift with

HIVV disease progression

.

Viro-immunologicall markers of HIV disease progression

Thee course of HIV infection and development of disease is characterized

byy a wide variation among infected individuals. Studies in the West indicated

thatt approximately 5% of HIV-infected persons develop AIDS within three years,

whereass approximately 12% of the HIV- infected persons are expected to

remainn AlDS-free for more than twenty years

"

. In contrast, HIV disease

progressionn in general is reported to be faster and the time of survival after the

onsett of AIDS is shorter in African patients

. Identification of factors which

aree changing with HIV infection and which predict and possibly contribute to the

outcomee of the infection and which could be useful in designing therapeutic

strategiess for appropriate patient care has been a major part of HIV

research-Severall cellular and serologic markers have been seen to have a strong

predictivee value for HIV disease progression. The level of HIV replication as

expressedd by viral loads is believed to be important in the immunologic decline

thatt characterize HIV disease and that have a major impact on the course of the

disease

.. Some studies have suggested that plasma HIV-1 RNA levels are

betterr predictors of progression to AIDS and patients with elevated plasma

HIV-RNAA levels are at high risk of poor outcome

. Consistent with those

findings,, it was reported that HIV disease progressors had significantly higher

HIV-- RNA copy numbers than did either slow progressors or long-term

asymptomaticc HIV-infected patients

. In contrast, a recent study by Rizzardini

eff al on HIV-infected Africans shows that viral load is lower in the Africans

comparedd to the Europeans, suggesting that HIV pathogenesis in this

populationn is mainly immunopahtologically driven

. The emergence of more

virulentt virus strains is also implicated to determine HIV disease

progression

.. These strains have syncytium-inducing (SI) capacity are

thoughtt to evolve from the non-syncytium-inducing (NSI) strains detected early

att infection. As the disease progresses SI viruses appear in approximately

50%% of patients. The appearance of SI viruses seems, however, to be rare in

HIV-11 subtype C infections and the majority of AIDS patients infected by these

genotypee harbor only NSI viruses

.

CD44 T cells are the main targets of HIV infection and their depletion is

thee hallmark of the deteriorating immune system

"

. The quantitative decline

off CD4

T cells expressed as an absolute number or percentage of total

lymphocytess is frequently used for staging of patients. Apart from progressive

declinee in the number of CD4

T cells, HIV infection is also associated with

changess in the representation of many different subset of T cells. Following

seroconversion,, the number of CD4

lymphocytes declines rapidly and less

rapidlyy thereafter, while the number of CD8

lymphocytes increases with similar

kinetics

.. However, within the CD8 population there are also subsets which

declinee in parallel with CD4

subsets. Furthermore, expansion of both CD4

andd CD8

T-cell memory subsets, loss of naive CD4

and CD8

T cells

,

increasee of CD4 and CD8 subsets expressing certain cell surface antigens,

especiallyy those reflecting immunologic activation have been implicated in

HIV-infectedd persons from studies in Europe and North America

"

. The

especiallyy on CD8

T cells increases dramatically with disease progression and

havee been shown to have a prognostic value for AIDS

. In addition to the

describedd quantitative changes, qualitative alterations of cells of the immune

systemm are observed in HIV infection. A functional impairment of T cells from

HIV-infectedd subjects can be detected early at infection. Loss or reduction of

T-cel!! proliferative capacity to in-vitro stimulation is one of these qualitative

changes

"

.. The loss of in-vitro recall antigen response is detected early at

infection

.. Also, it has been reported that T-cell proliferation in response to

stimulationn with CD3 and CD3

CD28 MoAbs decreases shortly after

seroconversion,, before the decline in CD4

T-cell number is observed

. The

responsee to mitogens, such as phytohaemaglutinin (PHA), remains unaffected

inn the early phases, but is significantly reduced later in infection

. It has also

beenn demonstrated that loss of T-cell reactivity to CD3 and CD3

CD28 MoAbs

inin vitro is a strong predictive marker for progression to AIDS, independent of

decliningg CD4 counts

. Thus, T-cell proliferative capacity not only has

beenn shown to be an important independent predictor of progression to HIV

disease,, but also has been used to monitor immunological improvement after

therapy

. .

Severall other potential factors, so called surrogate markers, including

inflammatoryy cytokines have also been suggested for use in monitoring disease

progressionn in HIV-infected patients. Neopterin, which is a metabolite of

guanosinee triphosphate is produced by macrophages when they are stimulated

byy interferon gamma from activated T cells

. The level of soluble

interleukin-22 receptor reflects the activation of T cells and that of R

-microglobulinn reflects lymphoid activation more generally

. The levels of B

-microglobulinn and neopterin are elevated in HIV infection and strongly

correlatedd with the risk of progression to AIDS

. Cytokines are integral

componentss of the immune response and their role in HIV disease progression

hass been extensively investigated

"

. Cytokines, such as IL-2 and IL-12, are

cruciall for cell-mediated immunity

, whereas it is well documented that

cytokines,, like tumor necrosis factor-a(TNF-a), upregulates HIV replication in

bothh T lymphocytes and monocytes/macro-phages via activation of cellular

transcriptionn factor

Tumor necrosis factor a (TNF-a) has two

specificc cell surface receptors and these two receptors, sTNFaRI and sTNFaRII

aree released from cells as a result of high level of TNF-a and are detectable in

solublee forms in body fluids

. Although sTNFaRII is not specific for HIV

infection,, serum levels of sTNFaRII are a strong predictor for disease

progressionn in asymptomatic HIV-positive persons

.

HIV-11 co-receptors and chemokines

Chemokinee receptors, which belong to a family of seven transmembrane

spanningg G-protein-coupled receptors also serve as co-receptors for HIV-1

entry

. It is now known that HIV-1 uses a number of chemokine receptors for

itss entry. The CC-chemokine receptor, CCR5, is used by macrophage-tropic

primaryy isolates, the viruses that predominate early in infection and are thought

too be important for transmission of HIV-1. The CXC-chemokine receptor,

CXCR4,, is used by T-cell-tropic or SI viruses that occur late during disease

progressionn to AIDS

. HIV can also use other chemokine receptors, CCR2b

andd CCR3, albeit to a lesser extent. Both CCR3 and CCR5 are expressed on

microgliaa and it is suggested that both receptors are involved in HIV infection of

thee central nervous system

. Bleul et al have shown that HIV co-receptors

aree differentially expressed on human T lymphocytes. CXCR4 is predominantly

expressedd on naïve T cells and CCR5 is mainly expressed on previously

activatedd memory cells

. The expression pattern of the HIV-1 co-receptors of

thee surface of CD4

T cells is believed to have an influence on susceptibility of

CD4

T cells to HIV-1 infection, viral tropism and rate of disease progression

.

AA study by Zhang et al indicated that CCR5 and CXCR4 serve as the major

co-receptorss for different HIV-1 subtypes and co-receptor usage is determined by

virall phenotype irrespective of viral genotype

. However, less frequent use of

CXCR44 is reported for subtype C virus

.

Thee natural ligands of the HIV-1 co-receptors are chemokines, which are

solublee factors thought to direct the migration of different leukocyte subsets to

sitess of inflammation

. They can be subdivided into two groups. The

a-chemokiness also known as CXC chemokines include SDF-1 and the

p-chemokiness or C-C-chemokines include RANTES, macrophage inhibitory

protein-1aa (MIP-1a) and MIP-1p. The CC-chemokines were shown to block

infectionn of susceptible cells into vitro by macrophage-tropic primary HIV

isolatess and SDF-1 was shown to inhibit T-cell-tropic viruses " .

I m m u n ee activation and HIV pathogenesis

Activationn of all components of the immune system is the major feature

off HIV infection

. The activated state of the immune system is reflected by

increasedd expression of antigens on cells which are otherwise expressed at a

reducedd level on resting cells and by increased level of soluble proteins which

aree released from activated cells

.

Severall in-vitro studies have established the role of cellular activation in

thee propagation of HIV infection in CD4 cells

. However, it seems

paradoxicall that on one hand immune activation is most probably involved in

thee immune control of HIV-1 infection

and, on the other hand, several lines

off evidence support that activation of immune cells may lead to enhanced HIV-1

replication

"

.. Activated CD4

lymphocytes are found to be more susceptible

too HIV infection compared to their resting state

and in-vitro activation of

latentlyy infected lymphocytes triggers active viral replication

, in vivo,

CCR55 is mainly expressed on memory or primed (CD45RO*) T cells

and

thesee cells are indicated to be selectively infected by HIV-1

. There is

evidencee that CCR5 expression on this subset of cells is associated with

HLA-DRR expression and increases with disease progression

.The strong

associationn observed between the decline of CD4

T cells and increased levels

off activation markers on CD4

T cells further support the view that cellular

activationn promotes HIV disease progression. Another study by Weissman et ai

demonstratedd that 100 times less virus is required to initiate HIV infection in cell

culturee from an individual after immunization than before immunization,

indicatingg the potential contribution of cellular activation associated with an

ongoingg antigen-specific immune response to the pathogenesis of HIV

disease

.. It is suggested that activation of CD4

T cells facilitates HIV

infectionn in a number of ways either by enhancing HIV entry into the cell,

triggeringg the completion of reverse transcription and viral integration or by

stimulatingg viral transcription from provira! DNA

. In light of these

observations,, chronic immune activation due to, among others, highly prevalent

parasiticc infections has been suggested to explain, at least in part, the reported

acceleratedd HIV disease progression in African patients

.

FinallyFinally there is evidence to support that there is persistent CD4

and

CD8

T-cell activation in HIV-infected individuals, which provides an optimal

environmentt for continuous HIV replication. CD4 and CD8 lymphocytes are

cruciall for the maintenance of appropriate immunological response against a

widee range of pathogens. In addition, CD4 cells secrete factors that affect the

growthh and differentiation of lymphoid cells and haematopoietic cells and the

functionn of non-lymphoid cells as well. Therefore, it is clear that quantitative or

qualitativee abnormalities of the CD4 and CD8 populations, as a resuit of HiV

diseasee and abnormal immune activation, can have profound effects on the

immunee system function.

HIVV infection susceptibility/resistance

Fromm the observation that some individuals remain uninfected despite

high-riskk exposure, it has been concluded that host factors exist that determine

susceptibilityy to HIV infection. Studies conducted in various population groups,

includingg commercial sex workers, discordant couples, infants born to

HIV-infectedd mothers and exposed health care workers, have described several host

factorss which possibly contribute to HIV infection resistance

"

.

CCR55 and CXCR4 are the main co-receptors used by HiV

.

Polymorphismm of the gene encoding CCR5 is one of the factors found to be

associatedd with resistance

. The A32 base pair deletion mutation in the CCR5

genee is shown to result in a premature stop codon and loss of HIV-1 co-receptor

activity.. However, some individuals with this mutation were found to be

HIV-infected,, indicating that the protection conferred by this mutation is not

absolute

.. In addition, HIV-1-infected individuals, heterozygous for the

A32bpp deletion mutation, have been shown to have slower rates of CD4

decline,, have lower viral loads and survive longer compared to individuals with

thee wild genotype

. Moreover, reduced cell surface expression of CCR5

hass been reported in individuals with heterozygous deletion compared to the

wildd genotype group

. The A32bp deletion is common in Caucasians but rare

inn Africans. Other mutations on the CCR5 than the A32bp deletion have also

beenn reported. A mutation in the promoter region of the CCR5, CCR5P1, has

beenn found to have a negative effect, in that infected individuals homozygous

forr this mutation progress rapidly to AIDS

. In contrast, polymorphism of

thee coding region of CCR2b has been suggested to be associated with a delay

inn disease progression but not with reduced transmission risk

. This

promotorr polymorphisms (CCR5-59653T). It is shown that CCR2-64I effect on

AIDSS progression is not mediated by a negative effect on the CCR5 co-receptor

function,, although a slightly reduced expression of CCR5 is reported in

individualss with CCR2-64) mutation

. Furthermore, a mutation on the 3

untranslatedd region of the SDF-1 chemokine gene (SDF1-3A) is indicated to be

associatedd with rapid disease progression

. No polymorphism is reported

forr CXCR4, which is mainly expressed on naive cells and is used by SI viruses.

Thee role of the cell surface expression levels of the co-receptors on

cellularr susceptibility and tropism of the virus is also implicated . A correlation

betweenn low expression of CCR5 and reduced infectability of T cells in vitro is

alsoo reported

'

.

In-vitroIn-vitro studies have shown that the CC-chemokines RANTES, MIP-1a

andd MIP-1p can block the entry of macrophage-tropic viruses into susceptible

cells

.. This inhibition is thought to be mediated by blocking env-driven HIV

fusionn through competition for the chemokine receptors or receptor

downregulation.. In relation to this, high level of chemokines was detected in

exposedd but uninfected individuals

. Other host genetic factors have also

beenn implicated to play a role and increased frequencies of certain HLA alleles

weree detected in exposed but uninfected individuals

. Furthermore, acquired

protectivee immunity after exposure to HIV was also suggested to play a role in

HIV-11 infection resistance. HIV-1-specific cytotoxic T lymphocytes are detected

inn exposed health workers, commercial sex workers and was indicated as one

off the mechanisms of natural protective immunity

. From studies on

commerciall sex workers in Kenya and Thailand mucosal immunity was also

shownn to be highly associated with HIV-infection resistance

. HlV-specific

mucosall IgA, in the absence of systemic IgG response, was detected in

mucosall sites of high proportions of HIV-1 exposed but uninfected women.

Thiss response was found rarely in HIV-infected women it's involvement in

mediatingg protection against HIV infection. Recently, another study by Mazzoli

etet ai reported HlV-specific IgA in the serum of exposed seronegative partners of

HIV-seropositivee persons, implying the involvement of not only mucosal but also

systemicc IgA-mediated immunity to HIV

.

E N A R PP a n d scope of this thesis

Thee Ethio-Netherlands AIDS Research Project (ENARP) is a

collaborativee project between the governments of Ethiopia and the Netherlands.

Itt started in 1994 with three main objectives:

i.. training of Ethiopian scientists

ii.. capacity development and

iii.conductingg research on HIV.

Thee project is based at the Ethiopian Health and Nutrition Research Institute

(EHNRl)) in Addis Ababa and is supported by three research groups in

Amsterdam,, the Netherlands: 1) The Division of Public Health and Environment,

Municipall Health Service (Prof Roel Coutinho, Epidemiology); 2) Department of

Viro-lmmunolgyy at CLB (Prof Frank Miedema, Immunology) and 3) The

Departmentt of Human Retrovirology, Acadamic Medical Center {Prof Jaap

Goudsmit,, Virology). The project has established a well-equipped laboratory.

ENARPP has also started a cohort study on HIV infection progression in two

factories,, Akaki and Wonji, 20 km and 100 km away from Addis Ababa,

respectively. .

Researchh for this thesis was conducted as part of the immunology research

programm of ENARP.

Scopee of this thesis

HIVV infection is associated with several changes of the immune system.

Inn chapter 2 CD4 and CD8 values as well as haematological parameters are

comparedd between Ethiopian and other populations. Since the base line values

off some of these parameters in Ethiopians were different in proportions and

numberss compared to Dutch and other published values for Africans,

immunohaematologicall reference ranges established for adult Ethiopians is

shownn also in chapter 2. The representation of several T-cell subsets are

studiedd in HIV-negative Ethiopians and compared to HIV-negative Dutch

subjectss in Chapter 3. Furthermore, HIV- associated changes in these subsets

aree analyzed in Ethiopians who are HIV-negative, HIV- positive with and without

AIDSS in chapter 3. Chapter 4 levels of soluble viro-immunological markers in

HIV-infectedd and -non-infected Ethiopians are presented. In addition, the

prognosticc value of these markers for HIV disease progression in Ethiopians is

discussed. .

AA study on the existence of possible host factors associated to HIV

infectionn resistance in high-risk HIV-1-negative Ethiopian commercial sex

workerss is presented in chapter 5. Chapter 6 shows the expression levels of

co-receptorss in different CD4 and CD8 T-cell subsets of HIV-infected and

-non-infectedd Ethiopian commercial sex workers. The co-receptor expression is

correlatedd to a polymorphism in the CCR2b gene. In chapter 7, the work

includedd in this thesis is discussed in relation to current literature.

References s

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