FUNDAMENTALS OF HIV

HIV virology, testing andmonitoringTemi Lampejo

Deenan Pillay

AbstractHuman immunodeficiency virus (HIV), the cause of AIDS, is widely

accepted as a cross-species infection descended from monkey simian im-

munodeficiency virus (SIV). Although originating in Africa, this virus has

spread to cause a worldwide pandemic and, along the way, has evolved

a number of mechanisms that enable HIV to evade host immune re-

sponses. It is hoped that vaccine developments may in the future provide

a means to effective immune control. Despite increasing global awareness

of HIV, in approximately 20e25% of individuals in the UK and USA the

infection remains undiagnosed, with higher observed rates in regions of

Asia and sub-Saharan Africa. As diagnostic methods continue to evolve,

the onus lies on medical practitioners to promote and facilitate increased

use of currently available tests. The most recent immunoassay technology

includes combined antigen (p24) and antibody techniques (4th genera-

tion tests) and rapid, portable point-of-care tests, which provide the op-

portunity for earlier diagnosis, initiation of therapy and reduced

transmission of HIV. Molecular techniques (detection of viral RNA or pro-

viral DNA) are vital in the diagnosis of early infection and vertical trans-

mission in addition to HIV monitoring and detection of drug resistance.

Along with current genotypic and phenotypic drug resistance assays,

readily available web-based systems aid in the clinical interpretation of

detected drug resistance mutations, and should ultimately facilitate

choice of therapy.

Keywords CD4; HIV transmission; resistance assay; tropism; viral

replication

Introduction

A number of controversial theories regarding the origins of the

human immunodeficiency virus (HIV) have been described. It is

now clear that it originates from simian immunodeficiency virus

(SIV), a virus that infects monkeys. SIV shares many genomic,

phylogenetic, and epidemiological characteristics with HIV,

strongly supporting the idea of cross-species transmission.1 The

precise origins of the pandemic HIV strain, HIV-1, have been

difficult to define but it is now generally accepted that HIV-1

derives from chimpanzee SIV (SIVcpz). Other SIVs have also

Temi Lampejo MBBS BSc(Hons) is a Virology Registrar at University College

London Hospitals NHS Foundation Trust, UK. Competing interests: none

declared.

Deenan Pillay MBBS PhD FRCPath is Professor of Virology, UCL, and Head

of Department of Infection, UCL, UK. Competing interests: none

declared.

MEDICINE 41:8 420

crossed species to man, the second most common being HIV-2,

which derives from SIVsm, a strain of SIV found to affect a

West African monkey known as the sooty mangabey.

Life cycle

The primary targets of HIV-1 are T lymphocytes and macro-

phages, including macroglial cells located within the central

nervous system. Through initial engagement of host cell re-

ceptors, a complex cascade of processes is set into motion that

ultimately results in the generation of further infectious virus

particles. The process by which HIV-1 replicates within a host

cell is illustrated in Figure 1.

Structure of HIV

The outermost structure of the spherical HIV-1 particle is the

host-cell derived envelope, which accommodates the trans-

membrane envelope proteins gp120 and gp41,2 appearing as

spikes, up to 72 in number, projecting outwards from the virion

(Figure 2). The gp120 protein, which binds the CD4 receptor, is

highly immunogenic and represents the target for most host

antibodies.3 These mostly strain-specific antibodies occupy the

CD4 binding site on gp120 blocking their interaction.3 Some

recently recognized broader-acting gp120 antibodies have very

high potency; they are considered a natural template for effective

antibody ‘design’.4,5

Lying beneath the outer lipid bilayer is the matrix, primarily

consisting of the Gag protein p17 (a cleavage product of the viral

Gag protein). The central area of the HIV-1 virion is occupied by

the cone-shaped core (or capsid), which consists of a shell of p24

protein (also a product of the Gag gene), and a core structure

consisting of two single strands of RNA and a third Gag

protein, p7.

Tropism

Cell entry requires binding to a co-receptor; for HIV-1 there are

two co-receptors, CCR5 or CXCR4. The viral specificity is deter-

mined by the V-3 loop of the gp120 molecule.6 Most HIV variants

use CCR5; a smaller proportion use CXCR4 and some use either

(dual tropic). Within an HIV-infected individual, strains of both

tropisms may co-exist. Advanced disease is associated with an

increased prevalence of CXCR4 tropic strains. CCR5 antagonists

such as maraviroc prevent interaction between gp120 and the

CCR5 co-receptor. They are therefore active against CCR5-tropic

strains but not against exclusively CXCR4 variants. Hence, HIV-1

tropism is routinely tested prior to starting these agents. Tropism

can be determined either by phenotypic assays with recombinant

viruses or by genotypic (sequence-based) methods.7

Within-host and between-host evolution

Evolution of HIV-1 occurs at such a rapid rate that individually

infected people have been shown to harbour genetically distinct

strains.8 Isolation of similar HIV-1 nucleotide sequences from

different individuals suggests they share a common source of

infection. However, the viral genome can change in chronic

HIV-1 infection by up to 1% per year: if an individual transmits

HIV several years after acquiring the infection, the transmitted

strain is likely to be distinctly different from the primary variant.8

� 2013 Elsevier Ltd. All rights reserved.

HIV-1 replication cycle AttachmentBinding to the CD4 receptor and one of the co-receptors (CXCR4 or CCR5)Entry inhibitors

FusionFusion of the virus with the host cell membrane

UncoatingUncoating of the viral genomic RNA

Reverse transcriptionViral RNA is converted to DNA by the enzyme reverse transcriptaseNucleoside and non-nucleoside reverse transcriptase inhibitors

Nuclear import The pre-integration complex is transported into the nucleus

IntegrationThe enzyme integrase catalyses the integration of proviral DNA into the host genomeIntegrase inhibitors

TranscriptionProviral DNA is transcribed to yield messenger RNA

Nuclear exportExport of viral messenger RNA from the nucleus

TranslationViral structural proteins are synthesized

AssemblyAssociation with the matrix protein Gag and assembly of the virus particle

BuddingThe immature virus particle buds from the membrane of the infected cell

ReleaseThe virus particle is released from the host cell

MaturationThe protease enzyme mediates maturation to an infectious virus particleProtease inhibitors

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13

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88

7

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5

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4

4

3

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2

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1

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Gag

Nucleus

Classes of antiretroviral agents blocking a particular stage of the cycle are shown in red.

CD4 CCR5CXCR4

Figure 1

Structure of the HIV-1 virion particle

p24 capsidprotein

Gag p17 matrix protein

RNA

gp120

gp41

Lipid membrane

Reverse transcriptase

Figure 2

FUNDAMENTALS OF HIV

MEDICINE 41:8 421

Phylogenetics is the study of the evolutionary descent of different

strains through nucleic acid sequence analysis. It has been

useful in identifying the sources of transmission in outbreaks

and in demonstrating routes of geographical spread, but its utility

in uncovering specific inter-individual transmission remains

limited.8

HIV diagnosis

HIV testing has significant benefits both for the individual and for

public health. Establishing an early diagnosis enables prompt to

access to care and treatment, resulting in improved patient

outcome. Testing is also important for the purposes of disease

prevention and reduction of transmission rates within a popu-

lation. Early screening tests for HIV diagnosis, based on immu-

noassays for immunoglobulin G (IgG) antibodies to HIV, detected

HIV antibodies only about 45e60 days after infection. The cur-

rent standard-of-care tests (fourth-generation assays) can detect

IgM and IgG antibodies against both HIV-1 and HIV-2 (which

usually appear within 20e30 days of infection9) as well as the

viral antigen p24. Viral p24 antigen can be detected approxi-

mately 5e7 days before the appearance of antibodies thereby

reducing the ‘window period’ between infection and a positive

test result (Figure 3).

A patient being tested for HIV should have an initial combined

HIV antigen/antibody screening test. If positive or indeterminate,

subsequent tests are performed for confirmation and also to

� 2013 Elsevier Ltd. All rights reserved.

080604020

HIV IgM Ab

HIV IgG AbHIV p24 Ag

HIV RNA

4th generationAg/Ab assay

3rd generationAb assay

2nd generationAb assay

1st generationAb assay

Days post-infection

Detectable markers of early HIV infection and the relative sensitivities of first-, second-, third- and fourth-generation immunoassays used in HIV diagnosis

Ab, antibody; Ag, antigen; HIV, human immunodeficiency virus; Ig, immunoglobulin.

Figure 3

FUNDAMENTALS OF HIV

differentiate HIV-1 from HIV-2. The patient is then asked to

provide a second blood sample in order to confirm the diagnosis.

Additional testing for viral RNA and/or proviral DNA may be

required in specific situations including:

� indeterminate antigen/antibody test results

� suspected HIV seroconversion

� high-risk exposure to HIV

� neonatal HIV testing.

Near-patient tests, also referred to as point-of-care tests

(POCTs), are now widely available. The benefits of these simple

and rapid screening tests are most evident in settings where

almost immediate results are needed to help clinical decision-

making (such as following an exposure incident), in the com-

munity without direct access to laboratory services, and in

circumstances where an individual declines a test or is unlikely

to return for their results.6 A result is available within 1e30

minutes of obtaining a capillary-blood fingerprick or mouth-swab

sample. Establishing POCT in a particular setting requires quality

assurance measures and accessible accredited laboratory testing.

Despite the commercial availability of point-of-care assays able

to detect HIV antibody and p24 antigen and to differentiate be-

tween HIV-1 and HIV-2, they remain less sensitive and specific

than laboratory screening tests. All patients with positive and

indeterminate POCT results and those suspected of acute HIV

infection therefore require laboratory testing to confirm HIV

status.

HIV monitoring

The best way to monitor the rate of viral replication is to

measure the number of virions in blood, the viral load. Viral

load predicts the long-term risk of disease progression.10 In

untreated patients, it guides the timing and choice of

MEDICINE 41:8 422

combination antiretroviral therapy (cART). In treated in-

dividuals, it is a marker of treatment efficacy and indicates po-

tential drug resistance or adherence issues. The primary tool for

measuring HIV RNA in disease monitoring is quantitative

reverse transcription-polymerase chain reaction (RT-PCR).

Follow-up monitoring should use the same laboratory assay.

Viral load assays are calibrated for blood, but can be adapted for

use in other body fluids such as CSF. However, there is currently

insufficient evidence to suggest that this contributes usefully to

ongoing monitoring in HIV.6

All patients with newly diagnosed infection should have a

baseline (pre-treatment) HIV viral load. Early or primary HIV

infection is characterized by high rates of viral replication and

correspondingly high levels of HIV RNA. Immune responses

subsequently suppress the virus and a decline in viraemia occurs

after a period of 4e6 months. An equilibrium is eventually

established between viral replication and the host immune sys-

tem, reaching a pseudo-steady-state with viral loads that gradu-

ally increase over time without therapy. The rate of disease

progression in the absence of therapy can be predicted by this

steady-state viral load. Clinically stable patients not having an-

tiretroviral therapy should have HIV viral load measurements

every 6 months.6 A minority of HIV-infected individuals control

viral replication spontaneously in the absence of ART at low (HIV

RNA <2000 copies/ml; ‘viraemic controllers’) or undetectable

(<50 copies/ml; ‘elite controllers’) viral loads.11 Elite and vir-

aemic controllers have heightened immunoregulatory responses

to HIV and slower rates of CD4 count decline, resulting in

reduced rates of disease progression and improved clinical

outcome.11 In patients who progress to advanced disease, a

dramatic rise in viral load occurs and correlates with clinical

complications such as opportunistic infections.

� 2013 Elsevier Ltd. All rights reserved.

FUNDAMENTALS OF HIV

Monitoring therapy

Antiretroviral therapy aims to achieve suppression of HIV RNA

below the limit of quantification. The widely used limit, as

determined by current commercially used PCR assays, has been

50 copies/ml. Novel assays are available providing lower limits

of quantification (as low as 20 copies/ml) but there is insufficient

evidence to support reliable interpretation of results at such low

levels. After commencing ART, viral load should be measured at

3- to 6-monthly intervals (depending on clinical status, adverse

effects and adherence) to confirm full HIV suppression below 50

copies/ml, which is the goal of therapy. The viral load serves as a

key marker for predicting survival and sustained HIV RNA sup-

pression is associated with a slower progression to AIDS.12

A rebound in viral load (greater than 50 copies/ml) may

indicate treatment failure. A confirmed rebound (through testing

of a second sample) should prompt further evaluation of the

patient’s clinical condition and relevant markers including CD4

counts. However, a rebound in viral load usually occurs before

clinical deterioration or falling CD4 counts and serves as an early

indicator of possible failure of therapy.

HIV resistance

Drug resistance is a major cause of treatment failure and disease

progression in HIV. The high rate of HIV-1 replication coupled

with the marked infidelity of HIV-1 reverse transcriptase results

in the generation of a vast array of mutations. In the presence of a

drug-selection pressure, mutations will be selected that enable

continued replication.

Primary resistance

‘Primary resistance’ occurs when an individual becomes infected

with a drug-resistant strain of HIV transmitted from another in-

dividual. Currently, in the UK, 11% of treatment-naı̈ve in-

dividuals demonstrate the presence of at least one mutation

conferring resistance to antiretroviral therapy.13 For this reason,

resistance testing should always be performed before starting

treatment to guide selection of a regimen likely to be successful.

This should be performed on a sample as close to the time of

diagnosis as possible (even if treatment is not yet indicated) to

maximize the likelihood of detecting drug resistance mutations,

as wild-type virus is likely to outgrow resistant strains in a newly

infected untreated host.

Secondary resistance

‘Secondary resistance’ occurs when viral loads rebound despite

continuing treatment. Usually this results from periods of poor

adherence; active replication in the presence of suboptimal drug

concentrations selects for resistant strains, which then come to

predominate. This is usually an indication to switch treatment, as

continuing therapy is likely to result in further resistance muta-

tions and a more extensively resistant virus, which will be more

difficult to treat. Antiretroviral drugs have a differing ‘genetic

barrier to resistance’ e the number of mutations required to

enable virus to replicate in the presence of that drug. Drugs with

a higher barrier are more robust in patients with poor adherence.

In this setting, resistance assays can guide choice of second-line

treatment. If there has been a period without ART in patients with

secondary resistance, reversion to the wild-type virus (with no

MEDICINE 41:8 423

resistance mutations) occurs within weeks following cessation of

antiretroviral therapy, as wild-type strains re-emerge from cells

harbouring thevirus.14Hence, resistance tests are best performedon

samples taken whilst the patient is still taking the failing regimen.

Viral resistance tests are either phenotypic, where viruses

are grown in drug-containing media, or genotypic, based on

interpretation of the RNA sequence. Genotypic assays are used

most widely, as they are simpler, cheaper and provide faster

results (1e2 weeks) compared to phenotypic testing (2e3

weeks). However, standard genotypic techniques underesti-

mate drug resistance mutation prevalence, as only 20% of

minority species (variants present at very low titres) are

detected. These minority variants, although present at low

level, may still be of clinical significance. Despite their limita-

tions, both genotypic and the less commonly used phenotypic

assays are predictive of virological response to antiretroviral

therapy. A number of website-based algorithms are available

for interpretation of sequence-based resistance tests.7,15e17 A

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