First BM Part 2 Principles of Pathology Masanori Watanabe

Human Immunodeficiency Virus

Introduction

- As of 2010 app. 34 million people have contracted HIV.

- Cases of newly infected people currently app. 2 million a year.
- Beginning of 1990s, there were only five drugs available for treatment of viral infections.
- Now there are over 7 times the number.
- Many new drug developments have been through continued research into the mechanisms
by which HIV infects and replicates in host cells.
- Incredibly fast evolution of HIV genome allows it to rapidly escape from immune and
therapeutic pressure.
- 2 forms of HIV:
o HIV-1 more virulent than HIV-2.
o HIV-1 further subdivided into groups according to polymorphisms in gag and env
genes:
 O group (outliers).
 M group (majority).
 Contains 10 Clades (strains) which differ in their epidemiology.
 50% of all HIV-1 in the world is Clade C – prominent in W. Africa, the
horn of Africa, and India (slightly more transmissible than other
clades).
 Clade B common in N. and S. America, and Europe.
 N group (non-O, non-M).

Discovery

- Barré-Sinoussi and Montagnier (Nobel Prize 2008):

o First isolated and characterised HIV.
o Cultured lymph node cells of patients with AIDS.
o Observed a retrovirus of the lentivirus group (bullet/coffin-shaped nucleus).
o Detected reverse transcriptase activity in cultured CD4+ lymphocytes.
o Electron microscopy identified retroviral particles budding from infected T cells.
o Caused the fusion of T cells and macrophages, followed by death (cytopathic).

HIV structure

- Envelope virus, buds out from host cell plasma membrane.

- 2 morphological forms:
o Immature (as virus is budding out).
o Mature (after fission event) – conical-shaped core, infectious.
- 2 positive single strand RNAs associated with a variety of core proteins.
- Matrix:
o Composed of an association of viral protein p17.
o Surround the capsid.

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- Capsid:
o Capsid protein P24.
o Protects replicative cassette.
o Responsible for conical-shaped core.
o Contains protease, reverse transcriptase, and integrase.
- Viral surface:
o Lipid envelope (derived from host cell when immature virion buds out).
o Contains surface glycoproteins GP120 and GP41 (trimeric configuration).
o Avirulent strains can be produced by knockout of either gene.

HIV genome

- Only has 9 genes.

- Grouped into 3 functional classes:
o Structural, regulatory, and accessory genes.
- Structural genes:
o gag:
 Encodes structural capsid proteins.
 p17, p9, p24.
o pol:
 Encodes enzymes required for replication and integration of viral RNA and
maturation of budding virions.
 Reverse transcriptase, protease, integrase.
o env:
 Encodes for surface glycoproteins.
 gp120, gp41.
- Regulatory genes:
o Facilitates HIV gene expression.
o Tat:
 Transactivator of HIV gene expression.
o Rev:
 Prevents the splicing of viral mRNAs as they are exported out of the nucleus.
- Accessory genes:
o Encode proteins allowing HIV to replicate in host cells whilst evading the immune
response.
o Vpu:
 Downregulates expression of CD4 via ubiquitin-proteasome axis.
 Renders naïve CD4 T cells unresponsive to antigen presentation via MHC
Class II.
o Vpr:
 Halts cells in G2.
 Facilitates transport of viral mRNA export out of nucleus.
o Vif:
 Similarly an essential component of viral replication within host cells.
o Nef:

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 Important immune evasion protein.

 Targets MHC Class I and CD4 for lysosomal degradation.
 Impairs ability of host cell ability to signal to immune cells that it is infected.
Knockout of some of these genes renders the virus avirulent, highlighting their
importance.

HIV life cycle

- Like all viruses, HIV is an obligate intracellular parasite.
- Uses the transcriptional and translational machinery of host CD4+ cells.
- Generates new viral proteins and ultimately gives rise to new virus particles.
- HIV life-cycle:

- Host cells expressing surface antigen CD4 are susceptible to infection: monocytes, dendritic
cells, memory CD4 T cells, naïve CD4 T cells.
- Entry mediated by interactions between gp41, gp120, and host cell surface molecules:
o gp120 attaches to CD4 receptors on host cell surface.
 Non-contiguous segments brought together to form 3D binding sites for
CD4).
o Causes conformational change to gp41.
 N-peptide translocates and inserts into cell membrane – ‘pre-hairpin
intermediate’.
o Further conformational changes occur, allowing the viral and cellular membranes to
be brought into close proximity for membrane fusion.
 CD4 alone is insufficient to allow entry of HIV-1 into cell, co-receptor
required.
 CCR5:

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o ‘macrophage-tropic’, non-syncytium-inducing HIV-1 (R5

viruses).
o CCR5-Δ32:
 Mutation found in app. 5% of people of N. European
descent.
 Protects against HIV R5 infection.
 Significant as R5 is the more transmissible form of
HIV-1.
 Deletion of 32 base pairs renders the CCR5 non-
functional.
 CXCR4:
o ‘T-cell-line-tropic’, syncytium-inducing HIV-1 (X4 viruses).
 In app. 50% of HIV-infected individuals:
o Observed that virus initially utilises CCR5 as a co-receptor,
then converts to using both, before finally using only CXCR4.
o Possibly an example of selective evolution based on loss of
target cells.
o After killing memory T cells, the virus must infect naïve T
cells which express CXCR4.
 Importance of co-receptors:
o Certain chemotactic cytokines (MIP-1α, MIP-1β, RANTES)
induce a dose-dependent inhibition of replication of some
strains of HIV.
o These utilise CCR5 as a receptor, and block the secondary
binding site for gp120, preventing infection.
- After the core is uncoated, viral RNA enters cytosol and viral RT catalyses the conversion of
viral RNA into double-stranded complementary DNA.
o Uses lysine-tRNA as a primer.
o RT lacks proof-reading activity – significant when considering the likelihood of
spontaneous errors and the implications this has for vaccine design and drug
resistance.
o Essential in life cycle of all retroviruses – one of the most active targets for drug
development.
- cDNA targeted to nucleus by nuclear-localisation signals contained in p17, Vpr, and Vif.
- In the nucleus, integrase inserts viral cDNA into host genome.
- In quiescent cells e.g. resting T cells, the rate of transcription is low and therefore viral
protein production will be low (and undetectable by the MHC Class I system).
- In active cells, transcription, nuclear export, and translation generate viral proteins which
are assembled at the membrane.
- New virions bud off, taking lipids from the host membrane as their envelope.
- Protease is responsible for generating mature viral proteins – cleaves pre-proteins into
functional products.

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How does HIV cause disease?

- In order to cause an infection, the virus must first cross an epithelial barrier e.g. the
urothelium.
- The virus infects tissue-resident CD4+ cells e.g. macrophages, dendritic cells, and replicates
within them, creating a small focus of infection.
o Evidence for the role of tissue-resident immune cells in spreading HIV:
 Comparison of HIV prevalence in circumcised and non-circumcised men.
 HIV prevalence is greater among non-circumcised group, possibly because
the foreskin contains large populations of dendritic cells.
- Infected cells then spread to local lymph nodes where they encounter CD4+ T cells.
- The formation of an immunological synapse between DCs and T cells may mediate direct
cell-cell spread of virions.
o Process can be imaged by tagging virion particles with fluorescent dye.
- From this point the infection can disseminate throughout the body.
- Circulation of lymph delivers infected cells to the venous circulation via the thoracic duct.
- This gives the virus access to the brain, lung, liver, spleen, gut, and other organs.
- Pathogenesis – two major mechanisms: (1) depletion of CD4+ T cells, (2) immune activation
o Depletion of CD4+ T cells:
 Impairs helper T cell response and therefore impaired neutralising antibody
response.
 Renders the host susceptible to infection from other pathogens.
 One potential mechanism for HIV killing cells:
 Through viroporin Lentiviral Lytic Peptide 1 (LLP-1).
 LLP-1 proposed to cause cell lysis by perforating the membrane and
causing the cell to undergo osmolysis.
 Experimental evidence – in vitro infection of cells:
o Leads to a characteristic increase in cell volume – ‘balloon
degeneration’.
o Causes an increase in permeability of cells to small
molecules and ions.
o Causes an increase in monovalent ion concentration in cells
(as measured by fluorescent ion dyes).
o However, it may not accurately represent in vivo
cytopathicity.
o Immune activation:
 A phenomenon whereby HIV causes immune dysfunction and subsequent
vulnerability to infection by over-stimulating the immune response.
 Consequences:
 Decreased half-life of circulating lymphocytes.
 Exhaustion of memory T cell pools.
 Fibrosis of lymph nodes (resulting in impaired lymphocyte
circulation).

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 Increased CD4+ cell expression of surface molecules (further

enhancing viral entry).
 Deleterious to fending off HIV infection and opportunistic pathogens.
 Caused by an interaction between several factors, but it is clear that the
damage to the gut mucosa and direct stimulation of IFN-secreting
plasmacytoid DCs by HIV RNA important.
 Damage to gut mucosa:
 Occurs as a result of excessive inflammation initiated by infection of
CD4+ cells in Peyer’s patches.
 Lymphocytes in GALT are highly active:
o Due to constant exposure to gut flora.
o Constitutively active subclinically.
o Therefore a very important site for HIV replication.
 Cells here express a special integrin (α4β7) which enhances ability of
virus to enter cells.
 Allows commensal bacteria to infiltrate through to the blood.
o Circulating levels of LPS are detectably higher in HIV-
infected individuals.
o Usually results in systemic immune activation.
 Evasion:
 HIV able to escape recognition by cytotoxic T lymphocytes.
 Due to error-prone RT – large number of mutations rapidly acquired.
 As waves of CTLs directed against new mutants are evaded, the cell
population depletes and the immune response fails.
- Over time, contribute to progression from acute infection to AIDS.
- Clinical latency ends app. 8 years after initial infection when the immune response finally
fails.

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Acquired immunodeficiency syndrome:

- First reported in 1981 in 5 N. American gay men.

- Very low CD4+ T cells (< 200/µL), high CD8+ T cell count (inverted ratio).
- Lymphadenopathy:
o Persistent, swollen lymph nodes.
o Wasting, fever.
- Opportunistic infections:
o Pneumocystis pneumonia:
 Caused by Pneumocystis jirovecii.
 Attacks interstitial, fibrous tissue of lungs.
 Marked thickening of alveolar septa and alveoli.
 Leads to hypoxia.
 Symptoms – fever, non-productive cough, shortness of breath, weight loss.
 First clue of existence of AIDS in early 1980s – physician began requesting
large quantities of pentamidine (rarely used antibiotic).
- Viral-induced sarcomas:
o Kaposi’s sarcoma:
 Caused by human herpesvirus-8.
 Cancerous tumour of connective tissue.
 Develops quicker in AIDS patients compared to cases seen before AIDS
epidemic.
 Mainly occurs in elderly Italian and Jewish men.

Anti-retroviral therapy

- Discovery of HIV has allowed for the rapid dissection of the viral replication cycle.
- The ability of HIV to generate drug-resistance to azidothymidine (AZT), the first RT inhibitor
to reach the market in 1987, suggested that therapy would require a combination of agents
with different targets involved in viral replication.
- Generally there are two types of targets:
o Cellular targets – afford drugs with a broader activity spectrum and less chance of
resistance, but greater toxicity.
o Viral proteins – include more specific, less toxic compounds with a greater chance of
resistance developing.

HAART (highly active anti-retroviral therapy)

- Principal method for preventing immune deterioration and concomitant opportunistic

infections.
- Protocol typically involves two NRTIs with either one NNRTI or one or two PIs.
- Resistance testing before deciding regimen allow it to be tailored specifically to them and
minimise the chance of resistance.
- Patient survival is greatly prolonged, although the virus is never fully eradicated.

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- Many drugs are inducers of cytochrome P450 enzymes and quickly metabolised by the liver,
must be taken frequently.
o One solution – ritonavir as an adjunct:
 Inhibits the cytochrome responsible for degradation, cyp3A4.
- Complexity of regimens makes compliance and drug interactions potentially problematic.

Stem cell transplantation

- A possible future treatment.

- Discovered in 2007 that transplant of stem cells carrying the CCR5-Δ32/Δ32 mutation to a
leukaemic patient rendered the virus undetectable and the person ‘cured’.

Vaccine

- Ultimate goal is an effective HIV vaccine.

- Vaccines induce two types of immune effector mechanisms:
o Antibody production.
o Activation of cytotoxic CD8+ T lymphocytes.
- Problems:
o Infidelity of RT:
 Produces app. 0.3 base changers per round of replication. As app. 1010 new
virions are produced daily, the drift caused by this facilitates multiple rounds
of neutralisation-escape.
o Glycan shield model:
 Proposed by Wei et al. which suggests that steric hindrance by bound
glycans on gp120 prevents neutralising antibodies from binding and provide
a ‘cloak of invisibility’. Furthermore, glycans are added by host cell
machinery and so we are immunologically tolerant. Experimental removal of
specific glycan sites on gp120 can dramatically increase viral sensitivity to
neutralising antibodies, offering hope for a protective vaccine therapy.
o Steric barriers:
 The CD4 binding site is exposed and relatively invariant due to its critical role
in virus-cell attachment, so is an attractive target for vaccine research.
However, the binding site has an awkward angle of approach which results
in steric clashes when antibodies attempt to bind, explaining why
neutralising CD4-binding site-specific antibodies are so rare.
- These barriers have precipitated in failed vaccines:
o VaxGen and STEP trials.
- However the RV144 Thai trial (2010) resulted in successful protection of 31% of volunteers.
o Prime and boost style vaccine suggested that antibodies are the major effector of
vaccine-mediated protection, because there was no change in viral load observed
and therefore no effective CTL response.
- Hessell et al., 2009:
o Infused monkeys with neutralising monoclonal antibodies and found that they
acquired immunity against mucosal infection with SIV, corroborating the idea that
short-lived antibody response is protective.

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First BM Part 2
Drug
Nucleoside RT inhibitors
Non-nucleoside RT inhibitors
Protease inhibitors
Fusion inhibitors
Co-receptor inhibitors
Integrase inhibitors
Principles of Pathology Masanori Watanabe
Example Mechanism Problems
Zidovudine Large group of nucleoside analogues. Mitochondrial γ-DNA polymerase is fairly
Abacavir Phosphorylated by host cell enzymes to sensitive.
give 5’-triphosphate derivative. Therapeutic response wanes with long
Competes with equivalent host cellular use.
triphosphate substrates for pro-viral DNA
synthesis.
Incorporation results in chain termination.
Mammalian α-DNA polymerase relatively
resistant.
Nevirapine Chemically diverse compounds which bind Monotherapy rapidly generates resistant
Efavirenz RT near catalytic site and denature it. strains.
Etravirine
Saquinavir Binds to cleavage sites of gag and gag-pol Minimum side effects.
Nelfinavir precursor polyproteins from which
Indinavir important structural and functional
Ritonavir proteins are derived.
Blocks the action of viral protease.
Prevents maturation of virus, renders it
avirulent.
Useful target as this protease does not
occur in hosts.
Enfuvirtide Synthetic gp41-derived peptides. Minimum side effects.
Interferes with gp41-mediated fusion, thus Given by subcutaneous injection in
blocking viral entry. combination with other drugs particularly
Broad activity against X4, R5, and dual- when resistance becomes a problem.
tropic variants. Expensive.
Maraviroc Numerous small molecular weight CCR5 Many are still in clinical trials (vicriviroc is a
and CXCR4 antagonists. promising future candidate).
Bind co-receptors and prevent fusion. Host cell receptor targeted – many
potential side effects.
Raltegravir Inhibit integrase, enzyme catalysing 3’- Developing resistance.
Elvitegravir processing and strand transfer of viral
cDNA.
Prevent integration into host genome.
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