1. University of Sheffield School of medicine and Biomedical Sciences.
HIV symposium
University of Sheffield School of medicine and Biomedical Sciences.

2. Aims of the HIV symposium:
Describe the basic features of the HIV-1 virus and its effect on the immune system.
Outline how we diagnose HIV and the ethical issues involved.
Discuss some of the major clinical features of HIV.
Review the global impact of HIV.

3. The Basic Science of Human Immunodeficiency Viruses
David H. Dockrell
University of Sheffield.

4. By the end of this lecture, you should be able to :
Understand the composition of the HIV genome.
Review viral life-cycle.
Understand theories on the origin of HIV-1 and HIV-2.
Understand the main effects of HIV on the immune system.

5. HIV-1 and HIV-2
Are 2 closely related retroviruses belonging to the lentivirus subfamily
Retroviruses are enveloped viruses.
Capable of reverse transcription. Viral RNA is copied into DNA which is incorporated into the host cell to allow gene transcription.
Other human retroviruses include HTLV-1 and -2.

6. HIV genome and proteins encoded.

7. Genomic structure
LTR
gag
pol
env
nef
vif
vpr
tat
rev
vpu
HIV proviral DNA
9kB RNA genome encodes 9 genes; Gag, Pol, Env, Tat, Rev, Nef, Vif, Vpr and Vpu.
Gag, Pol, Env, Tat, Rev essential for infectivity.

8. HIV proteins-early
Tat contributes to viral replication. Enhances production of host transcription factors e.g NF-kB.
Rev binds to viral RNA and allows export from nucleus and also regulates RNA splicing.
Nef is non-essential but increases infectivity

9. HIV proteins-late
Gag encodes structural proteins. Made as a polyprotein and cleaved by HIV protease.
Pol encodes the enzymes: reverse transcriptase, integrase and polymerase.
Env encodes the envelope proteins.

10. HIV proteins- Vif
Host restriction factors aim to block viral replication
APOBEC3G : during reverse transcription first strand DNA is subject to hypermutation and degradation.viral vif mediates its degradation

11. HIV-1 Structure
gp120
gp41
p17
p24 RT
Viral RNA

12. HIV life-cycle

13. Simplified replication strategy of HIV
Retrovirus
Membrane fusion
Integration
Uncoating
CD4
Co-receptor
gp120
DNA duplex,
made by RT
INT
Mature virus contains
host membrane components
as well as HIV-specified
proteins
Nucleus
Genomic and mRNA
made by cellular
RNA polymerase

14. Replication of HIV HIV replication consists of 9 steps: Attachment
Entry
Uncoating
Reverse transcription (error prone so genomic variability)
Genome integration
Transcription of viral RNA
Splicing of mRNA and translation into proteins
Assembly of new virions
Budding

15. SIVcpz from chimpanzees in wild is closely related to HIV-1
Santiago ML et al. Science 2002: 295; 465

16. HIV origins
HIV-1 arose from transmission of SIV cpz from chimpanzees to humans sometime pre-1950.
HIV-2 arose from SIV sm

17. HIV types M (main), O (outlying) and N (new) groups.
Main group separated into clades A-D, F-H, J-K.
Clade B predominates in Europe, USA.
Clade A in West and Central Africa.
Clade C in Southern Africa.
Some subtypes don’t seem to circulate on their own but as part of a mosaic virus due to genetic recombination eg. A/E virus in SE Asia.

18. HIV and the Immune System

19. HIV receptors.
HIV infects cells that express CD4. The interaction between CD4 and gp120 is conserved among all primate lentiviruses.
Binding of gp120 to CD4 induces a conformational change in gp120..
The co-receptor binding site includes a conserved bridging sheet and also amino acids in the V3 loop (influence cell tropism and co-receptor usage).
The co-receptors are chemokine receptors, CCR5 for M-tropic (R5 strains) strains and CXCR4 for T-tropic strins which can predominate in some individuals.
Mutations in the CCR5 gene can influence risk of infection.

20. ZFN-modified autologous CD4 T-cells
Two domains
Nuclease domain of Fokl restriction enzyme
Engineered zinc finger domain provides specific DNA binding to 24bp
Cleaves as a heterodimer with a 5-6bp gap
Delivered in an adenoviral vector to transiently express ZFN

21. SB-728-T: Engraftment of ZFN modified CCR5 disrupted autologous CD4 T-cells in HIV aviremic individuals on HAART

22. Cell tropism
CD4 T cells. Memory CD45RO+ cells infected preferentially early on. Naïve CD45RA cells can be infected later on in infection by X4 virus.
Macrophages infected by HIV.
Controversial if dendritic cells infected on early on but can trap virus via DC-SIGN and transport virus to lymph nodes to infect T-cells.
Occasionally other cells such Brain microvascular endothelial cells, CD34+ bone marrow progenitors, astrocytes and renal epithelial cells can be infected.

23. HIV Immunology
Despite vigorous humoral and cell-mediated responses clear correlates of protective immunity have not been observed.
The lack of identification of protective immune responses remains a major barrier to development of an effective vaccine against HIV-1.
Animal models such as SIV infection of rhesus macaques have identified both immune responses which help limit viral replication and also HIV proteins which induce these responses.
HIV is adept at escaping the selective pressure imposed by the immune system by immune evasion.

24. Problems in developing an immune response to HIV-1
Neutralising antibodies against primary isolates are of low magnitude.
The envelope glycoprotein is poorly immuogenic and shows genetic diversity.
MHC class I-restricted CTL are observed within peripheral blood within a few months of infection and persist during chronic phase, play a role in early decline in virus but are incomplete. Virus escapes from CTL responses through mutations. CTL responses are quantitatively and qualitatively poor
Unlike most viral infections HIV infection results in a paucity of virus specific CD4+ T-lymphocyte responses.
Failure of CD4+ T-lymphocyte proliferation.

25. Long term non-progressors
Heterogenous group. Give clues to elements of an effective immune response.
No symptoms of infection or signs of AIDS after at least 7 years infection with CD4 count >600 cells/ml in the absence of treatment.
Genetic factors: e.g. CCR5 D32.
Host immune responses e.g. Vigorous CTL responses.
Viral mutants Dnef and Vpr R77Q.

26. Adapted from Fauci, A. S. et. al. Ann Intern Med 1996;124:654-663
Long term non-progressor
1200
1000
400
200
800
600
Asymptomatic
Primary infection
Constitutional symptoms
Bacterial pneumonia
AIDS
Death
CD4 count 12 1 10
wks
years
Adapted from Fauci, A. S. et. al. Ann Intern Med 1996;124:

27. Reservoirs of HIV replication
Despite effective suppression of HIV with treatment most individuals have ongoing replication.
Sanctuary sites; Genital tract, Central nervous system, gastrointestinal system, bone marrow.
Particular cells e.g. macrophages, microglia.
Resting T-cells such as CD4+ CD45RO+ memory cells only support replication when activated.

28. Immune dysfunction associated with HIV (CD4+ T-lymphocytes )
Excessive and inappropriate activation
Decreased proliferation with antigens or mitogens.
Impaired production of IL-2 and IL-2R.
Preferential involvement of memory T- cells.
Skewing of CD4+T-cell receptor Vb repertoire.
Progressive decline in number and function of CD4 T-lymphocytes characterises HIV infection and leads to susceptibility to infection.

29. Potential echanisms of CD4+ T-lymphocyte depletion
Direct cytotoxicity of directly infected cells-however only 1:1, ,000 cells in peripherary directly infected.
Potential mechanisms: impaired cell homeostasis, syncytia formation, apoptosis and immune-mediated.
Bystander cell killing.
Apoptosis; gp120 binding to CD4 sensitising cells to apoptosis. Fas Ligand upregulation by tat.

30. Mechanisms of CD4+ T-lymphocyte depletion
Decreased production.
Infection of CD34+ progenitors in bone marrow.
Infection of Thymocyte progenitors and disruption of thymic microenvironment.
Redistribution.
Significant trafficking of CD4+ T-cells from peripherary to lymphoid tissue.

31. Other aspects of immune dysfunction.
CD8+ T-cells show enhanced activation and decreased cytolytic and non-cytolytic function.
B-cells show enhanced activation and decreased proliferation resulting in increased non-specific but decreased specific Ab production.
Decreased NK, neutrophil and Macrophage function.
Perturbed cytokine networks: ↓Th1 responses, ↑Th2/Th0.

32. Overview of immune response to HIV
Vigorous immune response but no demonstrable protective immunity with rare exceptions.
Excessive immune activation which favours viral replication.
Immunological dysfunction with involvement of all elements of host defence.
Ongoing viral replication with progressive immunological impairment leading to clinical manifestations of immunodeficiency.

33. Adapted from Fauci, A. S. et. al. Ann Intern Med 1996;124:654-663
CD4 count
Log viral Load
Time
Adapted from Fauci, A. S. et. al. Ann Intern Med 1996;124:

34. The pathogenesis of AIDS
HIV is a chronic disease that leads to progressive CD4 depletion.
This ultimately leads to increased immunosuppression which after a long latent period results in increased infections and other complications.
The major factor in CD4 depletion is the amount of virus in the blood.
This is influenced by viral and host factors.
When the individual has a CD4 count < 200 cells per mm3 they are at risk of opportunistic infections and said to have AIDS.
The immune system is unable to clear the infection due to the plasticity of the viral genome and viral mechanisms of immune evasion

35. Summary
HIV-1 is retrovirus that evolved from a simian immunodeficiency virus in chimpanzees.
It replicates in CD4 positive cells.
The virus copies its RNA into DNA and uses the host cell for gene transcription.
It results in gradual damage to the immune system mainly through depletion of CD4 T-cells.