1. HIV Pathogenesis and Natural Course of the Disease
HIV Care and ART: A Course for Physicians
Unit 4 should take approximately 1 hour and 30 minutes to complete
Step 1: Overview of Learning Objectives (Slides 1 – 2) – 5 minutes
Step 2: Virology and Immunology (Slides 3 – 23) – 40 minutes
Step 3: Pathogenesis and HIV Infection (Slides 24 – 43) – 40 minutes
Step 4: Key Points (Slides 44 – 45) – 5 minutes

2. Learning Objectives Describe the origin and basic virology of HIV-1
Describe the normal immunological response to HIV-1
List the mechanisms used by HIV-1 to evade the normal immune responses
Explain the principles of HIV-1 pathogenesis
Describe the natural course of HIV-1
Step 1: Overview of Learning Objectives (Slides 1 – 2) – 5 minutes

3. HIV-1 Virology
Step 2: Virology and Immunology (Slides 3 – 23) – 40 minutes

4. Transfer of SIV to Humans
“Natural transfer” theory (Science 2000)
SIV was transferred to humans through hunting and handling of chimpanzees
The epidemic required urbanization and increased population mobility
Most scientific-based theory
The “natural transfer” theory is widely acknowledged as the most plausible theory. While natural transfer of this virus has probably been occurring for thousands of years, the start and maintenance of the HIV epidemic required a lot of people interacting, and therefore urbanization and mobilization. In Africa, the start of the epidemic coincided with the emergence of these factors.
Source: Hahn et al. Science 2000; 287:

5. Transfer of SIV to Humans (2)
“Human error” theory (Edward Hooper,“The River” 2000)
Oral polio vaccine used in West Africa during the late 1950s may have been contaminated with SIV
SIV has not been recovered from this vaccine in subsequent studies
This theory is NOT widely accepted by the scientific community, but is presented here because it addresses an alternative theory sometimes seen in public press.
Source: Hooper E. “The River: A journey to the source of HIV and AIDS.” New York, NY; Little, Brown and Co, 2000

6. Spread of HIV in Africa, 1990-2005
This series of pictures shows how the prevalence of HIV changed according to time and location.
Source: UNAIDS report on the global AIDS pandemic. Available at
Source: UNAIDS, 2006

7. The HIV Epidemic Unfolds
Sudden outbreak in USA of opportunistic infections and cancers in homosexual men in 1981
Pneumocystis carinii pneumonia (PCP), Kaposi’s sarcoma, and non-Hodkins lymphoma
HIV isolated in Luc Montanier (Pasteur Institute, Paris) and Robert Gallo (NIH, Bethesda, USA)
HIV diagnostic tests developed in 1985
First antiretroviral drug, zidovudine, developed in 1986
Exploding pandemic
Has infected more than 50 million people around the world
Has killed over 22 million people
The epidemic was first recognized in major US cities (Los Angeles and New York) as an epidemic of severe opportunistic infections (OIs) in homosexual men in 1981.
HIV was isolated in France in 1984 by growing the virus in cell cultures. The isolation of the virus was challenging. Selective loss of T4 helper (CD4+) cells suggested a virus, but the causative agent was difficult to identify because it did not grow on resting T4 cells. Discovery depended on the ability to grow the virus in vitro which required the use of activated T4 cells.
Diagnostic tests which detected antibodies in the blood of infected patients were developed in in 1985.
UNAIDS/WHO, AIDS Epidemic Update: December 2000 (Joint United Nations Programme on HIV/AIDS, Geneva, Switzerland, 2000). Available online2.The World Health Report 2000 Health Systems: Improving Performance (World Health Organization, Geneva, Switzerland, 2000).

8. Classification of HIV HIV class: Lentivirus
Retrovirus: single stranded RNA transcribed to double stranded DNA by reverse transcriptase
Integrates into host genome
High potential for genetic diversity
Can lie dormant within a cell for many years, especially in resting (memory) CD4+ T4 lymphocytes
HIV type (distinguished genetically)
HIV-1 -> worldwide pandemic (current ~ 40 M people)
HIV-2 -> isolated in West Africa; causes AIDS much more slowly than HIV-1 but otherwise clinically similar
HIV belongs to the family of retroviruses which have an unusual life cycle that requires transcription of RNA to DNA - hence “retro” or backwards.
Two separate epidemics of HIV are recognized (HIV-1 and HIV-2). We will focus on HIV-1 which is the cause of the major epidemic throughout the world, but HIV-2 is also found in West Africa and some persons are infected with both viruses.

9. Classification of HIV-1
HIV-1 groups
M (major): cause of current worldwide epidemic
O (outlier) and N (Cameroon): rare HIV-1 groups that arose separately
HIV-1 M subgroups (clades)
>10 identified (named with letters A to K)
Descended from common HIV ancestor
One clade tends to dominate in a geographic region
Clades differ from each other genetically
Different clades have different clinical and biologic behavior
Of the three groups of HIV-1, M is the most common. The other two HIV-1 groups infect a small number of people in West Africa.
Clades differ from each other genetically: 30% in the env gene code sequences; 14% of the gag gene code sequences
Clade C, which is the most common clade in Ethiopia, causes a fast-progressing infection.

10. Origin and Distribution of HIV-1 Clades
HIV-1 rapidly evolves by two mechanisms:
Mutation: changes in single nucleosides of the RNA
Recombination: combinations of RNA sequences from two distinct HIV strains
Several common clades (e.g., A/G ad A/E) are recombinants
Geographic distribution of HIV group M clades
A in Central Africa
B in North American, Australia, and Europe
C in Southern and Eastern Africa (Ethiopia)
Clades have evolved by two mechanisms, mutation and recombination
Recombination occurs when long RNA segments from two distinct HIV strains are combined during reverse transcription in a human cell infected by two HIV strains. Recombinant clades (e.g., A/G ad A/E) are common.
Distinct clades are concentrated geographically. Ethiopians are infected mostly with clade C, which is common in Southern Africa.

11. The phenotype (or physical appearance/structure) of HIV

12. Steps in HIV replication are illustrated in the diagram.
A more detailed description of the steps are below:
Binding of gp120 to CD4 and co-receptor on the cell surface
Fusion of the viral envelope with the cell membrane
Release and disassembly of the viral core in the cytoplasm
Reverse transcription (Reverse transcriptase enzyme translates HIV’s single stranded RNA into a provirus made of double stranded DNA)
Viral DNA moves into cell nucleus
Viral DNA is integrated (by Integrase enzyme) into host genome to form HIV provirus
HIV provirus DNA is transcribed back to both viral genomic RNA and viral mRNA , the latter which is translated to HIV polyproteins.
The RNA virus and polyproteins are assembled beneath the cell membrane
The assembled package becomes enveloped in the host cell membrane as it buds off to form an HIV virion.
Further assembly and maturation occurs outside the cell by the protease enzyme, rendering the HIV virion infectious.

13. HIV at Surface of CD4 Lymphocyte
This transmission electron micrograph of an HIV virion on the surface of a CD4 lymphocyte shows both immature and mature virions.
Courtesy of CDC

14. How HIV Enters Cells gp120 env protein binds to CD4 molecule
CD4 found on T-cells macrophages, and microglial cells
Binding to CD4 is not sufficient for entry
V3 loop of gp120 env protein binds to co-receptor
CCR5 receptor - used by macrophage-tropic HIV variants
CXCR4 receptor - used by lymphocyte-tropic HIV variants
Binding of virus to cell surface results in fusion of viral envelope with cell membrane
Viral core is released into cell cytoplasm
Now a review of the lifecycle steps in more detail: For attachment and entry into the human cells, HIV use normal proteins on the surface of human cells.
HIV attaches to the CD4 cell protein. These receptors occur on CD4 cells, monocyte, and macrophages
As second step in entry, HIV binds to co-receptors, which are normally receptors for chemokines. A mutant CCR5 receptor gene that prevents the virus from binding to the cell has been discovered. Homozygosity for this mutant gene is strongly protective against HIV infection. Heterozygous people are not protected from infection but the disease may take longer to develop.

15. HIV and Cellular Receptors
HIV Receptors
HIV Receptors
HIV and Cellular Receptors
This picture shows HIV’s attachment and entry into a host cell. The gp-120 protein attaches to a CD4 receptor. The gp-41 is exposed for attachment to the host cell, and fusion of the cell membrane with the viral envelope starts.
Source: Levy JA. Infection by human immunodeficiency virus--CD4 is not enough. New Eng J Med, 335(20);
Copyright © 1996 Massachusetts Medical Society. All rights reserved.

16. Viral-host Dynamics About 1010 (10 billion) virions are produced daily
Average life-span of an HIV virion in plasma is ~6 hours
Average life-span of an HIV-infected CD4 lymphocytes is ~1.6 days
HIV can lie dormant within a cell for many years, especially in resting (memory) CD4 cells, unlike other retroviruses
The extremely high rates of viral replication results in every possible point mutation in the viral genome arising daily. In any given patient, the virus usually varies by 1-6% in the env gene, for example.

17. HIV Immunology

18. Overview of Adaptive Immune Response
Extracellular infection
APC
Intracellular infection
Free antigen
MHC I presentation of endogenous antigen
MHC II presentation of exogenous antigen
Naïve T8 cell
Naïve B-Cell
Naïve T4 helper cell
This is an important slide representing the adaptive immune response, which is the main response to HIV (as opposed to the innate immune response).
The adaptive immune response is divided into two types: the cell-mediated (cytotoxic t-cell) type and the humoral (antibody-mediated) type. In general, the location of the infection (intracellular or extracellular) determines the type of adaptive immune response.
Intracellular infections stimulate a cell-mediated response that will ultimately kill the infected cell. This is mediated by T8 cells, and utilizes the MHC I system.
Extracellular infections stimulate a humoral response that will help contain these free antigens. Some extracellular antigens will be picked up by APC and be presented by way of MHCII to the Thc, which will further differentiate into either TH1 or TH2.
TH1 in turn will augment the cell-mediated response and Th2 augments the humoral.
CENTRAL TO THE ADAPTIVE IMMUNE RESPONSE IS THE TH4 CELL. BECAUSE HIV DEPLETES AND DISTRUPTS THE FUNCTION OF THIS CELL, ADAPTIVE IMMUNITY IS IMPAIRED.
APC = antigen presenting cell
Humoral (plasma cells / antibodies)
Cell-mediated (CTLs)
Th1
Th2
Diagram courtesy of Dr. Samuel Anderson

19. General Principles of Viral-host Interactions:
Host: mounts HIV-specific immune responses
Cellular (cell-mediated) - most important
Humoral (antibody-mediated)
Virus: subverts the immune system
Infects CD4 cells that control normal immune responses
Integrates into host DNA
High rate of mutation
Hides in tissue not readily accessible to immune system
Induces a cytokine environment that the virus uses to its own replicative advantage
Achieved by “activation” of the immune system
While the host mounts an immune response, the virus employ mechanisms to evade the host’s response.

20. Cellular Immune Responses to HIV
CD8 Cytotoxic T lymphocyte (CTL)
Critical for containment of HIV
Derived from naïve T8 cells, which recognize viral antigens in context of MHC class I presentation
Directly destroy infected cell
Activity augmented by Th1 response
The next few slides will focus on the cellular immune response to HIV.

21. Cellular Immune Responses to HIV
CD4 Helper T Lymphocyte (Th)
Plays an important role in cell-mediated response
Recognizes viral antigens by an antigen presenting cell (APC)
Utilizes major histocompatibility complex (MHC) class II
Differentiated according to the type of “help”
Th1 - activate Tc (CD8) lymphocytes, promoting cell-mediated immunity
Th2 - activate B lymphocytes, promoting antibody mediated immunity
The CD4 Th cell is also involved in the cell-mediated response (in addition to humoral).
The Th1 response is mediated by certain interleukins such IL-2, interferon-gamma (IFN-gamma), and tumor necrosis factor-beta (TNF-beta)

22. Humoral Immune Response to HIV
Neutralization
Antibodies bind to surface of virus to prevent attachment to target cell
Antibody-dependent cell-mediated cytotoxicity (ADCC)
Fc portion of antibody binds to NK cell
Stimulates NK cell to destroy infected cell
Antibodies have many roles in HIV infection but overall they appear to be less effective in controlling HIV infection compared to cellular immunity. Neutralization and ADCC are two mechanisms by which antibodies can help contain HIV infection.
Antibodies made against proteins on surface of virus block attachment of the virus to the host cell receptor
Antibodies bind to infected cell, interact with NK cells, and indirectly affect destruction of the infected cell by stimulating the NK cell.

23. HIV Evasion Methods
Makes 10 billion copies/day -> rapid mutation of HIV antigens
Integrates into host DNA
Depletes CD4 lymphocytes
Down-regulation of MHC-I process
Impairs Th1 response of CD4 helper T lymphocyte
Infects cells in regions of the body where antibodies penetrate poorly, e.g., the central nervous system
So why doesn’t this immune response clear the infection?
The rapid rate of mutations enable the virus to escape immune recognition. Additionally, the virus integrates into the host’s DNA, where it can remain hidden in resting cells. Finally, the immune system is disrupted by HIV– especially the aspects important for controlling intracellular infection like HIV.
These reasons also contribute to the difficulty of HIV vaccine research.

24. Pathogenesis of HIV
Step 3: Pathogenesis and HIV Infection (Slides 24 – 43) – 40 minutes

25. Cells Infected by HIV Numerous organ systems are infected by HIV:
Brain: macrophages and glial cells
Lymph nodes and thymus: lymphocytes and dendritic cells
Blood, semen, vaginal fluids: macrophages
Bone marrow: lymphocytes
Skin: langerhans cells
Colon, duodenum, rectum: chromaffin cells
Lung: alveolar macriphages

26. General Mechanisms of HIV Pathogenesis
Direct injury
Nervous (encephalopathy and peripheral neuropathy)
Kidney (HIVAN = HIV-associated nephropathy)
Cardiac (HIV cardiomyopathy)
Endocrine (hypogonadism in both sexes)
GI tract (dysmotility and malabsorption)
Indirect injury
Opportunistic infections and tumors as a consequence of immunosuppression
Pathogenesis can be grouped by general mechanism of injury

27. General Principles of Immune Dysfunction in HIV
All elements of immune system are affected
Advanced stages of HIV are associated with substantial disruption of lymphoid tissue
Impaired ability to mount immune response to new antigen
Impaired ability to maintain memory responses
Loss of containment of HIV replication
Susceptibility to opportunistic infections
The effects of HIV on the human immune system are extensive and complex, resulting in both depletion and dysfunction of all elements of the immune system.

28. Mechanisms of CD4 Depletion and Dysfunction
Direct
Elimination of HIV-infected cells by virus-specific immune responses
Loss of plasma membrane integrity because of viral budding
Interference with cellular RNA processing
Indirect
Syncytium formation
Apoptosis
Autoimmunity

29. Syncytium Formation
Observed in HIV infection, most commonly in the brain
Uninfected cells may then bind to infected cells due to viral gp 120
This results in fusion of the cell membranes and subsequent syncytium formation.
These syncytium are highly unstable, and die quickly.
Viral gp 120 can be found on the surface of infected host cells after fusion of viral envelope and cell membrane, with retention of viral proteins at the cell surface.

30. Apoptosis Courtesy of CDC
Apoptosis (pronounced “apoh-toh-sis”) means programmed cell death. CD4 cells may undergo apoptosis in the presence of HIV infection when they cross-link with other CD4 molecules either by gp-120 alone or gp-120 in complex with anti-gp120 antibodies.
This cross linking provides the first of two signals required for apoptosis.
The second signal is the activation of the cell through the T-cell antigen receptor by either conventional antigen or superantigen.
Courtesy of CDC

31. Role of Cellular Activation in Pathogenesis of HIV
HIV induces immune activation
Which may seem paradoxical because HIV ultimately results in severe immunosuppression
Activated T-cells support HIV replication
Intercurrent infections are associated with transient increases in viremia
The magnitude of this increase correlates inversely with stage of HIV disease
Accounts for why TB worsens underlying HIV disease
Not only does the virus destroy and disrupt the immune system, the virus can manipulate the immune system to its own replicative advantage. This is achieved by immune activation. Clinically, this is demonstrated by the observation that viral load transiently increases in the presence of intercurrent illnesses, such as TB.

32. Role of Cytokine Dysregulation in Pathogenesis of HIV
HIV is associated with increased expression of pro-inflammatory cytokines
TNF-alpha, IL-1,IL-6, IL-10, IFN-gamma
Associated with up-regulation of HIV replication
HIV results in disruption and loss of immunoregulatory cytokines
IL-2, IL-12
Necessary for modulating effective cell-mediated immune responses (CTLs and NK cells)
The immune system activation (and disruption) by HIV is mediated by various cytokines

33. Consequence of Cell-mediated Immune Dysfunction
Inability to respond to intracellular infections and malignancy
Mycobacteria, Salmonella, Legionella
Leishmania, Toxoplama, Cryptosporidium, Microsporidium
PCP, Histoplamosis
HSV, VZV, JC virus, pox viruses
EBV-related lymphomas
Decline in immune status parallels the decline in CD4 number and function. Loss of these cells results in failure of normal Th1 response and cell-mediated immunity that is necessary for controlling intracellular infections.

34. Natural History of HIV Infection

35. Transmission Modes of infection Viral tropism
Sexual transmission at genital or colonic mucosa
Blood transfusion
Mother to infant
Accidental occupational exposure
Viral tropism
Transmitted viruses is usually macrophage-tropic
Typically utilizes the chemokine receptor CCR5 to gain cell entry
Patients homozygous for the CCR5 mutation are relatively resistant to transmission
Study of HIV ”exposed uninfected” individuals revealed the presence of a mutated CCR5 receptor in some people. Patients homozygous for this mutation are relatively resistant to the virus.

36. Early Phases of HIV Infection of Mucosal Surfaces
Cell free HIV
T-cell
Immature Dendritic cell
PEP
Skin or mucosa
Via lymphatics or circulation
Burst of HIV replication
24 hours
48 hours
HIV establishes infection across the skin or mucosal surfaces like the cervix or urethra within in 72 hours of its introduction.
This information suggests that PEP (post-exposure prophylaxis with antiretroviral drugs after high-risk blood or sexual contacts) should be immediate.
Source: I-TECH staff
HIV co-receptors, CD4 + chemokine receptor CC5
Mature Dendritic cell in regional LN undergoes a single replication, which transfers HIV to T-cell
Selective of macrophage-tropic HIV

37. Laboratory Markers of HIV Infection
Viral load
Marker of HIV replication rate
Number of HIV RNA copies/mm3 plasma
CD4 count
Marker of immunologic damage
Number of CD4 T-lymphocytes cells/mm3 plasma
Median CD4 count in HIV negative Ethiopians is significantly lower than that seen in Dutch controls
Female 762 cells/mm3 (IQR )
Male 684 cells/mm3 (IQR )
The average CD4 T cell count in HIV-1 uninfected Ethiopians reportedly ranged from 591 × 10*6 to 775 × 10*6 cells/L

38. Spread of HIV in Host Tissues
Early Events in Transmucosal HIV-1 Infection
The arrows indicate the path of the virus. The viral-envelope protein binds to the CD4 molecule on dendritic cells. Entry into the cells requires the presence of CCR5, a surface chemokine receptor. Dendritic cells, which express the viral co-receptors CD4 and CCR5, are selectively infected by R5 (macrophage-tropic) strains. Within two days after mucosal exposure, virus can be detected in lymph nodes. Within another three days, it can be cultured from plasma.
Source: Kahn and Walker. NEJM. 1998; 339:33-39
Copyright © 1998 Massachusetts Medical Society. All rights reserved.

39. Primary HIV Infection
The period immediately after infection characterized by high level of viremia (>1 million) for a duration of a few weeks
Associated with a transient fall in CD4
Nearly half of patients experience some mononucleosis-like symptoms (fever, rash, swollen lymph glands)
Primary infection resolves as body mounts HIV-specific adaptive immune response
Cell-mediated response (CTL) followed by humoral
Patient enters “clinical latency”
For about half of patients, in the period immediately after infection, virus titer rises (about 4 to 11 days after infection) and continues at a high level over a period of a few weeks. The patient often experiences some mononucleosis-like symptoms (fever, rash, swollen lymph glands) but none of this is life-threatening. Level of viremia associated with severity of symptoms, degree of initial CD4 depletion, viral set point during chronic infection.
By this time, infection is already established. Viral replication is already occurring in infected T-cells, T-cells are dying and latent virus is already hidden in the sanctuaries of the lymphoid organs, the central nervous system, and latent CD4 cells. Chronic and persistent infection has been established, and without intervention the patient will eventually die.

40. Window Period: Untreated Clinical Course
Acute HIV syndrome
antibody
Primary HIV infection
Asymptomatic
viremia
PCR
P24
ELISA a b
Time from a to b is the window period
The window period begins at the time of infection and can last 4 to 8 weeks.
During this period, a person is infected, infectious and viremic, with a high viral load and a negative HIV antibody test.
The point when the HIV antibody test becomes positive is called the point of seroconversion.
Source of graph: S Conway and J.G Bartlett, 2003 2 3 4
years
Weeks since infection
Source: S Conway and J.G Bartlett, 2003

41. Natural History of HIV-1
Acute (primary) retroviral syndrome is the initial event after infection, which is characterized by a rapid decline in CD4 cell count and high plasma viremia.
Development of cytotoxic T-cell (CTL) response results in clinical recovery of acute infection and a reduction in plasma viremia. The virus reaches “set point” as a result of this immune response. The viral load at this “set point” correlates with the rate of CD4 decline and disease progression. Overtime, HIV RNA levels gradually increase.
In parallel, the CD4 cell count gradually declines over several years, but rapidly drops 1.5 to 2 years before an AIDS-defining diagnosis.
When the CD4 count falls below 200, patients develop opportunistic infections, tumors, and neurological complications. The median survival after the CD4 count has fallen to <200 is 3.7 years, if untreated.
Source: Fauci AS, Pantaleo G, Stanley, Weissman D. Immunopathogenic mechanisms of HIV infection. Ann Intern Med 1996;124: Galens Curriculum, Module 8, p. 17.
Fauci As, 1996

42. HIV RNA Set Point Predicts Progression to AIDS
HIV RNA viral loads after infection can be used in the following ways:
To assess the viral set point
To predict the likelihood of progression to AIDS in the next 5 years
The higher the viral set point:
The more rapid the CD4 count fall
The more rapid the disease progression to AIDS
The rate of disease progression - is determined by the patient’s viral load.
With levels between 1,000 and 10,000 viral copies, the likelihood of AIDS in 5 years is 8%.
At 10,000 to approximately 50,000, the likelihood is 26%.
At 50,000 to <100,000 it is 49%.
Between 100,000 to 1,000,000 the likelihood is 62% at 5 years.

43. CD4 T-cell Count and Progression to AIDS
In contrast to VL, baseline CD4 is not a good predictor of time to progression to AIDS
Unless CD4<321 cells/ml
However, as the CD4 count declines over time, patients will develop opportunistic infections
Develop in a sequence predictable according to CD4 count
WHO Staging system

44. Key Points
HIV is a retrovirus, capable of integrating into host genome and establishing chronic infection
HIV can be classified into subgroups (clades) which have characteristic geographic distribution
The important steps in the lifecycle of HIV include cell entry, reverse transcription, integration, and maturation/assembly
Cell-mediated immunity is critical for containment of HIV infection and other intracellular infections
HIV evades host immunity by a variety of mechanisms
Step 4: Key Points (Slides 44 – 45) – 5 minutes

45. Key Points (2)
HIV activates the immune system to increase its own replication
CD4 count declines by both direct and indirect mechanisms
HIV RNA set point predicts rate of progression to AIDS
CD4 count decline is associated with a predictable sequence of opportunistic infections