Basics of Immunology - Lecture Course by M. V. Skok - Kyiv 2002

Chapter III. Immune Processes at the Whole Organism Level

Lecture 15. Acquired Immunodeficiency Syndrome (AIDS)

In the early 1980s, a disease was identified and classified as an acute secondary immunodeficiency. It was characterized by a generalized decline in all immune Functions, leading to death from common infections, most frequently Pneumonia. Concurrently, neurological disorders and a malignant condition, Kaposi's Sarcoma, were quite often observed.

A risk group for this disease was established, comprising drug users and homosexuals. It was subsequently discovered that AIDS is endemic in several African countries (such as Zaire and Uganda), where up to 90% of the population is infected.

The route by which this infection was introduced into America and Europe was debated for quite some time (there was even Structure/133.html">Discussion about the artificial creation of this virus as a biological weapon). It is most apparent that it was brought from African countries by migrants who joined the ranks of drug users and homosexuals.

As the circle of infected individuals widened, it became evident that the virus could also be transmitted through standard sexual contact, as well as via Blood, for instance, during organ transplantations and surgical Procedures.

The Nature of the pathogen causing AIDS was elucidated quite rapidly. Research laboratories headed by R. Gallo (USA) and L. Montagnier (France) demonstrated, almost simultaneously, that the causative agent is an RNA retrovirus related to other previously known immunodeficiency Viruses. It was named the HUMAN IMMUNODEFICIENCY VIRUS (HIV). Similar to other Retroviruses, upon entering a Cell, it utilizes Reverse Transcriptase to convert its RNA into cDNA, which then integrates into The Genome of the infected cell. METABOLISM/31.html">Transcription of this DNA leads to The production of new Viral Particles that bud off from The surface of the infected cell and infect new targets. It was shown that the virus infects CD4⁺ T Cells (i.e., helper T cells) and thereby severely compromises overall immune status. The viral surface protein gp120, responsible for binding to T cells, was identified fairly quickly. Following the Discovery of the virus's nature, hopes arose for the rapid eradication of the disease through (1) anti-retroviral therapy and (2) The Development of a vaccine. However, nearly 20 years have passed, and it is still premature to speak of victory over AIDS, despite the dedicated efforts of the world's leading laboratories past and present. During this time, a great deal has been learned about the virus itself, and this knowledge makes it clear why it is so difficult to overcome this infection and prevent it through vaccination.

It turned out that the disease features a very long asymptomatic period: months or even years pass from the time of infection to the appearance of symptoms. Initially, it was believed that the virus remained in a latent state during this period and was somehow activated later. However, it was established that the Immune Response to the virus manifests very rapidly: Antibodies to HIV can be detected in the blood of infected individuals as early as 10–12 days post-infection. On this basis, immunodiagnostic assays were developed and subsequently used to test both individuals and donor blood intended for transplantation. The ability to diagnose the disease played a major role in halting (or slowing down) the spread of the infection (by discarding infected blood units). Coupled with public awareness campaigns regarding transmission routes, these measures successfully reduced The rate of new infections in the USA. Unfortunately, infection rates continued and continue to rise in countries of Africa, Southeast Asia, and Eastern Europe. Sexual contact and The Use of non-sterile instruments (including syringes) became the primary routes of transmission. Today, there are over 30 million infected individuals worldwide, 90% of whom live in the developing world. In 1997, approximately 600,000 children acquired HIV from their mothers at birth. The main Treatment modality is anti-retroviral therapy. However, its efficacy is limited due to the specifics of viral metabolism within the body (see below). Furthermore, anti-viral drugs are quite expensive and not accessible to everyone. Crucially, they are unavailable to patients in developing countries—precisely where the prevalence of infection is highest.

How the virus enters The Cell.

HIV enters the cell with the help of its envelope protein. It is synthesized as a precursor, gp160, which is glycosylated in target cells and cleaved by cellular convertase into two subunits: gp120 (surface) and gp41 (transmembrane). The subunits remain non-covalently linked and oligomerize into trimers on the virion surface. Binding to the cell is mediated by gp120, whereupon gp41 undergoes conformational changes and mediates virus-Cell Fusion. gp41 features a so-called "fusion peptide" at its N-terminus, which is hydrophobic and Glycine-rich. This is followed by two hydrophobic repeats characteristic of coiled-coil Proteins. Upon binding of gp120 to the cell surface, the fusion Peptides, previously concealed within the interior of the molecule, are exposed and insert into the membrane of the target cell. This intermediate structure then transforms such that six helices of the trimer pierce the membrane like a hairpin. They form a pore through which the viral and cellular membranes fuse. This mechanism resembles that of Influenza and murine leukemia viruses.

In the mid-1980s, it was established that HIV interacts with CD4 On the surface of T helper cells. It was later discovered that CD4 is expressed not only on T helpers, but also on monocytes, macrophages, and dendritic cells.

Further studies revealed the existence of two viral strains differing in their cell tropism: the first infected activated and transformed T cells, while the second infected macrophages and resting T helpers. The difference between these two strains lay in The sequence of the V₃ domain of the gp120 protein.

These studies made it clear that the virus recognizes something else on the surface of infected cells In addition to CD4. Such molecules were successfully identified and proved to be chemokine receptors:

CXCR4 — expressed on activated and transformed T lymphocytes,

CCR5 — expressed on macrophages and naive T lymphocytes.

Accordingly, the viral strains were designated X4 and R5 based on their tropism for these cells. There are also dual-tropic viruses, designated R5X4.

The gp120 protein directly interacts with the CXCR4 and CCR5 receptors. Binding to CD4 induces Conformational Changes in gp120 and increases its affinity for chemokine receptors.

Thus, the chemokine receptor serves as the primary receptor that links the viral envelope protein to the cell. CD4 is necessary to concentrate the virus on the cell surface and facilitate interaction with the chemokine receptor; in other words, it functions essentially as a coreceptor for the chemokine receptor.

CXCR4 and CCR5 receptors are seven-transmembrane domain proteins (serpentine receptors) coupled to G proteins. It is now known that several chemokine receptors can serve as receptors for simian and human immunodeficiency viruses: 5 major and 4 minor ones. In addition to T cells and monocytes, they are expressed on eosinophils, microglial cells, rectal cells, and placental cells. Viral entry into the cell is mediated by G protein activity, and the transmitted signal resembles chemokine signaling (increased Ca²⁺ concentration, involvement of Tyrosine Kinases). Chemokines that normally bind to these receptors prevent viral binding (by competing with it). Interestingly, during the Cytology/cytology/16.html">Early stages of infection, the attack is initiated by the R5 virus, whereas viral strains with X4 or R5X4 tropism appear later—indicating an expansion of viral tropism.

A homozygous deletion in the CCR5 Gene, found in 1% of Caucasians, renders them resistant to HIV.

The expression of HIV-susceptible chemokine receptors on microglial cells explains the frequency of neurological disorders accompanying AIDS, while their expression in the rectum helps elucidate the route of infection in homosexuals.

Accessory proteins.

For Replication within an infected cell, HIV utilizes accessory proteins. In addition to the products of the gag, pol, and env genes utilized by other known retroviruses, HIV expresses genes for auxiliary small proteins: tat, rev, nef, vif, vpr, vpu. Each of these gene products performs specific functions that enhance the efficiency of viral replication. For instance, Nef releases the Lck tyrosine kinase bound to CD4 and instead recruits a protein kinase to The Plasma Membrane that phosphorylates proteins at Serine and Threonine residues. Thus, Nef directly affects signal Transduction from the T-cell receptor. Vpu interacts with specific residues on the cytoplasmic tail of CD4 while it is still in The Endoplasmic reticulum. The simultaneous synthesis of gp160 and CD4 in infected cells can lead to their binding even before reaching the membrane. Vpu binds CD4 molecules and targets them for degradation. This allows gp120 to exit the Endoplasmic reticulum and become incorporated into HIV virions.

How the virus behaves upon entering the cell and where it persists.

The virus can persist within cells:

1) as integrated into cellular DNA (activated T cells, macrophages, memory cells);

2) as unintegrated into cellular DNA (resting T cells);

3) as virions presented on follicular dendritic cells within the germinal centers of Lymphoid Organs.

Viral replication occurs exclusively in activated cells. Consequently, memory cells, resting T cells, and macrophages serve as viral reservoirs where the virus remains latent and undetectable.

In infected individuals, the proportion of activated cells increases in response to infection, and it is precisely within these cells that the virus actively replicates.

Thus, the spread of infection is facilitated by the activation of the immune response itself. The virus selectively destroys T cells activated in response to it; in effect, clonal deletion of virus-specific cells takes place. T cells activated by other Antigens are destroyed in the same manner, leading to a state of immunodeficiency.

The persistence of the virus in reservoirs in an inactive state explained the limited efficacy of antiretroviral therapy. The use of drug cocktails that interfere with viral replication (azacitidine, azathymidine) combined with a viral protease inhibitor successfully prolonged the asymptomatic period, increased CD4⁺ cell counts, and alleviated patients' conditions. However, these drugs did not eradicate non-replicating virus and rapidly selected for resistant strains. Furthermore, whenever patients experienced an increase in CD4⁺ cell counts, the virus began to replicate efficiently within these very cells.

It follows from the above that the prolonged asymptomatic phase of AIDS is a state of equilibrium between viral production and its clearance by The Immune System. An active response to the virus leads to immune system exhaustion and the loss of immune control. During this time, the virus persists in cellular reservoirs—chiefly memory cells—where it is invulnerable to antiretroviral therapy because it is not replicating.

Why the immune response to HIV declines.

The half-life of viral particles is less than 6 hours. During primary infection,

very high plasma viral loads are initially observed—~10⁶ RNA copies/mL. As the immune response develops, these levels may drop to 10 copies/mL and remain stable throughout the asymptomatic period. The rate of disease progression is proportional to the viral load in the blood.

Thus, a certain equilibrium between viral production and clearance is established and maintained during the asymptomatic stage. This indicates that the immune system remains capable of controlling the infection at this time.

Infected individuals initially exhibit extremely high levels of virus-specific CD8⁺ CTLs (up to 10% of all peripheral CD8⁺ cells). These appear earlier than antibodies, but subsequently decline sharply. This decline is driven by features of viral metabolism:

1) persistence in cells inaccessible to CTLs (e.g., microglia protected by the blood-Brain barrier);

2) persistence in reservoirs without active replication;

3) downregulation of MHC Class I expression in infected cells;

4) Upregulation of FasL expression on infected cells: upon contact with Fas⁺ CTLs, the latter undergo apoptosis;

5) deletion of CD4⁺ Th cells leads to a failure in activating new CTL precursors.

As a result, existing CTLs become exhausted without being replenished from precursors.

Challenges in developing an HIV vaccine.

The identification of the gp120 protein as key to viral binding to the target cell raised hopes for the rapid development of an AIDS vaccine. An epitope was quickly discovered against which antibodies supposedly neutralized the virus and protected animals from HIV infection. However, for humans, a vaccine based on monomeric gp120 proved ineffective: it induced low levels of antibodies that failed to neutralize the virus. It turned out that antibodies raised against gp120 peptides recognized different epitopes than antibodies isolated from patients (linear rather than conformational) and failed to account for the glycosylation of native viral gp120. Moreover, antibodies generated in individuals infected with live virus, when passively transferred to other individuals, also failed to confer Immunity or reduce viral titers in the infected hosts. This meant that the antibodies produced in response to the virus caused it no harm. It was demonstrated that they do not recognize the native virus; it is believed they are elicited not by live virions, but by their degradation products.

Consequently, the task facing vaccine developers proved exceptionally difficult: to elicit the production of antibodies more effective than those generated during a live viral infection. Currently, only 3 Monoclonal Antibodies capable of neutralizing the virus are known: two of them bind different epitopes on gp120, while the third recognizes a region of gp41. The task is further complicated by the fact that laboratory-adapted strains of the virus differ from wild-type strains, with laboratory strains being more sensitive to neutralization.

Thus, despite major advances in understanding the pathways of viral Metabolism in the Body, an effective HIV vaccine has not yet been created. Instead, alternative approaches to preventing infection have been proposed.

1) The use of soluble CD4 — prevents the interaction of gp120 with CD4⁺ cells.

2) Administration of chemokines that block the interaction of gp120 with the chemokine receptor and downregulate its surface expression on the cell; short positively charged peptides behave similarly;

3) Administration of gp41 peptides: they interact with The cell membrane and prevent the binding of viral gp41;

4) Use of antibodies against all elements of virus-cell interaction that hinder The formation of the fusogenic complex.

Understandably, these Methods are effective as prophylactics and act for a relatively short duration after administration, unlike Vaccines, which are capable of eliciting long-term protection. Therefore, the development of a vaccine against SIV remains one of the most critical challenges in combating this disease. Together with the improvement of antiretroviral therapy, this offers hope for overcoming one of the most severe Diseases of the 20th century.