Immunopathogenesis of HIV Infection

Written by cypher. Posted Saturday, January 17, 2009 @ 08:04:17 by Webmaster

INTRODUCTION

AIDS was recognized in the United States in 1981, when scientists at the Centers for Disease Control and Prevention (CDC) noted a cluster of cases of Pneumocystis carinii pneumonia and Kaposi’s sarcoma in homosexual men in New York City and Los Angeles.

HIV was isolated in 1983 and was demonstrated to be the agent of AIDS in 1984.

HIV is a retrovirus of the lentivirus family, which !include!s the human lymphotropic viruses (human T-cell lymphoma virus [HTLV]-I and II) and HIV-1 and -2.

HIV-2 infection is largely confined to West Africa.

HIV-1 is responsible for the world AIDS pandemic, which is now the number one cause of death owing to infectious disease in the world.

Currently, 34 million people are estimated to be infected with HIV-1, and 14 million have died from AIDS.

HIV DISEASE

Etiology

HIV-1 is divided into subtypes or clades.

The major clades, group M, are designated A–I; the less common group O has been largely confined to West and Central Africa.

Based on maps of genetic diversity between clades and compared with the simian immunodeficiency viruses (SIVs) that are endemic in African m!onkey!s, HIV-1 is believed to have derived from mutation of an SIV, whose original host was probably a chimpanzee.

HIV-1 is thought to have then infected human groups that lived in close proximity to infected chimpanzees, and possibly kept them as pets or hunted them.

Most likely, HIV remained for decades a disease largely confined to rural African villages, until urbanization of Africa eventually permitted worldwide spread.

The disease is spread through contact of infected body fluids, usually blood, semen or breast milk, by the mucous membranes or directly into the recipient’s blood or an open wound.

The vast majority of cases of HIV-1 infection in the world are the result of heterosexual intercourse.

In the United States, the disease was originally largely confined to homosexual men and then spread into intravenous drug users through the sharing of needles.

HIV infection is now rapidly increasing among women, both through intravenous drug use and via sexual intercourse with infected men.

Pediatric HIV infection usually occurs during labor and delivery from an infected mother but may also occur earlier in gestation or later, as a result of breast feeding.

Scope of the Epidemic

Worldwide, approximately 1 in every 100 adults aged 15–49 years is HIV-1-infected.

At least 1.2 million children under the age of 15 years are also infected. In 1998, approximately 16,000 new HIV infections occurred each day, more than 95% in developing countries.

The greatest risk factor for HIV infection is heterosexual intercourse, which has been responsible for 75% of the infections in the world.

The epidemic is especially concentrated in sub-Saharan Africa, where approximately 80% of the infections have occurred.

The disease is increasing most rapidly in South Africa.

The epidemic spread along truck routes from West to East Africa and from there to India and the Orient.

India has the largest number of HIV-infected people in any one country in the world.

The disease has spread through Southeast Asia and into China and Indonesia. In Europe, especially since the end of the Soviet Union, the disease has become particularly concentrated in some of the former Eastern Block nations, where economic collapse has fostered the drug trade and prostitution.

In the United States, up to 900,000 people are currently living with HIV infection and 688,200 cases of AIDS were reported to the CDC as of December, 1998.

The proportion of new AIDS cases diagnosed in women increased from 7% in 1985, to 23% in 1998. Of the U.S.

AIDS cases reported in 1998, 45% were among blacks, 33% among whites, and 20% among Hispanics.

Heterosexual transmission accounts for an increasing proportion of AIDS cases in the United States. From 1994 to 1997, the estimated proportion of adult U.S.

AIDS cases attributed to heterosexual contact grew from 8.5% to 22.1%.

Throughout the world, HIV infection is particularly a scourge of the most impoverished and disenfranchised nations and members of society.

It has greatly reduced life expectancy in many developing countries, created millions of orphans, reduced the healthy labor force, and placed huge burdens on businesses and health care structures.

It is fed by and contributes to social, political, and economic instability.

Throughout most of the world, the worst consequences of the HIV epidemic will not be felt for at least another decade.

Typical Disease Course

HIV-1 disease typically follows a course of acute HIV syndrome, which occurs in the weeks immediately after primary infection, and then years of clinical latency, with AIDS usually manifesting 6–10 years later.

Plasma viremia is greatest during the period of acute infection and at end-stage disease, and most transmission probably occurs during the acute and early infection phase. HIV-1 replication occurs primarily in activated CD4 T-lymphocytes.

During acute infection, the CD4 T-cell count falls from the normal level of about 1000 cells/mL to about half that level, accompanied by wide dissemination of virus and the seeding of lymphoid organs; it then usually rises again to about 75% of baseline as the plasma viremia falls.

The virus becomes largely sequestered in lymphoid tissue, with the plasma viral burden reflecting only a small fraction of total body viral burden.

A small fraction of the activated CD4 T-cells that have been infected with HIV revert to an inactive state, continuing to harbor the HIV provirus in their chromosomes.

This “latent pool” of HIV infected CD4 memory T-cells is extremely long lived and can release HIV at any time the cells become reactivated.

Viral replication continues within lymphoid tissue during the years of clinical latency, and the CD4 T-cell count gradually falls.

As the lymphoid architecture becomes disrupted and the host immune defenses become exhausted, the virus reemerges.

The patient experiences constitutional symptoms when the CD4 T-cell count falls to about 300 cells/mL.

Opportunistic infections, wasting disease, and rare cancers occur when the CD4 T-cell count drops below 200 cells/mL. If this pattern is not reversed by potent antiretroviral therapy, death typically follows within 2 years.

Variant Disease Courses

Although progression from time of HIV infection to end-stage disease typically takes 8–10 years in the absence of potent antiretroviral therapy, there are also cases of either very rapid or slow disease progression.

This variation has sometimes been linked to the characteristics of the infecting virus but more often seems to be a function of host immune response.

Rapid progressors have sometimes been infected with an overwhelmingly large burden of virus, for instance, in the case of transfusion with heavily contaminated blood products.

Other cases of rapid progression have been associated with primary HIV infection with strains that usually only arise late in disease course and that are able to bind to the beta-chemokine receptor CXCR4 and induce syncytium formation.

Failure to mount a broad enough host immunologic defense is a risk factor for rapid progression.

At the other end of the spectrum are those rare individuals who exhibit long-term non-progression, maintaining low levels of plasma viremia and elevated CD4 T-cell counts in the absence of antiretroviral therapy, despite 10 or more years of infection.

In a few cases, this has been associated with infection with a virus strain defective in essential viral genes.

More often, these individuals are found to have competent viruses, but also a more preserved immune response, particularly characterized by retention of HIV-specific T-helper lymphocyte activity.

Relative resistance to HIV infection or disease progression has been associated with different HLA groups and with expression of mutant cell surface receptors for HIV, particularly the beta-chemokine receptors.

Pediatric HIV infection is also characterized by variation in rate of disease progression, with rapid progression to AIDS occurring about onethird of the time in the absence of potent therapy.

Biology and Life Cycle of the Virus

HIV-1 is icosahedral in structure, with an inner (p18) and outer membrane, a protein core (p24) containing two strands of genomic RNA bound to reverse transcriptase, and glycoprotein spikes extending from the outer membrane.

The glycoprotein spikes are the two major viral envelope proteins, gp120 and gp41.

Most of the outer envelope consists of host cell-derived proteins, including major histocompatibility complex antigens, acquired as the virus particle buds from the cell.

The genome of HIV-1 is similar to that of other retroviruses, with gag encoding virion core proteins, env encoding envelope glycoproteins, and pol encoding the reverse transcriptase and integrase enzymes.

In addition, the HIV-1 genome contains the regulatory genes nef, rev, tat, vif, vpr, and vpu.

Regulatory elements are located in the long terminal repeats that flank the other genes.

HIV infection begins with the binding of the gp120 V1 region to the cellular CD4 molecule, found predominantly on T-helper lymphocytes and monocytes/macrophages.

This then results in a conformational change that exposes the gp120 V3 loop.

Second receptor binding by the V3 loop is the next key step, which confers infectious tropism depending on the host receptor that the virus is able to utilize.

Early in HIV infection, the infecting strains are typically best able to bind to the receptor CCR5 and are macrophage-tropic.

With disease progression, more pathogenic strains arise that are able to bind to CXCR4.

These strains are able to replicate in transformed T-cell lines that express CXCR4, but not CCR5, and they induce syncytium formation.

Other chemokine receptors have also been identified that HIV strains are able to utilize.

Resistance to HIV infection has been linked to production of high levels of the natural ligands for these receptors, competing for binding with HIV, and with mutations in the genes coding for the receptors, yielding a poor match for HIV binding.

Following binding by gp120 to both primary and secondary receptors, gp41 binding leads to fusion of viral and host cell membranes, uncoating of the HIV genomic RNA and its associated proteins, and its entry into the cell.

HIV reverse transcriptase then makes a double-stranded DNA copy of the viral RNA, which is transported to the nucleus and integrated into the host cell chromosome by the viral integrase enzyme.

The relative infidelity of the reverse transcriptase enzyme to the RNA template leads to a high mutation rate.

Transcription of the integrated provirus is dependent on host cell activation and DNA-dependent RNA-polymerase activity. Initially, double-spliced viral mRNA is produced, coding for viral proteins.

Later, as a result of the action of the HIV rev gene product, single-spliced and full-length HIV genomic RNA is produced and transported to the cytoplasm, where it is encapsulated in viral proteins.

The virion buds from the host cell membrane and then matures into an infectious virus particle after cleavage of immature viral proteins by HIV protease.

Each step of this complex life cycle presents opportunities for intervention with antiviral agents.

PATHOLOGIC MANIFESTATIONS

Host Response

HIV disease is characterized by immune activation, which becomes chronic owing to its failure to clear the infection.

This eventually leads to exhaustion of immunologic resistance and vulnerability to opportunistic disease.

The unremitting inflammatory immune response also results in tissue damage, contributing to wasting, renal disease, cardiac disease, dementia, and neuropathy.

Proinflammatory cytokines have been shown to stimulate HIV replication; therefore this response, which is elicited by HIV antigens, contributes to persistence of infection.

The viremia during acute HIV infection falls as HIV is sequestered in lymphoid tissue, largely bound to follicular dendritic cells (FDCs), and as cytotoxic lymphocyte (CTL) response to HIV arises.

Both infected and uninfected T-lymphocytes are also sequestered in the lymphoid tissues, in response to cytokine signaling and adhesion molecule expression.

A significant amount of neutralizing antibody to HIV is usually detectable in the peripheral blood weeks after the plasma viral burden has fallen, suggesting that cell-mediated immunity is the more important initial host immune response.

Studies of the breadth of CTL receptor V-beta repertoire demonstrated more rapid disease progression when the repertoire was most limited.

In contrast to the fall in CD4 T-cell numbers and function, CD8 T-cells are increased in both number and activation state throughout most of the course of HIV disease.

This produces the characteristic reversal of CD4 /CD8 cell ratio.

CD8 T-cells suppress HIV replication through CTL activity and through noncytolytic suppressor action.

Much of the latter activity is thought to be due to production of the beta-chemokines that are the natural ligands for the second receptors utilized by HIV during binding to target cells, although additional suppressor factors also seem to be involved.

Late in the course of HIV disease, the numbers of circulating CD8 T-cells fall, heralding much more rapid disease progression.

Although clinical manifestations of HIV disease may not occur for a decade after infection, HIV replication in lymphoid tissues continues throughout this time.

The high mutation rate of the virus leads to steady escape from immunologic containment, as well as development of resistance to antiretroviral drugs. With progression to AIDS, the architecture of the lymphoid tissue collapses, as both T- and B-cell regions involute and the FDC network is disrupted. HIV previously contained in lymphoid tissue is then released, with a sharp increase in plasma viremia.

In the absence of potent antiretroviral therapy, any condition that causes an inflammatory immune response is likely to induce increased HIV replication in the infected host.

This has been observed with a relatively mild stimulus, such as vaccination, as well as with the more potent stimulus of intercurrent illness, such as influenza.

As the disease progresses to AIDS, the opportunistic infections that follow may do the added damage of driving HIV expression by the inflammatory response they provoke, in addition to the harm the infection itself causes.

Globally, infection with both HIV and tuberculosis continues to be the most difficult public health problem complicating the HIV epidemic.

HIV disease progresses much more rapidly in persons infected with tuberculosis, who are also at greater risk of harboring multidrug-resistant tuberculosis.

Chronic parasitic infections also frequently accompany HIV infection, particularly in Africa.

Successful treatment of the parasite disease has been shown to ameliorate the course of the HIV coinfection.

Coinfection at the cellular level with herpesviruses and HIV may also directly drive HIV replication, through promotor stimulation.

Immune Dysfunctions in HIV Disease

AIDS is characterized by the progressive loss of reaction to antigenic stimulation and vulnerability to infection. Response is first lost to recall antigen, next to alloantigen, and finally to mitogen.

In pediatric AIDS, failure to resist common bacterial infections is frequently seen, whereas in adults, this is less common, reflecting the adult’s more mature humoral immunity.

In both populations, loss of resistance to intracellular parasites, viruses, protozoa, fungi, and mycobacteria demonstrates impaired cell-mediated immunity.

Polyclonal B-cell activation contributes to inappropriate antibody production, autoimmune disease, and B-cell lymphomas.

The primary target for HIV infection is the activated CD4 T-cell.

The central role of this cell type in coordinating both the humoral and cell-mediated immune response means that physical or functional loss of these cells leads to a broad array of immune dysfunctions.

B-cells that encounter a matching antigen engulf it, digest it, and display antigen fragments on their surface in complex with MHC molecules.

A mature CD4 T-cell with a matching receptor for the antigen and MHC display must next supply lymphokines to allow the B-cell to multiply and mature into antibody-producing plasma cells.

Failure of this T-helper cell function leads to loss of humoral response to the antigen against which the T-cell was primed.

Similarly, cell-mediated immunity depends on antigen display by an antigen-presenting cell (APC) such as a B-cell, macrophage, or circulating dendritic cell, encounter with a matching receptor on a mobilized T-cell, stimulation of the T-cell by second receptor binding and lymphokines from the APC, and appropriate activation of the T-cell.

The activated cell then secretes lymphokines that may attract immune cells (including macrophages, granulocytes, and other lymphocytes), stimulate the growth of T-cells, and induce killer cell activity. Defects in any of these steps leads to failure of all the subsequent responses.

Both the number and function of CD4 T-cells is compromised by HIV infection.

Many factors seem to contribute to the fall in CD4 T-cell number, including lysis by HIV itself, lysis by HIV-specific CTL, syncytia formation, apoptosis, and reduced rate of T-cell synthesis. Sequestration in lymphoid tissue also reduces the number of CD4 T-cells in the peripheral blood.

The rate of CD4 T-cell infection is inadequate to account for most of the cell loss, particularly early in HIV disease.

Apoptosis seems to contribute significantly to this cell loss, which affects uninfected as well as infected cells.

Many auxiliary HIV proteins, such as Nef, Tat, and Vpr, which have regulatory functions in HIV maturation, also appear to contribute to this immune dysfunction.

Linking of gp120, which is shed by HIV, with CD4 can program cells for apoptosis upon receipt of a second stimulatory signal delivered via the T-cell receptor.

Thus cells exposed to soluble HIV proteins, but uninfected by HIV, may undergo apoptosis.

This may lead to deletion of clones of memory cells at the moment they are activated by the antigen to which they are programmed to respond. It is not surprising, then, in the constant presence of HIV antigen, that HIV-specific CD4 T-helper cells are rapidly depleted. The same mechanism may underlie the loss of response to recall antigens, with accompanying vulnerability to other infectious agents.

Binding of HIV-induced proinflammatory cytokines with the apoptosis-inducing CD95 or tumor necrosis factor receptor 1 (TNFR-1) receptors may also contribute to cell death.

The rate of synthesis of T-cells has been shown to be reduced by HIV infection and to increase when HIV replication is suppressed by antiviral drugs. The reason for this inhibition of T-cell synthesis is unclear, but it may involve more than one mechanism.

The maturation of thymus-derived naive T-cells is probably inhibited by effects of HIV on both thymic epithelial cells and immature thymic precursor cells.

The extrathymic expansion of T-cells is inhibited by the disruption of cytokine signaling, in particular by the reduced expression of interleukin-2 (IL-2) and the IL-2 receptor.

The failure of CD4 T-cell function seems to be due to disruption of the normal cellular and intercellular signaling mechanisms.

CD4 T-cell anergy can result from inappropriate signaling after gp120 binding to CD4.

Stimulation by superantigen binding nonspecifically to the T-cell receptor may cause the massive overexpansion of T-cell subsets and may also cause deletion of these subsets if they are already primed for apoptosis.

APC interaction with T-cells may fail, if the proper cytokine signal does not accompany antigen presentation. HIV-infected monocytes/macrophages express decreased MHC class II, CD80/86 costimulatory molecule, and IL-12 and increased IL-10, Fas (CD-95), and Fas ligand (CD-95L).

Interaction of such APCs with CD4 T-cells predisposes to T-cell death, either through apoptosis or HIV infection In the absence of appropriate APC signaling, CD4 T-helper function will not be induced, leading to poor development of HIV-specific CD8 T-cell CTLs and noncytolytic suppressor activity. In addition to defective APC activity, HIV-infected monocytes/macrophages are also impaired in migration, phagocytosis, oxidative burst, and tumor surveillance.

This contributes to the vulnerability to opportunistic infections and cancer seen in AIDS.

These cells also seem to play a key role in HIV spread across tissue barriers, especially during primary infection and in infection of the central nervous system.

Microglial cells in central nervous system are of monocytic lineage and can be infected by HIV.

Expression of proinflammatory cytokines by HIV-infected microglia, as well as from invading macrophages, seems to contribute to neurotoxicity.

In summary, the failure of the immune system to clear HIV, although it may successfully contain the infection for many years, coupled with the central importance of the primary target cells in regulating the immune response, leads to chronic immune activation and immune dysregulation.

Initially, the lesions in the immune repertoire are those directed at HIV itself, especially the loss of HIV-specific CD4 T-helper cell function.

Chronic immune activation and apoptosis eventually lead to loss of cell-mediated immunity directed against ubiquitous opportunistic agents.

The chronic inflammation causes bystander damage, leading to complications such as dementia and wasting.

Successful therapy with antiviral drugs leads to rapid clearance of HIV from the peripheral blood and from most tissue sites.

This is followed by reduced immune activation and partial restoration of immune function.

Although resistance to many opportunistic infections are frequently restored by successful potent antiretroviral therapy, resistance to HIV itself remains an illusive goal.

THERAPY

Range of Possible Therapeutic Modalities

As will be discussed in detail in the chapters that follow, a variety of strategies are being explored in attempts to halt and reverse the immune dysfunction caused by HIV disease.

Foremost has been the use of antiviral agents to suppress HIV replication and the use of antibiotic prophylaxis to prevent the emergence of opportunistic infections. With the recent advent of potent antiretroviral therapy, the ability of the immune system to recover spontaneously has been demonstrated, and the limits of this recovery have also been seen.

Other strategies being tested involve modulation of the immune response, to reduce the excessive activation.

Supplementation of cytokines depressed by HIV disease, to restore the number and function of T-cells and monocytic cells, may yield improved resistance to opportunistic disease, and conceivably to HIV itself.

Therapeutic vaccines and strategies of treatment interruption to deliberately permit reexposure of the immune system to HIV antigen, in an effort to boost host immune response to HIV, are being tried.

Attempts are being made to reduce the size of the pool of cells latently infected with HIV, or to make it more difficult for these cells to become activated and to express HIV.

Gene-based therapies are being developed to confer resistance to HIV infection at the cellular level.

As these and other therapeutic interventions are developed, they present great challenges in clinical trial design.

Challenges of Therapeutic Trial Design

The limitations of the available animal models of HIV infection have forced researchers to go to human trials with more limited data than we would prefer to have.

Only chimpanzees can be infected with HIV, and the development of immunodeficiency following their infection is as slow as in human disease, if in fact it occurs at all.

They are therefore used primarily in testing vaccines, since the prevention of infection can be measured, but the impact of a therapy on disease course cannot.

Their use is further complicated by the fact that they are an intelligent, endangered species, whose use as a laboratory animal is tightly restricted and very expensive.

The macaque model is the next best choice.

Strains of simian immunodeficiency virus (SIV) have been developed that produce a predictable range of immunodeficiency disease course, from months to years.

Recently, the simian/human immunodeficiency virus, engineered to express antigens of both SIV and HIV (SHIV), has been used in the macaque model to test vaccines.

Unfortunately, there are sufficient differences between some of the SIV and HIV proteins that are the targets of antiviral drugs to make it impossible to use the potent antiretroviral cocktails that have been developed against HIV in the macaque model.

The expense of caring for macaques restricts the size of experiments using this model.

There are no good small animal models for HIV.

The use of genetically immunodeficient mice, in which human tissues have been implanted (the SCID-hu mouse model) has limited application and is very labor intensive.

The feline immunodeficiency (FIV) model is likewise too far removed from HIV for much data to be gleaned about therapy.

Human clinical trials are therefore the setting in which therapeutic interventions for HIV disease are generally first tested.

Clinical trials of therapies to reverse or prevent the immunopathology of HIV disease must be carefully designed to account for practical and ethical considerations.

Once trials have grown beyond the pilot stage, in which interventions in small numbers of subjects yield data that help to guide the planning of larger trials, sufficient numbers of participants must be enrolled so that the outcome can be reliably attributed to something other than chance.

The choice of end points is critically important to make sure that meaningful results are eventually obtained.

In the past, disease progression and survival were the outcomes most frequently used to judge effectiveness of therapeutic interventions for HIV disease.

However, the slow rate of progression of the disease required very large trials with long-term follow-up before sufficient numbers of events could display a significant difference between arms in a protocol.

The correlation of fall in CD4 T-cell count with disease progression led to that measure being viewed as the first surrogate marker in therapeutic trials. With the development of reliable techniques for quantitatively measuring HIV in the peripheral blood, and the demonstration of the correlation between viral load and risk of disease progression, HIV plasma viral load has become accepted as a partial surrogate for clinical progression.

However, CD4 T-cell count and HIV plasma viral load taken together still do not account for the full risk of disease progression.

Markers of immune activation, especially CD8 CD38 phenotype, seem to be at least as powerful predictors.

With the development of immune-based therapies given with a background of potent antiviral therapy, and the ensuing rate of disease progression being as low as 1% per year or less, surrogate marker end points are essential.

At the same time, although interventions that may result in change in viral load can be tested against that measure, it is quite conceivable that an intervention could confer significant immunologic benefit with little impact on viral load.

Interventions that reverse immune dysregulation, increase CD4 T-cell levels, modulate excessive immune activation, or decrease apoptosis all might fall into this category.

Validation of appropriate surrogate markers for immune-based therapies is the next hurdle in the advancement of this field.

The choice of the population in which to test interventions is also an important consideration in clinical trial design.

Patients with advanced disease, who have failed potent antiretroviral therapy, are eager to find alternate therapies, and their outcome might be relatively quickly learned.

Unfortunately, many of the interventions being tried are the least effective and most toxic in subjects with advanced disease.

Populations with a more intact immune response are therefore currently favored for trials of immune-based therapies.

At the same time, if surrogate markers are being relied on for end points, there must be something to measure in the population chosen.

For example, if change in viral load is chosen, then either the subjects must not have their viral load suppressed below the level of detection to begin with, or must have a likelihood of sufficient numbers of participants to experience viral breakthrough to be able to measure benefit from the intervention.

An alternate model being explored is to withdraw therapy at some time and measure the rate or the magnitude of viral load resurgence as an end point.

The possible risks to participants of this study design are being carefully examined.

Further complicating the design of clinical trials is the rapid evolution of the standard of care of HIV disease.

In trials that may take years to develop, enroll, and then follow to end points, care must be devoted to considering incorporation of the use of new antiviral drugs, new measures of efficacy of therapy, and new techniques for determining suitable antiviral regimens. (Examples are the routine use of potent antiretroviral cocktails, plasma viral load for assessing efficacy of therapy, and screening for antiviral resistance.)

Otherwise, the outcome of the trial may not be relevant in the context of the current standard of care at the trial’s conclusion.

Ethical considerations are extremely important in clinical trial design.

Consideration must be given not only to the risk to the individual participant but also to the benefit to the community from which participants are recruited.

In the United States, for instance, consideration must be given to including women and minorities in the participants in clinical trials and to not unnecessarily barring participation by pregnant women.

The greatest challenge is in designing trials suitable for developing countries.

The data gathered from such trials must be relevant to the population of that country and the prospect must exist for the therapy being tested to be available there if found to be effective.
An exquisite tension exists between the dire need for therapies in developing nations and the barriers of cost that may be insurmountable.

The unavailability of potent antiretroviral therapy in developing nations and the rapid rate of HIV disease progression still seen there makes this setting suitable for therapeutic trials with clinical end points.

However, the lack of therapeutic options outside the clinical trials mechanism makes this group especially vulnerable to exploitation, and careful ethical review of clinical trials planned for developing nations is extremely important.