Understanding AIDS & Its Virus.

AIDS or Acquired Immuno Deficiency Syndrome is a disease that is troubling this world for more than 3 decades since it’s discovery in 1981. More than 20.9 million people are inflicted by the disease as reported by WHO in mid-2017. The disease is caused by a Virus named Human Immuno Deficiency Virus, which attacks our immune system making us vulnerable to diseases. AIDS patients suffer from a range of secondary diseases like Tuberculosis, Hepatitis or Cancer and eventually die of them.  Owing to the seriousness of the disease, the World Health Organization ( headquartered in Geneva ), has designated December 1 as a World AIDS Day in order to highlight the menace of this disease and make people aware of it.

However, proper knowledge of the disease requires a thorough understanding of the HIV virus. So, today we are going for an in-depth analysis of HIV virus, its modes of action and what it means for us.

HUMAN IMMUNODEFICIENCY  VIRUS (HIV)

INTRODUCTION

hiv
HIV Virus

Human immunodeficiency virus (HIV) is a retrovirus belonging to the category lentivirus. This virus is the causative agent of the disease AIDS (Acquired Immuno Deficiency Syndrome). HIV is one of a group of atypical viruses called retroviruses that maintain their genetic information in the form of ribonucleic acid (RNA). Through the use of an enzyme known as reverse transcriptase, HIV and other retroviruses are capable of producing deoxyribonucleic acid (DNA) from RNA, whereas most cells carry out the opposite process, transcribing the genetic material of DNA into RNA. The activity of the enzyme enables the genetic information of HIV to become integrated permanently into the genome (chromosomes) of a host cell.The disease was first recognized in the USA in 1981.The virus was first isolated by US scientist Robert Gallo and French scientist Luc Montagnier in 1983. In 1986 AIDS was identified as an unknown disease in India. HIV infections may be caused by one of two retroviruses, HIV-1 or HIV-2. HIV-1 has caused a worldwide epidemic, but HIV-2 tends to be limited to West Africa. HIV is a complex virus which has RNA as the genetic material, the enzyme reverse transcriptase, capsid and a viral envelope containing glycoprotein surface receptors gp120 and gp41.

HIV GENOMIC STRUCTURE

The HIV genome has the following genes:

Structural genes:

hiv1
The HIV Genomic Structure
  • gag: It is a group of antigen gene found in all retroviruses. It makes various proteins necessary to protect the virus. In HIV it has 3 parts MA (matrix)p17, CA (capsid)p24 and nucleocapsid.
  • pol: This is the polymerase gene found in all retroviruses. It makes enzymes necessary for viral replication. In HIV it has 3 parts: PR (protease), EN(endonuclease). RT(reverse transcriptase) and IN(integrase).
  • env: this is the envelope gene which makes the viral envelope protein. In HIV it has 2 parts: SU(surface envelope) and TM(transmembrane envelope).

Regulatory genes:

  • tat: the transactivator gene influences the functions of genes some distance away. It controls the transactivation of all HIV proteins.
  • rev: a differential regulator of expression of viral protein genes.

Accessory genes:

  • vif : the virus infectivity factor gene is required for infectivity.
  • nef : the negative regulator factor gene which retards HIV replication.
  • vpr: the virus protein R gene has an undetermined function.
  • vpu: the virus protein U gene is required for efficient viral replication and release. It is found only in HIV-1
  • vpx: the virus protein X gene has an undetermined function. It is found only in HIV-2 and SIV.
hiv genome
The Genome Organization of HIV

THE VIRAL INFECTION

viral infectionThe primary hosts for HIV are the white blood cells variously called helper T lymphocytes, helper T cells, or CD4+ T cells. These cells are important components of the immune system that are normally responsible for activating the responses of many other immune cells. Helper T cells that become infected with HIV rapidly die. Soon after primary infection, the body is usually able to compensate for the loss of infected T cells by producing them in greater quantities, at which time individuals infected with HIV are asymptomatic. Over time, however, the virus becomes increasingly acute and there is a slow decline of helper T cells. Consequently, the number of helper T cells in the body (termed the CD4 count) is generally utilized to gauge the advance of the virus. A CD4 count of less than approximately 200 cells per microliter of blood may be accompanied by a variety of opportunistic infections and is considered the final stage of infection. The persistent barrage of such infections is what typically leads to the death of AIDS patients.

Mechanism of entry of virus into the cell

HIV is a relatively complex virus that is able to infect helper T cells chiefly due to a glycoprotein embedded in its envelope called gp120 (see Figure 1) that attaches to CD4, a protein found on the surfaces of the T cells. Entry of HIV into a host cell is also thought to involve a co-receptor on the cell surface, either CCR5 or CXCR4, which typically function as receptors of chemokines. The HIV virus envelope is a derivative of the plasma membrane of a host cell, obtained via budding. When HIV attempts to enter a cell, interactions between cell surface molecules and viral envelope proteins allow the envelope to fuse with the cell membrane. The envelope protein called gp41 is known to play an important role in this process.

entry mechanism
Attachment, Fusion and Viral Genome Entry

Mechanism of Infection

Once in the body, HIV attaches to several types of white blood cells. The most important are certain helper T lymphocytes. Helper T lymphocytes activate and coordinate other cells of the immune system. On their surface, these lymphocytes have a receptor called CD4, which enables HIV to attach to them. Thus, these helper lymphocytes are designated as CD4+.

life cycle
The Viral Life Cycle

HIV stores its genetic information as ribonucleic acid (RNA). Once inside a CD4+ lymphocyte, the virus uses an enzyme called reverse transcriptase to make a copy of its RNA, but the copy is made as deoxyribonucleic acid (DNA). HIV mutates easily at this point because reverse transcriptase is prone to making errors during the conversion of viral RNA to DNA. These mutations make HIV more difficult to control—by the body’s immune system and by drugs.

The viral DNA copy is incorporated into the DNA of the infected lymphocyte. The lymphocyte’s own genetic machinery then reproduces (replicates) the virus. Eventually, the lymphocyte is destroyed. The thousands of new viruses produced by each infected cell infect other lymphocytes and can destroy them as well. Within a few days or weeks, the blood and genital fluids contain many viruses, and the number of CD4+ lymphocytes may be reduced substantially. Because the number of viruses in blood and genital fluids is so large so soon after HIV infection, newly infected people can readily spread HIV to other people.

When HIV infection destroys CD4+ lymphocytes, it weakens the body’s immune system, which protects against many infections and cancers. This weakening is part of the reason that the body is unable to eliminate HIV infection once it has started. However, the immune system is able to mount some response. Within a month or two after infection, the body produces lymphocytes and antibodies that help lower the amount of HIV in the blood and keep the infection under control. For this reason, untreated HIV infection continues for an average of about 10 years (ranging from 2 to 20 years) before causing symptoms.

CD4 COUNT:

The number of CD4+ lymphocytes in the blood (the CD4 count) helps determine how well the immune system can protect the body from infections and how severe the damage done by the HIV is. Healthy people have a CD4 count of about 800 to 1,300 cells per microliter of blood. Typically, 40 to 60% of CD4+ lymphocytes are destroyed in the first few months of infection. After about 3 to 6 months, the CD4 count stops falling so quickly, but without treatment, it usually continues to decline at rates that vary from slow to rapid.

If the CD4 count falls below about 200 cells per microliter of blood, the immune system becomes less able to fight certain infections (such as the fungal infection that causes Pneumocystis jirovecii pneumonia). These infections do not usually appear in people with a healthy immune system. Such infections are called opportunistic infections because they take advantage of a weakened immune system. A count below about 50 cells per microliter of blood is particularly dangerous because additional opportunistic infections that can rapidly cause severe weight loss, blindness, or death commonly occur. CD4 count can be measured by Flow-cytometry.

VIRAL LOAD:

The amount of HIV in the blood (specifically the number of copies of HIV RNA) is called the viral load. Viral load represents how quickly HIV is replicating. When people are first infected, the viral load increases rapidly. Then, even without treatment, it drops to a lower level, which remains fairly constant, called the setpoint. This level varies widely from person to person. Viral load also indicates how contagious the infection is and how fast the infection is likely to worsen.

During successful treatment, the viral load decreases to a very low or undetectable level. However, inactive (latent) HIV is still present within cells, and if treatment is stopped, HIV starts replicating. An increase in the viral load during treatment indicates that the HIV has developed resistance to drug treatment, that people are not taking the drugs or both.

MODES OF TRANSMISSION

  • Sexual transmission, the presence of STD increases the likelihood of transmission.
  • Exposure to infected blood or blood products.
  • Sharing contaminated needles (IV drug users).
  • Transplantation of infected tissues or organs.
  • Mother to the fetus, perinatal transmission variable, dependent on viral load and mother’s CD 4 count.
Transmission
Transmission of the virus

SYMPTOMS

When initially infected, many people have no noticeable symptoms, but within a few weeks, fever, rashes, swollen lymph nodes, fatigue, and a variety of less common symptoms may develop. Symptoms of initial (primary) HIV infection last from a few days to 1 to 2 weeks. The symptoms disappear, but lymph nodes often remain enlarged, felt as small, painless lumps in the neck, under the arms, or in the groin. People can be infected with HIV infection for years—even a decade or longer—before developing symptoms. However, the first symptoms may be those of AIDS. AIDS is defined as the development of very serious opportunistic infections or cancer—the ones that usually develop only in people with a CD count of less than 200. Before AIDS develops, many people feel well, although some develop a variety of vague symptoms such as weight loss, fatigue, recurring fever or diarrhea, anemia, and thrush (a fungal infection of the mouth or vagina).

cdc-hiv-and-youth-infographic

Symptoms of AIDS are usually those of the specific opportunistic infections and cancers that develop. For example, people may have white patches in their mouth due to thrush or pain and rash due to herpes zoster.

However, HIV can also cause symptoms when it directly infects parts of the body:

  • Brain:

    Memory loss, difficulty thinking and concentrating, or both, eventually resulting in dementia, as well as weakness, tremor, or difficulty walking

  • Kidneys:

    Swelling in the legs and face, fatigue, and changes in urination (more common in blacks than in whites), but often not until the infection is severe

  • Heart:

    Shortness of breath, cough, wheezing, and fatigue (uncommon)

  • Genital organs:

    Decreased levels of sex hormones, which, for men, leads to a decreased interest in sex (common).

Common Opportunistic Infections Associated with AIDS
Infection Description Symptoms
Candidal esophagitis A yeast infection of the esophagus Painful swallowing and burning in the chest
Pneumocystis jirovecii pneumonia An infection of the lungs with the fungusPneumocystis jirovecii Difficulty breathing, cough, and fever
Toxoplasmosis Infection with the parasite Toxoplasma gondii, usually in the brain Headache, confusion, lethargy, and seizures
Tuberculosis Infection of the lungs and sometimes other organs with tuberculosis bacteria Cough, fevers, night sweats, weight loss, and chest pain
Mycobacterium avium complex infection Infection of the intestine or lungs with bacteria that resemble tuberculosis bacteria Fever, weight loss, diarrhea, and cough
Cryptosporidiosis Infection of the intestine with the parasiteCryptosporidium Diarrhea, abdominal pain, and weight loss
Cryptococcal meningitis Infection of the lining of the brain with the yeast Cryptococcus Headache, fever, and confusion
Cytomegalovirus infection Infection of the eyes or intestinal tract with cytomegalovirus Eye: Clouding of vision or blindness

Intestinal tract: Diarrhea and weight loss

DIAGNOSIS

Simple, screening tests that detect antibodies to HIV are done. Tests may be done on a blood sample in the laboratory or on a blood or saliva sample in the doctor’s office. If screening test results are positive, they are confirmed by a more accurate, specific test such as the Western blot. Often, these tests are not positive in the first weeks up to 2 months after initial HIV infection because antibodies to HIV are not yet being produced. Tests include the following:

  • Enzyme-linked immunosorbent assay (ELISA):

    ELisa
    Method of ELISA test

    This screening test is often used to detect HIV antibodies, but it requires complex equipment.Antibodies detected in ELISA include those directed against: p24, gp120, gp160 and gp41.Different types of ELISA techniques used:

    indirect

    competitive

    sandwich

ELISAs are for screening only, false positives do occur and may be due to AI disease, alcoholism, syphilis, and immunoproliferative diseases.

Other screening tests:.

Agglutination tests

Agglutination test
Particle Agglutination Test

using latex particles, gelatin particles or microbeads are coated with HIV antigen and will agglutinate in the presence of antibody.

Dot-Blot

dit bot

Testing utilizes paper or nitrocellulose impregnated with antigen, patient serum is filtered through, and anti-antibody is added with an enzyme label, the color change is positive.

Western blot:

This test is usually done to confirm the diagnosis when screening test results are positive. It is more difficult to do than the screening tests but is more accurate.It utilizes a lysate prepared from HIV virus.The lysate is electrophoresed to separate out the HIV proteins (antigens).The paper is cut into strips and reacted with test sera.After incubation and washing anti-antibody tagged with radioisotope or enzyme is added.Specific bands form where the antibody has reacted with different antigens.The following antigens must be present: p17, p24, p31, gp41, p51, p55, p66, gp120 and gp160. Depending on the particular antibodies in the sample, reactivities with the separated antigenic components result in band profiles.

western blot
Interpretation of Western Blot

The type of profile (the combination and intensity of bands that are present) determines whether the individual is considered positive for antibodies to HIV. The classification of Western blot results is determined by certain criteria. Most institutions now follow the CDC guidelines, which require reactivity to at least 2 of the following antigens: p24, gp41, gp120/160 for a positive  It is now universally accepted that a negative result is the absence of all bands. Two organizations, however, including the World Health Organization (WHO), suggest that results also can be reported as negative if there is only a very weak p17 band. Indeterminate classifications occur when there is reactivity to 1 or more antigens, but not fulfilling the criteria for positivity.

  • Real-time PCR:

    This is a laboratory technique which directly estimates the presence of the viral genome over the cycle of PCR (Polymerase Chain Reaction, a method replicating DNA/RNA in vitro i.e., in the lab).

PCR
PCR Diagnosis of HIV

TREATMENTS

Different types of  HIV/AIDS management drugs work uniquely to counter the virus. Combining several types of these drugs help minimize chances of the virus developing resistance top one drug (Briz et. al., 2006).

Types of the HIV Drugs

inhibitors
HIV Drugs and their targets
  • Entry or fusion inhibitors

  •  Non-nucleoside reverse transcriptase inhibitors (NNRTIs)

  •  Protease inhibitors (PIs)

  • Nucleoside reverse transcriptase inhibitors (NRTIs)

  • Integrase inhibitors

Entry or fusion inhibitors

 

Mechanism of action of fusion inhibitors

fusion inhibitors
Fusion Inhibitors

Entry or fusion inhibitors prevent HIV from entering CD4 cells. Contact between gp120-CD4 complex and chemokine receptor CCR5 or CXCR4 causes a change that the viral membrane to a fusogenic state which control the fusion process. Meanwhile, gp41 reveals its N-terminal, which goes through the fusion peptide into the cell outer covering. Gp41 restructure and this causes the HR1 and HR2 to come together and a thermostable six-helix structure necessary in viral and cellular membrane fusion forms. It is this change in free energy that goes with the emergence of the six-helix bundle that enables the fusion pore to form leading to viral capsid’s entry into the cell of interest (Poveda, Briz & Soriano, 2005).

Non-nucleoside reverse transcriptase inhibitors (NNRTIs)

 

Mechanism of action

NNRTIs work by disabling a protein that HIV needs to replicate. The reverse transcriptase consequently blocks it from altering the RNA to DNA. In effect, this strengthens the genetic material of the cell such that the genetic information of HIV cannot cause it to replicate the virus.

nnrti
NNRTI mode of action

Protease Inhibitors (PIs)

 

Mechanism of action

protease inhibitor

Protease inhibitors serve to prevent infected T-cells from replicating the virus. They cover the active sites of the protease enzyme in order to inhibit the HIV polyprotein. This brings down the level of the virus in the blood, boosting the patient’s immune system.

Resistance to protease inhibitors comes from mutations of the viral protease enzyme (Reeves & Piefer, 2005).

Nucleoside reverse transcriptase inhibitors (NRTIs)

 

Mechanism of action

nrti
NRTI mode of action

NRTIs are carried by the host cell and are later phosphorylated by cellular enzymes, which change them to their active form – NRTI triphosphates. Stability of NRTI and how efficiently it is converted into NRTI triphosphate are key determinants of the level of the inhibitor in the bloodstream required for the effect on HIV (Briz et. al., 2006).

Integrase inhibitors

 

Mechanism of action

Integrase inhibitors embed themselves on reverse transcriptase after it has created the viral DNA strand and hooks onto the last two nucleotides from the end of the DNA strand. This is termed as 3A2 processing. After this, integrase inhibitor forms a pre-integrase complex that carries a ring-shaped viral DNA and host proteins. It is this pre-integrase complex that helps the viral DNA pass through cell cytoplasm and into the cell nucleus. The integrase inhibitor prevents strand transfer and viral replication (Reeves & Piefer, 2005).

integrase inhibitors
Sit of action for integrase inhibitors

The AIDS Cocktail

 

  • highly active antiretroviral therapy, or HAART. HAART is a potent combination of at least three active antiretroviral medications (ARV).
  • HAART is popularly called the “AIDS cocktail” because of its mix of drugs.
  • Mainly a mix of 3 types of drugs is used NNRTIs, protease and integrase inhibitors.
  • Example: Raltegravir (integrase inhibitor)+ Tenofovir (NRTIs) + Emtricitabine  (NRTIs).

Viral Infection in case of CCR5 mutation :

  • CCR5 has multiple variants in its coding region, the deletion of a 32-bp segment results in a nonfunctional receptor, thus preventing HIV R5 entry
  • 2 copies of this allele provide strong protection against HIV infection. This allele is found in 5–14% of Europeans but is rare in Africans and Asians.
  • CCR5-Δ32 decreases the number of CCR5 proteins on the outside of the CD4 cell, which can have a large effect on the HIV disease progression rates.
  • Multiple studies of HIV-infected persons have shown that presence of one copy of this allele delays progression to the condition of AIDS by about two years.
  • It is possible that a person with the CCR5-Δ32 receptor allele will not be infected with HIV R5 strains.
  • A genetic approach involving intrabodies that block CCR5 expression has been proposed as a treatment for HIV-1 infected individuals.[When T-cells modified so they no longer express CCR5 were mixed with unmodified T-cells expressing CCR5 and then challenged by infection with HIV-1, the modified T-cells that do not express CCR5 eventually take over the culture, as HIV-1 kills the non-modified T-cells. This same method might be used in vivo to establish a virus resistant cell pool in infected individuals.

This hypothesis was tested in an AIDS patient who had also developed myeloid leukemia, and was treated with chemotherapy to suppress cancer. A bone marrow transplant containing stem cells from a matched donor was then used to restore the immune system. However, the transplant was performed from a donor with 2 copies of CCR5-Δ32 mutation gene. After 600 days, the patient was healthy and had undetectable levels of HIV in the blood and in the examined brain and rectal tissues. Before the transplant, low levels of HIV X4, which does not use the CCR5 receptor, were also detected. Following the transplant, however, this type of HIV was not detected either, further baffling doctors. However, this is consistent with the observation that cells expressing the CCR5-Δ32 variant protein lack both the CCR5 and CXCR4 receptors on their surfaces, thereby conferring resistance to a broad range of HIV variants including HIV X4. After three years, the patient has maintained the resistance to HIV and has been pronounced cured of the HIV infection.

Enrollment of HIV-positive patients in a clinical trial was started in 2009 in which the patients’ cells were genetically modified with a zinc finger nuclease to carry the CCR5-Δ32 trait and then reintroduced into the body as a potential HIV treatment

aids

suniti
Dr. Suniti Solomon with Richard Gere

Hence, HIV has many checks and balance which makes treatment for the disease quite difficult but with increasing time it has become more and easier. With proper treatment a sustainable life is possible, even marriage is possible if the match-making is done by comparing their CD4 count, as shown by Dr. Suniti Solomon, the pioneer who discovered the disease in Tamil Nadu, India. The documentary “Lovesick” highlights this aspect.

Although, Padma Shri Dr. Suniti Solomom her institute YRG Care survives giving hope to millions. One just needs to reach out! Dial 044-33125000 to get any kind of assistance. http://www.yrgcare.org/contact-us/

And for those looking for a platform to meet people from the community Positive Sathi, a matrimonial website for HIV positive people is here. 

 

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