Human Immunodeficiency Virus

presented by Rughoobur chitra Group 2(a) Faculty of foreign students

Human Immunodeficiency Virus

Acquired Immunodeficiency syndrome first described in 1981

HIV-1 isolated in 1984, and HIV-2 in 1986 Belong to the lentivirus subfamily of the retroviridae Enveloped RNA virus, 120nm in diameter HIV-2 shares 40% nucleotide homology with HIV-1 Genome consists of 9200 nucleotides (HIV-1): gag core proteins - p15, p17 and p24

pol - p16 (protease), p31 (integrase/endonuclease)

env - gp160 (gp120:outer membrane part, gp41: transmembrane part) Other regulatory genes ie. tat, rev, vif, nef, vpr and vpu

Types of HIV
For human immunodeficiency virus characterized by a high frequency of genetic changes that occur during self-renewal. Error Rate for HIV is 10 -3 - 10 -4 errors / (* genome replication cycle), which is several orders of magnitude more similar in eukaryotes. The length of the HIV genome is approximately 10 4 nucleotides. From this it follows that almost every virus on at least one nucleotide different from its predecessor. In nature, HIV exists as a set of quasi-species, being a single taxonomic unit. In the study of HIV yet been discovered species that differed significantly from each other on several grounds, including different structure of the genome. Varieties of HIV identified in Arabic numerals. To date, known HIV-1, HIV-2, HIV-3 HIV-4.

HIV-1 - the first representative of the group, which opened in 1983 . The most common form. HIV-2 - the kind of human immunodeficiency virus identified in 1986 [28] . Compared with HIV1, HIV-2 was studied to a much lesser degree. HIV-2 differs from HIV-1 genome structure. It is known that HIV-2 is less pathogenic and is transmitted is less likely than HIV-1. It was noted that people infected with HIV-2 have low immunity to HIV-1. HIV-3 - a rare species, the discovery of which was reported in 1988 [29] . Detected the virus did not react with antibodies of other known groups, and also had significant differences in genome structure. More common name for this species - HIV-1 subtype O [30] . HIV-4 - a rare variant of the virus, discovered in 1986 [31] . The global epidemic of HIV infection is mainly caused by the spread of HIV-1. HIV-2 prevalent mainly in West Africa . HIV and HIV-3-4 does not play a significant role in the spread of the epidemic. In most cases, unless otherwise stated, under the assumed HIV HIV-1

HIV particles

HIV Genome

HIV-1 genome and proteins encoded by them

Genome of HIV-1 HIV's genetic material is presented in two unrelated strings of positive-sense (positivesense, or (+)) RNA . HIV-1 genome has a length of 9.000 nucleotides .The ends of the genome are long terminal repeats (LTR), which control the production of new viruses and can be activated and the proteins of the virus and infected cell proteins. 9 genes of HIV-1 encodes at least 15 proteins [ . pol - encodes enzymes: reverse transcriptase (RT), integrase (IN) and protease (PR). gag - encodes a polyprotein Gag/p55, viral protease fissionable (PR) to structural proteins P6 , P7 , p17 , p24 . env - a protein gp160 , fissionable endoproteazoy Furin at the cell structural proteins gp41 and gp120 . The other six genes - tat, rev, nef, vif, vpr, vpu (vpx in HIV-2) - encode proteins that are responsible for the ability of HIV-1 to infect cells and produce new copies of the virus. Replication of HIV-1 in vitro is possible without gene NEF, VIF, vpr, vpu . However, these proteins are required for full infection in vivo .
]

Replication

The first step of infection is the binding of gp120 to the CD4 receptor of the cell, which is followed by penetration and uncoating.

The RNA genome is then reverse transcribed into a DNA provirus which is integrated into the cell genome.
This is followed by the synthesis and maturation of virus progeny.

HIV-1 Genotypes

There are 3 HIV-1 genotypes; M (Main), O (Outlayer), and N (New)

M group comprises of a large number subtypes and recombinant forms

Subtypes - (A, A2, B, C, D, F1, F2, G, H, J and K) Recombinant forms - AE, AG, AB, DF, BC, CD

O and N group subtypes not clearly defined, especially since there are so few N group isolates. As yet, different HIV-1 genotypes are not associated with different courses of disease nor response to antiviral therapy. However, certain subgroups may be difficult to detect by certain commercial assays.

Schematic of HIV Replication

HIV-1 Replication Cycle

Reverse Transcription Attachment CD4 Uncoating Integration
Integrase
Reverse Transcriptase

Assembly

Budding

CCR5

Maturation
Protease

CXCR4

The genes of Retroviruses with clinical consequences

Table
65-2

=genes common to all retroviruses

Clinical stages classification

1. 2. 3. 4. 5.

Incubation period (stage 1) Stage of primary manifestations (stage 2) Latent stage (stage 3) Stage of secondary manifestations (stage 4) End stage (stage 5)

Incubation stage (stage 1): From the moment of infection until clinical signs of acute infection and / or antibody production (average of 3 weeks to 3 months). Stage of primary manifestations (stage 2): 2 "A" - asymptomatic when clinical manifestations of HIV infection or opportunistic infections are not available, a response to the introduction of HIV is the antibody. 2 "B" - an acute HIV infection without secondary diseases (various clinical manifestations, most of them similar to symptoms of other infections). 2 C" - an acute HIV infection with secondary disease (compared to a temporary decline of T-4 lymphocytes develop secondary diseases - angina, bacterial pneumonia, candidiasis, herpes - a regular, well-treatable). The duration of clinical manifestations of acute HIV infection is usually 2 - 3 weeks.

Latent stage (stage 3): Slow the progression of immunodeficiency. The only clinical sign is swollen lymph nodes, which can not be removed. Duration of the latent stage of 2 - 3 to 20 years or more, on average 6 - 7 years. Has been a gradual decline in T-4 lymphocytes. Stage of secondary diseases (Stage 4): Ongoing HIV replication, resulting in the death of the T-4 lymphocytes and the development of secondary immunodeficiency in the background (opportunistic) diseases, infectious and / or cancer. Symptoms at this stage are reversible, meaning they can pass on their own or with treatment. Depending on the severity of secondary diseases are the following stage. 4 "A" - it is characterized by bacterial, fungal and viral lesions of the mucous and skin, inflammatory disease of the upper respiratory tract. 4 "B" - a more severe and prolonged skin lesions, Kaposi's sarcoma, weight loss, lesions of the peripheral nervous system and internal organs. 4 C" - severe, life-threatening opportunistic infections.

End-stage (Stage 5): The defeat of the organs and systems are irreversible trend. Even adequately performed antiviral therapy and treatment for opportunistic infections are not effective and the patient dies within a few months. Keep in mind that the HIV epidemic in Russia is young and the sick in the later stages are only a small percentage of all Russians living with HIV.

Clinical Features
1. Seroconversion illness - seen in 10% of individuals a few weeks after exposure and coincides with seroconversion. Presents with an infectious mononucleosis like illness. 2. Incubation period - this is the period when the patient is completely asymptomatic and may vary from a few months to a more than 10 years. The median incubation period is 8-10 years. 3. AIDS-related complex or persistent generalized lymphadenopathy. 4. Full-blown AIDS.

HIV classification system

Clinical categories
CD4 cell category > 500/mm3 200- 499/mm3 <200/mm3 A A1 A2 A3 B B1 B2 B3 C C1 C2 C3

(the equivalence between CD4 counts and CD4 % of total lymphocytes is [>500=>29%], [200-499= 14-28%], [<200= <14%] )

Clinical category A

Asymptomatic HIV infection. Persistant generalised lymphadenopathy (PGL) Acute (primary) HIV illness PGL: nodes in 2 or more extrainguinal sites, atleast 1cm in diameter for > 3months

Clinical category B
Symptomatic, not A or C conditions examples include but not limited to :Bacillary angiomatosis, candidiasis, vulvovaginal: Persistant > 1month, poorly responsive to reaction Candidiasis, oropharyngeal, cervical dysplasia, severe or carcinoma in situ Constitutional symptoms e.g fever (38.50c) or diarrhea >1 month The above must be attributed to HIV infection or have a clinical course or management complicated by HIV If weight loss >10% suspect HIV

Clinical category C
Candidiosis esophageal, trachea, bronchi, coccidiodomycosis, extrapulmonary, cryptococcosis, extrapulmonary cervical cancer, invasive Cryptococcosis, chronic intestinal (>5 months) CMV retinitis, or CMV in other than liver, spleen, node. HIV encephalopathy. Herpes simplex with mucocutaneous ulcer >1 month. Bronchitis, pneumonia. Histoplasmosis- disseminated, extrapulmonary isosporiasis, chronic >1 month. Kaposis sarcoma. Lymphoma: burkitts , immunoblastic, primary brownin. M. avium or M. kansasii, extrapulmonary. M.tuberculosis, pulmonary or extrapulmonary. Pneumocystis carinii pneumonia. Pneumonia recurrent (> 2 episodes in 1 year) Progressive multifocal leukocephalopathy. Salmonella bacteremia, recurrent toxoplasmosis, cerebral. Wasting syndrome due to HIV All conditions with HIV (1)CD4 T-lymphocytes count <200/mm 3 (CD4<14%) (2)Pulmonary tuberculosis (3)Recurrent pneumonia (>2 episodes within 1 year) (4)Invasive cervical carcinoma

Natural Course of HIV-1 Infection

Opportunistic Infections
Protozoal pneumocystis carinii (now thought to be a fungi), toxoplasmosis, crytosporidosis candidiasis, crytococcosis histoplasmosis, coccidiodomycosis Mycobacterium avium complex, MTB atypical mycobacterial disease salmonella septicaemia multiple or recurrent pyogenic bacterial infection CMV, HSV, VZV, JCV

Fungal

Bacterial

Viral

Opportunistic Tumours

The most frequent opportunistic tumour, Kaposi's sarcoma, is observed in 20% of patients with AIDS. KS is observed mostly in homosexuals and its relative incidence is declining. It is now associated with a human herpes virus 8 (HHV-8). Malignant lymphomas are also frequently seen in AIDS patients.

Kaposis Sarcoma

Other Manifestations

It is now recognised that HIV-infected patients may develop a number of manifestations that are not explained by opportunistic infections or tumours.

The most frequent neurological disorder is AIDS encephalopathy which is seen in two thirds of cases.
Other manifestations include characteristic skin eruptions and persistent diarrhoea.

Epidemiology
1. Sexual transmission - male homosexuals and constitute the largest risk group in N. America and Western Europe. In developing countries, heterosexual spread constitute the most important means of transmission. 2. Blood/blood products - IV drug abusers represent the second largest AIDS patient groups in the US and Europe. Haemophiliacs were one of the first risk groups to be identified: they were infected through contaminated factor VIII. 3. Vertical transmission - the transmission rate from mother to the newborn varies from around 15% in Western Europe to up to 50% in Africa. Vertical transmission may occur transplacentally route, perinatally during the birth process, or postnatally through breast milk. 4. In 2003, an estimated 4.8 million people (range: 4.26.3 million) became newly infected with HIV. This is more than in any one year before. Today, some 37.8 million people (range: 34.642.3 million) are living with HIV, which killed 2.9 million (range: 2.63.3 million) in 2003, and over 20 million since the first cases of AIDS were identified in 1981.

2007

HIV Pathogenesis

The profound immunosuppression seen in AIDS is due to the depletion of T4 helper lymphocytes. In the immediate period following exposure, HIV is present at a high level in the blood (as detected by HIV Antigen and HIV-RNA assays). It then settles down to a certain low level (set-point) during the incubation period. During the incubation period, there is a massive turnover of CD4 cells, whereby CD4 cells killed by HIV are replaced efficiently.

Eventually, the immune system succumbs and AIDS develop when killed CD4 cells can no longer be replaced (witnessed by high HIVRNA, HIV-antigen, and low CD4 counts).

HIV half-lives

Activated cells that become infected with HIV produce virus immediately and die within one to two days.
Production of virus by short-lived, activated cells accounts for the vast majority of virus present in the plasma. The time required to complete a single HIV life-cycle is approximately 1.5 days. Resting cells that become infected produce virus only after immune stimulation; these cells have a half-life of at least 5-6 months. Some cells are infected with defective virus that cannot complete the virus life-cycle. Such cells are very long lived, and have an estimated half-life of approximately three to six months. Such long-lived cell populations present a major challenge for antiretroviral therapy.

HIV pathogenesis

Laboratory Diagnosis

Serology is the usual method for diagnosing HIV infection. Serological tests can be divided into screening and confirmatory assays. Screening assays should be as sensitive whereas confirmatory assays should be as specific as possible. Screening assays - ELISAs are the most frequently used screening assays. The sensitivity and specificity of the presently available commercial systems now approaches 100% but false positive and negative reactions occur. Some assays have problems in detecting HIV-1 subtype O. Confirmatory assays - Western blot is regarded as the gold standard for serological diagnosis. However, its sensitivity is lower than screening EIAs. Line immunoassays incorporate various HIV antigens on nitrocellulose strips. The interpretation of results is similar to Western blot it is more sensitive and specific.

Immunological markers for HIV

Viral RNA is detectable in the blood from approximately 11 days post-infection. There is a rapid rise in viral RNA levels (viral load) until the immune system responds, after which the RNA levels decrease to a viral set point that is maintained for a prolonged period. HIV DNA and p24 become detectable in the blood from approximately 16 days post-infection. p24 le-vels then become undetectable until the end stage of disease, at which time p24 levels rise. HIV antibodies are detectable from approximately 24 days post-infection

ELISA for HIV antibody

Microplate ELISA for HIV antibody: coloured wells indicate reactivity

Western blot for HIV antibody

There are different criteria for the interpretation of HIV Western blot results e.g. CDC, WHO, American Red Cross.
The most important antibodies are those against the envelope glycoproteins gp120, gp160, and gp41 p24 antibody is usually present but may be absent in the later stages of HIV infection

Western Blot was previously used as a confirmatory test. Antibodies directed against HIV proteins bind to relevant areas on a strip and are detected by the addition of an enzyme-labelled secondary enzyme and substrate. Most laboratories no longer use Western Blot, as it is expensive and labour intensive, and 3rd and 4th generation ELISA assays are more reliable than those previously available. The Western Blot is however a useful tool for differentiating between infections with HIV-1 and HIV-2.

Other diagnostic assays

It normally takes 4-6 weeks before HIV-antibody appears following exposure. A diagnosis of HIV infection made be made earlier by the detection of HIV antigen, pro-DNA, and RNA. However, there are very few circumstances when this is justified e.g. diagnosis of HIV infection in babies born to HIV-infected mothers.

Prognostic tests
Once a diagnosis of HIV infection had been made, it is important to monitor the patient at regularly for signs of disease progression and response to antiviral chemotherapy.
HIV viral load - HIV viral load in serum may be measured by assays which detect HIV-RNA e.g. RT-PCR, NASBA, or bDNA. HIV viral load has now been established as having good prognostic value, and in monitoring response to antiviral chemotherapy.

HIV Antigen tests - they were widely used as prognostic assays. It was soon apparent that detection of HIV p24 antigen was not as good as serial CD4 counts. The use of HIV p24 antigen assays for prognosis has now been superseded by HIV-RNA assays.

MONITORING DISEASE PROGRESSION AND RESPONSE TO THERAPY

Monitoring of HIV-1 infection should not rely on any single marker. The patients clinical condition needs to be considered in addition to the HIV-1 viral load and the CD4+ T lymphocyte count.

HIV-1 Viral Load

Quantitative HIV-1 RNA testing (HIV-1 viral load) detects and quantifies the HIV-1 RNA in plasma. The viral load is given as a value representing RNA copies per millilitre, as well as a log value, e.g. 100 = 2 log, 1000 = 3 log, 10000 = 4 log etc. The log value provides an easier number to work with and better indicates significant changes in viral load. This test serves a number of purposes: It determines the viral setpoint and acts as a long-term predictor of prognosis. It acts as a guide for deciding when to initiate HAART. Certain guidelines recommend starting treatment if the viral load is greater than 100 000 copies/ml, unless the patient has been recently infected and has not yet reached a viral setpoint. It is a measure of viral response to therapy. An acceptable response to therapy is considered to be: o a viral load reduction of more than 1 log from the baseline value after 6-8 weeks of therapy. o a viral load of 5000 copies or less after 12 weeks of therapy. o a viral load below detection limit of the assay after 12-24 weeks of therapy. It is an indicator of the development of viral resistance to therapy: o Primary failure of therapy is indicated by a viral load that does

not decrease as expected after initiation of HAART. o Secondary failure of therapy refers to an increase in the viral load of 1 log or greater on two separate specimens at least two weeks apart after an initial adequate response to HAART 13, 14, 15. It must be noted that the HIV-1 viral load should not be used for the diagnosis of HIV-1 infection. This is because low false positive results (usually less than 2000 copies/ml) can occur with quantitative HIV-1 RNA testing. Such results can lead to confusion and misdiagnosis.

CD4+ T lymphocyte count

For completeness, a mention must be made of the CD4+ T lymphocyte count. The CD4+ T lymphocyte count declines with acute infection. Once the viral load has reached its setpoint, the CD4+ T lymphocyte level increases, but rarely returns to pre-infection levels. As the immune system becomes exhausted, the CD4+ T lymphocyte count begins to decrease. There are a number of different guidelines available recommending when to start HAART. The World Health Organisation recommends initiating HAART when the CD4+ T lymphocyte count is less than 200 cells/ml or higher if the patient has an AIDS-defining illness. The South African National Antiretroviral Treatment Guidelines agree with the WHO. The Southern African HIV Clinicians Society recommends initiating HAART as above, but also advises initiation if the CD4+ T lymphocyte count is 200-350 and has been shown to be decreasing rapidly. North American and European Guidelines offer HAART for CD4+ T lymphocyte counts less than 350 cells/ml 16. The CD4+ T lymphocyte count should be tested after diagnosis of HIV infection. If HAART is not yet indicated, the CD4+ T lymphocyte count should be repeated every 3-6 months. Once on treatment, the CD4+ T lymphocyte count should be monitored every 6 months. A patient requiring HAART should be managed by a medical practitioner with experience in the care of patients with HIV.

Anti-Retroviral Susceptibility Testing

It is now generally accepted that anti-viral susceptibility testing should be a routine part of the management of HIV-infected patients. It is reported that the outcome would be better if the results are interpreted by an expert in this area. There are two types of antiviral susceptibility assays:

Phenotypic very difficult and expensive to carry out. Thought to give a better idea of the actual situation in vivo. Genotypic the RT and Protease genes are sequenced. This can be done in-house and the results interpreted automatically by the HIV sequence database in the US. Commercial systems (Trugene, ABI and others) available which relies on their own database and interpretation by a panel of experts that meet regularly.

Treatment

Zidovudine (AZT) was the first anti-viral agent shown to have beneficial effect against HIV infection. However, after prolonged use, AZT-resistant strains rapidly appears which limits the effect of AZT.

Combination therapy has now been shown to be effective, especially for trials involving multiple agents including protease inhibitors. (HAART - highly active anti-retroviral therapy)
The rationale for this approach is that by combining drugs that are synergistic, non-cross-resistant and no overlapping toxicity, it may be possible to reduce toxicity, improve efficacy and prevent resistance from arising.

Anti-Retroviral Agents

Nucleoside analogue reverse transcriptase inhibitors e.g. AZT, ddI, lamivudine Non-nucleoside analoque reverse transcriptase, inhibitors e.g. Nevirapine Protease Inhibitors e.g. Indinavir, Ritonavir Fusion inhibitors e.g. Fuzeon (IM only) HAART (highly active anti-retroviral therapy) regimens normally comprise 2 nucleoside reverse transcriptase inhibitors and a protease inhibitor. e.g. AZT, lamivudine and indinavir. Since the use of HAART, mortality from HIV has declined dramatically in the developed world.

Prevention

The risk of contracting HIV increases with the number of sexual partners. A change in the lifestyle would obviously reduce the risk. The spread of HIV through blood transfusion and blood products had virtually been eliminated since the introduction of blood donor screening in many countries. AZT had been shown to be effective in preventing transmission of HIV from the mother to the fetus. The incidence of HIV infection in the baby was reduced by two-thirds. The management of health care workers exposed to HIV through inoculation accidents is controversial. Anti-viral prophylaxis had been shown to be of some benefit but it is uncertain what is the optimal regimen. Vaccines are being developed at present but progress is hampered by the high variability of HIV. Since 1987, more than 30 HIV candidate vaccines have been tested in approximately 60 Phase I/II trails, involving more than 10,000 healthy volunteers. A phase III trial involving a recombinant gp120 of HIV subtype B was reported in Feb 2005 to be ineffective in preventing HIV infection.