This web text will specifically focus on HIV-1 and HIV-2.

1	Background / History

• HIV is thought to have originated in non-human primates in Sub-Saharan Africa with the transferal to humans sometime in the 20th Century.
• An epidemic of HIV was first recorded on 5th June 1981.
• Two species of HIV are currently prevalent globally, HIV-1 and HIV-2, both of which are likely to have originated from Western and Central Africa.
• Both species of HIV have crossed the boundary or jumped species from animal to man which consitutes a Zoonosis.
• HIV-1 is broadly understood to have originated from wild Chimpanzees in the Cameroon in Africa, having evolved from the Simian Immunodeficiency virus, (SIV).
• HIV-2 is thought to have originated from the Sooty Mangabey monkey, (Cercocebus atyus), an old world monkey from Guinea-Bissau, the Cameroon and Gabon.
• The Hunter theory - This goes someway towards explaining how SIV could have been transferred from animal to man, in other words, a zoonosis. Chimpanzees infected with HIV-1 may have bitten hunters or the dead animals blood may have come into contact with hunters open wounds. It has been postulated that the HIV-1 jump from Chimpanzee to man occured sometime during the French colonial period in the Cameroon as a result of slave labour lowering the immunity of slaves. This enabled the speedy transmission of HIV-1. 
• Oldest recorded case of AIDS - between 1955 and 1957 from a British Printer who had travelled in the Navy and suffered symptoms of Auto Immune Disease Syndrome, (AIDS). At this time AIDS was not known.
• 1959 - A Congolese man's preserved blood sample was later confirmed to contain HIV-1. 
• Transmission - Owing to the spread of HIV-1 virus among homosexual men in California, USA in 1981-2 the disease was termed, "Gay related Immune Deficiency", (GRID).
• By August 1982  GRID was renamed Auto Immune Deficiency Syndrome, (AIDS) owing to the increasing heterosexual transmission of the virus.
• After being named, Human T-Lymphotropic Virus Type III, (HTLVIII) in the USA and Lymphadenopathy Associated Virus, (LAV) in France, taxonomists ruled in May 1986 that the generic term for the virus should be, Human Immunodeficiency Virus, (HIV).        

 

2	Classification

• Lentiviruses, for example, (HIV) have long incubation periods, and are classified within the Lentiviruses, "Lenti", being Latin for slow.
• Retroviruses are enveloped viruses possessing an RNA genome, replicating via a DNA intermediate.
• HIV-1 is more virulent and pathogenic than HIV-2 and is more easily transmitted than HIV-2, which has been until recent years, confined to West Africa, (see epidemiology).
• Lentiviruses - Viruses in this genus share common characteristics, for example;
  • not directly linked to malignancy.
  • cause infections which are slow, having a lengthy incubatin period or may cause no illness at all, (asymptomatic).
  • Similar accessory and functional genes down to sequence level.
  • All Lentiviruses are exogenous.
• Retroviridae family - these viruses share common characteristics, for example;
  • replication based on reverse transcriptase.
  • common structure of virions, identified by Electron Microscopy, (E.M).
  • Instability of Genome structure - altered by recombination and mutation.
  • Similar genome structure; positive sense, single stranded RNA.
  • Common structural genes, for example, env, pol, pro and gag.
• Retroviruses, for example, (HIV) can also be classified by using criteria based on molecular biology
  • Host cell receptors.
  • detailed structure of the virion.
  • endogenous / exogenous nature of the virus.

 

Table 1. Classification scheme for HIV

Classification	HIV
Family	Retroviridae
Genus	Lentivirus
Species group	HIV-1, HIV-2

 

 

 

 

 

 

3	Morphology

• Virion Size - 80-100nm in diameter
• Envelope - HIV is an enveloped virus through which 72 glycoprotein spikes protrude. These are densely dispersed across the surface of the virion. The envelope protein, (env) is composed of two subunits, the first of which is the outer glycoprotein Knob, (gp120) and a transmembrane segment, (gp41) connects the Knob to the viral envelope. Surface projections are approximately 8nm in length.
• Receptor binding site - The site that binds to CD4 receptors is located on gp120 plus the V3 loop.
• Matrix protein - This is denoted by p17 a protein which lines the inside of the lipid envelope.
• Cellular Proteins - these include the major Histocompatability Complex, (MHC) proteins for both class I and class II antigens. These are located in the viral envelope.
• Viral capsid -  for HIV-1 the viral protein capsid is icosahedral and is called, (p24).
• Protein core - the protein core, which can be described as vase, coffin or cone shaped, is contained within the viral capsid.
• Ribonucleic Acid, (RNA) molecules - within the protein core there are two, (diploid), molecules of positive sense, single stranded RNA to which are attached copies of; reverse transcriptase, integrase and protease enzymes. The abbreviation for the diploid RNA is p7 and p9.
• Lipids - Lipids are located in the envelope, each virion containing 35% lipid by mass. Viral lipids are derived from host cell plasma membranes.
• Proteins - 60% of the HIV virion is protein.
• Polyamines  - Carbohydrates form 3% of HIV virions.  

Fig.1. A Diagram to show the Structure of HIV.

 

Genome Structure

• Genome size -  approximately 10kb for HIV-1.
• oncogenic gene - the HIV-1 genome contains no oncogenic,(onc) genes. However, other viruses in the family contain the onc gene.
• Control genes - HIV-1 possesses specific genes that improve viral replication, for example;
  • tat  - transactivates transcription.
  • rev - transports complete unspliced mRNA. 
  • vif  - promotes virus infectivity.
  • nef - promotes virus infectivity.

Table 2. Functions of HIV Genes.

Gene	Virus Protein	Function
env	Envelope glycoprotein	Structural
gag	Matrix protein, p17	Structural
	Capsid protein, p24	Structural
	Nucleocapsid, p6, p7	Structural
pol	Reverse Transcriptase	Enzyme
	Integrase	Enzyme
	Protease	Enzyme
tat		Transcription transactivator
rev		Transports uncoupled RNA
vif		Viral infectivity promoter
nef		Viral infectivity promoter
vpu		Promotion of virus release
vpr		Promotion of viral entry into cell nucleus
vpx		Promotion of virus replication

 

 

4	Replication / Life Cycle

• Attachment - The HIV-1 gp120 binds to the CD4 receptor located on T-helper lymphocytes facilitating a structural change in viral envelope glycoproteins thus allowing fusion of both viral and cellular proteins. HIV-1 also infects other cells including, B Lymphocytes, some nerve cells, for example, dendritic cells. HIV-1 can also infect macrophages, etc. This may be because these cells also possess CD4 receptors.
• Co-receptor - HIV-1 viral strains that are T-cell tropic may utilise the chemokine receptor, CXCR-4 as a co-receptor. Macrophage tropic, m-tropic strains may use the chemokine, CCR-5 as a co-receptor. It is understood that both co-receptors, CXCR-4 and CCR-5 facilitate the efficient transmission of HIV-1 to the central nervous system, (CNS).
• Host Cell Penetration - This may be pH independant and endocytosis is assisted by gp41 where the virion core enters the cell cytoplasm and viral RNA is then transported to the host cell nucleus.
• Transcription - Once the ss+RNA has entered the host cell nucleus, the reverse transcriptase transcribes viral ss+RNA to produce a copy of the viral dsDNA. This DNA is circular in form and before integration it becomes linear.   
• Integration - This occurs in dividing lymphocytes, where reverse transcription and integration of DNA provirus into host cell chromosomal DNA take place, mediated by integrase. Integrase has been shown to be essential for retroviral replication. Although, integration is not necessary for viral replication it is necessary for viral latency. The viral DNA can remain in the host cell chromosome for many years and it's activation can depend on many factors.
• Transcription -  There are cell transcription factors, for example, NF-KB having the function of binding to regulatory sequences in the proviral long terminal repeat, (LTR). This stimulates the formation of tat  which sequentialy binds to the tar region of the LTR amplifying the transcription of all viral genes.
• Translation of Virion Proteins -The rev gene is one of the controling genes for the expression of complete and spliced viral mRNA's. A pre production of multiply spliced mRNA's for the regulatory proteins, tat, rev and nef are available at an early stage in the infection. There is then a later production of singly spliced and unspliced mRNA for the Structural genes, gag, env and pol plus the regulatory genes vif, vpr and vpu.
• Virion Assembly - env proteins are transported to the host cell plasma membrane whilst the viral genome assembles in the host cell cytoplasm. By way of N-terminal myristylation and host protein to viral protein interactions, gag precursor molecules assemle on the inner surface of the plasma membrane.
• Budding and Maturation - Progeny virions are exocytosed through the host cell plasma membrane. Once free of the host cell, virions then undergo a process of maturation.

Fig. 2. Diagrammatic representation of the life cycle of HIV 

 

 

5	Clinical Disease

• Incubation period - HIV antibody can be detected 2-4 weeks after exposure.
• Primary Illness - This can develop 6-8 weeks after exposure. Patients usually recover completely after 3 weeks after suffering any of the following symptoms;
  • Lethargy
  • fever
  • lympadenopathy
  • sore throat
  • mucosal ulcers
  • myalgia
  • maculopapular rash
• There are many AIDS defining conditions, (see below)
  • Candidiasis
  • cervical carcinoma
  • Coccidioidomycosis
  • Cryptococcosis
  • CMV / CMV Retinitis
  • Encephalopathy
  • HSV
  • Histoplasmosis
  • Isoporiasis
  • Kaposi's Sarcoma
  • Burkitt's lymphoma
  • Mycobacterial infection
  • Toxoplasmosis
  • Progressive multifocal leucoencephalopathy
  • Wasting syndrome L
• Laboratory anomalies associated with AIDS
  • High levels of HIV viral RNA
  • Circulating CD4 lypmocytes reduced
  • CD4:CD8 ratio reversed
  • raised levels of liver enzymes
  • Lymphopaenia - atypical reactive lymphocytes on blood film
  • Thrombocytopaenia
• Clinical Latency - After initial exposure and subsequent infection AIDS can take up to and beyond 10 years to manifest itself. Many factors affect disease progression.         

                                                            

Classification of HIV Disease Progression

Absolute CD4 Count (1mm3)	Conditions (A)	Conditions (B)	Conditions (C)
>500	A1	B1	C1
200-499	A2	B2	C2
<200	A3	B3	C4

A - Asymptomatic or persistent generalised lymphadenopathy or acute seroconversion

B - HIV related conditions

C - Clinical conditions

 

• Death rate - most deaths associated with HIV are a result of opportunistic infections or malignancies associated with the progressive failure of the immune system.
• Immunocompromised AIDS patients - Such patients can easily contract Tuberculosis, Pneumonia, esophagitis and toxoplasmosis, etc.
• Tumour related infections - AIDS patients can become infected with oncogenic viruses, for example, EBV, Kaposi's Sarcoma-associated Herpes Virus, (KSHV) or HPV. 

 

 

6	Pathogenicity

• HIV Transmission - HIV-1 and HIV-2 enter the bloodstream via a number of routes;
  • Sexual intercourse
  • anal intercourse
  • IVDA
  • Blood Transfusion / transplant surgery
  • Needlestick injuries
  • Vertical transmission, (mother to child)
• Sexual transmission - HIV infected lymphocytes in semen and other body fluids can cause infections if there is a weakness in the endothelial lining of the rectum or vagina.
• Clinical Infection - HIV-1 and HIV-2 may infect CD4+ lymphocytes, macrophages and dendritic cells, etc in the first stages of infection.
• Cytopathis effect - HIV is highly cytopathic and may cause cell fusion producing giant syncytia and eventually cell death in vitro. HIV glycoprotein can interact with CD4 receptors on uninfected cells to greatly enhance this effect. No synctia have been found in AIDS patients in vivo.
• Bystander cells - This is a term given to CD4+ lyphocytes that die but show no apparent signs of HIV infection giving rise to the hypothesis that there may well be an autoimmune component in HIV pathogenesis.
• Replication - It has bee proposed that high rates of viral replication occur post HIV infection with significant lympocyte destruction and cellular regeneration.
• HIV genome Replication - This is enhanced in antigen stimulated T cells. Persons presenting with infections that stimulate T cell production are at a greater risk of developing AIDS.
• Macrophages - These cells act as a reservoir of HIV as well as T Lymphocytes, etc.
• Opportunistic Organisms - These are allowed to proliferate owing t the progressive destruction of CD4+ Helper cells.

 

7	Immunology

• Host Response to HIV-1 Infection (see Fig. 3)
• Anti env antibodies - There is an abundance of anti env antibodies in the bloodstream of an HIV-1 infected person which last the duration of the the infection. Diagnostic kit manufacturers therefore target anti-env antibodies as this can provide an accurate diagnosis. These antibodies are slowly raised from the onset of infection and continue to proliferate throughout the course of infection.
• P24 Potein - These core proteins rise abruptly very early on in the HIV-1 infection and sharply decline as the onset of clinical symptoms appear at btween 1-2 months.
• Cytotoxic Response - Host antibodies give rise to a cytotoxic response against HIV-1 infection plus a host restricted Cytotoxic T-cell response to the structural proteins gag and env. Cytotoxic T-cell response to gag protein may confer antiviral immunity. Anti-env antibodies against HIV-1 and HIV-2 can be utilised for distinguishin between the two forms of HIV.   

 

 

Product	Product Code	Details
HIV 1&2 ELISA	CB7/003	96 tests

 

8	Epidemiology

• HIV has many routes of transmission via blood, tissue and body fluids, (see below);
  • Blood transfusion
  • Childbirth
  • Intravenous Drug Abuse, (IVDA)
  • Percutaneous needlestick
  • anal intercourse
  • vaginal intercourse
  • oral sexual activity
  • Organ transplantation
  • Vertical transmission, (childbirth / breast feeding)
  • saliva, (very slight risk)
• Transmission incidence - most cases of HIV transmission are through heterosexual, unprotected sexual intercourse leading to transmission through mucosal membranes. Sexually transmitted diseases, (STI's) can facilitate transmission of HIV owing to disruption of the normal epithelial barrier.
• Control - The most effective way to reduce HIV transmission in sexual practice is by the use of condoms. However, some religions may not advocate the use of condoms. This can therefore increase the risk of contacting HIV and other STI's.
• Death rate - 2.1 million people died of AIDS in 2007, 0.33 million of which were under the age of 15.
• Global incidence - It has been estimated that 32.2 million people were reported to be infected with HIV in 2007 of which 2.5 million people may have contrated HIV in 2007. Of these 2.5 million HIV sufferers, 0.42 million people are beleived to be children.
• Incidence - Sub Saharan Africa has the highest level of HIV incidence globally with approximately 68% of all known HIV infected persons. Around 76% of all AIDS related deaths occur in this region. It is estimated that South East Asia has the second highest incidence of HIV of 18% with approximately 300,000 deaths from AIDS in 2007.
• Life expectancy - This is dramatically reduced in the worst affected areas, described above, owing to the lack of treatment available.  

Fig. 4. Global prevalence of HIV

 

  

9	Laboratory Diagnosis

• HIV testing - There are many HIV positive persons unaware that they are infected and less than 1% of the sexually active, urban population in Africa have been tested for HIV.
• General HIV Testing Scheme;
  • Screening - ELISA for HIV-1 antibody
    • positives confirmed with ELISA.
    • If still positive, confirmed with Western Blot and or molecular technique, (PCR). If positive the person is deemed to be HIV positive.
    • Intermediate results, confirmed with a molecular technique, (PCR) or Immunofluorescent Antibody technique, (IFA).
• Antibody tests, (serology). Cannot normally detect antibody before 1 month after exposure
  • ELISA kits for antibody testing, (usually up 100% sensitive and 100% specific). These diagnostic kits are low cost, reliable and automatable and the most frequently used screening test for HIV.
  • Western Blot tests - These are more expensive and more accurate than ELISA tests and are used primarily for confirmation of a positive ELISA result.
  • RIPA / IFA / Passive Particle Agglutination tests - can all be used as confirmatory tests to confirm positive HIV results obtained by ELISA.
• Antigen tests - (can be detected before antibody detection tests).
  • Can be used to detect HIV only a few days after exposure.
  • Molecular tests - used to detect viral RNA which provides an accurate, highly sensitive, specific and reliable result.
• Virus Isolation - Patient lymphocytes are cultured in fresh, healthy blood with compatible culture lines, for example, T-Lymphomas to test fro the presence of HIV in cells. A reverse transcriptase assay or serological test is performed on the cells. The test is time consuming, taking up to 4 weeks and is only 70% to 90% successful.
• Prognostic tests - aids viral diagnosis and HIV disease progression;
  • CD4 counts
  • viral load 

 

10	Recent Developments

New Antivirals

• Fuzeon, (T-20), New, FDA approved antiretroviral drug
  • significant improvement for multi drug resistant HIV patients
  • Needs to be injected
  • Increases possibility of more effective combination therapy
• Maraviroc
  • Inhibits the CCR5 receptor on human cells
  • maybe possible negative immune consequences
  • Requires expensive profile test to be carried out to identify drug effectiveness

Gene Therapies in development

• VRX 496
  • This is a genetic factor
  • Infects T-Cells and attacks the HIV genetic code
  • Phase I Clinical trials, (05/08)
• M870
  • Gene therapy product
  • Causes T-Cells to become HIV resistant
  • Phase I clinical trials, (05/08)
• HGTV43
  • Antisense therapy
  • May make T-Cells HIV resistant
  • Phase I clinical trials, (05/08)
• RRZ2
  • This is a Ribosome
  • Attacks tat gene of HIV
  • Phase II clinical trials, (05/08)

Further developments

• Modified CD4 / CD8 Cells
  • Genetically modified
  • Blocks HIV attachment
• Immune Stimulators - Increase the bodies immune response to HIV.
• Cytokines - The use of chemical messengers to increase the bodies immune response.
• Nucleoside Analogue Reverse Transcriptase Inhibitors - Prevents HIV multiplying by blocking the reverse transcriptase enzyme.
• Protease Inhibitors - Inhibits the construction of new virions.
• Maturation inhibitor, (PA-457)
  • New drug action
  • Disrupts viral progeny budding
• Integrase inhibitors - Prevent HIV entering cells

 

 

 

 

11	Prevention / Control

Prphylaxis

• Prevention - There are many methods of preventing HIV transmission, see below;
  • Screening blood donors for HIV and other STI's
  • Condoms and Femidoms - provide an effective barrier
  • Chemoprophylaxis - Administered to prevent opportunitic infections
• Chemoprophlaxis - recommended for immunocompromised patients for prevention of opportunistic infections, for example;
  • CMV
  • Fungal infections
  • Mycobacterium avium complex
• Chemoprophylaxis - Recommended for immunosupressed individuals with CD4 countsof <200
• Biosafety categorisation - HIV is categorised as a biosafety 3 level pathogen and the following safety guidelines must be adhered to;
  • Avoidance of breakable glassware - cutting risk!
  • HIV propogation only in laboratories that can handle their containment
  • Use of Class II biosafety cabinets
  • Disposable gloves, gowns and masks should be worn
  • Minimum laboratory traffic where HIV samples are being manipulated
  • In case of spillage - liberal clean up and disinfection with a 10-30% Chlorine based disinfectant.
• Education - Aimed at high risk groups in endemic areas, for example prostitutes in Sub Saharan Africa, etc
  • Distribute clean needles to IVDA's to avoid transmission of HIV and other STI's
  • Distribute condoms to Prostitutes
  • Guidelines for sterilisation for, (Dentists / Tattoo parlours)
  • Advice on safe sex for heterosexuals and homosexuals
• HIV Vaccine - there is currently no effective, licensed vaccine available for HIV. There are plausable reasons for this;
  • Epitopes of the viral envelope are too variable
  • Functionality of certain epitopes of gp120 are masked by;
    • Glycosylation
    • Trimerisation
    • Receptor induced conformation changes
• AIDSVAX - This potential vaccine was trialled in North America and Thailand and acted upon gp120 but it failed owing to the following;
  • Trial vaccines produced an immune response but several frequent booster vaccines had to be administered to maintain the level of gp120 circulating antibodies
  • Many potential vaccines for HIV target the viral protein coat, for example, gp120 and gp160
• Current Vaccines in development - There are some potential HIV vaccines in Phase I clinical trials looking at;
  • Peptide responses
  • Lipopeptide responses
  • DNA responses
  • P24 responses
• Cost of HIV vaccine development - It is estimated that there is an annual spend of 682 million dollars on AIDS vaccine development.

 

12

Treatment

Antiretroviral therapy - One of the most effective drug treatment regimens currently available is, highly active antiretroviral therapy, (HAART).

Chemotherapy - HAART therapy is used as a long term, effective and suppressive treatment for AIDS patients. There is still no complete cure for HIV.

HIV cannot be removed from various intercellular pools in the body and drugs can produce serious side effects in patients.

HAART therapy - this is combination therapy in which 5 different classes of drugs are used in the U.K. 

Most effective antiretroviral drugs - the two most effective antiretroviral drugs are;

• Reverse transcriptase inhibitors
• Protease inhibitors

Protease inhibitors

• Act competitively on the virus aspartyl protease enzyme
• This enzyme assists with viral completion in preparation for exocytosis from the host cell
• However, HIV is becoming immune to some of these drugs, for example, 
  • Amprenavir
  • Atazanavir
  • Ritonavir

Reverse Transcriptase Inhibitors

• Nucletide analogues
  • Binds to viral DNA causing the DNA chain to become terminated
  • Do not require intracellular phosporylation for action, for example, Tenofovir
• Nucleoside analogues
  • Act in a similar manner to nucleotide analogues
  • Require phosporylation in the cell to become active
  • One of the first HIV treatments available
  • There are problems associated with mitochondrial activity and lactic acidosis with
    • Abacavir 
    • Didanosine
    • Stavudine
    • Zalcitabine
    • Zidovudine
• Non nucleoside analogues
  • Bind directly to reverse transriptase
  • Long half life / bioavailability
  • Innefective against HIV-2
  • Cross resistance to drugs is high
  • Side effects - rashes / raised liver enzymes common in;
    • Efavirenz
    • Nevirapine

Fusion Inhibitors

• Enfurvitide is currently the only drug licensed in the U.K
• It inhibits GP41 which mediates fusion of HIV with the target cell
• Synergistic efect with nucleoside analogues and protease inhibitors
• Administered subcutaneously
• Side efects are limited to the site of infection owing to the extracellular method of action, for example, Enfurvitide

13	Acknowledgements

 

14	References

 

15

Websites

en.wikipedia.org/wiki/HIV

www.hiv.com

hivinsite.ucsf.edu

www.hivsymptoms.org

www.cdc.gov/hiv/

www.hiv.lanl.gov/

www.medicinenet.com/human_immunodeficiency_virus_hiv_aids/article.htm

www.nhsdirect.nhs.uk/articles/article.aspx?articleID=196

www.webmd.com/hiv-aids/default.htm

www.labtestsonline.org/understanding/conditions/hiv.html

www.aidsinfo.nih.gov/

www.niaid.nih.gov/factsheets/hivinf.htm

www.mayoclinic.com/health/hiv-aids/DS00005/DSECTION=2

www.kidshealth.org/teen/sexual_health/stds/std_hiv.html

www.emedicinehealth.com/hivaids/page3_em.htm

www.hc-sc.gc.ca/dc-ma/aids-sida/index_e.

16	Disclaimer

The information contained in the Education Series text was obtained through collaboration with laboratories identifying and diagnosing HIV as well as scientific publications and relevant text books. We believe the information specified in this text to be correct although it is the responsibility of the participant to confirm information validity. Cosmos Biomedical Ltd claim no responsibility for misinterpretation of information. Copyright ^© Cosmos Biomedical Ltd, 2006.

17

Copyright

The MES Scheme, which includes question cards and web text is both Copyrighted and Trademarked and should not be reproduced under any circumstances in any way and at any time now or in the future without the express permission of Cosmos Biomedical Ltd. Only persons registered by Cosmos Biomedical Ltd are legally allowed to participate in the MES Scheme.  

Copyright ^© Cosmos Biomedical Ltd, 2006.

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