1. Lecture 5: serology Human Immunodeficiency Virus Dr. Dalia Galal

2. Human Immunodeficiency Virus Acquired Immunodeficiency syndrome first described in 1981 Belong to the lentivirus subfamily of the retroviridae Enveloped RNA virus, Genome consists nucleotides 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

3. HIV particles

4. 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.

5. 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 which is integrated into the cell genome. This is followed by the synthesis and maturation of virus progeny.

6. Schematic of HIV Replication

7. Clinical Features 1. Seroconversion - seen in 10% of individuals a few weeks after exposure and coincides with seroconversion. 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.

8. Opportunistic Infections Protozoalpneumocystis carinii toxoplasmosis, crytosporidosis Fungalcandidiasis, crytococcosis histoplasmosis, coccidiodomycosis BacterialMycobacterium avium complex atypical mycobacterial disease salmonella septicaemia multiple or recurrent pyogenic bacterial infection Viralcytomegalovirus (CMV), herpes simplex virus (HSV)

9. Opportunistic Tumours The most frequent opportunistic tumour, Kaposi's sarcoma, is observed in 20% of patients with AIDS. KS is observed mostly in sexuals and it is now associated with a human herpes virus 8 (HHV-8). Malignant lymphomas are also frequently seen in AIDS patients.

10. 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 which is seen in two thirds of cases. Other manifestations include characteristic skin eruptions and persistent diarrhoea.

11. Epidemiology 1. Sexual transmission - male homosexuals and constitute the largest risk group. 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. Haemophiliacs were one of the first risk groups to be identified: they were infected through contaminated factor. 3. Vertical transmission - the transmission rate from mother to the newborn varies from around 15% up to 50%. Vertical transmission may occur transplacentally route, perinatally during the birth process, or postnatally through breast milk.

12. 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, AIDS develop when killed CD4 cells can no longer be replaced (witnessed by high HIV-RNA, HIV-antigen, and low CD4 counts).

13. 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.

14. Practical Laboratory diagnosis of HIV

15. Practical Detection of antibodies Screening tests Enzyme immunosorbent assays (EIAs) Simple/rapid immuno-diagnostics assays Confirmatory or supplemental tests Western blot (WB) Alternatives to confirmatory tests Repetitive EIA or rapid assays/ Current HIV technologies

16. Practical This term describes a variety of assays that are based on the binding of antibodies with their antigens and the detection of this reaction using a component conjugated with an active enzyme. This enzyme acts on its substrate to produce a colour change.Test results are measured by measuring this colour. Four immunologic principles Indirect Competition Sandwich Immuno-capture EIAs (Enzyme Immunosorbent Assays)

17. Practical

18. Competitive EIA A measured amount of known enzyme-labeled component (being measured) is added to the reaction at the same time patient sample is added. The labeled component therefore competes against the unlabeled component in the patient sample for binding sites. Results Negative Reaction has color change Positive Reaction no color change

19. Practical

20. This test supplied as kit Easy to Perform Use to screen large number of sample Sensitive Specific Cost effective Reason for EIA

21. Practical MULTIPLE PREGNANCY MULTIPLE TRANSFUSION AUTO IMMUNE DISORDER CHRONIC HEPATITIS, CHRONIC ALCOHOLIC HBV VACCINATION ANTIBODY TO POLYSTERENE Source of False Positive Results

22. Simple/rapid immuno-diagnostics assays One class of rapid tests is the "dot blot" or "immunoblot"; they produce a well circumscribed colored dot on the solid phase surface if the test is positive. Most of these rapid assays now incorporate a built-in control that indicates that the test was performed correctly. This control is an anti-human immunoglobulin that binds any immunoglobulin in the sample and produces a separate indicator when all reagents are added appropriately. In addition, several varieties are available that include two "dots", which allow the differentiation of HIV-1 Practical

24. Advantages of Rapid HIV Antibody Tests Fast (10-20 min) and simple Typically Inexpensive ($1-2/test) Immediate result for subject Whole blood can be used No equipment, refrigeration required No electricity required No multiple timing steps Built in controls Practical

25. Western Blot (Immunoblotting) Solid-phase EIA with immobilized viral antigens to detect antibodies to specific HIV proteins. Practical

26. Principle AIDS is caused by at least 2 etiological agents HIV-1 & HIV-2 Inactivated and denatured protein of HIV-1 are fractioned by polyacrylamide gel electrophoresis Protein bands are transferred into nitrocellulose strips HIV-1 sample diluted with buffer are then incubated with the strip Conjugate peroxidase labeled anti human IgG is added It will bind to the antibodies already bound to the strip Chromogen is then added forming color reaction Reaction is then stopped by aspiration and reaction Practical

27. Creating Western Blot Strips HIV lysate proteins are separated by size using gel electrophoresis Proteins are transferred (blotted) onto the surface of a membrane Strips are incubated with patient serum and antihuman IgG conjugated with an enzyme (and chromagen) The membrane is cut into strips Practical

28. HIV Western Blot Banding Pattern envgp160 gp120 gp 41 gagp55 p18 p24 polp65 p51 p31 Interpretation of Results Negative: No bands present Positive: 2 ENV band present (WHO Guidelines) Indeterminate Any bands present but do not meet criteria for positive Practical

29. When should WB be used? Western Blot assay should not be used as a screening test. WB should be viewed as a supplemental test which can be used to confirm positive results obtained from EIA. HOWEVER: Specificity is less than that of EIA A significant number of indeterminate blots are seen in low risk populations Practical

30. Advantages of WB Specific interaction of antibody and antigen can be directly visualized. Disadvantages of WB Technically demanding Expensive Subject to interpretation Presence or absence of bands Intensity of those bands Practical