Isolation of HIV from AIDS patients has helped to determine etiologic association of HIV and AIDS. PBMCs from healthy donor are stimulated with medium containing PHA-P, which activates the T-cells and IL2 receptors. After three days, these activated cells are co-cultivated with an equal number PBMCs from the patient. The cultures are continued for 28 days. Fresh PHA-P stimulated cells are added to the co-culture once a week. The co-culture supernatant is tested once a week for p24 antigen production, a marker used for monitoring  HIV-1 replication. If culture supernatant is positive for p24 antigen on two subsequent weeks, the cultures are continued further, otherwise discontinued.

Procedure:

Stimulation of normal lymphocytes with PHA-P (Phytohaemagglutinin-phosphate)

1. Quickly thaw Normal lymphocytes stored in Liquid N2 and wash twice with washing medium (2%RPMI).
2. Resuspend the cell-pellet in 1ml PHA-P medium and take cell count using trypan blue and Neubauer’s chamber.
3. Make up the volume with PHA-P medium to get 1-2x106 cells/ml and transfer the cell suspension to T-25 flask. Incubate the cultures in presence of 5% CO2 atmosphere at 370C.
4. After 48-72 hr of incubation, use the stimulated cells for virus isolation by co-cultivation method.(The cells are referred to as PHA Blasts.)

VIRUS ISOLATION (CO-CULTIVATION METHOD)

1. Take 40 x 106 fresh PHA-P stimulated normal PBMCs in two 50ml tubes (20 x 106 PHA blasts per tube).
2. Centrifuge at 1200 rpm for 10 min. to pellet the cells.
3. Add 500 µl of culture supernate (cs) from primary culture on each pellet. (select the earliest PI day showing the p24 Ag detection test’s result O.D. greater than 1).
4. Keep at 370C in presence of 5% CO2 overnight to allow adsorption to take place (in a 50 ml tube with loose cap).
5. Next morning, wash the cells twice with 10 ml washing medium so as to remove the unadsorbed viral particles, if any.
6. Transfer to two T-75 flasks with 20 ml IL-2 medium each.
7. Collect the supernate for 0 PID p24 Ag detection test.(This result must be negative)
8. Test for the presence p24 antigen on 4 PID.
9. If it is positive for p24 antigen (OD greater than 1), scale up the culture volume to 40ml in each flask. Feed the culture with 20x106 PHA Blasts.

VIRUS ISOLATION (CO-CULTIVATION METHOD)

Procedure:

1. Take 40 x 106 fresh PHA-P stimulated normal PBMCs in two 50ml tubes (20 x 106 PHA blasts per tube).
2. Centrifuge at 1200 rpm for 10 min. to pellet the cells.
3. Add 500 µl of culture supernate (cs) from primary culture on each pellet.(select the earliest PI day showing the p24 Ag detection test’s result O.D. greater than 1).
4. Keep at 370C in presence of 5% CO2 overnight to allow adsorption to take place (in a 50 ml tube with loose cap).
5. Next morning, wash the cells twice with 10 ml washing medium so as to remove the unadsorbed viral particles, if any.
6. Transfer to two T-75 flasks with 20 ml IL-2 medium each.
7. Collect the supernate for 0 PID p24 Ag detection test. (This result must be negative)
8. Test for the presence p24 antigen on 4 PID.
9. If it is positive for p24 antigen (OD greater than 1), scale up the culture volume to 40ml in each flask. Feed the culture with 20x106 PHA Blasts.
10. If negative for p24 Ag on the 4 PID, remove 10 ml of the culture supernate and feed with the culture with equal amount of IL2 medium. Continue the culture till 7 PID days.
11. Test again for the presence of p24 Ag on 7th PID. If the p24 Ag is positive on 7th PID, follow the same procedure as in point no. 9 so as to generate a stock of 80 ml. If negative on 7th PID, follow step 10.
12. Discard those cultures that are repeatedly negative, as judged p24 Ag detection test, till 18 PID.

Store the 80ml stock (supernate) in cryovials (1ml/cryovial), and the cells in freezing medium.

HIV TITRATION - TISSUE CULTURE INFECTIVITY DOSE (TCID 50)

Procedure:

1. Take culture supernate from the developed virus stock and thaw it quickly.
2. Label the flat bottom 96-well plate as follows

Fig 1: Schematic representation of the 96-well plate :

3. Add 150 ml IL2 medium in all the wells except the ones marked 4-2.
4. Take the thawed virus and dilute it 1:12 in IL2 medium (Add 0.1 ml virus stock in 1.1 ml IL2 medium.)
5. Take the diluted virus and add 200 ml of it in wells marked 4-2. (Virus 1 in columns 1, 2 & 3 as shown in the fig. 1) and mix well.
6. With a multi-channel pipette transfer 50 ml of the mixed contents from the wells marked 4-2 to the wells marked 4-3. Go on with the serial dilution till row H is reached.
7. Take about 20 x106 PHA stimulated normal PBMC in 5 ml IL2 medium.
8. Transfer to two T-75 flasks with 20 ml IL-2 medium each.
9. Cover the plate and incubate it at 37oC, 5% CO2.
10. Re suspend the cells on day 4, discard 125 ml of cell suspension and replenish with 125 ml of fresh IL2 medium.
11. On day 7, collect the supernatants from all the wells separately and carry out p24 Ag detection test.

Calculation of TCID50

The Spearman-Karber formula for the calculation of TCID50 is as follows:

M= xk +d[0.5-(1/n)(r)]

wherein, xk= dose of highest dilution (8 as per the scheme),
r = sum of negative responses,
d = spacing between dilutions ( 1 as per  the scheme),
n = wells per dilution (3 as per  the scheme.)

Thus the 50% endpoint is 4M. This can be converted to 10x.
10x is the 50% titre. x= M ( log 4)

10x is TCID50 per 200 ml.

hence, TCID50  = 10x X 5.

Eg:-   if,  xk = 8, r=5, d = 1, n =3,

M = 8+1[0.5- (1/3) x 5]
   =  8 + (0.5-1.6)
   =  8 - 1.1
   =  6.9
hence  50 %  titre = 6.9 x log 4
=  6.9 x 0.602
=   4.15
=    104.15

TCID 50 /ml  = 5 x 104.15

=  100.7 x 104.15

=   104.85