2.13 N EUTRALIZING A NTIBODY A SSAY P ROTOCOL FOR H ETEROLOGOUS R ESPONSES
2.13.1 Envelope Clones
Thereafter, 100l of pre-incubated pseudovirus/plasma was added in duplicate to all intended cells and was incubated for 48 hrs at 37°C.
2.12.5 Day 4- Terminating Assay And Reading Luciferase On The Luminometer
After 48 hrs, the media or supernatant from the infected wells were aspirated, one plate at a time. Lysis buffer (75 µl) was added to each well. Each plate was wrapped with tape to prevent the lid from slipping off during the freeze/thaw process. Plates were frozen at -80°C for 30 min (until opaque) and then shaken at RT for 30 mins (until clear). This freeze/thaw cycle was repeated for a total of two freeze/thaw cycles. Luciferase reagent was reconstituted according to the manufacturer‘s protocol and kept in the dark until use. A volume of 20l of lysed cells was added to each of the 96 wells on a white luminometer 96-well plate. To this 100µl/well of luciferase assay reagent was added, and measured on the luminometer. Data was exported to Microsoft Excel and was then analyzed.
2.13 Neutralizing Antibody Assay Protocol for Heterologous Responses
These included eight subtype C, seven subtype B and five subtype A envelope pseudoviruses as depicted in table 2.12. Tier 1, Tier 2 and Tier 3 viruses were stratified previously based on clustering analysis of sensitivity patterns- with Tier 1 being the most sensitive, Tier 2 displaying moderate to low sensitivity and Tier 3 displaying the lowest sensitivity to neutralization (Seaman et al., 2010). The ConC plasmid carrying a consensus of all the HIV- 1 subtype C sequences from the Los Alamos database by 2001 (Kothe et al., 2006) and the envelope plasmids containing single point mutations (as described in Gray et al. (2011), were obtained from Lynn Morris. Table 2.12. depicts the envelope clones used, the subtype of the clone, the tiered categorization and the references for the viral isolates.
Table 2.12. HIV-1 Env pseudovirus panel of subtype C, B and A reference strains
Using the format of a 96-well flat-bottom culture plate as illustrated in Figure 2.6; 150 l of HIV-1 Isolate Subtype C, B or
A
Tiered Category
Reference
MW965.25 C 1 NIH ARRRP
ZM197 M.PB7 C 1 (Li et al., 2006b)
ConC C 2 (Kothe et al., 2006)
DU156.12 C 2 (Li et al., 2006b)
DU172.17 C 2 (Li et al., 2006b)
ZM214 M.PL15 C 2 (Li et al., 2006b)
CAP45.G3 C 2 (Li et al., 2006b)
CAP239.G3 C 2 (Gray et al., 2007)
SF162.LS B 1 (Stamatatos and Cheng-Mayer, 1998)
6535.3 B 1 (Li et al., 2005)
AC10.0.29 B 2 (Li et al., 2005)
QHO692.42 B 2 (Li et al., 2005)
WITO 4160.33 B 2 (Li et al., 2005)
TRO.11 B 2 (Li et al., 2005)
PVO.4 B 3 (Li et al., 2005)
Q23ENV17 A 2 (Blish et al., 2007)
Q842ENVd12 A 2 (Blish et al., 2007)
Q168ENVa2 A 2 (Blish et al., 2007)
Q461ENVe2 A 2 (Blish et al., 2007)
Q769ENVd22 A 2 (Blish et al., 2007)
added in all wells of column one (cell control). For columns two to twelve 100 l was added in all wells (column two - virus control). Depending on dilution of test serum or plasma sample (see standard dilution algorithm- below in Tables 2.13 and 2.14): an additional amount of growth medium (GM) was added to all wells of columns three to twelve, row-H so that for example if your starting dilution of plasma was 1:45, the volume of test plasma added was 5 l and the corresponding total amount of GM was 45l.
This format was designed to assay five samples in duplicate at each serum dilution (Figure 2.6. Template A). Adjustments may be made to test a larger number of samples per plate (Ten samples, Figure 2.6. Template B).
A positive control with a known neutralization titer against the target virus was included on at least one plate in series each time assays were performed. Also, at least one negative control sample was used. The required number of vials of virus was thawed by placing them in an ambient temperature water bath. When the viruses were completely thawed, the virus was diluted in GM to achieve a concentration of 4,000 TCID50/ml.
To each well in columns three to four, row H only, 5 l of test plasma sample was added.
Serial dilutions of the test plasma was done by pipetting 50 µl of this dilution to row G, and this process was repeated until eight serial dilutions were done to achieve a final dilution of 1:98415. To all wells in columns two to twelve, rows A through H, 50 l of cell-free virus was dispensed. and mixed by pipette action after each transfer. Pipette tips were rinsed in a reagent reservoir containing sterile PBS between each transfer to avoid carry-over. The plate was covered and incubated for 1 hr, at 37°C.
A suspension of TZM-bl cells at a density of 10,000 cells/ml in GM containing DEAE dextran was prepared. Thereafter, 100 l of cell suspension was dispensed (10,000 cells per
reservoir containing sterile PBS between each transfer to avoid carry-over. Plates were covered and incubated for 48 hrs, at 37°C.
From each well, 150 l of culture medium was removed and 100 l of Bright Glo Reagent was then dispensed to each well and incubated at RT for 2 mins to allow complete cell lysis and was mixed by pipetting action (two strokes) and 150 l of this mixture was then transferred to a corresponding 96-well black plate. The plate was read immediately in a luminometer.
Percent neutralization was determined by calculating the difference in average relative luminescence units (RLU) between test wells (cells + serum sample + virus) and cell control wells (cells only, column one), and dividing this result by the difference in average RLU between virus control (cell + virus, column two) and cell control wells (column one), subtracting from one and multiplying by 100.
nAb IC50 or nAb ID50 was defined as the neutralizing antibody titers that were expressed as the reciprocal of the serum dilution required to inhibit 50% virus inhibition (or reduce the RLU by 50%). Each experiment was performed independently at least twice with duplicate wells.