HIV RNA testing of plasma from 3 cohorts
A total of 196 seronegative plasma samples from 133 subjects (122 non-seroconverters and 11 seroconverters) from the SFMHS were tested for low-level HIV RNA using 921 TMA assays (). Additionally, nine HIV antibody positive plasma samples from subjects who had seroconverted within the previous six months (i.e., prior bleed was seronegative) were also tested. The nine seropositive plasma samples were all strongly HIV RNA positive, with all TMA replicate tests yielding S/CO values of > 15. Plasma samples from eleven seronegative subjects who seroconverted during the following six months (i.e., were seropositive at the subsequent bleed) were included among the 196 seronegative samples. Two of these eleven subjects were found to be in their primary HIV infection phase with similarly strong HIV RNA positive signals in all TMA replicate assays. In contrast none of the 25 samples collected more than six months prior to seroconversion in these 11 subjects were viremic.
The 122 highly exposed non-seroconverters from the SFMHS were also tested using multiple replicate TMA tests per sample. All samples except one (ID 50320) were TMA negative in all replicate assays (S/CO<1). A single sample had one out of five TMA replicate assays with a positive S/CO of 2.0. In order to further assess the viremic nature of this sample collected in February 1986, 8 ml of additional plasma from the same subject and time point was tested using 16 TMA replicate assays. All 16 replicate TMA assays were negative.
Of the 42 seronegative plasma samples provided from 42 SFCCC participants tested using 109 replicate TMA assays only one was HIV RNA positive (SFCCC 5093) (). This sample was strongly positive with all four TMA replicate positive with S/CO>15. A new aliquot of this plasma sample was acquired from the SFCCC repository. The sample again tested strongly HIV RNA positive in 2/2 TMA replicates. The sample was collected in August 1984, from a subject who remained seronegative at all four subsequent time points (March 1985, October 1985, February 1987, and April 1988), but was seropositive in March 1989 and at five subsequent time points through August 1990. In order to rule out any sample mix-up we analyzed 12 highly polymorphic human DNA alleles [30
] from the August 1984 plasma sample, PBMC DNA from the same time point, and PBMC DNA from both a February 1987 and a February 1990 clinic visit. The August 1984 plasma and PBMC human DNA matched at all 12 polymorphic alleles, but only 2/12 loci matched with the 1987 and 1990 PBMC samples. The 1987 and 1990 samples matched at all 12 loci. These results indicated that the 1984 plasma and PBMC came from a different person than the 1987 and 1990 samples. We concluded that the strongly HIV RNA positive 1984 plasma sample could not be assigned to the individual who sero-converted between April 1988 and March 1989, and represented a sample mix up from a viremic individual. PBMC derived DNA from the 1984 collected specimen was also tested for HIV proviral DNA, and found to be HIV DNA positive.
A total of 136 seronegative women in the WIHS with the most exposure to HIV were selected. Two hundreds and eighty-six plasma samples from the time points when exposure was highest for these subjects were then tested using 1043 TMA assays (). Seven of 286 plasma samples yielded positive TMA test results. Five of the positive plasma samples were from the time points collected immediately prior to seroconversion, and therefore represented typical primary HIV infection (in these 5 cases 4/4 replicate TMA were strongly positive with S/CO>15). The other two samples (WIHS 60101030 and WIHS 20204650) were from women who did not seroconvert at the subsequent bleed. These two samples had very low-level HIV RNA reactivity, in both cases only one out of four replicate TMA assays were positive with positive yet low S/CO of 1 to 2. Further plasma aliquots from these two subjects at the same time points were acquired and re-analyzed by replicate TMA assays. None of four replicate TMA tests run on each of these two samples were positive.
Negative proviral DNA testing of cases with possible transient viremia
In order to test for the presence of proviral DNA in the 3 potentially transiently infected subjects (SFMHS 50320, WIHS 20204650, WIHS 60101030), we used a nested PCR protocol targeting the gag region.
PBMC were not available from the same time point as the weakly TMA positive 1986 plasma sample from SFMHS subject 50320. PBMC collected at two time points in 1989, when the subject was still seronegative, were therefore selected for HIV proviral DNA testing. Ten repeat nPCR tests were performed on PBMC DNA from both 1989 samples. None of the 20 nPCRs were positive ().
The second sample showing minimal positive evidence of HIV viremia was from WIHS subject 60101030 and had been collected in August, 1999. DNA was extracted from PBMC collected from this time point as well as on March, 1999, February, 2000, and November, 2005 and a total of 36, 17, 35, and 7 nPCRs were performed. No proviral HIV DNA was detected.
The third sample showing possible evidence of HIV viremia was collected from WIHS subject 20204650 on February, 1999. DNA was extracted from PBMC collected on that date as well as September, 1999, February, 2000 and January, 2006 and a total of 33, 41, 57, and 8 nPCR for proviral DNA were performed. No proviral HIV DNA was detected. Proviral HIV DNA was therefore not detected in the PBMC from the same or later time points from any of the subjects with possible transient viremia.
From a total of 524 seronegative plasma samples collected from 311 highly exposed men and women, only 4 samples showed any evidence of HIV RNA more than six months prior to seroconversion. One case was the consequence of a sample mix up. In the remaining three cases only a single out of four or five replicate TMA assays were positive and then only with very low-level S/CO ratios. Further replicate TMA testing of more plasma failed to yield any positive results. Because very low levels HIV proviral DNA might represent an archive of a past transient infection [11
] we tested these 3 subjects for proviral DNA using nPCR. No PBMC proviral reservoirs were detected.
Our negative results indicate that if transient viremia occurs, it must be either at levels too low to be reproducibly detected and/or of too short duration to have been sampled in the plasma tested here. It also remains conceivable that viral replication is restricted near mucosal surfaces or in draining lymphoid tissues and therefore not able to reach detectable plasma levels [31
-34]. Alternatively the cellular immune responses reported in highly exposed seronegatives may be due to their continued exposure to non-infectious HIV proteins rather than to viral replication.