All results are based on RT analysis using tritiated thymidine, and are given in counts per minute (cpm/μL). Cultures with no HIV added served as the negative control for the determination of background radioactivity. All of these control cultures had mean RT values of less than 100 cpm/μL on all days with no difference between vaccinated and unvaccinated subjects.
Figure shows the results from cultures infected with R5 HIV-1ADA. Without autologous serum pre-treatment (Figure ), a statistically significant mean reduction of HIV-1ADA replication was observed in cultures from vaccinated subjects when compared to unvaccinated subjects on days 10 (nearly 3 fold, p = 0.035) and 13 (4 fold, p = 0.017). Similar results were observed in cultures started with autologous serum pretreatment (Figure ), with a greater than 3 fold decrease by day 10 (p = 0.013) and a 5 fold decrease by day 13 (p = 0.008). R5 HIV-1 replication in cells from unvaccinated subjects with autologous serum pretreatment was greater on nearly all days compared to viral replication in cells from the same subjects without autologous serum. This is likely due to the activating effects of the serum on the cultured PBMCs. No such activation of viral replication occurred in cultures from vaccinated subjects with autologous serum pre-treatment, which remained nearly identical to that of cultures without the autologous serum, suggesting that vaccination prevented the enhancement of viral replication by the serum. This enhancement of HIV-1 replication in cultures infected in the presence of autologous serum in the unvaccinated subjects is responsible for the greater divergence seen between vaccinated and unvaccinated subjects in Figure compared to Figure .
Figure 1 Comparison of R5 HIV-1ADA replication in PBMC cultures from vaccinia naïve (N-10) and vaccinia vaccinated (N = 9) subjects. Figure 1a shows cultures without and figure 1b shows cultures with pretreatment with autologous serum. A reduction in HIV (more ...)
In cultures infected with X4 HIV-1NL4-3 (Figure ) no statistically significant difference in viral replication between cells from vaccinated and unvaccinated subjects is observed although there is a trend toward reduction in HIV replication in vaccinated subjects. Pretreatment with autologous serum (Figure ) does not appear to make any difference in the replication of HIV-1NL4-3 when compared to non-pretreated cultures (Figure ). These findings suggest little if any effect by vaccinia immunization on replication of CXCR4-tropic HIV-1.
Figure 2 Comparison of X4 HIV-1NL4-3 in PBMC cultures from vaccinia naïve (N = 10) and vaccinia vaccinated (N = 9) subjects. Figure 2a shows cultures without and figure 2b shows cultures with pretreatment with autologous serum. No statistically significant (more ...)
Within the narrow 3-6 month time frame of this study, there did not appear to be any relationship between the time since vaccination and the level of viral replication (data not shown). Cells from subjects vaccinated 6 months prior to the study showed similar reductions of viral replication to those in cells from subjects vaccinated 3 months before the study. This prolonged effect of vaccination is significantly different from that seen with other viruses known to inhibit HIV replication (measles, dengue fever virus and GBV-C), where such inhibition can only be demonstrated during the life of the actual co-infection and disappears when the co-infecting virus is no longer detectable [11
]. Additionally, subsequent to this study, two of our co-authors independently repeated this study as part of a much larger investigation looking primarily at long term chemokine production after multiple immunizations [25
]. In their study, they were able to demonstrate reduced CCR5-tropic HIV-1 replication in PBMC cultures from vaccinia immunized subjects vaccinated up to 14 months prior to their study. A statistically significant reduction in replication did not occur in cultures infected with a CXCR4-tropic HIV-1, although there was a trend toward reduced replication. Their results are nearly identical to those reported in this study, suggesting that an as-yet-to-be-identified suppressive effect on HIV replication is associated with vaccinia immunization, and persists for an extended time following vaccination, long after the vaccinia would be expected to be cleared from the host.
In addition, Brichacek, et al.
in the above referenced study [25
] demonstrated long term elevations of MIP-1α, MIP-1β and IL-8 in the serum of vaccinia immunized subjects compared to vaccinia naïve subjects. It is possible that these long term chemokine elevations may play some role in the observed resistance of PBMCs from vaccinated subjects to HIV-1ADA
replication. In the present study we collected culture supernatant on days 2 and 5 following in vitro
infection with HIV-1ADA
for analysis of MIP-1α, MIP-1β and RANTES. Despite an observed trend towards higher levels of MIP-1α and MIP-1β in the cultures of vaccinated subjects, no statistically significant differences in the concentrations of those chemokines were found between the PBMC culture supernatants of vaccinated and unvaccinated subjects under our experimental conditions (Figure ). While it is possible that a long term elevation in baseline chemokine production may confer some protection against HIV infection and/or replication in vivo
, an alteration in the ability of the PBMCs from vaccinated subjects to secrete an excess of these chemokines as a rapid response to an HIV-1 challenge does not appear to play a role under these culture conditions. Interestingly, the levels of these chemokines measured in the culture supernatant were generally much lower in the uninfected control cultures (Figure ), with the exception of RANTES which demonstrated levels equivalent to the HIV-1ADA
infected cultures on culture day 5, but only for the vaccinated subjects. Though not statistically significant, there was also a trend toward higher baseline levels of all 3 chemokines in the vaccinated subjects, however the lack of statistical power may be related to the fact that only 2 vaccinated and 2 unvaccinated subjects underwent chemokine analysis for this control. The lack of statistical power prevents drawing any conclusions concerning this finding.
Figure 3 Chemokine analysis in culture supernatants. Comparison of MIP-1α (a), MIP-1β (b) and RANTES (c) release between the PBMCs from vaccinated (N = 9) and unvaccinated (N = 10) subjects on days 2 and 5 post culture inoculation with HIV-1ADA (more ...)