 |
|
|
| |
A vaccine strategy against AIDS: An HIV gp41 peptide immunization prevents NKp44L expression and CD4+ T cell depletion in SHIV-infected macaques
|
| |
| |
Download the PDF here
"In summary, anti-3S immunization preceding SHIV infection of cynomolgus macaques prevented both NKp44L expression on CD4+ T cells and the activation and cytotoxicity of NK cells. It therefore prevented a decline in CD4+ T cells but had no effect on viral load.......anti-3S immunization appears to prevent CD4+ T cell depletion in pathogenic infection. Additional experiments should demonstrate whether anti-3S vaccines or monoclonal antibodies or both have a therapeutic effect in infected individuals, by limiting CD4+ T cell depletion or promoting immune restoration during continued viral replication......In conclusion, our results raise questions about our understanding of HIV pathogenesis and present opportunities for prevention and treatment of the CD4 immune depression induced by HIV-1"
A vaccine strategy against AIDS: An HIV gp41 peptide immunization prevents NKp44L expression and CD4+ T cell depletion in SHIV-infected macaques
PNAS 2008
http://www.pnas.org/content/105/6/2100.full?sid=66be7d3c-3062-41df-88af-eddc79cd3265
Abstract
We previously showed that a gp41 peptide (3S) induces expression of a natural killer (NK) ligand (NKp44L) on CD4+ T cells during HIV-1 infection and that those cells are highly sensitive to NK lysis. In HIV-infected patients, anti-3S antibodies are associated with the maintenance of CD4+ T cell counts close to their baseline values, and CD4+ T cells decrease with the antibody titer. This study sought to determine whether anti-3S immunization could prevent NKp44L expression on these CD4+ T cells in vivo and inhibits the subsequent decline in CD4+ T cell counts by immunizing macaques with 3S and then infecting them with simian HIV162P3. The results show that anti-3S antibodies inhibited NKp44L expression and NK activity and cytotoxicity. They also decreased the apoptosis rate of CD4+ T cells in peripheral blood and lymph nodes. These data raise questions about the pathogenesis of HIV and present opportunities for both preventive and therapeutic HIV vaccine strategies.
Although CD4+ T cell depletion appears to be the principal component of HIV disease, its underlying mechanisms remain controversial. Numerous reports offer a wide variety of tentative explanations for immune depression, including cell activation, the virus's direct cytopathic effects, and toxicity caused by its pathogenic determinants (1-4). One of the most intriguing phenomenons, however, is that many of the CD4+ T cells that die during HIV infection are not infected (5). These cells must have died or been killed by a collateral effector mechanism not directly linked to viral replication. One plausible such mechanism is the expression of a natural killer (NK) ligand on CD4+ T cells during HIV infection: we (6) demonstrated that NKp44L, the ligand of NKp44 NK receptor, is expressed on CD4+ T cells of HIV-infected patients and showed that cells expressing NKp44L are highly sensitive to NK lysis. Ward et al. (7) have recently confirmed the specific expression of NKp44L on CD4+ T cells after in vitro infection with HIV-1.
We also showed that a highly conserved motif of HIV gp41 envelope protein interacts with CD4+ T cells to induce NKp44L (6). Humans at early stages of HIV infection produce antibodies against this peptide motif (called 3S) that can inhibit its in vitro expression, but this anti-3S antibody production decreases sharply thereafter. Although these antibodies did not neutralize the virus, they were associated with CD4+ T cell counts and their rates of decrease. The antibody titer was also inversely associated with NKp44L expression (8). Together these results question the feasibility of immune intervention against gp41 to prevent the consequences of HIV infection.
Most studies, which have attempted to stimulate specific immune responses against gp41 HIV protein, have been applied to induce HIV neutralization. Many of those studies demonstrate that neutralizing Abs can protect against HIV-1 infection in vitro and in animal models, but in vivo proof of their activity in infected humans remains circumstantial (9, 10). Trkola et al. (11) showed that HIV-1 delay rebounded rapidly after cessation of antiretroviral therapy through passive transfer of the neutralizing Abs 2G12, 2F5, and 4E10 against gp41 epitopes. During the natural course of HIV infection, fully functional variants continuously emerge and compete for outgrowth in the presence of a rapidly evolving neutralizing Ab response, which exerts a high level of selective pressure. Non-neutralizing epitopes, which are usually very conserved, could also be the targets of immune intervention (10, 12). In that respect, the sharp conservation of the 3S-motif among all viral isolates (6) suggests that 3S-based peptides provide a major B-cell epitope that should be considered for use to limit virus pathogenicity, independently of pathogen replication.
The present study sought to determine whether anti-3S immunization could prevent NKp44L expression on these CD4+ T cells in vivo and inhibit the subsequent decline in CD4+ T cell counts by immunizing macaques with 3S-peptide and then infecting them with simian HIV (SHIV)162P3.
Results and Discussion
Anti-3S Production After 3S-Keyhole Limpet Hemocyanin (KLH) Immunization in Macaques.
Uninfected macaques were immunized with 3S peptide coupled to KLH carrier protein or KLH alone and subsequently infected by i.v. injection of SHIV162P3, a CCR5-tropic virus. Profiles of viremia and CD4 count in SHIV162P3-infected cynomolgus macaques closely resembles naturally transmitted HIV strains in human patients (13, 14). All animals immunized with 3S-KLH, initially at monthly intervals, developed much stronger immune responses against 3S, whereas the animals immunized with KLH alone did not (Fig. 1 A). Furthermore the macaque anti-3S antibodies had the functional capacity to prevent in vitro NKp44L expression on CD4+ T cells, as previously reported for antibodies from HIV-infected patients (8). Sera from animals immunized against 3S (hereafter referred to as the immunized animals), but not from animals immunized by KLH alone (hereafter the controls), totally inhibited NKp44L expression on normal CD4+ T cells incubated with 3S-peptide (Fig. 1 B).
3S-KLH Immunization Decreases NKp44L Expression on CD4+ T Cells of SHIV-Infected Macaques.
Next, we compared viral load and NKp44L expression on CD4+ T cell depletion in the immunized and control animals after they were infected by SHIV. The infection slightly enhanced anti-3S Ab responses of the immunized animals, which remained at high titers (Fig. 2 A). In contrast, the antibody level in control animals was lower by a factor of ≈10. These antibodies appeared also later and decreased with time. As expected given that anti-3S antibodies had no neutralizing effect on viral infection in vitro (8), plasmatic viral load did not differ between the two groups of animals (Fig. 2 A): all animals showed a similar peak followed by a drop in viral load. Similarly, viral load did not significantly differ in lymph nodes at day 21 in the immunized and control animals (P = 0.20; data not shown). Importantly, NKp44L expression on CD4+ T cells differed significantly. In control animals, the percentage of cells expressing NKp44L increased over time, reaching 40% at day 42 and remained constant. In contrast, immunization before SHIV infection drastically inhibited NKp44L expression on CD4 T cells. However in one animal (no. 14599), NKp44L increased consistently after day 80 while remaining lower than controls. Therefore, although anti-3S antibodies did not affect viral replication, they suppressed in vivo NKp44L expression on CD4+ T cells of SHIV-infected animals, as CD4+ T cells incubated with anti-3S Abs did in vitro (Fig. 2 B). The 3S-gp41 motif is localized between the N-terminal heptad repeat 1 (HR1) and the HR2 domains. Several models indicate that this region may have contact with the host cell membrane during the formation of the ectodomain core structure (15-17).
Although anti-3S Abs had no neutralizing activities, our data also indicate that this 3S motif is transiently accessible during in vivo infection and is recognized by anti-3S Abs, which prevent its pathogenic effect on CD4+ T cells.
Protective Effect of 3S-KLH Immunization on CD4+ T Cells Decline.
Hypothesizing that anti-3S antibodies would subsequently affect the decline in the CD4+ T cells after SHIV infection, we compared the two groups to assess any effects on percentage and counts of CD4+ T cells in peripheral blood and lymph nodes. The percentage of CD4+ T cells observed over time after infection differed very significantly in the two groups (Fig. 2 C). In control animals, the frequency of peripheral blood CD4+ T cells decreased progressively; the difference compared with basal values increased with time (P = 0.03 at day 42 and P = 0.05 at day 188). In immunized animals, on the other hand, those percentages remained stable, never differing significantly from the values observed before infection (P = 0.12 at days 42 and 188). Similar differences were observed in lymph node CD4+ T cells in the two groups (data not shown). Note that the peripheral blood CD4+ T cell count reflected these differences. Counts fell significantly in control animals, whereas in immunized animals they never differed significantly from preinfection values (Fig. 2 D). These results, which relate NKp44L expression to CD4+ T cell depletion, were consistent with our earlier findings in HIV-infected patients and led us to hypothesize that NKp44L expression, by augmenting NK cell cytotoxicity, might be indirectly responsible for CD4+ T cell depletion (6).
3S-KLH Immunization Protects CD4+ T Cells from Apoptosis.
Because clear differences were observed as early as 42 days after infection, we sought further proof by comparing apoptosis of CD4+ T cells in immunized and control animals infected by SHIV during the period. Interestingly, anti-3S immunization induced a clear and significant reduction in CD4+ T cell apoptosis, which persisted over time, together with the high level of anti-3S response (Fig. 3 A). Similar significant differences were observed in lymph nodes (Fig. 3 A). Furthermore, nearly all apoptotic cells expressed NKp44L, and this expression was clearly related to cell death. No significant cell apoptosis was detected in peripheral blood or lymph node cells that did not express this ligand (Fig. 3 B). We note, however, that neither apoptosis alone nor 3S-associated CD4+ T cell apoptosis induced NKp44L expression in CD4+ T cells (data not shown).
Taken together, these results suggest that NKp44L is strongly associated in vivo with cell death during HIV infection and that the 3S motif is indirectly responsible for this cell death, because anti-3S antibodies control apoptosis. Similar correlation between NKp44L expression and apoptosis was also observed in CD4+ T cells of HIV patients (unpublished data). However, although NKp44L expression appears closely related to apoptosis, not all NKp44L+ T cells are apoptotic. Indeed, apoptotic cells accounted for <20% of NKp44L+ cells on day 14 in control animals, a percentage that increased with time and reached 40% by the end of the study period (Fig. 3 C). The same phenomenon was observed to a lesser degree in immunized animals, even though their rate of CD4+ T cell apoptosis was significantly lower (Fig. 3 C). These data suggest that a second event might be required to induce apoptosis.
3S-KLH Immunization Decreases NK Activation and Cytotoxicity.
We next postulated that this second event necessary to induce the death of CD4+NKp44L+ cells was caused by the effect of NK activation and cytotoxicity directed at cells expressing NKp44L. To test this hypothesis, we compared these indicators in immunized and control animals. As Fig. 4 shows, even though the number of NK cells did not differ in the two groups (Fig. 4 A), activation (CD3-CD8+CD69+; Fig. 4 B) and cytotoxicity (Fig. 4 C) of NK cells were clearly lower in immunized than control animals. This was the case in both peripheral blood and lymph nodes (data not shown). In three of four animals, NK cytotoxicity in immunized animals did not differ from the level observed in both groups before SHIV infection. In animal 14599, which showed an increase of NKp44L after day 80, there was a parallel increase of NK cytotoxicity after such a period (Fig. 4 C). NK cytotoxicity was highly correlated with both NK cell activation (Fig. 4 D) and NKp44L expression (Fig. 4 E) in both peripheral blood and lymph node cells (data not shown). These results considered together suggest that apoptosis induced by SHIV infection is the consequence of two sets of events, the first related to NKp44L expression, the second related to NK cell activation and cytotoxicity. Most important, they also demonstrate that anti-3S antibodies can prevent these related phenomena.
In summary, anti-3S immunization preceding SHIV infection of cynomolgus macaques prevented both NKp44L expression on CD4+ T cells and the activation and cytotoxicity of NK cells. It therefore prevented a decline in CD4+ T cells but had no effect on viral load. This effect was long lasting for three of four animals. In one animal, however, there was an escape phenomenon after day 80: a late increase of NKp44L was accompanied by CD4 cells drop and increase of NK cytotoxicity, emphasizing the relationship between these parameters. Taken globally, these results confirm in a macaque model of AIDS what we previously showed in humans, that the 3S-gp41 epitope can induce NKp44L expression (6). They also show clearly that in vivo ligand expression on CD4+ T cells is related to this 3S interaction. The finding that all apoptotic cells expressed NKp44L demonstrates that expression of this ligand by target cells is a prerequisite for their in vivo apoptosis during SHIV infection. Because the 3S peptide by itself did not induce apoptosis, nor did apoptosis induce NKp44L expression, our results also indicate that NKp44L may increase the susceptibility of a population of CD4+ T cells to an external death signal.
These findings, which show the strong relation of NKp44L expression and apoptosis to NK activation and cytotoxicity, provide support for the hypothesis that this second external event is caused by NK cytotoxicity. Numerous reports show that NK cells exhibit a variety of different behaviours during HIV-1 infection (18). Interestingly, the increased NK activity in some HIV-exposed patients suggests that NK cells may help protect against infection (19). Conversely, several studies show alterations in the number and function of NK cells during HIV infection and progression to AIDS (20, 21). On the other hand, various distinct allelic combinations of the NK inhibitory receptor KIR3DL1 and HLA-B loci significantly and strongly influence both AIDS progression and plasma HIV RNA abundance (22). Yet, none of the above studies referred to a correlation between the level of NK activity and the NK ligand expression. It is clear that further experiments are needed to conclusively demonstrate the role that NK cells may have in vivo. However, our previous in vitro data, together with the results reported herein, support the hypothesis of NK cells' deleterious effect and the importance of NKp44L in AIDS pathogenesis.
Our results also emphasize the fact that the 3S motif is a pathogenic viral determinant, which strongly affects disease progression, most especially the CD4+ T cell depletion. These results demonstrate that a key epitope induces such pathogenic phenomena in vivo. Although these data do not totally rule out the possibility that other factors contribute to CD4+ T cell depletion, they strongly support the hypothesis that the 3S viral peptide plays a major role in the immune depression of HIV and SHIV infection. They may provide insight to improve our understanding of the lack of pathogenicity of natural SIV lentivirus infection in African green monkeys, a pathogenic lentivirus to the natural host, and the different factors that might control viral burden and pathogenicity (23, 24).
More importantly, anti-3S immunization appears to prevent CD4+ T cell depletion in pathogenic infection. Additional experiments should demonstrate whether anti-3S vaccines or monoclonal antibodies or both have a therapeutic effect in infected individuals, by limiting CD4+ T cell depletion or promoting immune restoration during continued viral replication. Further studies are also needed to understand mechanisms of escape observed in one animal after day 80. The results reported herein open the way for additional strategies of immune intervention aimed at controlling disease development. Yet rather than choosing between a vaccine strategy against pathogenicity proved effective against tetanus, diphtheria and cholera bacteria, that is, inoculation against toxins, and a different strategy aiming at neutralizing the virus (25-27), we submit that these both types of preventive vaccinations should be envisioned as complements.
In conclusion, our results raise questions about our understanding of HIV pathogenesis and present opportunities for prevention and treatment of the CD4 immune depression induced by HIV-1.
|
| |
|
|
|
|
|
