Grouppe Kurosawa: HIV - The Folly of Modeling HIV Vaccine Efficacy in Chimpanzees (print)

The Folly of Modeling HIV Vaccine Efficacy in Chimpanzees

Chimpanzees are the only non-human primates in which the HIV naturally replicates. HIV vaccines that stimulate the production of neutralizing antibodies and promote the development of cell-mediated immune responses in chimpanzees are considered logical candidates for human clinical trials. Unfortunately, there is probably no correlation between vaccine efficacy in chimpanzees and their eventual effectiveness in preventing human HIV infections. Although chimpanzees are 99% genetically identical to humans, chimpanzees normally do not develop AIDS. When immunized, they induce a persistently high titer of neutralizing antibodies to viral envelope and core proteins and induce group-specific cytotoxic cell-mediated immune responses against the virus. Further, HIV viral challenge does not induce programmed cell death or damage the follicular architecture of chimpanzee lymph nodes (1-5). This aggressive immune response against viral proteins does not happen during most natural HIV infections in humans. The obvious question is why?

Under optimal conditions, immune cells release the pro-inflammatory cytokines TNF-alpha and IL-12 upon initial exposure to intracellular pathogens (6). TNF-alpha activates macrophages, while IL-12 stimulates the secretion of gamma interferon and the expansion of Th1 CD4 T cells. The generation of Th2 cells is inhibited. It has long been argued that Th2 CD4 cells predominate in AIDS. The reason for this cellular imbalance has never been adequately explained. In this essay, we will argue that the environmental and genetic background of the host, at the time of infection, determines whether the virus will be acutely cleared or reduced to a chronic infection.

TNF-alpha selectively destroys cells expressing higher levels of HIV viral antigens (7). In addition, TNF-alpha inhibits HIV-1 replication by inducing the production of RANTES and decreasing CCR5 co-receptor expression (8). This complementary inhibition pathway blocks the infection of naïve cells by monocytotropic HIV-1 viruses, the strains that have a selective advantage in infecting monocytes and dendritic cells at the initial portal of entry into the body (9-11). The secretion of TNF-alpha, and indeed IL-12, is subject to numerous environmental and genetic controls. Immunosuppressive immune hormones, such as IL-10, TGF-beta and the prostaglandin PGE2, all inhibit cellular immunity by blocking the release of pro-inflammatory hormones. If a virus, such as HIV-1, enters the body during a period of immunosuppression, the virus is unlikely to be completely cleared from the tissues. The immune response against the virus will be sluggish, prolonged and eventually ineffective in preventing the spread of the virus to cellular reservoirs, such as macrophages.

In this essay, we will argue four genetic factors, and numerous environmental factors determine, in large part, the extent of HIV-1 infectivity in humans. The genetic factors are sensitivity to complement destruction, sensitivity to viral Tat-mediated immunosuppression, IL-10 gene promoter polymorphism, and autoimmune phenomenon. Autoimmune phenomenon will be discussed in another essay.

Most experimental HIV vaccines attempt to induce the production of neutralizing antibodies against the envelope spikes of the virus (12). The rational is straightforward. The gp120/gp160 envelope protein of the HIV virus contains the CD4 and chemokine receptor binding sites. Antibodies that bind these sites theoretically neutralize the virus, thereby preventing infection. It is naturally assumed that these antibodies also activate complement. This would result in the targeted destruction of free virus and virally infected cells. In HIV infected humans, this does not occur (13-16). Antibodies against gp120 and gp41 can activate the complement cascade (indeed free virus can activate complement by the alternative pathway in the absence of antibody), but the cascade is inhibited by Factor H, a plasma protein that inhibits soluble C3b in the blood. Factor H directly binds both gp120 and gp41 and inactivates C3b virtually as soon as it is deposited on these antigens (13,14). Both gp120 and gp41 contain sequences that are similar to one of the Factor H binding sites on C3b (17-19). When Factor H is deposited on gp120/gp41, it interacts with covalently bound C3b and promotes its cleavage into inactive fragments C3dg and iC3b. To different degrees, all envelope viruses escape complete complement destruction by incorporating cell-derived complement control proteins CD55 (DAF or decay accelerating factor) and CD59 into their membranes as they bud from the membranes of host cells (20,21). HIV is the only virus that directly binds Factor H to its envelope proteins. The binding of Factor H to the HIV virus/infected cells protects them destruction by the humoral arm of the immune system. The HIV virus can be destroyed in human serum by inhibiting the activity of DAF and Factor H by monoclonal antibodies (22).

The inactive C3b fragments C3dg and iC3b, covalently bound to gp120/gp41, bind the complement receptors CR1, CR2, and CR3 (13,14). These receptors are found on dendritic cells, monocytes/macrophages, B cells, T cells and FDC or follicular dendritic cells. Under normal circumstances, intact, live virus does not become concentrated in lymph nodes or thymus. C3dg and iC3b are usually only bound to viral (pathogen) fragments that have previously been destroyed by complement. When localized to lymph nodes, these fragments bind FDCs and initiate the activation of naïve T and B cells. In HIV infections, live virus is concentrated in lymph nodes and thymus. It is very likely this live virus is targeted to FDC cells, T/B cells and macrophages by bound C3dg/iC3b. Interestingly, when inactive C3dg/iC3b triggers macrophage CR1 and CR3 receptors, the cellular transcription factor NF-kB becomes activated resulting in enhanced viral replication in latently infected cells (23). CR1 and CR3 also mediate infection of monocytes/macrophages and thymic T cells with complement-opsonized HIV (24-26). Finally, the activation of the CR3 receptor by C3dg/iC3b results in the suppression of IL-12 and gamma interferon release in vivo (27).

We are aware of no experimental evidence that indicates chimpanzees have any trouble destroying the HIV virus by complement fixation. When the HIV virus/infected cells are incubated in NON-heat inactivated mouse, rat, guinea pig, rabbit or feline serum, they are rapidly destroyed (13,14). Antibodies previously bound to the virus/infected cells mediate this destruction. This simple experiment has not been conducted with NON-heat inactivated chimpanzee serum. If fresh chimpanzee serum destroys HIV virus/infected cells in vitro, this suggests chimpanzee Factor H, in addition to rodent, rabbit, and feline Factor H, does not bind gp120/gp41. In the absence of Factor H binding, complement activation can proceed unabated to pore formation and eventual viral/cellular destruction. These simple studies need to be conducted utilizing fresh chimpanzee serum.

A number of neutralizing antibody-binding sites has been published in the literature. One prominent, non-V3 binding site is found on gp41. This sequence, ELDKWA, is highly conserved on numerous viral strains (28-31). Antibodies (monoclonal antibody 2F5) that recognize this site are broadly neutralizing. This gp41 sequence binds Factor H (13). One interpretation of the data is that these antibodies are neutralizing because they block Factor H binding to gp41. This permits complement activation and eventual destruction of the virus/infected cell.

The HIV viral protein Tat is probably the most immunosuppressive protein ever described in the scientific literature (32-34). It is synthesized early during viral synthesis and secreted from infected cells. Non-infected bystander cells readily take up Tat. The biochemistry of Tat is complex, and includes and activation and repression of numerous cellular and viral genes. Tat has been reported to:

1. Inhibit cellular (host), but not viral, mRNA translation (35)
2. Activates the HIV viral LTR by recruiting a Tat1-CyclinT1 complex to the viral TAR RNA (36)
3. Down-regulates bcl-2 and induces apoptosis in hematopoietic cells (37)
4. Induces neuronal death (38)
5. Decreases the ability of accessory cells to organize T cell clusters (39)
6. Activates B cells and induces B cell lymphoma (40,41)
7. Induces immunoglobulin synthesis by stimulation of IL-6 release (42)
8. Blocks dihydropyridine-sensitive (L-type) calcium channels in dendritic cells (thereby blocking their maturation) (43).
9. Inhibits CD26 or dipeptidylaminopeptidase IV activity on T cell membranes (thereby blocking recall activation of T cells)(44).
10. Blocks phagolysosomal fusion in monocytes (45).
11. Inhibits IL-2 and IL2R expression in CD4 cells (46).
12. Induces NF-kappaB activation (47,48).
13. Amplifies inflammatory redox state (oxidative stress) (49).
14. Stimulates TGF-beta release (additional immunosuppression) (50-53).
15. Represses transcription of MHC I genes (54).
16. Activates JNK and ERK/MAPK pathways in non-infected CD4 cells (55).
17. Stimulates monocyte chemotaxis (56).
18. Represses beta 2-microglobulin promotor (57).
19. Inhibits IL-12 synthesis (58).
20. Induces HIV-1 co-receptor synthesis (CCR5 and CXCR4) (promotes infectivity of both macrophage and T cell tropic viral strains) (59,60).
21. Hyperactivates T cells via the CD28 pathway (61).

Tat is probably best known for inducing apoptosis in bystander T cells (62-64). The HIV virus up-regulates fas ligand expression in macrophages (65,66). T cells expressing membrane fas are killed when they come in contact with these macrophages. Tat is now known to stimulate fas ligand expression in uninfected macrophages at concentrations 1,000 fold lower (500 pM) than those capable of stimulating T cells. (34). Soluble Tat, released from infected cells in sequestered areas, such as lymph nodes, induces the migration, chemotaxis, of monocytes into the areas of infection. Upon arrival, the monocytes differentiate into macrophages, express fas ligand on their membrane, and destroy naïve T cells.

Chimpanzees are resistant to the immunosuppressive effects of Tat (67). The reasons are unknown. Tat treated chimpanzee T cells do not show signs of oxidative stress, and are resistant to Tat-induced apoptosis, ACID (activation induced cell death) and CD95 (fas) ligand upregulation. Clearly, HIV infections are handled very differently by the immune systems of humans and chimpanzees.

Interleukin 10 (IL-10) and transforming growth factor beta (TGF-beta) are extremely immunosuppressive hormones, and will be discussed in greater depth in another essay. The IL-10 promoter is heterogeneous in the human population (68). Patients predisposed to a high IL-10 production have a poor response to interferon alpha, an important anti-viral agent. In addition to genetic factors, cocaine, opiates, ethanol, marijuana, and stress (69-75) regulate IL-10 and TGF-beta concentrations in the blood. In the case of physical or mental stress, glucocorticoids induce an immunological shift to a type 2 cytokine antigenic response (IL-10 predominant). IL-10 increases the sensitivity of glucocorticoid binding to monocytes, while TNF-alpha decreases it (76).

SUMMARY

Tat does not immunologically impair the chimpanzee immune response against the HIV virus, nor is there evidence that chimpanzees cannot fix complement to the surface of HIV virions or infected cells. Chimpanzees do not take illicit drugs, they do not smoke, they are not malnourished, they are not feed a diet rich in omega 6 fatty acids that can be readily converted to arachidonic acid, a precursor of immunosuppressive prostaglandins such as PGE2, nor do they live in the same stressful environment as humans. Vaccine studies in chimpanzees provide important data on the immunological responsiveness of various HIV antigens. However, the data derived from these studies are not predictive of the human immune response to live virus.

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