VCU M-1 Immunology HIV Immunopathogenesis

1. VCUM-1 ImmunologyHIV Immunopathogenesis Daniel Nixon DO, PhD Associate Professor of Medicine VCU - School of Medicine Director – VCU HIV/AIDS Center DNixon@mcvh-vcu.edu

2. Human Immunodeficiency Virus (HIV) • HIV is the causative agent of Acquired Immune Deficiency Syndrome (AIDS) • HIV (a Retrovirus) causes a chronic infection of CD4+ T-cells that results in progressive immune system impairment that after several years (typically 2-15) • Results in greatly increased susceptibility to “opportunistic” infections and malignancies. • Transmitted sexually or via blood • Over 34,000,000 infected worldwide, with over a million in the U.S. • Without treatment, all but a handful will die

3. HIV Life Cycle

4. The Trojan Horse...Dendritic cells and DC-SIGN • Recent evidence points to dendritic cells (DC) playing a role in HIV transmission. • The DC specific surface C-lectin “DC-SIGN” appears to bind HIV, not allowing infection of the DC but instead enhancing its presentation to/infection of CD4+ T-lymphocytes. • Follicular dendritic cells (FDCs) in lymph node “germinal centers” can bind infectious HIV and protect it from antibody, while in close proximity to CD4+ T-cells

5. Dodging antibodies and chemokines..HIV enters the host cell • HIV infects CD4+ T-lymphocytes, macrophages, and a few other cell types using the CD4 cell surface markers a receptor. • Chemokine receptors (such as CXCR4 or CCR5) function as an co-receptors for HIV-1. • Other routes of HIV entry into the cells exist. • Certain HIV Isolates are macrophage “trophic” and appear to be preferentially transmitted to new hosts • CCR5 is the chemokine receptor used by these HIV

6. Once in the cell, HIV finds a hostile environment.. Intracellular defenses against retroviruses • Mammalian evolution has resulted in at least three independent acquisitions of genes whose primary role appears to be an intrinsic defense against retroviral infection • One group of genes, exemplified by APOBEC3G, act primarily by deaminating cytidines in nascent retroviral DNA (Bishop et al., 2004, Harris et al., 2003, Lecossier et al., 2003, Wiegand et al., 2004, Yu et al., 2004, Zhang et al., 2003 and Zheng et al., 2004).. (HIV makes a protein Vif that binds and deactivates) • Fv1 and tripartite motif-5 (TRIM5 alpha) appear to target incoming retroviral capsids to block infection by as yet poorly defined mechanisms that may involve accelerated disassembly (Kozak and Chakraborti, 1996, Sebastian and Luban, 2005, and Stremlau et al., 2006).

7. Exposure to HIV at mucosal surface (sex) Day 0 Virus collected by dendritic cells, carried to lymph node Day 0-2 HIV replicates in CD4 cells, released into blood Day 4-11 Day 11 on Virus spreads to other organs Kahn JO, Walker BD. N Engl J Med. 1998;339:33-39.

8. Primary HIV Infection: Pathogenesis Anti-HIV T-cell response Sero-conversion Antibody response CD4 count(cells/mm³) Plasma RNA Viral Load Viral set point 1,000 CD4 Cell Count 500 4-8 Weeks Up to 12 Years 2-3 Years A lot of important stuff happens here

9. Early destruction of gut CD4+ cells with SIV..implications for HIV? • In early SIV infections, gut-associated lymphatic tissue (GALT), the largest component of the lymphoid organ system, is a principal site of virus production • Within weeks, CD4+ lymphocytes in GALT in primates is depleted by as much as 80% • In this primate study, non-activated memoryCCR5+ CD4 + lymphocytes preferentially eliminated early • In non-progressor SIV models, GALT CD4s + lymphs reconstitute..in progressors, they do NOT • Qingsheng L et al. Nature 434, 1148-1152 (28 April 2005)

10. An acute HIV seroconverter: terminal ileum is striking in its almost complete absence of discernible lymphoid tissue • Brenchley JM et al J Exp Med. 2004 Sep 20;200(6):749-59.

11. Early destruction of GALT CD4+ lymphocytes means massive losses • Brenchley JM and Douek DD. Nature Immunology 2006. 7, 235 - 239

12. Early on, HIV- specific CD4+ cells are activated and preferentially destroyed… Activated CD4 Cell CD4 Cell APC HIV HIV Picture Adapted from: Cohen DE, Walker BD. Clin Infect Dis 2001;32:1756-68

13. Which leads to loss of HIV-specific Cytotoxic T-Lymphocyte Response CD4 Cell HIV HIV-Infected CD4 Activated CD4 Cell Lymphokines Antigen-Presenting Cells Picture Mature CD8 CTL CD8 Cell Activated CD8 Pre CTL HIV Adapted from: Cohen DE, Walker BD. Clin Infect Dis 2001;32:1756-68

14. Despite these problems, host cellular immune system responds to HIV • Virus specific CD8+ cytotoxic T-lymphocytes (CTLs) are strongly implicated in control of HIV infection. • HIV specific CTLs found in large numbers in tissues with HIV • CTLs inhibit HIV replication in vitro • Association between emergence of CTLs that kill HIV infected cells and decline of plasma HIV • Certain MHC-1 haplotypes (e,g. HLA-B27 and B57) associated with slower progression to AIDS • CTL depleted primates have in higher SIV loads and rapid progression to “AIDS”

15. CTL depletion in primates results in increased SIV replication

16. Cellular Immune Response to Acute HIV Infection Acute HIV Weak CTL Rapid Progression Moderate CTL Moderate Progression Strong CTL Slow Progression 6 months Slide courtesy David Spach, MD From: Walker BD. Nature 2000;407:313-4.

17. Humoral responses to HIV • Current evidence suggests that antibodies play only a limited role is controlling HIV infection (unlike many other viral infections) • Most antibody formed is non neutralizing • B cell depletion in primate models does not alter viral clearance • Viral clearance in infected individuals occurs before neutralizing antibody • Most HIV neutralizing antibody formed is to a specific region of a viral surface glycoprotein. • This region is relatively immunologically “silent” • Variability in sequence leads to “Immune Escape”

18. HIV gp120 surface glycoprotein - a tough antibody target! • A) The CCR5 receptor is a tempting but difficult neutralizing Ab target • C) The target is inside the “hypervariable” V3 loop • D) The target area is glycosalated to make it relatively “stealth” to the immune system

21. Antibody (B12) binding gp120 CD4 binding domain • Wedged between the glycan-shielded silent face and a flexible inner domain/bridging sheet, with its projecting variable loops, the identified site is recessed • It must be accessible to a molecule as large as CD4 to serve its function..so Ab may have a chance.. • Recent serological analysis of long-term non-progressors attributes the broad and potent neutralizing properties of some of these sera to antibodies directed against this site Zhou T. et al. Nature. 2007 Feb 15;445(7129):732-7.

22. HIV “Escapes” from Neutralizing Antibody

23. HIV Persistence • So how does HIV persist despite an effective CTL response? • Amazing HIV mutational capability results in CTL and humoral immune “escape” • Selective elimination of anti-HIV CD4+ T-cells • Downregulation of MHC and CD4 molecules • Immune “sanctuaries” (eg. gut, brain, testes) • Ability of HIV to integrate into host cell genome

24. HIV NEF downregulates CD4+ and MHC-1

25. HIV effects on the Immune System • Initially after infection, many patients experience the “acute retroviral syndrome” which is similar to the flu or mononucleosis • Serologic testing this early may be negative • A measure of “viral load” by RT-PCR at this time shows impressive titers of virus • Next comes a period of clinical (but NOT virologic) “latency” as symptoms resolve but a relentless attack on the immune system ensues…

26. HIV and T cells go to “war” • Continuous and highly productive replication of HIV occurs in all infected individuals, although the rates of virus production vary by up to 70-fold in different individuals • Average T 1/2 of an HIV infected cell in vivo is 2.1 days • About 10 billion HIV particles are produced daily • About 2.6 billion CD4+ T-cells are produced daily

27. How does HIV deplete CD4+ T cells? • While HIV can (but does not always) kill the CD4+ T-lymphocytes that it infects, typically only a small % of the total CD4+ T-cell population are infected at any given time • Decreased thymic output of new naïve CD4+ T-cells is documented & may play a role • Chronic immune system activation independently predicts CD4 depletion/AIDS & induces apoptosis of uninfected cells….how? • ? > Immune cell “senescence = aging” = > activation, shorter telomeres, > inflammatory cytokines • ? Circulating lipopolysaccharide (LPS) and bacterial DNA from damaged GALT/gut mucosa

28. Plasma LPS is increased in HIV/SIV Brenchley, JM et al. Nature Medicine - 12, 1365 - 1371 (2006)

29. Plasma LPS levels correlate with HIV related T-lymphocyte activation Brenchley, JM et al. Nature Medicine - 12, 1365 - 1371 (2006)

30. HIV-1 - a disease marked by chronic T-cell activation • Following emergence from the thymus, T cells are in a “naïve” configuration in a resting (G0) state. • When appropriate secondary signals that are required to complete cell cycle are and differentiate are absent, the T-cell may end up in a state of “frozen” activation…know as anergy • Activation markers on CD8+ lymphocytes include CD38 and HLA-DR • HIV associated with increased CD8+ lymphocyte activation • Reduced CD25+ FOX P3 expressing Treg CD4+ lymphocytes may play a role • General consensus is that there is some positive correlation between HIV-1 viral load and degree of immune activation… • In SIV this appears NOT to be the case..

31. With SIV, immunologic activation, NOT SIV viral load, correlates with progression to AIDS • A very intriguing feature of SIV infection in natural host species, such as Sooty Mangebys and African green monkeys, is that CD4+ T cell depletion and AIDS do NOT arise despite high level levels of viremia • Decreased CD4+ T Cell Counts/AIDS in (cross species) SIV-Infected Sooty Mangebys Are Correlated with increased levels of T Cell activation • Some evidence suggests that this is true in HIV infected persons as well.. • Silvestri et al. Immunity 2003, 18:3 441-452

32. AIDS – more than just immunodeficiency: Evidence for immune suppression due to chronic activation as central in disease progression • T-cell activation is predictive of HIV disease progression in untreated patients, independent of HIV viral load(Giorgi JV et al. JID 1999:179;859-970 Calbone J et al. AIDS 2000; 14:2823-29 ) • CD4 depletion is directly linked to immune activation with HIV-1 and HIV-2 but only indirectly with viral load (Sousa AE et al. J Immunolo. 2002:169:3400-3406) • Several studies have shown that in patients with HIV viral load fully suppressed (by treatment) with blunted CD4 recovery, immune activation is a consistent feature(Anthony KB et al. JAIDS. 2003;33:125-133) (Hunt PW et al. JID 2003;187:1534-43))

33. HIV induces syncitia formation

34. HIV may induce Th1 to Th2 cytokine shift

35. HIV destroys lymph node germinal centers

36. HIV ultimately reduces T-cell help • T-cell destruction via excessive immune activation, apoptosis, reduced T-cell production and function, and loss/fibrosis of lymph node architecture result in reduced CD4+ T-lymphocyte help. • As T cell help is central to many aspects of immune function the is decreased or altered function of ALL arms of the immune system. • Predominant effects in patients involve susceptibility to infections and tumors normally dealt with by the cellular arm of the immune system. …

37. CMV Retinitis…note, for test you do not need to know the stuff on these clinical slides

38. Toxoplasmosis Encephalitis

39. Pneumocystis jiroveci (carinii) pneumonia

40. Kaposi’s Sarcoma (HHV8)

41. Systemic Inflammation in HIVBaseline Biomarkers and *All Cause Mortality in a large Clinical Trial (Kuller LH et al. PNAS 2008) *CVD, Malignancy, Liver Disease, Opportunistic Infection

42. Loss of CD4 help means B cell dysregulation & autoimmunity • B-cell polyclonal activation/dysregulation with hypergammaglobinemia is typical • Numerous autoimmune diseases and autoantibodies have been associated with HIV, including: • Immune thrombocytopenic purpura - up to 40%, anti RBC or WBC antibodies • Vasculitis • Anticardiolipin antibodies • Graves’ disease and anti-thyroid antibodies • Some diseases like SLE and sarcoidosis improve as CD4+ T-cell counts drop • False positive serologies can occur

43. Effects of long term antiretroviral therapy • Initially, improved CD4+ T-cell survival and expansion of the “memory” CD4 T-cell pool • Peripheral recruitment & proliferation of lymphoid tissue sequestered cells (during first several months) • Reconstitution of naïve T-cells due to increased thymic output (over several years) • Reduced T-cell activation (though often remains above “normal”) • Reduced gut leak and systemic inflammation/coagulopathy (though often remains above “normal”) • Less morbidity and improved survival (though often remains below “normal”) • Increased lymphoproliferative response to recall antigens are observed as well…except to HIV. • Interestingly, while effective suppression of HIV results in improved CTL responses to many antigens, HIV specific immunity actually declines.

44. Future Research… • Correlates of protective immunity have not yet been well defined. CTL activity (CD4+ and CD8+ cell) appears important and simulation of mucosal immunity & NK cell activity needs to be considered • A solution to the vast heterogeneity and mutational capability of HIV remains to be discovered. • Characterization of HIV sanctuaries • Other active areas of HIV “applied” immunology research include: • cytokine immune system restoration (using various interleukins, thymopoetic growth factors) • modulating the global immune system hyperactivation associated with disease progression.