Acquired Immunodeficiency Syndrome

1. Acquired Immunodeficiency Syndrome • Chapter 44

2. HIV and AIDS • HIV infection usually leads to • Immunosuppression • Subsequent susceptibility to opportunistic infections • Cancers • Wasting • Central nervous system degeneration

3. Molecular and Biologic Features of HIV • HIV structure and genes • HIV is a diploid RNA virus • Genome is 9 kb • 9 genes • 16 proteins • Protein core • Enveloped • Long-terminal repeats (LTR) flank the genome and contain cis-elements recognized by cellular transcription factors • The cellular transcription factors are expressed only when the T cell participates in an immune response

4. Molecular and Biologic Features of HIV • Viral life cycle • Viral glycoproteins gp120 and gp41 protruding from the envelope bind to CD4 and a chemokine receptor (CCR), respectively, on the target cell surface • Only cells that express both CD4 and CCR are susceptible to HIV infection • The viral envelope fuses with the cell's membrane and the core is dumped into the cytoplasm • The core proteins dissociate, liberating the viral genome and several enzymes into the cytoplasm • The viral reverse transcriptase (RT) synthesizes a double-stranded DNA copy of the viral genome • The viral DNA is then integrated into the host cell chromosome • Viral enzyme is integrase • Integrated viral genome is called a provirus

5. Molecular and Biologic Features of HIV • Viral Life Cycle (cont.) • It is evident that viral gene expression requires cell activation • Cellular transcription factors (trans-elements) recognize the proviral LTRs and stimulate viral gene expression • Cellular NFκB • Cellular Sp1 • Several viral polypeptides are post-translationally cleaved into functional proteins by the viral protease • env polypeptide (gp160) to gp41 and gp120 • gag polypeptide (core proteins) to p17, p24, p7, p6 • Viral proteins and nucleic acids assemble into a core • gp120 and gp41 protrude from the cell's surface • The core travels to the surface where it buds through the membrane, picking up its envelope containing gp120 and gp41 proteins • When the rate of budding exceeds the rate of membrane-synthesis, the infected cell begins to die

7. Course of HIV Disease • Early on, HIV infection is contained by the adaptive immune response • Infected individuals often have a flu-like illness • Viral loads are high • Immune response clears the majority of infected cells and virus • Some virus is sequestered in the lymphoid tissues • Follicular dendritic cells trap HIV (but they are not infected) • Virus remains viable for more than 1 year • Many T helper cells become infected with the virus, but the virus remains dormant because the majority of T cells are not activated • As infected Th cells engage a pathogen, the virus becomes active and produces more viruses and killing the T cell

8. Course of HIV Disease (cont.) • This results in a hole in the T cell repertoire • Subsequent exposure to the microbe goes unchecked because all the T cells to the microbe have been depleted • Loss of Th cells compromises the immune system's ability to contain pathogens • Other cells can be infected without CPE and act as a reservoir of virus • Macrophages • Dendritic cells

9. Immune evasion by HIV • High mutation rate changes immunogenicity of the virus • Antagonistic HIV peptides often emerge that induce T cell anergy • HIV nef protein downregulates MHC class I expression • nef also downregulates CD4 expression • vif inhibits APOBEC3G, an antiretrovirus protein (induces C > U mutations)

10. Long-Term Nonprogressors • A small percentage of infected persons fail to develop disease • Some are HIV+ but do not progress to AIDS • It is unknown why most of these individuals remain healthy (but infectious), but... • Some of them are heterozygous for the CCR5Δ32 allele of chemokine receptor 5. • This mutation results in a substantially-reduced infectivity of cells because HIV cannot induce a membrane fusion event • Therefore, virus does not enter the cells

11. Treatment and Prevention • Multi-drug cocktail (highly active antiretroviral therapy [HAART]) • Nucleotide analogs impair viral DNA synthesis • Protease inhibitors prevent viral maturation • Multi-drug resistant HIV strains are already appearing • Life expectancy • 1985 - 18 months from diagnosis • 2006 - 25 years from diagnosis • 2012 - Diagnosis at age 25 • Expect to live another 51 years • Uninfected person expected to live another 52 years • Vaccine (0/26) • Must be safe • Must be effective • Must be cheap (i.e. 3rd world) • Must not require special care and handling (e.g. refrigeration)

12. Clinical Features • AIDS is defined by: • HIV+ • T helper count below 200 cells/μl (normal is 500+) • When opportunistic infections occur • Oral candidiasis and tuberculosis; 250-500 cells/μl • Cryptotsporidiosis; 200 cells/μl • <200 cells/μl • Pneumocystis carinii pneumonia • Disseminated Mycobacterium avium • Cryptococcosis • Toxoplasmosis • Visceral herpes simplex viruses • Cytomegalovirus retinitis; <50 cells/μl

13. The Global AIDS Crisis • AIDS is altering the social structure of Africa • Many African towns have lost their entire adult populations to AIDS • This has resulted in many orphans • These children, without parental guidance and financial support, face substantial challenges • No education • Many die • Some are conscripted into rebel armies • African economies cannot support adequate HIV/AIDS management • Societal collapse may occur • It is the only continent where life expectancy is declining

14. African AIDS Orphans

15. HIV Vaccines

16. HIV gp120/gp41

17. Obstacles to the Development of HIV Vaccines • Antigenic diversity and hypervariability of the virus • Transmission of disease by mucosal route • Transmission of the virus by infected cells • Resistance of wild-type virus to seroneutralization • Integration of the virus genome into the host cell chromosomes • Latency of the virus in resting memory cells • Rapid emergence of viral escape mutants in the host • Down-regulation of MHC class I antigens • No spontaneous recovery from natural infection in spite of high-level immune responses of the host

18. Principal Roles of the HIV Accessory Proteins • Vpr • Together with matrix protein, targets the viral preintegration complex to the nucleus • Arrests dividing cells in G2 of the cell cycle • Enhances HIV replication in macrophages • Vpu • Down-regulates expression of CD4 molecules by targeting them to the proteasome, leading to their degradation in the endoplasmic reticulum • Forms ion channels in the cell membrane, thus helping to promote the release of virions from the cell • Neutralizes APOBEC3G, an innate antiretroviral mechanism (C>U mutase) • Vif • Plays a role in provirus formation and stabilizes newly synthesized DNA intermediates • Associates with cytoskeleton intermediate filaments and helps transport incoming virions to the nucleus • Neutralizes an APOBEC3G antiviral mechanism

19. Principal Roles of the HIV Accessory Proteins • Nef • Associates with cellular protein kinases (PAK65, p56tck, p59Hck) • Stimulates viral DNA synthesis and enhances virus infectivity in primary T cells and macrophages • Enhances virus replication in vivo, contributing to high viral loads and pathogenesis • Binds CD4 molecules at the plasma membrane and mediates their rapid endocytosis and lysosomal degradation • Down-regulates cell surface expression of major histocompatibility complex class I antigens and of immune costimulatory molecules of CD80 and CD86, a cytotoxic T-lymphocyte escape mechanism • Activates the expression of FasL, which induces apoptosis in bystander cells that express Fas

20. Clinical Progression of HIV Disease

21. Antigenic determinants and epitopes of interest • A major obstacle in effective vaccines is the elicitation of Nabs • The principal neutralizing determinant appears to be the hypervariable V3 loop of gp120 • Antibodies against one strain often are not cross reactive to other strains • HIV X4 vs. HIV R5 strains • Other epitopes include V1, V2 and CD4-binding domain (competitive) • gp41 has a three epitopes • Antibodies to each inhibit gp41-mediated fusion PDB ID 2B4C

22. Antigenic determinants and epitopes of interest • T cell epitopes are not restricted to virus surface antigens • Any HIV polypeptide can contain T cell epitopes • An effective antiviral immune response requires CD8+ CTLs • CTL responses are sustained by CD4+ Th responses • Unfortunately, this CD4+ T cell activation is precisely what HIV requires for virus replication • Thus, an effective Th cell response leads to the depletion of those Th cells • CTL escape mutants (peptide epitopes) always arise during HIV infection • Since T cells cannot undergo affinity maturation, like B cells, these escape mutants evade the CTL response

23. Candidate HIV vaccines • The development of an HIV vaccine is a formidable challenge • Most classical vaccines provide protection from disease but do not actually prevent infection • They allow a limited but controlled replication of the pathogen at the portal of entry • This raises the question of whether an HIV vaccine, if unable to prevent infection, could prevent development of disease • The difficulties of HIV vaccine testing include • Testing must be done in humans • Since controlled experiments cannot be done, must rely on retrospective analysis • Test population must be a high-risk group • Must be able to document their activities • Must be able to follow them for years (decades?)

24. Whole inactivated vaccines and virus-like particles • Not currently being considered • Regulatory and safety concerns (alloantigens, xenoantigens) • All HIV strains are propagated in transformed human cell lines • Transformed human cells cannot be used for virus propagation for vaccines • Formalin damages HIV epitopes, such that they fail to elicit strong Nabs • Virus-like particles • Canarypox virus with all HIV proteins except RT • Produced in primary cell culture • Elicit strong Nab response when administered with cholera toxin B subunit (adjuvant)

25. Live, attenuated vaccines • All work has been conducted with strains of simian immunodeficiency virus (SIV) in monkeys • SIVmac • Directed nef deletion mutant • Administration results in low-grade infection without clearance • Provided protection against challenge with 1,000 ID50 of virulent SIV • However: • Some adult monkeys developed AIDS from vaccine • Fatal to neonatal monkeys • Additional deletions of LTR and vif are currently being tested • Hybrid SHIV vaccines are also being examined

26. Live recombinant vaccines Potential vectors for HIV vaccine

27. Subunit vaccines • Subunit vaccines are composed of polypeptides from the virus that are antigenic and neutralizing • Typically produced by recombinant DNA technology • E. coli or other bacteria • Yeast • Baculovirus • DNA vaccines • Host cells for production must be able to perform post-translational modifications • Soluble envelope glycoproteins • Exclude transmembrane domains • Coexpress gp120 and gp41 • Easy to include antigens from multiple HIV strains • ΔV2gp120 is currently being tested in humans • Deletion provides better exposure of V3 loop

28. DNA Vaccines • Genetically engineered DNA molecules with nontransformingeukaryotic cis-elements • Typically plasmids • Easy to produce in large quantities • Extremely heat stable • Requires multiple boosters • Often require delivery by “gene gun” • Microscopic beads coated with DNA • Injected into the skin/muscle with high pressure • Presumably, the beads carry the DNA into cells, which then activate gene expression from the plasmid • This produces antigen that is processed by the class I and class II pathways • No DNA vaccine has been approved

29. Human Clinical Trials • More than 20 have been completed; all have failed • Many others are ongoing • Trials to date (completed and ongoing) • Subunit vaccines - 35 trials • Viral vector vaccines - 35 • Bacterial vector (Salmonella typhi) - 3 • DNA vaccine - 27

30. Human Clinical Trials • VaxGen gp120 phase II trial (VAX004 candidate) • 5,095 volunteers • Immunized with recombinant soluble gp120 from MN and GNA8 strains • Injections at 0, 1, 6, 12, 18, 24 and 30 months • Assessment at 36 months • Objectives • Does vaccine protect against HIV infection? • What differences between vaccinated and placebo infections? • Results • Vaccine did not protect (6.7% vaccinated vs. 7.0 % placebo) • No differences between between groups in regard to infection responses