Acquired Immunodeficiency

SyndromeChapter 44

HIV and AIDS

• HIV infection usually leads to

• Immunosuppression

• Subsequent susceptibility to opportunistic infections

• Cancers

• Wasting

• Central nervous system degeneration

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

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

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

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

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

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)

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

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)

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

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

African AIDS Orphans

Nation Orphans (2005)

South Africa 1,200,000

Tanzania 1,100,000

Zimbabwe 1,100,000

Kenya 1,100,000

Uganda 1,000,000

Nigeria 930,000

Zambia 710,000

DR Congo 680,000

Malawi 550,000

HIV Vaccines

HIV gp120/gp41

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

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

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

Clinical Progression of HIV Disease

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

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

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?)

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)

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

Live recombinant vaccines

Viruses BacteriaVaccinia virus Bacillus sp.

Canarypox, Fowlpox SalmonellaAdenovirus Lactobacillus

Alphaviruses (VEEV, SFV)

Streptococcus

Flaviviruses (YF17D) ListeriaRhabdoviruses (VSV)

OrthomyxovirusesParamyxoviruses

Picornaviruses

Potential vectors for HIV vaccine

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

DNA Vaccines• Genetically engineered DNA molecules with

nontransforming eukaryotic 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

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

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