ASSIGNMENT WORK ON

HIV
(VACCINE DEVELOPMENT & PRODUCTION)

INTRODUCTION
H.I.V. stands for Human Immunodeficiency Virus. Discovered in 1984 and named HIV in 1986. A retrovirus (capable of converting RNA into DNA by Reverse transcriptase enzyme) Causes A.I.D.S. (Acquired Immune Deficiency Syndrome) in humans in which the immune system of the body fails leading to life threatening opportunistic infections and various un-usual tumors. HIV infects the Helper T (CD4) cells to which it binds First reported in young homosexual males but later found in nonhomosexuals possibly by blood transfusion.

HIV types and sub-types

2 types of HIV: HIV-1 and HIV-2. Show 40% to 60% amino acid homology. HIV-1 is subdivided into subtypes or clades designated A to K(M Clades) and O. M and O show 55- 70% similarity/homology. N clade is a new group recently reported.

Structure of HIV

VIRAL ENVELOPE
Outer coat of the virus .Diameter of 1/10000 of a millimeter. Spherical in shape. Composed of 2 layers of fat, called lipid. Recent evidence suggests HIV may Enter or exit cells through LIPID RAFTS present on cell membrane. 72 spikes on average made of complex protein protrude from the virion surface. This protein is called ENV made of a Cap of Glycoprotein 120 and a stem of GP41. Primary focus of VACCINE development

Viral Core
Bullet shaped core called Capsid made of 2000 copies of a protein called P24. Capsid protects 2 single strands of HIV RNA,each with a copy of 9 genes. Gag, Pol and Env gene encode for structural proteins. 6 regulatory genes control viral replication. Include; Vif, Rev,Nef,Tat,Vpu,Vpr.

Other core protein include: p7- HIV nucleocapsid protein, and 3 enzymes- Protease, Reverse transcriptase,integrase. p17 lies between protein core and envelope and is embedded in the internal portion of the envelope. Two additional p55 products, p7 and p9, are nucleic acid binding proteins closely associated with the RNA

Genome

Structural genes
gag-group specific antigen gene, encodes viral nucleopcapsid proteins: p24, several internal proteins, p7, p15, p17 and p55. pol-polymerase gene; encodes the viral enzyme, protease (p10), reverse transcriptase (p66/55; alpha and beta subunits) and integrase (p32). env-envelope gene; encodes the viral envelope glyocproteins gp120 (extracellular glycoprotein) and gp41 (transmembrane glycoprotein).

Regulatory genes
tat: encodes transactivator protein rev: encodes a regulator of expression of viral protein vif: associated with viral infectivity vpu: encodes viral protein U vpr: encode viral protein R nef: encodes a 'so-called' negative regulator protein

LTR (long terminal repeats)

repetitive sequence of bases they are sticky ends which the integrase protein uses to insert the HIV genome into host DNA they act as promoter/enhancers when integrated into the host genome, they influence the cell machinery which transcribes DNA, to alter the amount of transcription which occurs.

Mechanism of pathogenesis

Viral infection
First step, HIV attaches to susceptible host cell.

Site of attachment is the CD4 antigen found on a variety of cells

     

helper T cells macrophages monocytes B cells microglial brain cells intestinal cells

T cells infected later on.

M-tropic strains of HIV-1

In early phase HIV infection, initial viruses are M-tropic. Macrophage (M-tropic) strains of HIV-1 or nonsyncitia-inducing strains use the beta-chemokine receptor CCR5 Viruses that use only the CCR5 receptor are termed R5

T-tropic strains of HIV-1

In late phase HIV infection, most of the viruses are T-tropic T-tropic isolates, or syncitia-inducing (SI) strains Use the alphachemokine receptor, CXCR4, for entry Viruse that only use CXCR4 are termed X4 ,

and those that use both, X4R5

ATTACHMENT
HIV binds to receptors on CD4 T-cell A message is sent to the CD4 T-cell to let the virus in

FUSION
Once bound, the virus is allowed to dump its contents into the CD4 T-cell Included in its contents are HIV RNA and reverse transcriptase

REVERSE TRANSCRIPTION
The HIV RNA is turned into double-stranded DNA within the CD4 Tcell The enzyme reverse transcriptase aids in this process

INTEGRATION
Once the DNA is formed, it hides itself in the human DNA housed in the CD4 TCell nucleus

TRANSCRIPTION
Copies of HIV DNA are made and released from the nucleus in small packages Each of the small packages contains information for creating a new HIV

ASSEMBLY
The protease enzyme in the cell combines the DNA packages to create active virus

BUDDING
Once the new HIV is formed, it pushes itself out of the CD4 T-cell The virus steals part of the CD4 T-cells protective coating

Viral-host Dynamics
About 1010 (10 billion) virions are produced daily Average life-span of an HIV virion in plasma is ~6 hours Average life-span of an HIV-infected CD4 lymphocytes is ~1.6 days HIV can lie dormant within a cell for many years, especially in resting (memory) CD4 cells, unlike other retroviruses

How does HIV kill cells?

Virus budding from cell membrane is not lethal. Cells die by autofusion, syncytial formation, and apoptosis. Other mechanisms? 1. Autofusion CD4 and gp120/41 proteins mediate fusion and intracellular vesicle formation.

2. Syncytium formation gp120/41 proteins on infected cells bind to CD4 receptors on normal cells, causing cell fusion. The giant multi-nucleated syncytium dies before long.

3.Apoptosis An infected helper T cell can direct an uninfected T helper cell to undergo apoptosis (programmed cell death). Apoptosis can be normal, for example, to eliminate auto-reactive T lymphocytes to establish self-tolerance

Modes of HIV/AIDS Transmission

Sexual transmission

Contaminated needles

Mother to fetus

Infected blood or blood products

Four Stages of HIV

Stage 1 - Primary
Short, flu-like illness - occurs one to six weeks after infection no symptoms at all Infected person can infect other people

Stage 2 - Asymptomatic
Lasts for an average of ten years This stage is free from symptoms There may be swollen glands The level of HIV in the blood drops to very low levels HIV antibodies are detectable in the blood

Stage 3 - Symptomatic
The symptoms are mild The immune system deteriorates emergence of opportunistic infections and cancers

Stage 4 - HIV @ AIDS

The immune system weakens The illnesses become more severe leading to an AIDS diagnosis

Molecular details of antigen

Viral Antigen MHC

Viral envelope glycoprotein

Gp120 and gp41 derived from the trimeric precursor gp160, which is cleaved by a cell endoprotease gp120 and gp41 subunits associate noncovalently and homotrimers of gp120gp41 are incorporated in the viral envelope Gp41 is bound to the virus through its C-terminal trans-envelope domain,whereas gp120 is not anchored to the viral envelope

Structure & function of Gp120

gene is around 1500 nucleotides long(500 amino acid residues) ve variable regions (V1V5). The rst four variable regions form surface-exposed loops that contain disulphide bonds at their bases. The conserved gp120 regions form discontinuous structures important for the interaction with the gp41 ectodomain and with the viral receptors on the target cell. Both conserved and variable gp120 regions are extensively glycosylated. CD4 has a linear ectodomain consisting of four immunoglobulin-like regions (D1D4) and binds to gp120 through its outermost domain (D1)

PDI & its role in HIV entry

The oxidoreductase protein disulfide isomerase (PDI). PDI binds to the CD4 domain D3, close to gp120binding site. PDI reduces gp120 disulfide bonds, which triggers the major conformational changes in gp120 & gp41required for virus entry. Inhibiting PDI prevents HIV-1 entry hence a potential target for drug.

HIV vaccine development

What Might a Successful Vaccine Do?

Antibodies Bind virus; neutralize or stop virus from infecting cells; eliminate virus Cytotoxic T lymphocytes (CTL) Recognize cells infected with virus and kill those cells

Challenges in HIV Vaccine Research

Viral Genetic Diversity: HIV is not just one specific virus.
Immune Protection: We dont know what immune responses are needed, or how strong they need to be. Neutralizing Antibody: Difficult to generate broadly neutralizing antibodies. Vaccine Testing: Slow process, very expensive

but on the Brightside

Precedent from other systems: Success against other viral infections Precedent from animal studies: Long-term control of infection in vaccinated monkeys Immune control of HIV-1: Infected individuals control infection Vaccine Trials: In progress

HIV Vaccine Approaches

Protein subunit Synthetic peptide Naked DNA Inactivated Virus Live-attenuated Virus

Vaccine strategies
peptide vaccine: made of tiny pieces of proteins from the HIV virus. recombinant subunit protein vaccine: made of bigger pieces of proteins that are on the surface of the HIV virus. Examples of a recombinant subunit protein are gp120,gp140, or gp160 produced by genetic engineering. DNA vaccine: uses copies of a small number of HIV genes which are inserted into pieces of DNA called plasmids. The HIV genes will produce proteins very similar to the ones from real HIV

live vector vaccine: non-HIV viruses engineered to carry genes encoding HIV proteins. The genes are inserted into another vector, which carries them into the body's cells. The genes in turn produce proteins that are normally found on the surface of the HIV virus. Vaccine combination: uses any two vaccines, one after another, to create a stronger immune response. Often referred to as "prime-boost strategy. Virus-like particle vaccine (pseudovirion vaccine): a non-infectious HIV look-alike that has one or more, but not all, HIV proteins.

HIV Vaccine Trial

Currently, there are no approved preventive vaccines for HIV infection since the first HIV vaccine trial Till date, only three vaccines have been successful to the phase III trial the first two failed, the third trial results that were carried in Thailand.

Protocol Number
HVTN 055 HVTN 065 HVTN 060

Number of Participants
150 120 144

Testing Locations
USA USA USA, Thailand

Producer
Therion GeoVax Wyeth Bavarian Nordic A/S Pharmexa-Epimmune NIH VRC Merck Epimmune

HVTN 067

108

USA

HVTN 204 HVTN 502 HVTN 064

480 3000 120

International International USA, Peru

HVTN 073

48

USA, South Africa

South African AIDS Vaccine Initiative (SAAVI)

HVTN 069 HVTN 049 HVTN 071 HVTN 050 HVTN 070 HVTN 063 HVTN 503

90 96 35 435 120 120 801

USA, Peru USA USA International USA USA, Brazil South Africa

NIH VRC Chiron Merck Merck UPenn/Wyeth Wyeth Merck

Thank You!