Flu vaccines

1
Flu vaccines

• WHO specifies the contents of the vaccine each
  year to contain the most likely strains of
  viruses that will attack the next year.
• Annually updated trivalent flu vaccine for
  2008-2009 season consisted of H3N2, H1N1 and B
  influenza viruses. (But the vaccine H1N1 strain
  doesnt confer protection to H1N1 swine flu.)
• Viruses are grown in hens eggs.
• Inactivated viruses are injected or live
  attenuated viruses are given as a nasal spray
  (not currently approved in children younger than
  5).

World Health Organization
2
H5N1 (Bird flu)

• Most flu viruses infect birds. Bird flu refers
  to influenza in birds and in humans in cases in
  which avian viruses cross the species barrier to
  infect humans.
• 2003-2004 Outbreaks of H5N1 in poultry in 8
  Asian countries and 100 cases of human infections
  with 50 fatalities.
• Few (maybe none) cases involved bird-to-human
  transmission. A pandemic would require
  human-to-human transmission. A change of only two
  amino acids would allow H5 to efficiently
  recognize human cells.
• H5N1 viruses are being tested for making a
  vaccine, but mass production of an H5N1 vaccine
  would require safety and efficacy trials that
  might take longer than viruses would spread in a
  pandemic.
• Antiviral drugs oseltamivir (Tamiflu) and
  zanamivir, but not amantadine, are active against
  H5N1. Supply not adequate for a global pandemic.
  Antiviral resistance occurs in N1 viruses during
  Tamiflu treatment.

3
Response to bird flu threat in Asia
Protect domestic ducks Burn chicken
carcasses Vaccinate chickens
from contact with wild ducks
http//pr.caltech.edu/periodicals/CaltechNews/arti
cles/v41/uneasypieces.html
4
CDC definition of AIDS

• A Acquired a virus received from someone else
• I Immune an individuals natural protection
  against disease-causing microorganisms
• D Deficiency a deterioration of the immune
  system
• S Syndrome a group of signs and symptoms that
  together define AIDS as a human disease

(Centers for Disease Control and Prevention
Atlanta, GA)
5
What is AIDS?

• HIV infection is not AIDS (is HIV disease)
• AIDS is umbrella term for 26 known diseases and
  symptoms
• AIDS diagnosis if meet three conditions
• Have one or more of known diseases/symptoms
• See next two slides
• CD4 T cell count lt200/µL
• What is CD4? What are T cells?
• Test positive for HIV What do HIV tests detect?

6
Figure 9-22
Opportunistic infections and malignancies in AIDS
patients
7
Symptoms of AIDS(each symptom can be caused by
another disease cant rely on symptoms to
diagnose AIDS)

• Rapid weight loss
• Dry cough
• Recurring fevers, night sweats
• Unexplained fatigue
• Swollen lymph glands
• Diarrhea that lasts more than a month
• White spots on tongue, in mouth, or throat
  (thrush)
• Pneumonia
• Red, brown, pink or purplish blotches on skin or
  inside mouth, nose, or eyelids (Kaposi Sarcoma)
• Memory loss, depression, other neurological
  disorders

8
DNA viruses follow the Central Dogma DNA --gt
RNA --gt Protein
transcription translation
Most RNA viruses also follow part of the Central
Dogma RNA --gt Protein
9
Clicker questionHoward Temins hypothesis
In the early 1960s, Howard Temin discovered
something strange about Rous sarcoma virus (RSV
a RNA virus that causes malignant bone tumors in
chickens). Temin noticed that RSV would not grow
in the presence of actinomycin D, an antibiotic
that inhibits DNA synthesis. What would you do
if you made this observation?

• Hypothesize that actinomycin D also inhibits
  translation of proteins from RNA -- test for new
  protein synthesis after adding actinomycin D
• Hypothesize that actinomycin D also inhibits
  viral RNA polymerases -- test for its effects on
  poliovirus RNA-dependent RNA polymerase
• Hypothesize that actinomycin D kills the host
  cell and therefore the RSV -- test uninfected
  cell viability in the presence of actinomycin D
• Hypothesize that DNA is an intermediate in RSV
  replication -- search for an enzyme that converts
  viral RNA into DNA
• Contact the media and prepare for a trip to
  Stockholm
• All of the above

10
HIV is a RetrovirusRetroviruses do NOT follow
the Central Dogma

• Retroviruses are a subset of RNA viruses that
  reverse usual flow of genetic information within
  host cell.
• Reverse transcription of viral RNA into viral
  DNA
• RNA --gt DNA --gt RNA --gt Protein
• Reverse transcriptase makes double-stranded DNA
  from RNA.
• RT is a RNA-dependent DNA polymerase

11
(For exogenous retrovirus start here)
Maturation
Binding Membrane Fusion
Overview of Retroviral Life Cycle
Uncoating Reverse Transcription
Budding
Expression Membrane Targeting
Nuclear transport Integration
(For endogenous retrovirus start here)
12
1975 Nobel Prize
Joined Max Delbrücks group at Caltech in 1949 to
work on bacteriophage. Switched to animal
oncoviruses.
Joined Dulbeccos group at Caltech in late 1950s.
Caltech PhD, 1959 (Animal Virology).
President of Caltech 1997 - 2006. Now
Professor of Biology at Caltech.
13
HIV structure (Beware some inaccuracies in
movie)

• Diameter 100 nm (1000 Å)
• Outer envelope (lipid bilayer membrane) contains
  gp120/gp41 spikes ) and random cellular (host)
  receptors acquired during budding
• gp glycoprotein refers to its molecular
  weight in kilodaltons
• Capsid (Bullet-shaped core)
• Protein arranged with symmetry related to an
  icosahedron
• RNA inside core 2 copies of positive sense RNA
  (9500 bases)
• Problems with movie i) only 14 spikes per
  virion
• ii) HIV does NOT contain an icosahedral matrix
  (other enveloped viruses, such as Herpes, do)

http//www.lifehouseproductions.com/hiv_anim.html
14
Figure 11-24
Genes and proteins of HIV (we will discuss gag,
pol, env, p24, tat, and rev)
HIV has an RNA genome flanked by two Long
Terminal Repeats (LTRs), which are involved in
viral integration and regulation of the viral
genome.
Includes p24
Remember tat
Remember rev
15
HIV gag, pol, env

• gag gene encodes structural proteins of the
  viral core as a poly-protein (cleaved into
  individual proteins by HIV protease).
• Matrix protein (p17)
• Capsid protein (p24) -- assays to determine
  whether HIV is present look for this abundant HIV
  capsid protein
• Nucleocapsid (nucleic acid binding protein)
  protein (p15)
• pol gene encodes enzymes involved in viral
  replication and integration (again, poly-protein
  cleaved into individual proteins by HIV
  protease).
• Reverse transcriptase
• Integrase
• HIV protease
• env gene encodes envelope glycoprotein gp160,
  which is cleaved by a host cell protease into
  gp120 and gp41.
• gp120 binds to CD4 (receptor) and CCR5
  (co-receptor)
• gp41 is responsible for fusion of viral and
  target cell membranes

Proteins often named as p, where the number
refers to the proteins MW in kilodaltons
16
First steps of the HIV lifecycle -- more details
17
The use of CD4 as a receptor targets HIV to cells
of the immune system

• HIV preferentially infects CD4 T cells because
  the cellular receptor for HIV is the T cell
  protein CD4. (Human macrophages and dendritic
  cells also express CD4.)
• CD4 T cells are a type of T lymphocyte (a white
  blood cell), which control other immune cells.
• HIV infection eventually results in low levels
  of CD4 T cells, resulting in opportunistic
  infections due to ineffective immune responses.

Peripheral Blood Lymphocytes
18
What happens in an untreated HIV infection

• CD4 T cells are lost during HIV infection. --
  Direct viral killing of infected cells --
  Increased susceptibility to programmed cell
  death -- CD8 T cells (killer T cells) kill
  infected cells
• Below 200 CD4 T cells/µl in peripheral blood,
  cell-mediated immunity is lost --gt infections
  with opportunistic microbes.
• -- Early infections include Candida species
  (thrush) and TB -- Activation of latent
  varicella zoster (chicken pox virus) --gt to
  shingles. -- Epstein Barr Virus (infectious
  mononucleosis) --gt B cell lymphomas
• -- Herpes virus HHV-8 --gt Kaposis sarcoma
• -- Fungus P. carinii --gt pneumonia
• -- Co-infection with hepatitis C
• -- Late stages often include cytomegalovirus or
  M. avium infections

19
Infection of a cell by HIV requires binding to
CD4 and a coreceptor on host cells

• Spike protein on HIV virion is a complex of two
  proteins gp120 and gp41.
• gp120 binds tightly to CD4 on target cell.
• Binding to CD4 causes a conformational change
  that enables gp120 to bind to a coreceptor on the
  target cell. All coreceptors are members of the
  chemokine receptor family.
• After binding of gp120 to CD4 and a chemokine
  receptor, gp41 inserts into the host cell
  membrane, causing fusion of the viral and host
  membranes.

CCR5 or CXCR4
A chemokine is a small protein that activates or
attracts (as in chemotaxis) leukocytes.
20
Fusion of viral and cellular membrane bilayers
21
Genetic deficiency of CCR5 confers resistance to
HIV infection

• ESN (exposed seronegative) A small group of
  individuals who remain seronegative despite
  exposure are homozygous for a non-functional
  variant of CCR5.
• Frequency in Caucasians of mutant allele is 10.
  1 are homozygous. Allele not found in Western
  or Central Africans or Japanese.
• No health problems in heterozygous or homozygous
  individuals.
• Resistance to infection in these individuals
  confirms that CCR5 is the major coreceptor used
  by HIV to establish primary infection in vivo.
• Possibility of using CCR5-blocking reagents
  prophylactically to prevent infection.

Often a switch to X4 viral phenotype (use CXCR4
as coreceptor) -- more T cells are infected
Usually infected with R5 viruses (use CCR5 as
coreceptor)
Macrophages become a reservoir for HIV after T
cell depletion.
22
Figure 11-23
HIV RNA is transcribed by viral reverse
transcriptase into DNA that integrates into the
host genome

• Viral genome enters cell after fusion of viral
  and host cell membranes.
• Viral reverse transcriptase protein packaged
  together with viral genome transcribes viral RNA
  into viral cDNA (complementary DNA).
• Viral cDNA integrated into host cell genome by
  viral integrase.
• Integrated cDNA is called the provirus.
• Analogous to integrated prophage in a phage ?
  lysogen

Integrase enters cell along with viral genome.
RT enters cell along with viral genome.
23
Consequences of integration of HIV genome into a
host cell chromosome

• HIV DNA that is integrated into a chromosome is
  duplicated when cell divides, therefore all
  progeny of the infected cell will contain HIV
  DNA.
• Individuals with HIV in its latent, proviral form
  are healthy and show no signs of AIDS.
• HIV can be passed among healthy individuals
  because HIV can residue as a provirus in T cells.
  Healthy individuals whose T cells contain
  integrated HIV DNA can transfer HIV in blood or
  semen, both of which contain T cells.

24
Figure 9-15 part 3 of 4
HIV lies dormant in resting T cells and
replicates in activated T cells
NFkB is normally supposed to be used by the cell
to transcribe genes related to host defense -- it
enters the nucleus to transcribe genes only under
conditions of stress (e.g., when T cells are
activated). Note that HIV is using the host
cells normal defense mechanisms to transcribe
its genes. Another example of evolution at work!
Tat and Rev promote viral replication in
activated T cells.
NFkB is a host cell transcription factor that
binds to the viral LTRs to initiate transcription
by the host cells RNA polymerase.
25
Tat is required for HIV-1 replication

• Tat transactivator protein
• Tat protein binds to start of a new HIV RNA
  strand
• Tat binds to TAR Transactivator Active Region
• Tat binds to TAR and activates transcription of
  HIV genes

26
Figure 9-15 part 4 of 4
Final steps in HIV assembly and budding from host
cell
HIV needs to export unspliced, singly spliced,
and multiply spliced mRNAs from the nucleus to
the cytoplasm in order to make all of its
proteins. Eukaryotic cells normally prevent
export of incompletely spliced mRNAs. Fully
spliced Rev mRNA leaves the nucleus and gets
translated in cytoplasm. Rev protein then enters
the nucleus and binds to a specific site on the
viral RNA and to a host transport protein to
force export of unspliced viral mRNA.
,
Note there are some inaccuracies in this
figure (maturation to a bullet-shaped capsid
occurs AFTER budding).