1. In thinking about vaccines, recall that there are two arms of the adaptive immune response
Cellular (Cytotoxic T Lymphocyte or CTL) response -- works well for controlling (most) viral infections because viruses replicate intracellularly. Less adapted for controlling extracellular pathogens such as bacteria or protozoa.
Humoral (antibody-mediated) response -- works well for controlling extracellular pathogens.
Passive transfer of antibodies alone (e.g., mother to offspring) can protect against viral infection.
Neutralizing antibodies can prevent reinfection by some viruses (e.g., influenza).

2. Vaccination
For most viruses, you are immune to reinfection by the same virus (e.g., chicken pox).
Immunization or vaccination: Process by which one is exposed to a live or inactivated virus, or to components of the virus, in order to establish a state of immunity.
Immunizations against smallpox introduced >1000 years ago. Variolation: introduce dried smallpox scabs into nose of an uninfected person, who then contracted a mild form of the disease but was immune to smallpox.
1-2% died after variolation compared with 30% after smallpox.
Vaccination against smallpox introduced by Edward Jenner in Jenner infected a boy with cowpox (a live-attenuated virus), then exposed him to smallpox, which he failed to contract.
Jenner later experimented on other children, including his son. These sorts of experiments are illegal today.

3. Clicker question
Most vaccines today are given in the form of whole-killed virus or live-attenuated virus. Whole-killed virus vaccines induce _____ immunity Live-attenuated virus vaccines induce ____ immunity.
cellular and humoral; cellular and humoral
cellular; cellular
humoral; humoral
cellular; cellular and humoral
humoral; cellular and humoral

4. Polio vaccine has been very successful in the US
Polio epidemics in 1950s affected >50,000 people in US.
Salk vaccine is an inactivated virus given in a series of injections. The vaccine induces circulating antibodies, but no cellular immunity. Prevents spread of virus from gastrointestinal tract to the central nervous system, but doesn’t prevent infection of the gastrointestinal tract by the virus.
Sabin vaccine is a live-attenuated virus given orally. Produces cellular immunity and circulating antibodies and prevents subsequent infection by wild-type virus. Worldwide use of Sabin vaccine has eradicated polio in the US and Americas.
We will discuss both of these methodologies (whole-killed virus and live-attenuated virus approaches) as potential ways to make an HIV vaccine.

5. Clicker question
What chemical did Jonas Salk use to inactivate poliovirus to make the famous vaccine?
Formaldehyde
Polyethylene Glycol
Methanol
Ammonia

6. Clicker question
What chemical did Jonas Salk use to inactivate poliovirus to make the famous vaccine?
1) Formaldehyde
2) Polyethylene Glycol (in many things, including in Dr. Pepper: prevents over-foaming)
3) Methanol
4) Ammonia

7. Clicker question
How many American children contracted polio in 1955 because of an incompletely inactivated batch of poliovirus vaccine produced by Cutter Laboratories?
1) 10
2) 100
3) 20000
4) 40000
Source: Offit, PA (2005) The Cutter Incident, 50 years later. New England Journal of Medicine

8. Clicker question
How many American children contracted polio in 1955 because of an incompletely inactivated batch of poliovirus vaccine produced by Cutter Laboratories?
1) 10
2) 100
3) 20000
4) ,000 doses contained live virus, resulting in 40, cases of abortive polio (headache, stiff neck, fever, muscle weakness). 51 children were permanently paralyzed, 5 died; 113 family members were paralyzed, 5 died.
Source: Offit, PA (2005) The Cutter Incident, 50 years later. New England Journal of Medicine

9. Cases of polio per year in US
From Morbidity and Mortality Weekly Report
(MMWR), Vol. 46, p. 79 (1997)

10. 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 season consisted of H3N2, H1N1 and B influenza viruses. (But the vaccine H1N1 strain doesn’t 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

11. Why we need an HIV vaccine: Anti-retroviral therapy hasn’t eradicated HIV
Anti-retroviral treatment regimens are complex, expensive, and can result in serious side effects.
Developing safe, effective and affordable vaccines that can prevent HIV infection in uninfected people is the best hope for controlling and/or ending the AIDS epidemic.
In 1984, Margaret Heckler (President Reagan’s Secretary of the Department of Health and Human Services) announced that the virus responsible for causing AIDS had been identified, and that a vaccine would be ready for testing within two years.
We still don’t have a vaccine. Why?

12. “CD4” means CD4 T cells; “CTL” means CD8 cytotoxic T lymphocytes; “VLP” means virus-like particle
Pantello and Koup, 2004, Nature Medicine 10:

13. Pantello and Koup, 2004, Nature Medicine 10: 806-810

14. Traditional approach to an HIV vaccine: whole-killed virus
Problems:
Safety issues: are the viruses really dead?
Production of strain-specific responses: would need to inject a mixture of MANY different killed HIVs.
A big problem: gp120 is shed from HIV, so what is being injected isn’t a form to which the host will make effective neutralizing antibodies.
Another big problem: where do you get the HIV to kill and then inject? Answer: grow in cells in the lab, but laboratory strains are antigenically different than primary HIV isolates.

15. Future research needs (from http://www. niaid. nih
• Identify HIV isolate(s) that retain sufficient envelope glycoprotein to effectively mimic wild-type HIV and generate a strong, protective immune response.
• Develop and test inactivation procedures that will retain antigenic integrity of the envelope glycoprotein.
• Develop cells and culture methods that will permit large-scale production of primary HIV isolates.
• Develop alternative methods that can circumvent some of the safety and technical concerns posed by manufacturing and then inactivating infectious HIV virions.

16. Live-attenuated virus approach to making an HIV vaccine
Initial optimism because live SIV with a deletion in the Nef gene* caused no disease in macaques and protected against initial infection by wild-type** SIV.
However, Nef-deletion mutants only slow disease progression, but don’t abrogate it completely
Adult macaques eventually showed immune dysfunction and 18% developed AIDS.
This vaccine shows limited or no cross protection against infection by other strains of SIV.
Live-attenuated HIV vaccines have not been tested in humans due to safety concerns.
Long-term growth of live-attenuated viruses would be done in culture, but laboratory strains of HIV are antigenically different than primary isolates.
*Nef down-regulates surface expression class I MHC molecules and CD4
**Wild-type: A normal, non-mutant form of a macromolecule, cell or organism

17. Possible vaccine approaches
Induction of antibodies by injection with whole-killed virus or viral proteins.
Induction of antibodies and cellular immunity using live-attenuated virus.
Induction of only cellular immunity (cytotoxic T lymphocyte (CTL) responses) by inducing expression of viral proteins.
DNA vaccines -- injection of purified DNA, usually into muscle, results in transient expression of proteins encoded by introduced genes. Peptides derived from these proteins are presented by MHC class I proteins to CD8 CTLs.
Note that these sorts of vaccines can never prevent infection because T cells do not recognize or kill free viruses. A strong anti-HIV CTL response, however, has the potential to reduce or eliminate virally-infected cells and therefore greatly reduce viral load.
DNA vaccines:

18. Possible HIV Vaccine Strategies
Peptide vaccines
Recombinant subunit protein vaccines
gp120, gp41, or gp160 produced by genetic engineering
Live vector vaccines
non-HIV viruses engineered to carry HIV genes
Virus-like particle vaccines
non-infectious HIV containing one or more HIV genes
DNA vaccines
HIV genes inserted into plasmids
Combinations of vaccines
Prime-boost regimens (e.g., DNA vaccine followed by injection of recombinant protein)
Current HIV Vaccine trials:

19. Merck’s HIV vaccine
Merck V520 ad5 vaccine -- a live attenuated virus designed to produce cellular and humoral immunity
Three stretches of HIV genome (from HIV gag, pol and nef genes)
Note HIV genes derived from only one HIV strain
Shell from adenovirus type 5 (normally causes cold symptoms)
Virus was replication incompetent -- could infect cells, but couldn’t produce more virus -- Vaccine cannot cause HIV infection.

20. STEP trial
1500 volunteers (high-risk for HIV infection) in Americas and Australia recruited in December volunteers by March 2007.
62% male, Average age = 29
Trial stopped in September 2007.
Vaccine conferred no protective effects against HIV infection and no effect on course of infection.
More infections in vaccine recipients (49) than placebo (33).
Much analysis now to figure out what went wrong.
Is difference in infection rates significant?
Did adenovirus immunity increase infection risk?

21. RV144 – the Thai Prime-Boost AIDS Vaccine Trial
Prime with ALVAC-HIV (to induce cellular immunity)
Recombinant canarypox vector vaccine genetically engineered to express HIV-1 gp120 (clade E) linked to the transmembrane anchoring portion of gp41 (clade B), and HIV-1 gag and protease (clade B)
Induces HIV-specific CD8 T cell responses in ~20% of recipients
Boost with AIDSVAX (to induce humoral immunity)
HIV gp120 envelope glycoprotein vaccine (one clade E gp120 and one clade B gp120)
Results in non-neutralizing antibodies in most recipients
Human trials in 2003 with AIDSVAX alone showed no protection
Reported in fall 2009 – First vaccine to show reduced risk of HIV infection

22. RV144 – the Thai Prime-Boost AIDS Vaccine Trial Cost: ~$105,000,000
Reported to lower rate of HIV infection by 1/3
16,402 participants (low to moderate risk for HIV infection); 1:1 vaccinated versus placebo (HIV testing every 6 months after vaccination)
74 in placebo group became infected
51 in vaccinated group became infected
31.2% efficiency; 96% confidence level (p < 0.05)
HIV replication, CD4 T cell loss same in HIV-infected vaccine and placebo groups

24. Is there any hope for an antibody-based therapeutic approach against HIV given that people don’t normally make broadly neutralizing antibodies against HIV?
HIV rapidly mutates so that antibodies are no longer effective.
HIV spike proteins are covered with carbohydrates, which are poorly or non-immunogenic.
Antibodies are too big to access some regions of the HIV spike.
Burton, Dennis R. et al. (2005) Proc. Natl. Acad. Sci. USA 102,

25. A limited number of “broadly” neutralizing antibodies have been isolated from HIV-infected individuals
Locations of antibody binding sites on schematic model of trimeric viral spike
Name
Epitope
Affinity (KD)
Structure
4E10
Conserved (98%) linear WFXI sequence of gp41 C-terminal to the 2F5 epitope
1 – 70 nM (n = 8)
2.2 Å
2F5
Conserved linear ELDKWA sequence of the membrane proximal ectodomain of gp41
0.5 – 5 nM (n = 8)
2.0 Å
b12
Discontinous epitope overlapping the CD4 binding site of gp120
0.2 – 8 nM (n = 5)
1.8 Å
2G12
a12 mannose cluster on the “silent” face of gp120
2 nM (n = 1)
1.8 Å 3.0 Å
17b
CD4-induced discontinuous epitope overlapping the coreceptor binding site
nM (n = 11)
2.5 Å
X5 (PD)
CD4-induced discontinuous epitope near the coreceptor binding site
0.1 – 100 nM (n = 3)
1.9 Å
WHAT I AM SHOWING HERE ARE REPRESENTATIVES OF THESE DIFFERENT CATEGORIES
AFFINITY AND NEUTRALIZATION IN THE CONTEXT OF HIV
Kwong, P.D. et al., J. Virol :
Burton, D.R., R.C. Desrosiers, et al., Nat Immunol, (3):
PD means “Phage display”, an in vitro method.

26. HIV has evolved to thwart the immune system it has to confront
Few, if any, HIV-infected individuals clear the virus.
We can, however, make neutralizing reagents (antibodies or antibody-like proteins) that prevent HIV infection.
What we cannot yet do is design an immunogen that will elicit broadly neutralizing reagents in a person (traditional vaccine approach).
Can we reprogram a human immune system so that it makes broadly neutralizing anti-HIV reagents?

27. Gene therapy -- could it be used to treat or prevent AIDS?
Adenovirus is not a retrovirus, so the inserted genes are not integrated into the host cell genome or inherited by the descendents of infected cell.
Use recombinant DNA technology to insert a new gene into a virus, then use that to infect cells. Infected cells will transcribe the new gene and make the encoded protein.

28. Gene therapy using retroviral vectors
Advantage: Don’t have to re-administer the recombinant virus (as required if using recombinant adenoviruses) because genetic material is incorporated into the host cell genome, so all descendents of infected cell will inherit it.
Disadvantage: Retroviral integrase can insert the viral genetic material anywhere in the host genome.
Insertion into the middle of a host cell gene will disrupt it.
Insertion upstream of a gene can disrupt its regulation.
Disruption of a gene regulating cell growth could lead to uncontrolled cell division (i.e., cancer).
Gene therapy trials in France to treat X-linked Severe Combined Immunodeficiency (X-SCID) were successful in curing the disease, but resulted in leukemia in several patients.

29. Gene therapy may be safer using lentiviral vectors than onco-retroviral* vectors
French trial treating X-SCID - 3 of 11 developed leukemia-like condition. Used murine leukemia virus (MLV)-based vector for ex vivo** infection of hematopoietic stem cells.
Lentiviral vectors (HIV,SIV) have different integration properties than the onco-retroviral vectors (MLV).
Infection with HIV is not known to cause integration-related cancers.
from Hematti, P., et al. PLoS Biol 2: e234 (2004)
* The prefix “onco” refers to oncology, which is the study of cancer.
**ex vivo: Pertaining to a biological process or reaction taking place outside of a living cell or organism.