1. Lab of Immunoregulation
Ira Berkower, M.D., Ph.D., Lab Chief
Carol D. Weiss, M.D., Ph.D.,
Section of Viral Envelope Glycoproteins
VRBPAC presentation, December 15, 2009

2. LAIR Mission
We provide scientific expertise in the review of viral vaccines for safety, efficacy and potency.
Regulatory responsibilities:
Evaluate new vaccines under IND and serve on licensing committees
Write regulatory guidance documents
Provide clinical oversight of HIV vaccine trials
Vaccines include:
- HIV: inactivated virions, live attenuated viral vectors, and viral replicons
DNA vaccines, and recombinant proteins
- Influenza: inactivated virions
- Hepatitis B: recombinant virus-like particles
Research areas:
- Virus-like particles for presentation of HIV envelope proteins
- Live attenuated viral vectors
- Virus entry pathways and antibody neutralization

3. HIV research areas: I. Virus-Like Particles expressing gp120
HBsAg
env
226/42
125/205
503
NH2
Berkower et. al. Virol 321:75 (2004)
VLP expressing gp120

4. The CD4bs is a target of broadly reactive neutralizing antibodies
20-21 loop
b15-a 3 loop
Open form gp120
Closed form/open form
Berkower, et al Virol 377:330 (2008)

5. Progress with gp120 VLPs
By deleting loop C, we exposed the CD4 binding site and
improved antibody binding by over 100- fold.
This is an important target of broadly reactive neutralizing
antibodies.
Results were published in Virology.
- confirmed by the Vaccine Center at NIH and used in their
vaccines.
- Loop C function has been tentatively identified as binding
the second exposed loop of the CCR5 coreceptor during
viral entry.
Recently, we have identified a hinge region where the
inner/outer domains meet: by modifying the hinge, we may
control the open/closed conformation of gp120 and further
improve antibody binding.

6. II. Virus-Like Particles expressing the membrane proximal
region (MPER) of gp41: an important target of neutralizing
antibodies
MPER
HBsAg
2F5 4E10 TM
Phogat et al Virol 373:72 (2008)

7. Progress with gp41 VLPs
HBsAg-MPER can assemble particles despite replacing one or two membrane spanning domains of HBsAg with a foreign TM domain of gp41.
These VLPs display MPER in its natural milieu on a lipid surface, anchored by its own TM domain.
They show enhanced antibody binding by monoclonals 2F5 and 4E10, and they induce broadly cross reactive anti-MPER antibodies.
We are currently using milder purification conditions to preserve MPER function and to elicit neutralizing antibodies.

8. III. Live attenuated viral vector
What if these VLP antigens are still not potent enough?
A live attenuated viral vector could present HIV antigens in the context of an acute infection.
This could also act in synergy with VLPs expressing the same antigens. As part of a prime and boost strategy, they could work together to elicit a much greater antibody response than either one separately
Ideal vector:
grow well enough to immunize
safe enough to use without further attenuation
host range would allow SIV challenge.

9. Rubella can express GFP or Influenza HA epitopes
zGFP
P90 C E2 E1
P150
A. Spadaccini et. al Vaccine (2009) in press
Living Vero cells
Fixed Vero cells

10. Progress with Rubella Vector
Achieved stable expression (>12 passages) of a
25KD protein in a live, replicating vector.
- Large enough and stable enough for vaccine applications.
- Suitable for monkey immunization and challenge studies
Results were published in Vaccine
Started using live attenuated vaccine strain of rubella
RA27/3: it grows like wild type rubella.
Established a collaboration to express MPER and Gag
antigens and to test growth and immunogenicity in vivo

11. Section of Viral Envelope Glycoproteins
Carol D. Weiss, M.D., Ph.D., Senior Scientist
Wei Wang, Ph.D., Staff Fellow
For conversion to staff scientist
Virus entry and antibody neutralization

12. peptide fusion inhibitors (?antibodies)
Background: HIV Env-mediated entry X
peptide fusion inhibitors (?antibodies)
antibodies
(pH trigger for HA)
HR1
HR2
CoR
CD4
CoR
CD4
gp120
fusion
gp41
native Env
fusion intermediate
six-helix bundle
HIV projects
-Elucidate Env entry mechanism with peptide fusion inhibitors
-Evaluate Env intermediates for antigenicity, immunogenicity,
and neutralization
You might guess that resistance to HR1 occurs in HR2, or HR1.

13. HIV research highlights
Two pathways of resistance for HR1 peptide fusion inhibitors that target fusion-intermediate conformations of Env
CD4bs mutations cluster with HR1, V3 mutations cluster with HR2, suggesting cross talk between these regions
Mutations confer cross-resistance to HR2 inhibitors (T20), but distinct from resistance that emerges to T20
Resistance mechanism involves global changes in Env function that enhance receptor use.
- Likely represents increase entry kinetics, decrease half-life of
fusion-intermediate target of peptides and explains cross-resistance to T20
Env antigens that mimic fusion-intermediates may be
useful targets for vaccine antigens.
Can enhance immunogenicity of gp41 regions containing broadly neutralizing determinants (2F5, 4E10)

14. Influenza project Established an influenza neutralization assay using
pseudotypes with HA on the outside and retroviral
capsid and a reporter gene on the inside to
Evaluate protective antibody titers and correlates of protection
Assess antigenic relatedness among influenza variants
Investigate effects of specific epitopes on infectivity & antigenicity

15. Influenza research highlights
Established methods for creating functional retroviral pseudotypes bearing HA from H1, H3, and H5 subtypes
Overcame technical hurdles to make functional pseudotypes for H1N1 and H3N2
Have shown:
- HA-pseudotype neutralization titers correlate well with
microneutralization titers
Cross-clade immunity among H5N1
In sum: HA pseudotypes provide a safe and versatile tool for evaluation neutralizing antibodies to seasonal and pandemic influenza
Being applied to H1N1

16. Conclusions Our research supports CBER’s mission by:
Advancing the field of HIV and influenza vaccines, maintaining our
expertise, and enabling us to provide high quality guidance
for sponsors of new vaccines.
There is synergy between VLPs and live vectors:
- The same antigenic determinant (gp120 or gp41) could be
primed on VLPs and boosted on a live viral vector.
There are also similarities between HIV and
influenza entry pathways:
- For HIV, understanding the fusion pathway leads to fusion
intermediates that can serve as vaccine targets, and
- For Influenza, the pseudotype assay allows us to evaluate
neutralizing antibodies and demonstrate relatedness or differences
among the seasonal and pandemic influenza strains: H1, H3, H5 and
H1N1.