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

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

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

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)

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.

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)

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.

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.

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

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

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

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.

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)

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

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 2009 H1N1

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.