What is a neutralizing antibody? (NAb)

• “An antibody that reacts with an infectious agent, usually a virus, and destroys or inhibits its infectiveness and virulence.” (Free Dictionary by Farley)

• “An antibody that keeps a virus from infecting a cell, usually by blocking receptors on the cell or the virus.”www.amfar.org/cgi-bin/iowa/bridge.html

Why don’t antibodies block HIV from infecting and/or clear HIV?

• Free virus and infected cells both display cell-surface spikes.

• Why doesn’t the body make effective anti-spike antibodies to prevent infection of host cells and clear free virus and virally-infected cells?

Envelopespike

CD4CCR5

Poignard et al. Annu. Rev. Immunol., 19: 253-274, 2001.Parren and Burton Adv. Immunol., 77:195-262, 2001.

Antibody could sterically interfere with viral attachment and/or fusion

Note the trimeric spikes shown on this

and subsequent slides are models, not

atomic resolution structures.

gp120

gp41

IgG

Need to understand role of gp120/gp41 in HIV entry into cells to understand why most antibodies are ineffective

against HIV

HIV spike (trimer of gp120/gp41 heterodimers) binds to CD4, then to CCR5 (co-receptor) on target cell.

gp41

gp120

receptor

co-receptor

Note that CD4 acts as the receptor for HIV, but it acts as a co-receptor in T cell-mediated cellular immune responses (in which case, the T cell receptor is the receptor).

HIV Envelope glycoprotein (gp160 -->gp120/gp41)

• gp160 assembles as trimer, then is cleaved into gp120 and gp41 during transport to surface of infected cell

– gp120 (soluble, no membrane-spanning region) contains CD4 and co-receptor (CCR5 or CXCR4) binding sites– gp41 is membrane-bound

• Cleavage of gp160 allows conformational changes upon binding CD4 and co-receptor

– Binding to CD4 triggers conformational changes in gp120 that open up binding site for co-receptor– Co-receptor binding leads to dissociation of gp120 from membrane-anchored gp41; gp41 then refolds resulting in fusion of viral and target cell membranes

Movie courtesy of Dennis Burton, Scripps Clinic

Available 3D structures

• Unliganded gp120 monomer (no CD4)• gp120 monomer + CD4 + antibody Fab• gp120 monomer + antibody Fab• gp41 post-fusion state

No high resolution structures of gp120 trimers or a gp120/gp41 complex

gp120* conformational states

Zhou, T., et al., Nature 445, 732 (2007)

http://www.childrenshospital.org/cfapps/research/data_admin/Site142/mainpageS142P3.html

*Crystal structures are presently available for gp120 monomers only -- no high resolution structural information about the trimeric spike.

Kwong et al., 2005, Science 310: 1025-1028

Modeled gp120 trimer on membraneNote: HIV envelope spikes are trimers of gp120/gp41 (gp41 anchors gp120 to the viral membrane and is responsible for fusion to the host cell membrane). gp41 not shown in this model because there are no structures of gp41 in a pre-fusion state.

Viral membrane

3D reconstruction of SIV virions by cryo-electron tomography Zanetti, G., et al., PLoS Pathogens 2, e83 (2006)

Trimeric HIV envelope spike structures have been examined on viruses using cryoelectron tomography

The HIV envelope spike is a trimer of gp120/gp41* heterodimers.*gp (glycoprotein) 120 is 120 kDa; gp41 is 41 kDa.

HIV-1 spike trimer structure from electron cryotomography

gp41

gp41

Liu et al., 2008, Nature 455: 109-113

gp120/gp41 evade antibodies in several ways

• Broadly neutralizing antibodies against gp120 or gp41 rarely found in infected patients

– Sequence variability in gp120

– gp120 is shed -- shed gp120 is conformationally different than trimeric gp120 spikes on the viral membrane. Shed gp120 acts as decoy because antibodies bind more tightly to it than to envelope (membrane-bound) gp120.

– gp120 carbohydrates hide protein epitopes (gp120 is 50% N-linked carbohydrate).

– Antibodies are too big to access conserved regions of gp120/gp41 spikes on a virion.

• CD4 binding site on gp120 is a narrow pocket.• Antibodies can’t access CCR5 binding site when HIV is bound to a

target cell membrane via CD4. • gp41 is inaccessible until gp120 dissociates.

gp120

Sequence variability: A variable loop on gp120 encourages the formation of strain-specific

antibodies

Infected cells release misfolded or

incompletely assembled gp120s as decoys

HIV keeps the immune system busy making ineffective antibodies

NAb

Antibodies produced against gp120 aren’t very useful against viruses because of “original antigenic sin”: once antibody responses are generated by vaccination (e.g., by shed gp120 or inactive gp120), host can’t make de novo Ab responses to envelope gp120 or gp120 in related viruses -- now have Abs with high affinity to shed gp120 or misfolded gp120, but low affinity to envelope gp120.

Most anti-gp120 antibodies don’t bind to gp120 trimers on a virus

gp120gp41

Clicker question: Env spikes are visible on free virus, but poorly accessible to antibodies, partly because gp120 is covered with carbohydrates. Carbohydrates are poorly immunogenic because:1) They are synthesized by host machinery2) They are charged3) They are hydrophobic4) They are sweet

EM image of SIV (HIV shows fewer spikes)

Clicker question: Although the co-receptor binding site on gp120 (i.e., the CCR5 binding site) is highly conserved, antibodies that bind to it are generally unable to neutralize the virus. This is because…

1) Blocking the co-receptor binding site won’t prevent infection if CD4 can still bind.2) The co-receptor binding site becomes accessible only after gp120 binds CD4 and is close to the membrane, so antibodies simply can’t fit.3) Antibodies that recognize the co-receptor binding site are unstable and easily degraded.

Clicker question: Although the co-receptor binding site on gp120 (i.e., the CCR5 binding site) is highly conserved, antibodies that bind to it are generally unable to neutralize the virus. This is because…

1) Blocking the co-receptor binding site won’t prevent infection if CD4 can still bind.2) The co-receptor binding site becomes accessible only after gp120 binds CD4 and is close to the membrane, so antibodies simply can’t fit.3) Antibodies that recognize the co-receptor binding site are unstable and easily degraded.

Labrijn, A. F., et al. (2003) J Virol 77, 10557-65.

In vitro neutralization assaysEvaluate infectivity of HIV in the presence of different concentrations of an Ab.

Which Ab is most effective at neutralization?1) Red 2) Blue 3) Green

scFv (single-chain Fv)

Intact IgG antibody

Smaller versions of antibodies against the CCR5 binding site neutralize HIV more effectively than intact

antibodies

These are idealized data from an in vitro neutralization assay.

The conserved coreceptor binding site is exposed on gp120 after CD4 binds, but there isn’t space for an intact antibody to fit both underneath gp120 and between the viral and target

cell membranes

Burton et al. (2005) PNAS 102, 14943-8

One subunit of gp120

Fab

FabFc

Steric restrictions on antibody access to the coreceptor binding site on gp120

Burton et al. (2005) PNAS 102, 14943-8

scFv and Fab versions of anti-CCR5 binding site antibodies can fit in space between gp120 and target cell membrane. Intact IgG is too big. Explains why scFv and Fabs of anti-CCR5 binding site antibodies neutralize virus better than IgG versions.

A limited number of potent, broadly NAbs have been isolated from HIV-infected individuals

Burton, Dennis R. et al. (2005) Proc. Natl. Acad. Sci. USA 102, 14943-14948

HIV-1 envelope spike

Broadly NAbs(until 2009):4E102F5b122G12

New broadly NAbs are more potent

Walker et al., 2009, Science 326: 289

PG9/PG16 NAbs against variable loops. Trimer-specific.

VRC01, a new anti-CD4 binding site NAb, is more potent than b12

Wu et al., 2010, Science 329: 856

Summary of broadly NAbs available 2010

Walker & Burton, 2010

Extra slides

Deglycosylated CD4-bound HIV gp120 core

Glycosylated unliganded SIV gp120 core

Conformational changes caused by CD4 binding to gp120

Chen et al., 2005, Nature 433: 834-841

V2

CD4 bindingsite

non-neutralizing

face: poorly

accessible on trimer

silent face: heavily

glycosylated outer domain

coreceptorbinding site

neutralizingface

V3

V1/V2

gp120 structurefrom Kwong et al

Burton et al. (2005) PNAS 102, 14943-8

gp120 spikes are covered in carbohydrates.Carbohydrates are poorly or nonimmunogenic.

Model of gp120 trimer based on structure of monomeric gp120.

Carbohydrates shown in yellow or blue stick representation.

Locations of neutralizing antibody epitopes indicated.

gp41 shown as pink cylinders

Viral membrane

Zanetti, G., et al., PLoS Pathogens 2, e83 (2006) Zhu, P. , et al., Nature 441, 847 (2006)

The resulting structure is low resolution, but shows that the trimer should be accessible to antibodies

Clicker question: Like the binding site on rhinovirus (common cold virus) for its cellular receptor, the CD4 binding site on gp120 is a narrow pocket. What is the evolutionary advantage for a virus of using a narrow pocket for a receptor binding site?

1) Recessed pockets allow for stronger binding interactions.2) Cellular receptors tend to fold into pointed structures.3) Antibody surfaces are too broad to access them, so the virus

can avoid the humoral immune response.

Luo, et al. 1987. Science. 235: 182-91.

Clicker question: Like the binding site on rhinovirus (common cold virus) for its cellular receptor, the CD4 binding site on gp120 is a narrow pocket. What is the evolutionary advantage for a virus of using a narrow pocket for a receptor binding site?

1) Recessed pockets allow for stronger binding interactions.2) Cellular receptors tend to fold into pointed structures .3) Antibody surfaces are too broad to access them, so the virus can avoid the humoral immune response.