Review Article Neutralization of Virus Infectivity by …downloads.hindawi.com/archive/2014/157895.pdfReview Article Neutralization of Virus Infectivity by Antibodies: Old Problems

Review ArticleNeutralization of Virus Infectivity by AntibodiesOld Problems in New Perspectives

P J Klasse

Department of Microbiology and Immunology Weill Cornell Medical College Cornell University New York NY 10065-4896 USA

Correspondence should be addressed to P J Klasse pek2003medcornelledu

Received 30 April 2014 Accepted 12 August 2014 Published 9 September 2014

Academic Editor Ma Luo

Copyright copy 2014 P J Klasse This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Neutralizing antibodies (NAbs) can be both sufficient and necessary for protection against viral infections although they sometimesact in concert with cellular immunity Successful vaccines against viruses induce NAbs but vaccine candidates against some majorviral pathogens including HIV-1 have failed to induce potent and effective such responses Theories of how antibodies neutralizevirus infectivity have been formulated and experimentally tested since the 1930s and controversies about the mechanistic andquantitative bases for neutralization have continually arisen Soluble versions of native oligomeric viral proteins that mimic thefunctional targets of neutralizing antibodies now allow themeasurement of the relevant affinities of NAbsThereby the neutralizingoccupancies on virions can be estimated and related to the potency of the NAbs Furthermore the kinetics and stoichiometry ofNAb binding can be compared with neutralizing efficacy Recently the fundamental discovery that the intracellular factor TRIM21determines the degree of neutralization of adenovirus has providednewmechanistic and quantitative insights SinceTRIM21 residesin the cytoplasm it would not affect the neutralization of enveloped viruses but its range of activity against naked viruses will beimportant to uncover These developments bring together the old problems of virus neutralizationmdashmechanism stoichiometrykinetics and efficacymdashfrom surprising new angles

1 Introduction

Neutralizing antibodies (NAbs) are the best correlate of pro-tection from viral infection after vaccination [1ndash8] Likewisethey are markers of immunity against reinfection after anacute infection has been cleared Such immunity can be life-long [9ndash11] Many vaccines against viral infections are goodinducers of protective neutralizing antibody responses butrecalcitrant problems remain in the field of viral vaccinationOne problem is antigenic variabilityThe antigenic targets forneutralizing antibodies on influenza virus vary abundantlyand therefore a new vaccine must be prepared every newseason [7] Hepatitis C virus (HCV) and human immunode-ficiency virus (HIV) are even more variable and after yearsof research we still cannot induce immune responses thatprotect against them [7 12ndash14]

Antibodies are the products of the adaptive humoralimmune response the molecules they recognize are calledantigens the molecules that elicit the antibody responseare immunogens Hence some proteins particularly foreign

ones are both immunogens inducing the production of spe-cific antibodies against themselves and antigens the targetsof the response Other molecules for example small organiccompounds may bind with high specificity to antibodieswithout being able to elicit those antibodies except whenconjugated to larger carriers [15] Such small moleculesare called haptens They illustrate the important distinctionbetween immunogenicity the capacity to elicit an immuneresponse and antigenicity the capacity to be recognized bythe immune response [15 16]Themore precise surface patchon an antigen that is directly contacted by the antibody isthe epitope and the corresponding surface on the antibodyin direct contact with the antigen is the paratope [17]

Some successful vaccines against virus infections havebeen based on attenuated but replicating variants of thepathogenic virus for example the smallpox vaccine and oneform of polio vaccine Attenuated variants of the simianimmunodeficiency virus (SIV) which is closely related toHIV have provided stronger protection than nonreplicatingexperimental vaccines against the wild-type virus but the

Hindawi Publishing CorporationAdvances in BiologyVolume 2014 Article ID 157895 24 pageshttpdxdoiorg1011552014157895

2 Advances in Biology

mechanism of protection is not completely elucidated [18ndash20] In other cases recombinant proteins representing sub-units of hepatitis B virus (HBV) and human papilloma virus(HPV) induce strong protection [21 22] The HPV vaccineconsists of virus-like particles that may have advantageousproperties both antigenically and immunogenically theymaypresent native neutralization epitopeswell and be seen bythe innate immune system as pathogen-associated molecularpatterns [23] But subunit immunizations have failed toprotect against HIV type 1 (HIV-1) [1 7 8 14 24] Onlyin the RV144 clinical trial which combined viral proteinsexpressed from a canarypox vector with recombinant subunitprotein boosts was some modest protection observed Butthe vaccine had not induced NAbs [8 25 26] Therefore thehunt is on for other antibodies and immune responses thatmight explain the limited protectionMany different antiviraleffects of antibodies have been described that do not qualifyas neutralization [27 28] This brings us to some semanticclarifications

2 The Definition of Virus Neutralization

Definitions are arbitrary and contain no deeper knowledgethan the proposed use of the defined term [29]Therefore theonly reason to adhere to a strict definition of neutralizationis that it may favor clarity and allow useful distinctions in thefield of antiviral research Neutralization as discussed here isdefined as the reduction in viral infectivity by the binding ofantibodies to the surface of viral particles (virions) therebyblocking a step in the viral replication cycle that precedesvirally encoded transcription or synthesis [30 31] Classicallythe term was applied only to antibodies and fragments ofantibodies Fab and F(ab1015840)2 but later it has naturally beenextended to single-domain antigen-binding recombinantfragments and natural nanobodies [32 33] Likewise thedefinition can be expanded to cover similar activities bysoluble forms of viral receptors [34 35] naturally occurringdefensins or other molecules of the innate immune system[36 37] it can be extended to lectins either derived fromplants or soluble recombinant and hybrid forms of mannoseC-type lectin receptors (MCLRs) [38] With regard to smallorganicmolecules if they are said to be neutralizing it shouldbe clarified whether that is meant to imply that they act bybinding to the surface of the virion [39ndash49] This reviewhowever only discusses antibody-mediated neutralization

What does the definition not include An antibodymightbind to a budding virion thus acting late in the viral cyclethereby blocking the release of newly formed virus from thesurface of an infected cell Examples of antibodies acting likethis are those directed to the neuraminidase on the influenzaviral surface [50] The enzyme releases progeny virus bydigesting the neuraminic-acid moiety of the receptor for thevirus We shall see later why such an antibody althoughits antigen decorates the virion surface does not interfereat the beginning of the replicative cycle and therefore perdefinition does not neutralize

Another semantic point is that an antibody to a receptorfor the virus on the cell surface may block viral infection but

does not neutralize according to the definition it does notbind to the virions each of which diffusing around in theextracellular space would be potentially as infectious as inthe absence of the antibody instead the target cells would berendered nonsusceptible Hence for clarity some other termsuch as infection-blocking antibody should be used in thatspecial case

The usefulness of a strict definition becomes obvi-ous in vaccine research when we ask by which mech-anism vaccine-induced antibodies protect from infectionAntibody-dependent cellular cytotoxicity (ADCC) is a well-established effect that requires effector cells and counteractsviral infection by killing the virus-producing cell [27 28]The antibodies involved may be neutralizing when tested inthe absence of effector cells (natural killer cells) but oftenare not And some neutralizing antibodies are not of theright isotype to mediate this effect Antibody-dependent cell-mediated viral inhibition (ADCVI) has a more complicatedrelationship to neutralization which may concomitantlyoccur in the assay used for measuring the effect But tothe extent that the inhibition depends on effector cells itis not neutralization Again antibody isotype will affect thecomponent in ADCVI that involves Fc-Fc-receptor interac-tions Furthermore the epitope location on the influenzavirus haemagglutinin determines whether an antibody ismerely neutralizing or also capable of mediating Fc-receptorinteractions and thereby ADCC and protection in vivo in amouse model [51]

HIV-1 can be transmitted directly from cell to cell via avirally induced connection known as the virological synapse[52ndash55] It is convenient however to call the inhibitionof cell-to-cell transfer something other than neutralizationeven when it is mediated by NAbs [56] The reason isthat non-NAbs including antibodies that interfere with theformation of the synapse by binding to cellular structuresand antibodies that counter virion formation or releasemight block this mode of transfer It is also noteworthy thatthe relative efficiency of neutralization and block of cell-to-cell transfer differs among NAbs [57] Observance of thesedistinctions makes for clarity

Normally neutralization is measured in the absenceof complement and the definition can certainly be mademore stringent by making that a criterion But it may bemore useful to allow complement-mediated enhancement ofneutralization as a legitimate category [58] regardless it isvaluable to quantify that effect [59 60] Again if complementfactors reduce viral infectivity by binding directly to the viralsurface rather than to antibodies already in complex withvirions [61] this could also qualifywithin thewider definitionof neutralization

One consideration of biological and medical importanceis that neutralization does not have to equate only whatis measured in the neutralization assays in vitro Althoughneutralization must be measured in vitro we can fruitfullydiscuss how it operates in vivo To infer that it is responsiblefor protection in vivo is more complicated and requiresthe use of antibodies without other effector functions orexperimental models in which cell-dependent mechanismsare knocked out by other means [62ndash64] And effects late

Advances in Biology 3

in the viral cycle may be harder to prevent in experimentsin vivo as always the hypotheses can only pass ever morestringent tests but never be proven true Semantically thoughthere should be no barriers to discussing neutralization thatoccurs both in vitro and in vivo

Thepart of the replicative cycle delimited by the definitionencompassesmany different steps some shared by all virusesothers specific for certain groups To infect a cell a virionmust attach itself to the cell surface This can occur viaancillary attachment factors or directly via themajor receptorused by the virus for entry some viruses then interact with asecond receptor or coreceptor [65ndash79] Naked viruses needto penetrate a cellular membrane to enter the cytoplasm orinstead they may inject their genome through a membrane[80] enveloped viruses must fuse their envelope with acellular membrane in order to translocate their core andgenome into the cytoplasm [79 81ndash85] Some viruses enterthe cytoplasm directly from the cell surface some dependon endocytosis for productive entry sometimes because thereduced pH in the endosome triggers penetration or fusion[86] If the virus enters directly from the cell surface thegenome-containing particle must again penetrate a barrierthe cortical cytoskeleton which is an actin mesh with maskssometimes smaller than naked virions or viral cores [7087] By entering after endocytosis the virus surmounts thisobstacle delivering the capsid to a more central locationAfter these steps the core may need to be uncoated or tomigrate to specific locations in the cell before transcriptionalor translational synthetic events take place beyond whichany inhibitory effects per definition no longer constituteneutralization

3 Mechanisms of Neutralization

Neutralization has many mechanistic aspects how the NAbsbind whether they induce conformational changes whetherthey irreversibly inactivate the viral proteins that mediateentry and whether they are most effective against virions insuspension or after virion attachment to cells Here howeverthe mechanism of neutralization refers specifically to whichearly step in the viral replicative cycle is blocked (Figure 1)

If the NAbs prevent the virions from attaching to the tar-gets cells that is themechanism of neutralization If the NAbsblock necessary receptor interactions after attachment thatis also a mechanism of neutralization so is interference withany other obligatory step in the entry of individual virusessuch as coreceptor engagement endocytosis fusion or pen-etration One way to explore postattachment neutralization(PAN) is to let virus adsorb to cells at a low temperaturethat does not permit fusion or internalization and then toadd the NAb It should not be inferred however that PANdoes not interfere with receptor interactions some receptorcontactsmay first be established at the lower temperature andthen the NAb binds and prevents further necessary receptorrecruitments during the warm-up [88 89] PAN might evenreverse attachment Still investigating the capacity for PANmay contribute to the characterization of a NAb That a NAbis capable of PAN does not however demonstrate PAN as the

Figure 1 The mechanism of neutralization Neutralization ofenveloped viruses blocks viral attachment and entry No othermechanisms are yet known but entry can be blocked at differentstages The three blue virions to the right represent enveloped virusparticles The first has an IgG bound to its receptor-binding protein(green for simplicity shown as a single copy) The bound NAbblocks the docking onto the receptor (grey) on the cell surface Thesecond virion has already established contact between its receptor-binding protein and the cell-surface receptor The NAb binds toan epitope on the envelope glycoprotein (viral proteins with thisfunction and topology are usually glycosylated) that may havebecome exposed after the receptor binding and blocks subsequentsteps these could be interactions with a second receptor or thefusogenic refolding of the envelope glycoprotein The third bluevirion is about to fuse with the cell membrane but NAbs boundto membrane proximal epitopes on fusogenic proteins (not shown)prevent the completion of this process The latter two interferenceswith entry could also occur in endosomes but hardly the firstunless there are alternative attachment proteins the virus can bindto and thereby get internalized The purple virion in the endosomeis prevented by NAbs from fusing its envelope with the vesicularmembrane Alternatively this purple virion could represent a nakedvirus particle the penetration of which is prevented by the NAbsThe block of infection in the endosome could properly be called apostinternalization block of entry for clarity entry should refer totransfer of the viral core or capsid or possibly only genome intothe cytoplasm The red virion on the cell surface depicts a nakedvirion that binds to a cell surface receptor and injects the genomeinto the cytoplasmThis process may occur in vesicles or semisealedinvaginations of the cell surface If the NAbs have not preventedreceptor interactions they may interfere with the extrusion ofthe genome The red virion in the cytoplasm has penetrated anendosomalmembrane in complexwith theNAb allowing binding toTRIM21 (yellow boxwith arrow) whichmediates the ubiquitinationof the complex targeting it for proteasomal degradation This fairlyrecently discovered effect constitutes the clearest example so far of apostentry mechanism of neutralization

overriding mechanism at more physiological temperaturesnor does the lack of PAN at low temperatures exclude thatit would occur at higher ones receptor-induced epitopeexposure on the entry-mediating viral proteins may requiretemperatures that do not block entry If nevertheless PAN isdetectable but less potent than preattachment neutralizationthere can be several explanations First it is conceivable thatwhen receptor contact is alreadymade a higher occupancy byNAb on the remaining receptor-binding proteins for stopping


entry is required than on the free virion Second kineticscome into play the on-rate for the NAb binding will needto be higher when the attachment-entry process has begunthan when the NAb binds in the absence of cells and hence ahigher NAb concentration is required if the on-rate constantis the same Third PAN may act partly by competition withreceptors thereby reversing attachment which may requirehigher concentrations of NAbs than preventing it becauseof the valency of the virus-receptor interactions and thestrengthening of initial attachment by subsequent receptorrecruitment

Many of these mechanisms are experimentally confirmedfor various viruses and for infection of target cells underdifferent conditions For example NAbs have been demon-strated to block attachment of rhinovirus to HeLa cells [90]The situation with poliovirus appears more complicated Butmore recent elucidation of the mechanisms of picornaviralentry may shed light on precisely which necessary stepsare prevented by neutralization and whether neutralizingmechanisms differ within the viral family [39ndash41 65 91 92]In the studies of polivirus neutralization some NAbs werefound not to block attachment but to block endocytosispartially to induce a pI shift in the virion or to preventtranscription [58 93ndash96] One study found that among NAbsinactivating unattached virus only the bivalently bindingminority were capable of PAN the others were renderedcapable of PAN through cross-linking of their Fc portionsFurthermore neutralization coincidedwith the prevention ofa structural change in the 135S RNA-containing virion andof the genomic extrusion that normally produces 80S emptycapsids in the uninhibited infection process [97] Finally thecapacity to aggregate poliovirus particles was recorded as anexception among the NAbs [97]

Aggregation of virions by antibody has been regardedas an effect to distinguish from neutralization [58] But inso far as it reduces infectivity it would qualify as a limitedneutralizing effect albeit with a complex dependence on anti-body and virion concentrations Typically virion aggregationas a function of antibody concentration describes a dome-shaped curve at low concentrations of antibodies cross-linking of antigens on individual virions is favored at higherconcentrations virions are bridged but when the epitopeson the virions come close to being saturated cross-linkingcan no longer occur A quantal assay may be required formeasuring the loss in infectivity a large aggregate woulddiffuse more slowly than virions but might sediment ontosusceptible cells fewer cells may end up being infected thanby a monodisperse suspension of virions but those that domay attain a highermultiplicity of infection In a quantal thatis focus-counting infectivity assay this could give a distinctreduction of infectivity [98] in assays based on the produc-tion of viral antigens or activation of reporter genes the effectcould be smaller or absent Still a reduced infectivity wouldcount as neutralization because it stems from how the virusis prevented from reaching and entering its target cells Invivo aggregation might have differential effects dependingon target cell availability and requirements for diffusionbut their phagocytosis of aggregates an ancillary inhibition

not attributable to neutralization itself might enhance theantibody-mediated inhibition

The situation is different when antibodies do not aggre-gate the virions but block attachment and entry by com-peting with receptor interactions of the virus or otherwisecounteract the function of the viral proteins that mediatefusion or penetration there would be no basis for a dome-shaped inhibition curve Then we might instead predictthat the higher the occupancy such antibodies reach on thesurface of the virions the less likely the viral infection willbe alternatively there is a definite threshold of occupancyabove which the infectivity is completely eliminated Theseconsiderations are formulated within the occupancy theory ofneutralization which has plausible links to the blocking ofdifferent attachment and entry steps as neutralizing mecha-nisms [31 99 100]

4 The Occupancy Theory of Neutralization

There are two kinds of scientific hypotheses some aretestable others are detestable Occupancy theories of neu-tralization come in different versions with varying potentialfor direct experimental testing Strong versions suggest thatIgG molecules because of their bulk impede the function ofviral attachment- or entry-mediating proteins One antibodymolecule per viral protein subunit or even per oligomermay be sufficient If a certain number of unoccupied suchviral proteins were required for infection then all virionswith higher occupancieswould be neutralizedTheminimumneutralizing occupancy would constitute the neutralizationthreshold So far so good but there are several complications

The binding to defective entry-mediating proteins onthe virions would not be directly relevant to neutralization(Figure 2) Antibodies that can only bind to nonfunctionalforms of entry-mediating proteins may never be neutralizingby themselves [101ndash104] although they might potentiateNAbs by making the sterically blocking coat on the virionthicker But some NAbs can bind both to functional anddefective targets and their added capacity to do the lattermay not be irrelevant to neutralization it may increase theiravidity and thereby indirectly enhance the occupancy on thefunctional targets

As outlined in Figure 2 the effects of binding to func-tional entry-mediating oligomers are also complex Theunoccupied functional sites might need to be clustered inone area of the virion surface in order to function Butthey may be moveable so that a sufficient number can berecruited into an entry complex together with receptors afterthe initial docking of the virion onto a single or too fewreceptor molecules [31 84 105ndash113] If these viral proteinsare not moveable however and they are unevenly distributedover the virion surface it may only be when NAbs bindwithin the clusters that they have a neutralizing effect All ofthese complications would be expected to soften the apparentthresholds of neutralization even if it is postulated that avirion is either completely neutralized or not [84 105 108ndash110]

A different view would be that any antibody occupancywould dent the propensity to infect and the fewer the


(a) (b)

Figure 2Neutralizing occupancies over heterogeneous populations of enveloped viruses Two enveloped virions are pictured Each has twelveglycoprotein spikes schematically displayed for clarity only at the circumference Functional spikes are shown in blue decayed or otherwisenonfunctional ones in grey Both virions have seven functional and five nonfunctional spikes but with different distributions over the twovirion surfaces If a virion requires a certain number of spikes in contiguity to form an entry complex and the spikes cannot move freely overthe virion surface the two different distributions will confer different neutralization sensitivities The virion to the left is neutralized threeNAb molecules inactivate the constellation of active spikes and one binds redundantly to an inactive spike The virion to the right is alsoneutralized but by only two NAbmolecules one inactivating a group of three spikes (three adjacent ones being postulated here to be the bareminimum for entry) and one binding redundantly to a spike that is functional but inert through lack of active neighbors Effects of this sortcould blur critical occupancy thresholds and reduce the steepness of neutralization curves in experiments with phenotypically mixed virusof which the virions carry random assortments of antigenic and nonantigenic subunits of the envelope glycoprotein oligomers Heterogeneityof the number (not shown) and distribution (shown) of functional entry-mediating viral proteins may explain how different occupancies arerequired for blocking viral entry Some of these considerations apply also to naked viruses

unoccupied functional entry-mediating molecules the lowerthe infectivity Such a proportional relationship applied to thefull range of occupancies we can call the incrementalmodelas opposed tomodels that postulate a threshold whichwe canname liminal [105 114] Now both models can be formulatedmathematically in their pure form but fitting them to datasuggests that mixedmodels are the most realistic [31 84 105ndash110] Thus there may be an absolute minimum of a closeconstellation of unoccupied entry-mediating molecules Butwhen the constellation is larger than necessary any antibodybinding still dents infectivity And if the redundancy of entrymolecules is huge that denting may be negligible in relativeterms In other words there is neither a strict proportionalitynor a sharp threshold

One version of the occupancy theory suggests thatneutralization will occur when NAbs achieve a sufficientlydense coat on the virions [99] This coating theory hasseveral distinct implications It invokes steric hindrance ofaccess not only to the occupied entry-mediating moleculebut to adjacent ones as well It implies a linear relationshipbetween virion surface area and the minimal number ofNAb molecules required for neutralization This linearityholds up approximately [99] It would explain why antibodiesto influenza virus neuraminidase do not neutralize [50

115] the density of that antigen is too low there are fourtimes asmany hemagglutinin as neuraminidasemolecules onthe virus surface [99] Some degree of neutralization doeshowever result from cross-linking the antibodies bound toneuraminidase by anti-Fc antibodies [116] Maybe the two-layered antibody coat gives greater steric interference

Likewise rabies virus is not neutralized by an antibodythat recognizes a minority conformation of its surface glyco-protein But when the majority is converted to the antigenicconformation by reduced pH or elevated temperature thevirus is neutralized by that antibody [117] This is strongsupport for the coating version of the occupancy theoryAnalogously the theory would explain why antibodies tosome cellular passenger antigens such as ICAM-1 and MHCon the surface of HIV or SIV virions do neutralize albeitin a largely complement-dependent manner [118ndash120] theantigen is so abundant that its coating partly impedes accessto the few envelope glycoprotein trimers

If steric interference can occur intermolecularly theblocking of a receptor-binding site by the binding of an IgGmolecule elsewhere on the same entry-mediating viral pro-tein is evenmore plausible NAbs that do not bind to themainreceptor-binding site on HIV-1 Env appear to block receptorinteractions if the target cells lack ancillary attachment


factors antibody binding anywhere on the receptor-bindingsubunit of Env suffices to block viral attachment to cells thatis when attachment is mediated by the primary receptorThus under those conditions steric or direct hindrance canprevent the first step in replication [121] Still it matters onwhich subunit the epitope is located Some NAbs are directedto epitopes close to the viralmembrane in the transmembraneprotein They do not interfere with attachment to cells thatlack ancillary attachment factors and hence apparently notwith receptor binding [121] In conclusion binding close tothe receptor-binding site may be required for an indirectblock of receptor interactions

The theory also implies that the potency of NAbs closelycorrelates with their affinity for the native oligomeric formof Env on the viral surface and this too largely holds upfor HIV-1 [100 103] Again an exception would be NAbsdirected to the membrane proximal region in the transmem-brane protein [122 123] Those epitopes may only becomeexposed or fully antigenic after receptor interactions haveinduced some conformational changes in Env Analogouslythe potency of peptide inhibitors binding to the transmem-brane protein does not correlate with affinity for the proteinbut with the on-rate constant [124] In summary affinity ofNAbs for native entry-mediating molecules correlates welloverall with neutralization potency but in some cases affinityfor a receptor-induced transient form of the viral proteindetermines neutralization

The coating theory furthermore implies that although thebinding of NAbs may induce conformational changes sucheffects should not be necessary for their neutralizing capacityThis prediction clashed with a long tradition of research par-ticularly on picornavirus [58 94ndash96] New data did indeedindicate a lack of correlation between neutralization andthe capacity to induce conformational changes in rhinovirus[125] which would suggest that the conformational changesare epiphenomena accompanying the binding of some NAbsbut not others A potent NAb against HIV-1 directed to anepitope separate from the CD4-binding site appears to blockCD4 interactions allosterically as detectedwith soluble formsof the Env trimer [126] whether such conformational changesare necessary also at the level of virions coated with the NAbwould be harder to investigate Perhaps allosteric interferencewith receptor interactions can add inhibitory power to meresteric hindrance

Yet another implication of the occupancy theory ingeneral and its coating version in particular is that non-NAbs should not be able to block NAbs Although studieson Japanese encephalitis virus suggested such interference[127] non-NAbs directed to the HIV-1 Env protein havebeen shown not to block neutralization by NAbs even whenthey are directed to overlapping epitopes What are theexplanations The occupancy theory implies that all NAbsthat bind to functional entry-mediating viral proteins shouldneutralize Indeed the non-NAbs that bind to epitopesoverlapping known neutralization epitopes on the HIV-1 Envsubunit gp120 have been shown not to bind to gp120 in itstrimeric native context [102 103]

For influenza hemagglutinin and poliovirus capsid pro-teins overlapping epitopes of NAbs and non-NAbs have

also been described but the crucial question is whetherthe non-NAbs really bind to the native protein on thesurface of infectious virions [93 128] Intriguingly it hasalso been observed that a mannose-binding lectin can blockneutralization by a NAb directed to a mannose epitope onHIV-1 Env [129] If the blocking of neutralization by non-NAbs ever occurs an explanation might have to be sought inconformational changes that the NAb confers and the non-NAb does not and vice versa The focus would shift backto whether particular conformational changes in the antigenare instrumental to neutralization Thus the non-NAb wouldprotect the antigen until it can be competed off by receptorsBut why would it not be competed off by the NAb

A very recent study indicates how neutralization-blocking non-NAbs might act although it reports effectsin the greyer area of shifts in potency and efficacy Certainmutations in HIV-1 Env reduce the degree of neutralizationby a broadly active and potent NAb (10E8 [122]) directedto a membrane-proximal epitope in the transmembraneprotein Although these mutations thus do not convert theNAb to a non-NAb it is possible to study how the bindingto the mutant virions affects their sensitivity to other NAbsthat is how the residually infectious virus in complex withthis still partly active NAb is neutralized [123] The NAbenhances the neutralization by some antibodies to otherepitopes in gp41 but reduces that by others including thosedirected to adjacent epitopes as well as those specific forthe CD4-binding site The stoichiometry of the binding ofthe NAb to mutant trimers is lower than for wild-type Envtwo instead of three paratopes bind [123] This low degree ofbinding stabilizes the trimer and may have distant allostericeffects such that the binding of other NAbs is facilitatedor impeded Hence this intriguing case may thus uniquelyshow that weakly neutralizing antibodies can counteractrather than add to the action of stronger ones If the allostericmechanism involves reduced affinity for the distant affectedepitopes it rather corroborates the occupancy theory thanundermines it

Also pertinent to the occupancy theory is which Abs cancapture virions Both NAbs and non-NAbs can capture HIV-1 virions but NAbs preferentially capture infectious virions[130] Paradoxically though non-NAbs which do not blockneutralization block capture [131] That raises the questionwhy the binding of NAbs to functional trimers alone doesnot mediate the capture of the virions Perhaps some NAbbinding to gp120makes it dissociate from the transmembraneprotein gp41 that anchors it in the viral membrane [132 133]Again if all NAb binding had that effect it would be hard toexplain the preferential capture of infectious virions byNAbs

As mentioned in the case of HIV-1 some neutralizationepitopes reside in the transmembrane protein but otherepitopes there are occluded by gp120 and although thelatter are immunogenic the antibodies directed to them donot neutralize because they can only bind to nonfunctionalstumps of trimers after the shedding of gp120 [103 134 135]This all agrees with the basic occupancy tenets Even theenhanced binding of NAbs during transient exposure ofthe epitopes close to the membrane agrees with the theoryAlthough these epitopes are present on functional trimers


they are only weakly antigenic in the native form of the Envspike

Several NAbs to enveloped viruses block late steps in theentry process that is the fusion of the viral with the cellularmembrane And this block does not have to occur at the cellsurface If the antibody binding allows receptor interactionsto some extent the virus may get endocytosed and the fusionthat is in some cases triggered by the lowering of the pH in theendosome is delayed and then the virus is shunted towardslysosomal destruction before it has fused and extruded itscore and genome into the cytoplasm This scenario has beenexemplified for West Nile virus [136]

HIV-1 may also depend on internalization for completefusion [83ndash85 137] and hence could be subject to neutraliza-tion by antibodies that permit endocytosis of the virion butinterfere with late fusion stepsWe could call this intracellularneutralization with the important distinction that it is stillentrymdashfusion preceding translocation of the core into thecytoplasmmdashthat is blocked But the definition of neutraliza-tion as outlined initially would allow for somewhat latersteps to be blocked namely at the early postentry stage inthe cytoplasm before the transcription of the viral genomeor translation of viral products Do such mechanisms everoccur

5 The Naked Truth aboutPostentry Neutralization

Postentry mechanisms of neutralization have been assertedrepeatedly about picornavirus and influenza virus [138 139]But definitive evidence was lacking and many a virologistmay have regarded neutralization as in practice synonymouswith antibody-mediated inhibition of attachment and entryThen a new intracytoplasmic mechanism of neutralizationwas discovered [140ndash142] Studying adenovirus James andcolleagues found that its neutralization is greatly dependenton the presence of tripartite motif-containing protein 21(TRIM21) in the target cells TRIM21 is located in thecytoplasm has a strong affinity for IgG and ubiquitinates theantibody-antigen complexes that it captures targeting themfor destruction by the proteasome [141 142]

One surprising aspect of how the intracytoplasmic neu-tralization works is that the adenovirus particle as an intactcomplex with the NAb must translocate across a vesicu-lar membrane into the cytoplasmic compartment At leastone important route of adenoviral entry is internalizationthrough macropinocytosis followed by penetration of thevesicular membrane Hence as long as the macropinocyticvesicle remains intact and the capsid ligated by the NAbhas not penetrated from there TRIM21 does not gain accessto the complex Only once penetration occurs can NAb-TRIM21 contact be established Quite conceivably a singleNAb molecule might be sufficient for targeting the complexto TRIM21 but it also seems possible that targeting wouldbe enhanced by a greater number of NAbs bound [143]Those considerations also raise questions of how many NAbmolecules can traverse the membrane together with the virusparticle one hypothesis would be that a high occupancyof NAbs prevents entry by blocking attachment receptor

interactions or a later penetration step but that TRIM21 actsas a safety net enabling the neutralization of virions withlow NAb occupancy That would however imply substan-tial TRIM21-independent neutralization at the highest NAbconcentrations which does not seem to happen [143] Thesenew problems will be further explored in the analyses ofstoichiometry and efficacy of neutralization below

How general could this mechanism be It would seemto be strictly limited to naked viruses For when envelopedviruses fuse NAbs bound to the viral surface proteins do notgain access to the cytoplasm And although capsid proteinsof enveloped viruses elicit strong antibody responses duringinfection and when expressed from vaccine vectors (see eg[144ndash146]) these antibodies cannot bind to their antigenswhen the virion is intact They would have to translocateinto the cytoplasm on their own Indeed the discovery ofthe TRIM21 mechanism seems to create a new dichotomy ofvirus neutralization a mechanistic divide between potentialNAb effects on naked and enveloped viruses But then theTRIM21mechanismmay not apply to all naked viruses eitherIf picornavirus injects its genome into the cytoplasm and thecapsid therefore never enters [80 97] the result would be anabsence of antibody-capsid complexes in the cytoplasm andhence a lack of targets for TRIM21

Some naked viruses and the capsids of enveloped onesneed to be uncoated after entry for replication to proceedIt is therefore not farfetched to imagine that the virus couldhave taken advantage of ubiquitination by TRIM21 and otherfactors to facilitate this step If degradation of the nakedvirion in complex with the NAb is too slow to preventescape of the genome towards the next replicative step or ifthe capsid of an enveloped virus gets ubiquitinated throughan alternative interaction the virus might benefit from thecellular assistance in its uncoating But the core of HIV-1 isdegraded by the proteasome to a large extent in uninhibitedinfection the degradation causes a net loss in infectivity [147]And at least with adenovirus the neutralizing effect seems todominate over any potential advantage to the virus

TRIM21-dependent neutralization also has implicationsfor whether some antibodies can block neutralization IfIgA and IgM specific for neutralization epitopes overlappingthose of IgG NAbs were incapable of ligating TRIM21they would conceivably be able to block TRIM21-dependentneutralization by IgG provided they were of high enoughaffinity and present at sufficient concentrations But evidencesuggests these Ab classes just like IgG can interact withTRIM21 [140 142]That capability would explain their inabil-ity to block this mechanism of neutralization a very differentexplanation from that of why antibodies rarely block theneutralization of enveloped viruses

Could no analogous mechanism operate againstenveloped virus One effect that might come closest is thebinding of NAb-virion complexes to Fc receptors followedby endocytosis and ultimately lysosomal degradation ofthe virus This antibody-dependent routing of virus wouldqualify as neutralization according to the definition WithHIV-1 for example internalization depending on low NAboccupancy and subsequent loss of infectivity were observedalbeit not categorized as neutralization perhaps because the


effect was too weak compared with regular neutralization[148] But it should be noted that this mechanism wouldprevent entry viable genome-containing cores would notenter the cytoplasm The routing to lysosomal degradationmay be inefficient because it is outcompeted by productiveentry which can occur across the endosomal membraneIndeed that may be the regular site of productive entry forHIV-1 [84 85 137] Conceivably the antibody in complexwith the Fc receptor would block the fusogenic Env-receptorinteractions by steric hindrance and thus delay fusion untildegradation in the lysosomal compartment starts Still itmight require lower occupancies by antibody moleculeson the virions than the regular entry block just as themuch more vigorous TRIM21 mechanism would Envelopedviruses among themselves also provide contrasting exampleslow occupancies on flaviviruses can mediate enhancement ofinfectivity whereas high occupancies by the same antibodiescause neutralization [81 149ndash155] We shall return tothese considerations when discussing stoichiometry morecomprehensively below

The TRIM21-dependent postentry mechanism of neu-tralization and the occupancy-limited entry-blocking mech-anisms have quite distinct implications for classical andnewly studied aspects of neutralization and will need to becontrasted continually As an illustration the occupancy the-ory might explain lack of neutralization by antibodies eventhough they bind to infectious virions theymight achieve toolowoccupancies or bind only to sites that are not functional inentry In contrast the TRIM21-dependent mechanism wouldnot explain such lack of neutralization as long as TRIM21recognizes the Fc portion of the antibody For ubiquitinationwould not seem to require threshold levels of antibodyoccupancy although quite plausibly the more the antibodybound the greater and faster would be the degradation ofthe capsid Nor would TRIM21 distinguish between antibodybound to functional entry-mediating molecules and otherantigens Perhaps some antibodies bound to capsids fail to gettranslocated into the cytoplasm they would dissociate and letthe unbound capsid enter or else the antibodywould neutral-ize at an earlier step Clearly the TRIM21 breakthrough notonly explains much but also raises intriguing new questions

In the context of the novel mechanism it is a provoca-tive observation that genetic antibody deficiencies seem topredispose for greater vulnerability to infection by nakedviruses than by enveloped ones [142] Is postentry inhibitionprevalent among naked viruses Does it provide a safetynet or constitute the major defence line Or are envelopedviruses more vulnerable to cellular immunity in addition tothe antibody responses

Those questions will have to be left unanswered here butthe TRIM21-dependent mechanism has intriguing connec-tions to the quantitative aspect of neutralization to which wedo not turn

6 Kinetics of Neutralization

The reason for this section is twofold first to try to undothe damage of erroneous inferences from the kinetics of theneutralization reaction itself and second to clarify how this

aspect is distinct from or related to other less trivial kineticaspects of neutralization

To view the neutralization of virions in suspension byantibody in solution as a chemical reaction requires somequestionable assumptions Thus it must be assumed that atsome point the binding events between the reactants convertthe virions from infectious to noninfectious an effect as blackand white as the formation or breakage of a covalent bondAs already alluded to virions (although here virus speciesare likely to differ substantially) may have a spectrum ofpropensities to infect and neutralization may be a shift insuch propensities that is not an all-or-nothing effect But ifwe accept the premise that neutralization is a complete loss ofinfectivity of the individual virion we could seek to know themolecularity of the neutralization reaction that is how manyantibody molecules must bind to achieve neutralizationMolecularity is related to but cannot be inferred from theorder of the reaction which is a kinetic concept The ordercan be empirically determined if the concentration of the freereactants aremonitored together with the ratesThus the rateof the neutralization reaction 119903 would be

119903 = 119896 [virus] lowast [119860119887]119899 (1)

where 119896 is the rate constant [virus] and [Ab] are the con-centrations of the free reactants (which hence both decrease)and 119899 is the order of the reaction in antibody concentrationwhich does not have to be an integer Since virions are alreadyassumed to act alone the reaction is first order in virionconcentration (neutralization through aggregation would becomplex in this scheme) What remains to be determinedis 119899 In attempts to infer the molecularity of the reactionthe rate of neutralization over time has been monitored asa function of antibody concentration Values of 119899 close to1 have been observed and the conclusion has been drawnthat a single antibody molecule inactivates one virion [156ndash158] The fallacy is that the free antibody concentration is notrecorded and its changes could not be measured because ofthe vast molar excess of antibody over virus It is a classicsituation of pseudo-first-order kinetics Since [Ab] does notchange significantly the following approximation is true

119903 asymp 1198961015840

[virus] (2)

where 1198961015840 asymp 119896 [Ab] is the pseudo-first-order rate constant forvirion concentrationThe data say nothing about the order inantibody concentration A second flaw is the assumption thatepitopes can be divided into critical and noncritical but thatsomehow only the binding to the critical epitopes would bereflected in the neutralization kinetics This does not makesense if binding is random and of equal affinity to the twokinds of epitopes a certain number of noncritical epitopeswould be bound with the same occupancy as for the criticalepitopes Hence the order was erroneously obtained fromkinetics and so-called single-hit molecularity was mistakenlyinferred from the order to cover these unjustified leapsthe term single-hit kinetics is sometimes used It should benoted that the original paper studied one naked and oneenveloped virus and observed similar kinetics for these Theenveloped virus was western equine encephalitis virus [159]


The naked virus was poliovirus Could these old suggestionshave anything to do with the new discovery of the TRIM21effect whichmight potentially apply to several naked viruses

No that would be a specious convergence TRIM21might not contribute to poliovirus neutralization anywayalthough the virus is naked if its genome is extruded bytransmembrane injection rather than translocation of thecapsid together with any bound NAb into the cytoplasmHence if it should turn out that TRIM21 allows singleantibodies to mediate neutralization of some viruses thatoutcome would be an important advance in knowledge Butit would shed no light on any surprising putative single-hitphenomena based on the kinetics of neutralization becausethere were no such phenomena only flawed interpretations

An earlier paper correctly described the consequence ofthe vast molar excess of antibody over virions (and overepitopes) as the virion concentration is varied over a widerange the proportion that is neutralized by a fixed antibodyconcentration remains constant This relationship was calledThe Percentage Law [160] Those observations illustratethe basis for pseudo-first-order kinetics the proportion ofantibody lost by binding is negligible

Another approach was taken to explore how many anti-bodymolecules must bind before neutralization occursThuson a curve for neutralization over time the first segmentof the curve was scrutinized for signs of any shoulderIf a shoulder was observed it was taken to suggest thatmore than one antibody had to bind before infectivity wasabolished for any virion Sometimes such a shoulder wasobserved sometimes not [161]The difficulty lies in obtainingthe requisite precision of data for a sufficiently early partof the curve And even with the most precise such datainterpretations are not incontrovertible The very method ofstopping the neutralization reaction namely rapid dilutionis unsatisfactory since it makes the results contingent uponthe degree of irreversibility And if the NAb is used at aconcentration below its 119870

119889 there might be a shoulder even

for very low occupancies because the rate of binding wouldbe so low Furthermore a single NAb bound to the smallestviruses would constitute a higher occupancy than severalNAbs bound to larger viruses Thus single- and multihitthresholds could look the same

A different possible cause of a shoulder is that an antibodyneeds to induce changes in the viral antigen that are slowerthan binding a lack of a shoulder could mean that virionslose some propensity to infect with the first binding eventsbut are not completely neutralized as the single-hit hypothesiswould suggest the data would not distinguish between apartial dent in the infectivity of many from a complete lossfor a few The reasoning here is analogous to the distinctionbetween incremental and liminal models Only a high andhomogeneous threshold might show up as a broad shoulderon the kinetic curve

The mistaken single-hit interpretations led to the searchfor mechanisms such as inactivating signals from boundNAbs to the interior of the virion for enveloped viruses andconformational shifts in the whole capsid for naked virusesGenerally the hunt was on for postentry mechanisms For ablock of receptor interactions and entry seemed less readily

explicable by single-hit molecularities at least in the case ofenveloped viruses

As a practical consequence of the single-hit hypothesesthe neutralizing occupancies will be low except if the virus isalso postulated to have only a single relevant antigenmolecule[84 105 106 111] And the lower the occupancy requiredthe easier would be the task of inducing protective bindingtiters of NAbs by vaccination titers and occupancies arethe products of antibody affinities and concentrations Themisinterpretations can misguide vaccine research

7 Kinetics of Binding The Example ofHIV-1 Env

The kinetics of the binding of NAbs and other antibodies tosurface proteins of viruses are more readily studied than thekinetics of neutralization One technique that allows kineticmeasurements is surface plasmon resonance (SPR) [162ndash167]With this technique the antigen or the antibody can beimmobilized to a sensor chip If the antigen is immobilizedthe antibody in solution is injected to flow over it Bindingproduces a change in the angle of the reflection of polarizedlight which is monitored and translated into a resonancesignal proportional to the mass of protein that has boundThis technique has been used formeasuring antibody bindingto the neutralization targets of various viruses including theHIV-1 Env glycoproteinsModeling of the binding at differentconcentrations gives the on-rate constant 119896on the off-rateconstant 119896off and their ratio 119896off119896on = 119870119889 the dissociationconstant a reciprocal measure of affinity furthermore sincethe maximum equilibrium binding is approached and canbe extrapolated the stoichiometry of binding can also beestimated Such studies on the binding to conformationallyflexible viral envelope glycoprotein oligomers that mediateentry into susceptible cells have recently become more rele-vant to neutralization through improvements in the mimicryof native antigens

The antigenicity of the receptor-binding subunit gp120of the HIV-1 Env trimer has been studied extensively butas mentioned many of the epitopes that gp120 exposes areshielded on the native trimer [168] Likewise the uncleavedprecursor of Env although it trimerizes differs antigeni-cally from native functional trimers [102] Still in orderto produce soluble trimers truncated N-terminally of thetransmembrane segment that do not disassemble a commonapproach is to delete the cleavage site between the subunitsand to add extra trimerization motifs C-terminally of thetruncation [169ndash174] But these uncleaved soluble trimersdo not adopt native-like structures and are therefore poorantigenic mimics of functional spikes

As an alternative approach proteolytic processing hasinstead been enhanced by modifying the cleavage site andby coexpression with the protease furin but to maintainthe integrity of the trimer of heterodimers a disulfide bondhas been added to link gp120 covalently to the truncatedtransmembrane protein gp41 [103 126 175ndash180] Thesesoluble trimers structurally mimic native trimers on thesurface of the virion as assessed by electron microscopy


[102] furthermore their three-dimensional structure hasbeen determined to near-atomic scale resolution in complexwith Fabs of different NAbs [181 182]

For SPR studies several dangers of artifactual results lurkin various approaches Even with trimers that mimic thenative spikes structurally if they are immobilized directlyto the SPR chips by covalent such as amide coupling theirantigenicity will be perturbed Hence it is advantageous toadd His or epitope tags C-terminally at the truncation sothat the trimers can be captured by Ni2+ or antibody that isimmobilized on the chip When the cleaved and stabilizedEnv trimers are immobilized by such capture they bindNAbs active against the corresponding strain of the virusexcellently and non-NAbs negligibly Take different antibod-ies directed to the CD4-binding site and to the variableV3 region as examples Both groups contain antibodies thatare neutralizing and others that are nonneutralizing againstparticular strains of HIV-1 They bind equally well to themonomeric Env subunit gp120 and to uncleavable mutanttrimeric forms of Env derived from the same strains Butonly the neutralizing ones bind well to the trimers derivedfrom the strain they neutralize Some NAbs to particularepitopes do not bind to monomeric or nonnative forms ofEnv they are trimer-specific whereas non-NAbs regardlessof epitope fail to bind the native-like trimers (Figure 3) [102103 175 183] Hence what NAbs have in common is that theyrecognize native-like entry-mediating viral proteins whatnon-NAbs have in common is that they do not How theydiffer or resemble each other in the recognition of other formsof Env is then irrelevantThis supports the occupancy theoryof neutralization [31 99 106 184] Possibly some antibodiesthat have been observed not to neutralize other viruses inspite of binding to virionsmay also turn out to recognize onlynonfunctional forms of the viral surface proteins

Because of the richness of the information obtained bySPR NAbs with similar affinity but widely different kineticsof binding can be identified Such characterization of bindinggoes beyond mere occupancy and ushers in the possibilityof testing more dynamic neutralization theories how do thekinetics of NAb and receptor binding together mold theefficacy of neutralization

When the binding of IgG and Fabs is compared by SPRthe specific models for bivalent or monovalent binding canbe explored We know little of the density of Env trimers oninfectious HIV-1 virions let alone the ratio of functional todefective or decayed trimers That ratio may also change asthe virions age But the immobilization of Env on the SPRship can be precisely controlled and translated into trimerdensities that can be compared with and adjusted to theobserved densities of Env on HIV-1 virions With improvedknowledge simulations of trimer densities on virions andthereby realistic average trimer distances may render themeasured degree of bivalency of binding to the antigen on theSPR chip relevant to neutralization SomeNAbs however canbind to both defective and native-like trimers and this wouldenhance binding through bivalency other NAbs recognizeonly the native-like trimers and that would limit their avidity

Other viruses than HIV-1 tend to have higher ratios ofhalf-maximal inhibitory concentrations of Fab over those of

IgG [185]The contribution of the bulk of the Fc portion pos-sibly through steric effects can be ascertained by comparingFab and (Fab1015840)2 in neutralization But the binding of IgG canalso be strengthened by Fc-Fc interactions and this couldbe evaluated by SPR [186 187] Likewise Fabs and smallersingle-chain constructs could be compared Through thesecombined comparisons the avidity and bulk effects would bedistinguished Such background knowledge can be comparedwith and corroborated by simulations of the trimer density onthe virion surface

It should be noted that the degree of bivalent bindingwhich enhances the potency of NAbs by reducing the off-rate of their binding is favored by high densities of antigenon the virion surface [185] Such an effect would counteractthe relative neutralization resistance stemming from a highredundancy of functional entry-mediating molecules [106188ndash191] But the counteracting selective forces are not sym-metric that some NAbs bind equally well to functional andnonfunctional entry-mediating molecules would increasebivalency when extra nonfunctional oligomers are presentbut the redundancy effect requires functional oligomersThese factors may play out in the evolution of natural viralvariants with varying degrees of neutralization sensitivity

One SPR-based study of the simian immunodeficiencyvirus (SIV) and its Env protein gave several surprisingresults NAbs and non-NAbs boundwith similar kinetics andtherefore similar affinity to recombinant soluble Env protein[192] But this was uncleaved Env protein later found toexpose nonneutralization epitopes and the SPRwas based onimmobilized antibody with trivalent Env in solution whichdoes not simulate the potentially bivalent but often monova-lent binding of IgG to virions Also surprising at the time ofpublicationwas that Env from a neutralization resistant strainbound with faster on- and off-rates to NAbs (and non-NAbs)than did Env from a sensitive variant The on- and off-ratedifferences canceled each other out yielding no net affinitydifference This would point to a greater importance of lowoff-rates in conferring neutralization sensitivity than high on-rates although the other caveats about the SPR conditionsmight invalidate comparisons with neutralization Certainlythe relative influence of the on- and off-rate constants mayvary But how they vary will be determined by the length ofthe preincubation with NAb in the neutralization assay Thatraises questions about which conditions are most relevant toprotection in vivo

8 Kinetics of Entry and Virion DecayMode of Neutralization

A classic neutralization assay comprises four stages Firstvirus and antibody are incubated together Second the virusis allowed to adsorb to target cells Third viral replicationproceeds to produce viral product or induce the expressionof a reporter molecule Fourth the product is measured inan assay and converted to a signal and compared with no-antibody and no-virus (background noise) controls [31]Thefirst two phases can be varied the first one can even beeliminated The second one can be performed at reducedtemperature so that internalization and entry are prevented


Glycan-dep CD4bs gp41800

600

400

200

0

0 300 600 900

Resp

diff

(RU

)

2G12

PGV04

PGT135

b6F240b12

Non-NAbTime (s)

V3 V3-glycan and -base

PGT128PGT121PGT123

14e19b

800

600

400

200

0

Resp

diff

(RU

)

0 300 600 900

Non-NAbTime (s)

V1V2 glycan200

150

100

50

0

PG16

PGT145PG9

0 300 600 900

Resp

diff

(RU

)

Time (s)

gp120

gp41

Figure 3 The kinetics of NAb binding Soluble envelope glycoprotein oligomers can be immobilized on SPR chips via His or epitope tags Ifthe trimers are good structural mimics of native functional oligomers and the density of trimers approximate that on the virion surface thenthe NAb binding involved in neutralization can be simulated and its kinetic constants can be determined by SPR Here a soluble stabilizedtrimer of the envelope glycoprotein of a Clade A isolate of HIV-1 was studied The subunits of the trimer are labeled in the schematic to thelower right the black bars represent engineered disulfide bonds that were introduced to stabilize each protomer of the trimer The trimerwas immobilized and the binding of NAbs and non-NAbs was compared The sensorgrams show the response (RU) over time (s) during anassociation phase (upward curve) and a dissociation phase (downward curve in several cases dissociation is very slow and barelymeasurable)Antibodies directed to different groups of epitopes as indicated for the three diagrams are compared The nonneutralizing antibodies aremarked with arrows Thus neutralization correlates eminently with binding to trimers that are native-like according to electron microscopyThe figure is reproduced from Sanders et al [103] with modifications

The outcome can differ greatly in accordance with suchvariations Some NAbs gain substantially in efficacy whenthey are preincubated with the virus for prolonged periodsDrastic examples areNAbs directed to internal epitopes in thepolivirus proteins VP1 and VP4 that are transiently exposedas the proteins ldquobreatherdquo at physiological temperature [193]Other NAbs appear to act after attachment of the virusto the target cells even after receptor interactions [31 99194] If epitopes are transiently exposed only after receptorinteractions whole IgG molecules can be partially occludedfrom access to such sights in the multimolecular entrycomplexes Fabs directed to such epitopes have been shownto be more potent than IgGs [195 196]

Hence we can distinguish different modes of neutraliza-tion induced decay in suspension and deceleration of entryat the cell surface or from an endosome Strictly mode is thendistinct from mechanism for the two modes may block thesame replicative step for example receptor interactions It isnotable however that the TRIM21-dependent mechanism ofneutralization of adenovirus and perhaps other naked viruses[142] would have a different relationship to the suggestedtwo modes of neutralization to the extent that the infectivityis reduced already in suspension or entry is delayed thecytoplasmic action of the NAbs becomes redundant Butit might constitute an important third layer of protectionapparently the dominant one under specific circumstances


[143] Intracytoplasmic inhibitory activities ofNAbswould bea third mode

The effects of NAbs acting on virions in suspensionvary with epitope specificity and virus and some of theseeffects are reversible others irreversible [95 160]The virionson their own usually have a specific infectious half-life insuspension and the suspension mode of neutralization canbe measured as the shortening of that half-life [194 197] Asan example for enveloped viruses with noncovalently linkedsubunits of the entry-mediating surface proteins the NAbeffect may be to induce the dissociation of an outer subunitso that the virus loses the capacity to bind to receptorsin the case of HIV-1 soluble forms of the main receptorCD4 induce the dissociation of the gp120 subunit fromthe envelope glycoprotein spike Such induced sheddingalso mediated by some NAbs may represent a prematuretriggering similar to or more drastic than what is inducedby CD4 and coreceptor interactions at the cell surface or inan endosome in the absence of antibody during uninhibitedentry [84 132 133 198]

Thus induced decay can be considered an aspect ofthe kinetics of neutralization it would constitute a secondinactivating step after NAb binding Its relevance to pro-tection in vivo will be contingent on prolonged periods ofviral exposure to antibody before encounters with susceptiblecells that relevance would also depend on whether theantibody binding has nonneutralizing inhibitory effects suchas opsonization of the viral particles for cellular destructionthrough phagocytosis The latter might dominate It shouldalso be noted that the spontaneous as well as the induceddecay may affect the number of entry-mediating moleculeson the virion that must be inactivated by NAbs in order toblock infection an IC

50value of neutralization is not fixed

but can vary with the preincubation time Apparently NAbscan nibble and chip away at the threshold of neutralization

Although the binding of antibody is reversible thedeceleration of entry might have an irreversible outcome asdoes the induced decay in suspension By slowing down theproductive entry the NAbs may by default shunt the virusonto an abortive pathway [199] Viruses that use penetrationor fusion at the cell surface as an obligatory step could berouted onto an abortive endocytic pathway by Fc-Fc-receptorinteractions or simply by delayed entry That would sealthe fate of the NAb-covered virion The threat of lysosomaldestruction lurks at the end of that route

With viruses that use the endocytic pathway for entry thesituation is different For example someNAbs bound toWestNile virus were reported to decelerate the internalization ofthe virus via the endocytic pathway and thus make the virusavailable for PAN longer [200] Conversely other NAbs orat least lower occupancies of NAbs can enhance infection ofWest Nile virus by favoring endocytosis and allowing fusionwith the endosomal membrane [151 201]

All other things being equal any irreversible aspect ofvirus neutralization whether through the induced-decaymode in suspension or the routing of virions towards destruc-tion by cells in the other modes must benefit host protectionQuestions remain whether irreversibility of inactivation isredundant or crucial

9 Molecularity How Many Hits

As we have seen erroneous interpretations of neutralizationkinetics have sown recalcitrant confusion about how manyNAb molecules are required for neutralization that is itsmolecularity the minimum number of molecules involved inthe rate-determining step of a reaction Kinetics of neutraliza-tionwill not reveal itsmolecularity but the kinetics of bindingcan give the affinity and hence allow legitimate estimates ofoccupancyOccupancy is the equivalent of stoichiometry andif the absolute number of functional neutralization-relevantentry-mediating viral protein sites is known themolecularityfollows

When the binding kinetics are studied by SPR stoichio-metric values can also be obtained but these refer to themaximum binding of for example paratopes per oligomericantigen those data will be useful but not sufficient for cor-rectly determining the molecularity at the level of the virionAnd there are caveats to SPR determinations for examplesome NAbs dissociate so slowly that significant values of 119896offcannot be obtained or 119896on is so high that mass-transportlimitation yields uncertainty Both affinity and stoichiometryhowever can also be determined by calorimetry whereasstoichiometry in addition can be ascertained by electronmicroscopy (Figure 4) [103 126 182] and those data wouldprovide necessary complements to and correctives of the SPRdata

Phenotypically mixed virus has been used in orderto determine the molecularity of entry and neutralizationmore directly Such virus preparations contain differentproportions of antigenic and nonantigenic or functional anddefective protomers of entry-mediating oligomeric proteins[111ndash114 202] Hence combinatorial mathematical equationsderived from the binomial theorem can be applied to inter-pret the data [105 107 108 110] But also this approach entailspitfalls If the entry protein is oligomeric and each virionas is usual has more than one oligomer the modeling mustsimultaneously juggle two levels of analysis each with its ownpotential threshold One consequence is that quite differentmodels can imply indistinguishable empirical data becausechanges in themodel premises at the oligomer and virion lev-els compensate each other [84 105] Notably the premise thateach virion only possesses a single entry-mediating oligomerand therefore only needs one translates into exactly the sameequation as the most extreme incremental model mentionedbefore namely that each oligomer contributes equally to theinfectivity no matter how many intact oligomers are left pervirion or how they are clustered

The premises that infectivity is binary all virions are fullyor not at all infectious and that there are no degrees ofneutralization of the individual virion should probably bothbemodified Already Andrewes and Elford came stumblinglyclose to predicating incremental neutralizing effects [203] iftheir analysis had been heeded some fallacies stemming fromall-or-nothing premiseswould not have proliferated Anotherextreme that is all unoccupied functional protein oligomerscontribute incrementally in exact proportion to their numbermay be equally unrealistic A virion can probably be half-neutralized but that does not mean that there cannot be


PGV04

gp120

PGV04

gp120PDB3SE9

Top view

Side view

(a)

17b

gp120

sCD4

17b

gp120 PDB1RZKsCD4

Top view

Side view

(b)

Top view

Side view

PGV04 PGT122 PG9 PGT135 sCD4 + 17b

(c)

Figure 4 NAbs bind with different stoichiometries to oligomeric antigens The images show electron-microscopy-based reconstructions ofsoluble trimers derived from the same Clade A isolate as in Figure 2 at sim23 A resolution (a) The propeller-like top view and the side viewof the trimer with one Fab directed to the CD4-binding site (PGV04) on each of the three gp120 subunits (b) The same trimer is shown incomplex with a soluble form of CD4 and the Fab of an antibody that binds to the coreceptor-binding site (17b) This NAb neutralizes poorlyas IgG and better as a Fab because the epitope is not constitutively present and is relatively inaccessible in the viral entry complex wheninduced by CD4 interactions Three copies each of CD4 and Fab molecules can be seen in the top view (c) The same combinations as in (a)and (b) are shown first and last in the row The second and fourth panels represent trimers in complex with the Fabs of two NAbs (PGT122and PGT135) that bind to different epitopes and with different angles from each other and from PGV04 and 17b In the middle is a NAb withthe unusual stoichiometry of one Fab per trimer PG9 which binds preferentially to trimers and at an oblique angle to an epitope with directcontributions from two subunits The blue mesh delineates the trimer itself The figure is reproduced from Sanders et al [103]

redundancy in the number of entry proteins on virions orthat all virions are proportionately infectious down to theirlast oligomeric spike or entry-mediating molecule [84 105]With these complexities in mind one can nevertheless deriveinformative inferences frommixed phenotype data And thatis important because it helps estimating what concentrationsand affinities of NAbs that vaccines must induce in order toprotect

Other approaches for determining neutralizing antibodyoccupancies are electron microscopy and various biochem-ical measurements of NAb binding to virions The lattermethods will only give an average degree of binding butanalogously neutralization assays merely provide data onthe average infectivity for the virion population Electronmicroscopy thus has an edge since it can describe the wholedistribution of NAb occupancies over the virion population

A source of artifacts with these binding assays collectivelyis that they do not distinguish between infectious andnoninfectious virions Knowledge of the ratio of infectious tononinfectious virionswill be informative the higher the ratiothe greater the relevance of the measurements But as withneutralization infectivity is not likely to be an all-or-nothingproperty

Studies based on radioactively labeled NAbs indicatedthat poliovirus particles with one bivalently bound IgGmolecule were neutralized [98] Adenovirus was suggestedto be fully neutralized with on average 14NAb moleculesbound per virion [204]This raises questions of the precisionor relevance of the measurements Actually neutralizationand binding were not carried out under the same conditionsin the latter study [204] virus and NAb were incubated for3 h at 37∘C and then for an additional 21 h at 4∘C before the


infectivity assay The infectivity results were plotted as loga-rithmic survival as a function of linear serum concentrationIn the binding analysis the NAb and virus were incubatedfor only 30min at 37∘C and then virion-NAb complexes wereseparated from free NAb by sucrose gradient centrifugationThe recovered virus was not infectious Quite plausibly theseparation procedure might perturb both equilibrium bind-ing and virion infectivity The analyses also pose theoreticalproblems

A Poisson analysis was attempted to account for thebinding-neutralization relationship under the two differentconditions the long and the short incubation There aretwo problems First single-hit neutralization with Poisson-distributed binding and an average of 14NAb moleculesper virion implies a residual infectivity of about 25 Sinceno infectivity was detected either the virions were non-specifically damaged by the procedure or the precision ofthe assay was insufficient for this kind of analysis Secondthe Poisson analysis of neutralization requires knowledgeof the number of NAbs bound and the ensuing residualinfectivity The transition from a mere serum concentrationto the multiplicity of bound NAbs would require much moreinformation namely the average affinity and concentration[106 205] And the theoretical curve for logarithmic residualinfectivity as a function of NAb concentration is then notlinear even for single-hit neutralization [106 205] Onlythe curve postulating single-hit neutralization with averageNAb multiplicity of binding on the 119909-axis is linear [205] Inpractice however an approximately linear curve may verywell arise if the lowest dilutions of the serum happen to yieldNAb concentrations far above the average 119870

119889value Hence

linearity observed under such conditions contrary to theargument invoked in the study [204] is not evidence forsingle-hit molecularity

The suggested single NAb binding to poliovirus men-tioned above was not based on Poisson analysis [98] Astudy that did apply Poisson analysis refuted single-hitneutralization of poliovirus and suggested aminimumof fourNAbs per virion [206] Notably first-order kinetics were alsoobserved and it should be clear by now that there is nothingcontradictory in those observations This study derived theminimum number of NAb molecules from reading the valueon the 119909-axis at 1e relative infectivity This procedure hasbeen common practice but is not justified in theory It is onlyfor the single-hit curve that 1e corresponds exactly to theminimum NAb number The higher the actual number thegreater the deviation Still a molecularity of 70 IgGmoleculesper influenza virion was inferred by this method [158]

The Poisson analysis would provide the best test of thedata by a comparison with the disparate theoretical curvesfor various molecularities one two three four five and soon hits [106 205]That way the correspondence of the data toa particularmodel can legitimately be assessed Or at least thepredicted relative infectivity for each model at the thresholdvalue could be calculated and the readings could be comparedwith the theoretical value With other molecularities than 1(a minimum of one NAb per virion required for completeneutralization) the reading on the 119909-axis at 119910 = 1e is as

theoretically arbitrary as it would be at say 50 neutraliza-tion which does notmean it is uninformative at least itmightrefute a single-hit molecularity [31 106]

For the record Poisson analysis involves the followingThe natural logarithm of the still infectious fraction of virusln(119868119868

0) is plotted on the 119910-axis as a function of the average

number of NAbs per virion 120582 on the 119909-axis The minimumnumber of NAbs per virion required for neutralization isstipulated to have the integer value 119871 The infectious fractionof virions will be equal to the cumulated fractions with fewerthan 119871 NAbs bound to them 119868119868

0= sum119871minus1

119903=0(120582119903119890minus120582)119903 Thus

with 119871 = 1 the contested single-hit molecularity and atan average of 1 NAb per virion that is 120582 = 1 119868119868

0= 119890minus1

so that ln(1198681198680) = minus1 if the single-hit hypothesis was true

approximately 37 of the infectivity would remain whenthe virions have on average one NAb bound to them Butobviously 119871 will differ more from the 119909 value at 119910 = 1e thehigher 119871 is

The prevalent errors in Poisson analyses may howeverdwarf in importance compared with the flawed premise ofthe approach that all virions have equal infectivity in theabsence of NAbs and that the threshold of neutralization isabsolute occupancies below it have zero effect once it isreached neutralization is complete and higher occupanciesmake zero difference

The reason for focusing on the pitfalls of inferencesfrom and errors in the execution of Poisson analysis ofneutralization is the great impact these flaws have hadon the neutralization field The single-hit molecularity hasbeen elevated to virtual dogma on spurious grounds it hasbeen disseminated by textbooks [207] The discovery ofthe TRIM21-dependent mechanism suggests that single-hitneutralization is molecularly plausible it would then havebeen believed previously for the wrong reasons and largelyabout the wrong viruses In reality the neutralization ofadenovirus infection of mouse embryonic fibroblasts andof the human epithelial cell line HeLa occurred at averagenumbers of NAbs per virion of 16 and 48 respectively whenthe NAb was murine Both cell lines were IFN-stimulatedto maximize TRIM21 expression The explanation for thediffering results between the cell lines would be that murineTRIM21 has a higher affinity for the murine NAbs thandoes human TRIM21 [143] At lower TRIM21 levels muchhigher occupancies were required Or at the extreme endeven saturating concentrations of NAbs yielded no measur-able neutralization likewise Fc mutants of NAbs unable tointeract with TRIM21 failed to neutralizeThese newfindingsshed much light on the context-dependent and nonabsolutemolecularities that are sufficient for neutralizationThey alsosuggest a remarkable inefficiency of neutralization at stepsbefore cytoplasmic entry Therefore older observations withwhich they sometimes seem to clash should be revisited

In an earlier study adenovirus particles bound by NAbsfailed to penetrate from the endosomal vesicle and wouldtherefore not have been subject to TRIM21-mediated taggingfor degradation the observed block was at an earlier stepThesuggested mechanism was that NAbs binding to the pentonbase prevented interactions with the endosomal membrane


and therefore blocked entry Antifiber antibodies aggregatedvirions and thereby afforded some degree of neutraliza-tion Antihexon NAbs blocked pH-induced conformationalchanges in the capsid thought to be conducive to penetrationAntihexon NAbs also mediated PAN [204]

Direct evidence supports multihit molecularities of neu-tralization of many viruses not only enveloped ones Thus4-5 NAb molecules are required to neutralize poliovirusone of the smallest viruses 36ndash38 for papillomavirus 70for influenza virus and 225 for rabies virus [99] Theseascending numbers suggest a roughly linear relationshipbetween surface area of the virion and the minimum numberof NAb molecules required for neutralization [99 201] Thiscorrelation agrees approximately with occupancy theories ofneutralization but falls short of demonstrating their generalvalidity There are two naked viruses in the comparison butas discussed different principles apply to mechanisms andmolecularities of the neutralization of naked and envelopedviruses Hypothetically the approximate linearity mightapply best to enveloped viruses but among them such factorsas the density functionality and fragility of the envelopeprotein would play in and host cell density can also deter-mine the efficiency that is Nab concentration dependenceof neutralization [208] In that regard the observations arenot based on comparable conditions for the different virusesTherefore the rough correlation between virion size and thenumber bound NAbs required for neutralization is all themore striking

The enveloped West Nile virus which belongs to theflavivirus genus of the Flaviridae family probably presentsthe quantitatively best understood example of antibody neu-tralization [149] The same antibodies can both neutralizeand enhance viral infection which effect they have dependson the occupancy achieved Around 120 epitopes for someparticular NAbs are available per virion When these NAbsoccupy 25 of the epitopes virion infectivity is reducedby half At lower occupancies these same antibodies areinstead capable of enhancing infection by routing the virusonto the endocytic pathway via Fc receptors thus withoutsterically blocking the ultimate fusion of the envelope withthe endosomal membrane [151 201]

Other NAbs both to West Nile and Dengue virus requirehigher occupancies on their epitopes for neutralization Theexplanation which elegantly illustrates the occupancy theoryis that fewer of those epitopes are accessible for NAb bindingso that to achieve a similar threshold number of NAbs or Fabsper virion a greater proportion of the exposed epitopes mustbe ligated [149]

Asmentioned it has been suggested that low occupanciesof the Env spikes on HIV-1 virions shunt the virus onto anonproductive endocytic pathway also through Fc-receptorinteractions [148] This mechanism of inactivation madesense when HIV-1 was thought to enter productively throughdirect fusion with the cell surface membrane But now thatstrong evidence suggests the endocytic pathway is productiveand indeed even obligatory [83ndash85 137 209ndash211] this effectof the low antibody occupancies has become intriguingEnhancing effects of lowNAboccupancies onHIV-1 have alsobeen observed [106] From the vaccine perspective it would

be reassuring to know what different numbers of NAbs onvirions do to the infectivity

10 Extent Efficacy and Persistent Fraction

The persistent fraction (PF) of viral infectivity is the plateauof infectivity that is asymptotically approached as the incu-bation with NAbs is prolonged or the NAb concentrationis increased (Figure 5) Here the focus will be on the latterbut some general remarks are warranted on the varioushypotheses that have been formulated over many years toaccount for these phenomena [58 99 156 184 212ndash214]

A plausible explanation might be that the virus is het-erogeneous and the PF simply represents a resistant variantAlthough generalizations would be rash at least in somecases the virus which expanded from persistent fractionshas however shown similar neutralization sensitivity tothat of the original virus [58 99 184 212 214] If genet-ically based causes are excluded epigenetic ones such asglycan-processing may be responsible for the persistence Ifneutralization required conformational changes in the viralproteins they might be induced to different degrees andsome virions might then be more resistant to the effect ofNAb binding [58] A spectrum of densities of functionallypreserved neutralization antigen molecules over the virionpopulationmight also yield if not absolute resistance at leasta tail of less sensitive virions

Before PFs were demonstrated with monoclonal NAbsheterogeneities among the antibodies in sera were impli-cated The existence of neutralization-blocking antibodieswas also invoked [58 127] Even monoclonal NAbs canbe heterogeneous because of variation in posttranslationalmodifications for example tyrosine sulfation that affectsaffinity [215] Still PFs aremore general phenomena and theiroccurrence cannot be tied to particular NAbs although theirsizes vary among NAbs

Much focus of early PF research was on aggregates andhow they would retain some infectivity and attach withgreater avidity than single virions [58] But PFs were alsoobserved with monodispersed virus [213] It should be notedthat the law of mass action does not imply any PF (Figure 5)only when a subset of target molecules have aberrant sub-stantially reduced affinity would the neutralization tail offBurnet fruitfully suggested that NAb dissociation is respon-sible for the PF [212] This is compatible with the findingsthat both the addition of secondary antibodies cross-linkingthose in the neutralizing serum and the combination ofNAbs to distinct epitopes can reduce the size of the PF [213]The dissociation hypothesis gains further plausibility whenapplied to the dynamic competition between receptors andNAbs at the site of entry It is also relevant that the degree ofmonovalent binding by IgG rises when binding approachessaturation as expected from the well-established prozoneeffect [216] Thus the avidity decreases and the competitivebinding to receptors is favored over that to paratopes

Recently the PF of HIV-1 was shown to correlate withthe off-rate constants of NAbs as measured with native HIV-1 Env trimers by SPR [183] In addition stoichiometry was


0 1 2 3 4 5 6 7000102030405060708091011

Log(Ab(nM))

Rela

tive i

nfec

tivity

(a)

1 2 3 4 5 6 70

1Log(Ab(nM))

Log(

relat

ive i

nfec

tivity

) minus1

minus2

minus3

minus4

minus5

minus6

minus7

minus8

(b)

Figure 5 Neutralization potency and efficacy (a) Potency conventional neutralization curves (relative infectivity as a function of thelogarithmic antibody concentration) for simulated data are shown The green and red curves describe neutralization with identical potencythat is IC

50values although the green curve has a higher slope coefficient Red black and blue curves represent decreasing potencies in

that order while having the same slope Neutralization represented by the grey curve falls between the black and blue in potency but hasmarkedly higher slope coefficient than both Antigenic heterogeneity reduces the slope coefficient and so does negative cooperativity Positivecooperativity would raise the slope coefficient (b) Efficacy exactly the same simulated data as in (a) are plotted in a log-log plot to illustratethe importance of the persistent fraction (PF) of infectivity after neutralizationThe twomost potent NAbs from (a) (red and green) are shownto have widely different efficacies the persistent fraction differs by about three logs Furthermore the curves for the less potent NAbs (blackand blue) cross the red curve and tend towards a greater efficacy by one or half a log respectively The greater slope of the grey curve thanof the others is apparent also in this plot but only here is the greater efficacy of the neutralization represented by the grey than by the greencurve evident another case of lower potency and greater efficacy

related to the PF (Figures 4 and 5) Most NAbs to the HIV-1 Env trimer bind with a stoichiometry approaching threeparatopes per trimer but some trimer-specific NAbs onlyhave a single epitope at the apex of the trimer (Figure 4)[175] Thus stoichiometry as assessed both by calorimetryand SPR correlated with the size of the PF the higher thestoichiometry the lower the PF [183]This finding might alsobe related to dissociation becausewith a stoichiometry of oneparatope per viral oligomer the vulnerability to dissociationwould be greater Indeed other evidence indicates that asingle IgG molecule per trimer of some NAbs is sufficientto block the function of that trimer [84 105 108 110 111114 202] Thus when three are bound two are redundantbut would act as safety nets particularly in the dynamiccompetitionwith receptors when the demands on occupancyare highest

Research into causes of the PF has been revived bythe revelations about the TRIM21-dependent neutralizationThus neutralization of adenovirus by the same NAb leaveshigher or lower PFs in inverse proportion to the levels ofTRIM21 in the target cell [143] But eventually a doubleplateau establishes itself increasing NAb occupancies andTRIM21 expression levels reach diminishing returns andthe residual infectivity plateaus out Indeed the higher theviral dose is the more easily the combined defense byNAbs and TRIM21 is overwhelmed Quite plausibly othercellular factors those responsible for the degradation ofNAb-virus complexes after the TRIM21-mediated ubiquitinationbecome limiting at this point Clearly the old problem of thePF has been greatly illuminated by the discovery of the role

of auxiliary cellular factors in neutralization The questionis how far these insights could extend beyond adenovirusneutralization [142]

11 Synergy and Cooperativity

At the molecular level two NAbs might potentially enhanceeach otherrsquos binding [217] That would imply synergy in neu-tralization benefitting the induction of antibody responsesto multiple epitopes by vaccination But synergy in neu-tralization has also been observed when there seems to benone at the level of antibody binding to the antigen moleculeand indeed when there could not be any synergy in bindingbecause the synergizing NAbs are directed to overlappingepitopes [218] Then the explanation must be different Aplausible one is the heterogeneity in the population of targetmolecules that is the antigens which can be extensive forsome viruses [219] This heterogeneity does not have tobe genetically determined alone but could also stem fromvariation in posttranslational modification Thus synergywould arise from how two NAbs complement each other bycovering different epitopes or sometimes different variants ofthe same epitope for which the NAbs have distinct affinitiesThis kind of explanation would apply equally to synergy inefficiency that is potency as in efficacy the latter beingreflected in a reduced PF

There have been conceptual as well as practical andmodeling errors in synergy determination But the classicalsynergy index which contrarymany claims does have generalvalidity can shed light on both synergistic and antagonistic


effects provided it is calculated with approaches that avoidsome prevalent sources of artifacts and errors [219 220] Stillit is purely a measure of synergy or antagonism in potencyEfficacy may be at least as important not least in vivo andwill require an equally rigorous framework for analysis

Cooperativity of single inhibitors acting on several siteson oligomeric target molecules has classically been measuredas slopes the Hill coefficients of the inhibition curves [221]It is clear however that heterogeneity can lower the slopesof those curves [219 222 223] And that the combinationof two NAbs with reduced slopes yields a higher slope mayexplain how antigenic heterogeneity gives rise to synergisticphenomena [219] The slope of neutralization curves hasbeen much less studied than the midpoint that is thepotency but it reflects an important property of the NAband is partly the product of epitope heterogeneity it can alsobe subject to paratope heterogeneity even for monoclonalantibodies when the intrinsic affinity is modulated by forexample tyrosine sulfation which will not be achieveduniformly over populations of NAbs produced naturally[215]

Generally in studies of inhibition when one moleculeis inert and the other is active but the combination is stillmore active the proper term for the combinatorial effectis potentiation [224] Regarding postentry neutralization itcan be noted that interferons may potentiate neutralizationby raising TRIM21 [141ndash143] It remains to be seen to whatextent the TRIM21-dependent inhibition merely provides asafety net for when NAbs fail to prevent entry or whetherit rather constitutes the main mechanism of neutralizationand against which naked viruses it is active This field ofstudy may present many new intricate problems of howcellular factors limit or enhance neutralization and whetherthe combinations yield additive synergistic or antagonisticnet effects

12 Conclusions Reductionism Redux

It would be a great advantage in vaccine development toknow that no other special feature is required of an antibodyto render it neutralizing than simply its capacity to ligatefunctional entry-mediating viral proteins on the surface ofthe virion vaccination becomes simpler if it is sufficientto create mimics of such viral proteins and make themimmunogenic For some viruses such as HIV-1 it is alreadyvery difficult to obtain sufficiently good mimics of thenative Env protein and to focus the immune response onconserved regions so that the antibodies induced will alsorecognize the Env trimers of naturally occurring divergentvariants of the virus Therefore any further complicationssuch as requirements for antibodies to induce particularconformational changes in the antigens or indeed elaborateposttranslational modifications required for the strongestbinding would be most unwelcome Likewise it is gratifyingif neutralization works in such a way that antibodies capableof blocking it do not readily arise it may still be impossibleto avoid inducing some irrelevant inert antibodies or withsome viruses antibodies that enhance infection

The comprehensive view of neutralization as interferencewith different steps of viral entry and as arising from acritical degree of coating of the virus particle with NAbs issupported by several lines of evidence for many viruses Butthe discovery of the role of the cytoplasmic factor TRIM21in adenovirus neutralization has provided an intriguingexample of genuine postentry neutralization and rationalexplanations for why very fewNAbs per virion are sometimessufficient for neutralization Whether this mechanism repre-sents an exception or engenders a more general dichotomybetween naked and enveloped viruses remains to be seenWhenever naked viral cores can penetrate cell membraneswhile retaining bound NAbs there is potential for intracy-toplasmic neutralization expedited by cellular factors

Improved biophysical characterization of NAb bindingto native-like viral antigens provides much information ofpotential relevance to the efficacy of neutralization in vitroand protection by NAbs in vivo The rough correlationbetween affinity and neutralization potency may howeveryield an oversimplified picture of the relationship betweenNAb binding and neutralization Stoichiometry and kineticsof binding may affect also the efficacy of neutralizationwhich may be more important than potency for preventingthe establishment of infection in an organism Althoughimproved knowledge of howNAbs protect would benefit vac-cine development any added requirements beyond sufficientbinding to functional antigens would again make the taskharder

Early attempts to reduce neutralization to chemical prin-ciples were flawed in part because infectivity and its inhi-bition were oversimplistically viewed as all-or-nothing phe-nomena But now an emerging quantitative understanding ofhow viral proteins contribute to infectivity together with thebiophysical characterization of the binding of NAbs to viralantigens may explain many aspects of virus neutralizationAlthough both the block of entry and postentry mechanismsneed to be understood in their distinct molecular detailscommon principles may prevail such as requisite occupan-cies and ubiquitous competition with the dynamic events ofviral replication

Conflict of Interests

The author declares that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

The authorrsquos work in this area is supported by the NIH grantR37 AI36082

References

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[2] P R Krause S R Bialek S B Boppana et al ldquoPriorities forCMV vaccine developmentrdquo Vaccine vol 32 no 1 pp 4ndash102013


[3] S A Plotkin ldquoImmunologic correlates of protection induced byvaccinationrdquo Pediatric Infectious Disease Journal vol 20 no 1pp 63ndash75 2001

[4] S A Plotkin ldquoCorrelates of vaccine-induced immunityrdquo Clini-cal Infectious Diseases vol 47 no 3 pp 401ndash409 2008

[5] S A Plotkin ldquoCorrelates of protection induced by vaccinationrdquoClinical and Vaccine Immunology vol 17 no 7 pp 1055ndash10652010

[6] S A Plotkin ldquoComplex correlates of protection after vaccina-tionrdquo Clinical Infectious Diseases vol 56 no 10 pp 1458ndash14652013

[7] D R Burton P Poignard R L Stanfield and I A WilsonldquoBroadly neutralizing antibodies present new prospects tocounter highly antigenically diverse virusesrdquo Science vol 337no 6091 pp 183ndash186 2012

[8] P J Klasse and J P Moore ldquoGood CoP bad CoP Interrogatingthe immune responses to primate lentiviral vaccinesrdquo Retrovi-rology vol 9 article 80 2012

[9] I J Amanna N E Carlson and M K Slifka ldquoDuration ofhumoral immunity to common viral and vaccine antigensrdquoNewEngland Journal ofMedicine vol 357 no 19 pp 1903ndash1915 2007

[10] M K Slifka R Antia J KWhitmire and R Ahmed ldquoHumoralimmunity due to long-lived plasma cellsrdquo Immunity vol 8 no3 pp 363ndash372 1998

[11] E Hammarlund M W Lewis S G Hansen et al ldquoDura-tion of antiviral immunity after smallpox vaccinationrdquo NatureMedicine vol 9 no 9 pp 1131ndash1137 2003

[12] P J Klasse R W Sanders A Cerutti and J P Moore ldquoHowcan HIV-type-1-Env immunogenicity be improved to facilitateantibody-based vaccine developmentrdquo AIDS Research andHuman Retroviruses vol 28 no 1 pp 1ndash15 2012

[13] F Klein H Mouquet P Dosenovic J F Scheid L Scharf andM C Nussenzweig ldquoAntibodies in HIV-1 vaccine developmentand therapyrdquo Science vol 341 pp 1199ndash1204 2013

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[15] N S Greenspan ldquoDimensions of antigen recognition and levelsof immunological specificityrdquoAdvances in Cancer Research vol80 pp 147ndash187 2001

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[17] N S Greenspan ldquoCohens conjecture Howards hypothesisand Ptashnes ptruth an exploration of the relationship betweenaffinity and specificityrdquo Trends in Immunology vol 31 no 4 pp138ndash143 2010

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[41] M G Rossmann Y He and R J Kuhn ldquoPicornavirus-receptorinteractionsrdquo Trends in Microbiology vol 10 no 7 pp 324ndash3312002


[42] YDKwonA Finzi XWu et al ldquoUnligandedHIV-1 gp120 corestructures assume the CD4-bound conformation with regula-tion by quaternary interactions and variable loopsrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 109 no 15 pp 5663ndash5668 2012

[43] N Madani A Schon A M Princiotto et al ldquoSmall-moleculeCD4 mimics interact with a highly conserved pocket on HIV-1gp120rdquo Structure vol 16 no 11 pp 1689ndash1701 2008

[44] A Repik andP R Clapham ldquoPlugging gp120sCavityrdquo Structurevol 16 no 11 pp 1603ndash1604 2008

[45] A Schon N Madani J C Klein et al ldquoThermodynamicsof binding of a low-molecular-weight CD4 mimetic to HIV-1gp120rdquo Biochemistry vol 45 no 36 pp 10973ndash10980 2006

[46] Q Zhao L Ma S Jiang et al ldquoIdentification of N-phenyl-N1015840-(2266-tetramethyl-piperidin-4-yl)-oxalamides as a new classof HIV-1 entry inhibitors that prevent gp120 binding to CD4rdquoVirology vol 339 no 2 pp 213ndash225 2005

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[49] Z Si N Madani J M Cox et al ldquoSmall-molecule inhibitorsof HIV-1 entry block receptor-induced conformational changesin the viral envelope glycoproteinsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 101 no14 pp 5036ndash5041 2004

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[60] E Mehlhop S Nelson C A Jost et al ldquoComplement pro-tein C1q reduces the stoichiometric threshold for antibody-mediated neutralization of West Nile virusrdquo Cell Host ampMicrobe vol 6 no 4 pp 381ndash391 2009

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[64] B Moldt M Shibata-Koyama E G Rakasz et al ldquoA nonfu-cosylated variant of the anti-HIV-1 monoclonal antibody b12has enhanced Fc120574riiia-Mediated antiviral activity in vitro butdoes not improve protection against mucosal SHIV challengein macaquesrdquo Journal of Virology vol 86 no 11 pp 6189ndash61962012

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[83] G B Melikyan ldquoCommon principles and intermediates of viralprotein-mediated fusion the HIV-1 paradigmrdquo Retrovirologyvol 5 article 111 2008

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[85] G B Melikyan ldquoHIV entry a game of hide-and-fuserdquo CurrentOpinion in Virology vol 4 pp 1ndash7 2014

[86] MMarsh andAHelenius ldquoVirus entry open sesamerdquoCell vol124 no 4 pp 729ndash740 2006

[87] M Marsh and R Bron ldquoSFV infection in CHO cells cell-typespecific restrictions to productive virus entry at the cell surfacerdquoJournal of Cell Science vol 110 part 1 pp 95ndash103 1997

[88] S Lu S D Putney and H L Robinson ldquoHuman immunode-ficiency virus type 1 entry into T cells more-rapid escape froman anti-V3 loop than from an antireceptor antibodyrdquo Journal ofVirology vol 66 no 4 pp 2547ndash2550 1992

[89] S Putney ldquoHow antibodies block HIV infection paths to anAIDS vaccinerdquo Trends in Biochemical Sciences vol 17 no 5 pp191ndash196 1992

[90] T J Smith N H Olson R H Cheng et al ldquoStructure of humanrhinovirus complexed with Fab fragments from a neutralizingantibodyrdquo Journal of Virology vol 67 no 3 pp 1148ndash1158 1993

[91] B Brandenburg L Y Lee M Lakadamyali M J Rust XZhuang and JMHogle ldquoImaging poliovirus entry in live cellsrdquoPLoS Biology vol 5 no 7 p e183 2007

[92] A Panjwani M Strauss S Gold et al ldquoCapsid protein VP4of human rhinovirus induces membrane permeability by theformation of a size-selective multimeric porerdquo PLoS Pathogensvol 10 no 8 Article ID e1004294 2014

[93] E A Emini S Y Kao A J Lewis R Crainic and E WimmerldquoFunctional basis of poliovirus neutralization determined withmonospecific neutralizing antibodiesrdquo Journal of Virology vol46 no 2 pp 466ndash474 1983

[94] B Mandel ldquoCharacterization of type 1 poliovirus by elec-trophoretic analysisrdquo Virology vol 44 no 3 pp 554ndash568 1971

[95] B Mandel ldquoAn analysis of the physical and chemical factorsinvolved in the reactivation of neutralized poliovirus by themethod of freezing and thawingrdquo Virology vol 51 no 2 pp358ndash369 1973

[96] BMandel ldquoNeutralization of poliovirus a hypothesis to explainthe mechanism and the one hit character of the neutralizationreactionrdquo Virology vol 69 no 2 pp 500ndash510 1976

[97] R Vrijsen A Mosser and A Boeye ldquoPostadsorption neutral-ization of poliovirusrdquo Journal of Virology vol 67 no 6 pp 3126ndash3133 1993

[98] K Wetz P Willingmann H Zeichhardt and K O HabermehlldquoNeutralization of poliovirus by polyclonal antibodies requiresbinding of a single IgG molecule per virionrdquo Archives ofVirology vol 91 no 3-4 pp 207ndash220 1986

[99] D R Burton E O Saphire and P W H I Parren ldquoA model forneutralization of viruses based on antibody coating of the virionsurfacerdquo Current Topics in Microbiology and Immunology vol260 pp 109ndash143 2001

[100] P W H I Parren I Mondor D Naniche et al ldquoNeutralizationof human immunodeficiency virus type 1 by antibody to gp120is determined primarily by occupancy of sites on the virionirrespective of epitope specificityrdquo Journal of Virology vol 72no 5 pp 3512ndash3519 1998

[101] P Poignard P J Klasse and Q J Sattentau ldquoAntibody neutral-ization of HIV-1rdquo Immunology Today vol 17 no 5 pp 239ndash2461996

[102] R P Ringe RW Sanders A Yasmeen et al ldquoCleavage stronglyinfluenceswhether solubleHIV-1 envelope glycoprotein trimersadopt a native-like conformationrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 110 pp18256ndash18261 2013

[103] R W Sanders R Derking A Cupo et al ldquoA next-generationcleaved soluble HIV-1 Env Trimer BG505 SOSIP664 gp140expresses multiple epitopes for broadly neutralizing but notnon-neutralizing antibodiesrdquo PLoS Pathogens vol 9 Article IDe1003618 2013

[104] C Wilson M S Reitz Jr K Aldrich et al ldquoThe site of animmune-selected pointmutation in the transmembrane proteinof human immunodeficiency virus type 1 does not constitutethe neutralization epitoperdquo Journal of Virology vol 64 no 7 pp3240ndash3248 1990

[105] P J Klasse ldquoModeling howmany envelope glycoprotein trimersper virion participate in human immunodeficiency virus infec-tivity and its neutralization by antibodyrdquo Virology vol 369 no2 pp 245ndash262 2007

[106] P J Klasse and J P Moore ldquoQuantitative model of antibody-and soluble CD4-mediated neutralization of primary isolatesand T-cell line-adapted strains of human immunodeficiencyvirus typerdquo Journal of Virology vol 70 no 6 pp 3668ndash36771996

[107] C Magnus O F Brandenberg P Rusert A Trkola and R RRegoes ldquoMathematical models a key to understanding HIVenvelope interactionsrdquo Journal of Immunological Methods vol398-399 pp 1ndash18 2013

[108] C Magnus and R R Regoes ldquoEstimating the stoichiometry ofHIV neutralizationrdquo PLoS Computational Biology vol 6 no 3Article ID e1000713 2010

[109] C Magnus and R R Regoes ldquoRestricted occupancy modelsfor neutralization of HIV virions and populationsrdquo Journal ofTheoretical Biology vol 283 pp 192ndash202 2011

[110] CMagnus P Rusert S Bonhoeffer A Trkola andR R RegoesldquoEstimating the stoichiometry of human immunodeficiencyvirus entryrdquo Journal of Virology vol 83 no 3 pp 1523ndash15312009

[111] X Yang S Kurteva S Lee and J Sodroski ldquoStoichiometry ofantibody neutralization of human immunodeficiency virus type1rdquo Journal of Virology vol 79 no 6 pp 3500ndash3508 2005

[112] X Yang S Kurteva X Ren S Lee and J Sodroski ldquoStoichiom-etry of envelope glycoprotein trimers in the entry of humanimmunodeficiency virus type 1rdquo Journal of Virology vol 79 no19 pp 12132ndash12147 2005


[113] X Yang S Kurteva X Ren S Lee and J Sodroski ldquoSubunit sto-ichiometry of human immunodeficiency virus type 1 envelopeglycoprotein trimers during virus entry into host cellsrdquo Journalof Virology vol 80 no 9 pp 4388ndash4395 2006

[114] K Schoslashnning O Lund O S Lund and J S Hansen ldquoStoi-chiometry ofmonoclonal antibody neutralization of T-cell line-adapted human immunodeficiency virus type 1rdquo Journal ofVirology vol 73 no 10 pp 8364ndash8370 1999

[115] C RMadeleyWHAllan andA P Kendal ldquoStudieswith avianinfluenza A viruses serological relations of the haemagglutininand neuraminidase antigens of ten virus isolatesrdquo Journal ofGeneral Virology vol 12 no 2 pp 69ndash78 1971

[116] M Majer and F Lik ldquoSensitization of influenza virus A2-Singapore by antineuraminidaserdquo Journal of General Virologyvol 13 no 2 pp 355ndash356 1971

[117] H Raux P Coulon F Lafay andA Flamand ldquoMonoclonal anti-bodies which recognize the acidic configuration of the rabiesglycoprotein at the surface of the virion can be neutralizingrdquoVirology vol 210 no 2 pp 400ndash408 1995

[118] CD Rizzuto and J G Sodroski ldquoContribution of virion ICAM-1 to human immunodeficiency virus infectivity and sensitivityto neutralizationrdquo Journal of Virology vol 71 no 6 pp 4847ndash4851 1997

[119] L O Arthur J W Bess Jr R C Sowder II et al ldquoCellularproteins bound to immunodeficiency viruses implications forpathogenesis and vaccinesrdquo Science vol 258 no 5090 pp 1935ndash1938 1992

[120] M Page RQuartey-PapafioM Robinson et al ldquoComplement-mediated virus infectivity neutralisation by HLA antibodiesis associated with sterilising immunity to siv challenge in themacaque model for HIVAIDSrdquo PLoS ONE vol 9 Article IDe88735 2014

[121] S Ugolini I Mondor P W H I Parren et al ldquoInhibition ofvirus attachment to CD4+ target cells is amajormechanism of Tcell line-adapted HIV-1 neutralizationrdquo Journal of ExperimentalMedicine vol 186 no 8 pp 1287ndash1298 1997

[122] J Huang G Ofek L Laub et al ldquoBroad and potent neutraliza-tion of HIV-1 by a gp41-specific human antibodyrdquo Nature vol491 no 7424 pp 406ndash412 2012

[123] A S Kim D P Leaman and M B Zwick ldquoAntibody togp41 MPER alters functional properties of HIV-1 Env withoutcomplete neutralizationrdquo PLoS Pathogens vol 10 Article IDe1004271 2014

[124] H K Steger andM J Root ldquoKinetic dependence toHIV-1 entryinhibitionrdquoThe Journal of Biological Chemistry vol 281 no 35pp 25813ndash25821 2006

[125] T J Smith ldquoAntibody interactions with rhinovirus Lessons formechanisms of neutralization and the role of immunity in viralevolutionrdquoCurrent Topics inMicrobiology and Immunology vol260 pp 1ndash28 2001

[126] J Julien D Sok R Khayat et al ldquoBroadly neutralizing antibodyPGT121 allosterically modulates CD4 binding via recognitionof the HIV-1 gp120 V3 base and multiple surrounding glycansrdquoPLoS Pathogens vol 9 no 5 Article ID e1003342 2013

[127] N Hashimoto and A M Prince ldquoKinetic studies on theneutralization reaction between Japanese encephalitis virus andantiserumrdquo Virology vol 19 no 3 pp 261ndash272 1963

[128] AM Breschkin J Ahern andDOWhite ldquoAntigenic determi-nants of influenza virus hemagglutinin VIII Topography of theantigenic regions of influenza virus hemagglutinin determinedby competitive radioimmunoassay with monoclonal antibod-iesrdquo Virology vol 113 no 1 pp 130ndash140 1981

[129] A Marzi T Gramberg G Simmons et al ldquoDC-SIGN and DC-SIGNR interact with the glycoprotein of marburg virus and theS protein of severe acute respiratory syndrome coronavirusrdquoJournal of Virology vol 78 no 21 pp 12090ndash12095 2004

[130] P Liu L D Williams X Shen et al ldquoCapacity for infectiousHIV-1 virion capture differs by envelope antibody specificityrdquoJournal of Virology vol 88 no 9 pp 5165ndash5170 2014

[131] P Poignard M Moulard E Golez et al ldquoHeterogeneity ofenvelope molecules expressed on primary human immun-odeficiency virus type 1 particles as probed by the bindingof neutralizing and nonneutralizing antibodiesrdquo Journal ofVirology vol 77 no 1 pp 353ndash365 2003

[132] P Poignard T Fouts DNaniche J PMoore andQ J SattentauldquoNeutralizing antibodies to human immunodeficiency virustype-1 gp120 induce envelope glycoprotein subunit dissocia-tionrdquo The Journal of Experimental Medicine vol 183 no 2 pp473ndash484 1996

[133] C R Ruprecht A Krarup L Reynell et al ldquoMPER-specificantibodies induce gp120 shedding and irreversibly neutralizeHIV-1rdquo Journal of Experimental Medicine vol 208 no 3 pp439ndash454 2011

[134] P J Klasse J A McKeating M Schutten M S Reitz Jr andM Robert-Guroff ldquoAn immune-selected point mutation in thetransmembrane protein of n immunodeficiency virus type 1(HXB2-EnvAla 582(rarr Thr)) decreases viral neutralization bymonoclonal antibodies to the CD4-binding siterdquo Virology vol196 no 1 pp 332ndash337 1993

[135] M Thali M Charles C Furman et al ldquoResistance to neutral-ization by broadly reactive antibodies to the human immunod-eficiency virus type 1 gp120 glycoprotein conferred by a gp41amino acid changerdquo Journal of Virology vol 68 no 2 pp 674ndash680 1994

[136] S W Gollins and J S Porterfield ldquoA new mechanism forthe neutralization of enveloped viruses by antiviral antibodyrdquoNature vol 321 no 6067 pp 244ndash246 1986

[137] K Miyauchi Y Kim O Latinovic V Morozov and G BMelikyan ldquoHIV enters cells via endocytosis and dynamin-dependent fusion with endosomesrdquoCell vol 137 no 3 pp 433ndash444 2009

[138] S J Armstrong andN J Dimmock ldquoNeutralization of influenzavirus by low concentrations of hemagglutinin-specific poly-meric immunoglobulin A inhibits viral fusion activity butactivation of the ribonucleoprotein is also inhibitedrdquo Journal ofVirology vol 66 no 6 pp 3823ndash3832 1992

[139] S A Reading and N J Dimmock ldquoNeutralization of animalvirus infectivity by antibodyrdquo Archives of Virology vol 152 no6 pp 1047ndash1059 2007

[140] A H Keeble Z Khan A Forster and L C James ldquoTRIM21is an IgG receptor that is structurally thermodynamically andkinetically conservedrdquo Proceedings of the National Academy ofSciences of the United States of America vol 105 no 16 pp6045ndash6050 2008

[141] D L Mallery W A McEwan S R Bidgood G J Towers CM Johnson and L C James ldquoAntibodies mediate intracellularimmunity through tripartite motif-containing 21 (TRIM21)rdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 107 no 46 pp 19985ndash19990 2010

[142] W AMcewan D L Mallery D A Rhodes J Trowsdale and LC James ldquoIntracellular antibody-mediated immunity and therole of TRIM21rdquo BioEssays vol 33 no 11 pp 803ndash809 2011


[143] W A McEwan F Hauler C R Williams et al ldquoRegulationof virus neutralization and the persistent fraction by TRIM21rdquoJournal of Virology vol 86 no 16 pp 8482ndash8491 2012

[144] A Kramer T Keitel K Winkler W Stocklein W Hohneand J Schneider-Mergener ldquoMolecular basis for the bindingpromiscuity of an anti-p24 (HIV-1) monoclonal antibodyrdquo Cellvol 91 no 6 pp 799ndash809 1997

[145] J M Binley P J Klasse Y Cao et al ldquoDifferential regulationof the antibody responses to Gag and Env proteins of humanimmunodeficiency virus type 1rdquo Journal of Virology vol 71 no4 pp 2799ndash2809 1997

[146] J N Weber R A Weiss and C Roberts ldquoHuman immunod-eficiency virus infection in two cohorts of homosexual menneutralising sera and association of anti-GAG antibody withprognosisrdquoThe Lancet vol 1 no 8525 pp 119ndash121 1987

[147] O Schwartz V Marechal B Friguet F Arenzana-Seisdedosand J Heard ldquoAntiviral activity of the proteasome on incominghuman immunodeficiency virus type 1rdquo Journal of Virology vol72 no 5 pp 3845ndash3850 1998

[148] V Holl M Peressin T Decoville et al ldquoNonneutralizingantibodies are able to inhibit human immunodeficiency virustype 1 replication inmacrophages and immature dendritic cellsrdquoJournal of Virology vol 80 no 12 pp 6177ndash6181 2006

[149] K A Dowd and T C Pierson ldquoAntibody-mediated neutraliza-tion of flaviviruses a reductionist viewrdquo Virology vol 411 no 2pp 306ndash315 2011

[150] T C Pierson D H Fremont R J Kuhn and M S DiamondldquoStructural insights into the mechanisms of antibody-mediatedneutralization of flavivirus infection implications for vaccinedevelopmentrdquo Cell Host amp Microbe vol 4 no 3 pp 229ndash2382008

[151] T C Pierson Q Xu S Nelson et al ldquoThe stoichiometry ofantibody-mediated neutralization and enhancement of WestNile virus infectionrdquoCell Host andMicrobe vol 1 no 2 pp 135ndash145 2007

[152] S B Halstead ldquoImmune enhancement of viral infectionrdquoProgress in Allergy vol 31 pp 301ndash364 1982

[153] S B Halstead J S Chow and N J Marchette ldquoImmunologicalenhancement of dengue virus replicationrdquo NATURE NEWBIOL vol 243 no 122 pp 24ndash26 1973

[154] S BHalstead andE JORourke ldquoDengue viruses andmononu-clear phagocytes I infection enhancement by non-neutralizingantibodyrdquo Journal of Experimental Medicine vol 146 no 1 pp201ndash217 1977

[155] S B Halstead J S Porterfield and E J OrsquoRourke ldquoEnhance-ment of dengue virus infection in monocytes by flavivirusantiserardquo American Journal of Tropical Medicine and Hygienevol 29 no 4 pp 638ndash642 1980

[156] R Dulbecco M Vogt and A G R Strickland ldquoA study of thebasic aspects of neutralization of two animal viruses Westernequine encephalitis virus and poliomyelitis virusrdquoVirology vol2 no 2 pp 162ndash205 1956

[157] L McLain and N J Dimmock ldquoSingle- and multi-hit kineticsof immunoglobulin G neutralization of human immunode-ficiency virus type 1 by monoclonal antibodiesrdquo Journal ofGeneral Virology vol 75 no 6 pp 1457ndash1460 1994

[158] H P Taylor S J Armstrong and N J Dimmock ldquoQuantitativerelationships between an influenza virus and neutralizing anti-bodyrdquo Virology vol 159 no 2 pp 288ndash298 1987

[159] M B Sherman and S CWeaver ldquoStructure of the recombinantalphavirus western equine encephalitis virus revealed by cry-oelectron microscopyrdquo Journal of Virology vol 84 no 19 pp9775ndash9782 2010

[160] C H Andrewes and W J Elford ldquoObservations on anti-phagesera I ldquoThe percentage lawrdquordquo British Journal of ExperimentalPathology vol 14 no 6 pp 368ndash376 1933

[161] A J Della-Porta and E GWestaway ldquoAmulti-hit model for theneutralization of animal virusesrdquo Journal of General Virologyvol 38 no 1 pp 1ndash19 1978

[162] T Berggard S Linse and P James ldquoMethods for the detectionand analysis of protein-protein interactionsrdquo Proteomics vol 7no 16 pp 2833ndash2842 2007

[163] L Jason-MollerMMurphy and J Bruno ldquoOverview of Biacoresystems and their applicationsrdquo in Current Protocols in ProteinScience vol 19 Chapter 19 Unit 19 13 p 13 2006

[164] D Nedelkov and R W Nelson ldquoSurface plasmon resonancemass spectrometry recent progress and outlooksrdquo Trends inBiotechnology vol 21 no 7 pp 301ndash305 2003

[165] M Piliarik H Vaisocherova and J Homola ldquoSurface plasmonresonance biosensingrdquo Methods in Molecular Biology vol 503pp 65ndash88 2009

[166] R L Rich and D G Myszka ldquoSpying on HIV with SPRrdquo Trendsin Microbiology vol 11 no 3 pp 124ndash133 2003

[167] F Vollmer and S Arnold ldquoWhispering-gallery-mode biosens-ing label-free detection down to single moleculesrdquo NatureMethods vol 5 no 7 pp 591ndash596 2008

[168] R Pantophlet and D R Burton ldquoGP120 target for neutralizingHIV-1 antibodiesrdquo Annual Review of Immunology vol 24 pp739ndash769 2006

[169] P L Earl C C Broder D Long et al ldquoNative oligomeric humanimmunodeficiency virus type 1 envelope glycoprotein elicitsdiverse monoclonal antibody reactivitiesrdquo Journal of Virologyvol 68 no 5 pp 3015ndash3026 1994

[170] F Gao E A Weaver Z Lu et al ldquoAntigenicity and immuno-genicity of a synthetic human immunodeficiency virus type 1group M consensus envelope glycoproteinrdquo Journal of Virologyvol 79 no 2 pp 1154ndash1163 2005

[171] J M Kovacs J P Nkolola H Peng et al ldquoHIV-1 envelopetrimer elicits more potent neutralizing antibody responses thanmonomeric gp120rdquo Proceedings of the National Academy ofSciences of theUnited States of America vol 109 no 30 pp 12111ndash12116 2012

[172] P Spearman M A Lally M Elizaga et al ldquoA trimeric V2-deleted HIV-1 envelope glycoprotein vaccine elicits potentneutralizing antibodies but limited breadth of neutralization inhuman volunteersrdquo Journal of Infectious Diseases vol 203 no8 pp 1165ndash1173 2011

[173] X Yang J Lee E M Mahony P D Kwong R Wyatt andJ Sodroski ldquoHighly stable trimers formed by human immun-odeficiency virus type 1 envelope glycoproteins fused with thetrimeric motif of T4 bacteriophage fibritinrdquo Journal of Virologyvol 76 no 9 pp 4634ndash4642 2002

[174] X Yang RWyatt and J Sodroski ldquoImproved elicitation of neu-tralizing antibodies against primary human immunodeficiencyviruses by soluble stabilized envelope glycoprotein trimersrdquoJournal of Virology vol 75 no 3 pp 1165ndash1171 2001

[175] J Julien J H Lee A Cupo et al ldquoAsymmetric recognitionof the HIV-1 trimer by broadly neutralizing antibody PG9rdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 110 no 11 pp 4351ndash4356 2013


[176] JM Binley RW Sanders B Clas et al ldquoA recombinant humanimmunodeficiency virus type 1 envelope glycoprotein complexstabilized by an intermolecular disulfide bond between thegp120 and gp41 subunits is an antigenic mimic of the trimericvirion-associated structurerdquo Journal of Virology vol 74 no 2pp 627ndash643 2000

[177] R W Sanders M Vesanen N Schuelke et al ldquoStabilization ofthe soluble cleaved trimeric form of the envelope glycoproteincomplex of human immunodeficiency virus type 1rdquo Journal ofVirology vol 76 no 17 pp 8875ndash8889 2002

[178] J M Binley R W Sanders A Master et al ldquoEnhancing theproteolytic maturation of human immunodeficiency virus type1 envelope glycoproteinsrdquo Journal of Virology vol 76 no 6 pp2606ndash2616 2002

[179] S Beddows M Franti A K Dey et al ldquoA comparativeimmunogenicity study in rabbits of disulfide-stabilized prote-olytically cleaved soluble trimeric human immunodeficiencyvirus type 1 gp140 trimeric cleavage-defective gp140 andmonomeric gp120rdquo Virology vol 360 no 2 pp 329ndash340 2007

[180] S Beddows N Schulke M Kirschner et al ldquoEvaluating theimmunogenicity of a disulfide-stabilized cleaved trimeric formof the envelope glycoprotein complex of human immunodefi-ciency virus type 1rdquo Journal of Virology vol 79 no 14 pp 8812ndash8827 2005

[181] J P Julien A Cupo D Sok et al ldquoCrystal structure of a solublecleaved HIV-1 envelope trimerrdquo Science vol 342 pp 1477ndash14832013

[182] D Lyumkis J P Julien N de Val et al ldquoCryo-EM structureof a fully glycosylated soluble cleaved HIV-1 envelope trimerrdquoScience vol 342 no 6165 pp 1484ndash1490 2013

[183] A Yasmeen R Ringe R Derking et al ldquoDifferential bindingof neutralizing and non-neutralizing antibodies to native-like soluble HIV-1 Env trimers uncleaved Env proteins andmonomeric subunitsrdquo Retrovirology vol 11 article 41 2014

[184] P J Klasse and Q J Sattentau ldquoMechanisms of virus neu-tralization by antibodyrdquo Current Topics in Microbiology andImmunology vol 260 pp 87ndash108 2001

[185] J S Klein and P J Bjorkman ldquoFew and far between how HIVmay be evading antibody avidityrdquo PLoS Pathogens vol 6 no 5pp 1ndash6 2010

[186] N S Greenspan ldquoAffinity complementarity cooperativity andspecificity in antibody recognitionrdquo Current Topics in Microbi-ology and Immunology vol 260 pp 65ndash85 2001

[187] N S Greenspan D A Dacek and L J N Cooper ldquoCooperativebinding of two antibodies to independent antigens by an Fc-dependent mechanismrdquo The FASEB Journal vol 3 no 10 pp2203ndash2207 1989

[188] C C LaBranche T LHoffman J Romano et al ldquoDeterminantsof CD4 independence for a human immunodeficiency virustype 1 variant map outside regions required for coreceptorspecificityrdquo Journal of Virology vol 73 no 12 pp 10310ndash103191999

[189] C C LaBranche M M Sauter B S Haggarty et al ldquoA singleamino acid change in the cytoplasmic domain of the simianimmunodeficiency virus transmembrane molecule increasesenvelope glycoprotein expression on infected cellsrdquo Journal ofVirology vol 69 no 9 pp 5217ndash5227 1995

[190] A N Vzorov K M Gernert and R W Compans ldquoMultipledomains of the SIV Env protein determine virus replicationefficiency and neutralization sensitivityrdquo Virology vol 332 no1 pp 89ndash101 2005

[191] E Yuste J D Reeves R W Doms and R C DesrosiersldquoModulation of Env content in virions of simian immunodefi-ciency virus Correlation with cell surface expression and virioninfectivityrdquo Journal of Virology vol 78 no 13 pp 6775ndash67852004

[192] J D Steckbeck I Orlov A Chow et al ldquoKinetic rates ofantibody binding correlate with neutralization sensitivity ofvariant simian immunodeficiency virus strainsrdquo Journal ofVirology vol 79 no 19 pp 12311ndash12320 2005

[193] Q Li A G Yafal Y M Lee J Hogle and M Chow ldquoPoliovirusneutralization by antibodies to internal epitopes of VP4 andVP1 results from reversible exposure of these sequences atphysiological temperaturerdquo Journal of Virology vol 68 no 6pp 3965ndash3970 1994

[194] E J Platt M M Gomes and D Kabat ldquoKinetic mechanismfor HIV-1 neutralization by antibody 2G12 entails reversibleglycan binding that slows cell entryrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 109 no20 pp 7829ndash7834 2012

[195] J S Klein P N P Gnanapragasam R P Galimidi C PFoglesong A P West Jr and P J Bjorkman ldquoExamination ofthe contributions of size and avidity to the neutralizationmech-anisms of the anti-HIV antibodies b12 and 4E10rdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 106 no 18 pp 7385ndash7390 2009

[196] A F Labrijn P Poignard A Raja et al ldquoAccess of anti-body molecules to the conserved coreceptor binding site onglycoprotein gp120 is sterically restricted on primary humanimmunodeficiency virus type 1rdquo Journal of Virology vol 77 no19 pp 10557ndash10565 2003

[197] S P Layne M J Merges M Dembo et al ldquoFactors underlyingspontaneous inactivation and susceptibility to neutralization ofhuman immunodeficiency virusrdquo Virology vol 189 no 2 pp695ndash714 1992

[198] J P Moore J A Mckeating R A Weiss and Q J SattentauldquoDissociation of gp120 from HIV-1 virions induced by solubleCD4rdquo Science vol 250 no 4984 pp 1139ndash1142 1990

[199] E J Platt J P Durnin and D Kabat ldquoKinetic factors controlefficiencies of cell entry efficacies of entry inhibitors andmechanisms of adaptation of human immunodeficiency virusrdquoJournal of Virology vol 79 no 7 pp 4347ndash4356 2005

[200] E G Westaway ldquoThe neutralization of arboviruses II neutral-ization in heterologous virus-serum mixtures with four groupB arbovirusesrdquo Virology vol 26 no 4 pp 528ndash537 1965

[201] P J Klasse and D R Burton ldquoAntibodies to West Nile virus adouble-edged swordrdquo Cell Host and Microbe vol 1 no 2 pp87ndash89 2007

[202] C Herrera P J Klasse C W Kibler E Michael J PMoore and S Beddows ldquoDominant-negative effect of hetero-oligomerization on the function of the human immunodefi-ciency virus type 1 envelope glycoprotein complexrdquo Virologyvol 351 no 1 pp 121ndash132 2006

[203] C H Andrewes and W J Elford ldquoObservations on anti-phagesera II properties of incompletely neutralized phagerdquo BritishJournal of Experimental Pathology vol 14 no 6 pp 376ndash3831933

[204] C Wohlfart ldquoNeutralization of adenoviruses kinetics stoi-chiometry and mechanismsrdquo Journal of Virology vol 62 no 7pp 2321ndash2328 1988

[205] P J Klasse ldquoNeutralization of infectivityrdquo in Encyclopedia ofVirology Elsevier-Academic Press 3rd edition 2008


[206] J Icenogle H Shiwen G Duke S Gilbert R Rueckert andJ Anderegg ldquoNeutralization of poliovirus by a monoclonalantibody kinetics and stoichiometryrdquo Virology vol 127 no 2pp 412ndash425 1983

[207] K M Murphy Janewayrsquos Immunobiology New York NY USA2012

[208] S P LayneM JMerges J L SpougeMDembo and P L NaraldquoBlocking of human immunodeficiency virus infection dependson cell density and viral stock agerdquo Journal of Virology vol 65no 6 pp 3293ndash3300 1991

[209] J Daecke O T Fackler M T Dittmar and H KrausslichldquoInvolvement of clathrin-mediated endocytosis in humanimmunodeficiency virus type 1 entryrdquo Journal of Virology vol79 no 3 pp 1581ndash1594 2005

[210] B M Dale G P McNerney D LThompson et al ldquoCell-to-celltransfer of HIV-1 via virological synapses leads to endosomalvirion maturation that activates viral membrane fusionrdquo CellHost and Microbe vol 10 no 6 pp 551ndash562 2011

[211] L von Kleist W Stahlschmidt H Bulut et al ldquoRole of theclathrin terminal domain in regulating coated pit dynamicsrevealed by small molecule inhibitionrdquo Cell vol 146 pp 471ndash484 2011

[212] F M Burnet E V Keogh and D Lush ldquoThe immunologicalreactions of the filterable virusesrdquo The Australian Journal ofExperimental Biology andMedical Science vol 15 no 3 pp 227ndash368 1937

[213] R M Iorio and M A Bratt ldquoNeutralization of Newcastledisease virus by monoclonal antibodies to the hemagglutinin-neuraminidase glycoprotein requirement for antibodies to foursites for complete neutralizationrdquo Journal of Virology vol 51 no2 pp 445ndash451 1984

[214] R M Iorio and M A Bratt ldquoSelection of unique antigenicvariants of Newcastle disease virus with neutralizing mono-clonal antibodies and anti-immunoglobulinrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 82 no 20 pp 7106ndash7110 1985

[215] R Pejchal L M Walker R L Stanfield et al ldquoStructureand function of broadly reactive antibody PG16 reveal anH3 subdomain that mediates potent neutralization of HIV-1rdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 107 no 25 pp 11483ndash11488 2010

[216] Q Vos E A Klasen and J J Haaijman ldquoThe effect of divalentand univalent binding on antibody titration curves in solid-phase ELISArdquo Journal of Immunological Methods vol 103 no1 pp 47ndash54 1987

[217] R Derking et al submitted 2014[218] S Laal S Burda M K Gorny S Karwowska A Buchbinder

and S Zolla-Pazner ldquoSynergistic neutralization of humanimmunodeficiency virus type 1 by combinations of humanmonoclonal antibodiesrdquo Journal of Virology vol 68 no 6 pp4001ndash4008 1994

[219] T J Ketas S Holuigue K Matthews J P Moore and P JKlasse ldquoEnv-glycoprotein heterogeneity as a source of apparentsynergy and enhanced cooperativity in inhibition of HIV-1 infection by neutralizing antibodies and entry inhibitorsrdquoVirology vol 422 no 1 pp 22ndash36 2012

[220] MC Berenbaum ldquoWhat is synergyrdquoPharmacological Reviewsvol 41 no 2 pp 93ndash141 1989

[221] A V Hill ldquoThe combinations of haemoglobin with oxygen andwith carbon monoxide Irdquo Biochemical Journal vol 7 no 5 pp471ndash480 1913

[222] A Hoffman and A Goldberg ldquoThe relationship betweenreceptor-effector unit heterogeneity and the shape of theconcentration-effect profile pharmacodynamic implicationsrdquoJournal of Pharmacokinetics and Biopharmaceutics vol 22 no6 pp 449ndash468 1994

[223] J N Weiss ldquoThe Hill equation revisited uses and misusesrdquoTheFASEB Journal vol 11 no 11 pp 835ndash841 1997

[224] W R Greco G Bravo and J C Parsons ldquoThe search forsynergy a critical review from a response surface perspectiverdquoPharmacological Reviews vol 47 no 2 pp 331ndash385 1995

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Enzyme Research



Microbiology


mechanism of protection is not completely elucidated [18ndash20] In other cases recombinant proteins representing sub-units of hepatitis B virus (HBV) and human papilloma virus(HPV) induce strong protection [21 22] The HPV vaccineconsists of virus-like particles that may have advantageousproperties both antigenically and immunogenically theymaypresent native neutralization epitopeswell and be seen bythe innate immune system as pathogen-associated molecularpatterns [23] But subunit immunizations have failed toprotect against HIV type 1 (HIV-1) [1 7 8 14 24] Onlyin the RV144 clinical trial which combined viral proteinsexpressed from a canarypox vector with recombinant subunitprotein boosts was some modest protection observed Butthe vaccine had not induced NAbs [8 25 26] Therefore thehunt is on for other antibodies and immune responses thatmight explain the limited protectionMany different antiviraleffects of antibodies have been described that do not qualifyas neutralization [27 28] This brings us to some semanticclarifications

2 The Definition of Virus Neutralization

Definitions are arbitrary and contain no deeper knowledgethan the proposed use of the defined term [29]Therefore theonly reason to adhere to a strict definition of neutralizationis that it may favor clarity and allow useful distinctions in thefield of antiviral research Neutralization as discussed here isdefined as the reduction in viral infectivity by the binding ofantibodies to the surface of viral particles (virions) therebyblocking a step in the viral replication cycle that precedesvirally encoded transcription or synthesis [30 31] Classicallythe term was applied only to antibodies and fragments ofantibodies Fab and F(ab1015840)2 but later it has naturally beenextended to single-domain antigen-binding recombinantfragments and natural nanobodies [32 33] Likewise thedefinition can be expanded to cover similar activities bysoluble forms of viral receptors [34 35] naturally occurringdefensins or other molecules of the innate immune system[36 37] it can be extended to lectins either derived fromplants or soluble recombinant and hybrid forms of mannoseC-type lectin receptors (MCLRs) [38] With regard to smallorganicmolecules if they are said to be neutralizing it shouldbe clarified whether that is meant to imply that they act bybinding to the surface of the virion [39ndash49] This reviewhowever only discusses antibody-mediated neutralization

What does the definition not include An antibodymightbind to a budding virion thus acting late in the viral cyclethereby blocking the release of newly formed virus from thesurface of an infected cell Examples of antibodies acting likethis are those directed to the neuraminidase on the influenzaviral surface [50] The enzyme releases progeny virus bydigesting the neuraminic-acid moiety of the receptor for thevirus We shall see later why such an antibody althoughits antigen decorates the virion surface does not interfereat the beginning of the replicative cycle and therefore perdefinition does not neutralize

Another semantic point is that an antibody to a receptorfor the virus on the cell surface may block viral infection but

does not neutralize according to the definition it does notbind to the virions each of which diffusing around in theextracellular space would be potentially as infectious as inthe absence of the antibody instead the target cells would berendered nonsusceptible Hence for clarity some other termsuch as infection-blocking antibody should be used in thatspecial case

The usefulness of a strict definition becomes obvi-ous in vaccine research when we ask by which mech-anism vaccine-induced antibodies protect from infectionAntibody-dependent cellular cytotoxicity (ADCC) is a well-established effect that requires effector cells and counteractsviral infection by killing the virus-producing cell [27 28]The antibodies involved may be neutralizing when tested inthe absence of effector cells (natural killer cells) but oftenare not And some neutralizing antibodies are not of theright isotype to mediate this effect Antibody-dependent cell-mediated viral inhibition (ADCVI) has a more complicatedrelationship to neutralization which may concomitantlyoccur in the assay used for measuring the effect But tothe extent that the inhibition depends on effector cells itis not neutralization Again antibody isotype will affect thecomponent in ADCVI that involves Fc-Fc-receptor interac-tions Furthermore the epitope location on the influenzavirus haemagglutinin determines whether an antibody ismerely neutralizing or also capable of mediating Fc-receptorinteractions and thereby ADCC and protection in vivo in amouse model [51]

HIV-1 can be transmitted directly from cell to cell via avirally induced connection known as the virological synapse[52ndash55] It is convenient however to call the inhibitionof cell-to-cell transfer something other than neutralizationeven when it is mediated by NAbs [56] The reason isthat non-NAbs including antibodies that interfere with theformation of the synapse by binding to cellular structuresand antibodies that counter virion formation or releasemight block this mode of transfer It is also noteworthy thatthe relative efficiency of neutralization and block of cell-to-cell transfer differs among NAbs [57] Observance of thesedistinctions makes for clarity

Normally neutralization is measured in the absenceof complement and the definition can certainly be mademore stringent by making that a criterion But it may bemore useful to allow complement-mediated enhancement ofneutralization as a legitimate category [58] regardless it isvaluable to quantify that effect [59 60] Again if complementfactors reduce viral infectivity by binding directly to the viralsurface rather than to antibodies already in complex withvirions [61] this could also qualifywithin thewider definitionof neutralization

One consideration of biological and medical importanceis that neutralization does not have to equate only whatis measured in the neutralization assays in vitro Althoughneutralization must be measured in vitro we can fruitfullydiscuss how it operates in vivo To infer that it is responsiblefor protection in vivo is more complicated and requiresthe use of antibodies without other effector functions orexperimental models in which cell-dependent mechanismsare knocked out by other means [62ndash64] And effects late





















(a) (b)






































119903 = 119896 [virus] lowast [119860119887]119899 (1)


119903 asymp 1198961015840

[virus] (2)






























600

400

200

0

0 300 600 900

Resp

diff

(RU

)

2G12

PGV04

PGT135

b6F240b12

Non-NAbTime (s)


PGT128PGT121PGT123

14e19b

800

600

400

200

0

Resp

diff

(RU

)

0 300 600 900

Non-NAbTime (s)

V1V2 glycan200

150

100

50

0

PG16

PGT145PG9

0 300 600 900

Resp

diff

(RU

)

Time (s)

gp120

gp41



















PGV04

gp120

PGV04

gp120PDB3SE9

Top view

Side view

(a)

17b

gp120

sCD4

17b

gp120 PDB1RZKsCD4

Top view

Side view

(b)

Top view

Side view


(c)









119889value Hence








0= sum119871minus1

119903=0(120582119903119890minus120582)119903 Thus


0= 119890minus1




















0 1 2 3 4 5 6 7000102030405060708091011

Log(Ab(nM))

Rela

tive i

nfec

tivity

(a)

1 2 3 4 5 6 70

1Log(Ab(nM))

Log(

relat

ive i

nfec

tivity

) minus1

minus2

minus3

minus4

minus5

minus6

minus7

minus8

(b)





















Acknowledgment


References















































































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





















(a) (b)






































119903 = 119896 [virus] lowast [119860119887]119899 (1)


119903 asymp 1198961015840

[virus] (2)






























600

400

200

0

0 300 600 900

Resp

diff

(RU

)

2G12

PGV04

PGT135

b6F240b12

Non-NAbTime (s)


PGT128PGT121PGT123

14e19b

800

600

400

200

0

Resp

diff

(RU

)

0 300 600 900

Non-NAbTime (s)

V1V2 glycan200

150

100

50

0

PG16

PGT145PG9

0 300 600 900

Resp

diff

(RU

)

Time (s)

gp120

gp41



















PGV04

gp120

PGV04

gp120PDB3SE9

Top view

Side view

(a)

17b

gp120

sCD4

17b

gp120 PDB1RZKsCD4

Top view

Side view

(b)

Top view

Side view


(c)









119889value Hence








0= sum119871minus1

119903=0(120582119903119890minus120582)119903 Thus


0= 119890minus1




















0 1 2 3 4 5 6 7000102030405060708091011

Log(Ab(nM))

Rela

tive i

nfec

tivity

(a)

1 2 3 4 5 6 70

1Log(Ab(nM))

Log(

relat

ive i

nfec

tivity

) minus1

minus2

minus3

minus4

minus5

minus6

minus7

minus8

(b)





















Acknowledgment


References















































































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology













(a) (b)






































119903 = 119896 [virus] lowast [119860119887]119899 (1)


119903 asymp 1198961015840

[virus] (2)






























600

400

200

0

0 300 600 900

Resp

diff

(RU

)

2G12

PGV04

PGT135

b6F240b12

Non-NAbTime (s)


PGT128PGT121PGT123

14e19b

800

600

400

200

0

Resp

diff

(RU

)

0 300 600 900

Non-NAbTime (s)

V1V2 glycan200

150

100

50

0

PG16

PGT145PG9

0 300 600 900

Resp

diff

(RU

)

Time (s)

gp120

gp41



















PGV04

gp120

PGV04

gp120PDB3SE9

Top view

Side view

(a)

17b

gp120

sCD4

17b

gp120 PDB1RZKsCD4

Top view

Side view

(b)

Top view

Side view


(c)









119889value Hence








0= sum119871minus1

119903=0(120582119903119890minus120582)119903 Thus


0= 119890minus1




















0 1 2 3 4 5 6 7000102030405060708091011

Log(Ab(nM))

Rela

tive i

nfec

tivity

(a)

1 2 3 4 5 6 70

1Log(Ab(nM))

Log(

relat

ive i

nfec

tivity

) minus1

minus2

minus3

minus4

minus5

minus6

minus7

minus8

(b)





















Acknowledgment


References















































































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a) (b)






































119903 = 119896 [virus] lowast [119860119887]119899 (1)


119903 asymp 1198961015840

[virus] (2)






























600

400

200

0

0 300 600 900

Resp

diff

(RU

)

2G12

PGV04

PGT135

b6F240b12

Non-NAbTime (s)


PGT128PGT121PGT123

14e19b

800

600

400

200

0

Resp

diff

(RU

)

0 300 600 900

Non-NAbTime (s)

V1V2 glycan200

150

100

50

0

PG16

PGT145PG9

0 300 600 900

Resp

diff

(RU

)

Time (s)

gp120

gp41



















PGV04

gp120

PGV04

gp120PDB3SE9

Top view

Side view

(a)

17b

gp120

sCD4

17b

gp120 PDB1RZKsCD4

Top view

Side view

(b)

Top view

Side view


(c)









119889value Hence








0= sum119871minus1

119903=0(120582119903119890minus120582)119903 Thus


0= 119890minus1




















0 1 2 3 4 5 6 7000102030405060708091011

Log(Ab(nM))

Rela

tive i

nfec

tivity

(a)

1 2 3 4 5 6 70

1Log(Ab(nM))

Log(

relat

ive i

nfec

tivity

) minus1

minus2

minus3

minus4

minus5

minus6

minus7

minus8

(b)





















Acknowledgment


References















































































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
































119903 = 119896 [virus] lowast [119860119887]119899 (1)


119903 asymp 1198961015840

[virus] (2)






























600

400

200

0

0 300 600 900

Resp

diff

(RU

)

2G12

PGV04

PGT135

b6F240b12

Non-NAbTime (s)


PGT128PGT121PGT123

14e19b

800

600

400

200

0

Resp

diff

(RU

)

0 300 600 900

Non-NAbTime (s)

V1V2 glycan200

150

100

50

0

PG16

PGT145PG9

0 300 600 900

Resp

diff

(RU

)

Time (s)

gp120

gp41



















PGV04

gp120

PGV04

gp120PDB3SE9

Top view

Side view

(a)

17b

gp120

sCD4

17b

gp120 PDB1RZKsCD4

Top view

Side view

(b)

Top view

Side view


(c)









119889value Hence








0= sum119871minus1

119903=0(120582119903119890minus120582)119903 Thus


0= 119890minus1




















0 1 2 3 4 5 6 7000102030405060708091011

Log(Ab(nM))

Rela

tive i

nfec

tivity

(a)

1 2 3 4 5 6 70

1Log(Ab(nM))

Log(

relat

ive i

nfec

tivity

) minus1

minus2

minus3

minus4

minus5

minus6

minus7

minus8

(b)





















Acknowledgment


References















































































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






















119903 = 119896 [virus] lowast [119860119887]119899 (1)


119903 asymp 1198961015840

[virus] (2)






























600

400

200

0

0 300 600 900

Resp

diff

(RU

)

2G12

PGV04

PGT135

b6F240b12

Non-NAbTime (s)


PGT128PGT121PGT123

14e19b

800

600

400

200

0

Resp

diff

(RU

)

0 300 600 900

Non-NAbTime (s)

V1V2 glycan200

150

100

50

0

PG16

PGT145PG9

0 300 600 900

Resp

diff

(RU

)

Time (s)

gp120

gp41



















PGV04

gp120

PGV04

gp120PDB3SE9

Top view

Side view

(a)

17b

gp120

sCD4

17b

gp120 PDB1RZKsCD4

Top view

Side view

(b)

Top view

Side view


(c)









119889value Hence








0= sum119871minus1

119903=0(120582119903119890minus120582)119903 Thus


0= 119890minus1




















0 1 2 3 4 5 6 7000102030405060708091011

Log(Ab(nM))

Rela

tive i

nfec

tivity

(a)

1 2 3 4 5 6 70

1Log(Ab(nM))

Log(

relat

ive i

nfec

tivity

) minus1

minus2

minus3

minus4

minus5

minus6

minus7

minus8

(b)





















Acknowledgment


References















































































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology










119903 = 119896 [virus] lowast [119860119887]119899 (1)


119903 asymp 1198961015840

[virus] (2)






























600

400

200

0

0 300 600 900

Resp

diff

(RU

)

2G12

PGV04

PGT135

b6F240b12

Non-NAbTime (s)


PGT128PGT121PGT123

14e19b

800

600

400

200

0

Resp

diff

(RU

)

0 300 600 900

Non-NAbTime (s)

V1V2 glycan200

150

100

50

0

PG16

PGT145PG9

0 300 600 900

Resp

diff

(RU

)

Time (s)

gp120

gp41



















PGV04

gp120

PGV04

gp120PDB3SE9

Top view

Side view

(a)

17b

gp120

sCD4

17b

gp120 PDB1RZKsCD4

Top view

Side view

(b)

Top view

Side view


(c)









119889value Hence








0= sum119871minus1

119903=0(120582119903119890minus120582)119903 Thus


0= 119890minus1




















0 1 2 3 4 5 6 7000102030405060708091011

Log(Ab(nM))

Rela

tive i

nfec

tivity

(a)

1 2 3 4 5 6 70

1Log(Ab(nM))

Log(

relat

ive i

nfec

tivity

) minus1

minus2

minus3

minus4

minus5

minus6

minus7

minus8

(b)





















Acknowledgment


References















































































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





























600

400

200

0

0 300 600 900

Resp

diff

(RU

)

2G12

PGV04

PGT135

b6F240b12

Non-NAbTime (s)


PGT128PGT121PGT123

14e19b

800

600

400

200

0

Resp

diff

(RU

)

0 300 600 900

Non-NAbTime (s)

V1V2 glycan200

150

100

50

0

PG16

PGT145PG9

0 300 600 900

Resp

diff

(RU

)

Time (s)

gp120

gp41



















PGV04

gp120

PGV04

gp120PDB3SE9

Top view

Side view

(a)

17b

gp120

sCD4

17b

gp120 PDB1RZKsCD4

Top view

Side view

(b)

Top view

Side view


(c)









119889value Hence








0= sum119871minus1

119903=0(120582119903119890minus120582)119903 Thus


0= 119890minus1




















0 1 2 3 4 5 6 7000102030405060708091011

Log(Ab(nM))

Rela

tive i

nfec

tivity

(a)

1 2 3 4 5 6 70

1Log(Ab(nM))

Log(

relat

ive i

nfec

tivity

) minus1

minus2

minus3

minus4

minus5

minus6

minus7

minus8

(b)





















Acknowledgment


References















































































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology














600

400

200

0

0 300 600 900

Resp

diff

(RU

)

2G12

PGV04

PGT135

b6F240b12

Non-NAbTime (s)


PGT128PGT121PGT123

14e19b

800

600

400

200

0

Resp

diff

(RU

)

0 300 600 900

Non-NAbTime (s)

V1V2 glycan200

150

100

50

0

PG16

PGT145PG9

0 300 600 900

Resp

diff

(RU

)

Time (s)

gp120

gp41



















PGV04

gp120

PGV04

gp120PDB3SE9

Top view

Side view

(a)

17b

gp120

sCD4

17b

gp120 PDB1RZKsCD4

Top view

Side view

(b)

Top view

Side view


(c)









119889value Hence








0= sum119871minus1

119903=0(120582119903119890minus120582)119903 Thus


0= 119890minus1




















0 1 2 3 4 5 6 7000102030405060708091011

Log(Ab(nM))

Rela

tive i

nfec

tivity

(a)

1 2 3 4 5 6 70

1Log(Ab(nM))

Log(

relat

ive i

nfec

tivity

) minus1

minus2

minus3

minus4

minus5

minus6

minus7

minus8

(b)





















Acknowledgment


References















































































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



600

400

200

0

0 300 600 900

Resp

diff

(RU

)

2G12

PGV04

PGT135

b6F240b12

Non-NAbTime (s)


PGT128PGT121PGT123

14e19b

800

600

400

200

0

Resp

diff

(RU

)

0 300 600 900

Non-NAbTime (s)

V1V2 glycan200

150

100

50

0

PG16

PGT145PG9

0 300 600 900

Resp

diff

(RU

)

Time (s)

gp120

gp41



















PGV04

gp120

PGV04

gp120PDB3SE9

Top view

Side view

(a)

17b

gp120

sCD4

17b

gp120 PDB1RZKsCD4

Top view

Side view

(b)

Top view

Side view


(c)









119889value Hence








0= sum119871minus1

119903=0(120582119903119890minus120582)119903 Thus


0= 119890minus1




















0 1 2 3 4 5 6 7000102030405060708091011

Log(Ab(nM))

Rela

tive i

nfec

tivity

(a)

1 2 3 4 5 6 70

1Log(Ab(nM))

Log(

relat

ive i

nfec

tivity

) minus1

minus2

minus3

minus4

minus5

minus6

minus7

minus8

(b)





















Acknowledgment


References















































































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
















PGV04

gp120

PGV04

gp120PDB3SE9

Top view

Side view

(a)

17b

gp120

sCD4

17b

gp120 PDB1RZKsCD4

Top view

Side view

(b)

Top view

Side view


(c)









119889value Hence








0= sum119871minus1

119903=0(120582119903119890minus120582)119903 Thus


0= 119890minus1




















0 1 2 3 4 5 6 7000102030405060708091011

Log(Ab(nM))

Rela

tive i

nfec

tivity

(a)

1 2 3 4 5 6 70

1Log(Ab(nM))

Log(

relat

ive i

nfec

tivity

) minus1

minus2

minus3

minus4

minus5

minus6

minus7

minus8

(b)





















Acknowledgment


References















































































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


PGV04

gp120

PGV04

gp120PDB3SE9

Top view

Side view

(a)

17b

gp120

sCD4

17b

gp120 PDB1RZKsCD4

Top view

Side view

(b)

Top view

Side view


(c)









119889value Hence








0= sum119871minus1

119903=0(120582119903119890minus120582)119903 Thus


0= 119890minus1




















0 1 2 3 4 5 6 7000102030405060708091011

Log(Ab(nM))

Rela

tive i

nfec

tivity

(a)

1 2 3 4 5 6 70

1Log(Ab(nM))

Log(

relat

ive i

nfec

tivity

) minus1

minus2

minus3

minus4

minus5

minus6

minus7

minus8

(b)





















Acknowledgment


References















































































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




119889value Hence








0= sum119871minus1

119903=0(120582119903119890minus120582)119903 Thus


0= 119890minus1




















0 1 2 3 4 5 6 7000102030405060708091011

Log(Ab(nM))

Rela

tive i

nfec

tivity

(a)

1 2 3 4 5 6 70

1Log(Ab(nM))

Log(

relat

ive i

nfec

tivity

) minus1

minus2

minus3

minus4

minus5

minus6

minus7

minus8

(b)





















Acknowledgment


References















































































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology















0 1 2 3 4 5 6 7000102030405060708091011

Log(Ab(nM))

Rela

tive i

nfec

tivity

(a)

1 2 3 4 5 6 70

1Log(Ab(nM))

Log(

relat

ive i

nfec

tivity

) minus1

minus2

minus3

minus4

minus5

minus6

minus7

minus8

(b)





















Acknowledgment


References















































































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0 1 2 3 4 5 6 7000102030405060708091011

Log(Ab(nM))

Rela

tive i

nfec

tivity

(a)

1 2 3 4 5 6 70

1Log(Ab(nM))

Log(

relat

ive i

nfec

tivity

) minus1

minus2

minus3

minus4

minus5

minus6

minus7

minus8

(b)





















Acknowledgment


References















































































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology












Acknowledgment


References















































































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology













































































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




























































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Review Article Neutralization of Virus Infectivity by …downloads.hindawi.com/archive/2014/157895.pdfReview Article Neutralization of Virus Infectivity by Antibodies: Old Problems