disabling complement regulatory activities of vaccinia virus complement control protein reduces...

Disabling complement regulatory activities of vaccinia viruscomplement control protein reduces vaccinia viruspathogenicity

John Bernet1, Muzammil Ahmad, Jayati Mullick2, Yogesh Panse, Akhilesh K. Singh,Pradeep B. Parab, and Arvind Sahu⁎

National Centre for Cell Science, Pune University Campus, Ganeshkhind, Pune 411007, India

AbstractPoxviruses encode a repertoire of immunomodulatory proteins to thwart the host immune system.One among this array is a homolog of the host complement regulatory proteins that is conserved invarious poxviruses including vaccinia (VACV) and variola. The vaccinia virus complementcontrol protein (VCP), which inhibits complement by decaying the classical pathway C3-convertase (decay-accelerating activity), and by supporting inactivation of C3b and C4b by serineprotease factor I (cofactor activity), was shown to play a role in viral pathogenesis. However, therole its individual complement regulatory activities impart in pathogenesis, have not yet beenelucidated. Here, we have generated monoclonal antibodies (mAbs) that block the VCP functionsand utilized them to evaluate the relative contribution of complement regulatory activities of VCPin viral pathogenesis by employing a rabbit intradermal model for VACV infection. TargetingVCP by mAbs that inhibited the decay-accelerating activity as well as cofactor activity of VCP orprimarily the cofactor activity of VCP, by injecting them at the site of infection, significantlyreduced VACV lesion size. This reduction however was not pronounced when VCP was targetedby a mAb that inhibited only the decay-accelerating activity. Further, the reduction in lesion sizeby mAbs was reversed when host complement was depleted by injecting cobra venom factor.Thus, our results suggest that targeting VCP by antibodies reduces VACV pathogenicity and thatprincipally the cofactor activity of VCP appears to contribute to the virulence.

KeywordsSmallpox vaccine; Vaccinia virus; VCP; Complement; Immune evasion

1 IntroductionThe first barriers that microorganisms including viruses must breach for being successfulpathogens are imposed by the innate immune system of which the complement systemconstitutes a major arm [1–4]. The complement system comprises of an intricate group ofboth soluble and cell-associated proteins activated through three major pathways, the

Â© 2011 Elsevier Ltd.⁎Corresponding author. Tel.: +91 20 2570 8083; fax: +91 20 2569 2259. [email protected][email protected] work was done in partial fulfillment of Ph.D. thesis of J.B. submitted to the University of Pune, Pune, India.2National Institute of Virology, Microbial Containment Complex, Pashan, Pune 411021, India.This document was posted here by permission of the publisher. At the time of deposit, it included all changes made during peerreview, copyediting, and publishing. The U.S. National Library of Medicine is responsible for all links within the document and forincorporating any publisher-supplied amendments or retractions issued subsequently. The published journal article, guaranteed to besuch by Elsevier, is available for free, on ScienceDirect.

Sponsored document fromVaccine

Published as: Vaccine. 2011 October 06; 29(43): 7435–7443.

Sponsored Docum

ent Sponsored D

ocument

Sponsored Docum

ent

classical, alternative and lectin pathways. Complement activation results in the generation ofactive components, including C3b and C4b, which aid in the assembly of enzymes called asC3/C5-convertases that facilitate downstream cleavage and formation of the membraneattack complex (MAC) capable of lysing pathogens. Additionally, the activation productsC3a and C5a show anaphylatoxic and chemotactic properties [5] and also play a role in Tcell activation [6], and surface bound complement components derived from C3 interactwith specific immune receptors, thus acting as a connecting link with the adaptive immunesystem [7]. Hence, the complement system exerts assault on pathogens directly by lysis andindirectly by boosting the pathogen-specific immune responses [8].

Variola virus, the etiological agent of smallpox, caused large-scale mortality and morbidityamong humans before its successful eradication. Even though smallpox has been eradicatedthere are two major concerns related to poxviruses, one of which is the possibility of usageof variola as a bioterrorism agent and the other being cross-species related infections, e.g.,monkeypox and cowpox virus infection of humans [9–11], requiring further understandingof the pathogenesis of this complex group of viruses.

Complement activation either through the alternative pathway or through the classicalpathway plays a pivotal role in the neutralization of poxviruses. Vaccinia virus (VACV), theprototypic poxvirus, has two major forms: the extracellular enveloped (EV) and theintracellular mature virus (MV). Among these, the EV form is more resistant toneutralization by antibodies, but this is reversed in the presence of complement [12]. This isfurther highlighted by the observation that both in vitro and in vivo neutralization of the EVform could be achieved with antibodies targeted against B5R, an EV form-specific protein,in the presence of complement [13]. These studies besides emphasizing the role of antigenspecific antibodies also identify the pivotal role complement plays in targeting andneutralizing poxviruses.

Viruses override the complement system by developing various mechanisms to maskthemselves against the host's complement assault [14–17]. Poxviruses in particular, havebeen shown to encode mimics of human regulator of complement activation (RCA) proteinsto target complement, besides the additional strategy of recruitment of human RCAs [18–21]. Vaccinia and variola viruses, the two important members of the genus Orthopoxvirus[22,23], encode soluble RCA homologs named vaccinia virus complement control protein(VCP) and smallpox inhibitor of complement enzymes (SPICE), respectively [24,25]. Botheffectively inhibit complement, with SPICE being more human specific than VCP [25,26].Other members of the pox family, like cowpox virus, monkeypox virus and ectromelia, alsoencode functional RCA mimics with marked identity among the homologs, exceptmonkeypox virus strains, which have been shown to either lack or have a truncated form ofthe homolog [20,27–31].

VCP is entirely formed by four complement control protein (CCP) domains separated byshort linkers, which is a characteristic of the RCA proteins [32–34] and exists either as asecreted or a cell associated form [24,35]. Functional studies revealed that it inhibits thecomplement-mediated neutralization of both the infectious forms of VACV i.e., MV as wellas EV [36,37]. Notably, VCP has been shown to be involved in modulating the humoral andT cell mediated responses to VACV infection [38]. Detailed studies on the mechanism ofcomplement inactivation showed that it binds to complement proteins C3b and C4b [39,40]and inhibits complement by targeting C3-convertases, by supporting factor I mediatedinactivation of C3b and C4b (cofactor activity) [39,41] and by accelerating irreversibledissociation of the classical pathway C3-convertase (decay-accelerating activity) [39,42].Structure–function analysis using monoclonal antibodies (mAbs), chimeric proteins anddeletion mutagenesis indicated that all the domains contribute to C3b and C4b binding and

Bernet et al.


Sponsored Docum

ent Sponsored D

ocument

Sponsored Docum

ent

functional activities [42–45]. Further, our recent domain swapping study with humancomplement regulators revealed that of the four domains, domain 1 imparts decay of theprotease subunit from the C3-convertase and domains 2 and 3 recruit factor I for impartingproteolytic inactivation of C3b and C4b [46].

The importance of VCP in VACV pathogenesis was addressed initially in a study usingrabbit and guinea pig intradermal models where it was shown that VACV devoid of VCPproduced comparatively smaller lesions than the wild type virus [36]. More recently, similarexperiments were also performed using mice ear pinnae model in complement sufficient andC3−/− mice [38]. Though these studies clearly demonstrated the importance of VCP inpathogenesis, the significance of complement inhibition by VCP as a contributing factor inthe pathogenesis based on the individual known functions is still not clear. In the presentstudy, we have generated a panel of mAbs against VCP and used them to dissect the regionson VCP that play a role in the complement regulatory activity. We then asked whether VCPcould serve as a virulence determinant by targeting complement and if so, whichcomplement regulatory activity of VCP plays a predominant role in virulence in vivo.

2 Materials and methods2.1 Virus and proteins

The vaccinia virus strain Western Reserve (VACV-WR) was a kind gift from Dr. SekharChakrabarti (National Institute of Cholera & Enteric Diseases, Kolkata, India). The viruswas cultured by infecting African green monkey kidney cells (CV-1) followed bypurification on a sucrose gradient. Recombinant VCP (rVCP) and its truncation mutants(CCP 1–3, CCP 2–4, CCP 1–2, CCP 2–3 and CCP 3–4) were expressed and purified aspreviously described [42]. The complement proteins C1, C2, C4, C4b and factor I werepurchased from Calbiochem (La Jolla, CA), and complement proteins factor H, C3 [42] andC3b [41] were purified as described earlier. Human soluble CR1 (sCR1) was a generous giftfrom Dr. Henry Marsh (AVANT Immunotherapeutics, Inc., Needham, MA). Cobra venomfactor (CVF) was purified from Naja naja kaouthia venom as described earlier with minormodifications [47]. In brief, the lyophilized venom was dissolved in 20 mM sodiumphosphate buffer, pH 7.4, loaded onto Source Q column (12 cm × 9.5 cm, GE HealthcareBio-Sciences) in the same buffer and eluted with a linear salt gradient to 1 M NaCl.Fractions containing CVF as judged by SDS-PAGE were then purified using Superose 12column (two columns linked in a series; GE Healthcare Bio-Sciences) followed by Mono Qcolumn. SDS-PAGE analysis showed purity of >95%. Its functionality was verified by itsability to form the stable C3-convertase [47].

2.2 Monoclonal antibodies (mAbs)The VCP specific mAbs were generated by immunizing 5–6 week old BALB/c mice withthe rVCP. In brief, mice were immunized with 20 μg of rVCP in Freund's completeadjuvant, followed by two boosts 15 days post prime at weekly intervals with the same dose,but in Freund's incomplete adjuvant. Following immunization, spleen was removed and thespleen cells were fused in-house with myeloma cells as per established protocols [48,49].The clones from the fusion were screened by ELISA and subcloned to isolate the individualclones. Antibody isotyping was performed by an ELISA-based hybridoma isotyping kit (BDBiosciences, San Diego, CA, USA). The IgG mAbs were purified by capryllic acidprecipitation method or by Hi-Trap affinity protein G column (GE Healthcare Bio-Sciences,Sweden). Homogeneity of mAbs was assured by SDS-PAGE analysis.

Bernet et al.


Sponsored Docum

ent Sponsored D

ocument

Sponsored Docum

ent

2.3 Rabbit infectionsVACV pathogenicity studies were performed in rabbits using skin lesion model [36]. Inbrief, 104 pfu of VACV-WR strain in sterile PBS in a total volume of 100 μl were injectedintradermally with or without the mAb on the shaved backs of two New Zealand Whiterabbits (age 6–7 months) in duplicate and lesions formed (scabs) were measured after every24 h using calipers. The mean of four measurements was used for graphical representationof individual time point per site. To study the role of complement during infection, similarexperiments were also performed in two additional rabbits depleted of complement byadministering 100 U/kg of cobra venom factor. All the results were grouped and statisticallyevaluated by performing Mann–Whitney Rank Sum test (SigmaStat). The experimentalprotocol was approved by the Institute's Animal Care and Use Committee.

2.4 Binding of mAbs to VCP and VCP truncation mutantsThe ELISA plates were coated overnight at 4 °C with rVCP or VCP mutants (CCP 1–3,CCP 2–4, CCP 1–2, CCP 2–3, CCP 3–4; 200 ng/well), blocked by adding 5% milk andincubated with mAbs (1 μg/well) for 1 h at room temperature. Binding was probed byadding 1:2000 diluted anti-mouse HRP conjugate (Biorad, Hercules, CA) and detected with2,2′-Azino-bis (3-ethylbenzthiazoline) 6-sulfonic acid (ABTS) (Roche, Mannheim,Germany) at 414 nm.

2.5 Inhibition of factor I cofactor activity of VCP by mAbsInhibition of factor I cofactor activity of VCP by mAbs was determined as described below.rVCP (0.5 μg) was mixed with 3 μg of mAb and incubated for 15 min at 37 °C. Thereafter,3 μg of C3b or C4b and 0.1 μg of factor I was added to the reaction mixture and the volumewas adjusted to 20 μl using PBS. It was then further incubated at 37 °C for 2 h. The reactionwas stopped by adding SDS-PAGE sample buffer containing DTT and C3b/C4b cleavageswere analyzed on a 10% SDS-PAGE gel [40].

2.6 Inhibition of classical pathway decay-accelerating activity of VCP by mAbsInhibition of the classical pathway decay-accelerating activity of VCP by mAbs wasdetermined by utilizing a hemolytic assay [42,50]. Briefly, 10 μl of EAC142 (classicalpathway C3-convertase; 1 × 109 cells/ml) was mixed with 0.2 μM of rVCP and 1 μM ofmAb in a total volume of 25 μl and incubated for 5 min at 22 °C. The remaining C3-convertase activity was determined by measuring hemolysis after incubating the reactionmixture for 30 min at 37 °C with 1:100 diluted guinea pig serum containing 40 mM EDTA.Hemolysis was measured at 405 nm.

2.7 Surface plasmon resonance measurementsThe kinetics of binding of the mAbs to VCP was determined on the SPR-based biosensorBIACORE 2000 (Biacore AB, Uppsala, Sweden). All the experiments were performed inPBS-T (10 mM sodium phosphate, 145 mM NaCl, pH 7.4 containing 0.05% Tween 20) at25 °C. About 2600 RUs of each of the mAbs was immobilized on test flow cells (Fc-2, Fc-3or Fc-4) of a CM5 chip using amine-coupling chemistry and non-immobilized flow cell(Fc-1) served as the control flow cell [40]. Various concentrations of rVCP were then flownover the chip at 30 μl/min for 120 s and dissociation was followed for an additional 180 s.The chip was regenerated by injecting brief pulses of 0.2 M sodium carbonate, pH 9.5. Dataobtained for the control flow cell were subtracted from those obtained for test flow cell andevaluated using BIAevaluation software version 4.1 using global fitting.

Bernet et al.


Sponsored Docum

ent Sponsored D

ocument

Sponsored Docum

ent

2.8 Measurement of half-life of mAbs in rabbitsThe half-life of two of the VCP neutralizing mAbs (NCCS 67.5 and 67.9) in rabbits wasassessed by radiolabeling the antibodies with 131I. One hundred microliters of the labeledmAbs at a dose of 100–200 μCi (∼65–100 μg) were injected intradermally on backs of NewZealand White rabbits and imaged using an ELGEMS “Millennium MPS” gamma raycamera (GE, USA) at the Department of Veterinary Medicine and Veterinary NuclearMedicine Center (Mumbai, India). A maximum of 250 kilocounts was set for acquiringimages and a medium energy collimator was used to capture emerging radiations. Theimages were acquired at various time points and analyzed using GENIE acquisition userinterface software (GE, USA). The first image acquired immediately after the injection wasconsidered as zero time point. The data obtained were normalized by considering the countsobtained at the zero time point as 100%.

3 Results3.1 Anti-VCP monoclonal antibody generation and affinity measurements

To re-examine the VCP domains responsible for complement modulation and to understandthe in vivo relevance of these complement regulatory functions in VACV pathogenesis, weraised a panel of mAbs against VCP by immunizing BALB/c mice followed by fusion, andcloning and subcloning of the candidate hybrids. The monoclonal antibodies thus generatedlargely belonged to IgG1κ isotype. Four antibodies, namely NCCS 67.5, NCCS 67.9, NCCS67.11 and NCCS 67.13, all belonging to IgG1κ isotype, were chosen for furthercharacterization as they differentially inhibited the functional activities of VCP (see below).Of the four, mAb 67.5 uniquely displayed two distinct light chains, which could be a resultof difference in their glycosylation states [51]. The light chains however did not show anybinding to Con A suggesting that the difference in their molecular weights was not a resultof difference in the presence of Con A binding carbohydrates (Supplementary Fig. 1).Although unusual, the clonal origin of an antibody containing two separate light chains hasbeen reported earlier [52,53]. Thus, it seems that mAb 67.5 may belong to such a category.

All the four antibodies bound to VCP in a direct ELISA (data not shown). Since surfaceplasmon resonance (SPR) offers more quantitative data on biomolecule interactions, weutilized this method to measure the affinities of these antibodies. In the SPR setup, theantibodies were immobilized onto the chip and rVCP was flowed over it to measure binding.All the antibodies bound to VCP in a dose dependent manner (Fig. 1). The equilibriumdissociation constant (KD) of mAbs 67.5 and 67.9 (5.35 × 10−9 M and 6.6 × 10−9 M) waslower compared to those of 67.11 (4.64 × 10−8 M) and 67.13 (2.32 × 10−7 M), respectively(Table 1). Interestingly, in a dot blot assay, these mAbs bound to VCP only under non-reducing conditions (data not shown) indicating that these antibodies recognize theconformational epitopes on VCP.

3.2 Mapping of VCP domains recognized by anti-VCP monoclonal antibodiesTo identify the VCP domains to which these mAbs bind we performed an indirect ELISAusing various truncation mutants of VCP (CCP 1–3, CCP 2–4, CCP 1–2, CCP 2–3 and CCP3–4) that were expressed earlier in our laboratory using the Pichia expression system [42].These expressed mutants were designed in such a way that they started with the first Cys ofthe domain of interest and ended with the last residue of the inter-domain linker. Thus, thisdesign kept the entire linker region at the C-terminal side of each of the mutants. The mAbs67.5 and 67.9 reacted with CCP 1–3, CCP 2–4, CCP 2–3 and CCP 3–4 mutants (Fig. 2A andB) indicating that they recognize either domain 3 or the linker between domains 3 and 4(Fig. 2F). The antibodies 67.11 and 67.13 on the other hand reacted only with truncationmutants CCP 2–4 and CCP 3–4 (Fig. 2C and D). Since the latter two antibodies did not

Bernet et al.


Sponsored Docum

ent Sponsored D

ocument

Sponsored Docum

ent

show binding to CCP 1–3, CCP 1–2 or CCP 2–3 it indicates that the binding epitopes forthese antibodies lie on CCP domain 4 (Fig. 2F).

3.3 Inhibition of C3b and C4b cofactor activities of VCP by anti-VCP monoclonalantibodies

One of the functions of VCP is to serve as a cofactor for the complement specific serineprotease factor I to mediate the inactivation of C3b (composed of α′ and β chains) and C4b(composed of α′, β and γ chains), the non-catalytic subunits of C3-convertases, which are thekey enzymes in activation of the complement cascades. This function results in the cleavageof the α′-chains of C3b and C4b leading to the generation of their inactivated forms (iC3b orC4c and C4d) which can no longer participate in the formation of C3-convertases. Asexpected, incubation of rVCP or human factor H (control) with C3b and factor I resulted incleavage of α′-chain of C3b (Fig. 3A). Similarly, incubation of rVCP or human sCR1(control) with C4b and factor I resulted in cleavage of α′-chain of C4b (Fig. 3B). WhenmAbs were analyzed for their ability to inhibit the C3b cofactor activity of VCP, only 67.5and 67.9 showed inhibition; neither 67.11 nor 67.13 could inhibit this activity (Fig. 3A).Essentially similar results were obtained for inhibition of C4b cofactor activity by themonoclonal antibodies. Only 67.5 and 67.9 showed inhibition, while 67.11 and 67.13 failedto inhibit the C4b cofactor activity (Fig. 3B). These data therefore revealed that CCP domain3 and/or linker between CCPs 3 and 4 of VCP play an essential role in imparting thecofactor activities.

3.4 Inhibition of decay-accelerating activity of VCP by anti-VCP monoclonal antibodiesBesides acting as a cofactor for C3b and C4b inactivation, VCP is also an efficient decayaccelerator of the classical/lectin pathway C3-convertase C4b,2a. Thus, to examine theeffect of mAbs on VCP-mediated decay of the convertase, we utilized a hemolytic assay. Inthis assay, C4b,2a was formed on antibody sensitized sheep erythrocytes using purifiedcomplement components and then the enzyme was allowed to decay in the presence ofrVCP or rVCP pre-incubated with each of the mAbs. The activity of the remaining enzymewas assayed by adding EDTA-sera (a source of C3-C9) and measuring hemolysis.Interestingly, the antibodies that inhibited the C3b and C4b cofactor activities (67.5 and67.9) also inhibited the decay-accelerating activity of VCP, albeit with 67.5 having muchless effect compared to 67.9. Among the remaining two antibodies 67.11 and 67.13, whichbound to CCP 4 domain, only the former had moderate inhibitory activity while the latterdid not inhibit the decay activity. The C3-convertase decay inhibition efficiency of themonoclonals followed the order 67.9 ≈ 67.11 > 67.5 with 67.13 having negligible inhibitorypotential (Fig. 4).

3.5 Blocking VCP function using monoclonal antibodies reduces VACV pathogenicity inrabbits

Since mAbs differentially inhibited the VCP functions it was intriguing to know if blockingVCP function in vivo with these mAbs would translate into differences in viralpathogenesis.

For in vivo disabling of VCP using mAbs, a prerequisite is that they should be retained atthe site of injection until VCP is secreted by the infected cells. To verify this, we determinedtheir half-life. The mAbs (67.5 and 67.9) were labeled with 131I, injected intradermally oneither flanks of New Zealand White rabbits and imaging was carried out with a γ-raycamera. The results showed that the labeled antibodies were retained at the site of injectioneven after 72 h. The half-life was found to be 8 h for both the antibodies (Fig. 5; data notshown for 67.9).

Bernet et al.


Sponsored Docum

ent Sponsored D

ocument

Sponsored Docum

ent

Next, in order to determine whether disabling of VCP using neutralizing mAb affectsVACV pathogenicity, we used a rabbit skin lesion model. In these experiments, VACV-WRwas injected intradermally (104 pfu) either alone or in combination with mAbs and thelesion size was measured over a period of time. Initially, the two blocking antibodies (67.5and 67.9) were titrated with VACV-WR to identify the optimal concentration required forreduction in lesion response. When varying concentrations of 67.5 (Fig. 6A) or 67.9 (datanot shown) were mixed with VACV-WR and injected in rabbits they showed a dosedependent inhibition of lesion formation. Maximum decrease in the lesion size was observedat 25 μg mAb concentration. We then performed experiments with all the four mAbs using afixed concentration (25 μg). There was a significant difference in the lesion size where 67.5or 67.9 was injected along with VACV-WR (Fig. 6B). Moderate decrease in the lesiondevelopment was also observed where 67.11 was injected, but 67.13 showed a negligibleeffect on the lesion development. These data therefore suggested that in vivo inhibition ofcomplement regulatory activities of VCP by neutralizing mAbs result in reduction in VACVpathogenesis.

3.6 Neutralizing monoclonal antibodies do not reduce VACV pathogenicity incomplement-depleted rabbits

Although the above results suggested that blocking of complement regulatory activities ofVCP by mAbs resulted in neutralization of virus and in turn its pathogenicity, it still did notprovide direct evidence of a role of host complement. Consequently, we performed similarexperiments in two complement-depleted animals.

Complement depletion in rabbits was achieved by injecting CVF. A bolus of 100 U/kgadministered through the ear vein completely depleted complement in rabbits in 4 h and thedepleted state was maintained till day 5, after which there was a gradual restoration ofcomplement activity (data not shown). Because it took approximately 4 h to completelydeplete complement in rabbits, in these experiments, we injected CVF 6 h prior to thechallenge in duplicate with VACV-WR or VACV-WR along with mAb in the back of eachrabbit. It is clear from our data that intradermal injection of VACV-WR (104 pfu) with orwithout mAb (25 μg of each) led to the formation of more similar sized lesions (Fig. 6C). Itcould therefore be suggested that inhibition of VCP-mediated regulation of host complementby neutralizing antibodies result in neutralization of VACV in a host complement dependentmanner.

4 DiscussionVCP is one of the most extensively studied pox viral RCA homologs [4,54,55]. It is nowclear that it possesses the ability to regulate the complement system in the fluid phase aswell as on the surface of the infected cells by binding to heparan sulfate proteoglycans [56]and the viral protein A56 [35]. Further, it has also been established that its deletion causesattenuation of VACV lesion and increase in specific inflammatory responses in mice[36,38]. However, the in vivo role of its complement interacting domains and importance ofits various inhibitory activities with relevance to in vivo pathogenesis is still not understood.In the present study, we have raised neutralizing mAbs against VCP, mapped the domainsthey recognize and utilized them to address the in vivo relevance of different functionalactivities of VCP in VACV pathogenesis.

Prior to this study, mAbs against VCP have been generated by Isaacs et al. [45] andLiszewski et al. [57]. The former study by Isaacs et al. [45] showed that mAbs directedagainst CCP domains 2 and 4 inhibit VCPs interaction with its target proteins C3b and C4band thereby its activity. And later Liszewski et al. [57] demonstrated that mAb thatrecognizes the linker between CCP domains 1 and 2 inhibit the cofactor as well as decay-

Bernet et al.


Sponsored Docum

ent Sponsored D

ocument

Sponsored Docum

ent

accelerating activity of VCP. Although these studies established the importance of CCPdomains 2 and 4 and the linker between domains 1 and 2 in VCPs target recognition andfunctional activities, no attempts were made in these studies to utilize the antibodies todissect the in vivo importance of complement regulatory activities of VCP in VACVvirulence.

In the present study, we have characterized four mAbs of which two (67.5 and 67.9)recognized domain 3 or the linker between domains 3 and 4, and the other two (67.11 and67.13) recognized domain 4. Of these four antibodies, 67.5, 67.9 and 67.11 inhibited thecomplement regulatory activities of VCP (Figs. 3 and 4) suggesting that domains 3 and 4 arecritical for the VCP function. This however is not surprising as domain mapping employingchimeric mutants, truncation mutants and mAbs indicated that all the four domains of VCPare important for its interaction with C3b and C4b [42–45]. In addition, we now also knowthat CCP domains 2 and 3 provide a docking surface for factor I and thus are critical for thecofactor activity, and CCP domain 1 is essential for displacement of C2a from the C3-convertase C4b,2a (decay activity) [46]. In light of these data on domain requirements inVCP for its functional activities, it is likely that the mAbs 67.5 and 67.9 exert their effect byinhibiting the interaction of VCP with C3b/C4b and/or factor I and mAb 67.11 exercises itseffect by inhibiting the interaction of VCP with C3b/C4b.

The mAbs characterized here displayed differential effect on the cofactor and decayactivities of VCP. The mAb 67.5 primarily inhibited the cofactor activity, 67.9 inhibitedboth the cofactor activity and the decay-accelerating activity, 67.11 inhibited only decay-accelerating activity and 67.13 did not inhibit any of the activities (Figs. 3 and 4). Hence,these were suitable to gain insight into the role of these activities of VCP in VACVpathogenesis. Here we employed the rabbit intradermal model to study the effect of theseneutralizing antibodies on VACV pathogenesis [36]. Injection of mAbs 67.5 and 67.9 alongwith VACV showed significant reduction in the lesion size when compared to the lesionsformed by VACV alone or VACV injected with the control antibody (67.13) (Fig. 6A andB), indicating that like deletion of VCP from VACV [38,46], disabling of VCP functionsalso leads to attenuation of VACV lesions. Interestingly, mAb 67.11 that inhibited only thedecay-accelerating activity of VCP had no significant effect on the lesion formationsuggesting thereby that the cofactor and not the decay-accelerating activity plays a majorrole in contributing to virulence (Fig. 6B). Nonetheless, there are a few caveats. The affinityof 67.11 for VCP is about 10-fold less compared to 67.9 (Fig. 1 and Table 1) and therefore itis also likely that the observed difference in lesion formation is due to the variation in theaffinities of these two antibodies for VCP. Interpretation of the viral pathogenicity dataemploying mAb 67.11 is further complicated by the fact that even in the in vitro DAA assay,its effect was a relatively modest one. A closer look at the data however show that mAb67.5, despite having comparatively poor DAA inhibitory activity than 67.9, is equallyefficient in inhibiting the lesion formation. Moreover, both these antibodies display similaraffinities for VCP (Fig. 1 and Table 1). Thus, it is likely that cofactor activity is a majorcontributor to VCP-mediated enhancement of VACV pathogenesis.

Monkeypox virus strains like Central African (Congo basin) strain encodes a complementregulator (ORF D14L), while the West African strain lacks this protein [28]. A comparisonof the West African strain versus the Congo basin strain shows the latter to be more virulentthan the former [28] suggesting that complement evasion may play a role in poxviruspathogenesis. Intriguingly, the Congo basin strain encodes a protein (MOPICE) that containsonly cofactor activity and lack the decay activity [21]. Thus, these observations also providesupport to our proposal that the cofactor activity of VCP could play a major role in thepoxvirus pathogenesis.

Bernet et al.


Sponsored Docum

ent Sponsored D

ocument

Sponsored Docum

ent

Although smallpox has been eradicated from the globe, the recent upsurge in terrorism hasincreased the concern of usage of variola virus as a biological weapon and has resulted indevelopment of new generation vaccines [11]. The important question therefore is shouldVCP be present in the new generation vaccine vectors? We show here that disabling ofcomplement regulatory activities of VCP by neutralizing mAbs result in reduction of VACVlesion size in rabbits and this reduction is dependent on the presence of host complement(Fig. 6). Although our study does not define the mechanism responsible for this, clearlythere are two possibilities: (i) the lack of VCP-mediated host complement regulation couldresult in complement-mediated neutralization of VACV in the presence of antibodies thatare produced against VACV during the infection and (ii) the lack of VCP-mediatedcomplement regulation could enhance the protective immune response against VACV. Boththese probabilities have been established by earlier studies. In support of the first possibility,in vitro studies have shown that complement has the ability to neutralize both MV [36,37]and EV [37] in the presence of anti-VACV antibodies. And an in vivo study hasdemonstrated complement to be protective against poxvirus infection [58]. Similarly, insupport of the second alternative, a recent study has elegantly shown that infection of VCP-null VACV in mice results in enhanced T cells at the site of infection, enhanced neutralizingantibody responses and reduced viral titers [38]. Thus, our study along with these studiessuggests that deletion of VCP from the new generation vaccine vectors would result in moresafe and effective vaccine vectors against smallpox.

ReferencesFinlay B.B. McFadden G. Anti-immunology: evasion of the host immune system by bacterial and viral

pathogens. Cell. 2006; 124(4):767–782. [PubMed: 16497587]Ram S. Cullinane M. Blom A.M. Gulati S. McQuillen D.P. Monks B.G. Binding of C4b-binding

protein to porin: a molecular mechanism of serum resistance of Neisseria gonorrhoeae. J Exp Med.2001; 193(3):281–295. [PubMed: 11157049]

Lee S.H. Jung J.U. Means R.E. ‘Complementing’ viral infection: mechanisms for evading innateimmunity. Trends Microbiol. 2003; 11(10):449–452. [PubMed: 14557025]

Lambris J.D. Ricklin D. Geisbrecht B.V. Complement evasion by human pathogens. Nat RevMicrobiol. 2008; 6(2):132–142. [PubMed: 18197169]

Volanakis, J.E.; Frank, M.M. Marcel Dekker, Inc.; New York: 1998.Strainic M.G. Liu J. Huang D. An F. Lalli P.N. Muqim N. Locally produced complement fragments

C5a and C3a provide both costimulatory and survival signals to naive CD4+ T cells. Immunity.2008; 28(3):425–435. [PubMed: 18328742]

Carroll M.C. The complement system in regulation of adaptive immunity. Nat Immunol. 2004; 5(10):981–986. [PubMed: 15454921]

Pyaram K. Yadav V.N. Reza M.J. Sahu A. Virus–complement interactions: an assiduous struggle fordominance. Future Virol. 2010; 5(6):709–730.

Weaver J.R. Isaacs S.N. Monkeypox virus and insights into its immunomodulatory proteins. ImmunolRev. 2008; 225:96–113. [PubMed: 18837778]

Vorou R.M. Papavassiliou V.G. Pierroutsakos I.N. Cowpox virus infection: an emerging health threat.Curr Opin Infect Dis. 2008; 21(2):153–156. [PubMed: 18317038]

Moss B. Smallpox vaccines: targets of protective immunity. Immunol Rev. 2011; 239(1):8–26.[PubMed: 21198662]

Lustig S. Fogg C. Whitbeck J.C. Moss B. Synergistic neutralizing activities of antibodies to outermembrane proteins of the two infectious forms of vaccinia virus in the presence of complement.Virology. 2004; 328(1):30–35. [PubMed: 15380355]

Benhnia M.R. McCausland M.M. Moyron J. Laudenslager J. Granger S. Rickert S. Vaccinia virusextracellular enveloped virion neutralization in vitro and protection in vivo depend oncomplement. J Virol. 2009; 83(3):1201–1215. [PubMed: 19019965]

Bernet et al.


Sponsored Docum

ent Sponsored D

ocument

Sponsored Docum

ent

Cooper N.R. Nemerow G.R. Complement, viruses, and virus-infected cells. Springer SeminImmunopathol. 1983; 6(4):327–347. [PubMed: 6364429]

Mullick J. Kadam A. Sahu A. Herpes and pox viral complement control proteins: ‘the mask of self’.Trends Immunol. 2003; 24(9):500–507. [PubMed: 12967674]

Stoiber H. Pruenster M. Ammann C.G. Dierich M.P. Complement-opsonized HIV: the free rider on itsway to infection. Mol Immunol. 2005; 42(2):153–160. [PubMed: 15488605]

Judson K.A. Lubinski J.M. Jiang M. Chang Y. Eisenberg R.J. Cohen G.H. Blocking immune evasionas a novel approach for prevention and treatment of herpes simplex virus infection. J Virol. 2003;77(23):12639–12645. [PubMed: 14610186]

Smith G.L. Symons J.A. Khanna A. Vanderplasschen A. Alcami A. Vaccinia virus immune evasion.Immunol Rev. 1997; 159:137–154. [PubMed: 9416508]

Lachmann P.J. Microbial subversion of the immune response. Proc Natl Acad Sci U S A. 2002;99(13):8461–8462. [PubMed: 12077314]

Ahmad M. Pyaram K. Mullick J. Sahu A. Viral complement regulators: the expert mimickingswindlers. Indian J Biochem Biophys. 2007; 44(5):331–343. [PubMed: 18341208]

Liszewski M.K. Leung M.K. Hauhart R. Buller R.M. Bertram P. Wang X. Structure and regulatoryprofile of the monkeypox inhibitor of complement: comparison to homologs in vaccinia andvariola and evidence for dimer formation. J Immunol. 2006; 176(6):3725–3734. [PubMed:16517741]

Moss, B. Fields virology. 4th ed.. Knipe, D.M.; Howley, P.M.; Griffin, D.E., editors. LippincottWilliams and Wilkins; Philadelphia: 2001. p. 2849-2883.

McFadden G. Poxvirus tropism. Nat Rev Microbiol. 2005; 3(3):201–213. [PubMed: 15738948]Kotwal G.J. Isaacs S.N. Mckenzie R. Frank M.M. Moss B. Inhibition of the complement cascade by

the major secretory protein of vaccinia virus. Science. 1990; 250(4982):827–830. [PubMed:2237434]

Rosengard A.M. Liu Y. Nie Z. Jimenez R. Variola virus immune evasion design: expression of ahighly efficient inhibitor of human complement. Proc Natl Acad Sci U S A. 2002; 99(13):8808–8813. [PubMed: 12034872]

Yadav V.N. Pyaram K. Mullick J. Sahu A. Identification of hot spots in the variola virus complementinhibitor (SPICE) for human complement regulation. J Virol. 2008; 82(7):3283–3294. [PubMed:18216095]

Miller C.G. Shchelkunov S.N. Kotwal G.J. The cowpox virus-encoded homolog of the vaccinia viruscomplement control protein is an inflammation modulatory protein. Virology. 1997; 229(1):126–133. [PubMed: 9123854]

Chen N. Li G. Liszewski M.K. Atkinson J.P. Jahrling P.B. Feng Z. Virulence differences betweenmonkeypox virus isolates from West Africa and the Congo basin. Virology. 2005; 340(1):46–63.[PubMed: 16023693]

Chen N. Danila M.I. Feng Z. Buller R.M. Wang C. Han X. The genomic sequence of ectromelia virus,the causative agent of mousepox. Virology. 2003; 317(1):165–186. [PubMed: 14675635]

Mukinda V.B. Mwema G. Kilundu M. Heymann D.L. Khan A.S. Esposito J.J. Re-emergence ofhuman monkeypox in Zaire in 1996. Monkeypox Epidemiologic Working Group. Lancet. 1997;349(9063):1449–1450. [PubMed: 9164323]

Uvarova E.A. Shchelkunov S.N. Species-specific differences in the structure of orthopoxviruscomplement-binding protein. Virus Res. 2001; 81(1–2):39–45. [PubMed: 11682123]

Kotwal G.J. Moss B. Vaccinia virus encodes a secretory polypeptide structurally related tocomplement control proteins. Nature. 1988; 335(6186):176–178. [PubMed: 3412473]

Henderson C.E. Bromek K. Mullin N.P. Smith B.O. Uhrin D. Barlow P.N. Solution structure anddynamics of the central CCP module pair of a poxvirus complement control protein. J Mol Biol.2001; 307(1):323–339. [PubMed: 11243823]

Murthy K.H. Smith S.A. Ganesh V.K. Judge K.W. Mullin N. Barlow P.N. Crystal structure of acomplement control protein that regulates both pathways of complement activation and bindsheparan sulfate proteoglycans. Cell. 2001; 104(2):301–311. [PubMed: 11207370]

Girgis N.M. Dehaven B.C. Fan X. Viner K.M. Shamim M. Isaacs S.N. Cell surface expression of thevaccinia virus complement control protein is mediated by interaction with the viral A56 protein

Bernet et al.


Sponsored Docum

ent Sponsored D

ocument

Sponsored Docum

ent

and protects infected cells from complement attack. J Virol. 2008; 82(9):4205–4214. [PubMed:18287241]

Isaacs S.N. Kotwal G.J. Moss B. Vaccinia virus complement-control protein prevents antibody-dependent complement-enhanced neutralization of infectivity and contributes to virulence. ProcNatl Acad Sci U S A. 1992; 89(2):628–632. [PubMed: 1731333]

Vanderplasschen A. Mathew E. Hollinshead M. Sim R.B. Smith G.L. Extracellular enveloped vacciniavirus is resistant to complement because of incorporation of host complement control proteins intoits envelope. Proc Natl Acad Sci U S A. 1998; 95(13):7544–7549. [PubMed: 9636186]

Girgis N.M. Dehaven B.C. Xiao Y. Alexander E. Viner K.M. Isaacs S.N. The vaccinia viruscomplement control protein modulates adaptive immune responses during infection. J Virol. 2010;85(6):2547–2556. [PubMed: 21191012]

Mckenzie R. Kotwal G.J. Moss B. Hammer C.H. Frank M.M. Regulation of complement activity byvaccinia virus complement-control protein. J Infect Dis. 1992; 166(6):1245–1250. [PubMed:1431243]

Bernet J. Mullick J. Panse Y. Parab P.B. Sahu A. Kinetic analysis of the interactions between vacciniavirus complement control protein and human complement proteins C3b and C4b. J Virol. 2004;78(17):9446–9457. [PubMed: 15308738]

Sahu A. Isaacs S.N. Soulika A.M. Lambris J.D. Interaction of vaccinia virus complement controlprotein with human complement proteins: factor I-mediated degradation of C3b to iC3b1inactivates the alternative complement pathway. J Immunol. 1998; 160(11):5596–5604. [PubMed:9605165]

Mullick J. Bernet J. Panse Y. Hallihosur S. Singh A.K. Sahu A. Identification of complementregulatory domains in vaccinia virus complement control protein. J Virol. 2005; 79(19):12382–12393. [PubMed: 16160165]

Rosengard A.M. Alonso L.C. Korb L.C. Baldwin W.M. III, Sanfilippo F. Turka L.A. Functionalcharacterization of soluble and membrane-bound forms of vaccinia virus complement controlprotein (VCP). Mol Immunol. 1999; 36(10):685–697. [PubMed: 10509819]

Smith S.A. Sreenivasan R. Krishnasamy G. Judge K.W. Murthy K.H. Arjunwadkar S.J. Mapping ofregions within the vaccinia virus complement control protein involved in dose-dependent bindingto key complement components and heparin using surface plasmon resonance. Biochim BiophysActa. 2003; 1650(1–2):30–39. [PubMed: 12922167]

Isaacs S.N. Argyropoulos E. Sfyroera G. Mohammad S. Lambris J.D. Restoration of complement-enhanced neutralization of vaccinia virus virions by novel monoclonal antibodies raised againstthe vaccinia virus complement control protein. J Virol. 2003; 77(15):8256–8262. [PubMed:12857894]

Ahmad M. Raut S. Pyaram K. Kamble A. Mullick J. Sahu A. Domain swapping reveals complementcontrol protein modules critical for imparting cofactor and decay-accelerating activities in vacciniavirus complement control protein. J Immunol. 2010; 185(10):6128–6137. [PubMed: 20956343]

Rawal N. Pangburn M.K. Functional role of the noncatalytic subunit of complement C5 convertase. JImmunol. 2000; 164(3):1379–1385. [PubMed: 10640753]

Galfre G. Milstein C. Preparation of monoclonal antibodies: strategies and procedures. MethodsEnzymol. 1981; 73(Pt. B):3–46. [PubMed: 7300683]

Prasad D.V. Parekh V.V. Joshi B.N. Banerjee P.P. Parab P.B. Chattopadhyay S. The Th1-specificcostimulatory molecule, m150, is a posttranslational isoform of lysosome-associated membraneprotein-1. J Immunol. 2002; 169(4):1801–1809. [PubMed: 12165502]

Pan Q. Ebanks R.O. Isenman D.E. Two clusters of acidic amino acids near the NH2 terminus ofcomplement component C4 alpha′-chain are important for C2 binding. J Immunol. 2000; 165(5):2518–2527. [PubMed: 10946278]

Krishnan I.S. Hansen H.J. Gold D.V. Goldenberg D.M. Leung S.O. Co-secretion of two distinct kappalight chains by the mu-9 hybridoma. Hybridoma. 1999; 18(4):325–333. [PubMed: 10571262]

Zack D.J. Wong A.L. Stempniak M. Weisbart R.H. Two kappa immunoglobulin light chains aresecreted by an anti-DNA hybridoma: implications for isotypic exclusion. Mol Immunol. 1995;32(17–18):1345–1353. [PubMed: 8643104]

Bernet et al.


Sponsored Docum

ent Sponsored D

ocument

Sponsored Docum

ent

Cherepakhin V.V. Petrosian M.N. Ibragimov A.R. Bogacheva G.T. Trakht I.N. Allelic exclusion inhybridomas. I. Isolation and properties of a hybrid clone producing two allelic variants of lightchain immunoglobulins in the rat. Mol Biol (Mosk). 1984; 18(2):474–480. [PubMed: 6425644]

Bernet J. Mullick J. Singh A.K. Sahu A. Viral mimicry of the complement system. J Biosci. 2003;28(3):249–264. [PubMed: 12734404]

Stoermer K.A. Morrison T.E. Complement and viral pathogenesis. Virology. 2011; 411(2):362–373.[PubMed: 21292294]

Liszewski M.K. Bertram P. Leung M.K. Hauhart R. Zhang L. Atkinson J.P. Smallpox inhibitor ofcomplement enzymes (SPICE): regulation of complement activation on cells and mechanism of itscellular attachment. J Immunol. 2008; 181(6):4199–4207. [PubMed: 18768877]

Liszewski M.K. Leung M.K. Hauhart R. Fang C.J. Bertram P. Atkinson J.P. Smallpox inhibitor ofcomplement enzymes (SPICE): dissecting functional sites and abrogating activity. J Immunol.2009; 183(5):3150–3159. [PubMed: 19667083]

Moulton E.A. Atkinson J.P. Buller R.M. Surviving mousepox infection requires the complementsystem. PLoS Pathog. 2008; 4(12):e1000249. [PubMed: 19112490]

Appendix A Supplementary dataRefer to Web version on PubMed Central for supplementary material.

AcknowledgmentsWe thank Dr. Sekhar Chakrabarti for providing the vaccinia virus (WR) strain, Dr. M.G.R. Rajan and Dr. P.R.Chaudhary for help with Gamma Ray Imaging, Dr. Ramanamurthy and Dr. Kohale for help with the in vivoexperiments, Dr. A.C. Mishra (Director, National Institute of Virology, Pune), Dr. C. G. Raut and Dr. D. Mitra forallowing to use their facilities for virus culture and in vivo experimentations. We remember with gratitude theexcellent technical assistance provided by Late Mr. Anand Bidlan.

Contributors: J.B. generated and characterized the mAbs, and performed pathogenesis experiments; M.A.performed the cofactor assays and lectin blot; J.M. and Y.P. constructed, expressed and purified the VCP truncationmutants; A.K.S. performed the decay-acceleration assay; P.B.P. supervised the mAb generation andcharacterization; A.S. conceived and supervised the entire work; and J.B. and A.S. wrote the manuscript. Conflict ofinterest statement: The authors have no financial conflicts of interest. Funding: This work was supported by theWellcome Trust Overseas Senior Research Fellowship and a project grant from the Department of Biotechnology,India to A.S. The authors also acknowledge the financial assistance to M.A. by the University Grant Commission,New Delhi.

Bernet et al.


Sponsored Docum

ent Sponsored D

ocument

Sponsored Docum

ent

Fig. 1.Surface plasmon resonance analysis of binding of anti-VCP monoclonal antibodies to VCP.Sensogram overlay plots of the binding of anti-VCP mAbs 67.5, 67.9, 67.11 and 67.13 toVCP. The anti-VCP mAbs were immobilized on the CM-5 sensor chips (Biacore AB) usingamine-coupling chemistry and various concentrations of VCP (indicated on right hand sideof each panel) were flown over the chip in PBS containing 0.05% Tween 20 to measurebinding. The solid black lines represent the best fit data to 1:1 Langmuir binding model(A + B ↔ AB; BIAevaluation 4.1).

Bernet et al.


Sponsored Docum

ent Sponsored D

ocument

Sponsored Docum

ent

Fig. 2.Mapping of VCP domains recognized by anti-VCP monoclonal antibodies. Identification ofVCP domains recognized by the mAb was performed by examining the binding of the mAbsto various VCP truncation mutants by ELISA. The filled bars represent binding of the mAbsto the control (BSA), while the empty bars represent binding of the mAbs to the indicatedVCP truncation mutant. Panels (A, B, C, and D) depict binding of mAbs 67.5, 67.9, 67.11and 67.13, respectively, and panel (E) represent binding of VCP to rabbit polyclonalantibody (control). The schematic summary of VCP domains recognized by the variousmAbs is presented in panel (F).

Bernet et al.


Sponsored Docum

ent Sponsored D

ocument

Sponsored Docum

ent

Fig. 3.Inhibition of factor I cofactor activity of VCP for C3b and C4b by anti-VCP monoclonalantibodies. (A) Inhibition of C3b cofactor activity. C3b (3 μg) was incubated with VCP(0.5 μg), factor H (1 μg; fH; control) or VCP (0.5 μg) pre-incubated with each of the mAbs(where indicated) and factor I (100 ng) at 37 °C for 2 h. The reactions were stopped byadding SDS-PAGE sample buffer containing DTT and ran on SDS-PAGE gels. (B)Inhibition of C4b cofactor activity. C4b (3 μg) was incubated with VCP (0.5 μg), solublecomplement receptor 1 (1 μg; sCR1; control), or VCP (0.5 μg) pre-incubated with each ofthe mAbs (where indicated) and factor I (100 ng) at 37 °C for 2 h. The reactions werestopped by adding SDS-PAGE sample buffer containing DTT and ran on SDS-PAGE gels.The gels were stained with Coomassie blue to visualize inhibition of α′-chain cleavage,which indicates inhibition of C3b or C4b cofactor activity.

Bernet et al.


Sponsored Docum

ent Sponsored D

ocument

Sponsored Docum

ent

Fig. 4.Inhibition of classical pathway decay-accelerating activity of VCP by anti-VCP monoclonalantibodies. The classical pathway (CP) C3-convertase was formed on sheep erythrocytesand allowed to decay in the presence of VCP (0.2 μM) or VCP (0.2 μM) and antibody(1 μM). The remaining C3-convertase activity was detected by adding EDTA-sera (a sourceof C3-C9). The percentage of CP C3-convertase activity was normalized by considering thepercentage of lysis in the absence of VCP as 100%. The dotted line was used as a cut off fordetermining the inhibition of VCP mediated decay of C3-convertase by mAb. NA indicatescontrols where no antibody was added.

Bernet et al.


Sponsored Docum

ent Sponsored D

ocument

Sponsored Docum

ent

Fig. 5.Half-life of anti-VCP mAb in rabbit. Anti-VCP mAb 67.5 was labeled with 131I and injectedintradermally at two sites in the back of New Zealand White rabbit (NZW) followed byGamma-ray imaging. The emitted radiation from the sedated animal was imaged with anELGEMS Millennium MPS gamma ray camera (GE, USA) using a medium energycollimator. The figure shows images of the residence of the labeled mAb at the indicatedtime points and the graph represents percentage of counts at the indicated time period. Thedata was normalized by setting 100% equal to the total counts present at 0 time point.

Bernet et al.


Sponsored Docum

ent Sponsored D

ocument

Sponsored Docum

ent

Fig. 6.Monoclonal antibody effect in vivo in a rabbit skin lesion VACV-WR challenge model. (A)Skin lesions developed after intradermal injection of 104 pfu of VACV-WR (extreme rightlesions marked as C) or 104 pfu of virus mixed with graded concentrations of mAb 67.5(25 μg, 12.5 μg, 6.3 μg and 3 μg, first four lesions from left). The image was taken 8 dayspost infection. (B) Skin lesion size after intradermal injection of 104 pfu of VACV-WRalone or in combination with indicated antibodies (25 μg) in rabbits. (C) Skin lesion sizeafter intradermal injection of 104 pfu of VACV-WR alone or mixed with 25 μg of theindicated antibodies in rabbits depleted of complement by injecting cobra venom factor 6 hprior to infection. Data points presented in panels (B and C) represent mean of fourreplicates performed in two rabbits. Statistical comparisons were performed between mAbtreated lesions and untreated lesions. *p < 0.01 and **p < 0.001 (Mann–Whitney Rank Sumtest, SigmaStat). Differences are also significant between 67.13 treated lesions and67.5/67.11 treated lesions between days 5 and 11 (p ≤ 0.001).

Bernet et al.


Sponsored Docum

ent Sponsored D

ocument

Sponsored Docum

ent

Sponsored Docum

ent Sponsored D

ocument

Sponsored Docum

ent

Bernet et al.

Table 1

Kinetic and affinity data for the interactions of monoclonal antibodies with VCP.a

Ligand Analyte kd (1/s)/ka (1/M s) SE (kd/ka) KD (M) χ2

mAb 67.5 VCP 0.0164/3.07 × 106 9.52 × 10−5/1.81 × 104 5.35 × 10−9 1.1b

mAb 67.9 VCP 0.0171/2.59 × 106 9.96 × 10−5/1.51 × 104 6.6 × 10−9 1.01b

mAb 67.11 VCP 0.034/7.32 × 105 1.73 × 10−4/7.32 × 105 4.64 × 10−8 0.468b

mAb 67.13 VCP 0.0418/1.8 × 105 5.22 × 10−4/3.94 × 103 2.32 × 10−7 0.218b

aka, association rate constant; kd, dissociation rate constant; KD, equilibrium rate constant; SE; standard error.

bData were calculated by global fitting to a 1:1 Langmuir binding model (BIAevaluation 4.1).


disabling complement regulatory activities of vaccinia virus complement control protein reduces...

Documents