JOURNAL OF VIROLOGY, Feb. 2011, p. 1820–1833 Vol. 85, No. 40022-538X/11/$12.00 doi:10.1128/JVI.02127-10Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Capturing the Natural Diversity of the Human Antibody Responseagainst Vaccinia Virus�

Johan Lantto,1* Margit Haahr Hansen,1 Søren Kofoed Rasmussen,1 Lucilla Steinaa,1†Tine R. Poulsen,1 Jackie Duggan,2 Mike Dennis,2 Irene Naylor,2 Linda Easterbrook,2

Søren Bregenholt,1 John Haurum,1‡ and Allan Jensen1

Symphogen A/S, Lyngby, Denmark,1 and Health Protection Agency, Porton Down, Wiltshire, United Kingdom2

Received 8 October 2010/Accepted 24 November 2010

The eradication of smallpox (variola) and the subsequent cessation of routine vaccination have leftmodern society vulnerable to bioterrorism employing this devastating contagious disease. The existing,licensed vaccines based on live vaccinia virus (VACV) are contraindicated for a substantial number ofpeople, and prophylactic vaccination of large populations is not reasonable when there is little risk ofexposure. Consequently, there is an emerging need to develop efficient and safe therapeutics to be usedshortly before or after exposure, either alone or in combination with vaccination. We have characterizedthe human antibody response to smallpox vaccine (VACV Lister) in immunized volunteers and isolated alarge number of VACV-specific antibodies that recognize a variety of different VACV antigens. Using thisbroad antibody panel, we have generated a fully human, recombinant analogue to plasma-derived vacciniaimmunoglobulin (VIG), which mirrors the diversity and specificity of the human antibody immuneresponse and offers the advantage of unlimited supply and reproducible specificity and activity. Therecombinant VIG was found to display a high specific binding activity toward VACV antigens, potent invitro VACV neutralizing activity, and a highly protective efficacy against VACV challenge in the mouse taillesion model when given either prophylactically or therapeutically. Altogether, the results suggest that thiscompound has the potential to be used as an effective postexposure prophylaxis or treatment of diseasecaused by orthopoxviruses.

Although smallpox (variola) was eradicated over 30 yearsago by a worldwide vaccination campaign, the threat of bio-terrorism has reintroduced this deadly and highly contagiousdisease as a serious hazard to public health and notably totoday’s unvaccinated inhabitants of crowded urban settings.Prophylactic vaccination employing the smallpox-related vac-cinia virus (VACV) is associated with rare, but potentiallylife-threatening, adverse events (27), and unfortunately vacci-nation is contraindicated for people (and their household con-tacts) with compromised immune systems or skin conditions(such as eczema, dermatitis, and varicella), pregnant women,infants, and those receiving immunosuppressive medicines.Complications due to VACV vaccination may be treated withplasma-derived vaccinia immunoglobulin (VIG) isolated fromvaccinated donors. However, since only a small fraction of theinjected immunoglobulin targets the antigens of interest, largeinjection volumes are required, and it is therefore probably notrealistic to use plasma-derived VIG in treating a generalizedsmallpox outbreak. Furthermore, since prophylactic vaccina-tion of large populations is not reasonable when there is littlerisk of exposure, the urgent concerns over the implications ofan accidental or intentional release of smallpox and also thepossible outbreak of zoonotic poxvirus diseases such as mon-keypox have led to a renewed interest in investigating antiviral

treatment options and in understanding the humoral immuneresponse to virus exposure.

Variola virus, which is the causative agent of smallpox,VACV, and monkeypox virus all belong to the Orthopoxvirusgenus of the Poxviridae family. Characteristically, these virusesare large (approximately 200-kb genome) and have a complexmode of assembly and appearance, including multiple viralmembranes and surface proteins with various functions. Inaddition, orthopoxviruses have two types of infectious virions:intracellular mature virions (IMV) and extracellular envelopedvirions (EEV). The IMV are assembled in the cytoplasm andconsist of a virally encoded membrane surrounding a coreparticle containing the genome. The IMV can either be re-leased from the infected cell by cellular lysis or be furtherprocessed by wrapping of virus particles in a host-derivedmembrane to generate EEV. Each type of virions has distinctfunctions, with IMV being involved in transmission betweenhosts and EEV thought to be primarily involved in dissemina-tion within the host (54). Neutralizing antibodies mainly exerttheir effect by recognizing surface proteins expressed on theouter virion membranes. These proteins are unique to eitherIMV or EEV, and the two virion types thus present differentsets of targets to the humoral defense (15, 54). When thepresent study was initiated in 2004, animal studies had identi-fied neutralizing antibodies against five VACV IMV-specificantigens (L1R, A27L, A17L, H3L, and D8L) and two EEV-specific antigens (B5R and A33R) (reviewed in reference 2).Although the exact biological function of these proteins re-mains unclear, essential functions during virion assembly andvirus entry have been assigned to specific proteins (15, 54).

* Corresponding author. Mailing address: Symphogen A/S, Elek-trovej, Building 375, DK-2800 Lyngby, Denmark. Phone: 45 4526 5050.Fax: 45 4526 5060. E-mail: jol@symphogen.com.

† Present address: ILRI, Biotechnology, Nairobi, Kenya.‡ Present address: ImClone Systems, New York, NY.� Published ahead of print on 8 December 2010.

1820

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/j

vi o

n 12

Feb

ruar

y 20

22 b

y 19

4.44

.50.

96.

Early attempts to use inactivated VACV preparations com-posed largely of IMV for vaccination resulted in poor protec-tion and led to the conclusion that a neutralizing antibodyresponse to VACV needs to comprise antibodies to both viralparticle types (6). More recent studies in animal models haveconfirmed that although some protection against virus chal-lenge can be obtained with single-protein vaccination or anti-bodies directed against individual IMV or EEV surface anti-gens, the best protection is afforded when a combinatoryapproach targeting both IMV and EEV is employed (24, 30,31, 38, 46). Lustig et al. have provided one explanation for theimproved protective effect of combinations of antibodies to thetwo virion types as they have shown that IgG against the A33REEV surface protein, which was not in vitro neutralizing on itsown, was capable of eliciting complement-mediated lysis of theouter EEV membrane, thereby making the inner IMV particlesusceptible to a neutralizing L1R-specific antibody (47). Re-cently, other protective mechanisms involving complement,such as direct neutralization through coating of the virionsurface and complement-dependent lysis of VACV-infectedcells, have been demonstrated for B5R-specific antibodies (4).The neutralization in this case did not involve virion lysis anddid not require the presence of a neutralizing IMV-specificantibody (4), thus further illustrating the complex nature ofantibody-mediated protection against VACV.

Altogether, given the complexity of VACV, which includestwo types of infectious virions and a multitude of antigens, itseems clear that the optimal therapeutic antibody againstVACV should not be limited to a single antigen but, rather,should consist of a combination of specificities. Antibody ther-apeutics targeting multiple epitopes, e.g., bispecific antibodiesor cocktails consisting of two or more separately producedantibodies, are increasingly being developed to raise the ther-apeutic effectiveness when large and complex entities are tar-geted (3, 55). Such antibody mixtures may constitute entirelynew treatment modalities but may also replace existing plasma-derived immunoglobulin products, which, as already noted,suffer certain drawbacks that make them a less attractive ther-apeutic option. In contrast, target-specific recombinant poly-clonal antibodies offer the advantage of unlimited supply, highand reproducible specificity and activity, and an improvedsafety profile (8).

In this study, we have characterized the antibody response tosmallpox vaccine (VACV Lister) in immunized human volun-teers and employed the Symplex technology (48, 49) to isolatea large number of VACV-specific antibodies recognizing avariety of different VACV antigens. Using this broad panel ofantibodies, we have generated a fully human recombinant VIGthat mirrors the diversity and specificity of the human antibodyimmune response and has the potential to be used as an ef-fective postexposure prophylaxis or treatment of diseasecaused by orthopoxviruses.

MATERIALS AND METHODS

Blood donors. Twelve voluntary blood donors were recruited from a smallpoxvaccination program of British first-responder health care workers. The partic-ipants in this study gave signed informed consent, and approval was obtainedfrom the Salisbury Research Ethics Committee (United Kingdom) and the Re-gional Research Ethics Committee of Copenhagen (Denmark) prior to initiationof the study. The blood donors included both first-time vaccinees and revaccinees

who received dried smallpox vaccine of the VACV Lister strain (batch 347;National Collection of Pathogenic Viruses, Health Protection Agency, Centrefor Emergency Preparedness and Response, Porton Down, United Kingdom),and blood was drawn in the range of 9 to 20 days after vaccination.

Viruses, antigens, and reagents. Live VACV of the NYCBOH and IHD-Wstrains was obtained from the ATCC (VR-1536 and VR-1441, respectively), andthe Lister strain was provided by the National Collection of Pathogenic Viruses,United Kingdom. Purified VACV particles of the Lister and IHD-W strainsinactivated by the UV-psoralen method (Advanced Biotechnologies, Columbia,MD) were used for enzyme-linked immunospot assay (ELISPOT) and bindingassays. Similarly inactivated particles of the IHD-J strain were kindly provided byGrant McFadden (University of Western Ontario, London, Ontario, Canada).

Recombinant VACV proteins were produced in the baculovirus expressionsystem. B5R and VACV complement protein (VCP or C3L; both Lister strain)were obtained from Qbiogene (Illkirch, France) and A27L, A33R, and L1R (allWR strain) were from the University of Pennsylvania (Philadelphia, PA).

In vitro translated proteins (e.g., A10L, A56R, D8L, and H3L) were obtainedby PCR amplification of the individual genes from genomic VACV Lister DNA,followed by expression using a TNT T7 Quick for PCR DNA coupled transcrip-tion/translation system (Promega, Madison, WI) with Transcend biotinylatedtRNA according to the manufacturer’s instructions.

Vaccinia immune globulin intravenous (human) ([VIG] lot number 17304043;Cangene, Winnipeg, MB, Canada) was obtained from Statens Serum Institut(Copenhagen, Denmark).

Characterization of the donor VACV-specific immune response. Followingisolation of peripheral blood mononuclear cells (PBMC) from donor blood usingLymphoprep (Axis-Shield, Oslo, Norway), the CD19� B cell population waspurified using magnetic cell sorting (MACS) as described by Meijer et al. (48).Serum (diluted 1:2 with phosphate-buffered saline [PBS]) was also collected andstored at �20°C. The frequency of plasma cells producing VACV-specific anti-bodies was determined using an ELISPOT assay. Briefly, MACS-purified CD19�

cells (1 � 105 cells per well) were added to nitrocellulose-bottomed (HA filtertype) plates (Millipore, Bedford, MA) coated with a solution of 20 �g/ml inac-tivated VACV particles. Following incubation and washing, immobilized vacciniavirus-specific human IgG and IgA were detected separately using horseradishperoxidase (HRP)-conjugated anti-human IgG (CalTag, Burlingame, CA) andHRP-conjugated anti-human IgA (AbD Serotec, Oxford, United Kingdom). Theplates were developed as described previously (48), and the number of spots wascounted manually using a stereomicroscope.

The serum IgG response against VACV particles and recombinant antigenswas characterized by enzyme-linked immunosorbent assay (ELISA). Ninety-six-well ELISA plates (MaxiSorp; Nunc A/S, Roskilde, Denmark) were coated withantigen, blocked, and washed according to standard protocols. Following incu-bation with a dilution series of the serum samples and additional washing, boundIgG was detected using HRP-conjugated anti-human IgG (CalTag), and theplates were developed as described before (48). Each serum sample was assayedin duplicate against each antigen on two separate plates. Endpoint titers weredetermined as the reciprocal dilution giving a signal 4-fold above background byinterpolation from the titration curves.

Generation of antibody repertoires. Plasma cells (PC) were selected from theCD19� cell population by fluorescence-activated cell sorting (FACS) based onthe expression profile of CD19, CD38, and CD45 cell surface proteins as de-scribed by Meijer et al. (48). The resulting cell population was single-cell sortedinto 96-well PCR plates containing PCR buffer (Phusion HF buffer; Finnzymes,Espoo, Finland), primers for reverse transcription-PCR (RT-PCR) (49), andRNase inhibitor (RNasin; Promega). Antibody repertoires were generated fromthe single-cell-sorted PC using the Symplex PCR (48). Cells from different blooddonors were processed separately, and the resulting antibody repertoires werealso kept separate. The overall Symplex PCR procedure was performed asdescribed elsewhere (49), except for the use of a different enzyme combinationin the multiplex overlap extension RT-PCR. The following was added to eachwell to obtain the given final concentration: deoxynucleoside triphosphate(dNTP) mix (200 �M each), RNasin (20 U/�l), Sensiscript reverse transcriptase(diluted 1:320; Qiagen, Hilden, Germany), and Phusion DNA polymerase (0.02U/�l; Finnzymes). Following reverse transcription for 1 h at 37°C, antibody geneswere amplified and linked using the following PCR cycle: initial denaturation at98°C for 30 s; 30 cycles of 98°C for 20 s, 60°C for 30 s, and 72°C for 45 s; finalextension at 72°C for 45 s. Following further amplification of the multiplexoverlap extension PCR products in a nested PCR (49), the linked antibody geneswere cloned into the Fab expression vector pJSK301 (48, 49). The generatedrepertoire of expression vectors was propagated in Escherichia coli (TOP10), andindividual bacterial colonies were arrayed in 384-well master plates and stored asglycerol stocks.

VOL. 85, 2011 RECOMBINANT POLYCLONAL ANTIBODY AGAINST VACCINIA VIRUS 1821

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/j

vi o

n 12

Feb

ruar

y 20

22 b

y 19

4.44

.50.

96.

Screening of cloned repertoires. E. coli expression of Fab fragments wasperformed in 384 deep-well plates essentially as described previously (48). TheFab-containing supernatants were cleared by centrifugation and used for screen-ing of individual clones in a bead-based fluorescence-linked immunosorbentassay (FLISA) (56). Briefly, inactivated virus particles of the Lister strain, theIHD-W strain, and the recombinant protein antigens B5R, VCP, and A33R wereimmobilized individually on polystyrene beads (6.79-�m diameter; SpherotechInc., Lake Forest, IL) by incubating 16.5 �g of protein or 20 �g of virus particleswith 100 �l of 5% (wt/vol) beads. The coated beads, goat anti-human IgG AlexaFluor 647 conjugate (Molecular Probes/Invitrogen, Carlsbad, CA), and super-natant containing Fab fragments were mixed in assay-compatible 384-well plates(Nunc 142761), and the fluorescence at the bead surface in individual wells wasrecorded using an 8200 Cellular Detection System (Applied Biosystems, FosterCity, CA). Cutoff was set at 50 counts (fluorescent beads) during the primaryscreening in order to identify as many clones reactive with viral particles orantigens as possible. The expressed Fab fragment repertoires were screenedagainst all five populations of coated beads.

Clones that were positive in the primary screening were retrieved from theoriginal master plates and reexpressed in 96-well plates and tested in a secondaryscreening against the same antigens by both FLISA and standard ELISA (48).

Sequence analysis. Plasmids encoding clones that were identified as positive inthe secondary screening were subjected to DNA sequencing (MWG Biotech,Ebersberg, Germany), and the obtained sequences were clustered based onsequence homology. Immunoglobulin variable (V), diversity (D), and joining (J)gene segment usage was determined for each sequence by comparison to germline sequences from the ImMunoGeneTics (IMGT [www.imgt.org]) referencedirectory (44). Antibody clones that share V and J segment usage, complemen-tarity determining region 3 (CDR3) lengths in the heavy chain, and consensussequences in the CDR3 of both the heavy and light chains (CDRH3 and CDRL3,respectively) were assigned to the same clonotype.

Expression of full-length recombinant human antibodies. Selected antibodyheavy and light V gene pairs were transferred to a mammalian antibody expres-sion vector, described in Meijer et al. (49), to allow for production of full-lengthIgG1 molecules. In the process of transferring the genes, mutations introducedby cross-priming during the Symplex PCR were also corrected. The genes en-coding the V genes of an antibody of interest were individually PCR amplifiedwith a primer set containing sequences corresponding to the identified variablegerm line genes, thereby correcting any changes introduced by cross-priming.The resulting products were digested and sequentially inserted into the mam-malian expression vector using conventional ligation procedures (57). A Fast-Start High Fidelity PCR system (Roche, Penzberg, Germany) was used for theamplification of the genes. All restriction enzymes and the T4 DNA ligase wereobtained from New England BioLabs (Ipswich, MA).

The resulting constructs were individually transfected into a subclone of theCHO Flp-In cell line (Invitrogen), CHO019 (57). Following selection of stablecell lines in the presence of Geneticin (Invitrogen), each cell line was adapted tosuspension culture in serum-free EX-CELL 302 medium (Sigma, St. Louis, MO),expanded, and cryopreserved at �150°C. Twenty-six of the individual cell lines(expressing antibody clones 029, 037, 058, 086, 147, 186, 188, 195, 197, 203, 211,229, 235, 286, 295, 303, 339, 461, 482, 488, 526, 551, 586, 589, 607, and 633) wereselected for the generation of a polyclonal cell line, and batches of recombinantpolyclonal VACV-specific antibody were produced in 5-liter bioreactors (B.Braun Biotech International, Melsungen, Germany), essentially as describedpreviously (57). Each cell line was also cultured in shaker flasks for productionof the individual antibodies. The monoclonal antibodies (MAbs) and polyclonalantibodies were individually purified from the culture supernatants by protein Aaffinity chromatography and analyzed by SDS-PAGE and cation exchange chro-matography (57).

Assessment of antibody reactivity. Following identification of unique antibodyclones, large-scale batches of Fab fragments of each clone were prepared forfinal confirmation of the reactivity with inactivated virus particles of the Lister,IHD-W, and IHD-J strains and the recombinant antigens A27L, A33R, B5R,L1R, and VCP by ELISA as outlined in previous sections. The subsequentlyproduced full-length antibodies were similarly analyzed.

Antigen specificities of the individual antibody clones were further analyzed byWestern blotting using acetone-precipitated VACV (Lister) particles, whichsubsequently were dissolved in SDS loading buffer containing 8 M urea to obtainoptimal resolution of the viral proteins. The proteins were separated onNuPAGE Bis-Tris gels (4 to 12% or 10%; Invitrogen) and transferred ontopolyvinylidene difluoride (PVDF) membranes (Invitrogen). After a blockingstep, the Western blots were probed with purified VACV-specific MAbs andHRP-conjugated anti-human IgG (CalTag) and developed using the SuperSignal

West Femto Maximum Sensitivity Chemiluminescent Substrate (Pierce, Rock-ford, IL) according to the manufacturer’s instructions.

A number of antibodies were also tested for reactivity with in vitro translatedVACV antigens. Purified antibodies were immobilized in ELISA plates at 1�g/ml and incubated with dilutions of the different in vitro translated antigens.Bound antigen was detected by streptavidin-peroxidase polymer (Sigma), and theplates were developed as described above.

A panel of purified MAbs (029, 037, 058, 086, 147, 186, 188, 195, 197, 203, 211,229, 235, 286, 295, 303, 339, 461, 482, 488, 526, 551, 586, 589, 607, and 633) wasalso tested for reactivity with VACV (WR strain) proteome arrays (VacciniaChip 9, versions 1,2, and 10b; Antigen Discovery, Irvine, CA). The arrays con-tained VACV proteins produced in a cell-free transcription and translationsystem, with or without disulfide bridge formation (20), and the 10b array alsocontained purified proteins produced in E. coli or mammalian cells. Each MAbunderwent duplicate analyses on the arrays. The data were averaged for eachsample, and the average negative-control values (plus 10 standard deviations)were subtracted.

Affinity measurements. The affinities of selected antibody clones were deter-mined by surface plasmon resonance using a Biacore 2000 (Biacore AB, Upp-sala, Sweden). The antigens (B5R, VCP, A33R, and A27L) were immobilizedindividually on a CM5 chip surface using standard amine coupling chemistry toa level resulting in a maximum resonance unit (RUmax) value of approximately100 RU or less. Purified antibody Fab fragments prepared by papain cleavage(ImmunoPure Fab preparation Kit; Pierce) were diluted serially in HBS-EP (10mM HEPES [pH 7.4], 150 mM NaCl, 3 mM EDTA, 0.005% P20 surfactant)running-buffer (Biacore AB) and passed over the chip at 10 to 50 �l/min. Bindingkinetics and affinity constants (KD) were determined using the BIAevaluationsoftware (Biacore AB) by global fitting of four to six different concentrationspassed over the same sensor surface.

Preparation of VACV for use in vitro and in vivo. Infectious VACV particleswere added to subconfluent B-SC-1 cells in serum-free Earle’s minimal essentialmedium (MEM; Invitrogen), and the virus was allowed to adsorb for 45 min at37°C. Following addition of MEM containing 2% (vol/vol) fetal calf serum (2%MEM), the cells were incubated at 37°C until they showed full cytopathic effect.The supernatant was discarded, and the cells were detached into PBS, centri-fuged, resuspended in PBS, and frozen at �80°C. After samples were thawed, thecell debris was removed by centrifugation, and aliquots of the supernatant werestored at �80°C. The titer was determined by a plaque reduction assay (seebelow).

Plaque reduction and neutralization assay. For the evaluation of in vitroneutralizing activity, antibodies were diluted in serum-free MEM and preincu-bated for 1 h at 37°C with 1 � 104 PFU of Lister strain virus particles. Themixture was applied to a monolayer of Vero cells preseeded in 24-well plates,which were incubated for 4 h at 37°C in 5% CO2. The infected cells were overlaidwith 2% MEM containing 2% carboxymethylcellulose, followed by incubationfor 3 days at 37°C in 5% CO2. The cells were fixed by incubation with 10%formalin in PBS and stained using 20% ethanol containing 0.1% crystal violet,and the number of plaques in each well was counted.

Tail lesion model. The mouse tail lesion model (7) was used to evaluate the invivo protective activity of the recombinant VIG. All experiments were performedaccording to the regulations of the United Kingdom Home Office Animals(Scientific Procedures) Act (1986).

Female BALB/c, 6- to 8-week-old mice (Health Protection Agency) werechallenged with infectious VACV particles of either the Lister strain (2 � 106

PFU) or the NYCBOH strain (5 � 105 PFU) by injection into the tail vein. Thevirus challenge doses were determined in separate sighting studies as the numberof PFU required to produce 30 to 40 lesions per tail in untreated mice. Twenty-four hours before or after challenge, groups of eight mice were treated intra-muscularly with different amounts of recombinant VIG or plasma-derived VIG(Cangene) or with 300 �g of a negative-control antibody (anti-rhesus D recom-binant polyclonal antibody [57 ]) in 500 �l of PBS. One group of mice was treatedintramuscularly with 2.5 mg of cidofovir (Vistide; Gilead Sciences, Inc., FosterCity, CA) as a positive control. Seven days after virus challenge, the lesions oneach tail were counted in a blinded fashion, and the number of lesions in thegroups treated with recombinant or plasma-derived VIG or with cidofovir wasexpressed as a percentage of the lesions in the mice treated with the negative-control antibody, which had no effect on the number of tail lesions. Statisticalsignificance was determined using a homoscedastic Student’s t test.

Nucleotide sequence accession numbers. Complete V region sequences of arepresentative antibody clone from each clonotype can be found in GenBankunder accession numbers HQ378325 to HQ378502.

1822 LANTTO ET AL. J. VIROL.

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/j

vi o

n 12

Feb

ruar

y 20

22 b

y 19

4.44

.50.

96.

RESULTS

Characterization of the immune response in smallpox vac-cinees. To characterize the human immune response to VACVand select the optimal starting material for isolation of VACV-specific antibodies, the blood samples obtained from the small-pox vaccinees were analyzed in terms of frequencies of plasmacells (PC) producing VACV-specific antibodies and serum an-tibody levels to VACV antigens.

Circulating PC were selected from the CD19� B cell popu-lation by FACS (48), and the percentages of PC secretingVACV-specific antibodies were estimated by ELISPOT assay.Most of the vaccinees had a clearly detectable frequency ofVACV-specific PC, and the revaccinees had in general thehighest frequencies, reaching up to 0.6% of the total number ofPC (Table 1). A detailed analysis of the kinetics of the B cellresponse was not possible since the majority of the bloodsamples were, for logistical reasons, obtained 14 or 15 daysafter the vaccination. However, the only sample that was ob-tained earlier (donor identification number [ID] 11; 9 daysafter vaccination) also had the highest frequency of VACV-specific IgG-secreting PC (Table 1), thus suggesting that the Bcell response induced by smallpox vaccination peaked beforethe time point where most of the blood samples were obtained.

The serum antibody titers against inactivated viral particles

from three virus strains (Lister, IHD-W, and IHD-J) and fiverecombinant antigens (B5R, VCP, A27L, A33R, and L1R)were determined by standard ELISA. The Lister strain wascommonly used in Western Europe during the WHO smallpoxeradication campaign (23) and was also used for vaccination inthis study. Both the IHD-J and IHD-W strains are derivedfrom the NYCBOH strain (www.atcc.org), which was used inthe United States during the eradication of smallpox (23).

The serum response against the different antigens variedgreatly both between and within individual donors, likely areflection of the differences in vaccination responsiveness, re-sponse period, vaccination history, and immunogenicity of thedifferent antigens. Four out of the six previously vaccinateddonors (IDs 01, 02, 07, and 08) exhibited strong serum re-sponses against both VACV particles and recombinant anti-gens, whereas the other two donors (IDs 05 and 11) had low oralmost undetectable titers against all antigens (Fig. 1). Asalready mentioned, donor 11 also had the shortest responseperiod, i.e., 9 days, which may explain its poor serum response.The antibody response in revaccinees has been reported toincrease rapidly from day 6 to 8 until it peaks around day 12 to15 after vaccination (26).

Due to the presence of multiple targets for antibodies on theVACV particles, the response was expected to be stronger

TABLE 1. Overview of the vaccinated donors, their immune responses, and the cloned antibody repertoires

Donor ID Previous vaccination(s)(yr)

Responseperiod (days)

% PC ofCD19� cells

% VACV-specific PC

Estimated repertoiresize (no. of PCR

products)

No. of confirmedVACV-specific

antibodiesIgG IgA

01 1950, 1970 14 12 0.40 0.20 600 1002 1966, 1979 14 2.6 0.32 0.15 400 903 20 1.7 0.21 0.0304 18 2.5 0.13 005 1978-1979 14 2.4 0.25 0.06 1,200 1807 1956-1958 15 NDb ND ND08 Yesa 15 15 ND ND 300 1409 14 20 0.07 0.0310 14 2.0 0.03 011 1966 9 7.2 0.47 0.01 1,050 38

a Date of previous vaccination(s) unknown.b ND, not determined.

FIG. 1. The natural antibody response to smallpox vaccination is highly variable. Serum reactivity with VACV particles (strains Lister, IHD-J,and IHD-W) and recombinant antigens (A27L, A33R, B5R, L1R, and VCP) was determined in 10 human smallpox vaccinees by ELISA. Theendpoint titer corresponds to the reciprocal dilution giving a signal 4-fold above background. All determinations were performed in duplicate, andthe results are presented as means � standard errors of the means.

VOL. 85, 2011 RECOMBINANT POLYCLONAL ANTIBODY AGAINST VACCINIA VIRUS 1823

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/j

vi o

n 12

Feb

ruar

y 20

22 b

y 19

4.44

.50.

96.

against these than against the single recombinant antigens.This was indeed the case for the IHD-J strain particles but notfor the other two virus strains. A part of the observed differ-ence may naturally be attributed to differences in coating ef-ficiency and uncertainties in the concentration determination,but there may also be differences in the recognition of domi-nant antigens, in particular for the IHD-W particles, whichwere poorly recognized by all sera. IHD-J has been shown tohave an increased EEV particle production (51), whereasIHD-W is a hemagglutinin (A56R)-negative strain derivedfrom the IHD-J strain (17). In agreement with previous stud-ies, the serum response to L1R was very weak, or nearly un-detectable, in all donors (5, 43, 52).

Generation and screening of antibody repertoires. Based onthe serum reactivity, the ELISPOT analysis, and the expecta-tion that the recall antibody response generated by repeatedvaccination will yield antibodies of higher affinity, it was de-cided to use cells from five of the revaccinees as the startingmaterial for the generation of antibody repertoires. The pro-tocol used for this study is based on the capture of immuno-globulin V genes from single B cells that have a high expressionof antibody (i.e., PC). Since donors 01, 02, 05, and 11 had thehighest frequencies of VACV-specific PC (Table 1), they con-stituted the prime candidates. Cells from donor 08 could not beanalyzed by ELISPOT due to the small amount of blood ob-tained, but based on the high overall frequency of PC in theCD19� cell population and the vaccination history (Table 1),this donor was also included.

The nucleic acids encoding the antibody repertoires wereisolated from the single-cell-sorted PC by the Symplex PCR,which creates a physical link between the heavy V region genefragment and the full-length light chain (48, 49). The protocolwas designed to amplify antibody genes of all human immu-noglobulin heavy chain and kappa light chain V region genefamilies by the use of two primer sets, one for heavy V region(IgG and IgA isotypes) amplification and one for kappa light-chain amplification. Using approximately 5,000 cells from eachdonor, repertoires of between 300 and 1,200 overlap productswere generated by the multiplex overlap extension RT-PCR(Table 1). The size of the repertoires was estimated by analyz-ing a representative number of reactions on agarose gels (49)(data not shown). The overlap products from each donor werepooled separately and inserted into a Fab expression vector forproduction and screening of the individual antibody specifici-ties.

Approximately three times as many antibody-expressingclones as the estimated number of input PCR products (Table1) were arrayed and screened on different VACV antigens,thus giving 95% likelihood for the representation of all uniqueV gene pairs amplified by the Symplex PCR (14). The largenumber of hits obtained in the screening of the five repertoireswas retested against the same VACV antigens to verify reac-tivities, and approximately 700 clones were subjected to se-quencing and clustering according to sequence similarity. Agroup of clones that shared V and J segment usage in both theheavy and light V region, heavy-chain CDR3 length, and con-sensus sequences in both CDRH3 and CDRL3 were assignedto the same cluster or clonotype. The number of clones as-signed to a clonotype varied from a single clone to more than30 in the case of one of the A33R-specific clonotypes (Table 2).

As shown in Fig. 2 and Table 2, the isolated repertoire ofVACV-specific antibodies displays a high degree of geneticdiversity. In fact, all human immunoglobulin heavy-chain vari-able region (IGHV) gene families, as well as the immunoglob-ulin kappa light-chain variable region (IGKV) gene families 1to 4 (44; IMGT [www.imgt.org]), were found among the con-firmed VACV-specific clones. Genes belonging to the IGKV5and IGKV6 families, which were not found among the con-firmed clones, are only rarely expressed (25). The heavy-chainV gene usage correlates well with what has been found previ-ously in human antibody repertoires (9, 22, 33), with a frequentusage of the IGHV3, IGHV1, and IGHV4 families. The light-chain V gene usage was dominated by the IGKV1 family,mainly due to the frequent usage of the IGKV1-39 genes (Fig.2 and Table 2). These genes are also among the most fre-quently used light-chain V genes in the human immunoglob-ulin repertoire (16, 22, 25). As a whole, the IGKV1 genes madeup 75% of the repertoire. The only other IGKV gene of prom-inent usage in the isolated repertoire was IGKV3-20, whichalso is the single most frequently expressed light-chain gene inhumans (22). The observed genetic diversity is indicative of anunbiased recovery of V genes by the Symplex PCR and sug-gests that the isolated antibody repertoire mirrors the diversityof the natural humoral response to VACV in humans.

In addition, the sequence analysis revealed that six clono-types that were isolated from three different repertoires utilizehighly similar VDJ and VJ rearrangements. The clonotypesrepresented by clones 160 and 250 were isolated from donors05 and 08, respectively, and the four clonotypes represented byclones 211, 214, 238, and 351 were all isolated from donor 11.These clonotypes all use the IGHV3-9 and IGKV1-39 genesand have highly similar CDRH3 sequences (Table 2). TheWestern blot analysis also showed that these clones all recog-nize the same antigen, subsequently identified as H3L (seebelow). However, the differences in CDRL3 sequence (Table2), the use of different D and J segment genes, and the distinctpatterns of somatic mutations (data not shown) demonstratethat these clonotypes have clearly arisen through independentrearrangements. In donor 11, this type of rearrangement hasapparently arisen four times.

Characterization of individual antibody specificities. Fol-lowing sequencing and identification of unique clonotypes, arepresentative clone from each cluster was tested as a Fabfragment or as a full-length IgG after transfer of the V genesto a mammalian expression vector. Together with the sequenceanalysis, this characterization revealed 89 clonotypes that werereactive with inactivated VACV particles of the Lister,IHD-W, or IHD-J strain or the recombinant antigens B5R andVCP (Table 2). All five donor repertoires contributed to dif-ferent extents to this panel of VACV-specific antibodies (Ta-ble 1).

The confirmed B5R- and VCP-specific clones were mainlyidentified by screening on the recombinant antigens, and thereactivities were verified by ELISA using the same antigens(data not shown). However, all five B5R-specific clones alsobound to inactivated VACV (Lister) particles to some extent.Some of the 11 VCP-specific clones also bound weakly toinactivated VACV particles (Lister or IHD-W strain), indicat-ing that VCP is associated with the viral particle preparationsdespite being a secreted protein. The two A33R-specific clono-

1824 LANTTO ET AL. J. VIROL.

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/j

vi o

n 12

Feb

ruar

y 20

22 b

y 19

4.44

.50.

96.

TABLE 2. Summary of the genetic origin and reactivity of the 89 confirmed VACV-specific antibodies

Antibodyclone

IGHVgene CDRH3 sequence IGKV

gene CDRL3 sequenceNo. of

clones incluster

Specificitya

029 1-24 CATDVWRRTPEGGTDWF-------DPW 3-11 CLQRSNWP-ITF 12 H3L031 4-34 CAGSRSFDLLTAYDLFHRKGNAM-DVW 1-39 CQQSYRTL-YTF 6 (H3L)037 6-1 CARGDVLRYF--------------DYW 1-5 CQQYNGYSEVTF 4 WR148058 3-30 CAKGGLGTNEF-------------DHW 1-5 CQQYDTYP-ITF 2 WR148086 5-51 CATLPRYDAYGARIR---------DYW 1-33 CQQYDNLP-PTF 16 I1L089 1-2 CARYCSSPTCS-------------IVW 3-20 CQQYGRSP-LTF 2 (WR148)112 2-5 CAHSSQRVVTGL------------DFW 1-39 CQQSYNTP-ATF 10 A27L113 2-70 CARIRTCYPDLYGDYNDAF-----DIW 1-33 CQQYENVP-YTF 2 VCP147 3-7 CARAADYGDY--------------VRP 2-28 CMQALQIP-RTF 8 H5R156 3-30 CAKHVAAGGTL-------------DYW 2-24 CMQTTHIP-HTF 3 (A14L)

159 1-46 CARVIRKYYTSSNSYLTEQAF---DIW 1-39 CQQTYGNP-LTF 9 A27L160 3-9 CVKETVAGRRGAF-----------DYW 1-39 CQQSFRTP-HTF 10 H3L166 4-34 CARGREWPSNF-------------DSW 3-11 CQQRAIWP-PEF 1169 5-51 CARRGSTYYY--------------DTW 1-39 CQQSFTSW-WTF 2 B5R172 3-21 CASKPYGGDFG-------------SYW 1-5 CQQYSNYP-ITF 2 B5R183 5-51 CARPPSNWDESF------------DIW 1-17 CLQHNSF--LTF 1 (H3L)186 1-3 CARDPTQWLLQGDVYDM-------DVW 2-30 CMQGTHWP-PAF 6 A27L188 4-30-4 CAREAWLGEPLLLGDDAF------DIW 1-17 CLQLHTFP-RTF 3 A27L189 3-30 CARDGAGEWDLLMRRDF-------DYW 1-39 CQQGYSTP-YTF 4 (H3L)195 1-18 CARDPARRPRSGYSVF--------EYW 3-15 CHQYNYWPPLAF 1 H3L

197 4-4 CARDNRQSSSWVEGFFYYYGM---DVW 3-20 CQQYGISP-RTF 1 H3L201 1-46 CARLRLGATIGRD-----------DYW 2-28 CMQALQTP-HTF 2 H3L203 4-30-4 CARDRASSGYDSRVWF--------DPW 1-33 CHQYDSLP-FTF 2 A27L205 3-30 CARTYRVYAKFDPF----------DVW 3-20 CQQFGYSPRFTF 5 H3L211 3-9 CGKDGVPGRRGYI-----------EDW 1-39 CQQTYITP-KSF 4 H3L214 3-9 CVKDNIAGRRGSF-----------DSW 1-39 CQQSYRTP-LTF 3 (H3L)215 1-2 CARDYIRATGATPSKYFIYYYGM-GVW 1-27 CQKYDSAP-YTF 12 (H3L)219 1-18 CARARRVTNSPNNWF---------DPW 1-39 CQQTYTIP-LTF 1 (H3L)225 4-34 CARAGERSGSGSFVLGRF------DFW 1-39 CQQSYSTL-RTF 2 A14L229 3-49 CTNTSSLAVA--------------GNW 4-1 CQQYYKTP-PTF 3 A27L

232 4-39 CARIPQQRVNYF------------DYW 1-5 CQQYNSYP-LTF 1 H3L235 4-39 CARLPGQRTTFF------------DYW 1-5 CQQYNFY--GTF 1 H3L237 6-1 CARGRRFEDDAF------------DIW 1-39 CQQSYSIP-RTF 3 (H3L)238 3-9 CVKDSVAGRRGGF-----------DHW 1-39 CQQSYSMSPYTF 3 (H3L)242 1-f CATRDGDF----------------DHW 1-16 CQQYNSFP-LTF 8243 5-51 CVRHGTRYSFGRSDII--------DIW 1-17 CLQQNNYP-WTF 9 (I1L)246 5-51 CTKTPARGAYGDYIS---------GSW 1-33 CQQYDNFP-YTF 3 (H3L)250 3-9 CAKSTKAVRRGSF-----------DYW 1-39 CQQTYIST-RTF 3 (H3L)267 1-69 CARGGKLYEGNGYYSFHYF-----DYW 1-33 CQQYDNLP--LF 1 VCP269 2-5 CAHTELAF----------------DYW 1-33 CQQYDNLI--TF 1 VCP

271 1-69 CARVKGTQNYYGM-----------DVW 1-5 CQQYESDI-FTF 2 VCP274 4-34 CHYYDSTGYYVS------------DFW 1-16 CQQYGRYP-LTF 1 VCP286 1-18 CARGVVLIQTILF-----------DYW 1-39 CQQSHRTP-YTF 4 VCP290 1-69 CARTVLDSGAYSYY----------DSR 1-5 CQQYQTY--STF 1 VCP291 1-69 CARSRGSQDYYGM-----------DVW 1-5 CQQYESDS-WTF 2 VCP294 3-23 CAKLRDSSVYSAYVFRVIF-----DCW 1-33 CQQYDNLP-FTF 1 VCP295 3-11 CATLTVASTY--------------DYW 2-30 CMQGTHWP-YSF 8 B5R297 3-9 CVKDTVALLTSRGGCM--------DVW 1-12 CQQAYSFP-WTF 12 VCP302 2-5 CAHSPPHGG---------------DYW 1-33 CQQYDNL--PTF 1 VCP303 3-30 CAKGLSQALNYYGSGS--------PFL 3-20 CQQYGTSP-WTF 3 B5R

335 3-30 CAKDRGVSAWYPRDAF--------DIW 1-39 CHQSYSLP-FTF 1 (H3L)339 4-59 CARVPLIEAGITIFAKIGAF----DIW 3-20 CQQYVASP-FTF 2 D8L349 5-51 CARGSPMIKFYF------------DYW 3-20 CHQYGTSP-RTF 2 (A14L)351 3-9 CARATGAGRRNPL-----------DYW 1-39 CQQSYITP-YTF 1 (H3L)431 1-46 CARPYRSYSSSPQ-----------DYW 3-20 CQQYGSSP-YTF 9 A56R437 4-31 CARYYYSSGPKF------------DYW 1-12 CQQANSFP-FTF 9446 1-69 CARNNRPLGALFGM----------DVW 1-9 CQQLDTYP-LTF 4461 1-18 CARGGFNRLV--------------DPW 3-20 CQQYASSP-YTF �30 A33R482 4-31 CARNTGIYLGGSPGGTRNNWF---DPW 1-39 CQHSSKTP-FTF 5 A33R488 5-51 CARQQAKTLYYDSSGSKSAF----DIW 1-5 CQQYSSY--LSF 2 A27L

Continued on following page

VOL. 85, 2011 RECOMBINANT POLYCLONAL ANTIBODY AGAINST VACCINIA VIRUS 1825

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/j

vi o

n 12

Feb

ruar

y 20

22 b

y 19

4.44

.50.

96.

types were identified by screening on the VACV Lister parti-cles, and screening on recombinant antigen did not yield anyadditional clones. The reactivity of both clones was determinedby ELISA using the recombinant antigen. The seven A27L-specific clonotypes were also identified by screening on virusparticles, and the specificity was subsequently identified byELISA using recombinant A27L.

In order to assign antigenic specificities to the clonotypesthat were not reactive with any of the recombinant antigens, anextensive analysis was performed using Western blotting, cap-ture ELISA with in vitro translated VACV antigens, andVACV protein array analysis. The Western blotting using ac-etone-precipitated VACV Lister particles revealed seven dis-tinct reactivities, arbitrarily labeled B, C, D, E, G, HI, and L,

TABLE 2—Continued

Antibodyclone

IGHVgene CDRH3 sequence IGKV

gene CDRL3 sequenceNo. of

clones incluster

Specificitya

515 4-31 CARVRGNIVATTAFYYYYGL----DAW 1-12 CQQANGFL-WTF 1516 3-11 CAKFDYGEGAYHF-----------DFW 1D-12 CQQAHSFP-VTF 2 A56R517 4-31 CARDPIALPGRGVF----------DYW 3-20 CQQYGSSP-LTF 1 (D8L)520 4-34 CSSGYYFAGGEF------------DYW 1-12 CQQANSFP-RTF 4526 3-49 CTRDRGYSDHTGLYTRFGF-----DSW 2-28 CMQSLQT--VTF 2 B5R532 1-69 CARWRGGYSGYGDYF---------DSW 1-39 CQHSYETPPYTF 3536 4-4 CARDPQKPRQHLWPNPYYYSGM--DVW 1-5 CQQYNFYP-WTF 2551 1-69 CARGRSWRGYL-------------DYW 3-20 CQQYGGSP-QTF 1 A56R559 1-46 CARDYGDYCGGDCPYDAF------DIW 1-27 CQKYNSAP-LTF 1572 1-69 CARDKGESDINGWQTGAFYYGM--DVW 1-12 CQQANSFP-VTF 3

575 2-26 CARIDSVGWPSSHYYGM-------DVW 1-12 CQQTHSFPPWTF 2586 4-59 CARDRITGYDSSGHAF--------DIW 1-5 CQQYHHYS-PTF 1589 4-34 CARGGKFCGSTSCFTEGRL-----DYW 1-39 CQQSYSTL-RTF 1 A14L607 3-30 CAKDSGRYSSLGHYYYYGM-----DVW 1-39 CQQGYSTP-PTF 1 A10L611 5-51 CARGYYYDTSGYRPGSF-------QHW 1-5 CQQYDSYP-YTF 2612 3-30 CARPLFYGAGDAF-----------DIW 1-39 CQQSYSTP-RTF 2614 1-69 CADWVVGNYNGL------------DVW 3-20 CQQYSDS--LTF 1617 1-46 CAREGKHDFWRGYFSPLGM-----DVW 3-20 CHQYGGAQ-GTF 5621 1-69 CATARNSSNWYEGHYYL-------AHW 1-33 CQQYDTLPPITF 1626 4-30-4 CANMVVVATQPKNWF---------DPW 1-39 CQQSHSSP-WTF 1

628 4-30-4 CASYGSGMGSEYYF----------GHW 1-39 CQQSYLAP-WTF 1 A56R632 1-2 CGRVGREAFYYYGM----------DVW 1-9 CQQFNNYP-YTF 3633 1-2 CARASRRLTTHNYF----------DGW 1-16 CQQYHTYP-FTF 3 H3L634 3-48 CARVRGGRYF--------------DYW 3-20 CQKYGRSPTWTF 1640 5-51 CASGSGYDSYYNM-----------DVW 1-39 CQQGFTTP-ITF 3643 7-4-1 CARDSSTVTGLMTEYNWF------DPW 1-39 CQQSYSTP-YTF 1649 4-31 CATEKGSGGDVGKF----------DNW 1-39 CQQSYTIPLWTF 1650 1-69 CATGQQLYSL--------------HYW 2-30 CMQGTHWP-PTF 1651 1-2 CAREGPQFYYDSGDYYSAHSPGDFDHW 1-39 CQQSDSTP-YTF 1 (H3L)

a Parentheses indicate that the specificity was assigned by comparison of reactivities determined by Western blotting.

FIG. 2. The captured repertoire of VACV-specific antibodies mirrors the natural genetic diversity of a human antibody response. Thefrequencies of IGHV (left panel) and IGKV (right panel) gene usage in the VACV-specific antibody repertoire were calculated based on thegenetic origin of each of the 89 confirmed VACV-specific antibodies (Table 2). The highly homologous gene pair IGKV1-12/IGKV1D-12 was forclarity treated as a single entity.

1826 LANTTO ET AL. J. VIROL.

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/j

vi o

n 12

Feb

ruar

y 20

22 b

y 19

4.44

.50.

96.

and also confirmed the already identified reactivities withA27L, A33R, B5R, and VCP. Figure 3 shows representativeresults from the analysis. The VCP-specific clones reactedstrongly with the recombinant protein in ELISA, but only a fewof them recognized the protein in Western blotting withVACV particles. As also noted by other investigators (37), acluster of proteins with molecular masses between 30 and 40kDa was recognized by the VACV-specific antibodies (C, E, G,and HI reactivities and B5R and VCP) (Fig. 3). The HI reac-tivity was found to be the predominant, with a total of 22clonotypes, including the six that use similar rearrangements ofIGHV3-9 and IGKV1-39 (160, 211, 214, 238, 250, and 351),displaying this profile. The other reactivities were representedby only a few clonotypes each. Approximately one-third of theclones that were reactive with VACV particles when tested byELISA and FLISA did not recognize a specific antigen in theWestern blot analysis.

Results from the capture ELISA using in vitro translatedVACV antigens demonstrated that the clones displaying theHI reactivity profile recognized the H3L protein of the Listerstrain (Fig. 4A). Similarly, it was shown that the antibodyclones displaying the E and D reactivities recognized D8L andA56R, respectively (Fig. 4A). These results correlate well withthe observed Western blotting reactivity profiles (Fig. 3) asH3L has been shown to migrate as a single band of 35 kDa(59), D8L as a single band of about 34 kDa (50), and A56R astwo bands with apparent sizes of 85 and 68 kDa (10). Clone

607, which did not recognize a specific antigen in the Westernblot analysis, was reactive with the in vitro translated A10L(major core protein) (Fig. 4A).

The VACV protein array analysis (20), which was performedwith a set of 26 antibody clones produced as full-length IgG,verified several of the already identified specificities and alsorevealed a few additional specificities (Fig. 4B and data notshown). All five A27L-specific antibodies that were tested, 186,188, 203, 229, and 488, were reactive with the in vitro translatedprotein on the VACV array. Three of the previously identifiedH3L-specific clones, 195, 197, and 211, also reacted with theprotein expressed in an E. coli-based cell-free system, and twoothers, 029 and 235, recognized the protein when it was ex-pressed in a system allowing for disulfide bond formation.Clone 633, which displayed weak reactivity in Western blotting,was also found to recognize H3L on the protein arrays (datanot shown). Clone 607 was also verified as being A10L specific,in accordance with the capture ELISA using in vitro translatedprotein (Fig. 4A).

Two clones that displayed the B reactivity profile in theWestern blot analysis (Fig. 3), 037 and 058, reacted stronglywith WR148, the VACV A-type inclusion (ATI) protein ho-mologue (Fig. 4B). This protein has no direct VACV Copen-hagen homologue, but is usually referred to as A25L (www.poxvirus.org). The B reactivity clones recognized an �82-kDaprotein in the Western blotting with Lister strain viral particles(Fig. 3), which is also the size reported for the ATI protein(37). Three additional reactivities were also discovered asclones 086, 147, and 589 were found to recognize I1L (DNA-binding core protein), H5R (viral late gene transcription fac-tor) and A14L (major IMV membrane component), respec-tively (Fig. 4B). The reactivities in Western blotting (clone 086,C; clone 147, G; and clone 589, L) (Fig. 3) correlate well withthe reported sizes of 36 kDa for both I1L and H5R (39, 40) and14 to 16 kDa for A14L (28).

A number of antibody clones, including three B5R-specificclones (295, 303, and 526), the two A33R-specific clones (461and 482), and one A56R-specific clone (551), recognized onlytheir respective targets as purified proteins expressed in mam-malian cells. The D8L-specific clone 339 recognized the targetgene product only when it was expressed in a cell-free systemwhich allowed for disulfide bond formation (data not shown).Not surprisingly, correct folding, disulfide bond formation,and/or posttranslational modifications appear to be requiredfor specific recognition by some antibodies.

The VACV protein array analysis failed to identify the tar-get antigen for only two antibody clones, 286 and 586. Neitherof these reacted specifically with any particular in vitro ex-pressed VACV protein, regardless of the expression systemused (Fig. 4B and data not shown). Clone 286 had already beenidentified as VCP specific and, as discussed further below,recognizes recombinant VCP expressed in insect cells withnanomolar affinity (Fig. 5). The lack of reactivity with theprotein arrays may be due to an improper conformation of thein vitro expressed protein. In fact, protein array analysis hasfailed to detect anti-VCP antibodies in both commercial, plas-ma-derived VIG, and hyperimmune serum (18, 20; this study).Clone 586 did not react specifically in any of the confirmatoryassays used, and the specificity of this clone remains unknown.

In summary, using a variety of assays with both natural and

FIG. 3. The confirmed VACV-specific antibodies display a numberof distinct reactivities with VACV proteins. Representative Westernblots of the identified reactivities B, C, HI, E, D, G, and L andrecombinant A33R, B5R, VCP, and A27L, as indicated above eachpanel, are shown. Virus particles or recombinant antigens were used astarget antigen and probed with different VACV-specific antibodies(clone numbers are given above the right lane in each panel). M,molecular size marker (kDa).

VOL. 85, 2011 RECOMBINANT POLYCLONAL ANTIBODY AGAINST VACCINIA VIRUS 1827

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/j

vi o

n 12

Feb

ruar

y 20

22 b

y 19

4.44

.50.

96.

recombinant VACV proteins, antigenic specificities have beenassigned to more than two-thirds of the 89 confirmed VACV-specific antibodies. The analysis showed that at least 12 differ-ent VACV proteins, including A10L, A14L, A27L, A33R,A56R, B5R, D8L, H3L, H5R, I1L, VCP, and the ATI protein,are recognized by the isolated human VACV-specific antibod-ies, thus further demonstrating the diversity of the capturedantibody repertoire. For a comparison, commercial, plasma-derived VIG reacted with A10L, A14L, A27L, A33R, A56R,D8L, D13L, F13L, H3L, H5R, I1L, I3L, L4R, and WR148(ATI protein) on the VACV proteome arrays (Fig. 4B). Inaddition, plasma-derived VIG also recognized recombinantA33R, B5R, L1R, and VCP in ELISA (Fig. 6 and data notshown), whereas the anti-rhesus D recombinant polyclonal an-tibody, which was used as a negative control, did not displayany reactivity toward the tested antigens (Fig. 6). Antigenicspecificities could not be assigned to the remaining antibodyclones as these did not recognize any particular antigen in anyof the applied assays. The cause for the absence of reactivitymay be that the epitopes recognized by these antibodies arepoorly represented in the antigenic preparations, either due to

FIG. 4. The confirmed anti-VACV antibodies recognize a wide range of VACV proteins. (A) Reactivity of selected antibody clones with in vitrotranslated VACV proteins as determined by capture ELISA. Each protein was incubated in serial dilutions (approximately 1:10 to 1:100 dilutionof the crude in vitro translation reaction) with the different immobilized anti-VACV antibodies, and the bound antigen was detected bystreptavidin-peroxidase polymer. (B) Representative heat map showing the interaction of monoclonal antibodies at 50 ng/ml and commercial,plasma-derived VIG at 5 �g/ml with VACV antigens on Vaccinia Chip 9, version 1 (Antigen Discovery). Each interaction was assigned a colorshade based on the signal intensity (see scale at the bottom of figure) after subtraction of the average control signal intensity plus 10 standarddeviations from the average raw signal intensity.

FIG. 5. Affinities of human VACV-specific antibodies isolated bythe Symplex technology. The affinity distributions among antibodyclones reactive with VACV antigens A27L, A33R, B5R, and VCP weredetermined by surface plasmon resonance. Each symbol represents aunique antibody clone, identified by number.

1828 LANTTO ET AL. J. VIROL.

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/j

vi o

n 12

Feb

ruar

y 20

22 b

y 19

4.44

.50.

96.

a lack of proper conformation or posttranslational modifica-tions or to the absence of complex formation in the case ofepitopes made up by more than one protein. For VACV, suchcomplexes have been shown to be formed by, e.g., A33R,A34R, and A36R; A34R and B5R; and A17L, A26L, and A27L(32, 53, 58). Judging from the binding characteristics, some ofthe unassigned antibodies appear to recognize antigens of lowabundance in the VACV particle preparations (data notshown). It should be noted that none of the confirmed VACV-specific antibody clones bound to Vero cells, i.e., the cells usedto produce the VACV particles, when tested at 10 �g/ml in aFACS-based assay (data not shown). All in all, the reactivity ofthe isolated antibody repertoire appears to be essentially thesame as that demonstrated for plasma-derived VIG.

Furthermore, in order to determine the quality of the iso-lated antibody repertoire, the binding kinetics of the interac-tion with the target antigen was determined for a selection ofantibodies. This analysis was for practical reasons restricted toantibodies specific for antigens that were available as purified,recombinant proteins (A27L, A33R, B5R, and VCP). The ki-

netic measurements revealed affinity constants (KD) between10�8 to 10�10 M for the majority of the investigated antibodies(Fig. 5), thus showing that the isolated antibodies reach intothe high-affinity range. In fact, the observed affinities are wellwithin the range of those of currently used therapeutic mono-clonal antibodies (11), suggesting that the captured antibodieshave therapeutic potential without further improvements interms of affinity. A number of antibodies reactive with A27Land VCP did not recognize the antigens immobilized to theBiacore chip and were for this reason excluded from the anal-ysis.

Generation of a recombinant VIG. Having identified a largenumber of antibodies reactive with different VACV proteins,we selected 26 antibody clones for the creation of a recombi-nant VIG with the broadest possible reactivity against VACVstrains and related orthopoxviruses. To allow for consistentproduction and easy characterization, the selected clones werecombined and expressed as a recombinant polyclonal antibodymixture using the Sympress technology. This technology hasalso been used to generate an anti-rhesus D recombinant poly-

FIG. 6. Recombinant VIG displays a high specific binding activity toward VACV antigens. The reactivity of purified recombinant VIG (orangecircles) against VACV particles (Lister and IHD-J) and recombinant antigens (A27L, A33R, B5R, and VCP) was evaluated by ELISA.Commercial, plasma-derived VIG (gray diamonds) and a negative-control polyclonal antibody (anti-rhesus D [57]; black squares) were similarlytested for comparison. All determinations were performed in duplicate, and the results are presented as means � standard errors of the means.

VOL. 85, 2011 RECOMBINANT POLYCLONAL ANTIBODY AGAINST VACCINIA VIRUS 1829

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/j

vi o

n 12

Feb

ruar

y 20

22 b

y 19

4.44

.50.

96.

clonal antibody comprised of 25 unique antibodies (57), whichis currently in clinical trials (identifier NCT00718692 [clinicaltrials.gov]). Due to the complexity of antibody-mediated pro-tection against VACV infection, which makes it difficult toassess the full impact of individual antibodies on the basis of invitro assays, antibody clones reactive with all the identifiedantigens were included in the recombinant VIG. Where pos-sible, more than one antibody clone per target was included toobtain optimal epitope coverage, and the numbers of antibod-ies against each antigen was also selected as to reflect thefrequency in the captured repertoire (Table 2) and, thus, thenatural antibody response toward each antigen (5, 20, 37, 52).Multiple antibodies against the dominating antigens, A27L andH3L, were for this reason included in the recombinant VIG.As described in the previous section, VCP also yielded numer-ous antibodies, but due to the secreted nature of the protein,only a single, high-affinity antibody (Fig. 5) specific for thisantigen was included.

The recombinant VIG thus contains the following reactivi-ties: A10L (clone 607), A14L (clone 589), A27L (clones 186,188, 203, 229, and 488), A33R (clones 461 and 482), A56R(clone 551), B5R (clones 295, 303, and 526), D8L (clone 339),H3L (clones 029, 195, 197, 211, 235, and 633), H5R (clone147), I1L (clone 086), VCP (clone 286), and the ATI protein(clones 037 and 058). In addition, a single antibody clone (586)for which no specificity could be assigned was also included asa representative of the group of antibody clones that react withunknown antigens of low abundance in the VACV particlepreparations (see above).

The resulting recombinant VIG was tested for reactivity withdifferent VACV strains and recombinant VACV antigens byELISA, and the in vitro neutralizing activity was determined ina plaque reduction neutralization assay against the Listerstrain. As shown in Fig. 6, the recombinant VIG displayed ahigh specific binding activity, approximately 250-fold higherthan the commercial, plasma-derived VIG, more or less irre-spective of the target antigen tested. Furthermore, the recom-binant VIG was found to exhibit an 800-fold higher virus neu-tralizing activity against the Lister VACV strain than theplasma-derived VIG (50% effective concentration [EC50],0.125 versus 100 �g/ml).

Once it was established that the recombinant VIG harborsboth high VACV-specific binding activity and in vitro neutral-izing activity, the in vivo antiviral activity was assessed using themouse tail lesion model against the Lister and NYCBOHVACV strains. Inoculation of VACV into the tail vein of micehas been shown to produce a nonlethal and self-limiting infec-tion that results in discrete dermal lesions along the entiresurface of the tail (7), in close resemblance to the reaction atthe vaccination site following successful smallpox vaccinationin humans. The mouse tail lesion model represents a simpleand reliable way of assessing protective efficacy and is generallymore ethically acceptable than lethal animal models. The an-tibodies were administered intramuscularly 1 day before or 1day after virus challenge to evaluate both prophylactic andtherapeutic efficacies in early infection. The effect of antibodytreatment on tail lesion formation was determined 7 days aftervirus challenge by manual counting of the lesions on each tail.A challenge dose that produced 30 to 40 lesions per tail in thegroups receiving the negative-control antibody was used in

both settings. As shown in Fig. 7, both the recombinant and theplasma-derived VIG were capable of inhibiting lesion forma-tion in both the prophylactic and therapeutic settings. How-ever, the recombinant VIG was found to be at least 200-foldmore potent than the commercial, plasma-derived VIG againstboth virus strains in both settings. In addition, at the highestdose tested (250 �g/mouse), the recombinant VIG almostcompletely abolished the formation of VACV lesions (Fig. 7).This was in stark contrast to the plasma-derived VIG, whichwas capable of inhibiting lesion formation only to 30 to 40% ofthe negative control at a 20 times higher dose (5 mg/mouse).For a comparison, prophylaxis and treatment with a standarddose of 2.5 mg/mouse of the small-molecule antiviral drugcidofovir (21) against both virus strains reduced lesion forma-tion to 25 to 28% of that in the control group (data not shown).The highest dose of recombinant VIG was significantly moreeffective at inhibiting lesion formation than both the commer-cial, plasma-derived VIG at 5 mg/mouse and cidofovir at 2.5mg/mouse in both settings (P � 0.02).

DISCUSSION

The concerns over the potential use of smallpox virus inbiological warfare or terrorism and the increasing risk of zoo-notic poxvirus outbreaks have revived the need for treatmentoptions to be used alone or in combination with vaccination.For this reason, we set out to characterize and capture thenatural human antibody response toward VACV in vaccinateddonors with the aim of creating a recombinant VIG that would

FIG. 7. Recombinant VIG protects mice against VACV challengein vivo. Groups of eight mice were treated with recombinant VIG(orange circles) or commercial, plasma-derived VIG (gray diamonds)at the indicated doses 24 h before (solid lines) or after (broken lines)challenge with VACV (Lister strain or NYCBOH strain). Tail lesionswere enumerated in a blinded fashion 7 days after virus challenge, andthe results are expressed relative to the negative control (anti-rhesus Dpolyclonal antibody [57]). Data are shown as means � standard errorsof the means.

1830 LANTTO ET AL. J. VIROL.

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/j

vi o

n 12

Feb

ruar

y 20

22 b

y 19

4.44

.50.

96.

mimic the natural antibody response but have a higher degreeof specificity and therapeutic efficacy and improved safety pro-file than plasma-derived products. Using the Symplex technol-ogy, a diverse repertoire consisting of 89 unique humanVACV-specific antibodies recognizing at least 12 differentVACV antigens with affinities similar to those of licensedmonoclonal antibody therapeutics was isolated. A thoroughanalysis of the sequences of the isolated VACV-specific anti-bodies showed that these are of a highly diverse genetic origin(Fig. 2), which corresponds well to that of normal humanantibody responses (9, 16, 22, 25, 33). Furthermore, the anti-gen recognition of the captured antibody repertoire also cor-responds to the specificity of the serum response induced byvaccination with VACV (5, 20, 37, 52), thus demonstrating aneffective and unbiased capture of VACV-specific antibodiesusing the Symplex technology.

The antigenic specificities identified in the captured VACV-specific repertoire and also included in the recombinant VIGinclude both IMV- and EEV-associated antigens and one se-creted antigen (VCP). Five of the recognized proteins aresurface proteins of the IMV (A27L, D8L, and H3L) or theEEV (A33R and B5R) that have been shown to induce neu-tralizing and/or in vivo protective antibodies in animal modelsor humans (for a review, see reference 2) and constitute thusobvious targets for a therapeutic antibody composition. Inagreement with the findings by other investigators (19), H3Lwas found to be the most frequently recognized antigen withinthe captured repertoire. Besides confirming the immunodomi-nance of H3L in humans, the results from this study also showthat H3L seems to select for a common antibody sequencemotif in different individuals, possibly indicating the presenceof an imprint in the germ line-encoded antibody repertoirerequired for recognition of this antigen. A similar bias in Vgene usage has been observed for a number of other antigens,including viral proteins, bacterial polysaccharides, and aller-gens, and may in part explain the immunodominance of certainantigens or epitopes (reviewed in reference 42). Two addi-tional surface proteins, A14L and A56R, were also recognizedby the isolated antibodies. A14L, which is an IMV membraneprotein, is a known target for antibodies, and it has been shownto evoke an antibody response that correlates with virus neu-tralization in smallpox vaccinees (5). A56R is the VACV hem-agglutinin protein, and it is considered a constituent of theEEV envelope (54). However, the role of A56R-specific anti-bodies in the protection against VACV infection is unclear.Depletion of A56R-reactive antibodies from hyperimmune hu-man serum does not affect the neutralizing activity (52), but, asdiscussed further below, this may be an effect of the redun-dancy in the neutralizing response toward VACV. It has alsobeen debated whether A56R is a true EEV envelope proteinsince it does not seem to accumulate intracellularly in virion-like structures, and approximately one-third of EEV particlesalso lack A56R (41, 45). Furthermore, not all VACV strainsexpress A56R, as in the case of IHD-W (17). The fact thatA56R is expressed on the surface of infected cells (45) suggeststhat it still may be of importance, e.g., for opsonization ofinfected cells.

In addition, a number of nonenvelope IMV proteins, includ-ing A10L (major core protein), I1L (DNA-binding core pro-tein), H5R (viral late gene transcription factor), and the ATI

protein, were recognized. It was not unexpected to retrievebinders against these proteins as it has been shown that asignificant part of the antibody response following smallpoxvaccination is directed against these antigens (20, 37). With theexception of H5R, they are also major components of IMV orare abundantly expressed in VACV-infected cells (13, 20).Recently, it was shown that polyclonal antibody raised againstthe ATI protein was capable of blocking VACV plaque for-mation in vitro, thus demonstrating that this protein is actuallya target of neutralizing antibodies (12). The ATI protein is notan integral envelope protein, but it appears to be associatedwith the surface of IMV through complex formation with otherproteins. Whereas it seems unlikely that the other three pro-teins (A10L, I1L, and H5R) also are targets for neutralizingantibodies, they are clearly being targeted by the natural hu-man antibody response, and these specificities were for thatreason included in the recombinant VIG.

VCP (C3L) is a secretory protein of VACV that is capableof inhibiting complement activation, thereby preventing anti-body-mediated virus neutralization and allowing the virus toevade the host immune response (35). Due to its homology tohuman proteins, it has been suggested that VCP is poorlyimmunogenic and will not elicit an antibody response (36). Asshown in this study, this is clearly not the case since a majorityof the tested donor sera contained high titers against the re-combinant VCP (Fig. 1), and multiple VCP-specific antibodieswere isolated from the cloned repertoires (Table 2). Otherinvestigators have also shown that VCP is the major secretoryVACV protein that is recognized by VIG (1). Being a secretedprotein, VCP is not a target for neutralizing antibodies, and ithas also been shown that the humoral response induced byvaccination with recombinant VCP fails to protect against le-thal VACV challenge in a mouse model (1). However, anti-bodies raised against VCP have been shown to block its activityin vitro (1, 34), and it is possible that anti-VCP antibodies havea role in ameliorating disease or enhancing the effect of neu-tralizing antibodies. VCP is thus a natural and potentially im-portant reactivity to include in a recombinant VIG.

As indicated by the size of the VACV genome and numberof reactivities against VACV in VIG and hyperimmune serumobserved in this study (Fig. 4) and elsewhere (18, 20), theantigens recognized by the captured antibody repertoire do notrepresent all the VACV antigens that the humoral immuneresponse is potentially capable of recognizing. However, pro-tein array profiling studies have shown that only a limitednumber of VACV proteins are consistently recognized by VIGand/or human hyperimmune serum. The list of recognizedproteins includes A10L, A13L, A17L, A27L, A33R, A56R,B5R, D8L, D13L, E3L, F13L, H3L, H5R, L4R, and WR148(ATI protein), and, when expressed under the proper condi-tions, L1R (18, 20). The majority of these antigens were alsorecognized by the members of the isolated VACV-specificrepertoire, including most of the surface antigens shown toinduce neutralizing or protective antibodies (2). One signifi-cant exception that was not found in the isolated repertoire isL1R. This is an IMV envelope protein that has been shown tobe the target of both neutralizing and in vivo protective anti-bodies (reviewed in reference 2). However, as demonstratedboth here (Fig. 1) and elsewhere (5, 43, 52), L1R appears to bepoorly immunogenic in humans, and the serum response to

VOL. 85, 2011 RECOMBINANT POLYCLONAL ANTIBODY AGAINST VACCINIA VIRUS 1831

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/j

vi o

n 12

Feb

ruar

y 20

22 b

y 19

4.44

.50.

96.

L1R is generally weak, or nearly undetectable, in vaccinatedhumans. The failure to identify any L1R-specific antibodies inthis study is therefore likely due to an absence or a very lowfrequency of L1R-specific B cells in the starting material forthe Symplex PCR. Dedicated screening using recombinant orpurified proteins might have yielded additional hits againstindividual proteins, including L1R. However, due to the abun-dance of antibodies against other IMV envelope proteins, thiswas not implemented for L1R. A number of studies havesuggested that there is a significant degree of redundancy inthe neutralizing antibody response toward IMV; i.e., in theabsence of one or more neutralizing specificities, other speci-ficities are able to compensate for the loss. For instance,Benhnia et al. have demonstrated that although purified anti-H3L IgG is sufficient to neutralize VACV, depletion of H3Lantibodies from serum does not significantly reduce the neu-tralizing activity (5). Comparable results have also been ob-tained for anti-L1R and also for a combination of L1R andH3L antibodies, thus leading to the proposal that the naturalimmune response to the IMV form of VACV is not directedtoward a single key antigen but is instead aimed at the devel-opment of a highly functionally redundant response of neu-tralizing antibodies to multiple different viral proteins (5). Putzand colleagues have also showed that depletion of hyperim-mune sera of antibodies against A27L, H3R, or L1R only hadlimited effect on the residual neutralizing activity (52), and Heet al. have presented similar results for A27L (29). Thus, thecollective IMV reactivity, which includes antibodies to A14L,A27L, D8L, and H3L, in the recombinant VIG is likely morethan able to substitute for the missing anti-L1R reactivity. Onthe other hand, similar depletion experiments with EEV anti-gens have suggested that B5R is the only target of importancefor EEV neutralization as depletion of A33R- and A56R-specific antibodies had no impact on the residual neutralizingactivity, whereas depletion of antibodies against B5R fully ab-rogated the neutralizing activity (52).

Since the recombinant VIG was designed to consist exclu-sively of VACV-specific antibodies, it did not come as a sur-prise that it displayed a very high specific binding activity to-ward VACV particles and proteins. The binding activity wasalso reflected in the in vitro and in vivo potency as the recom-binant VIG displayed an 800-fold higher in vitro neutralizingactivity and at least 200-fold higher in vivo protective capacitythan the commercial, plasma-derived VIG. Based on the bind-ing activity and the in vitro neutralizing activity, it was expectedthat prophylactically administered recombinant VIG wouldelicit a profound antiviral effect. However, it was not antici-pated that the effect of postchallenge treatment would benearly identical to the effect of prophylaxis for all of the ad-ministered therapeutics, including cidofovir. This result prob-ably reflects a limitation of the mouse model used, in which alarge number of infectious virus particles (�105 PFU) areinjected into the tail vein, thereby creating a temporary highlocal virus concentration, which gives rise to 30 to 40 lesions inuntreated mice. The temporary high concentration of virus willlikely exceed the concentration of the systemically adminis-tered VACV-specific antibody, which thereby becomes morelikely to influence continued virus dissemination rather thanblocking of the primary infection. If this is the dynamic of thein vivo model used in this study, no profound differentiation

between prophylactic and therapeutic administration ofVACV-specific antibody can be expected, in correlation withthe observed result. However, potent prophylactic and thera-peutic efficacy of the recombinant VIG has also been demon-strated in a lethal mouse model based on intranasal challenge,thus corroborating the observed protective efficacy (H. S.Nielsen et al., unpublished data).

Taken together, the in vitro and in vivo data clearly demon-strate that a high degree of relevant antigenic specificities isincluded in the recombinant VIG and that it contains a hightiter of protective antibodies. However, as suggested by thestudies demonstrating a redundancy in the neutralizing anti-body response toward VACV (5, 29, 52) and the fact that anumber of antigenic specificities with no clear role in the pro-tection against VACV were included in the antibody compo-sition, it is possible that a less complex antibody compositionwith equal or even higher efficacy could be created by carefulselection of the appropriate specificities. It should be kept inmind, though, that several studies have suggested that multiplespecificities targeting antigens of both the IMV and EEV arerequired for optimal protection against the virus (24, 30, 31, 38,46). In addition, the requirements for effective protectionagainst other orthopoxviruses such as variola virus or monkey-pox virus have not been investigated in detail, and it seemsreasonable to believe that multiple specificities are needed inorder to achieve broad and efficacious protection against thevarious viruses of the genus.

In conclusion, we have demonstrated the generation of arecombinant VIG that mirrors the diversity and specificity ofthe natural human antibody immune response to VACV anddisplays a high protective efficacy against VACV. We believethat this recombinant VIG has the potential to be used againstorthopoxvirus infections either as postexposure prophylaxis ortreatment. A detailed characterization of the biological activityof the recombinant VIG against different viruses both in vitroand in animal models will be presented elsewhere (Nielsen etal., unpublished).

ACKNOWLEDGMENTS

We thank Barbara T. Andersen, Berit Mikkelsen, Bettina C. Over-gaard, Camilla Holst Dahl, Elisabeth V. Andersen, Finn C. Wiberg,Hanne Wagner, Maria Schmidt, Vincent W. Coljee, and YvonneBerger Larsen for excellent technical assistance and Ulla Jessen foreditorial assistance.

The study was supported by award U01AI070378 from the NationalInstitute of Allergy and Infectious Diseases.

The content is solely the responsibility of the authors and does notnecessarily represent the official views of the National Institute ofAllergy and Infectious Diseases or the National Institutes of Health.

A.J., J.L., M.H.H., S.K.R., and L.S. are inventors on a patent appli-cation on the recombinant VIG described here.

REFERENCES

1. Adamo, J. E., C. A. Meseda, J. P. Weir, and M. J. Merchlinsky. 2009.Smallpox vaccines induce antibodies to the immunomodulatory, secretedvaccinia virus complement control protein. J. Gen. Virol. 90:2604–2608.

2. Amanna, I. J., M. K. Slifka, and S. Crotty. 2006. Immunity and immunolog-ical memory following smallpox vaccination. Immunol. Rev. 211:320–337.

3. Beck, A., T. Wurch, C. Bailly, and N. Corvaia. 2010. Strategies and chal-lenges for the next generation of therapeutic antibodies. Nat. Rev. Immunol.10:345–352.

4. Benhnia, M. R., et al. 2009. Vaccinia virus extracellular enveloped virionneutralization in vitro and protection in vivo depend on complement. J. Vi-rol. 83:1201–1215.

5. Benhnia, M. R., et al. 2008. Redundancy and plasticity of neutralizing anti-

1832 LANTTO ET AL. J. VIROL.

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/j

vi o

n 12

Feb

ruar

y 20

22 b

y 19

4.44

.50.

96.

body responses are cornerstone attributes of the human immune response tothe smallpox vaccine. J. Virol. 82:3751–3768.

6. Boulter, E. A., H. T. Zwartouw, D. H. Titmuss, and H. B. Maber. 1971. Thenature of the immune state produced by inactivated vaccinia virus in rabbits.Am. J. Epidemiol. 94:612–620.

7. Boyle, J. J., R. F. Haff, and R. C. Stewart. 1966. Evaluation of antiviralcompounds by suppression of tail lesions in vaccinia-infected mice. Antimi-crob. Agents Chemother. 6:536–539.

8. Bregenholt, S., A. Jensen, J. Lantto, S. Hyldig, and J. S. Haurum. 2006.Recombinant human polyclonal antibodies: a new class of therapeutic anti-bodies against viral infections. Curr. Pharm. Des. 12:2007–2015.

9. Brezinschek, H. P., et al. 1997. Analysis of the human VH gene repertoire.Differential effects of selection and somatic hypermutation on human pe-ripheral CD5�/IgM� and CD5�/IgM� B cells. J. Clin. Invest. 99:2488–2501.

10. Brown, C. K., P. C. Turner, and R. W. Moyer. 1991. Molecular character-ization of the vaccinia virus hemagglutinin gene. J. Virol. 65:3598–3606.

11. Carter, P. J. 2006. Potent antibody therapeutics by design. Nat. Rev. Immu-nol. 6:343–357.

12. Chang, S. J., Y. X. Chang, R. Izmailyan, Y. L. Tang, and W. Chang. 2010.Vaccinia virus A25 and A26 proteins are fusion suppressors for maturevirions and determine strain-specific virus entry pathways into HeLa, CHO-K1, and L cells. J. Virol. 84:8422–8432.

13. Chung, C. S., et al. 2006. Vaccinia virus proteome: identification of proteinsin vaccinia virus intracellular mature virion particles. J. Virol. 80:2127–2140.

14. Clarke, L., and J. Carbon. 1976. A colony bank containing synthetic Col Elhybrid plasmids representative of the entire E. coli genome. Cell 9:91–99.

15. Condit, R. C., N. Moussatche, and P. Traktman. 2006. In a nutshell: struc-ture and assembly of the vaccinia virion. Adv. Virus Res. 66:31–124.

16. Cox, J. P., I. M. Tomlinson, and G. Winter. 1994. A directory of humangerm-line V kappa segments reveals a strong bias in their usage. Eur. J. Im-munol. 24:827–836.

17. Dales, S., et al. 1978. Biogenesis of vaccinia: isolation of conditional lethalmutants and electron microscopic characterization of their phenotypicallyexpressed defects. Virology 84:403–428.

18. Davies, D. H., et al. 2005. Profiling the humoral immune response to infec-tion by using proteome microarrays: high-throughput vaccine and diagnosticantigen discovery. Proc. Natl. Acad. Sci. U. S. A. 102:547–552.

19. Davies, D. H., et al. 2005. Vaccinia virus H3L envelope protein is a majortarget of neutralizing antibodies in humans and elicits protection againstlethal challenge in mice. J. Virol. 79:11724–11733.

20. Davies, D. H., et al. 2007. Proteome-wide analysis of the serological responseto vaccinia and smallpox. Proteomics 7:1678–1686.

21. De Clercq, E. 2002. Cidofovir in the treatment of poxvirus infections. Anti-viral Res. 55:1–13.

22. de Wildt, R. M., R. M. Hoet, W. J. van Venrooij, I. M. Tomlinson, and G.Winter. 1999. Analysis of heavy and light chain pairings indicates that re-ceptor editing shapes the human antibody repertoire. J. Mol. Biol. 285:895–901.

23. Fenner, F., D. A. Henderson, I. Arita, Z. Jezek, and I. D. Ladnyi. 1988.Smallpox and its eradication. World Health Organization, Geneva, Switzer-land.

24. Fogg, C., et al. 2004. Protective immunity to vaccinia virus induced byvaccination with multiple recombinant outer membrane proteins of intracel-lular and extracellular virions. J. Virol. 78:10230–10237.

25. Foster, S. J., H. P. Brezinschek, R. I. Brezinschek, and P. E. Lipsky. 1997.Molecular mechanisms and selective influences that shape the kappa generepertoire of IgM� B cells. J. Clin. Invest. 99:1614–1627.

26. Frey, S. E., F. K. Newman, L. Yan, K. R. Lottenbach, and R. B. Belshe. 2003.Response to smallpox vaccine in persons immunized in the distant past.JAMA 289:3295–3299.

27. Fulginiti, V. A., A. Papier, J. M. Lane, J. M. Neff, and D. A. Henderson. 2003.Smallpox vaccination: a review, part II. Adverse events. Clin. Infect. Dis.37:251–271.

28. Grosenbach, D. W., S. G. Hansen, and D. E. Hruby. 2000. Identification andanalysis of vaccinia virus palmitylproteins. Virology 275:193–206.

29. He, Y., et al. 2007. Antibodies to the A27 protein of vaccinia virus neutralizeand protect against infection but represent a minor component of Dryvaxvaccine-induced immunity. J. Infect. Dis. 196:1026–1032.

30. Hooper, J. W., D. M. Custer, and E. Thompson. 2003. Four-gene-combina-tion DNA vaccine protects mice against a lethal vaccinia virus challenge andelicits appropriate antibody responses in nonhuman primates. Virology 306:181–195.

31. Hooper, J. W., et al. 2004. Smallpox DNA vaccine protects nonhuman pri-mates against lethal monkeypox. J. Virol. 78:4433–4443.

32. Howard, A. R., T. G. Senkevich, and B. Moss. 2008. Vaccinia virus A26 andA27 proteins form a stable complex tethered to mature virions by associationwith the A17 transmembrane protein. J. Virol. 82:12384–12391.

33. Huang, S. C., R. Jiang, A. M. Glas, and E. C. Milner. 1996. Non-stochastic

utilization of Ig V region genes in unselected human peripheral B cells. Mol.Immunol. 33:553–560.

34. Isaacs, S. N., E. Argyropoulos, G. Sfyroera, S. Mohammad, and J. D. Lam-bris. 2003. Restoration of complement-enhanced neutralization of vacciniavirus virions by novel monoclonal antibodies raised against the vaccinia viruscomplement control protein. J. Virol. 77:8256–8262.

35. Isaacs, S. N., G. J. Kotwal, and B. Moss. 1992. Vaccinia virus complement-control protein prevents antibody-dependent complement-enhanced neu-tralization of infectivity and contributes to virulence. Proc. Natl. Acad. Sci.U. S. A. 89:628–632.

36. Jha, P., and G. J. Kotwal. 2003. Vaccinia complement control protein:multi-functional protein and a potential wonder drug. J. Biosci. 28:265–271.

37. Jones-Trower, A., et al. 2005. Identification and preliminary characterizationof vaccinia virus (Dryvax) antigens recognized by vaccinia immune globulin.Virology 343:128–140.

38. Kaufman, D. R., et al. 2008. Differential antigen requirements for protectionagainst systemic and intranasal vaccinia virus challenges in mice. J. Virol.82:6829–6837.

39. Klemperer, N., J. Ward, E. Evans, and P. Traktman. 1997. The vaccinia virusI1 protein is essential for the assembly of mature virions. J. Virol. 71:9285–9294.

40. Kovacs, G. R., and B. Moss. 1996. The vaccinia virus H5R gene encodes lategene transcription factor 4: purification, cloning, and overexpression. J. Vi-rol. 70:6796–6802.

41. Krauss, O., R. Hollinshead, M. Hollinshead, and G. L. Smith. 2002. Aninvestigation of incorporation of cellular antigens into vaccinia virus parti-cles. J. Gen. Virol. 83:2347–2359.

42. Lantto, J. 2002. Antibody evolution and repertoire development. Ph.D.thesis. Lund University, Lund, Sweden.

43. Lawrence, S. J., et al. 2007. Antibody responses to vaccinia membraneproteins after smallpox vaccination. J. Infect. Dis. 196:220–229.

44. Lefranc, M.-P., and G. Lefranc. 2001. The immunoglobulin factsbook. Ac-ademic Press, San Diego, CA.

45. Lorenzo, M. M., I. Galindo, G. Griffiths, and R. Blasco. 2000. Intracellularlocalization of vaccinia virus extracellular enveloped virus envelope proteinsindividually expressed using a Semliki Forest virus replicon. J. Virol. 74:10535–10550.

46. Lustig, S., et al. 2005. Combinations of polyclonal or monoclonal antibodiesto proteins of the outer membranes of the two infectious forms of vacciniavirus protect mice against a lethal respiratory challenge. J. Virol. 79:13454–13462.

47. Lustig, S., C. Fogg, J. C. Whitbeck, and B. Moss. 2004. Synergistic neutral-izing activities of antibodies to outer membrane proteins of the two infec-tious forms of vaccinia virus in the presence of complement. Virology 328:30–35.

48. Meijer, P. J., et al. 2006. Isolation of human antibody repertoires withpreservation of the natural heavy and light chain pairing. J. Mol. Biol.358:764–772.

49. Meijer, P. J., L. S. Nielsen, J. Lantto, and A. Jensen. 2009. Human antibodyrepertoires. Methods Mol. Biol. 525:261–277.

50. Niles, E. G., and J. Seto. 1988. Vaccinia virus gene D8 encodes a viriontransmembrane protein. J. Virol. 62:3772–3778.

51. Payne, L. G. 1979. Identification of the vaccinia hemagglutinin polypeptidefrom a cell system yielding large amounts of extracellular enveloped virus.J. Virol. 31:147–155.

52. Putz, M. M., C. M. Midgley, M. Law, and G. L. Smith. 2006. Quantificationof antibody responses against multiple antigens of the two infectious formsof vaccinia virus provides a benchmark for smallpox vaccination. Nat. Med.12:1310–1315.

53. Rottger, S., F. Frischknecht, I. Reckmann, G. L. Smith, and M. Way. 1999.Interactions between vaccinia virus IEV membrane proteins and their rolesin IEV assembly and actin tail formation. J. Virol. 73:2863–2875.

54. Smith, G. L., A. Vanderplasschen, and M. Law. 2002. The formation andfunction of extracellular enveloped vaccinia virus. J. Gen. Virol. 83:2915–2931.

55. Swann, P. G., et al. 2008. Considerations for the development of therapeuticmonoclonal antibodies. Curr. Opin. Immunol. 20:493–499.

56. Swartzman, E. E., S. J. Miraglia, J. Mellentin-Michelotti, L. Evangelista,and P. M. Yuan. 1999. A homogeneous and multiplexed immunoassay forhigh-throughput screening using fluorometric microvolume assay technol-ogy. Anal. Biochem. 271:143–151.

57. Wiberg, F. C., et al. 2006. Production of target-specific recombinant humanpolyclonal antibodies in mammalian cells. Biotechnol. Bioeng. 94:396–405.

58. Wolffe, E. J., A. S. Weisberg, and B. Moss. 2001. The vaccinia virus A33Rprotein provides a chaperone function for viral membrane localization andtyrosine phosphorylation of the A36R protein. J. Virol. 75:303–310.

59. Zinoviev, V. V., N. A. Tchikaev, O. Y. Chertov, and E. G. Malygin. 1994.Identification of the gene encoding vaccinia virus immunodominant proteinp35. Gene 147:209–214.

VOL. 85, 2011 RECOMBINANT POLYCLONAL ANTIBODY AGAINST VACCINIA VIRUS 1833

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/j

vi o

n 12

Feb

ruar

y 20

22 b

y 19

4.44

.50.

96.