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1 Arbovirus Diseases Branch, Division of Vector-Borne Infectious Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Public Health Service, US Department of Health and Human Services, PO Box 2087, Fort Collins, CO 80522, USA
2 Alexion Antibody Technologies, San Diego, CA 92121, USA
Correspondence
Ann R. Hunt
arh4{at}cdc.gov
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ABSTRACT |
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Published online ahead of print on 7 June 2006 as DOI 10.1099/vir.0.81925-0.
The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are DQ487205–DQ487208.
Supplementary tables are available in JGV Online.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Monath & Trent, 1981). Equine epizootics have high mortality (38–83 %) and often lead to human epidemics involving thousands of cases (Groot, 1972; Rivas et al., 1997). Human disease is usually self-limiting with 1–4 % of cases progressing to severe encephalitis (Bronze et al., 2002). VEEV has also caused laboratory-acquired human infections via parenteral or airborne inoculation.
Experimental VEEV vaccines have been developed primarily for protection of laboratory workers and military troops (Pittman et al., 1996). A live-attenuated vaccine, TC-83, was developed by passage of Trinidad donkey (TrD) virus in guinea pig heart cells (Berge et al., 1961; Lord, 1974; McKinney, 1972; Sharman, 1972). The murine response to TC-83 vaccination develops rapidly. Protection against subcutaneous or airborne challenge from virulent TrD virus occurs at 4 days post-vaccination, and is associated with production of VEEV neutralizing antibodies (Burke et al., 1977; Ferguson et al., 1978; Fillis & Calisher, 1979; Johnson & Martin, 1974; Walton & Johnson, 1972). TC-83 vaccine is not available for general human use (Phillpotts et al., 2002).
VEEV contains two major surface glycoproteins, E1 and E2. Similar to Sindbis virus, the VEEV virion likely contains protein spikes organized as trimers of E1–E2 heterodimers (Parades et al., 2001; Phinney et al., 2000; Zhang et al., 2002). We have previously used murine monoclonal antibodies (mAbs) to analyse the antigenic structure of the VEEV E2 glycoprotein. These studies identified six epitopes (E2c–h) that form a critical viral neutralization domain (E2 aa 182–207). Anti-E2c mAbs, such as 3B4C-4, exhibit high virus neutralizing and protective activity by blocking virus attachment to cells (Roehrig et al., 1988; Roehrig & Mathews, 1985). More recently, anti-E2c and anti-E2g mAbs (1A4A-1 and 1A3A-9, respectively) were shown to protect mice prophylactically from an aerosol challenge with VEEV and also to cure mice from infection 24 h after virus inoculation (Phillpotts et al., 2002).
Based on the foregoing evidence, murine mAbs specific for epitopes within the critical neutralization domain may be useful in the prevention and treatment of VEEV infection in humans. However, rodent antibodies are highly immunogenic in humans and therefore limited in their clinical application. Antibody humanization is a process used to decrease the immunogenicity of rodent mAbs by replacing much of the murine amino acid sequence with human amino acids, while maintaining the original antigenic specificity. Clinical studies have indicated that humanized antibodies are less immunogenic than murine or chimeric antibodies and thus have more therapeutic potential (Hwang & Foote, 2005; Tsurushita et al., 2005). We have humanized mAb 3B4C-4 by using phage-display technology and rational antibody design (Kang et al., 1991; McCafferty et al., 1990; Rader et al., 1998; Scott & Smith, 1990; Smith, 1985). The resultant humanized mAb, designated Hy4-26C (Hy4 IgG), maintained the important biological activities of 3B4C-4 and was able to passively protect mice before both intraperitoneal (i.p.) and intranasal (i.n.) challenge with virulent VEEV and to cure mice after i.p. challenge.
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METHODS |
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), was used for library construction and panning in the bacterial strain ER2537 (New England BioLabs) that suppresses an amber stop between the end of the heavy (H) chain and the phage coat protein III to generate Fab-phage. This same vector was used with non-suppressor strain Top10F' (Invitrogen) to produce soluble Fab containing both HA-epitope and His6-purification tags. Hybridoma cell line 3B4C-4 has been described previously (Roehrig et al., 1982). Stable cell lines expressing Hy4 IgG were made in 293 EBNA cells using the Effectene reagent (Qiagen) and selected with puromycin concentrations of 5.0 or 20.0 µg ml–1.
The VEE complex viruses used in this study were TC-83 (variety 1AB), TrD (1AB), P676 (1C), 3880 (1D), Mena II (1E) and Everglades (EVE, strain Fe3-7C, subtype 2) and were from the Division of Vector-Borne Infectious Diseases (DVBID) stocks. Viruses grown in Vero or BHK-21 cells were purified by equilibrium density-gradient centrifugation (Obijeski et al., 1976). Purified TC-83 virus, used for panning phage-display libraries, was inactivated by treatment with 0.3 % 
-propiolactone in 0.1 M Trizma, pH 9, for 48 h at 4 °C. Virus inactivation was verified by inoculation of Vero cell culture or 4-day-old sucking ICR mice (intracerebrally) and monitored for cytopathic effect or signs of illness, respectively. Inactivated virus was evaluated for preservation of important epitopes (Roehrig & Mathews, 1985).
cDNA cloning of Fab genes from murine mAb 3B4C-4.
Total RNA isolated from 3B4C-4 hybridomas was used with First Strand cDNA kit (Boehringer Mannheim Biochemical) for the generation of oligo(dT)-primed cDNA. Forward primers were pooled into three or four mixes and then used in combination with the single reverse primer for either 
- or H-chain genes (Barbas et al., 2001). The light (L) chain products were digested by SacI/XbaI and cloned into pRL5, followed by XhoI/SpeI digestion and insertion of the H chains. The murine L and H chains in pRL5 were subjected to several rounds of phage-display selection on TC-83 antigen to ease the identification of the correct chains, essentially as described previously (Rader et al., 1998).
Generating chimeric 3B4C-4 H chain.
The murine variable region was amplified by PCR from a plasmid containing murine Fab 3B4C-4 cDNA using a murine forward primer (MHyVH1) (Barbas et al., 2001) and a chimeric H chain reverse primer annealing to the murine framework (FR) 4 region and having a tail of human constant H (CH)-chain region 1 (ChimHy4-B; Supplementary Table S1 available in JGV Online). The human CH1 domain was derived from a human Fab clone using PCR. The murine variable H (VH)-chain region and human CH1 domains were fused by overlap PCR and then cloned into pRL5. During the humanization process mAb amino acid composition was modified to include as many amino acids of human origin as possible, but maintaining the original epitope-binding specificity. Supplementary Table S2 (available in JGV Online) delineates the humanization steps and amino acid source found in intermediate Fab clones as well as in the final Hy4 Fab.
Humanization of the L chain.
The murine L chain complementarity-determining region (CDR) 3 was grafted into a library of rearranged human L chains. Human bone marrow mononuclear cells were obtained from Poietics/BioWhittaker. RNA was isolated and first strand cDNA was made as described above. The first PCR reactions were set up with human variable region forward primers (FR1 specific primers; Barbas et al., 2001), and reverse primers annealing at the end of FR3 containing a tail of 3B4C-4's CDR3 sequence [Hy4LCDR3b1, Hy4LCDR3b2, Hy4LCDR3b3 and Hy4LCDR3b4 (Supplementary Table S1 available in JGV Online)]. The resulting product included the front half of the humanized L chain library containing a portion of the 3B4C-4-specific CDR3. The back half of the humanized L chain library was generated using an FR4 forward primer Hy4LCDR3-F (Supplementary Table S1) containing a tail of 3B4C-4's CDR3 in combination with human constant region reverse primer CK1dX (Supplementary Table S1). A fusion PCR in which the CDR3 region provided the overlap was set up using these two halves of the L chain library, using the same protocol described above, with primers RSC-F (Supplementary Table S1) and CK1dX. The fusion PCR product was digested by SacI/XbaI and ligated into vector pRL5, which already contained the 3B4C-4 chimeric H chain. A phage-display library was created and panned on TC-83 antigen.
Humanization of the H chain.
The murine H chain gene was compared to human germline sequences using the VBase database (http://vbase.mrc-cpe.cam.ac.uk/) to identify the nearest match, which were the VH1 gene at locus 1-f and the J-gene JH3a. The human germline amino acids were used at all positions except the following: (i) CDR3 was entirely murine amino acid sequence, (ii) where choice of murine or human CDR1 and CDR2 was given, or (iii) where choice of amino acid was given due to divergence of human and murine amino acid at selected FR positions [Vernier zone (Foote & Winter, 1992) or VH/variable L (VL)-chain interface (Santos & Padlan, 1998) at amino acid positions 37, 48, 67, 69, 71 and 91] (Kabat et al., 1991).
The humanized H chain library was constructed using oligonucleotide-ligation assembly to create separate front- and back-halves of the VH-gene (Chalmers & Curnow, 2001; Sutton et al., 1995). Each half of the gene contained a portion of the CDR2 that was used for full VH-gene assembly by overlap PCR. For cloning purposes, the assembled VH-genes contained an XhoI restriction site just prior to the +1 amino acid, as well as a small portion of the CH1 region containing the native ApaI restriction site. Oligonucleotides were synthesized and then combined so that six complementary sets could be assembled (Table 1). Each oligonucleotide set was separately added to reaction mixtures containing 1x ligation buffer and Ampligase Thermostable DNA Ligase (Epicentre Technologies). Reactions were thermocycled for 30 cycles of 95 °C for 30 s, 60 °C for 30 s, 65 °C for 15 min (decreasing 1.0 min per cycle to a minimum of 1 min); followed by 65 °C for 15 min.
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Modification of Hy4-26 L chain.
DNA cassettes from FR1 to the beginning of FR3, created by Aptagen, incorporated human germline DPK9 sequence with bacterial codon preference, murine CDR1 and CDR2, as well as two positions of degeneracy where human germline and murine amino acid sequences differed in a Vernier zone and a VH/VL interface (Kabat #4 and #43). The two positions of choice resulted in a total of four cassettes. The cassettes were cloned into the L chain of Hy4-26 by using SacI/PpuMI. The four Fabs, and their corresponding amino acid at positions 4 and 43, were Hy4-26A (M4, P43), Hy4-26B (M4, A43), Hy4-26C (L4, A43) and Hy4-26D (L4, P43).
Conversion of Fab to IgG.
The humanized Hy4-26C Fab was cloned into a single vector expression system that allowed mammalian expression of the respective whole IgG1/. The first step involved placement of mammalian control elements between the Fab L and H chains. The Fab sequences were then moved into the final IgG expression vector that contained all remaining elements necessary for expression in mammalian cells. DNA was sequenced to confirm construction of the desired Hy4 IgG plasmid.
Antibody purification.
Fabs with HA-epitope and His6-purification tags were expressed in Top10F' bacteria. Small-scale purification was done using supernatants from overnight bacterial cultures with the Ni-NTA Spin kit (Qiagen) under native conditions according to kit instructions. Large-scale Fab purifications were performed on a nickel charged HiTrap 5 ml chelating HP column (Amersham Biosciences) using the periplasmic fraction of 2 l overnight bacterial cultures. Complete Hy4 IgG antibody was expressed and purified from a stable 293 cell line. This cell line was adapted to serum-free medium and then grown in a hollow fibre system. IgG expressed in the culture supernatant was purified by fast protein liquid chromatography over a protein A column.
Mice.
Swiss Webster mice were used for all passive antibody protection studies. The use of animals for research purposes complied with all relevant federal guidelines and specific protocols were approved by the DVBID Institutional Animal Care and Use Committee.
Enzyme immunoassays.
Indirect ELISAs for assaying antiviral murine sera or humanized antibody were performed essentially as described previously (Roehrig et al., 1980). Antibody binding to purified virus was detected by goat anti-species IgG–alkaline phosphatase (AP) conjugates (Jackson ImmunoResearch Laboratories). An absorbance ratio (A405 test sample/A405 negative control) of greater than two was considered to be positive. For the Fab ELISA, 200 ng inactivated TC-83 virus in 25 µl 0.1 M NaHCO3 coating buffer, pH 8.6, was applied to one-half area microtitre wells (CoStar High Bind; Corning). After overnight incubation at 4 °C the wells were blocked with 1 % BSA and bacterial supernatants were then added for 1 h at 37 °C. Subsequent 1 h incubations of anti-HA tag antibody 12CA5 (Roche Diagnostics) and anti-mouse IgG–AP conjugate (Sigma) were performed followed by addition of Sigma 104 substrate.
For the competition ELISA, purified Fabs containing an HA-epitope tag were used at a constant concentration corresponding to 80 % of maximum TC-83-binding activity. HA-tagged Fabs were mixed with increasing amounts of unlabelled competitor 3B4C-4 Fab. Binding of the HA-tagged Fabs to TC-83 virus was detected as described for the Fab ELISA.
Neutralization assays.
The plaque-reduction neutralization test (PRNT) for complete IgG antibodies was done according to established protocols in Vero cells (Monath, 1976), using approximately 60–100 p.f.u. of TC-83 virus; end-point titres corresponded to a 70 % reduction in the number of plaques. The PRNT assay for Fab antibody fragments included an incubation with 0.025 ml of a secondary antibody, anti-mouse or anti-human F(ab')2, for 30 min at 37 °C after incubation of the purified Fab (0.05 ml) virus (0.025 ml) mixture for 30 min at 37 °C (Mathews et al., 1985). The secondary-antibody incubation was done to promote cross-linking of Fab fragments to enhance neutralization.
Passive antibody transfer and virus challenge in mice.
For studies using prophylactic antibody treatment, 6–8-week-old Swiss Webster mice were inoculated i.p. with 100 µl of specified amounts of purified murine mAb 3B4C-4 or its humanized equivalent Hy4 IgG approximately 24 h prior to virus challenge. For therapeutic treatment, mice were inoculated i.p. with 10 µg mAb within 1 h of virus inoculation, or 24 or 48 h after virus infection. Mice were challenged i.p. with 100 mean morbidity doses (100 i.p. MD50) of VEEV (TrD) (126 p.f.u.) or VEEV IC (P676) (126 p.f.u.). Hy4 IgG-treated mice were also challenged i.p. with either 103, 105 or 107 i.p. MD50 or intranasally (i.n.) with 100 i.n. MD50 (1350 p.f.u. in 5 µl BA-1) of VEEV (TrD) to determine antibody protective capacity. Mice were monitored for signs of illness for 2 weeks; survivors were bled 14 days post-challenge. Although some animals died as a result of virus challenge, death was not a required end point. Animals were euthanized when they became severely ill or paralysed.
To determine the amount of passive antibody remaining in the peritoneal cavity at the time of virus inoculation, groups of five mice inoculated i.p. with either 100, 10, 1 or 0.1 µg Hy4 IgG in 100 µl vols were subjected to peritoneal lavage with 7 ml PBS 24 h following antibody transfer. The recovered lavage fluid was clarified and an aliquot was concentrated 10 times by ultrafiltration (Millipore), and was monitored for Hy4 IgG binding to TC-83 virus by ELISA. Absorbance values (A405) were compared to a standard curve of purified Hy4 IgG to estimate the amount of antibody in both lavage fluid and in total blood volume of animals given the same dose of mAb. The mean blood volume was estimated at 7.17 ml per 100 g body weight (Sluiter et al., 1984). The mean body weight for 8-week-old female mice was 32.72 g; therefore, the mean blood volume was 2.35 ml.
Nucleotide sequence accession numbers.
The nucleotide sequence data for murine mAb 3B4C-4 and humanized Hy4 IgG were assigned the following GenBank accession numbers: (i) 3B4C-4 murine L chain (DQ487205
[GenBank]
), (ii) 3B4C-4 murine V-gene and CH1 of H chain (DQ487206
[GenBank]
), (iii) Hy4-26C L chain (DQ487207
[GenBank]
) and (iv) Hy4-26C H chain (DQ487208
[GenBank]
).
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). This resulted in identification of three humanized L chain clones, Hy4-11, Hy4-14 and Hy4-43. For the H chain humanization, Fabs Hy4-26, -53 and -63 with three H chain murine CDRs were selected from a small rationally designed library. A competition ELISA showed that the HA-tagged humanized Fabs Hy4-26, -53 and -63 were competed by unlabelled 3B4C-4 Fab for binding to TC-83 virus, indicating that they bound at or near the original 3B4C-4 epitope on the virus (data not shown). Purified Fabs Hy4-26, -53 and -63 were also analysed by Western blot on reduced and non-reduced TC-83 virus and, like the murine mAb, specifically recognized the viral E2 glycoprotein (data not shown).
Modification of Hy4-26 L chain and ELISA activity of the modified Hy4-26 Fabs
To determine if a humanized Fab retaining all six murine CDRs would have improved activity, modifications were made to the Hy4-26 L chain. In addition to grafting in murine CDR1 and CDR2, two positions of degeneracy were allowed on the L chain where human germline and murine amino acid sequences differed. This generated four versions of the modified L chain: Fabs Hy4-26A, Hy4-26B, Hy4-26C and Hy4-26D.
The modified Fabs were purified and tested for binding to TC-83 virus by ELISA (data not shown). Fab Hy4-26A, as a representative of the panel, was then tested in a competition ELISA against murine 3B4C-4 Fab for binding to TC-83 virus. The results showed that Fabs Hy4-26A and 3B4C-4 competed for the same (or similar) TC-83 virus epitope (Fig. 1).
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). The Hy4-26 Fab and its four variants were able to neutralize virus efficiently compared with the unaltered, bacterially expressed murine 3B4C-4 Fab, indicating that for these Fabs the humanization process had not adversely affected this important biological function. Based on both the antigen-binding and neutralization results, Fab Hy4-26C was converted to whole IgG antibody for further study.
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). The Hy4 IgG ELISA and PRNT end-point titres on epizootic VEEV varieties 1AB and 1C demonstrated that the cross-reactivity pattern of 3B4C-4 had been generally maintained (Table 3) (Mathews & Roehrig, 1982; Roehrig & Mathews, 1985). Moreover, the neutralization titres of the humanized IgG antibody were as high as those of its murine counterpart on these VEEV varieties. However, the reactivity of Hy4 IgG was much reduced for the enzootic VEEV variety 1D and subtype 2, both by ELISA and PRNT, compared with 3B4C-4, suggesting that some alteration in antibody reactivity had occurred during the humanization process.
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). The 5 day clearance pattern was similar to that found for mAb 3B4C-4 given by the intravenous route (Mathews & Roehrig, 1982). Hy4 IgG was still detectable 2 weeks after transfer and the titre had decreased by a maximum of approximately threefold.
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). The survival rate was 70–100 % for the antibody-treated groups, including mice given as little as 100 ng Hy4 IgG (P<0.05, two-tailed Fisher's exact test). None of the control mice survived. The mean survival time (m.s.t.) for mice in the PBS control group was 6.0 days. Overall, just four mice died in two different antibody treatment groups, and these deaths occurred 6–10 days post-challenge. None of the surviving mice in any group, including those mice that mounted an anti-VEEV antibody response, showed signs of virus-induced morbidity (hair ruffling, ataxia). For comparison purposes, one group of mice was given 10 µg murine mAb 3B4C-4 and 90 % of these mice survived TrD virus challenge.
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). Of mice inoculated with 500 µg mAb 80 % survived the challenge. The dose of TrD virus, 1350 p.f.u., required for the i.n. challenge was nearly 11-fold greater than that used for i.p. challenge to produce similar morbidity in naïve mice. The m.s.t. for unprotected control mice that received an i.n. virus challenge was 4.9 days. Non-survivors that had an extended m.s.t. (6 days) were from the group that received 500 µg Hy4 IgG.
The survivor sera were tested by ELISA on TC-83 virus to determine the residual human antiviral IgG titre as well as to detect any de novo murine antibody response to the challenge virus (Table 4). The level of humanized IgG antibody remaining 2 weeks after challenge appeared to be generally related to the amount of antibody that was transferred, with log10 geometric mean titres (g.m.t.) ranging from a high of 3.83 to a low of 0.36 (corresponding to doses of 100 and 0.1 µg passive antibody, respectively). The titre of murine antiviral antibody was inversely related to the amount of the passive antibody dose; only those mice given the smallest dose of passive antibody (0.1 µg) had significant immune responses to the challenge virus (murine antiviral titres of 
1 : 12 800 for seven mice). For two mice inoculated with 100 µg Hy4 IgG, there was an apparent problem with the antibody transfer, since very little humanized IgG could be detected 14 days post-challenge (Table 4, row 2). These two mice evidently received enough passive antibody to allow them time to mount a significant protective antibody response (log10 g.m.t. of 5.05) and they showed no signs of illness. Based on the results of the titration of passive antibody (Table 4), a standard dose of 10 µg Hy4 IgG was used for all subsequent passive antibody–virus challenge experiments. The capacity of a 10 µg dose of Hy4 to protect mice from a range of TrD virus challenge doses (102–107 i.p. MD50) was examined (Table 5). Significant numbers of mice were protected at each virus challenge dose, up to the maximum possible challenge of 107 i.p. MD50 (2.26x107 p.f.u.) (P<0.001 for mAb-treated groups challenged with 102, 103 or 107 i.p. MD50 TrD virus, and P<0.005 for the group challenged with 105 i.p. MD50 TrD virus). None of the surviving mice, even those that mounted an anti-VEEV response, showed any overt signs of illness. Survivor sera were evaluated by ELISA on TC-83 virus for both humanized and murine antiviral antibodies (Table 5). Residual Hy4 IgG was detected only in survivors given the two lowest virus challenge doses (102 and 103 i.p. MD50); conversely, higher levels of induced murine antibodies were found only in survivors of the two highest virus challenge doses (105 and 107 i.p. MD50). A 10 µg dose of Hy4 IgG, administered 24 h prior to virus challenge, was also able to protect significant numbers of mice from lethal infection with 100 i.p. MD50 of epizootic VEEV variety 1C (P676) (10 of 10 mice survived; P<0.05).
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Therapeutic administration of Hy4 IgG protected mice up to 24 h after TrD virus infection
Ten micrograms of humanized Hy4 IgG or murine mAb 3B4C-4 were inoculated i.p. at different times following TrD virus infection (100 i.p. MD50) in Swiss Webster mice to determine the curative capacity of these antibodies (Table 6). mAb 3B4C-4 was able to protect mice if given within 1 h of virus challenge (P<0.05); however, Hy4 IgG was able to cure mice up to 24 h following TrD virus infection (P<0.05). Although some mice did survive virus infection when treated with 3B4C-4 24 h later, or with Hy4 IgG 48 h later, the numbers were not statistically significant. None of the surviving mice showed clinical signs of infection at any time during the 14 day observation period.
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DISCUSSION |
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; Men et al., 2004; Rader et al., 1998, 2000; Santos & Padlan, 1998; Steinberger et al., 2000). The humanized Fab Hy4-26 had the best PRNT titres of the three clones tested. Modification of the L chain of Fab Hy4-26 was performed to further improve its activity, and these changes increased PRNT titres to within fourfold of 3B4C-4 Fab. Competition ELISA indicated that the humanized Fabs had retained the murine mAb's epitope specificity. After converting the modified Fab Hy4-26C to whole IgG, the resulting complete, humanized antibody contained about 9.7 % murine residues.
The association of in vitro neutralizing activity with in vivo protection for immunoglobulins used in passive immunization is well known (Parren & Burton, 2001). Hy4 IgG and murine mAb 3B4C-4 were nearly equal in concentration of antibody required to achieve a 70 % PRNT end point (Table 3). Although variation in testing protocols used by other investigators makes it difficult to directly compare antibody concentrations required to achieve in vitro neutralization end points, the 70 % PRNT end-point concentration of 39 ng ml–1 for Hy4 IgG makes this antibody one of the most effective compared with other antiviral recombinant humanized or human IgGs (Higo-Moriguchi et al., 2004; Johnson et al., 1997; Maruyama et al., 1999; Men et al., 2004; Tempest et al., 1991; Tsui et al., 1996). Hy4 IgG also demonstrated other desirable characteristics for a recombinant, humanized antibody: (i) retaining reactivity of parental mAb with epizootic VEEV strains, (ii) not enhancing virus replication nor interfering with the host immune response, and (iii) exhibiting protective capacity in an established animal model for VEEV infection.
The Hy4 IgG prophylactic and therapeutic capacities are the best indicator of its potential effectiveness for human use (Tables 4–6). This protective capacity was also shown to extend to the epizootic 1C VEEV variety. Hy4 IgG showed effective protection, even at a dose of 100 ng, versus a robust TrD virus challenge dose (Table 4). This result was not surprising since our previous studies with 3B4C-4 determined that no TrD virus was detected in most samples of spleen, brain or serum from passively protected mice on days 1 through 5 after virus challenge, nor was there any significant decrease in passive antibody levels in these tissues over the 5 day critical infectious period (Mathews & Roehrig, 1982).
Prophylactic Hy4 IgG also protected Swiss Webster mice from i.n. virus challenge, although a significantly higher antibody dose (500 µg) was required compared with that needed for protection from virus inoculated by the i.p. route (Table 4). This was likely due to the 10-fold higher dose of TrD virus needed for successful i.n. challenge versus i.p. challenge. The i.n. route also resulted in more rapid morbidity, with an m.s.t. of 4.9 days in untreated, control mice. Although Phillpotts et al. (2002) found that 100 µg of an anti-VEEV E2c mAb (1A4A-1) protected mice from aerosol challenge with TrD virus, the mice used were the inbred BALB/c strain, which may be more readily protected against an airborne infection (Hart et al., 1997). In Phillpotts study, 100 aerosol LD50 doses of TrD virus corresponded to approximately 89 p.f.u., a viral dose similar to that used in the current study (126 p.f.u.) for outbred mice, which were protected from i.p. VEEV challenge with a 100 µg dose of passive antibody.
Passive antibody persisted in the serum for at least 14 days, and the amount of Hy4 IgG remaining was generally related to the amount transferred and inversely related to the virus challenge dose (Tables 4 and 5). At the higher challenge doses (105 and 107 i.p. MD50) no Hy4 IgG was detected after 14 days post-challenge, but significant numbers of mice were protected. This type of survival data versus increasing i.p. challenge doses were very similar to passive doses (5 and 20 µg) of murine 3B4C-4 mAb transferred by the intravenous route (Mathews & Roehrig, 1982). It is probable that the 10 µg dose of Hy4 IgG reduced the initial high viraemia and allowed mice to mount an effective response to the infection. This hypothesis is supported by the presence of high-titred murine antiviral antibody in day 14 sera from these survivors.
The failure to detect de novo murine IgG production in some protected animals suggests development of nearly complete sterilizing immunity following transfer of 1–100 µg Hy4 IgG with a challenge dose of 100 i.p. MD50 TrD virus, and 500 µg Hy4 IgG with a challenge of 100 i.n. MD50 TrD virus (Table 4). It is likely that there was substantial virus replication in mice immunized with 100 ng Hy4 IgG, as evidenced by the high-titred murine antiviral response. The amount of Hy4 IgG transferred ranged from a high of approximately 3 mg (kg body weight)–1 to a low of 3 µg kg–1. Since significant protection from i.p. challenge was afforded at all antibody doses, Hy4 IgG compares favourably with anti-Ebola virus neutralizing recombinant mAb KZ52, which protected five of five guinea pigs from lethal Ebola Zaire virus challenge at 25 mg antibody (kg body weight)–1, but protected only two of three animals at 5 mg kg–1 (Parren et al., 2002). For mice challenged by the i.n. route, 15 mg antibody (kg body weight)–1 protected 80 % of the animals.
The persistence of Hy4 serum titre in mice for the 2 week observation period (Fig. 2) indicated that passive antibody could be an effective prophylactic for at least several weeks in mice. In human clinical trials, humanized antibodies have been shown to have reduced immunogenicity compared with murine mAbs and also to have prolonged serum half-lives (Nishibori et al., 2006). Reported pharmacokinetics of a variety of humanized mAbs used in clinical trials reveal that serum half-life can vary from 6 to 7 days [HuOKT3 (Woodle et al., 1999) and HuM195 (Caron et al., 1998)] to longer than 20 days [rhuMAbVEGF (Gordon et al., 2001) and epratuzumab (Leonard et al., 2004)].
Only recently has it been reported that anti-VEEV mAbs can be used therapeutically to cure mice from an established VEEV infection (Phillpotts et al., 2002). In our study, successful post-exposure antibody treatment with Hy4 IgG probably helped to control the initial course of infection, thus allowing the host to mount an effective immune response. It is probable that passive antibody could act at the level of the infected cell, as well as with free virus particles, to help control infection-related tissue damage (Parren et al., 2002). The relatively short m.s.t. (5–6 days) of naïve mice infected with VEEV, as well as the development of peak virus titres in the brain within 3 days of peripheral infection, probably limits the window of opportunity for successful post-exposure treatment (Mathews & Roehrig, 1982). We were able to demonstrate, however, that passive antibody protected mice up to 24 h after infection (Table 6). Further evaluation of the protective power of this humanized antibody in non-human primates challenged with virulent VEEV will be necessary to establish clinical relevance of this approach.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Berge, T. O., Banks, I. S. & Tiggert, W. D. (1961). Attenuation of Venezuelan equine encephalomyelitis virus by in vitro cultivation in guinea-pig heart cells. Am J Hyg 73, 209–218.
Bronze, M. S., Huycke, M. M., Machado, L. J., Voskuhl, G. W. & Greenfield, R. A. (2002). Viral agents as biological weapons and agents of bioterrorism. Am J Med Sci 323, 316–325.[CrossRef][Medline]
Burke, D. S., Ramsburg, H. H. & Edelman, R. (1977). Persistence in humans of antibody to subtypes of Venezuelan equine encephalomyelitis (VEE) virus after immunization with attenuated (TC-83) VEE virus vaccine. J Infect Dis 136, 354–359.[Medline]
Caron, P. C., Dumont, L. & Scheinberg, D. A. (1998). Supersaturating infusional humanized anti-CD33 monoclonal antibody HuM195 in myelogenous leukemia. Clin Cancer Res 4, 1421–1428.[Abstract]
Chalmers, F. M. & Curnow, K. M. (2001). Scaling up the ligase chain reaction-based approach to gene synthesis. Biotechniques 30, 249–252.[Medline]
Ferguson, J. A., Reeves, W. C., Milby, M. M. & Hardy, J. L. (1978). Study of homologous and heterologous antibody response in California horses vaccinated with attenuated Venezuelan equine encephalomyelitis vaccine (strain TC-83). Am J Vet Res 39, 371–376.[Medline]
Fillis, C. A. & Calisher, C. H. (1979). Neutralizing antibody responses of humans and mice to vaccination with Venezuelan encephalitis (TC-83) virus. J Clin Microbiol 10, 544–549.
Foote, J. & Winter, G. (1992). Antibody framework residues affecting the conformation of the hypervariable loops. J Mol Biol 224, 487–499.[CrossRef][Medline]
Gordon, M. S., Margolin, K., Talpaz, M., Sledge, G. W. Jr, Holmgren, E., Benjamin, R., Stalter, S., Shak, S. & Adelman, D. (2001). Phase I safety and pharmacokinetic study of recombinant human anti-vascular endothelial growth factor in patients with advanced cancer. J Clin Oncol 19, 843–850.
Groot, H. (1972). The health and economic impact of Venezuelan equine encephalitis (VEE). In Venezuelan Encephalitis, pp. 7–16. Edited by PAH Organization. Washington, DC.
Hart, M. K., Pratt, W., Panelo, F., Tammariello, R. & Dertzbaugh, M. (1997). Venezuelan equine encephalitis virus vaccines induce mucosal IgA responses and protection from airborne infection in BALB/c, but not C3H/HeN mice. Vaccine 15, 363–369.[CrossRef][Medline]
Higo-Moriguchi, K., Akahori, Y., Iba, Y., Kurosawa, Y. & Taniguchi, K. (2004). Isolation of human monoclonal antibodies that neutralize human rotavirus. J Virol 78, 3325–3332.
Hwang, W. Y. K. & Foote, J. (2005). Immunogenicity of engineered antibodies. Methods 36, 3–10.[CrossRef][Medline]
Johnson, K. M. & Martin, D. H. (1974). Venezuelan equine encephalitis. Adv Vet Sci Comp Med 18, 79–116.[Medline]
Johnson, S., Oliver, C., Prince, G. A. & 13 other authors (1997). Development of a humanized monoclonal antibody (MEDI-493) with potent in vitro and in vivo activity against respiratory syncytial virus. J Infect Dis 176, 1215–1224.[Medline]
Johnstone, R. E. & Peters, C. J. (1995). Alphaviruses. In Fields Virology, 3rd edn, pp. 843–898. Edited by B. N. Fields, D. M. Knipe & P. M. Howley. Philadelphia, PA: Lippincott–Raven.
Kabat, E. A., Wu, T. T., Perry, H. M., Gottesman, K. S. & Foeller, C. (1991). Sequences of Proteins of Immunological Interest, 5th edn. Edited by US Department of Health and Health Services. Bethesda, MD.
Kang, A. S., Barbas, C. F., Janda, K. D., Benkovic, S. J. & Lerner, R. A. (1991). Linkage of recognition and replication functions by assembling combinatorial antibody Fab libraries along phage surfaces. Proc Natl Acad Sci U S A 88, 4363–4366.
Leonard, J. P., Coleman, M., Ketas, J. C. & 14 other authors (2004). Epratuzumab, a humanized anti-CD22 antibody, in aggressive non-Hodgkin's lymphoma: phase I/II clinical trial results. Clin Cancer Res 10, 5297–5298.
Lord, R. D. (1974). History and geographic distribution of Venezuelan equine encephalitis. Bull Pan Am Health Organ 8, 100–110.[Medline]
Maruyama, T., Rodriguez, L. L., Jahrling, P. B., Sanchez, A., Khan, A. S., Nichol, S. T., Peters, C. J., Parren, P. W. H. I. & Burton, D. R. (1999). Ebola virus can be effectively neutralized by antibody produced in natural human infection. J Virol 73, 6024–6030.
Mathews, J. H. & Roehrig, J. T. (1982). Determination of the protective epitopes on the glycoproteins of Venezuelan equine encephalomyelitis virus by passive transfer of monoclonal antibodies. J Immunol 129, 2763–2767.[Abstract]
Mathews, J. H., Roehrig, J. T. & Trent, D. W. (1985). Role of complement and the Fc portion of immunoglobulin G in immunity to Venezuelan equine encephalomyelitis virus infection with glycoprotein-specific monoclonal antibodies. J Virol 55, 594–600.
McCafferty, J., Griffiths, A. D., Winter, G. & Criswell, D. J. (1990). Phage antibodies: filamentous phage displaying antibody variable domains. Nature 348, 552–554.[CrossRef][Medline]
McKinney, R. W. (1972). Inactivated and live VEE vaccines – a review. In Venezuelan Encephalitis, pp. 369–376. Edited by PAH Organization. Washington DC.
Men, R., Yamashiro, T., Goncalvez, A. P., Wernly, C., Schofield, D. J., Emerson, S. U., Purcell, R. H. & Lai, C. J. (2004). Identification of chimpanzee Fab fragments by repertoire cloning and production of a full-length humanized immunoglobulin G1 antibody that is highly efficient for neutralization of dengue type 4 virus. J Virol 78, 4665–4674.
Monath, T. P. (1976). Togaviruses, bunyaviruses, and Colorado tick fever. In Handbook of Clinical Microbiology, pp. 597–603. Edited by N. R. Rose & H. Friedman. Washington, DC: American Society for Microbiology.
Monath, T. P. & Trent, D. W. (1981). Togaviral diseases of domestic animals. In Comparative Diagnosis of Viral Diseases, pp. 331–440. Edited by E. Kurstak & C. Kurstak. New York: Academic Press.
Nishibori, N., Horiuchi, H., Furusawa, S. & Matsuda, H. (2006). Humanization of chicken monoclonal antibody using phage-display system. Mol Immunol 43, 634–642.[CrossRef][Medline]
Obijeski, J. F., Bishop, D. H., Murphy, F. A. & Palmer, E. L. (1976). Structural proteins of La Crosse virus. J Virol 19, 985–997.
Paredes, A., Alwell-Warda, K., Weaver, S. C., Chiu, W. & Watowich, S. J. (2001). Venezuelan equine encephalomyelitis virus structure and its divergence from old world alphaviruses. J Virol 75, 9532–9537.
Parren, P. W. H. I. & Burton, D. R. (2001). The anti-viral activity of antibodies in vitro and in vivo. In Advances in Immunology, pp. 195–262. New York: Academic Press.
Parren, P. W. H. I., Gieisbert, T. W., Maruyama, T., Jahrling, P. B. & Burton, D. R. (2002). Pre- and postexposure prophylaxis for Ebola virus infection in an animal model by passive transfer of a neutralizing human antibody. J Virol 76, 6408–6412.
Phillpotts, R. J., Jones, L. D. & Howard, S. C. (2002). Monoclonal antibody protects mice against infection and disease when given either before or up to 24 h after airborne challenge with virulent Venezuelan equine encephalitis virus. Vaccine 20, 1497–1504.[CrossRef][Medline]
Phinney, B. S., Blackburn, K. & Brown, D. T. (2000). The surface conformation of Sindbis virus glycoproteins E1 and E2 at neutral and low pH, as determined by mass spectrometry-based mapping. J Virol 74, 5667–5678.
Pittman, P. R., Makuch, R. S., Mangiafico, J. A., Cannon, T. L., Gibbs, P. H. & Peters, C. J. (1996). Long-term duration of detectable neutralizing antibodies after administration of live-attenuated VEE vaccine and following booster vaccination with inactivated VEE vaccine. Vaccine 14, 337–343.[CrossRef][Medline]
Rader, C., Cheresh, D. A. & Barbas, C. F. III (1998). A phage display approach for rapid antibody humanization: designed combinatorial V gene libraries. Proc Natl Acad Sci U S A 95, 8910–8915.
Rader, C., Ritter, G., Nathan, S. & 7 other authors (2000). The rabbit antibody repertoire as a novel source for the generation of therapeutic human antibodies. J Biol Chem 275, 13668–13676.
Rivas, F., Diaz, L. A., Cardenas, V. M. & 18 other authors (1997). Epidemic Venezuelan equine encephalitis in La Guajira, Colombia, 1995. J Infect Dis 175, 828–832.[Medline]
Roehrig, J. T. & Mathews, J. H. (1985). The neutralization site on the E2 glycoprotein of Venezuelan equine encephalomyelitis (TC-83) virus is composed of multiple conformationally stable epitopes. Virology 142, 347–356.[CrossRef][Medline]
Roehrig, J. T., Corser, J. A. & Schlesinger, M. J. (1980). Isolation and characterization of hybrid cell lines producing monoclonal antibodies directed against the structural proteins of Sindbis virus. Virology 101, 41–49.[CrossRef][Medline]
Roehrig, J. T., Day, J. W. & Kinney, R. M. (1982). Antigenic analysis of the surface glycoproteins of a Venezuelan equine encephalomyelitis virus (TC-83) using monoclonal antibodies. Virology 118, 269–278.[CrossRef][Medline]
Roehrig, J. T., Hunt, A. R., Kinney, R. M. & Mathews, J. H. (1988). In vitro mechanisms of monoclonal antibody neutralization of alphaviruses. Virology 165, 66–73.[CrossRef][Medline]
Santos, A. D. & Padlan, E. A. (1998). Development of more efficacious antibodies for medical therapy and diagnosis. Prog Nucleic Acid Res Mol Biol 60, 169–194.[Medline]
Scott, J. K. & Smith, G. P. (1990). Searching for peptide ligands with an epitope library. Science 249, 386–390.
Sharman, R. (1972). Equine diseases. In Venezuelan Encephalitis, pp. 221–223. Edited by PAH Organization. Washington, DC.
Sluiter, W., Oomens, L. W., Brand, A. & Van Furth, R. (1984). Determination of blood volume in the mouse with 51chromium-labelled erythrocytes. J Immunol Methods 73, 221–225.[CrossRef][Medline]
Smith, G. P. (1985). Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228, 1315–1317.
Steinberger, P., Sutton, J. K., Rader, C., Elia, M. & Barbas, C. F. III (2000). Generation and characterization of a recombinant human CCR5-specific antibody. A phage display approach for rabbit antibody humanization. J Biol Chem 275, 36073–36078.
Sutton, D. W., Havstad, P. K. & Kemp, J. D. (1995). Rapid gene synthesis using ampligase thermostable DNA ligase. Epicentre Forum 2, 1–2.
Tempest, P. R., Bremmer, P., Lambert, M., Taylor, G., Furze, J. M., Carr, F. J. & Harris, W. J. (1991). Reshaping a human monoclonal antibody to inhibit human respiratory syncytial virus infection in vivo. Biotechnology 9, 266–276.[CrossRef][Medline]
Tsui, P., Tornetta, M. A., Ames, R. S., Bankosky, B. C., Griego, S., Silverman, C., Porter, T., Moore, G. & Sweet, R. W. (1996). Isolation of neutralizing human RSV antibody from a dominant, non-neutralizing immune repertoire by epitope-blocked panning. J Immunol 157, 772–780.[Abstract]
Tsurushita, N., Hinton, P. R. & Kumar, S. (2005). Design of humanized antibodies: from anti-Tac to Zenapax. Methods 36, 69–83.[CrossRef][Medline]
Walton, T. E. & Johnson, K. M. (1972). Epizootiology of Venezuelan equine encephalomyelitis in the Americas. J Am Vet Med Assoc 161, 1509–1515.[Medline]
Woodle, E. S., Xu, D., Zivin, R. A. & 8 other authors (1999). Phase I trial of a humanized, Fc receptor nonbinding OKT3 antibody, huOKT3gamma (Ala-Ala) in the treatment of acute renal allograft rejection. Transplantation 68, 608–616.[CrossRef][Medline]
Zhang, W., Mukhopadhyay, S., Pletnev, S. V., Baker, T. S., Kuhn, R. J. & Rossman, M. G. (2002). Placement of the structural proteins of Sindbis virus. J Virol 76, 11645–11658.
Received 8 February 2006;
accepted 9 May 2006.
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) or murine Fab 3B4C-4 (
) on TC-83 virus. HA-tagged Fab concentrations were held constant and were separately mixed with increasing concentrations of untagged competing 3B4C-4 Fab. HA-tagged Fab bound to virus was detected using an anti-HA secondary antibody.
), (n=10); 10 µg Hy4 IgG (