Department of Molecular and Cellular Biology, Centro Nacional de Biotecnología, CSIC, Campus Universidad Autónoma, E-28049 Madrid, Spain1
Author for correspondence: Mariano Esteban. Fax +34 91 585 4506. e-mail mesteban{at}cnb.uam.es
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Abstract |
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Introduction |
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Methods |
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Purification of mAb C3.
In order to isolate mAb C3 IgG (subtype G2a) present in ascites fluid, we used the MAb Trap GII kit (Amersham Pharmacia Biotech), following the manufacturer's instructions. Protein content of the eluted fractions was measured with the bicinchoninic acid (BCA) protein assay kit (Pierce). The starting material, the pooled flow-through and fractions containing IgGs were analysed by SDSPAGE (see Fig. 1).
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Inoculation of mice.
Female BALB/c mice (H-2d) (68 weeks old) were infected intraperitoneally (i.p.) with different doses (indicated as p.f.u.) of WRluc virus in 200 µl sterile PBS. For the administration of purified mAb C3, mice were injected i.p. with the indicated amounts of mAb C3 in 200 µl PBS. Control animals were injected with the same amount of BSA (Sigma) in PBS.
Measurement of luciferase activity in mouse tissues.
Gene expression of recombinant virus in different mouse tissues was followed by the highly sensitive luciferase assay, as described previously (Rodríguez et al., 1988 ; Ramírez et al., 2000
). At the times indicated post-inoculation, animals were sacrificed and the spleen and ovaries were removed aseptically, washed with sterile PBS and stored at -70 °C. Tissues from individual animals were homogenized in luciferase extraction buffer (300 µl per spleen and 200 µl per ovary) (Promega). Luciferase activity was measured in the presence of luciferin and ATP according to the manufacturers instructions using a Lumat LB 9501 Berthold luminometer and expressed as relative luciferase units (RLU) per mg protein. Protein content in the samples was measured as described above.
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Results |
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mAb C3 neutralizes VV infection in vivo
In order to investigate the in vivo neutralizing capacity of mAb C3 over VV, we used an animal model system based on BALB/c mice and, as the virus system, a recombinant VV (WRluc) that expresses the luciferase reporter gene in the TK locus under the control of the early-late VV promoter p7.5. We have described previously that this recombinant virus provides a highly sensitive indicator to follow virus replication in tissues of infected BALB/c mice, detecting one infected cell over a background of 106 non-infected cells, and that luciferase levels correlate with virus titres in different organs (Rodríguez et al., 1988 ). Since TK- viruses are 10-fold less virulent by the i.p. route in the mouse than wild-type virus (Buller et al., 1985
), we used doses of recombinant virus that either kill or do not kill the animals. The advantage of the luciferase assay over virus titration in tissues is that it provides a precise, quantitative measurement of the extent of virus replication. To examine the ability of mAb C3 to protect against a lethal challenge with VV, groups of four animals were injected by the i.p. route with 10 or 100 µg purified mAb C3 in PBS (corresponding to approximately 1000 and 10000 in vitro NT50 units). Control mice were injected with BSA (50 or 500 µg per mouse). One hour later, animals were challenged by the i.p. route with a lethal dose of 1x108 p.f.u. purified recombinant WRluc virus. In the purified virus stock, IMVs are the major infectious form present in the inoculum, and this virus preparation was used throughout this work. At the high dose used for virus inoculation, all control mice died within 3 days, with characteristic signs of illness, such as skin necrosis and ruffled hair. In contrast, animals pre-treated with 10 or 100 µg of mAb C3 survived following virus inoculation, with no signs of illness (Fig. 2a
). Lower doses of mAb C3 were not tested in this lethal model.
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Definition of the interval when mAb C3 prevents VV replication in mice target tissues under prophylactic conditions
Next, we investigated the length of time during which passive transfer of mAb C3 could be used to limit the replication of VV. Three groups of four female BALB/c mice, 68 weeks old, were injected i.p. with 10 µg purified mAb C3 or with BSA control and, at 1, 2 and 3 days after the administration of the antibody, each group was inoculated i.p. with a sub-lethal dose of 2·5x107 p.f.u. WRluc per mouse. The scheme for mAb C3 treatment, the time of virus infection and measurements of luciferase levels in ovaries and spleen are given in Fig. 3. As observed, pre-treatment with mAb C3 for 1 or 2 days was efficient in neutralizing VV infection, since luciferase levels in ovaries and spleens were close to background compared with the untreated control group. However, the neutralizing activity was negligible in mice given mAb C3 3 days before virus infection, as no differences on luciferase levels were observed between this group and the control group. The findings shown in Fig. 3(b)
revealed that mAb C3 was quite effective in blocking VV replication when administered in prophylactic schedule up to 2 days before VV challenge. Its efficacy decreased if given 3 days before VV inoculation, which might be due to the life-span of the antibody in the animal.
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Discussion |
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While VV encodes about 200 proteins, only a few of those polypeptides have been shown to induce neutralizing antibodies (the products of A27L, B5R, D8L, H3L and L1R). Of the two infectious forms of the virus, the most potent neutralizing antibodies are found against the IMV form (Czerny & Mahnel, 1990 ; Ichihashi, 1996
; Law & Smith, 2001
). This is interesting, considering that EEV is the form involved in virus dissemination through the host, thus contributing to distant virus spread, whereas IMV is probably more efficient in local cell-to-cell transmission by cellular fusion (Boulter & Appleyard, 1973
; Blasco & Moss, 1992
; Smith & Vanderplasschen, 1998
). Because of the infection characteristics of the two virus forms, antibodies in circulation should, in principle, be less efficient in vivo in neutralizing IMVs than EEVs. Indeed, early experiments in animal models using inactivated virus as vaccine showed that this non-replicating virus induced high neutralization titres against IMV but it protected the animals poorly against live virus challenge. In contrast, antiserum raised against a live virus could trigger protection efficiently (Turner & Squires, 1971
; Boulter & Appleyard, 1973
; Appleyard & Andrews, 1974
). These observations indicate that antigen presentation and the way that specific host-cell-mediated immune responses are activated by live virus are critical events in the immune mechanisms that lead to protection against a poxvirus infection. It is remarkable that little is known about epitopes in poxvirus antigens that activate specific cell immune responses. Virus proteins targeted for neutralization have been mapped in IMV (Gordon et al., 1991
; Rodríguez et al., 1987
; Hsiao et al., 1999
; Wolffe et al., 1995
) and immune protection with the purified 14-kDa protein has been obtained in mice upon challenge with VV (Lai et al., 1991
).
In this study, we have demonstrated in a mouse model that both prophylactic and therapeutic administration of a mAb directed against the 14-kDa IMV protein (mAb C3) can control, to differing extents, a systemic poxvirus infection in the host through inhibition of virus replication in target tissues. Administration of the neutralizing antibody prior to challenge with VV prevented early infection of target tissues to the extent that virus replication was nearly undetectable following infection. Indeed, survival after VV infection was observed in mice pre-treated with mAb C3 (Figs 2 and 5
). The protected animals did not experience weight loss and all survived a lethal challenge of the virus (Fig. 5
). The efficacy of mAb C3 administered prior to virus challenge is probably caused by direct neutralization of the virus inoculum. A non-specific response elicited in the mouse by the addition of IgG is unlikely, as other workers have shown that some antibodies to VV antigens inoculated into mice had no effect on a lethal virus challenge (Galmiche et al., 1999
). Importantly, after the onset of virus infection, mAb C3 was able to limit the infection, at least if the mAb was administered during the 3 days following infection with a sub-lethal dose of the virus. When a lethal dose of the virus was employed, the therapeutic intervention had reduced efficacy (Fig. 5
). The time of administration and dose of antibody in target tissues might be critical for efficient inhibition of virus replication. As shown in Fig. 3
, pre-incubation 1 or 2 days before infection with 10 µg mAb C3 protected mice against virus replication in the ovaries. Pre-treatment of mice with the same amount of antibody for 1 h and infection with only twice as much virus resulted in 10000 times greater luciferase expression in the ovaries (Fig. 2
). These data imply that when the antibody is applied and the concentration reached in target tissues are critical. Distinct features of the spleen and ovary (organized lymphoid and peripheral solid tissue, respectively) may have had a major influence on the effector mechanisms involved in the clearance of virus mediated by mAb C3. Whether this is due to accessibility of molecules and cells remains unclear, but this has been proposed in other virus systems (Zinkernagel et al., 1997
). In addition, the half-life of the antibody in circulation may limit its efficacy; this problem could be overcome by the use of higher concentrations of antibody or by repeated injections during the course of infection. Alternatively, it is also possible that the apparent absence of effectiveness when administered 3 days prior to VV challenge might have been underestimated, and collecting data at more than just 3 days p.i. could help to clarify this point. Finally, it will be interesting to address the effectiveness of mAb C3 administration when VV is inoculated by the intranasal or intradermal route, closer to natural poxvirus infection.
When mAb C3 was administered after VV infection, inhibition of virus replication in ovaries occurred after a refractory period (Fig. 4). Virus clearance was observed after 3 days p.i., regardless of the timing of mAb C3 administration (1, 2 or 3 days p.i.). Our findings showed clearly that neutralization of an IMV protein by mAb C3 controls the replication of VV efficiently in target tissues. As mAb C3 neutralizes through binding to the 14-kDa protein, which is exposed in IMV but not in EEV particles (Czerny & Mahnel, 1990
; Sodeik et al., 1995
; Vázquez et al., 1998
), these results suggest that, at 13 days p.i., the bulk of virus released from cells is probably EEV, and this is not neutralizable by mAb C3, whereas, at later times, the majority of virus released is probably IMV, as a result of cell lysis, and this form is sensitive to neutralization by mAb C3. However, other components of the immune system, like complement-dependent specific neutralization of VV infectivity, might contribute to virus clearance.
In conclusion, we have shown in this investigation that mAb C3 can be used in vivo as a prophylactic and therapeutic product to limit the infectious process of VV effectively. In the treated animals, the replication of a sub-lethal dose of virus was inhibited almost completely in the spleen and ovaries. This was observed even when the mAb C3 was administered 3 days p.i., the time when virus replication peaks in the ovaries. More importantly, pre-treatment with mAb C3 efficiently prevented VV replication at sub-lethal and lethal doses, since almost no virus gene expression was detectable in treated, infected animals and all animals survived a lethal virus challenge. Our data indicate that, during a prophylactic intervention, the incoming virus is neutralized rapidly by the circulating mAb C3, while, during therapeutic intervention, the virus produced by an early infection can be controlled only after a refractory period, during which the infectious process, probably the EEV form, is not sensitive to neutralization by mAb C3. In the therapeutic case, the extent of protection will be determined by the dose of virus inoculated. The sequence conservation of the 14-kDa protein in orthopoxviruses is greater than 95% at the amino acid level for the Bangladesh (BSH) and India (IND) strains of variola virus (Shchelkunov et al., 1995 ). There is a single amino acid substitution at position 40 (E to G) in the mAb C3-binding domain, amino acids 2943 (Vázquez et al., 1998
), between the 14-kDa proteins of VV Copenhagen strain (A27L) and variola BSH (A31L) and two substitutions at position 40 (E to G) and 42 (D to N) in variola IND (A30L) (http://www.poxviruses.org/). The role that the EEV and IMV forms of variola have in the initiation and spread of infection needs to be addressed. Due to sequence conservation of the 14-kDa protein between members of the orthopoxvirus group, this antiviral therapy may be used to control infection by pathogenic human poxviruses. This is the first report of an effective prophylactic and therapeutic intervention against a poxvirus infection by a neutralizing mAb. Humanizing mAb C3 could provide an antiviral compound against orthopoxviruses, particularly in the event of a monkeypox outbreak or bioterrorism attack with variola.
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Acknowledgments |
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References |
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Received 13 November 2001;
accepted 17 January 2002.