Administration to mice of a monoclonal antibody that neutralizes the intracellular mature virus form of vaccinia virus limits virus replication efficiently under prophylactic and therapeutic conditions

Juan C. Ramírez1, Esther Tapia1 and Mariano Esteban1

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


   Abstract
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Abstract
Introduction
Methods
Results
Discussion
References
 
The WHO smallpox eradication program was concluded 21 years ago and the non-vaccinated population is now at risk of poxvirus infections, either by contact with monkeypox or through bioterrorism. Since drugs specific against poxvirus infections are limited, neutralizing monoclonal antibodies (mAbs) that are effective in vivo may be an important tool in controlling poxvirus infections. To this end, we studied the efficacy of the mAb C3, reactive against the trimeric 14-kDa protein of vaccinia virus (VV) localized in the membrane of the intracellular form of mature virus, for its ability to neutralize VV infection in mice. The results show that prophylactic as well as therapeutic administration of mAb C3 can be an effective means of control of VV replication within the host. The interval of antibody efficacy following a single administration, before and after VV inoculation, has been defined. This study reinforces the notion that neutralizing mAbs should be developed to control health-related human infections by poxviruses.


   Introduction
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Abstract
Introduction
Methods
Results
Discussion
References
 
Twenty-one years after the official certification of worldwide smallpox eradication (WHO, 1980 ), there are still concerns for safety and risks of poxvirus outbreaks (Breman & Henderson, 1998 ). The current risk lies in the fact that the population under 25 years of age is susceptible to infection with variola and the fact that only limited amounts of human vaccine and neutralizing hyperimmune serum are available. The recent outbreaks of human cases of monkeypox (Mukinda et al., 1997 ; Breman, 2000 ) and the potential use of virulent poxviruses as bioterrorist weapons make it imperative to find alternative methods that can be used rapidly to control outbreaks of human poxvirus infection. Although passive transfer of antibodies prior to or during an infection has been shown to reduce the severity of the disease or to inhibit secondary spread of the virus (Kempe, 1960 ), current knowledge is limited with regard to the target proteins in variola and vaccinia viruses that harbour neutralizing epitopes that are capable of directing and efficiently controlling the virus infectious process. Mouse monoclonal antisera raised against these virus components could be useful to evaluate efficacy in animal models, before developing monoclonal antibodies (mAbs) for human use. Among the nearly 200 proteins encoded by vaccinia virus (VV), five virus products, encoded by the genes H3L (Gordon et al., 1991 ), A27L (Rodríguez et al., 1985 ), B5R (Galmiche et al., 1999 ), D8L (Hsiao et al., 1999 ) and L1R (Wolffe et al., 1995 ), are known to contain epitopes that elicit neutralizing antibodies, while other proteins can induce different protective immune responses (Hooper et al., 2000 ). One of the best-characterized and most promising proteins for immune protective responses against human poxvirus infection is the product of the A27L gene of VV, the 14-kDa protein. This is a product synthesized late during virus infection. It forms covalently linked trimers and is localized in the envelope of the intracellular mature virus (IMV) form (Rodríguez et al., 1987 ; Sodeik et al., 1995 ). The 14-kDa protein plays an important role in the biology of the virus, acting in virus-to-cell and cell-to-cell fusion (Dallo et al., 1987 ; Gong et al., 1990 ; Rodríguez et al., 1987 ; Rodríguez & Smith, 1990 ). The N terminus of the 14-kDa protein contains a heparin-binding domain, a fusion domain and a domain responsible for interacting with proteins or lipids in the Golgi stacks for extracellular enveloped virus (EEV) formation and virus spread (Vázquez & Esteban, 1999 ). The 14-kDa protein interacts with the 21-kDa protein (A17L gene) through a C-terminal domain (Rodríguez et al., 1993 ), and a structural model of the trimeric form has been proposed (Vázquez et al., 1998 ). A mAb, C3, raised against this protein neutralizes the virus in vitro (Rodríguez et al., 1985 ) and immunization of mice with the purified protein elicits protection after lethal challenge with VV (Lai et al., 1991 ). In the course of the infection, VV produces two well-recognized infectious particles, IMV and EEV, the latter composed of IMV wrapped by a double membrane acquired from the Golgi apparatus (Hiller & Weber, 1985 ; reviewed in Moss, 1996 ). Both forms of the virus are neutralizable with antibodies (Appleyard & Andrews, 1974 ; Law & Smith, 2001 ; Turner & Squires, 1971 ) and they have different immunological and biological properties (Vanderplasschen et al., 1997 ). In this investigation, we have focused on the effect that systemic administration of mAb C3 (passive transfer) in BALB/c mice has on replication of the Western Reserve (WR) strain of VV. The results show that passive transfer of mAb C3 is an efficient prophylactic and therapeutic intervention to limit a poxvirus infection.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Viruses and cells.
VV recombinants employed in this study were derived from the WR strain. WRluc, expressing the luciferase and {beta}-galactosidase genes inserted in the thymidine kinase (TK) gene, was generated according to standard methods and has been described previously (Rodríguez et al., 1988 ). Intracellular WRluc was grown in HeLa cells, purified through a sucrose cushion and titrated in African green monkey kidney (BSC-40) cells as described previously (Ramírez et al., 2000 ).

{blacksquare} 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 SDS–PAGE (see Fig. 1).



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Fig. 1. Purification of mAb C3 and in vitro neutralization activity. (a) Coomassie blue-stained SDS–PAGE gel loaded with 1:100 dilution of eluted fractions containing IgGs from the HiTrap rProtein A column (Amersham Pharmacia Biotech). The flow-through as well as the ascites fluid are shown. Molecular mass markers are indicated on the left. (b) Neutralization titres of the eluted fraction #13 ({bullet}) and the original ascites fluid ({square}). The indicated dilutions of the IgG-containing fraction and ascites fluid were incubated for 1 h at 37 °C with 400 p.f.u. wild-type WR virus and the inhibition of plaque formation on BSC-40 cells was then measured. Values represent mean percentages of neutralization [(p.f.u. with sample/p.f.u. with pre-immune serum)x100] obtained in triplicate. Arrows indicate the NT50.

 
{blacksquare} Neutralization assay.
Sera obtained from animals were inactivated at 56 °C for 30 min, serial dilutions were made in PBS supplemented with 2% FCS and samples were incubated with 400 p.f.u. WRluc at 37 °C for 1 h. Confluent BSC-40 cell monolayers were infected in triplicate with the different virus dilutions and the resulting plaques were visualized at 48 h post-infection (p.i.) after crystal violet staining and counted. As a control, inactivated serum from mock-infected mice was employed. The neutralization titre (NT50) is the reciprocal dilution of antibody that results in a 50% reduction in the number of virus plaques.

{blacksquare} Inoculation of mice.
Female BALB/c mice (H-2d) (6–8 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.

{blacksquare} 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 manufacturer’s 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.


   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
In vitro neutralization of VV by purified mAb C3
Before carrying out experiments in vivo, we first sought to purify mAb C3 to avoid problems associated with the administration of the high protein content present in the original ascites fluid. Thus, ascites fluid was applied to a Protein A–Sepharose column and, after washing, the eluted fractions containing the IgGs (Fig. 1a) were used to study the in vitro neutralization activity of VV in comparison with the original ascites fluid. The neutralizing titre (NT50) was 1:5000 for the ascites fluid and 1:3000 for purified mAb C3 (Fig. 1b), in agreement with previous data (Rodríguez et al., 1985 ). To estimate the specific neutralizing activity [expressed as the lowest concentration (µg/ml) of protein that gives the NT50], we measured the protein content of the ascites fluid and the IgG fractions, and these were 3 and 0·067 µg/ml, respectively. Thus, after IgG purification, a 45-fold enrichment was achieved in the neutralizing activity of mAb C3 from ascites fluid.

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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Fig. 2. In vivo neutralization of vaccinia virus with mAb C3. (a) Four BALB/c mice were injected i.p. with either 10 ({blacksquare}) or 100 ({diamondsuit}) µg purified mAb C3 diluted in PBS or with control BSA ({circ}) and inoculated i.p. 1 h later with 1x108 p.f.u. WRluc per mouse. Dead animals were scored daily and represented as the percentage of surviving animals. (b) Three BALB/c mice per group were injected i.p. with either 0 ({circ}), 1 ({blacktriangleup}) or 10 ({blacksquare}) µg purified mAb C3 diluted in PBS and inoculated i.p. 1 h later with 5x107 p.f.u. WRluc per animal. At the times p.i. indicated, virus gene expression was evaluated by the luciferase assay. Luciferase activity, related to the amount of protein present in tissue extracts (RLU/mg protein), is represented. Background levels found in tissues from control uninfected mice are shown as shaded rectangles. Results represent mean values from samples of three animals per day and group with standard deviations.

 
We next studied virus replication in target tissues from mice that received an i.p. injection of 1, 10 or 100 µg mAb C3 and, 1 h later, were inoculated with WRluc (5x107 p.f.u. per mouse). The dose of WRluc was reduced to ensure survival of untreated infected animals and to allow virus to spread to tissues (Rodríguez et al., 1988 ). By comparing luciferase levels in untreated and mAb C3-treated animals, the effect of the antibody on VV replication could be defined. Virus replication was followed in the spleens of 6- to 8-week-old female BALB/c mice at 1–3 days p.i. and in the ovaries at 1–5 days p.i. VV replicates very efficiently in these tissues, but it persists longer in ovaries than in the spleen. On the various days p.i., the spleen and ovaries were removed, the tissues were homogenized and luciferase activity was measured in cleared supernatants. Tissues from mice that received 100 µg mAb C3 were analysed only at 3 and 5 days p.i. The results are shown in Fig. 2(b). Luciferase activity was reduced markedly in the spleens and ovaries of mice given mAb C3. This inhibition was observed at a low concentration (1 µg per mouse) of mAb C3. The results with 100 µg mAb C3 per mouse were similar to those obtained with mice injected with 10 µg mAb C3 (Fig. 2 and data not shown). In the spleen, a 30-fold lower luciferase activity was found at 3 days p.i. with 1 and 10 µg mAb C3 per mouse, when compared with control BSA-inoculated animals. In fact, in the spleens of protected animals, the values for luciferase activity were near to background levels, indicating that virus replication was extensively blocked. In the ovaries, larger differences were observed in the treated animals compared with the untreated, control group; at 5 days p.i., a 100-fold reduction was observed in animals receiving 1 µg mAb C3 while, when the dose was 10 µg per mouse, a 1000-fold reduction in the level of luciferase was observed. It is interesting that dose-dependent inhibition of virus replication by antibody was not observed in the spleen, and the levels of luciferase expression were maintained with time. In the ovaries, dose-dependent inhibition by antibody was observed (Fig. 2) and levels of luciferase expression increase 100-fold over the initial value and were consistently much higher than those observed in the spleen. The qualitative differences in the results from the spleen and ovaries could be explained by the extent of virus replication in those tissues and by the distribution of the antibody in target tissues, since mAb C3 was injected by the i.p. route and animals were inoculated i.p. 1 h later with WRluc. From the results shown in Fig. 2, we conclude that a dose of 10 µg mAb C3 is quite efficient in limiting VV replication in target tissues. We chose this dose for passive transfer studies.

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, 6–8 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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Fig. 3. Pre-treatment with mAb C3 precludes VV replication in mice. Groups of three BALB/c mice, 6–8 weeks old, were injected i.p. with 10 µg purified mAb C3 per mouse and mice were inoculated i.p. on day 1, 2 or 3 with 2·5x107 p.f.u. WRluc per animal, following the scheme indicated in (a). (b) Luciferase activity was measured in ovaries and spleen at the indicated times p.i. as described in the legend to Fig. 2. Values are measurements from individual mice.

 
mAb C3 can control VV replication when administered several days after virus inoculation
Having established the prophylactic efficacy of mAb C3 against VV, we were interested to determine whether the antibody could be used therapeutically by administration following VV infection. Thus, four groups of BALB/c mice were inoculated i.p. with a sub-lethal dose of 2·5x107 p.f.u. WRluc per mouse and, at different times (1, 2 or 3 days p.i.), three groups received an i.p. injection of 10 µg mAb C3, while a control group was left untreated. The scheme for VV infection and mAb C3 treatment is indicated in Fig. 4(a). Three mice per group were sacrificed at daily intervals and luciferase levels were determined in ovaries and in the spleen. The values for luciferase activity in the infected but untreated animals were taken as the reference for all the groups that received mAb C3. As shown in Fig. 4(b) for ovaries, when mAb C3 was added 1 day p.i. (filled circles in Fig. 4b), the levels of luciferase at 3 days p.i. were the same as those in the untreated group; by day 4, a 2-log reduction was observed in luciferase levels compared with the untreated group. When mAb C3 was added at 2 days p.i. (filled triangles in Fig. 4b), the levels of luciferase at 3 days p.i. were the same as those in the control group while, at 4 days p.i., a 1-log reduction was observed compared with the untreated group. When mAb C3 was added at 3 days p.i. (filled squares in Fig. 4b), the levels of luciferase at 4 days p.i. were reduced by 3 logs compared with the untreated group. Thus, it appears that virus forms that are sensitive to neutralization by mAb C3 are accessible in the ovaries from 3 days p.i., but not before. Significantly, mice given mAb C3 at 3 days p.i. cleared the virus faster from the ovaries than those that received the antibody at 1 or 2 days p.i. (filled circles and triangles, respectively; Fig. 4b). In the spleen (Fig. 4b, inset), the main difference in levels of luciferase was observed at 4 days p.i. between untreated and mAb C3-treated groups.



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Fig. 4. mAb C3 administered after VV infection protects mice. Groups of four BALB/c mice, 6–8 weeks old, were infected i.p. with 2·5x107 p.f.u. WRluc per animal and the animals were injected at day 1, 2 or 3 p.i. with 10 µg purified mAb C3 per mouse or with BSA (control group). Luciferase activity (b) was measured in ovaries and spleen of treated animals as depicted in the scheme shown in (a). Values represented are means with standard deviations.

 
Prophylactic intervention with mAb C3 leads to full protection, while therapeutic intervention results in limited protection after challenge with a lethal dose of VV
In order to assess whether mice treated pre- or post-infection with mAb C3 could survive a lethal infection for a long time, groups of animals (10 per group) were either pre-treated for 1 day with 10 µg antibody per mouse and then infected i.p. with a lethal dose of WRluc (1x108 p.f.u. per animal) or were first infected with the same dose of virus and, at 1 day p.i., were treated with 10 µg mAb C3 per mouse. As a control, we used animals infected with the lethal dose of WRluc. Protection was followed daily in the three groups by measuring weight loss, signs of illness (reduced mobility, ruffled fur, eye necrosis and arched backs) and survival. As expected, almost all untreated animals rapidly developed severe weight loss and clear signs of illness (Fig. 5a) and died by 4 days p.i. (Fig. 5b). Animals pre-treated with the antibody did not experience weight loss and showed healthy status (Fig. 5a) and all survived virus infection (Fig. 5b). In contrast, animals infected for 1 day and treated thereafter with mAb C3 developed weight loss (Fig. 5a) with mild signs of illness (data not shown) and showed limited survival (Fig. 5b). The protected animals in the antibody-treated groups recovered fully from the infection and survived, as did uninfected animals.



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Fig. 5. Protection from a lethal virus challenge after prophylactic and therapeutic interventions. Groups of BALB/c mice (10 animals per group) were either pre-treated with 10 µg mAb C3 per mouse for 1 day and then infected with a lethal dose of WRluc (1x108 p.f.u. per animal) ({blacksquare}), infected with a lethal dose of WRluc and treated 1 day p.i. with 10 µg mAb C3 per mouse ({blacktriangleup}) or not treated and infected with a lethal dose of WRluc ({circ}). (a) Mice were weighed individually and monitored daily for signs of illness, scored from zero to five. Mean percentage weight loss of each group (with standard deviation) compared with the weight immediately prior to infection and the mean score of signs of illness in each group (inset) are shown. (b) Dead animals were scored daily and are represented as the percentage of animals surviving.

 

   Discussion
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Abstract
Introduction
Methods
Results
Discussion
References
 
Infection with poxviruses is known to trigger potent cellular and humoral immune responses (Buller & Palumbo, 1991 ; Moss, 1996 ). The production of virus-specific antibodies is a component of the long-term memory immune response to prevent re-infection (Buller & Palumbo, 1991 ). Passive administration of specific antibodies prior to or during an infection has been shown to reduce the severity of the smallpox disease (Kempe, 1960 ). Moreover, passive administration of neutralizing sera in animal models has been shown to lead to protection upon challenge with a poxvirus (Boulter & Appleyard, 1973 ). Due to the success of the worldwide smallpox eradication campaign and the availability of an effective vaccine, no major effort has been invested in the development of additional strategies to control poxvirus infections. This is particularly important should a human infection re-emerge, either as a result of infection with the closely related poxvirus monkeypox (Mukinda et al., 1997 ) or through bioterrorism (Breman & Henderson, 1998 ). The availability of neutralizing antibodies would be an effective means of rapid intervention.

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 1–3 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 29–43 (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.


   Acknowledgments
 
We thank Ros Bablanian for critical reading of the manuscript and Victoria Jimenez for expert technical assistance. This investigation was supported, in part, by grants PM98-0112 of Spain and EU (BIO4-CT98-0456). J.C.R. was supported by a post-doctoral fellowship from Comunidad Autónoma de Madrid (CAM).


   References
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
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Received 13 November 2001; accepted 17 January 2002.