Using whole-body bioimaging, we followed the dissemination of recombinant vaccinia viruses in live mice and analyzed the mechanism of protection conferred by prophylaxis with anti-vaccinia virus IgG. Three types of human immunoglobulin therapies were tested: VIGIV, an anti-vaccinia virus IgG licensed for human use (Cangene, Canada); a new investigational fully human rVIG (Symphogen, Denmark); and subset antibody compositions of this product targeting different combinations of MV and EV proteins. The main findings were as follows: (i) BALB/c mice challenged with vaccinia virus strains that differ in the quantity of released EV particles exhibited similar lethality curves; (ii) prophylactic treatments with either VIGIV (30 mg per animal) or rVIG (100 μg per animal) protected animals from lethality following challenge with either WRvFire or IHD-J-Luc viruses; (iii) using daily bioimaging of individual mice, we demonstrated that neither prophylactic treatment prevented initial virus replication at the site of inoculation (nasal cavity) or dissemination to the lower respiratory tract (lungs) and internal organs (spleen and liver), even in surviving animals; (iv) protection from lethality correlated with significant reductions in viral loads (as measured by total fluxes) in at least three organs on days 3 to 5 postchallenge, while transient reduction for 1 or 2 days did not correlate with survival; and (v) at a 30-μg dose, a subset of HuMAbs targeting only key proteins of EV (A33 and B5) protected animals from lethal challenge with both viruses, whereas at a 100-μg dose, subsets of both anti-EV and anti-MV HuMAbs were protective (data not shown).
VIGIV and the rVIG target proteins in both MV and EV isoforms of vaccinia virus. Therefore, WRvFire and IHD-J-Luc were both used in the current study, since they both produce similar levels of MV but differ in the amounts of released EV. The more stable MV was suggested to play a key role in the virus spread between animals, whereas EV is important for dissemination within the infected host (34
). In our experiments, in the absence of antibody treatment, total fluxes in WRvFire-infected animals were 0.5 to 1.0 log higher in the nasal cavity and lungs than in IHD-J-Luc-infected mice (C and D), thus confirming that WRvFire replicated at higher levels than IHD-J-Luc in the upper and lower respiratory tracts but not in spleen and liver. Since the times to death were identical after WRvFire and IHD-J-Luc challenge, the data suggested that the levels of viral replication in internal organs might play a primary role in determining the kinetics of lethality. For both viruses, bioluminescence measurements showed that the level of viral replication in the nasal cavity 24 h postinfection was 2 to 3 log higher than in the lungs, liver, and spleen. Thus, whole-body bioimaging revealed that the nasal cavity is a primary site of vaccinia virus replication following intranasal challenge and not only the lungs as previously reported (19
We used two different sources of anti-vaccinia virus IgG; a licensed VIGIV from Cangene (human IgG purified from plasma of subjects vaccinated with Dryvax vaccine), and fully human recombinant VIG generated by single-cell PCR using B cells taken from human volunteers that had been vaccinated with the Lister vaccine (18
). In preliminary studies, the neutralizing activities of both immunoglobulin preparations were evaluated using the β-galactosidase (β-Gal) reporter gene assay for MV neutralization (WR strain) (23
) and the comet assay for EV neutralization (IHD-J strain) (A. Garcia, personal communication). In the MV neutralization assay, the 50% infectious dose (ID50
) for rVIG was 0.2 μg/ml compared with 18 μg/ml for VIGIV (90-fold difference). In the comet assay, the lowest inhibitory doses were 0.23 μg/ml and 62 μg/ml, for rVIG and VIGIV, respectively (270-fold difference). These in vitro
results were predictive of the higher in vivo
activity of the rVIG reported in the current study. The two products were evaluated as prophylactic treatments for protection from lethality, weight loss, reduction in viral replication at the site of inoculation, and dissemination of virus to the lungs, spleen, and liver. Our results showed that both VIGIV and rVIG protected mice from lethality following WRvFire and IHD-J-Luc challenge. In the case of VIGIV, a complete protection from lethality in both viral infections was achieved at 30 mg/animal dose (or 178,800 U/kg) and was in the same range as the dose used for treatment of progressive vaccinia virus in a military smallpox vaccinee who received a cumulative dose of 186,000 U/kg given by multiple injections over a period of time (20
). At the same time, 100 μg/animal of rVIG exerted full protection irrespective of the challenge virus. The difference between these two immunoglobulin preparations primarily reflects the fact that VIGIV is a preparation of total IgG, with only a fraction of vaccinia virus-specific antibodies. The 26 HuMAbs in the rVIG are vaccinia virus specific, targeting vaccinia virus MV and EV epitopes () (18
). Interestingly, at the fully protective dose, the rVIG and VIGIV had opposite effects on viral replication in the respiratory tract: the rVIG significantly reduced viral loads in the nasal cavity (but not in the lungs), while VIGIV was effective in reducing viral loads in the lungs but not in the nasal cavity ( and ). The reason for the difference in the activities of these products in the upper and lower respiratory tracts is not clear at this point.
Several vaccinia virus proteins have been mapped as targets of neutralizing antibodies. The EV B5 and A33 proteins were identified as the main targets for protective antibodies in animal models (1
), and only B5 was confirmed as a target for the EV-neutralizing activity in sera derived from humans vaccinated with the Lister strain of vaccinia virus (31
). In MV, five membrane proteins were identified as targets for neutralizing antibodies: A27, L1, H3, D8, and A17. Immunizations with A27 (13
), L1 (9
), H3 (30
), and D8 (30
) conferred protection in mice and/or macaques. A subunit vaccine targeting the L1 (MV) and B5 (EV) protected mice against lethal intranasal challenge, confirming the benefit of combined anti-MV- and anti-EV-specific responses (17
). In agreement with studies in animal models, Dryvax vaccination has been shown to induce neutralizing Abs against membrane glycoproteins of both MV (L1, A27, and H3) and EV (B5 and A33) (6
The rVIG contains a mixture of 26 HuMAbs targeting proteins in both MV and EV, and we showed that the 100-μg dose fully protected mice from WRvFire- and IHD-J-Luc-induced lethality. The 30-μg dose of the rVIG completely protected animals from lethality following IHD-J-Luc challenge, but only partially protected animals from WRvFire challenge (). These differences in the effects of rVIG could not be attributed to the difference in the composition of the WRvFire and IHD-J-Luc viral stocks, as both of them contained similar 1:10 ratios of infections to noninfectious particles (see Materials and Methods). Therefore, it was possible that in our experiments rVIG more efficiently controlled IHD-J-Luc than WRvFire dissemination. To further address this possibility, antibody compositions that contained subsets of HuMAbs in the rVIG were evaluated. The selected HuMAbs were targeting major neutralizing epitopes in MV (composition C1), EV (composition C2), or both (composition C) (A). At the 30-μg dose, composition C2 containing four HuMAbs targeting A33 and B5 proteins in EV protected mice from lethality following infection with both WRvFire and IHD-J-Luc. Therefore, in the case of WRvFire infection, C2 was fully protective at a dose 3-fold lower than that for the original rVIG product containing a mixture of MV and EV MAbs. These findings are in agreement with a previous report showing that protection against lethal challenge was improved when EV-specific antibodies were present along with anti-MV antibodies (19
). It was somewhat unexpected that the anti-MV cocktail (composition C1) did not protect from challenge with WRvFire at the 30-μg dose. It should be noted that 30 μg is a very low dose (~1.5 mg/kg of body weight), and as the C1 composition protected at the higher dose of 100 μg (not shown), the lack of protection at this low dose likely reflects the dose dependency of the anti-MV-mediated protection. Another contributing factor might be the lack of anti-L1 antibodies in the C1 mixture. However, other investigators have shown that there is a significant degree of redundancy in the neutralizing antibody response toward MV: following depletion of one or more neutralizing specificities, such as anti-L1, other specificities have been found to compensate for the loss (3
). Since C1 was protective at a higher (100 μg/animal) but not at a lower (30 μg/animal) dose, future studies using fractionated composition C1 will help identify protective MAbs that might be diluted in the composition C1 by other MAbs that are not effective.
Antibodies play a crucial role in preventing secondary infection and even primary infection after active vaccination. Several mechanisms have been identified through which antibodies curtail viral replication, including blocking of the viral attachment to the cell membrane (anti-MV), inhibition of the release of EV from infected cells (8
), complement-dependent lysis of the EV membrane with a subsequent exposure of MV to neutralizing antibody (22
), complement-dependent neutralization of the virus, and/or complement-directed lysis of infected cells (anti-EV) (2
). In preclinical animal models, the protective efficacy of antibodies is traditionally evaluated by indirect assays, such as protection from weight loss and/or from lethality and by measuring of viral loads in infected organs using sensitive cell lines. Here we took advantage of bioimaging of live animals that allowed us to follow dissemination of the virus in individual animals throughout the observation period. This approach eliminates the animal-to-animal variability related to the collection of organs from different animals at multiple time points. Using bioluminescence recorded from internal organs of individual mice, we compared the mean total fluxes between IgG-treated and control mice by using the t
statistic. It was found that in all cases when mice were protected from lethality, mean viral loads (fluxes) were significantly reduced in three organs simultaneously: in the respiratory tract (nasal cavity or lungs) and in the spleen and liver on days 3 to 5 postinfection. Therefore, our data indirectly confirmed that the administration of VIGIV or rVIG 2 days before virus challenge resulted in the distribution of the antibodies to all key sites of viral replication. On the other hand, our data also suggested that there is no single organ where the infection can be completely blocked using passively transferred antibodies, and initial virus dissemination could not be prevented. This finding is different from active vaccination, where dissemination to the internal organs was completely blocked, although the dose used for challenge was the same as that in the current study (39
). In addition, at the fully protective doses, all three types of passive immunoglobulin treatments significantly reduced viral loads in spleen and liver, in agreement with our earlier study showing that the viral load at these sites provided the most accurate prediction of lethality versus survival outcome (39
Eradication of smallpox was a major public health achievement that led to cessation of the immunization of general public against vaccinia virus. Nevertheless, the need for protection against smallpox has not been eliminated. An efficient and safe vaccine against smallpox requires the continuation of immunization of designated personnel in case of a bioterror attack, and as recently shown, could be useful in protection against monkeypox in populations in central Africa, who are at risk of infections due to frequent contact with animals (32
Together, these findings provide valuable information on the impact of anti-vaccinia virus immunoglobulins on virus replication in internal organs. The same tools will be applied for the evaluation of new antiviral products (alone and in combinations) for pre- and postexposure treatments.