It is striking that VIG treatment can prevent the development of PV in mice that are completely lacking in adaptive immunity, if given early enough in the course of disease. We observed dose-dependent frequencies of lesion regression and long-term survival when VIG treatment was started at 2 days postinfection, and a proportion of the mice survived disease free for at least 120 days postinfection without receiving VIG beyond day 15. Results of combination treatment with VIG and topical cidofovir provide the first demonstration that an established vaccinia virus infection is cleared in SCID mice after the cessation of treatment by any regimen. Cellular immunity has been deemed necessary for effective vaccinia virus clearance since the 1960s, when it was recognized that PV patients had cellular, as well as humoral, immunodeficiencies. Supporting the contention that antibodies are not essential for controlling primary vaccinia virus infections are observations that the same patients with agammaglobulinemia have been vaccinated and have not developed PV, that mice can clear poxvirus infections in the absence of B cells, and that mice and humans with defective cellular immunity seem unable to resolve vaccinia virus infection (3
). In immunocompetent mice, both antibodies and T cells are involved in the resolution of primary vaccinia virus infections, although in NYCBOH strain-scarified mice, no single immune cell type among CD4+
, B, and NK cells is essential (3
). However, an absolute role for cellular immunity is controversial since B cells, but not CD4+
T cells, are required for vaccine-induced protection from monkeypox virus in a nonhuman primate model. Moreover, passive transfer of anti-vaccinia virus antibodies protects primates from severe monkeypox disease (11
). This divergence of opinion may be partially explained by the diversity of experimental models used to explore the relative importance of adaptive immune responses. Model systems, as well as human observations, vary with respect to the type of underlying immunodeficiency. In animal studies, the vaccinia virus strain, dose, and route of challenge also differ. Additionally, a strong innate immune response may be beneficial in the clearance of ongoing disease (a treatment scenario) while an adaptive immune response may be sufficient for prevention of disease. Our observations suggest that aggressive antibody treatment, even in the absence of adaptive cellular immune responses, can treat a developing indolent vaccinia virus infection. Since the use of VIG can be considered late prophylaxis or early treatment in our model, the high levels of antibodies being administered may delay or limit infection to the extent that innate effectors are sufficient for clearance of virus.
The precise mechanism of action of VIG is not known; however, the correlation between primary lesion regression and survival and the benefits of topical over parenteral cidofovir support Neyts' contention that the skin is an important platform for the eventual spread of vaccinia virus to organs (25
). The vaccinia virus dose-response relationship to survival in mice treated with the same dose of VIG suggests that early antibody treatment limits virus spread from the skin and also that early postexposure prophylaxis in humans, as soon as immunocompromised status is recognized and when the viral burden is relatively light, may be prudent. It is possible that antibody Fc
region functions such as complement binding or accessory cell activation (macrophages, NK, or mast cells) help to resolve local or systemic vaccinia virus infection in SCID mice. Complement-dependent efficacy of an anti-B5 monoclonal antibody has recently been demonstrated (4
), and NK cells can be important for limiting vaccinia virus infection (14
). Our ongoing work is focused on Fc
region functions of VIG to identify effector mechanisms. If Fc
-mediated effects are important, VIG product potency enhancement strategies could be envisioned and potency could be further characterized and assured for new antibody preparations.
In SCID mice inoculated with the NYCBOH strain of vaccinia virus, the expanding primary lesion, development of distal lesions, and slow pace of infection mimic clinical descriptions of PV in humans (5
). Similar to reported human lesions, SCID mouse lesions contained few inflammatory cells other than neutrophils in underlying ulcerated skin in later infection. Local satellite lesions were not observed in SCID mice; these are variably observed in PV patients. It is difficult to know whether the time course of disease is different in SCID mice and human patients, in part because the more well-documented human cases had interventional treatments and medical support for recent cases was intensive. Unfortunately, further comparisons of the human and murine versions of the disease cannot be made because specimens from historical PV cases are unavailable for study using modern techniques. Important information that would further support the relevance of the SCID model to human PV includes the timing of the distribution of vaccinia virus infection beyond the skin and understanding of the fundamental mechanism(s) of death. In the SCID mouse, dissemination appears to be related to the inoculum dose. A small challenge dose (104
PFU) results in a very limited dissemination of virus, while a larger challenge dose (106
PFU) results in a more widespread, albeit sporadic, distribution of virus, with titers in the affected organs apparently much lower than those described for other mouse orthopox disease models (12
). In humans, very little information has been acquired with respect to virus dissemination to organs, and we are unable to find any references where reproductive tissue was analyzed. In some cases, vaccinia virus in nonskin tissues was sparse, and in others, vaccinia virus detection was widespread in multiple organs and tissues, including brain, gastrointestinal, and lymphoid tissues. Patients with widespread vaccinia virus at autopsy were also those with the most severe T cell defects (16
), which is more consistent with the SCID model. The cause of death is not well understood in mice or in humans, although some affected patients probably died of non-vaccinia virus secondary infections (16
). In contrast to human cases, opportunistic or bacterial infections as a cause of death in SCID mice are unlikely since untreated mice died over a narrow and predictable time window within and across experiments, tissue examination did not reveal signs of bacterial infection, and control mice had prolonged survival. Mice with PV become cachectic, and preliminary evidence suggests increased levels of proinflammatory cytokines, such as tumor necrosis factor alpha, in blood, which suggests that innate immune activation could contribute to death (data not shown). As far as we are aware, no similar data exist for human PV.
The striking failure to recruit macrophages and neutrophils early after infection suggests that local innate responses could synergize with VIG to limit PV. A potential strategy to address this possibility would be local inhibition of the ubiquitin/proteosome pathway, which in vitro
inhibits vaccinia virus replication and enhances release of chemoattractants, including interleukin-8 and RANTES (17
). Also, as observed for vaccinia virus and other orthopoxviruses, we found hyperproliferation of epidermal cells and sebaceous glands infected by vaccinia virus, consistent with the effects of virus-encoded vaccinia virus-induced epidermal growth factor and F1L (29
). These findings may suggest a role for small-molecule sensitizers (e.g., epigallocatechin gallate [1
]) in combination with VIG to blunt infected cell proliferation and potentially boost apoptosis during PV. We are exploring the possibilities of examining small-molecule synergies in combination treatments with VIG.
The increase in mast cells observed at late time points in this model, similar to findings in vaccinia virus-scarified guinea pigs (10
), is of uncertain significance and possibly specific to the rodent models selected. There are no data regarding mast cell recruitment to PV lesions in humans and limited information on how mast cells may impact the course of viral infections generally. Recent studies show that mast cells can speed the development of adaptive immunity (9
), which could enhance antipathogen responses in immunocompetent animals. Moreover, mast cells bear FcR (8
) and can be activated and/or degranulated in the presence of antibody, raising the possibility of synergy with VIG. Additional work is required to explore these possibilities.
In comparing our studies to prior studies of VIG in immunocompromised mice, we note a number of differences that might explain the unexpected observation of long-term survival of VIG-treated SCID mice. Variations include different routes of administration which are shown to impact vaccinia virus pathogenesis (23
), different strains of vaccinia virus (19
), different VIG preparations whose potencies relative to that of the VIG we used or relative to each other are not known (19
), and different models of immunocompromised status. One previous study showed that combining VIG treatment with a nucleoside derivative resulted in prolonged survival of scarified SCID mice while treatment was continued, although vaccinia virus persisted at the inoculation site and the mice were not followed long term off therapy(19
). Prior work demonstrated that cidofovir alone and VIG alone can both delay death, which provided a rationale for attempting combination treatments (19
Given the limited knowledge of human PV pathogenesis, use of the SCID mouse in conjunction with licensed smallpox vaccine given intradermally seems to be a relevant and informative model for studying PV antibody therapy, with or without additional drugs. This study further confirms the in vivo effects of VIG, provides new information about the efficacy potential of combination treatments in severely immunocompromised patients, and supports the SCID mouse model as one that should be useful in understanding how VIG suppresses vaccinia virus in the immunocompromised host.