VACV Infection Research Articles

Protection of Rabbits and Immunodeficient Mice against Lethal Poxvirus Infections by Human Monoclonal Antibodies

Smallpox (variola virus) is a bioweapon concern. Monkeypox is a growing zoonotic poxvirus threat. These problems have resulted in extensive efforts to develop potential therapeutics that can prevent or treat potentially lethal poxvirus infections in humans. Monoclonal antibodies (mAbs) against smallpox are a conservative approach to this problem, as the licensed human smallpox vaccine (vaccinia virus, VACV) primarily works on the basis of protective antibody responses against smallpox. Fully human mAbs (hmAbs) against vaccinia H3 (H3L) and B5 (B5R), targeting both the mature virion (MV) and extracellular enveloped virion (EV) forms, have been developed as potential therapeutics for use in humans. Post-exposure prophylaxis was assessed in both murine and rabbit animal models. Therapeutic efficacy of the mAbs was assessed in three good laboratory practices (GLP) studies examining severe combined immunodeficiency mice (SCID) given a lethal VACV infection. Pre-exposure combination hmAb therapy provided significantly better protection against disease and death than either single hmAb or vaccinia immune globulin (VIG). Post-exposure combination mAb therapy provided significant protection against disease and death, and appeared to fully cure the VACV infection in ≥50% of SCID mice. Therapeutic efficacy was then assessed in two rabbit studies examining post-exposure hmAb prophylaxis against rabbitpox (RPXV). In the first study, rabbits were infected with RPVX and then provided hmAbs at 48 hrs post-infection, or 1 hr and 72 hrs post-infection. Rabbits in both groups receiving hmAbs were 100% protected from death. In the second rabbitpox study, 100% of animal treated with combination hmAb therapy and 100% of animals treated with anti-B5 hmAb were protected. These findings suggest that combination hmAb treatment may be effective at controlling smallpox disease in immunocompetent or immunodeficient humans.

CD11b+, Ly6G+ Cells Produce Type I Interferon and Exhibit Tissue Protective Properties Following Peripheral Virus Infection

The goal of the innate immune system is containment of a pathogen at the site of infection prior to the initiation of an effective adaptive immune response. However, effector mechanisms must be kept in check to combat the pathogen while simultaneously limiting undesirable destruction of tissue resulting from these actions. Here we demonstrate that innate immune effector cells contain a peripheral poxvirus infection, preventing systemic spread of the virus. These innate immune effector cells are comprised primarily of CD11b+Ly6C+Ly6G- monocytes that accumulate initially at the site of infection, and are then supplemented and eventually replaced by CD11b+Ly6C+Ly6G+ cells. The phenotype of the CD11b+Ly6C+Ly6G+ cells resembles neutrophils, but the infiltration of neutrophils typically occurs prior to, rather than following, accumulation of monocytes. Indeed, it appears that the CD11b+Ly6C+Ly6G+ cells that infiltrated the site of VACV infection in the ear are phenotypically distinct from the classical description of both neutrophils and monocyte/macrophages. We found that CD11b+Ly6C+Ly6G+ cells produce Type I interferons and large quantities of reactive oxygen species. We also observed that depletion of Ly6G+ cells results in a dramatic increase in tissue damage at the site of infection. Tissue damage is also increased in the absence of reactive oxygen species, although reactive oxygen species are typically thought to be damaging to tissue rather than protective. These data indicate the existence of a specialized population of CD11b+Ly6C+Ly6G+ cells that infiltrates a site of virus infection late and protects the infected tissue from immune-mediated damage via production of reactive oxygen species. Regulation of the action of this population of cells may provide an intervention to prevent innate immune-mediated tissue destruction.

Permissivity of the NCI-60 cancer cell lines to oncolytic Vaccinia Virus GLV-1h68

BackgroundOncolytic viral therapy represents an alternative therapeutic strategy for the treatment of cancer. We previously described GLV-1h68, a modified Vaccinia Virus with exclusive tropism for tumor cells, and we observed a cell line-specific relationship between the ability of GLV-1h68 to replicate in vitro and its ability to colonize and eliminate tumor in vivo.MethodsIn the current study we surveyed the in vitro permissivity to GLV-1h68 replication of the NCI-60 panel of cell lines. Selected cell lines were also tested for permissivity to another Vaccinia Virus and a vesicular stomatitis virus (VSV) strain. In order to identify correlates of permissity to viral infection, we measured transcriptional profiles of the cell lines prior infection.ResultsWe observed highly heterogeneous permissivity to VACV infection amongst the cell lines. The heterogeneity of permissivity was independent of tissue with the exception of B cell derivation. Cell lines were also tested for permissivity to another Vaccinia Virus and a vesicular stomatitis virus (VSV) strain and a significant correlation was found suggesting a common permissive phenotype. While no clear transcriptional pattern could be identified as predictor of permissivity to infection, some associations were observed suggesting multifactorial basis permissivity to viral infection.ConclusionsOur findings have implications for the design of oncolytic therapies for cancer and offer insights into the nature of permissivity of tumor cells to viral infection.

The Adaptor Molecule MyD88 Directly Promotes CD8 T Cell Responses to Vaccinia Virus

Vaccinia virus (VACV) elicits a robust CD8 T cell response that plays an important role in host resistance. To date, there is little information on the molecules that are essential to generate large pools of VACV-specific effector CD8 T cells. In this study, we show that the adaptor molecule MyD88 is critical for the magnitude of primary CD8 T cell responses to both dominant and subdominant VACV epitopes. MyD88(-/-) mice exhibit profound reduction in CD8 T cell expansion and antiviral cytokine production. Surprisingly, the defect was not due to impaired APC function, as MyD88(-/-) dendritic cells matured normally and were able to promote strong CD8 T cell priming following VACV infection. Rather, adoptive transfer experiments demonstrated that intrinsic MyD88-dependent pathways in CD8 T cells were critical. MyD88-deficient CD8 T cells failed to accumulate in wild-type hosts and poor expansion of MyD88-deficient VACV-specific CD8 T cells resulted after virus infection. In contrast, no defect was evident in the absence of TRIF, TLR2, TLR4, TLR9, and IL-1R. Together, our results highlight an important role for MyD88 in initial antiviral CD8 T cell responses and suggest that targeting this pathway may be useful in promoting and sustaining anti-VACV immunity.

The adaptor molecule MyD88 directly promotes CD8 T cell responses to vaccinia virus (128.12)

Abstract Vaccinia Virus (VACV) elicits a robust CD8 T cell response that plays an important role in protection. To date, there is little information on the molecules that are essential to generate large pools of VACV-specific CD8 T cells. Here we show that the adaptor molecule myeloid differentiation primary-response gene 88 (MyD88) is critical for the magnitude of primary CD8 T cell responses to both dominant and subdominant VACV epitopes, with MyD88-/- mice exhibiting profoundly reduced CD8 T cell expansion and anti-viral cytokine production. Interestingly, the defect was not due to impaired antigen-presenting cell function. MyD88-/- DC matured normally and were able to promote strong CD8 T cell priming following VACV infection. Rather, adoptive transfer experiments demonstrated that MyD88-dependent pathways in CD8 T cells were critical. MyD88-deficient CD8 T cells were defective in surviving in wild-type hosts, and poor expansion of MyD88-deficient VACV-specific CD8 T cells resulted after virus infection. In contrast, no defect was evident in the absence of TRIF, TLR2, TLR9, and IL-1R. Together, our results highlight an important role for MyD88 in initial anti-viral CD8 T cell responses and suggest that targeting this pathway may be useful in promoting and sustaining anti-VACV immunity. This work was supported by NIH grants AI77079 to S.S.A and AI67341 to M.C.

OX40 Drives Protective Vaccinia Virus-Specific CD8 T Cells

Vaccinia virus (VACV) affords long-lasting protection against variola virus, the agent of smallpox. VACV-reactive CD8 T cells contribute to protection but their molecular control is unknown. We show that the TNFR molecule OX40 (CD134) controls primary VACV-specific CD8 T cell expansion and antiviral cytokine production and dictates development of strong memory to both dominant and subdominant VACV epitopes. Using adoptive transfer of OX40-deficient CD8 TCR-transgenic T cells responding to Ag in the context of VACV infection, we found that this reflects a direct action of OX40 expressed by CD8 T cells. Furthermore, CD8 T cells that can protect against lethal VACV challenge do not develop in mice deficient in OX40. Thus, OX40, which has been found to play little if any role in the generation of CD8 T cells to several viruses, including lymphocytic choriomeningitis virus and influenza, plays a dominant role in shaping the CD8 T cell response to VACV. These data suggest that unique costimulatory pathways might control alternate antiviral CD8 responses, demonstrating the plasticity of the immune response in utilizing different mechanisms to achieve similar ultimate goals.

T cell independent B cell response to an RNA‐binding viral protein (Vaccinia E3)

How the innate immune system impact and shape the adaptive immune system is not totally understood; the adjuvant properties of the molecules dedicated to the recognition of Pathogen‐Associated Molecular Patterns (PAMPs) is currently subject to intense research. Using mice deficient in MHC II, we found that all but one of the IgG antibody specificities induced after VACV infection are dependent on CD4 T cell help, the only exception is E3, a double stranded RNA (dsRNA) binding protein to which antibodies of the IgG3 isotype can be detected as early as day 5 post‐infection. The anti‐E3 IgG response is not dependent of MyD88, the adaptor protein involved in the signaling of all know Toll‐like receptors (TLR) but TLR3. However, TRIF deficient mice have a severely diminished antibody response against E3; TRIF is the adaptor for TLR3. TLR3 is a member of the TLR family of PAMPs receptors and recognizes dsRNA inducing the production of type I interferons. As both TLR3 and E3 bind dsRNA, we hypothesize that E3‐dsRNA complex is bridging the signals of the BCR with the signals of TLR3, resulting in a T cell independent IgG response.

A quantitative analysis of the variables affecting the repertoire of T cell specificities recognized following VACV infection (43.56)

Abstract Many components contribute to immunodominance in the response to a complex virus, but their relative importance is unclear. This was addressed using vaccinia virus and HLA-A*0201 as the model system. A comprehensive analysis of 18 viral proteins recognized by CD8+ T cell responses demonstrated that about 1/40 of all possible 9–10 mer peptides were high-affinity A*0201 binders. Peptide immunization and T cell recognition data generated from 91 peptides indicated that about half of the binders were capable of eliciting T cell responses, and that 1/7 immunogenic peptides are generated by natural processing. Based on these results, we estimate that vaccinia virus encodes about 150 dominant and subdominant epitopes restricted in the context of HLA-A*0201. However, of all these potential epitopes, only 15 are immunodominant and actually recognized in vivo during vaccinia virus infection of HLA-A*0201 transgenic mice. Neither peptide binding affinity, complex stability, TCR avidity nor amount of processed epitope appeared to strictly correlate with immunodominance status. Further experiments suggested that vaccinia infection impairs the development of responses directed against subdominant epitopes. This suggested that immunoregulatory factors restrict the repertoire of T cell specificities following vaccinia infection by at least a factor of ten.