The ability of antibody (Ab) to modulate HSV pathogenesis is well recognized but the mechanisms by which HSV-specific IgG antibodies protect against genital HSV-2 disease are not well understood. The requirement for Ab interactions with Fcgamma receptors (FcgammaR) in protection was examined using a murine model of genital HSV-2 infection. IgG antibodies isolated from the serum of HSV-immune mice protected normal mice against HSV-2 disease when administered prior to genital HSV-2 inoculation. However, protection was significantly diminished in recipient mice lacking the gamma chain subunit utilized in FcgammaRI, FcgammaRIII, FcgammaRIV and FcepsilonRI receptors and in normal mice depleted of Gr-1(+) immune cell populations known to express FcgammaR, suggesting protection was largely mediated by an FcgammaR-dependent mechanism. To test whether neutralizing Ab might provide superior protection, a highly neutralizing HSV glycoprotein D (gD)-specific monoclonal antibody (mAb) was utilized. Similar to results with HSV-specific polyclonal IgG, administration of the gD-specific mAb did not prevent initial infection of the genital tract but resulted in lower virus loads in the vaginal epithelium and provided significant protection against disease and acute infection of the sensory ganglia; however, this protection was independent of host FcgammaR expression and was manifest in mice depleted of Gr-1(+) immune cells. Together, these data demonstrate that substantial Ab-mediated protection against genital HSV-2 disease could be achieved by either FcgammaR-dependent or -independent mechanisms. These studies suggest that HSV vaccines might need to elicit multiple, diverse antibody effector mechanisms to achieve optimal protection.
The tissue sites of long-term herpes simplex virus type 2 (HSV-2)-specific antibody production in mice and guinea pigs were identified. In addition to secondary lymphoid tissue and bone marrow, HSV-specific plasma cells were detected in spinal cords of mice up to 10 months after intravaginal inoculation with a thymidine kinase-deficient HSV-2 strain and in lumbosacral ganglia and spinal cords of guinea pigs inoculated with HSV-2 strain MS. The long-term retention of virus-specific plasma cells in the peripheral and central nervous systems following HSV infection may be important for resistance to reinfection of neuronal tissues or may play a role in modulation of reactivation from latency.
ß-L-1-[5-(E-2-bromovinyl)-2-(hydroxymethyl)-1,3-(dioxolan-4-yl)] uracil (L-BHDU) prevents varicella-zoster virus (VZV) replication in cultured cells and in vivo. Its mechanism of action was investigated by evaluating its activity against related herpesviruses and by analyzing resistant VZV strains. L-BHDU was effective against herpes simplex virus type 1 (HSV-1) with an EC50 of 0.22 µM in human foreskin fibroblast (HFF) cells. L-BHDU also inhibited HSV-2 and simian varicella virus (SVV) to a lesser extent. VZV mutants resistant to L-BHDU and other antiviral compounds were obtained by serial passage of the wild type VZV pOka and VZV Ellen strains in the presence of increasing drug concentrations. VZV strains resistant to L-BHDU (L-BHDU R ) were cross-resistant to acyclovir (ACV) and brivudin (BVdU) but not to foscarnet (PFA) and cidofovir (CDV). Conversely, ACV-resistant strains were also resistant to L-BHDU. Whole genome sequencing of L-BHDU R strains identified mutations in ATP-binding (G22R) and nucleoside binding (R130Q) domains of VZV thymidine kinase (TK). The wild type and mutant forms of VZV TK were cloned as GST fusion proteins and expressed in E. coli. The partially purified TK G22R -GST and TK R130Q -GST proteins failed to convert thymidine to thymidine monophosphate whereas the wild type TK-GST protein was enzymatically active. Similarly, L-BHDU R virus TK did not phosphorylate the drug. As expected, wild type VZV converted L-BHDU to L-BHDU monophosphate and diphosphate forms. In conclusion, L-BHDU effectiveness against VZV and HSV-1 depends on thymidine kinase activity.Parental Oka (POka, Accession number: AB097933) strain and VZV Ellen, a standard laboratory strain passaged more than 100 times since its isolation (Accession number: JQ972913.1), were propagated in HFFs. Simian varicella virus (SVV, Delta herpesvirus strain), kindly provided by Dr. Ravi Mahalingam (University of Colorado School of Medicine, Denver), was propagated in Vero cells and virus stock was prepared as described previously (Mahalingam et al., 1992). Wild type HSV-1 KOS and TK mutant HSV-1 KOS (LTRZ1) were a kind gift from Dr. Donald Coen (Harvard University). All the above HSV-1 strains were propagated and quantified in Vero cells according to standard protocol (Blaho et al., 2005). The E-377 and G strains of HSV-1 and HSV-2 respectively were obtained from American Type Culture Collection (ATCC, Manassas, VA) and characterized as reported previously (Prichard et al., 2009). The HCMV (AD169) was also obtained from ATCC. The HSV-1 F strain expressing luciferase (R8411) under the control of the ICP27 promoter was a gift from Dr. Bernard Roizman (University of Chicago). CompoundsL-BHDU was synthesized as described before . Acyclovir (ACV, A669, Sigma), (E)-5-(2-bromovinyl)-20-deoxyuridine (BVdU, B9647, Sigma) and sodium phosphonoformate tribasic hexahydrate (PFA, P6801, Sigma) were purchased from Sigma Aldrich, St. Louis, MO. Cidofovir (CDV) was kindly provided by Southern Research Institute, Birmingham, AL, USA. Stock solutions of ...
In primary infection, CD8+ T cells are important for clearance of infectious HSV from sensory ganglia. We present evidence for CD4+ T cell‐mediated clearance of infectious HSV‐1 from genital tract and neuronal tissues. HSV‐specific CD4+ T cells were present in genital and neuronal tissues coincident with HSV‐1 clearance. Neuronal CD4+ T cells secreted IFN‐γ, TNF‐α, IL‐2 or IL‐4 after stimulation with HSV antigen. By adoptively transferring CD4+ T cells to Rag1−/ − mice, we showed that CD4+ T cells were sufficient for clearance of HSV‐1 from the genital tract. Compared to controls that did not receive T cells, CD4‐recipient mice had dramatically lower viral titers in the spinal cord and sensory ganglia, suggesting CD4+ T cells were active in clearing infectious HSV‐1 from neuronal tissue. To examine possible mechanisms by which CD4+ T cells resolve the neuronal HSV‐1 infection, CD4+ T cells from either perforin‐ or FasL‐deficient mice were adoptively transferred to Rag1−/ − mice. Clearance of infectious virus from genital and neuronal tissues was not significantly different in mice receiving these deficient T cells, compared to mice receiving wild‐type cells. These results suggest CD4+ T cells are important for resolution of primary HSV‐1 infection at both genital and neuronal sites, possibly via a non‐lytic mechanism. Supported by NIH grants AI42815, AI054444. Vale‐Asche Predoctoral Fellowship to AJJ.
We examined the influence of early IFN‐γ exposure on CD8+ T cell effector function. Immunization of C57BL/6 mice with a thymidine kinase deficient herpes simplex virus type 2 (HSV‐2 TK‐) elicited CD8+ T cells secreting mainly IFN‐γ and TNF‐α (Tc1), whereas CD8+ T cells from immunized IFN‐γ−/−mice secreted mainly TNF‐α with a lesser population secreting type 2 cytokines (Tc2). To facilitate studies of the effector function of these cells we cultured OT‐I CD8+ T cells in conditions that generated Tc1 or Tc2. Like CD8+ T cells from IFN‐γ−/−mice, Tc2 secreted type 2 cytokines, IL‐4, IL‐5 and IL‐10 with a distinct population producing IFN‐γ and TNF‐α. Tc1 produced both IFN‐γ and TNF‐α, like B6 CD8+ T cells. Tc1 and Tc2 expressed similar surface activation and differentiation markers. Tc2 exhibited delayed proliferation and reduced CTL activity in vivo, when compared to Tc1. These differences correlated with a delay in clearance of an OVA expressing HSV‐2 strain by Tc2 recipients, relative to Tc1 recipients. Despite the ability to secrete regulatory cytokines, such as IL‐10, co‐transfer of Tc2 with Tc1 did not interfere with the Tc1‐mediated virus clearance. Together these results suggest that early exposure to IFN‐γnot only imprints type 1 cytokine secretion, but also affects proliferation and cytolysis. Supported by NIH AI42815 and AI054444. MHN supported by a Sealy Center for Vaccine Development Predoctoral Fellowship.
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