No abstract

No abstract

No abstract

The ion permeability of planar lipid bilayers, as measured electrically, was found to increase modestly upon treatment with purified complement complex C5b,6 and complement components C7 and C8. The subsequent addition of C9 greatly amplified this change. No permeability changes occurred when components were added individually to the membrane, or when they were used in paired combinations, or when C5b,6, C7, C8, and C9 were admixed prior to addition. Thus, there is a significant parallel between the permeability changes induced in the model membrane and damage produced in biological membranes by the C5b-9 complement attack sequence. The efficiency of membrane action by C5b-9 was critically dependent on the order in which components were added to the membrane. There were also differences in the electrical properties of membranes treated with C5b-8 and C5b-9, though in both cases the enhanced bilayer permeability is best attributed to the formation of trans-membrane channels. Collectively, the data are consistent with the hypothesis that the mechanism of membrane action by complement involves the production of a stable channel across the lipid bilayer, resulting in cell death by colloid-osmotic lysis. The term "complement" refers to a group of proteins in normal blood serum that plays an important role as a mediator of both immune and allergic reactions. The sequential interaction of the nine complement proteins is illustrated in Fig. 1. One consequence of this enzymatic cascade is the drastic alteration in the permeability of biomembranes to substances of low molecular weight (1). This activity is a result of the interaction of the five terminal components (C5b-9) with the lipid moiety of the cell membrane, as has been learned from experiments in which C5b-9 released trapped marker from phospholipid liposomes concomitant with the production of lesions visible by electron microscopy (2). In studies with erythrocytes, we have found that a single membrane lesion suffices for lysis of a cell (1). In a recent study we have presented evidence which indicates that polypeptide chains from the C5b and C7 subunits of the C5b,6,7 complex become inserted in the phospholipid bilayer of the erythrocyte membrane (3). We believe that this process represents the initial stage of the cytolytic attack and that subsequent reaction of C8 and C9 with the inserted C5b,6,7 opens a channel extending through the phospholipid bilayer (4).We now present an exploration of this mechanism by use of planar lipid bilayers (black lipid membranes, BLM). This is an atrractive system because the morphology of BLM is known to be bimolecular and because changes in membrane permeability (1968) 39, 935-9381. to ions can be measured electrically. There have been several earlier studies of the effect of antigens, antibodies, and fresh blood serum as a source of complement on BLM in which changes in electrical conductance were observed (5-7). In the present experiments, we have used purified complement components and initiated the terminal r...

Recombinant vesicular stomatitis virus (rVSV) vectors offer an attractive approach for the induction of robust cellular and humoral immune responses directed against human pathogen target antigens. We evaluated rVSV vectors expressing full-length glycoprotein D (gD) from herpes simplex virus type 2 (HSV-2) in mice and guinea pigs for immunogenicity and protective efficacy against genital challenge with wild-type HSV-2. Robust Th1-polarized anti-gD immune responses were demonstrated in the murine model as measured by induction of gD-specific cytotoxic T lymphocytes and increased gamma interferon expression. The isotype makeup of the serum anti-gD immunoglobulin G (IgG) response was consistent with the presence of a Th1-CD4 ؉ anti-gD response, characterized by a high IgG2a/IgG1 IgG subclass ratio. Functional anti-HSV-2 neutralizing serum antibody responses were readily demonstrated in both guinea pigs and mice that had been immunized with rVSV-gD vaccines. Furthermore, guinea pigs and mice were prophylactically protected from genital challenge with high doses of wild-type HSV-2. In addition, guinea pigs were highly protected against the establishment of latent infection as evidenced by low or absent HSV-2 genome copies in dorsal root ganglia after virus challenge. In summary, rVSV-gD vectors were successfully used to elicit potent anti-gD Th1-like cellular and humoral immune responses that were protective against HSV-2 disease in guinea pigs and mice.Herpes simplex virus type 2 (HSV-2) infections remain a serious public health problem worldwide. HSV-2 genital lesions are not only painful and disfiguring but also facilitate the transmission of human immunodeficiency virus (HIV) (7). The seroprevalence in the United States has increased by 30% between 1976 and 1994, and roughly one of every five people over the age of 12 in the United States is infected with HSV-2 (15). Individuals latently infected with HSV-2 remain infected for life and can exhibit asymptomatic viral shedding. It is therefore believed that, without intervention, such as the development of prophylactic and/or therapeutic HSV-2 vaccines, HSV-2 prevalence will continue to rise in the future.Small experimental animal vaginal challenge models in mice and guinea pigs have been used for preclinical evaluation of a number of HSV-2 vaccine strategies, including subunit vaccines (gB and/or gD with or without interleukin-12 [IL-12]), plasmid HSV DNA vaccines (gD and/or gB with or without cytokine DNA (IL-2, IL-4, IL-10, IL-12, IL-15, or IL-18), attenuated HSV-2 vaccines (TKϪ, BlacZ, dl5-29, RAV 9395, ICP10⌬PK, or AD472), and virus-vectored HSV-2 vaccines (adenovirus, varicella-zoster virus, or vaccinia virus) (1,9,12,17,21,22, 34, 39, 40,45,60,62,66). Various degrees of success have been achieved in these preclinical studies, but limited success has carried over to the clinical setting, where the experience with HSV-2 subunit vaccines has had mixed results (10). Nonetheless, an adjuvanted gD subunit approach has achieved some success in early clinical trials...