We tested the ability of 20 synthetic defensins to protect cells from infection by type 1 and type 2 herpes simplex viruses (HSV-1 and -2, respectively). The peptides included rhesus defensins (RTDs) 1 to 3, originally isolated from rhesus macaque leukocytes, and three peptides (retrocyclins 1 to 3) whose sequences were inferred from human -defensin (DEFT) pseudogenes. We also tested 14 retrocyclin analogues, including the retro, enantio, and retroenantio forms of retrocyclin 1. Retrocyclins 1 and 2 and RTD 3 protected cervical epithelial cells from infection by both HSV serotypes, but only retrocyclin 2 did so without causing cytotoxicity or requiring preincubation with the virus. Surface plasmon resonance studies revealed that retrocyclin 2 bound to immobilized HSV-2 glycoprotein B (gB2) with high affinity (K d , 13.3 nM) and that it did not bind to enzymatically deglycosylated gB2. Temperature shift experiments indicated that retrocyclin 2 and human ␣ defensins human neutrophil peptide 1 (HNP 1) to HNP 3 protected human cells from HSV-2 by different mechanisms. Retrocyclin 2 blocked viral attachment, and its addition during the binding or penetration phases of HSV-2 infection markedly diminished nuclear translocation of VP16 and expression of ICP4. In contrast, HNPs 1 to 3 had little effect on binding but reduced both VP16 transport and ICP4 expression if added during the postbinding (penetration) period. We recently reported that defensins are miniature lectins that bind gp120 of human immunodeficiency virus type 1 (HIV-1) with high affinity and inhibit the entry of R5 and X4 isolates of HIV-1. Given its small size (18 residues), minimal cytotoxicity, lack of activity against vaginal lactobacilli, and effectiveness against both HSV-2 and HIV-1, retrocyclin 2 provides an intriguing prototype for future topical microbicide development.The worldwide AIDS epidemic has intensified interest in identifying naturally occurring antiviral molecules (5,29,34,57). Certain rabbit and human ␣ defensins were shown to protect cells from infection by herpes simplex virus type 1 (HSV-1) and HSV-2 almost 20 years ago (17,30), and more recent studies indicated that rabbit ␣ defensin NP-1 blocks HSV infection at a very early stage (46). Even adenoviruses, which are nonenveloped, are susceptible to ␣ defensins (3, 21), although the mechanism of this effect is unknown. In vitro, human ␣ defensins human neutrophil peptide 1 (HNP 1) to HNP 3 can protect cells from infection by human immunodeficiency virus type 1 (HIV-1), and release of these defensins from the ␣, ␤ CD8-positive T cells of HIV-infected subjects may (60) or may not (11) correlate with their long-term clinical stability.Defensin peptides belonging to three subfamilies, designated ␣, ␤, and defensins, have been identified in leukocytes and other cells of humans or nonhuman primates. ␣ defensins contain 29 to 35 residues and are produced as ϳ100-residue prepropeptides (16). Human neutrophils (polymorphonuclear leukocytes) contain four ␣ defensins, called HNPs 1, 2, 3, an...

Genomic DNA libraries were constructed for Chlamydia trachomatis serovars B and C by using BamHI fragments, and recombinants that contained the major outer membrane protein (ompl) gene for each serovar were identified and sequenced. Comparisons between these gene sequences and the gene from serovar L2 demonstrated fewer base pair differences between serovars L2 and B than between L2 and C; this finding is consistent with the serologic and antigenic relationships among these serovars. The translated amino acid sequence for the major outer membrane proteins (MOMPs) contained the same number of amino acids for serovars L2 and B, whereas the serovar C MOMP contained three additional amino acids. The antigenic diversity of the chlamydial MOMP was reflected in four sequence-variable domains, and two of these domains were candidates for the putative type-specific antigenic determinant. The molecular basis of ompl gene diversity among C. trachomatis serovars was observed to be clustered nucleotide substitutions for closely related serovars and insertions or deletions for distantly related serovars.Chlamydia trachomatis is a procaryote that is the cause of a wide spectrum of human diseases affecting hundreds of millions of people worldwide. The most notable diseases are trachoma, a blinding eye disease, and genitourinary tract diseases that often result in sterility (7). Chlamydiae are obligate intracellular bacteria that have a unique biphasic growth cycle which facilitates their survival in two discontinuous habitats. The major outer membrane protein (MOMP) of chlamydiae is one of the principal cell wall surface components that is responsible for the structural integrity of the extracellular infectious elementary body and the developmental conversion to the plastic and fragile intracellular reticulate body (8). This protein also has poreforming capabilities that permit exchange of nutrients for the reticulate body form (2). The structural and porin functions of the MOMP are mediated by disulfide bond interactions within and between MOMP molecules and other components (16).Surface components of chlamydiae are presumed to modulate the essential biological events of attachment, induced phagocytosis, inhibition of phagolysosomal fusion, infectivity, toxicity, and the host immune responses that contribute to immunity and pathogenesis (21). The immunodominant MOMP has been implicated in some of these important functions, primarily by association of many of these processes with a type-specific antigen. The predominant typespecific determinant for chlamydiae has been attributed to the MOMP by using monospecific (4) and monoclonal (25, 27) antibodies.C. trachomatis has been extensively characterized serologically, with more than 15 serovars defined by polyvalent antisera (28) and monoclonal antibodies (29). Monoclonal antibody specificities to MOMP define species-, subspecies-, and type-specific determinants; thus, the MOMP represents a serological matrix of epitopes such that a single molecule possesses both constant and varia...

A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide microplate assay was adapted to screen for the ability of 20 host-defense peptides to inactivate herpes simplex virus type 1 and type 2. The procedure required minimal amounts of material, was reproducible, and was confirmed with standard antiviral testing techniques. In screening tests, with the exception of melittin, a highly cytotoxic and hemolytic peptide found in bee venom, the alpha-helical peptides in our test panel (magainins, cecropins, clavanins, and LL-37) caused little viral inactivation. Several beta-sheet peptides (defensins, tachyplesin, and protegrins) inactivated one or both viruses, sometimes with remarkable selectivity. Two peptides were identified as having antiviral activity against both viruses, indolicidin (a tryptophan-rich peptide from bovine neutrophils) and brevinin-1 (a peptide found in frog skin). The antiviral activity of these two peptides was confirmed with standard antiviral assays. Interestingly, the antiviral activity of brevinin-1 was maintained after reduction and carboxamidomethylation, procedures that abolished its otherwise prominent hemolytic and cytotoxic effects.

The principal surface protein antigen of Chlamydia trachomatis is the major outer membrane protein (MOMP). The MOMP is antigenically complex. Among the 15 serovars of C. trachomatis, mAbs define serovar-, subspecies-, and species-specific determinants on MOMP. The molecular basis of the antigenic diversity of these proteins is reflected in amino acid variable sequence domains. We have mapped the dominant topographic antigenic determinants of MOMP that are defined by mAbs. Using recombinant DNA approaches we have identified the linear distribution of two antigenic domains. One domain contains a serovar-specific determinant and the other contains subspecies- and species-specific determinants. These antigenic domains correspond to two amino acid sequence variable domains. Synthetic peptides were immunogenic and these resolved the serovar-specific determinant within a 14-amino acid peptide. The subspecies- and species-specific determinants were overlapping within a 16-amino acid peptide.

New vaccine strategies are needed for prevention of leptospirosis, a widespread human and veterinary disease caused by invasive spirochetes belonging to the genus Leptospira. We have examined the immunoprotective capacity of the leptospiral porin OmpL1 and the leptospiral outer membrane lipoprotein LipL41 in the Golden Syrian hamster model of leptospirosis. Specialized expression plasmids were developed to facilitate expression of leptospiral proteins inEscherichia coli as the membrane-associated proteins OmpL1-M and LipL41-M. Although OmpL1-M expression is highly toxic inE. coli, this was accomplished by using plasmid pMMB66-OmpL1, which has undetectable background expression without induction. LipL41-M expression and processing were enhanced by altering its lipoprotein signal peptidase cleavage site to mimic that of the murein lipoprotein. Active immunization of hamsters with E. coli membrane fractions containing a combination of OmpL1-M and LipL41-M was found to provide significant protection against homologous challenge with Leptospira kirschneri serovar grippotyphosa. At 28 days after intraperitoneal inoculation, survival in animals vaccinated with both proteins was 71% (95% confidence interval [CI], 53 to 89%), compared with only 25% (95% CI, 8 to 42%) in the control group (P < 0.001). On the basis of serological, histological, and microbiological assays, no evidence of infection was found in the vaccinated survivors. The protective effects of immunization with OmpL1-M and LipL41-M were synergistic, since significant levels of protection were not observed in animals immunized with either OmpL1-M or LipL41-M alone. In contrast to immunization with the membrane-associated forms of leptospiral proteins, hamsters immunized with His6-OmpL1 and His6-LipL41 fusion proteins, either alone or in combination, were not protected. These data indicate that the manner in which OmpL1 and LipL41 associates with membranes is an important determinant of immunoprotection.