Amy L. Cole

Cationic polypeptides are required for anti-HIV-1 activity of human vaginal fluid.

Mucosal surfaces of the vagina are the portals for heterosexual transmission of HIV-1 and therefore play a fundamental role in the pathogenesis of primary infection. In the search for direct biological evidence for the role of human vaginal fluid in innate host defense, we characterized the anti-HIV-1 function of cationic polypeptides within minimally manipulated vaginal fluid. In the current study we revealed that vaginal fluid confers intrinsic anti-HIV-1 properties against both X4 and R5 strains of HIV-1 and could protect against HIV-1 infection and reduce proviral genome integration in organotypic cultures of human cervicovaginal tissue. The majority of this activity was contained in the cationic polypeptide fraction, and the depletion of cationic polypeptides using a selective cation exchange resin ablated most of the intrinsic activity against HIV-1. By adding the cationic polypeptide fraction to depleted vaginal fluid, we were able to restore activity against HIV-1. Using a proteomic approach, we identified 18 cationic polypeptides within vaginal fluid, nearly all of which are either known antimicrobials or have other purported roles in host defense. Interestingly, physiologic concentrations of 13 of the cationic polypeptides were not active alone against HIV-1, yet in concert they partially restored the anti-HIV-1 activity of cation-depleted vaginal fluid. These results suggest that synergism between cationic polypeptides is complex, and full anti-HIV-1 activity probably involves the aggregate of the cationic peptides and proteins in vaginal fluid.

PRO 2000 elicits a decline in genital tract immune mediators without compromising intrinsic antimicrobial activity

Objective:Vaginal microbicides should protect against infection without disrupting the mucosal environment or its mediators of host defense. The objective of this study was to examine the effect of 14 daily applications of 0.5% PRO 2000 or placebo gel on mediators of mucosal immunity and intrinsic antimicrobial activity. Design and methods:A randomized, prospective, double-blind, placebo-controlled study was conducted among 24 healthy, abstinent women. Levels of cytokines, chemokines, defensins, and other protective factors and intrinsic antimicrobial activity were determined in cervicovaginal lavage samples collected on study days 0, 7, 14, and 21. Results:No increase in pro-inflammatory cytokines was observed. Rather cytokines and protective factors including interleukin (IL)-1 receptor antagonist, immunoglobulins and human beta-defensin 2 were lower in the drug compared with the placebo group. All of the mediators returned towards baseline on day 21. Women who were cycling had lower levels of most proteins on study days 7 and/or 14 compared with women on oral contraceptives; however, the magnitude of decline was greater in women who received PRO 2000 compared with placebo gel. The reduction in protective factors was not associated with a loss in the intrinsic anti-viral (HIV or herpes simplex virus) activity or anti-bacterial activity (Escherichia coli or Staphylococcus aureus). Conclusion:In contrast to experience with nonoxynol-9, PRO 2000 did not trigger an inflammatory response in cervicovaginal secretions. There was a modest reduction in mucosal immune mediators, but this loss was not associated with a reduction in intrinsic antimicrobial activity.

Reawakening retrocyclins: ancestral human defensins active against HIV-1.

Human alpha and beta defensins contribute substantially to innate immune defenses against microbial and viral infections. Certain nonhuman primates also produce theta-defensins—18 residue cyclic peptides that act as HIV-1 entry inhibitors. Multiple human theta-defensin genes exist, but they harbor a premature termination codon that blocks translation. Consequently, the theta-defensins (retrocyclins) encoded within the human genome are not expressed as peptides. In vivo production of theta-defensins in rhesus macaques involves the post-translational ligation of two nonapeptides, each derived from a 12-residue “demidefensin” precursor. Neither the mechanism of this unique process nor its existence in human cells is known. To ascertain if human cells retained the ability to process demidefensins, we transfected human promyelocytic cells with plasmids containing repaired retrocyclin-like genes. The expected peptides were isolated, their sequences were verified by mass spectrometric analyses, and their anti-HIV-1 activity was confirmed in vitro. Our study reveals for the first time, to our knowledge, that human cells have the ability to make cyclic theta-defensins. Given this evidence that human cells could make theta-defensins, we attempted to restore endogenous expression of retrocyclin peptides. Since human theta-defensin genes are transcribed, we used aminoglycosides to read-through the premature termination codon found in the mRNA transcripts. This treatment induced the production of intact, bioactive retrocyclin-1 peptide by human epithelial cells and cervicovaginal tissues. The ability to reawaken retrocyclin genes from their 7 million years of slumber using aminoglycosides could provide a novel way to secure enhanced resistance to HIV-1 infection.

Antimicrobial polypeptides are key anti-HIV-1 effector molecules of cervicovaginal host defense.

Mucosal surfaces of the cervix and vagina are portals for heterosexual transmission of human immunodeficiency virus type 1 (HIV‐1) and, therefore, play a fundamental role in the pathogenesis of primary infection. Cationic antimicrobial polypeptides including defensins are the principal effector molecules of mucosal innate immunity against microbes and viruses such as HIV. In cervicovaginal secretions, antimicrobial polypeptides constitute the majority of the intrinsic anti‐HIV‐1 activity, synergism between cationic polypeptides is complex, and full anti‐HIV‐1 activity involves the complete complement of cationic polypeptides. Periods in which cationic antimicrobial polypeptide expression is reduced are likely associated with increased susceptibility to HIV‐1 infection. This review provides an overview of the role of cationic antimicrobial polypeptides in innate cervicovaginal anti‐HIV‐1 host defense, and discusses how hormones and bacterial infections can regulate their expression. Emphasis is placed on the θ‐defensin (retrocyclin) class of anti‐HIV‐1 peptides and their potential for development as topical microbicides to prevent HIV‐1 transmission.

REVIEW ARTICLE: Antimicrobial Polypeptides are Key Anti‐HIV‐1 Effector Molecules of Cervicovaginal Host Defense

Mucosal surfaces of the cervix and vagina are portals for heterosexual transmission of human immunodeficiency virus type 1 (HIV‐1) and, therefore, play a fundamental role in the pathogenesis of primary infection. Cationic antimicrobial polypeptides including defensins are the principal effector molecules of mucosal innate immunity against microbes and viruses such as HIV. In cervicovaginal secretions, antimicrobial polypeptides constitute the majority of the intrinsic anti‐HIV‐1 activity, synergism between cationic polypeptides is complex, and full anti‐HIV‐1 activity involves the complete complement of cationic polypeptides. Periods in which cationic antimicrobial polypeptide expression is reduced are likely associated with increased susceptibility to HIV‐1 infection. This review provides an overview of the role of cationic antimicrobial polypeptides in innate cervicovaginal anti‐HIV‐1 host defense, and discusses how hormones and bacterial infections can regulate their expression. Emphasis is placed on the θ‐defensin (retrocyclin) class of anti‐HIV‐1 peptides and their potential for development as topical microbicides to prevent HIV‐1 transmission.

Exoproteome of Staphylococcus aureus reveals putative determinants of nasal carriage.

Due to the increasing prevalence of nosocomial and community-acquired antibiotic resistant Staphylococcus aureus (SA), understanding the determinants of SA nasal carriage has become a major imperative. Previous research has revealed many host and bacterial factors that contribute to SA nasal carriage. To assess bacterial factors that facilitate nasal carriage, we compared the exoproteome of a nasal carrier strain of SA to a genetically similar noncarrier strain. Additionally, the carrier strain biofilm exoproteome was also compared against its planktonic counterpart. Using high throughput proteomics, it was observed that the carrier strain of SA secretes a greater number of proteins that may promote successful colonization of the human nose, including cell attachment and immunoevasive proteins, than the noncarrier strain. Similarly, SA carrier strain biofilm exoproteome contains a greater number of immunoevasive proteins than its planktonic counterpart. Analysis of the most abundant immunoevasive proteins revealed that Staphylococcal protein A was present at significantly higher levels in carrier than in noncarrier strains of SA, suggesting an association with nasal carriage. While further analyses of specific differences between carrier and noncarrier strains of SA are required, many of the differentially expressed proteins identified can be considered to be putative determinants of nasal carriage.

Cationic polypeptides contribute to the anti-HIV-1 activity of human seminal plasma

Mucosal surfaces of the reproductive tract as well as their secretions have important roles in preventing sexual transmission of HIV‐1. In the current study, the majority of the intrinsic anti‐HIV‐1 activity of human seminal plasma (SP) was determined to reside in the cationic polypeptide fraction. Antiviral assays utilizing luciferase reporter cells and lymphocytic cells revealed the ability of whole SP to prevent HIV‐1 infection, even when SP was diluted 3200‐fold. Subsequent fractionation by continuous flow acid‐urea (AU)‐PAGE and antiviral testing revealed that cationic polypeptides within SP were responsible for the majority of anti‐HIV‐1 activity. A proteomic approach was utilized to resolve and identify 52 individual cationic polypeptides that contribute to the aggregate anti‐HIV‐1 activity of SP. One peptide fragment of semenogelin I, termed SG‐1, was purified from SP by a multistep chromatographic approach, protein sequenced, and determined to exhibit anti‐HIV‐1 activity against HIV‐1. Anti‐HIV‐1 activity was transient, as whole SP incubated for prolonged time intervals exhibited a proportional decrease in anti‐HIV‐1 activity that was directly attributed to the degradation of semenogelin I peptides. Collectively, these results indicate that the cationic polypeptide fraction of SP is active against HIV‐1, and that semenogelin‐derived peptides contribute to the intrinsic anti‐HIV‐1 activity of SP.—Martellini, J. A., Cole, A. C., Venkataraman, N., Quinn, G. A., Svoboda, P., Gangrade, B. K., Pohl, J., Sørensen, O. E., Cole, A. M. Cationic polypeptides contribute to the anti‐HIV‐1 activity of human seminal plasma. FASEB J. 23, 3609–3618 (2009). www.fasebj.org

The retrocyclin analogue RC‐101 prevents human immunodeficiency virus type 1 infection of a model human cervicovaginal tissue construct

Retrocyclins are cyclic antimicrobial peptides that exhibit potent activity towards a broad range of primary and laboratory‐adapted strains of human immunodeficiency virus type 1 (HIV‐1) in vitro. The current study shows that RC‐101, an analogue of retrocyclin, prevented HIV‐1 infection in an organ‐like construct of human cervicovaginal tissue and retained full activity in the presence of vaginal fluid. The peptide remained within the cervicovaginal tissues throughout the 9‐day incubation period without altering tissue viability, inducing damage or inducing the release of inflammatory cytokines. Collectively, these data support the potential development of RC‐101 as a topical microbicide to prevent HIV‐1 infection and transmission.

The Formulated Microbicide RC-101 Was Safe and Antivirally Active Following Intravaginal Application in Pigtailed Macaques

Background RC-101 is a congener of the antiretroviral peptide retrocyclin, which we and others have reported is active against clinical HIV-1 isolates from all major clades, does not hemagglutinate, and is non-toxic and non-inflammatory in cervicovaginal cell culture. Herein, film-formulated RC-101 was assessed for its antiviral activity in vitro, safety in vivo, retention in the cervix and vagina, and ability to remain active against HIV-1 and SHIV after intravaginal application in macaques. Methodology/Principal Findings RC-101 was formulated as a quick-dissolving film (2000 µg/film), retained complete activity in vitro as compared to unformulated peptide, and was applied intravaginally in six pigtailed macaques daily for four days. At one and four days following the final application, the presence of RC-101 was assessed in peripheral blood, cervicovaginal lavage, cytobrushed cervicovaginal cells, and biopsied cervical and vaginal tissues by quantitative western blots. One day following the last film application, cervical biopsies from RC-101-exposed and placebo-controlled macaques were collected and were subjected to challenge with RT-SHIV in an ex vivo organ culture model. RC-101 peptide was detected primarily in the cytobrush and biopsied cervical and vaginal tissues, with little to no peptide detected in lavage samples, suggesting that the peptide was associated with the cervicovaginal epithelia. RC-101 remained in the tissues and cytobrush samples up to four days post-application, yet was not detected in any sera or plasma samples. RC-101, extracted from cytobrushes obtained one day post-application, remained active against HIV-1 BaL. Importantly, cervical biopsies from RC-101-treated animals reduced RT-SHIV replication in ex vivo organ culture as compared to placebo-treated animals. Conclusions/Significance Formulated RC-101 was stable in vivo and was retained in the mucosa. The presence of antivirally active RC-101 after five days in vivo suggests that RC-101 would be an important molecule to develop further as a topical microbicide to prevent HIV-1 transmission.

HIV-1 Adapts to a Retrocyclin with Cationic Amino Acid Substitutions That Reduce Fusion Efficiency of gp41

Retrocyclin (RC)-101 is a cationic θ-defensin that inhibits HIV-1 entry. Passaging HIV-1BAL under selective pressure by this cyclic minidefensin resulted in only a 5- to 10-fold decrease in viral susceptibility to RC-101. Emergent viral isolates had three amino acid substitutions in their envelope glycoprotein. One was in a CD4-binding region of gp120, and the others were in the heptad repeat (HR) domains of gp41 (HR1 and HR2). Each mutation replaced an electroneutral or electronegative residue with one that was positively charged. These mutations were evaluated either alone or in combination in a single-round viral entry assay. Although the mutation in gp120 did not affect viral entry, the mutation in HR1 of gp41 conferred relative resistance to RC-101. Interestingly, the envelope with the HR2 mutation was less efficient and became codependent on the presence of RC-101 for entry. The adaptive response of HIV-1 to this cationic host defense peptide resembles the responses of bacteria that modulate their surface or membrane charge to evade analogous host defense peptides. These findings also suggest that interactions between θ-defensins and gp41 may contribute to the ability of these cyclic minidefensins to prevent HIV-1 entry into target cells.