Fulvia Di Marzo Veronese

Characterization of an HIV-1 point mutant blocked in envelope glycoprotein cleavage

The envelope proteins of retroviruses are derived from a polypeptide precursor protein by cleavage adjacent to a cluster of basic amino acids. Site-specific mutagenesis was used to construct a mutant of the human immunodeficiency virus type 1 (HIV-1) in which the arginine residue at the carboxy-terminus of the gp120 was changed to a threonine residue. This single substitution was sufficient to abolish all detectable cleavage of the gp160 envelope precursor polypeptide as well as virus infectivity. The gp160 was produced in normal quantities from a biologically active clone of the mutant virus after transfection into cos-1 cells. The mutant gp160 contained N-linked oligosaccharide chains with mannose-rich cores similar to those of the gp160 produced by the wild-type clone. Immunofluorescence assays showed that gp160 was transported to the surface of transfected CD4+ HeLa cells. No envelope proteins of known size could be detected in the media of cells transfected with the mutant virus, suggesting that functional virions were not formed. Binding of the mutant gp160 to the CD4 receptor molecule was unimpaired. Despite this and the presence of gp160 on the cell surface, neither growth of mutant-transfected CD4+ HeLa cells nor cocultivation of transfected cos-1 cells with H9 cells resulted in significant syncytium formation. The data indicate that the carboxy-terminal arginine residue of HIV-1 gp120 is necessary for envelope protein cleavage and suggest cleavage is important in the virus life cycle in both functional virus release and membrane fusion.

Characterization of a Neutralizing Monoclonal Antibody to the External Glycoprotein of HIV-1

The major neutralizing epitope on the external glycoprotein of HIV-1 was studied with an envelope-specific monoclonal antibody and with a human serum positive for antibodies to HIV-1 proteins, both of which were able to neutralize virus infectivity. The monoclonal antibody reacted specifically with gp120 from HIV-1IIIB, and was shown to neutralize infection of CEM cells by cell-free virions, and inhibited the formation of syncytia normally observed when uninfected cells are cocultured with HIV-1-infected cells. Similar neutralization of viral infection and inhibition of syncytia formation was also demonstrated by the HIV-1-antibody-positive human serum. By examining a number of overlapping peptides from a region of HIV-1 gp120 known to contain a neutralizing epitope, this epitope was localized between amino acids 307 and 320 (V3 loop) in the external glycoprotein molecule. The monoclonal antibody did not interfere with the binding of gp120 to CD4, or with the subsequent step of CD4-induced shedding of gp120 from the viral envelope. However, it blocked the proteolytic cleavage of the V3 loop by thrombin, suggesting that the antibody may be inhibiting the interaction of the loop with other membrane-bound proteins.

Hormone regulation of the mucosal environment in the reproductive tract and the prevention of HIV infection.

On May 28, 29, 2013, a workshop entitled ‘Hormone Regulation of the Mucosal Environment in the Reproductive Tract and the Prevention of HIV Infection’ was organized and sponsored by the National Institute of Allergy and Infectious Diseases (NIAID) of the National Institutes of Health (NIH) in collaboration with the Geisel School of Medicine at Dartmouth in Boston, MA. This event was planned in conjunction with the 12th Congress of the International Society for Immunology of Reproduction (ISIR) hosted by the American Society of Reproductive Immunology (ASRI). The goal of this workshop was to build bridges between the HIV and reproductive immunology fields, two scientific communities that have not historically talked to each other. The workshop followed on a major theme emerging from a previous meeting also organized by NIAID in collaboration with the Dartmouth Medical School in 2010 entitled ‘Mucosal Immunity in the Male and Female Reproductive Tract and Prevention of HIV Transmission’ (AJRI, Volume 65, Issue 3, Pages 181–376, March 2011. Special Issue: Sexual Transmission of HIV in the 21st Century). Participants in that meeting strongly felt that the HIV field had an incomplete understanding of the impact of endogenous and exogenous hormones on the mucosal environment of the FRT and on HIV susceptibility. Indeed, the role of hormones and hormonal contraception in HIV transmission is an ongoing controversy. The observational evidence regarding hormonal contraceptive use and HIV acquisition risk is inconsistent in method, quality, and conclusion. The majority of results for both oral and injectable contraceptives found no significant change in HIV acquisition risk associated with using these methods, compared to using no hormonal contraception. In contrast, recent results from the HIV prevention trials Partners PrEP and VOICE found that both oral and injectable contraceptive use increased HIV acquisition risk, reigniting the debate. Biological studies strongly suggest that both exogenous and endogenous hormones are likely to affect the human female reproductive tract (FRT) environment and immunity in ways that could plausibly have an influence on HIV acquisition risk. However, most of the discussions around this debate have focused on identifying design of clinical trials that would inform on the issue of contraception and HIV risk, without any focus on the potential biological mechanisms that are involved in the interaction. Therefore, the main purpose of this workshop was to ‘go back to the basics’ and have a conversation on the complexity of biological interactions between sex hormones, mucosal immunity, and increased susceptibility to HIV infection. The first of its kind, this meeting brought together leaders in HIV research, reproductive biology and immunology to exchange information, identify gaps in knowledge, and initiate avenues of collaboration with the ultimate goal of creating interest in and expanding on this critical area of HIV research. The scientific program included five plenary sessions with ample time for discussion between speakers and participants that numbered 125 attendees. The organizing committee, composed of Drs. Charles R. Wira, Geisel School of Medicine at Dartmouth; Fulvia Veronese, NIAID; Jim Turpin, Division of AIDS, NIAID; Susan Cu-Uvin, the Warren Alpert Medical School of Brown University; Ashley Haase, University of Minnesota; Charu Kaushic, McMaster University; Alan Landay, Rush University Medical Center; and Jiri Mestecky, University of Alabama at Birmingham, developed a scientific agenda that focused on a broad range of cross-cutting topics to be addressed by leaders in the field. The topics of these sessions were as follows: Landscape of HIV prevention. Role of endogenous hormones in regulation of the FRT immune function. Role of mucosal (secretion) microenvironment in HIV prevention and risk. Role of mucosal (tissue) environment in HIV prevention and risk. Hormonal contraceptives and their impact on HIV. The first session of the meeting set the stage and provided the context for the subsequent sessions by presenting the landscape of HIV prevention research. Participants came from different disciplines and provided a set of presentations broadly covering key topics in HIV prevention: the mechanisms of early virus infection and dissemination, control of HIV through vaccination, and the development of preventive interventions like microbicides and PrEP. The following presentations throughout the 2 days focused on identifying what we know about the role of endogenous hormones in FRT physiology, immunity, and susceptibility to infection; defining the role of secretions throughout the genital tract, their origins, regulation by sex hormones and the microbiome, and their roles in immune protection; identifying the role of the tissue environment throughout the FRT in protecting from or increasing susceptibility to HIV infection. Discussions at the end of each session were vibrant and thought provoking with great audience participation. Main topics of discussion included: the early events in HIV infection, the upper reproductive tract and particularly the ovaries as hot spots for infection, the differential immune response depending on the location of the immune cells in the FRT and its influence on the overall response to infection, the role of stromal fibroblasts in maintaining a microenvironment for regulating immune protection, the pharmacology of contraceptive hormones and their tissue pharmacokinetics (PK), and the different susceptibility to infection depending on the stage of the menstrual cycle. At the end of the workshop, we were humbled by the complexity and nuances of the hormones/ HIV susceptibility interaction, but came away with much better understanding of the opportunities, challenges, and areas of research that should be pursued. Critical to this effort is the identification of preclinical models to study the biological mechanisms underpinning the hormones/HIV interaction and relevant populations for the studies. The trans-gender population is very important in this regard because of the range of hormones used and the vulnerability of these individuals to HIV infection. Included in this workshop was the examination of the links between sexual violence, genito-anal injury, and HIV, which is grossly understudied but very significant for understanding the HIV epidemics disproportionate impacts on young women and girls. The unique and innovative nature of the workshop attracted participants from all over the world. While the majority of participants were from the United States, we had representation from Germany, Hungary, Europe, Australia, South Africa, Tanzania, India, and Japan. It was very rewarding and exciting to see that many participants were young researchers just entering the field. The social events around the workshop were key in promoting the interaction of graduate students and postdoctoral fellows with leaders in the fields of HIV, sex hormones, and reproductive immunology. Lunches were filled with animated discussions and plenty of opportunities for exchange of data, opinions, and ideas for collaborations. Owing to an overwhelming response of our invited speakers for Review Articles, we are able to present this special issue, dedicated exclusively to the hormone regulation of the mucosal environment in the reproductive tract and the prevention of HIV infection. As indicated in Contents, Review Articles have been categorized according to the plenary sessions within which they were presented. The organizers are grateful for the fruitful collaboration with AJRI. Hopefully, the information shared during the workshop and in this special issue will shed some light and attract interest to the critical interaction between sex hormones, the female reproductive tract, and HIV transmission with particular focus on contraceptive hormones. Moreover, the biological mechanisms discussed during the meeting provide a solid foundation for understanding the risk of HIV acquisition associated with pregnancy, menopause, sexually transmitted diseases, or other endocrine changes during the life cycle in women. Throughout the world, fertility regulation and contraception are among the most important issues for women, far more important than their risk of acquiring HIV. It is imperative that we provide women with the safest and most effective methods to prevent unwanted pregnancies and HIV infection. We are hopeful that this workshop contributed toward this goal.

Proteins Encoded by the Human T-Lymphotropic Virus Type III/ Lymphadenopathy Associated Virus (HTLV-III/LAV) Genes

Human T-lymphotropic virus-type III/lymphadenopathy associated virus (HTLV-III/LAV) has been identified as the primary etiologic agent of human acquired immune deficiency syndrome (AIDS) (1–4). Among members of epidemiologically identified risk groups of AIDS, there has been a high prevalence of HTLV-III/LAV infection irrespective of whether they have clinical AIDS or have only some signs and symptoms associated with AIDS [AIDS-related complex (ARC)], or are completely free of any disease symptoms (4–6). Thus a significant proportion (up to 50 or 60 %), depending on where they live) of homosexual males at risk for AIDS have serum antibodies to HTLV-III/LAV proteins (6,7). Infectious virus has been isolated from lymphocyte cultures derived from many of these asymptomatic antibody-positive individuals (2,8). HTLV-III/LAV-infection through contaminated blood has been identified as the cause of AIDS in transfusion recipients with no other risk factor for AIDS (9,10).