Robin J. Shattock

Prevention of virus transmission to macaque monkeys by a vaginally applied monoclonal antibody to HIV-1 gp120

A topical microbicide reduces the probability of virus transmission when applied to the vagina or rectum of a person at risk of sexually acquiring HIV-1 infection. An effective microbicide could significantly reduce the global spread of HIV-1, particularly if women were able to use it covertly to protect themselves. A microbicide could target the incoming virus and either permanently inactivate it or reduce its infectivity, or it could block receptors on susceptible cells near the sites of transmission. We describe here how vaginal administration of the broadly neutralizing human monoclonal antibody b12 can protect macaques from simian-human immunodeficiency virus (SHIV) infection through the vagina. Only 3 of 12 animals receiving 5 mg b12 vaginally in either saline or a gel and then challenged vaginally (up to 2 h later) with SHIV-162P4 became infected. In contrast, infection occurred in 12 of 13 animals given various control agents under similar conditions. Lower amounts of b12 were less effective, suggesting that protection was dose dependent. These observations support the concept that viral entry inhibitors can help prevent the sexual transmission of HIV-1 to humans.

Inhibiting sexual transmission of HIV-1 infection

The worldwide infection rate for HIV-1 is estimated to be 14,000 per day, but only now, more than 20 years into the epidemic, are the immediate events between exposure to infectious virus and the establishment of infection becoming clear. Defining the mechanisms of HIV-1 transmission, the target cells involved and how the virus attaches to and fuses with these cells, could reveal ways to block the sexual spread of the virus. In this review, we will discuss how our increasing knowledge of the ways in which HIV-1 is transmitted is shaping the development of new, more sophisticated intervention strategies based on the application of vaginal or rectal microbicides.

Target cells in vaginal HIV transmission.

Understanding the mechanisms of HIV transmission to women will be crucial to the development of effective strategies to curb this epidemic. Current data suggest that HIV has at least two routes to penetrate the vaginal epithelium and reach lymphoid tissues, trans-epithelial migration of infected Langerhans cells or virus penetration into the lamina propria through loss of epithelial integrity resulting in direct infection of lymphocytes, dendritic cells and macrophages.

Mycobacterial 65‐kD heat shock protein induces release of proinflammatory cytokines from human monocytic cells

Monocytes having phagocytosed mycobacteria are known to present the bacterial 65‐kD heat shock protein (hsp) on their cell surface to αβ and γδ T lymphocytes. Cytotoxic CD4+ cells may then lyse monocytes expressing mycobacterial 65‐kD hsp. However, it is not known whether 65‐kD hsp directly stimulates monocyte functions other than antigen presentation. This study has demonstrated that following extraction of bacterial lipopolysaccharide, purified recombinant mycobacterial 65‐kD hsp may directly activate THP‐1 cells, a human monocytic line, to accumulate mRNA for and secrete tumour necrosis factor (TNF), a cytokine important in granuloma formation, the characteristic host immune response to mycobacterial infection. TNF gene expression and secretion following stimulation by hsp was dose‐dependent and abolished by heat‐induced proteolysis. Subsequently, THP‐1 cells secreted IL‐6 and IL‐8, cytokines involved in recruitment and differentiation of T lymphocytes. The data indicate that secretion of proinflammatory cytokines from monocytes activated by mycobacterial 65‐kD hsp may be important in the host immune response and in the development of antigen‐specific T cell‐mediated immunity.

IFN-gamma at the site of infection determines rate of clearance of infection in cryptococcal meningitis.

In animal models, immunity to cryptococcal infection, as in many chronic fungal and bacterial infections, is associated with a granulomatous inflammatory response, intact cell-mediated immunity, and a Th1 pattern of cytokine release. To examine the correlates of human immunity to cryptococcal infection in vivo, we analyzed immune parameters at the site of infection over time and assessed the rate of clearance of infection by serial quantitative cerebrospinal fluid (CSF) fungal cultures in 62 patients in a trial of antifungal therapy for HIV-associated cryptococcal meningitis. CSF IL-6, IFN-γ, TNF-α, and IL-8 were significantly higher in survivors compared with nonsurvivors. There were negative correlations between log TNF-α, IFN-γ, and IL-6 levels and baseline cryptococcal CFU. Log IFN-γ, G-CSF, TNF-α, and IL-6 were correlated positively with the rate of fall in log CFU/ml CSF/day. In a linear regression model including antifungal treatment group, baseline CFU, and these cytokines, only treatment group and log IFN-γ remained independently associated with rate of clearance of infection. The results provide direct in vivo evidence for the importance of quantitative differences in IFN-γ secretion in human immune control of granulomatous infections, and increase the rationale for adjunctive IFN-γ in the treatment of refractory HIV-associated cryptococcosis.

Inhibition of Human Immunodeficiency Virus Type 1 Infection by the Candidate Microbicide Dapivirine, a Nonnucleoside Reverse Transcriptase Inhibitor

ABSTRACT Heterosexual transmission of human immunodeficiency virus (HIV) remains the major route of infection worldwide; thus, there is an urgent need for additional prevention strategies, particularly strategies that could be controlled by women, such as topical microbicides. Potential microbicide candidates must be both safe and effective. Using cellular and tissue explant models, we have evaluated the activity of the nonnucleoside reverse transcriptase inhibitor (NNRTI) dapivirine as a vaginal microbicide. In tissue compatibility studies, dapivirine was well tolerated by epithelial cells, T cells, macrophages, and cervical tissue explants. Dapivirine demonstrated potent dose-dependent inhibitory effects against a broad panel of HIV type 1 isolates from different clades. Furthermore, dapivirine demonstrated potent activity against a wide range of NNRTI-resistant isolates. In human cervical explant cultures, dapivirine was able not only to inhibit direct infection of mucosal tissue but also to prevent the dissemination of the virus by migratory cells. Activity was retained in the presence of semen or a cervical mucus simulant. Furthermore, dapivirine demonstrated prolonged inhibitory effects: it was able to prevent both localized and disseminated infection for as long as 6 days posttreatment. The prolonged protection observed following pretreatment of genital tissue and the lack of observable toxicity suggest that dapivirine has considerable promise as a potential microbicide candidate.

Cyanovirin-N potently inhibits human immunodeficiency virus type 1 infection in cellular and cervical explant models

In the absence of a protective vaccine against human immunodeficiency virus (HIV), there is an urgent need for the development of effective topical microbicides to prevent HIV infection. Candidate vaginal microbicides should provide protection against circulating strains, be cheap, stable on storage, safe and easy to use. Here we describe a detailed study of the safety and efficacy of Cyanovirin-N (CV-N) in vitro, and in an ex vivo model of female genital tissue explants. CV-N demonstrated potent activity in the low nanomolar range against laboratory and primary isolates. Activity was related to the affinity of CV-N for binding to whole virions as determined by acoustic resonance. Potent activity was also observed against cell-associated HIV-1, although slightly reduced. CV-N activity in the presence of whole semen was reduced by 7-10-fold, although it remained in the low nanomolar range and was minimally modified by the presence of Candida albicans. Furthermore, CV-N potently inhibited infection of ectocervical explants and virus dissemination by tissue-emigrating cells. In peripheral blood mononuclear cell (PBMC) assays, CV-N was shown to have some mitogenic activity following 3 days exposure to compound, and this was associated with a modest increase in expression of gamma interferon, stromal cell-derived factor 1beta and interleukin 4. However, 2 h exposure to CV-N had no effect on cytokine expression in PBMC or tissue explant culture over a 24 h period, suggesting that the potential for inflammation is low. Data presented here indicate that targeting HIV envelope glycoproteins may provide an effective strategy to prevent HIV-1 infection mediated by either cell-free virus or infected cells.

Sustained plasma TNF-alpha and HIV-1 load despite resolution of other parameters of immune activation during treatment of tuberculosis in Africans.

OBJECTIVEnTo determine the impact of treatment of tuberculosis on plasma HIV-1 load in African subjects and to correlate viral load with response to treatment and changes in immune activation.nnnDESIGNnClinical and microbiological responses, immune activation parameters and plasma HIV-1 load were determined in 20 patients with pulmonary tuberculosis and HIV-1 coinfection in Ghana, West Africa during the first 3 months of anti-tuberculosis treatment.nnnMETHODSnPlasma HIV-1 load and markers of immune activation were determined by commercially available assays. Human leukocyte antigen (HLA)-DR incorporation into the HIV-1 envelope was measured by using an immunomagnetic capture technique.nnnRESULTSnTreatment of tuberculosis resulted in significant improvements in weight and haemoglobin, a high sputum smear conversion rate and marked reductions in mean plasma tumour necrosis factor (TNF) receptor-1, interleukin-6 and C-reactive protein. Furthermore, incorporation of host HLA-DR into the HIV-1 envelope decreased; this also suggested a reduction in immune activation of the cells supporting viral replication. However, of importance with regard to AIDS pathogenesis, neither mean plasma TNF-alpha nor HIV-1 load decreased significantly.nnnCONCLUSIONSnThe failure of HIV-1 plasma load to decline significantly during the initial months of anti-tuberculosis treatment is associated with high, sustained systemic levels of TNF-alpha. The dissociation between the sustained levels of plasma TNF-alpha and the major reductions in other, diverse immune activation parameters may represent dysregulation of cytokine production in these African patients.

Transgenic plant production of Cyanovirin-N, an HIV microbicide

Cyanovirin‐N (CV‐N) is a microbicide candidate that inactivates a wide range of HIV strains by binding to gp120. Production of CV‐N, or any protein microbicide, needs to be at extremely high levels and low cost to have an impact on global health. Thus, it is unlikely that fermentor‐based systems will be suitable, including recombinant E. coli, where CV‐N aggregates and dimers have consistently been found. Transgenic plants may provide a suitable expression system for protein microbicides, as production can be easily and economically scaled up. Here, Nicotiana tabacum was transformed with a gene encoding CV‐N to explore proof of concept for the production of CV‐N in transgenic plants. Plant‐derived rCV‐N was recoverable at levels of 130 ng/mg of fresh leaf tissue, or at least 0.85% of total soluble plant protein. Western blot analysis demonstrated that virtually all of the rCV‐N was expressed in the desired monomeric form. Functionality was demonstrated by specific binding to gp120 and protection of T‐cells from in vitro HIV infection. Hydroponic culturing of transgenic plants demonstrated CV‐N rhizosecretion at levels of 0.64 μg/ml hydroponic media after 24 days. Therefore, we suggest that transgenic plants have the potential to provide strategies for large‐scale protein microbicide production.

Inhibition of ex vivo proinflammatory cytokine secretion in fatal Mycobacterium tuberculosis infection

Tuberculosis is characterized by fever, weight loss, a prolonged acute‐phase protein response and granuloma formation. These characteristics may partly be due to action of proinflammatory cytokines tumour necrosis factor (TNF), IL‐6 and IL‐8. We investigated plasma concentrations of these cytokines before and after ex vivo lipopolysaccharide stimulation of whole blood leucocytes from 41 Zambian patients with tuberculosis, 32 of whom were also HIV+. Although patients had a reduced weight, were more anaemic and had higher erythrocyte sedimentation rate compared with controls (all P < 0·0005), clinical and laboratory measurements of disease state were similar in those who died and survivors. In contrast, plasma IL‐6 and IL‐8 concentrations were higher in patients who died (P < 0·05). There was no detectable cytokine mRNA in unstimulated leucocytes. There was reduced secretion of TNF (P < 0·005 at 2h), IL‐6 (P < 0·005 at 8 h) and IL‐8 (P < 0·005 at 24 h) after ex vivo stimulation of whole blood leucocytes from patients who died compared with survivors. This was partly due to a soluble inhibitory factor present in plasma. The only additional effect of concurrent infection by HIV with Myco. tuberculosis was decreased IL‐6 secretion following ex vivo stimulation of leucocytes. Reduced proinflammatory cytokine release may represent a critical impairment of host immune defences important in determining outcome in tuberculosis.