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New Microbcide Discovery in Monkeys from Ashley Haase
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"We show that GML (glycerol monolaurate) can break this vicious cycle of signalling and inflammatory responses in the cervix and vagina to prevent acute SIV infection in five out of five animals with repeated intra-vaginal challenges of 105 TCID50 of SIV, and particularly notably, in three out of three animals challenged four times with this high dose. This result represents a highly encouraging new lead in the search for an effective microbicide to prevent HIV-1 transmission that meets the criteria of safety, affordability and efficacy20. GML is a US Federal Drug Administration (FDA) generally recognized as safe (GRAS)7 agent that has been applied daily intra-vaginally in K-Y warming gel, an FDA-approved vehicle for human vaginal use, for 6 months in rhesus macaques with no evidence of pathological effects or alteration of resident Lactobacilli16. GML is inexpensive (each dose used here cost less than 1 cent), and is efficacious in preventing acute systemic infection. Certainly, longer-term and well-powered studies with larger numbers of animals will be needed to definitively establish efficacy, and efficacy against occult infections, reportedly manifest as long as a year after repeated low-dose intravaginal inoculations21, and for which we now have preliminary evidence in this repeated high-dose model in one of the three animals with previously undetectable virus. Even conservative estimates of efficacy greater than or equal to60% (see Methods) extrapolate, according to mathematical models, to 2.5 million averted HIV infections over a 3-year period22, thus providing rationale and motivation for human trials of GML alone as a microbicide, and/or combined with other agents that specifically inhibit HIV-1 replication23. More generally, other microbes may exploit mucosal signalling and the innate inflammatory response to establish infection, so that GML may be the first example of a class of compounds that provide protection by interfering with these responses."
News
Microbicide protects monkeys from HIV-like virus
Published online 4 March 2009 | Nature | doi:10.1038/news.2009.135
Gel may fight virus by suppressing counterproductive immune responses.
Heidi Ledford
A microbicide made from glycerol monolaurate - an ingredient in some foods and cosmetics - can protect female monkeys from contracting an HIV-like virus, researchers have found. The compound may act by suppressing an unfortunate immune response that helps the virus rather than fights it.
In a report published in Nature1, immunologist Ashley Haase of the University of Minnesota in Minneapolis and his colleagues propose a new approach to microbicide development. Other candidate microbicides cripple the virus itself, or its interactions with its favoured target - immune cells called CD4+ cells.
But while characterizing the earliest stages of infection, Haase and his colleagues found how and when the immune system recruits more CD4+ cells to the site of infection - a response that helps the virus to spread. Inhibiting this immune response, Haase reasoned, could stop the virus in its tracks.
"It is a new idea and sort of counterintuitive," says Haase. "You would think we should induce the innate immune response. But it turns out these viruses have not only learned to live with that immune response, they relish it."
"It turns out these viruses have not only learned to live with that immune response, they relish it."
Ashley Haase
University of Minnesota
The results could provide a further boost for the field of HIV microbicides. In February, preliminary results from a clinical trial of the candidate microbicide Pro 2000 suggested that the gel reduced HIV infection in women who used it (see 'Microbicide gel may help against HIV'). But although the results were promising, they were not statistically significant, leaving researchers to wait on findings from a larger trial due later this year.
Nevertheless, that flash of hope might have stimulated renewed investment in the field: soon afterwards, the Bill & Melinda Gates Foundation and the UK Department for International Development together awarded nearly US$130 million for microbicide development.
Counterproductive countermeasures
The early stages of HIV infection are difficult to study in humans, because the time of initial infection is often not known. So Haase and his colleagues looked at a related virus - called simian immunodeficiency virus (SIV) - in female rhesus macaques (Macaca mulatta). The researchers monitored the small population of CD4+ cells that are initially infected in the cervix and vagina, then watched as more of these cells arrived and expanded the site of infection.
The team also found that the immune system generated several proteins during the first few days of infection. These proteins, the chemokines MIP-1_, MIP-1b, and Mip-3_, have previously been shown to attract CD4+ cells.
This finding reminded Haase of work done by a fellow immunologist at the University of Minnesota, Patrick Schlievert. Schlievert's lab had found that glycerol monolaurate inhibits the production of some deadly bacterial toxins, including anthrax toxin and toxins responsible for toxic shock syndrome. The compound also inhibits some immune responses to a toxic shock syndrome toxin2.
So Haase mixed glycerol monolaurate with a gel, and tested its ability to block SIV infection in the macaques. Two weeks later, four of the five control monkeys that were treated with the gel alone were infected with the virus. None of the macaques treated with glycerol monolaurate showed evidence of infection in these first two weeks, although Haase notes that one of the monkeys did later develop an infection.
Those treated with glycerol monolaurate also produced less Mip-3_ than the control animals - suggesting that the treatment reduces the immune response that helps HIV to spread.
Battle-scarred
Despite those results, some researchers are reluctant to back the idea of taking glycerol monolaurate into human trials. The compound is a surfactant, and trials with another surfactant - the spermicide nonoxynol-9 - not only failed to protect women from HIV, but increased their risk of infection. "At the moment, the jury would be quite sceptical of a surfactant," says Ian McGowan, an immunologist at the University of Pittsburgh School of Medicine in Pennsylvania who has studied microbicides as a principal investigator in the Microbicide Trials Network. "This product would have an uphill path to get to the clinic."
Haase has heard such criticism before, most notably in reviews of his applications for research funding. He counters that repeated use of nonoxynol-9 was found to cause inflammation and lesions in the vagina and cervix, yet his safety studies have thus far shown no evidence that glycerol monolaurate causes such damage1.
Meanwhile, Robin Shattock, an HIV researcher at St George's, University of London, says that the latest results do not rule out the possibility that glycerol monolaurate is simply destroying the virus directly, as most surfactants are thought to do. An approach that targets the immune system would be interesting, says Shattock, "but the evidence as presented is at best circumstantial".
Nevertheless, the results should stimulate more research aimed at tackling the early events after infection says Sharon Hillier, another principal investigator in the Microbicide Trials Network. "They're provocative," she says. As for glycerol monolaurate, Hillier says the compound is "going to require more attention before we know whether or not it has promise as a microbicide".
References
1. Li, Q. et al. Nature Advanced online publication doi:10.1038/nature07831 (2009).
2. Witcher, K. J. , Novick, R.P. & Schlievert, P. M. Clin. Diag. Lab.
Immunol. 3, 10- 13 (1996).
Letters to Nature
Nature , | doi:10.1038/nature07831; Received 20 November 2008; Accepted 20 January 2009; Published online 4 March 2009
Glycerol monolaurate prevents mucosal SIV transmission
Qingsheng Li1, Jacob D. Estes2, Patrick M. Schlievert1, Lijie Duan1, Amanda J. Brosnahan1, Peter J. Southern1, Cavan S. Reilly3, Marnie L. Peterson4, Nancy Schultz-Darken5, Kevin G. Brunner5, Karla R. Nephew5, Stefan Pambuccian6, Jeffrey D. Lifson2, John V. Carlis7 & Ashley T. Haase1
1. Department of Microbiology, Medical School, University of Minnesota, MMC 196, 420 Delaware Street S.E., Minneapolis, Minnesota 55455, USA
2. AIDS and Cancer Virus Program, Science Applications International Corporation-Frederick, Inc., National Cancer Institute, Frederick, Maryland 21702, USA
3. Division of Biostatistics, School of Public Health, University of Minnesota, MMC 303, 420 Delaware Street S.E., Minneapolis, Minnesota 55455, USA
4. Department of Experimental and Clinical Pharmacology, College of Pharmacy, University of Minnesota, 2001 Sixth Street S.E., Minneapolis, Minnesota 55455, USA
5. Wisconsin National Primate Research Center, University of Wisconsin, 1220 Capitol Court, Madison, Wisconsin 53715, USA
6. Department of Laboratory Medicine and Pathology, Medical School, University of Minnesota, MMC 76, 420 Delaware Street S.E., Minneapolis, Minnesota 55455, USA
7. Department of Computer Science and Engineering, Institute of Technology, University of Minnesota, 200 Union Street S.E., Minneapolis, Minnesota 55455, USA
Abstract
Although there has been great progress in treating human immunodeficiency virus 1 (HIV-1) infection1, preventing transmission has thus far proven an elusive goal. Indeed, recent trials of a candidate vaccine and microbicide have been disappointing, both for want of efficacy and concerns about increased rates of transmission2, 3, 4. Nonetheless, studies of vaginal transmission in the simian immunodeficiency virus (SIV)-rhesus macaque (Macacca mulatta) model point to opportunities at the earliest stages of infection in which a vaccine or microbicide might be protective, by limiting the expansion of infected founder populations at the portal of entry5, 6. Here we show in this SIV-macaque model, that an outside-in endocervical mucosal signalling system, involving MIP-3alpha (also known as CCL20), plasmacytoid dendritic cells and CCR5+ cell-attracting chemokines produced by these cells, in combination with the innate immune and inflammatory responses to infection in both cervix and vagina, recruits CD4+ T cells to fuel this obligate expansion. We then show that glycerol monolaurate-a widely used antimicrobial compound7 with inhibitory activity against the production of MIP-3alpha and other proinflammatory cytokines8-can inhibit mucosal signalling and the innate and inflammatory response to HIV-1 and SIV in vitro, and in vivo it can protect rhesus macaques from acute infection despite repeated intra-vaginal exposure to high doses of SIV. This new approach, plausibly linked to interfering with innate host responses that recruit the target cells necessary to establish systemic infection, opens a promising new avenue for the development of effective interventions to block HIV-1 mucosal transmission.
To understand how SIV infection in a small founder population of cells at the portal of entry transitions in less than two weeks to systemic infection, with massive levels of viral replication and depletion of gut CD4+ T cells5, 6, 9, 10, we analysed the anatomical and temporal expansion of these small founder cell populations. We created atlases of the numbers and locations of SIV RNA+ cells detected by in situ hybridization in cervical and vaginal tissues from animals at 4-10 days post-inoculation (d.p.i.), with the rationale that by locating sites that initially had the largest numbers of infected cells, and then determining how infection expanded and spread from these infected founder populations, we would gain insight into the sites of virus entry and subsequent events underlying the expansion on which systemic infection depends.
In screening 20-40 sections of cervical and vaginal tissues from each animal in this 4-10 d.p.i. time frame, we identified sections with SIV RNA+ cells in nine animals, and in each animal we found one predominant focus of infected cells in the endocervix. There were further clusters of infected cells in the transformation zone (the junction of ecto- and endocervix) adjoining the endocervical and vaginal foci in three animals. We illustrate at the bottom of Fig. 1a the thumbnail representative images of the montages created from the captured images of sections from these animals, and in Fig. 1b a small cluster of SIV RNA+ cells found at 4 d.p.i. only in endocervix, and then in 1 out of 40 sections in one isolated area, as reported previously6. We mapped onto a two-dimensional grid the positions of cell centres (centroids) of SIV RNA+ cells in this focus (Fig. 1c), and predominant foci at 6-10 d.p.i. that were again found in endocervix.
These atlases showed that infection expands by accretion of new infections around an initial cluster of infected cells in endocervix, rather than by diffuse spread of infection in the submucosa, and that the successive influxes of new CD4+ T target cells in inflammatory infiltrates fuel local expansion. The marked growth of SIV RNA+ clusters is evident from comparisons of the map dimensions from 4 to 10 d.p.i. (Fig. 1d, e and Supplementary Fig. 1a-c), and from the growth of clusters amid inflammatory cell infiltrates at 6 d.p.i. (Fig. 1f), in which SIV RNA+ cells are located among dark staining nuclei of cells in inflammatory infiltrates. These focal infiltrates contained increased numbers of CD4+ T cells compared to uninfected animals or at 1 d.p.i., and were apparent at 4 d.p.i. (Fig. 2a-c and Supplementary Fig. 2). Virtually all of the infected cells were CD3+ CD4+ T cells (Fig. 2d).
The isolated focus at 4 d.p.i. seemed unlikely by itself to have induced such an extensive influx of CD4+ T cells, and indeed we found evidence implicating endocervical epithelium and plasmacytoid dendritic cells (pDCs) in the initial recruitment of target cells to the endocervical submucosa. We had previously stained these tissues for a pDC marker11, CD123 (also known as IL3RA), to investigate the possible role of pDCs in a 'premature' T-regulatory response to infection12, and now noted areas with CD123+ pDCs aligned just beneath the endocervical epithelium. These subepithelial pDC collections were observed at 1 d.p.i., and were not seen in the same numbers or location in uninfected animals (Fig. 3a-c). The pDCs also stained positive for the specific marker BDCA2 (also known as CLEC4C)11 (data not shown), were strongly positive for interferons alpha (Fig. 3d) and beta (data not shown), and expressed the CCR5+ cell-attracting chemokines MIP-1alpha (CCL3) and MIP-1beta (CCL4) (Fig. 3e), which could thus serve as one mechanism to quickly recruit CD4+ T cells to the endocervix. We also found increased expression at 1 and 3 d.p.i. of cervical MIP-3alpha, the principal chemokine known to induce pDC migration and T cells into peripheral issues13, in microarray comparisons of uninfected and infected animals (Supplementary Table 1), and increased MIP-3alpha staining in endocervical epithelium (Fig. 3f). These findings demonstrate an outside-in signalling pathway triggered by exposure to the viral inoculum that recruits pDCs and T cells to create an environment rich in target cells at the sites of initial infection.
This initial influx of CD4+ T cells was followed by a secondary inflammatory process, probably driven by RANTES and other chemokine-producing cells within inflammatory infiltrates (Supplementary Fig. 3), in which SIV RNA+ cells were clearly concentrated at 10 d.p.i. (Supplementary Fig. 1d). Unlike endocervix, we saw no evidence for a signalling pathway capable of recruiting additional CD4+ T cells in the foci of SIV RNA+ cells in the transformation zone and vagina in three animals. However, an inflammatory response provided susceptible target cells for expansion of the infection at these sites as well, because infected cells (Supplementary Fig. 4a) were generally in areas of inflammation containing IL-8+ cells, with associated epithelial thinning and disruption (Supplementary Fig. 4b, c). Thus, inflammation with increases in susceptible target populations is the common denominator across sites.
The importance of the innate immune and inflammatory response in providing new target cells for local expansion and systemic dissemination suggested that inhibiting this immunoinflammatory process might prevent transmission and systemic infection. We focused on glycerol monolaurate (GML) because of the compound's documented relevant activities in inhibiting immune activation and chemokine and cytokine production by human vaginal epithelial cell cultures (HVECs) on exposure to staphylococcal toxins8, 14. We showed that GML inhibited the production of MIP-3alpha and IL-8 (as a general marker of inflammation and increased susceptibility to HIV-1 infection in female genital tissues15) by HVECs in response to the more relevant exposure to HIV-1 (Fig. 4a, b). MIP-3alpha and IL-8 levels were also reduced in cervical and vaginal fluids collected in a safety study16 from rhesus macaques treated intra-vaginally with 5% GML daily for 6 months (Fig. 4c, d).
Encouraged by these results, we tested the potential efficacy of GML against repeated high dose intra-vaginal SIV challenges in ten animals, in an extension of the GML safety study16. We first evaluated its efficacy in a pilot study in which we could examine cervical and vaginal and lymphatic tissues obtained at the expected peak of viral replication at 14 d.p.i.6. Two animals from the safety study that were treated daily with 5% GML in K-Y warming gel, and two animals that received K-Y warming gel alone as a vehicle control, were challenged intra-vaginally 1 h after compound introduction with 105 50% tissue-culture infective dose units (TCID50) of SIV. Four hours later they were again given either GML or K-Y warming gel, and challenged after 1 h with an equivalent dose of SIV, and then continued on daily doses of either GML or K-Y warming gel.
Both of the GML-treated animals were completely protected from this high dose SIV challenge. Using in situ hybridization there was no evidence for SIV RNA+ cells in cervical, vaginal (Supplementary Fig. 5a, b) or lymphatic tissues (data not shown), and no evidence of inflammation (Supplementary Fig. 5a, b) or virus detectable in plasma (Fig. 5a). In contrast, in one of the two controls, SIV RNA+ cells were detected in endocervical, vaginal (Supplementary Fig. 5c, d) and lymphatic tissues (data not shown) and there was an influx of inflammatory cells associated with infection in the endocervix and vagina (Supplementary Fig. 5c, d), and high levels of virus in plasma (Fig. 5a) were all readily apparent. We then challenged three other GML-treated animals and three K-Y warming gel controls, repeating the challenges 4 weeks later if the animals showed no evidence of systemic infection (plasma levels of <20 copies of SIV RNA per ml). Again, GML prevented acute systemic infection after four exposures to this high dose vaginal challenge, whereas all three control animals became infected (Fig. 5b).
In seeking interventions to prevent vaginal transmission in a SIV-macaque model, we have focused on the critical window of opportunity at the earliest stages of infection when infected founder cell populations are small, and the virus must overcome the limited availability of susceptible target cells to sustain and sufficiently expand the initially infected founder cell populations to disseminate and establish a self-propagating infection in secondary lymphoid organs5. Here we show that SIV exploits the innate immune and inflammatory response to overcome this inherent limitation in the availability of target cells in the endocervix-the predominant site of the initial infected cell clusters. We document the growth of clusters by accretion of new infections in influxes of CD4+ T cell targets, and provide evidence plausibly linking the first influx to an outside-in mucosal signalling pathway in which the exposure of endocervical epithelium to the viral inoculum increases the expression of MIP3-alpha to recruit pDCs, which in turn produce MIP-1alpha and MIP-1beta to recruit CCR5+ targets.
The discovery reported here of in vivo induction of MIP3-alpha in endocervical epithelium, together with our in vitro results and the previous report of the induction of MIP3-alpha in uterine epithelial cultures by microbial-related stimuli17, point to outside-in signalling as a general feature of mucosal epithelium of the upper female genital tract. This signalling pathway and the production of interferons and virus-inhibiting chemokines by pDCs, support the concept that the mucosal lining of the upper female genital tract is truly the front line of the innate mucosal immune system18. Although our conclusion that innate defences there are actually critical to the establishment and spread of infection may thus at first seem counterintuitive, it is in keeping with the previous report of possibly enhanced vaginal transmission with agonists used to stimulate innate immunity19, and with the concept advanced here: although interferons and anti-viral chemokines produced locally by pDCs may protect themselves and contribute to limiting infection initially, on balance, SIV's greater immediate need is for target cells, which is served by the inflammatory component of the innate immune response.
We show that GML can break this vicious cycle of signalling and inflammatory responses in the cervix and vagina to prevent acute SIV infection in five out of five animals with repeated intra-vaginal challenges of 105 TCID50 of SIV, and particularly notably, in three out of three animals challenged four times with this high dose. This result represents a highly encouraging new lead in the search for an effective microbicide to prevent HIV-1 transmission that meets the criteria of safety, affordability and efficacy20. GML is a US Federal Drug Administration (FDA) generally recognized as safe (GRAS)7 agent that has been applied daily intra-vaginally in K-Y warming gel, an FDA-approved vehicle for human vaginal use, for 6 months in rhesus macaques with no evidence of pathological effects or alteration of resident Lactobacilli16. GML is inexpensive (each dose used here cost less than 1 cent), and is efficacious in preventing acute systemic infection. Certainly, longer-term and well-powered studies with larger numbers of animals will be needed to definitively establish efficacy, and efficacy against occult infections, reportedly manifest as long as a year after repeated low-dose intravaginal inoculations21, and for which we now have preliminary evidence in this repeated high-dose model in one of the three animals with previously undetectable virus. Even conservative estimates of efficacy greater than or equal to60% (see Methods) extrapolate, according to mathematical models, to 2.5 million averted HIV infections over a 3-year period22, thus providing rationale and motivation for human trials of GML alone as a microbicide, and/or combined with other agents that specifically inhibit HIV-1 replication23. More generally, other microbes may exploit mucosal signalling and the innate inflammatory response to establish infection, so that GML may be the first example of a class of compounds that provide protection by interfering with these responses.
Methods Summary
Animals, inoculation of SIV, GML and K-Y warming gel
Adult female rhesus macaque monkeys (Macacca mulatta), housed in accordance with the regulations of the American Association of Accreditation of Laboratory Animal Care standards, were inoculated twice intra-vaginally with 1 ml of 105 TCID50 per ml SIVmac 251 (ref. 6). One-ml of K-Y warming gel plusminus 5% GML was administered atraumatically into the vagina daily and before viral challenges.
SIV RNA in plasma
SIV RNA copy equivalents per ml (Eq ml-1) in plasma was determined using a quantitative PCR with reverse transcription (qRT-PCR) assay24.
In situ hybridization and immunohistochemistry
Blood, cervical, vaginal and lymphoid tissues were collected from euthanized animals, fixed and then embedded in paraffin. In situ hybridization combined with immunohistochemical staining and immunochemistry were performed as described9, 12.
Digital atlases
Images of fields with SIV RNA+ cells were acquired, merged (Photoshop 7.0 automerge), and, after using Photoshop Action procedures to delineate SIV RNA+ cells, centroid x, y coordinates were assigned using MetaMorph software, and the coordinates were plotted with Excel.
Induction and measurement of MIP-3alpha and IL-8
HIV-1 plusminus GML was added to HVECs cultured as described25. Chemokines in the supernatants were measured by ELISA25.
Microarray analysis
Gene expression profiles in cervix before and after intravaginal SIV inoculation were analysed with the Affymetrix GeneChip Rhesus Macaque Genome Array as described26.
Statistical methods
The negative binomial distribution was used to model repeated challenges. The model assumes that outcomes for distinct animals are independent, and that the probability of being infected differs between the two groups. The use of maximum likelihood or Bayesian methods (which don't assume the sample size is large) both indicate that the efficacy of GML against transmission is at least 65%, in which the posterior probability that GML is more likely to prevent infection than K-Y warming gel is 0.98, and the P-value that the probability differs between groups is 0.04.
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