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

Correspondence to: Ashley T. Haase¹ Correspondence and requests for materials should be addressed to A.T.H. (Email: haase001@umn.edu).

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 cells^{5, 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.

Figure 1: Mapping early expansion of infection in endocervix.

SIV RNA⁺ cells appear black in transmitted light, green in reflected light and in maps. a–c, The arrow from the thumbnail montage images (bottom of a) of cervix and vagina (4–10 d.p.i.) points to an enlarged image and map of a single focus (box) of SIV RNA⁺ cells in endocervix (4 d.p.i.). Anticlockwise-rotated image of focus (box) (b) and map of x, y coordinates (m) (c) of cell centroids to the right. d, e, Endocervical focus (d) and map (e) (7 d.p.i.) are shown. f, Endocervical focus (6 d.p.i.) SIV RNA⁺ cells (green) are concentrated in an inflammatory infiltrate (cells with dark staining nuclei). Original magnification for all images, 10.

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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).

Figure 2: Influx and infection of CD4⁺ T cells in cervix in early infection.

a–c, Sections stained with anti-CD4 antibody. Note the relative paucity of CD4⁺ cells in an SIV^- (negative animal) (a), or an SIV-inoculated animal 1 d.p.i. (b), compared to increased numbers of CD4⁺ cells seen in an infected animal at 4 d.p.i. (c). d, SIV RNA⁺ cells in infiltrates are CD3⁺ T cells. Encircled SIV RNA⁺ cells (overlying black silver grains) are stained brown with anti-CD3. Original magnification, 10 (a–c) and 20 (d).

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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 (Fig. 3d) and (data not shown), and expressed the CCR5⁺ cell-attracting chemokines MIP-1 (CCL3) and MIP-1 (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-3, 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-3 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.

Figure 3: pDCs, cytokines and chemokines associated with endocervical epithelium after exposure to SIV.

a, Uninfected animal, original magnification 10. b, c, Rapid accumulation of pDCs beneath endocervical epithelium at 1 d.p.i. shown at 10 (b) and 40 (c) original magnifications. pDCs stained brown with anti-CD123 antibody. Arrow in c points to the location of pDCs beneath the epithelium. d, e, Arrows point to subepithelial pDCs stained red with anti-interferon- antibody at 1 d.p.i. (d, 20 magnification) or with anti-MIP-1 antibody (e, 10 magnification). f, Arrow points to MIP-3⁺ endocervical epithelium (red) at 1 d.p.i. Original magnification in f, 10.

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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 toxins^{8, 14}. We showed that GML inhibited the production of MIP-3 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-3 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).

Figure 4: GML inhibits HIV-1 induced expression of MIP-3 and IL-8 in HVECs and in cervical and vaginal fluids.

a, b, R5 isolate of HIV-1 added to HVECs in the amounts indicated  GML. MIP-3 (a) and IL-8 (b) release from HVECs was measured and expressed as the difference from control. c, d, At the end of a 6-month safety study, cervical and vaginal fluids were collected with a swab that reproducibly adsorbed 0.1 ml of fluid from animals that received GML or K-Y warming gel in the a.m. and p.m. of two successive days. MIP-3 (c) and IL-8 (d) were measured by ELISA. Bars indicate s.e.m.

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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).

Figure 5: GML prevents mucosal transmission and acute infection.

a, Pilot experiment continuation of daily dosing safety study. Two animals treated with GML in K-Y warming gel (circles) and two treated with gel only (squares) were challenged twice (two arrows), 1 h after treatment, with 10⁵ TCID₅₀ of SIV. Colours indicate individual animals. SIV RNA in plasma was measured to peak viremia, 14 d.p.i. b, Three animals treated with GML and three given K-Y warming gel were challenged as described in a. The animals that were not infected were treated and challenged again 4 weeks later, shown at the right.

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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- to recruit pDCs, which in turn produce MIP-1 and MIP-1 to recruit CCR5⁺ targets.

The discovery reported here of in vivo induction of MIP3- in endocervical epithelium, together with our in vitro results and the previous report of the induction of MIP3- 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 60% (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.

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Supplementary Information

Supplementary information accompanies this paper.

Acknowledgements

We thank C. Miller and D. Lu at the California National Primate Research Center, for helpful discussion and virus stocks, J. Kemnitz at the Wisconsin National Primate Research Center, for discussion and administrative support, and C. O'Neill and T. Leonard for help with the manuscript and figures. This work was supported in part by National Institute of Health (NIH) grants R21 AI071976 and P01 AI066314 (A.T.H.), funds from the National Cancer Institute, NIH, under contracts N01-CO-12400 and HHSN266200400088C (J.D.L.), and grant number P51 RR000167 from the National Center for Research Resources, a component of the NIH, to the Wisconsin National Primate Research Center. This research was conducted in part at a facility constructed with support from Research Facilities Improvement Program grant numbers RR15459-01 and RR020141-01. This publication's contents are solely the responsibility of the authors and do not necessarily represent the official views of the NCRR or NIH.

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