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 analyzed the anatomic and temporal expansion of these small founder populations. We created atlases of the numbers and locations of SIV RNA+cells detected by in situ
hybridization in cervical vaginal tissues from animals at 4–10 days post-inoculation (dpi), 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 sites of virus entry and subsequent events underlying the expansion on which systemic infection depends.
In screening 20–40 sections of cervical vaginal tissues from each animal in this 4–10 dpi time frame, we identified sections with SIV RNA+cells in 9 animals, and in each animal found one predominant focus of infected cells in the endocervix. There were additional clusters of infected cells in the transformation zone (junction of ecto- and endocervix) adjoining the endocervical and vaginal foci in three animals. We illustrate at the bottom of the thumbnail images representative of the montages created from the captured images of sections from these animals, and in a small cluster of SIV RNA+cells found at 4 dpi only in endocervix, and then in one of forty sections in one isolated area, as reported previously6
. We mapped onto a two-dimensional grid the positions of cell centers (centroids) of SIV RNA+cells in this focus (), and predominant foci at 6 to 10 dpi 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 thumbnail montage images (bottom of 1a) of cervix and vagina, 4–10dpi, points (more ...)
These atlases revealed 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 successive influxes of new CD4+T target cells in inflammatory infiltrates fuels local expansion. The dramatic growth of SIV RNA+clusters is evident from comparisons of the map dimensions from 4 to 10 dpi ( and Supplemental Figure 1a–c
), and the growth of clusters amidst inflammatory cell infiltrates illustrated in at 6 dpi, where SIV RNA+cells are located amidst 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 dpi, and were apparent at 4 dpi ( and Supplemental Figure 2
). Virtually all of the infected cells were CD3+CD4+T cells ().
Figure 2 Influx and infection of CD4+ T cells in cervix in early infection. a–c. Sections stained with anti-CD4. Note relative paucity of CD4+ cells in SIV-(negative animal) (a), or SIV inoculated animal 1 dpi (b), compared to increased numbers of CD4+ (more ...)
The isolated focus at 4 dpi 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 initially recruiting target cells to the endocervical submucosa. We had previously stained these tissues for a pDC marker 11
, CD123, in investigating 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 1 dpi, and were not seen in the same numbers or location in uninfected animals (). The pDCs also stained with the specific marker BDCA211
(not shown), were strongly positive for interferons α () and β (not shown), and expressed the CCR5+cell-attracting chemokines MIP-1α and MIP-1β (), 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 dpi of cervical MIP-3α/CCL20, the principal chemokine known to induce pDC migration and T cells into peripheral issues13
, in microarray comparisons (Supplemental Table 1
) of uninfected and infected animals, and increased MIP-3α/CCL20 staining in endocervical epithelium (). These findings reveal 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 following exposure to SIV. a. Uninfected animal. b–c. Rapid accumulation of pDCs beneath endocervical epithelium at 1 dpi shown at 10X (b) and 40X (c) original magnifications. (more ...)
This initial influx of CD4+T cells was followed by a secondary inflammatory process, likely driven by RANTES and other chemokine- producing cells within inflammatory infiltrates (Supplemental Figure 3
) in which SIV RNA+cells were clearly concentrated at 10dpi (Supplemental Figure 1d
). Unlike endocervix, we saw no evidence of a signaling 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 (Supplemental Figure 4a
), were generally in areas of inflammation containing IL-8+cells, with associated epithelial thinning and disruption (Supplemental Figure 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 focussed 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 production of MIP-3α/CCL20 and IL-8 (as a general marker of inflammation and increased susceptibility to HIV-1 infection in female genital tissues 15
) by HVECs in response to the more relevant exposure to HIV-1 (), and reduced MIP-3α/IL-8 levels () in cervical vaginal fluids collected in a safety study16
from rhesus macaques treated intra-vaginally daily for six months with 5% GML.
Figure 4 GML inhibits HIV-1 induced expression of MIP-3α and IL-8 in HVECs and in cervical vaginal fluids. a–b. R5 isolate of HIV-1 added to HVECs in the amounts indicated± GML. c–d. At the end of a six-month safety study, cervical (more ...)
Encouraged by these results, we tested GML’s potential efficacy against repeated high dose intra-vaginal SIV challenges in 10 animals, in an extension of the GML safety study16
. We first evaluated GML’s efficacy in a pilot study in which we could examine cervical vaginal and lymphatic tissues obtained at the expected peak of viral replication at 14 dpi6
. Two animals from the safety study that were treated daily with 5% GML in K-Y warming gel and two animals that had received just K-Y warming gel, as a vehicle control, were challenged intra-vaginally one hour after compound introduction with 105
of SIV. Four hours later, they were again given either GML or K-Y warming gel, and challenged one hour later with an equivalent dose of SIV, and then continued on daily GML or K-Y warming gel.
Both GML-treated animals were completely protected from this high dose challenge. There was no evidence by in situ
hybridization for SIV RNA+cells in cervical vaginal (Supplemental Figure 5a, b
) or lymphatic tissues (not shown), and no evidence of inflammation (Supplemental Figure 5a, b
) or virus detectable in plasma (). By contrast, in one of the two controls, SIV RNA+ cells were detected in endocervical, vaginal (Supplemental Figure 5c, d
) and lymphatic tissues (not shown) and an influx of inflammatory cells associated with infection in the endocervix and vagina (Supplemental Figure 5c, d
), and high levels of virus in plasma () were all readily apparent. We then challenged three additional 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/ml). Again, GML prevented acute systemic infection after four exposures to this high dose vaginal challenge, whereas all three-control animals became infected ().
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), (more ...)
In seeking interventions to prevent vaginal transmission in a SIV-macaque model, we have focused on that critical window of opportunity in the earliest stages of infection when infected founder populations are small, and 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
. We show here 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 where 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 exposure of endocervical epithelium to the viral inoculum increases expression of MIP3-α to recruit pDCs, which, in turn produce MIP-1α and MIP-1β to recruit CCR5+targets.
The discovery reported here of in vivo
induction of MIP3-α in endocervical epithelium, together with in vitro
results here and report of induction of MIP3-α in uterine epithelial culturesby microbial-related stimuli 17
, point to outside-in signalling as a general feature of mucosal epithelium of the upper female genital tract. This signalling pathway and the evidence of 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
. While our conclusion, that innate defenses there are actually critical to the establishment and spread of infection, may thus at first seem counter intuitive, that conclusion is in keeping with report of possibly-enhanced vaginal transmission with agonists used to stimulate innate immunity19
, and with the concept advanced here: while interferons and anti-viral chemokines locally produced 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 cervix and vagina to prevent acute SIV infection in five of five animals with repeated intra-vaginal challenges of 105
of SIV, and, particularly impressively, in three of three animals challenged four times with this high dose. This result represents a novel and highly encouraging new lead in the search for an effective microbicide to prevent HIV-1 transmission that meets the criteria of safety, affordability and efficacy 20
. GML is a US Federal Drug Administration (FDA) -generally recognized as safe (GRAS) 7
agent that has been daily-applied 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 Lactobacilli
. GML is inexpensive (each dose used here cost less than one 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 following 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 percent (see statistical 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 through interfering with these responses.