Women comprise over half of all individuals living with HIV-1 worldwide with intravaginal sex as the predominant route of transmission.1 However, the rate of infection among exposed females is statistically low, indicating that only a fraction of viral exposures result in productive infection.2–5 At each exposure, HIV can interact with the cervical epithelium6,7 mediating cellular infiltration6,8 and potentially engaging Toll-like receptor (TLR) 7 on dendritic cells.8–10 The effects of nonproductive HIV-1 or simian immunodeficiency virus (SIV) cervicovaginal exposures (absent infection) on subsequent infection outcomes remain unclear.
The CAPRISA004 trial found a positive association between a pre-existing inflammatory cytokine profile within cervicovaginal fluids and subsequent HIV infection,11 supporting previously held views that vaginal immune activation increases HIV-1 infectivity. In parallel, a study examining SIV infectivity in rhesus macaques after direct topical application of TLR-7/9 ligands to the cervicovaginal mucosa showed increased localized inflammation and increased viral load after challenge.12 However, there are well-documented cases of women with a history of chronic multiyear HIV-1 exposure without infection indicating that exposure-initiated mechanisms may reduce future infectivity on re-exposure.13–15 Of interest, studies of resistance in these women have largely failed to identify HIV-specific responses as a determinant of protection15 but a loss of HIV-1 “resistance” in a subset of these women after reduced sex work (ie, HIV-1 exposure) has suggested that resistance is sustained by innate rather than adaptive responses.16
Acute cellular changes after cervicovaginal mucosal infection have been studied. Infection is established within CD4+ T cells and other permissive cells of the lamina propria17–20 following a robust cellular infiltrate of plasmacytoid dendritic cells (pDCs) and CD4 T cells,6 and increased mRNA expression of proinflammatory cytokines/chemokines by day 3 post infection (DPI).21 Of interest, Li et al demonstrated that infiltrating cervical pDCs produce interferon-alpha (IFN-α) within hours of SIV exposure, preceding CD4+ T-cell infiltrates by 72 hours.6
The anti-HIV properties of pDCs and type I IFN have been documented and involve intrinsic and secondary antiviral mechanisms, such as the induction of tetherin or APOPBEC family members, and the modulation of innate and adaptive effectors.22–27 In a recent clinical study in individuals with chronic HIV infection, IFN immunotherapy sustained viral suppression during antiretroviral therapy interruption in a subset of subjects supporting the interpretation that IFN can inhibit HIV-1 production/replication.28 However, the role of infiltrating pDCs as a source of type I IFNs and its impact on acute SIV infection if present before infection as a result of repeated noninfectious viral exposures remains unclear. Taking advantage of a replication-deficient SIVsmB7 clone derived from SIVsmH3, able to bind CD4 and fuse with its target without subsequent viral replication,29–31 we tested the hypothesis that repeated vaginal exposures to SIV in the absence of productive infection would decrease viral infection potential and/or decrease replication kinetics as a consequence of sustained cervical pDC infiltration despite the increase in local CD4+ T-cell infiltrates.
Ethics Statement and Animal Procedures
Healthy Indian Rhesus monkeys (Macaca mulata) were acquired from the Caribbean Primate Research Center of the University of Puerto Rico (UPR)—Medical Sciences Campus (MSC). Animals were quarantined for 6 months and maintained at the AAALAC-accredited facilities of the Animal Resources Center, UPR-MSC.
All animal studies were approved by the UPR-MSC, Institutional Animal Care and Use Committee (IACUC), and comply with the Guide for the Care and Use of Laboratory Animals. Animal Welfare Assurance number: A3421, Protocol number: 3380308.
In addition, steps were taken to reduce suffering in accordance with the recommendations of the Weatherall report, “The Use of Nonhuman Primates in Research.” For instance, all procedures were conducted under anesthesia by using ketamine 10–20 mg/kg, delivered intramuscular.
Phase I: 10 (6 and 4) macaques were given either intravaginal SIVsmB7 or CEM mock control inoculations. Inoculations were given twice daily for 3 days in either the follicular or luteal phase, as shown in Figure 1 and Figure S1 (see Supplemental Digital Content,http://links.lww.com/QAI/A482). Macaques were euthanized on the fourth day.
Phase II: female macaques were housed together before use in the study to allow the animals to reach menstrual synchrony. Twenty-four macaques, 12 macaques per group, were given either intravaginal SIVsmB7 or CEM mock control inoculations (Table 1 and Fig. 1). Inoculations were given twice daily for 3 days during the luteal phase. On the fourth day after inoculation, each macaque was given a single 1 mL challenge dose SIVmac251. Blood was taken before the challenge and on days 7, 14, 21, and 42 to determine plasma viral load and CD4 count. Macaques were euthanized on days 42–46 based on the analysis focused on early viral kinetics after a single exposure and infection.
Euthanasia was performed only on fully anesthetized animals by injection of pentobarbital sodium at 390 mg/mL; 1 cc/10 lbs IV. Vaginal and cervical tissues were taken for analysis.
SIVmac251 was diluted 1:2 from a 20,000 TCID50 per milliliter viral stock grown in specific pathogen free rhesus macaque peripheral blood mononuclear cell produced by Dr Ron Desrosiers (New England National Primate Research Center, Harvard Medical School) and kindly provided by Dr Nancy Miller (NIAID) through contract #N01-AI-30018. Animals were challenged with 1 mL of the diluted viral stock. In vivo, titration was performed as described in the Supplemental Methods (see Supplemental Digital Content,http://links.lww.com/QAI/A482).
SIVsmB7 is a virus-like particle derived from a clone of a CEMx174 cell line stably infected with SIVsmH3. SIVsmB7 is noninfectious because of a 1.6 kbp deletion, including integrase, vif, vpr, and vpx genes. Cell-free SIVsmB7 and CEMx174 supernatant (CEM mock control) were isolated by standard 20% (wt/vol) sucrose gradient ultracentrifugation. P27 enzyme-linked immunosorbent assay was used to determine 500 μg P27 SIVsmB7 dose. CEMx174 dose was established by equal protein quantification with SIVsmB7 dose.
CD4 and CD8 Counts
CD4 and CD8 counts were monitored by TruCount Absolute Count Kits (BD Bioscience, San Jose, CA) used according to manufacture protocol.
SIV Viral Loads
Plasma viral load was assessed by quantitative reverse transcriptase-polymerase chain reaction as previously described.32 Full method can be found in the Supplemental Methods (see Supplemental Digital Content,http://links.lww.com/QAI/A482).
Samples from the vagina and cervix were harvested postmortem from each animal and fixed in 4% paraformaldehyde and embedded in paraffin for sectioning, SafeFix II (Fisher Scientific, Pittsburgh, PA), or frozen. Immunohistochemical staining for CD123, CD68, CD4, and Mx1 was conducted as previously reported6 (For complete method, see Supplemental Methods,http://links.lww.com/QAI/A482). CD123+ cells were considered pDC as a previous report by Li et al6 has shown that CD123+ cells infiltrating into the endocervical subepithelium 3 days after SIV exposure stain positive for both HLA-DR and IFN-α confirming these cells are pDC.
Cross-sectional phase 1 and phase 2 two group comparisons between SIVsmB7-exposed and CEM mock control–inoculated animals were made using Wilcoxon rank-sum tests, Fisher test or Student t tests. Two-tailed P values less than 0.05 were considered statistically significant.
To evaluate the rate of change for LogVL and CD4, a change-point model with random intercept terms for each monkey and fixed effects for baseline CD4, days-postinfection (DPI) (overall and after 14 DPI), a group indicator for SIVsmB7 exposure, and 2-way interaction terms between DPI and group, was fitted. This model accounts for the within individual correlation in repeated measurements and allows for a different slope before and after 14 DPI. The models are listed below, with model variables estimates described in the results. The function (DPI−14)+ = 0 when DPI ≤ 14 and (DPI−14)+ = DPI−14 when DPI > 14.
Model Equations for SIVsmB7 or CEM-Inoculated Animals
Statistical analysis was performed using R 2.14.1 and Prism.
Phase I: Acute Cellular Infiltrates After 72 Hours of SIVsmB7/CEM Mock Control Exposure
Ten female macaques followed during their natural luteal or follicular phases were used to initiate 3-day twice-daily SIVsmB7/CEM mock control dosing within a 3-day period after the start of a menstrual phase (as exemplified for 2 luteal-staged animals in Figure S1, Supplemental Digital Content,http://links.lww.com/QAI/A482). Animals were inoculated with CEM mock or SIVsmB7 as described in Figure 1 and in the methods.
Consistent with previously published data showing that infectious SIV exposure resulted in acute endocervical pDC and CD4+ T-cell infiltration within 3 days of SIV exposure/infection,6 replication-deficient SIVsmB7-treated animals had a significant higher number of endocervical CD123+ pDC (P = 0.0317, mean: 1328 cells/mm2 vs 435 cells/mm2) and CD4 T cells (P = 0.0357, CD4 mean: 141.2 cells/mm2 vs 17.92 cells/mm2) as compared with CEM mock control–inoculated animals at 72 hours (Fig. 2A) irrespective of hormonal phase. Moreover, in animals exposed to SIVsmB7, we detected a trend in increased expression of Mx1 (an IFN-stimulated gene; Fig. 2A) localized in areas enriched for CD123+ cells. This is consistent with recruitment of CD123+ pDCs to the endocervix and local production of type I IFNs. Although exposure to SIVsmB7 did not result in increased vaginal pDC infiltration (Fig. 2B), it did result in higher levels of CD4+ cells in vaginal tissues (Fig. 2B; P = 0.0476; mean, 164.8 cells/mm2 vs 37.95 cells/mm2). Similar to pDCs, SIVsmB7-treated animals had higher (although not statistically significant) levels of CD68 macrophages than control animals in the cervix, but not in the vaginal tissue. Altogether, results confirm that exposure to replication-deficient SIV can modulate the tissue microenvironment, allowing us to directly test how these local viral-induced changes (ie, pDC and CD4 T-cell infiltrates) would impact a challenge with infectious SIVmac251.
Phase II: Outcome of SIVmac 251 Infection After 72-Hour Preconditioning With SIVsmB7 or CEM Control
Twenty-four female macaques were challenged with SIVmac251 after undergoing 72-hour exposure (as described above) to either SIVsmB7 (n = 12) or CEM control (n = 12). The groups were similar in weight and inoculation date relative to menstrual cycle (Table 1; see Figure S2, Supplemental Digital Content,http://links.lww.com/QAI/A482), and there was no significant difference in major histocompatibility complex (MHC) class I allele distribution (Fisher exact test, P > 0.05) or for those associated with spontaneous control between groups33–35 (see Table S1, Supplemental Digital Content,http://links.lww.com/QAI/A482).
Endogenous estrogen levels at the time of challenge between SIVsmB7 and CEM mock control arms were not significantly different (P = 0.84) (Table 1), indicating the absence of differences in estrogen-related structural epithelial factors known to impact viral infectivity.36–38
As it has been shown that cervicovaginal inflammation is common among captive rhesus macaques,39 a 28-plex nonhuman primate luminex assay was performed within 1 week of challenge to assess levels of proinflammatory mediators in cervicovaginal lavages (CVL) in 13 of the 24 macaques (6 SIVsmB7/7 CEM mock). Twelve of the cytokines/chemokines tested were below the limit of detection and no cytokine/chemokine tested showed any significant difference between groups. Results for selected cytokines and chemokines are summarized in Figure S3 (see Supplemental Digital Content,http://links.lww.com/QAI/A482).
Following a single, intravaginal SIVmac251 challenge using an infectious dose previously determined through an intravaginal titration (see Supplemental Methods,http://links.lww.com/QAI/A482), all 24 macaques became infected, with a detectable viral load within 7 or 14 days postinfection (DPI) (Fig. 3B). Viral load peaked in all animals by 14–21 DPI, with a mean viral load of 6.63 and 8.02 for CEM mock control- and SIVsmB7-treated, respectively (Student t test: P = 0.0720).
Although we didn't detect a significant difference in peak viral load, there was a trend toward increased viral load in SIVsmB7-treated macaques as compared with CEM mock–treated animals (Fig. 3B). We did detect a statistically significant decline in CD4+ T cells at 14 DPI in SIVsmB7-treated macaques as compared with control animals (Student t test: P = 0.0268, mean: −355.8 CD4/μl vs CEM—120.4 CD4/μl) (Fig. 3D) consistent with a more severe CD4 depletion after infection in the SIVsmB7-treated females. Notably, 4 of the macaques in the control group (animals 8D4, M847, 31R, and 530) had lower plasma viral loads. These animals were similar to the other animals in the control and experimental groups in terms of MHC genotypes, batch of doses used, day of manipulation, estrogen levels, weight, age, and history of parity (data not shown). Analysis of baseline CVL samples from 2 of these macaques, 53O and 31R, showed no difference to other infected control macaques.
Model of Log Viral Load Rise and CD4 Decline in Phase II Macaques
As our data detected that SIVsmB7 exposures before an infectious challenge caused a significant decline in CD4 at peak viral load after infection, we generated linear models for CD4 or log viral load kinetics over days (DPI) and included a spline (or change-point) at peak viral load day 14 representing the end of the acute change in variables after infection (illustrated in both viral load and CD4 decline data in Figs. 3C, D). As expected, both models showed that (1) DPI is a significant determinant of both CD4 decline and viral load rise for animals in either group as exemplified by its coefficient β3 in control (P = 0.0436 for CD4 and P < 0.0001 for logVL; Methods and Table 2) and β3′ in SIVsmB7 treated (P = 0.0408 for CD4 and P = 0.0036 for logVL) and (2) day 14 is a valid change-point as illustrated by the reverse of direction of estimates for β3 and β4 for the control and β3′ and β4′ for the SIVsmB7 group.
The model for CD4 T-cell kinetics closely approximated our CD4 data and indicated a significantly different change rate between groups before day 14 (β3′, P = 0.0408; Fig. 3 and Table 2). The effect of infection in the CEM mock control animals showed a loss of 10.06 CD4+ T cells per microliter each day until peak viremia (140.8 loss at peak viral load). SIVsmB7-treated macaques had a 2.5-fold higher daily loss of CD4+ T cells totaling 25.0 CD4+ T cells per microliter per day (342.8 loss at peak viral load).
As for viral load, the model showed a significantly higher rate of change in viral load for the SIVsmB7 over CEM control before day 14 (β3′, P = 0.0036; Fig. 3 and Table 2) in support of greater CD4 T-cell loss. Specifically, log viral load rise each day after infection was predicted to be 0.553 log per day in SIVsmB7-treated macaques as compared with 0.446 log per day for CEM mock control animals. Taking into account baseline CD4 count, CD8 count, CD4/CD8 ratio, age, or weight, as independent factors did not change the model's output for viral load rise.
Altogether, our results suggest that noninfectious SIV exposures and associated conditioning of the local microenvironment result in a greater CD4+ T-cell decline with higher viral load.
We show for the first time that an early, local, innate, and CD4 T-cell response to replication-deficient SIV particles can enhance the rate of CD4+ T-cell decline and viral load rise on a subsequent productive infection. Our results were contrary to our original hypothesis that the observed increase in SIV-induced cervical pDC infiltrates and local IFN-mediated mechanisms would inhibit productive infection. Instead, our data are consistent with viral exposures acutely modulating levels of vaginal and cervical CD4+ T-cell infiltrates, providing a greater pre-existing “substrate” for viral infection, potentially overwhelming any inhibition provided by pDC and resulting in an accelerated decrease of CD4+ T cells in association with viral replication. Importantly, our data assessing the impact of recruited pDC were collected during natural progesterone high periods independent of pharmacological treatments commonly used to synchronize animals as these have been shown to impair pDC function.40,41
Our results parallel a report by Wang et al where female macaques were pre-exposed to TLR-9 or TLR-7 ligands, CpG, or imiquimod, respectively, before and during SIVmac251 challenge.12 Wang et al did not quantify differences in cellular infiltrates before challenge nor model viral kinetics to peak viral load, but did observe increased inflammatory cytokines and infiltrate after dosing and a higher viral load 8 weeks after infection. Their reported data are consistent with our observed accelerated CD4 loss and viral load rise in SIVsmB7 pre-exposed animals. We speculate that the Wang et al study showed a more marked increase in viral replication than our study due to use to 2 challenges using a SIVmac251 one log higher (greater viral inoculum42) and use of optimized TLR-7/9 ligands, which may have induced a greater inflammatory environment (greater local immune activation43).
Importantly, our results are directly relevant to both low-dose SIV repeated challenge study designs commonly used in vaccine studies and to settings of repeated HIV exposures in women. Regarding SIV study designs, our data suggest that early SIV challenges may encounter a different microenvironment than later challenges as a result of immune cell infiltration. Infections after repeated exposures may increase local cell targets and result in accelerated infection kinetics and/or greater gut-associated lymphoid tissue depletion when compared with infections occurring at the start of exposures. Per human cohorts, our data suggest that a sex worker in an area with high HIV-1 prevalence may have different levels of intravaginal immune cell infiltration, independent of coinfections, as compared with women with low levels of HIV-1 exposure. The presence of increased CD4+ T cells and CD68 cells in women at higher risk of HIV-1 infection or in nonhuman primates after multiple SIV exposures may decrease the efficiency of prophylactic interventions by increasing infection efficiency. However, it would be of interest to determine if vaccine-elicited antiviral antibodies in the presence of an increase in cellular infiltration could also provide a greater effector population to harness potentially protective antibody-depended cell-mediated cytotoxicity mechanisms in high-risk uninfected women. Taken together our data suggest that SIV/HIV exposures can contribute to local immune modulation affecting the host tissue response to future viral infection as already established for other STD coinfections.4,16,44,45 Future experiments using a lower infectious dose or repeated low to high-dose escalation of virus would be needed to directly show if SIVsmB7 pre-exposures would increase infection rate over controls as suggested by our data.
Our study had limitations that we addressed. First, our replication-deficient virus, SIVsmB7, was derived from the human cell line, CEMx174. Previous studies have shown that xeno/allogeneic adaptive immune responses to MHC alone can block infection.46 We found that both SIVsmB7 and CEMx174 supernatants had MHC class I and class II proteins (see Figure S4, Supplemental Digital Content,http://links.lww.com/QAI/A482). The 72-hours time frame used for this experiment would limit development of adaptive xenoantigenic responses; moreover, we used a challenge virus derived from a rhesus macaque cell line to avoid this possible confounder response (Methods). It has been shown that innate allogenic responses can be rapid in response to live tissue grafts,47 yet little is known about the speed and strength of such responses through mucosal administration of MHC proteins. Mucosal studies to date have only examined such responses after the induction of the adaptive response.48,49 We interpret that because MHC proteins are present in both SIVsmB7 and control, it is unlikely there would be a differential response yet to be detected based on MHC alone. Importantly, CD4+ T-cell and pDC infiltration was significantly greater in SIVsmB7-treated animals indicating the control proteins did not induce change in the absence of SIV proteins. As for other proteins shown in Figure S4 and Table S2 (see Supplemental Digital Content,http://links.lww.com/QAI/A482), proteomic analysis of both CEM mock and SIVsmB7 doses had comparable protein content to SIVsmB7 outside the presence of viral proteins or Mov10. Interestingly, the presence of Mov10 (see Table S2, Supplemental Digital Content,http://links.lww.com/QAI/A482) in SIVsmB7 proteomic analysis is consistent with enriched particle generation, as previous reports have shown that Mov10 incorporates into viral particles.50 It has also been shown that ectopic expression of Mov10 is an inhibitor of HIV-1/SIV infectivity50–53; however, our data together with a recent report showing that basal levels of Mov10 had no impact on HIV-1 replication or infectivity argues against its antiviral potential.51
Second, previous studies have shown that captive rhesus macaques can have divergent pre-existing cervicovaginal inflammation.39,54 To control for this, we did random testing of CVL from rhesus macaques and found no difference in inflammatory cytokine levels between treatment groups (see Figure S3, Supplemental Digital Content,http://links.lww.com/QAI/A482) despite observing differential CD4 changes between groups as described. The only difference we did detect was a 1-year difference in age between groups (Table 1). We do not expect this minor difference impacts our data as previous research has evidenced no significant difference in immune parameters, such as immune activation or serum cytokine level, among young to middle-age macaques.55
Third, our conclusions address the cervicovaginal mucosa and not other mucosal routes of exposure. Future studies will need to establish whether repeated replication-deficient rectal exposures induce CD4+ T-cell infiltration and affect the severity of a productive infection. Intriguingly, sustained SIV-resistance has been reported following repeated low-dose intra-rectal SIV challenges, highlighting a potential difference in compartments or difference between 72 hours versus chronic exposure effects or both.56
In conclusion, our data support a model where viral exposures in the absence of productive infection induce changes in cervicovaginal cellular infiltrates that do not prevent infection. Furthermore, our data may indicate an added risk to repeated short-term exposures not previously identified and introduces an unaccounted tissue change variable that may impact the outcome of SIV challenge in studies evaluating protection by low-dose repeated challenge formats using female macaques.
The authors acknowledge the critical support received from the UPenn CFAR Viral/Molecular and Nonhuman Primate Core and the TNPRC Flow Cytometry Core Laboratory for immune and viral assays during the study. This study was also made possible by support from the Virology Laboratory, UPR-MSC (T. Arana, P. Pantoja, and R. Medina); the Wistar Institute Cancer Center Proteomics and Histotechnology Core Laboratories (K. Speicher, N. Gorman, T. Beer, and R. Delgiacco). The authors acknowledge additional support from J. Dubin, L. Azzoni, PhD, A. Mackiewicz, and M. Fuller.
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