Following exposure to HIV, viral RNA in the blood is detected only after a lag time, which is referred to as an eclipse time, during which there is a window of opportunity for early interventions.1 On HIV-1 vaginal exposure, the eclipse time is approximately 10 days in humans, and 7 days in simian immunodeficiency virus macaque models.1,2 The eclipse time following rectal exposure appeared shorter in some studies because of the more rapid dissemination of SIV,3,4 whereas the others showed that the challenge dose affected the eclipse time, with high to low challenge doses leading to 4–8.5 days of eclipse time.5
A HIV vaccine aiming at achieving sterile protection would need to block or eradicate the virus in the mucosa during or even before the eclipse phase of infection.6 Despite the importance of rectal infection in the AIDS epidemic, the eclipse time and the factors that affect the colorectal eclipse time have not been fully elucidated. In this study, we examined the eclipse time of 40 macaques after rectal inoculation of SIVmac251, and investigated the factors that might modulate the eclipse time. Additionally, we measured the early viral loads (VLs) in the rectum and vagina, and explored the associations between VLs in these mucosal tissues and plasma. An understanding of these would be helpful in the designing of vaccines to prevent anal HIV transmission.
Animals, Viral Challenge, and VL Measurements
Forty adult Indian rhesus macaques (Macaca mulatta) were used with the approval of the National Cancer Institute Animal Care and Use Committee. There were 3 males and 37 females and all were either Mamu-A*01 or A*02 positive, and negative for Mamu-B*08 and B*17. Of total 40 macaques 26 were intracolorectally immunized with a SIV/HIV peptide prime/MVA boost regimen, adjuvanted with TLRLs (D-type CpG oligodeoxynucleotide, MALP2, PolyI:C), and recombinant human IL-15. Among them, 6 macaques received the above-mentioned base vaccine, whereas the rest of them received base vaccine with B7-DC-Ig (n = 6) alone, α-GalCer (n = 6) alone, or both (n = 8). Six macaques received adjuvants only. The detailed immunization was performed as previously described.7–9 Eight naive macaques were included. Ten weeks after the last boost, all animals received intrarectal inoculations of 1:100; 1:100; and 1:50 diluted SIVmac251 stock (provided by Nancy Miller from NIAID, Desrosiers stock-batch number February 17, 2006. Based on the previous animal titration study, 1 animal ID50 is about 1:500 dilution) sequentially at 2-week intervals until infected. After challenge, plasma SIV RNA levels were measured at days 4, 7, 11, 14, 21, 28 and 2–14 months postinfection. Rectal/vaginal tissue VLs were measured at day 14 postinfection. Fifty copies per milliliter (or per milligram) was the cutoff threshold for detection.
Flow Cytometric Analysis of Viral Target Cells in the Colorectal Tissues
Seven weeks before the first viral challenge, an open laparotomy was performed on each of the vaccinated and adjuvanted animals to obtain a colorectal wedge. For the naive animals, 10 rectal pinches were collected. The colonic wedge was rinsed with Hanks balanced salt solution to remove the mucus, and then cut into 2″ pieces. The colonic tissue pieces/rectal pinches were incubated with Hanks balanced salt solution containing 5 mM EDTA and 2 mM DTT at room temperature for 15 minutes twice. The supernatants, which contain intraepithelial lymphocyte cells, were collected, pooled and filtered through 100 μM cell strainers. The collected colorectal intraepithelial lymphocytes were washed and subjected to fluorescence-activated cell sorting, staining with viability dye and antibody mixtures including anti-CD45 to exclude the dead and CD45-negative epithelial cells. The frequencies of ki67, CD69, CD38, and HLA-DR within CD4+ and CD8+ T cells were further assessed. Approximately 8000, 6000, and 2000 CD4+T cells (mean) were analyzed for vaccinated, adjuvanted, and naive animals (see Figure S1, Supplemental Digital Content, http://links.lww.com/QAI/A767). The following antibodies were purchased: CD3-PE-Cy7, CD8-APC-Cy7, Ki67-APC, HLA-DR PE-Cy5, and CCR5-PE (from BD Pharmingen, San Diego, CA); CD69-Alexa Fluor 700 (from Biolegend, San Diego, CA); CD38-FITC (from STEMCELL Technologies, Vancouver, BC, Canada); and CD4-qdot 605 (from eBioscience, San Diego, CA).
We performed statistical analyses with Prism (GraphPad) and SAS. A 2-sided significance level of 0.05 was used for all analyses. The Mann–Whitney or Jonckheere–Terpstra trend tests were used as shown in the figures. Spearman analysis was used for correlations.
More Rapid Viral Dissemination After Rectal SIVmac251 Exposure Was Associated With High Frequency of Colorectal Ki67+CCR5+CD4+T Cells
Forty macaques were rectally challenged by SIVmac251 at 2-week intervals until infected. Plasma viral RNA was monitored at day 4, 7, 11, and 14 after each challenge, and if the macaques were confirmed infected, they were not challenged further. After 3 challenges, all the macaques were infected. Of 40 macaques, 26 (65%) had first detectable viral RNAs in the blood at day 7 after the challenge that led to the productive infection. Strikingly, 6 animals (15%) had detectable viral RNA in the blood at day 4, whereas another 8 did not show infection until day 11 or 14. When plotting the frequencies of the macaque eclipse times, we found that the middle transmitters (65%) had a 7-day eclipse time, whereas early transmitters had an eclipse time of 4 days (15%), and we could define 2 populations of late transmitters with 11 and 14 days of eclipse time (Figs. 1A, B). Investigating whether the eclipse time affected the VLs, we found that day14 plasma VL was highest in the early-transmitter group, and then middle > late 1 > late 2 transmitter groups, respectively (Fig. 2A), as predicted.7 However, there was no significant difference in the set-point plasma VLs (from day 42–147 after infection), or in the rectal and vaginal tissue VLs at day 14 postinfection among the early, middle, and late transmitters (Figs. 2B–D), suggesting that the eclipse time did not affect these VLs.
Whereas immune activation promotes HIV-1 disease progression,10,11 a number of studies have suggested that low levels of immune activation at the time of exposure also reduced the risk of HIV-1 infection.12–15 In our previous study, the level of immune activation in the rectal mucosa, rather than in peripheral blood mononuclear cells, inversely correlated with acute plasma VLs.7 However, whether immune activation affects eclipse time is less clear. Before challenge, the macaques had varied levels of CCR5+CD4+ T cells in the colorectal mucosa. The Ki67+ proliferating viral target CCR5+CD4+ T cells in the colorectal mucosa were significantly higher in the early or middle-transmitter groups than those in the late-transmitter groups, and inversely correlated with eclipse time in both the naive and vaccinated populations, as well as the animals infected after the first inoculation (Figs. 2E–H). This trend was not found in the CD69+, or HLA-DR+, or CD38+ T cells that were CCR5+CD4+ (data not shown). Thus, the abundance of preexisting Ki67+ CCR5+CD4+ T cells in the rectum/colon predicted rapid viral dissemination. To have a long enough eclipse time for a vaccine to mount a protective recall response, it seems essential to reduce/limit these cells at the portal of viral entry.
Acute Rectal VLs Correlated With Plasma VLs in a Delayed Manner
After vaginal inoculation, the virus must first establish a small founder population of infected cells at the portal of entry, and then a self-propagating infection in the draining lymph nodes before it is detected in the blood.1 On rectal inoculation, whether similar events are needed is an open question. One difference between the rectum and vagina is that the former has more abundant viral target CD4+T cells than the latter. Indeed, both at 14 days after the viral challenge and at the chronic stage, the median rectal VL was more than 1 log higher than the median vaginal VL (Figs. 3A, B). If the virus was first expanded in the rectum before it disseminated to the blood, and the mucosal tissues and blood were well equilibrated, a positive correlation would be expected. However, the day 14 rectal VL did not correlate with blood plasma VL 14 days postinoculation (Fig. 4A). Instead, day14 rectal VL positively correlated with days 21 and 28, but not day 7 or day 14 plasma VLs (Figs. 4B–D). This lag in correlation suggests that initial local viral propagation in the rectal mucosa was important for subsequent dissemination; any interventions that could reduce the rectal VLs at an early time point might affect the plasma VLs at later time points. Moreover, at day 14, an equilibration between virus in rectum and in blood had not been fully established yet. This was in sharp contrast to the scenario at the chronic infection stage, where the correlation between rectal VL and plasma VL was highly significant (R = 0.85, P < 0.0001; Fig. 4E). In contrast, the plasma VL did correlate with vaginal VL at day 14 (Fig. 4F). At 6-month postinfection, most macaques had undetectable vaginal VL, but those with detectable VL had higher plasma VL (P = 0.011). The fact that the viral equilibration between colorectal and plasma was delayed, whereas that between plasma and vaginal mucosa was not at the acute stage likely reflected the site and route of infection and dissemination, from colorectal mucosa to blood and then to vaginal mucosa. If the animals were infected intravaginally, the reverse pattern might have been seen.
To develop a HIV vaccine to prevent anal transmission, information on the eclipse time, and the factors that affect the eclipse time, is desirable. Intrarectally inoculated SIV in macaque models is the best available model of human rectal transmission of HIV-1.16 Small numbers of animals, and high inoculation doses pose a big challenge for studies on eclipse time on mucosal infections. High-dose SIV affected not only the dissemination profile,4 but also the eclipse time.5 In particular, higher doses of virus resulted in a shorter eclipse time, and more than 10 transmitted/founder viruses.5 The latter did not recapitulate natural HIV-1 transmission in humans, where typically only 1 or a few transmitted/founder viruses are found.17 We addressed this question using multiple doses of SIVmac251 intrarectal inoculations. We enumerated the viral variants in the infected animals, and found that of the 12 animals analyzed, 9 had only 1 variant and 3 had 2 variants,7 suggesting that the challenge was recapitulating human mucosal HIV-1 transmission much more closely than conventional high-dose challenge. Based on the number of variants transmitted, it seems that the dose administered was an intermediate dose.18
Two potentially confounding factors may complicate the interpretation of this study. One was that 26 of 40 macaques were vaccinated with an SIV/HIV peptide-prime/MVA-boost vaccine that induced SIV-specific T-cell responses. Another was that up to 3 viral inoculations were given to the animals, which might induce protective viral-specific immune responses and harmful immune activation, and affect the subsequent viral dissemination (10 animals received 2, and 4 animals received 3 viral inoculations). However, consistent with no/low antigen-specific antibody responses induced using this regimen,9 we did not observe a difference in viral acquisition time between the vaccinated and the naive animals (Fig. 1A). As the time interval between the last boost and the first viral challenge was 10 weeks, any direct effect of adjuvants on rectal mucosa is negligible. Antigen-specific mucosal T-cell responses induced by the vaccine and/or previous viral exposure(s) could potentially suppress the viral replication, and thus lengthen the eclipse time. This, however, could be counteracted by the immune activation induced by the previous inoculations. Nevertheless, the distribution of the eclipse time of the vaccinated and the naive animals was similar, suggesting that this regimen of vaccination did not significantly affect the eclipse time (Fig. 1A). Furthermore, the inverse correlation between eclipse time and number of activated CD4+ T cells in the mucosa held up even when only the naive animals were examined (Fig. 2G), showing that this correlation did not depend on the vaccination. Moreover, we did not find any association of the number of intrarectal viral challenges with the distribution of the eclipse time (see Figure S2, Supplemental Digital Content, http://links.lww.com/QAI/A767), and the distribution of eclipse time of the animals infected after the first inoculation did not significantly differ from that of the animals infected after 2 or 3 inoculations, suggesting that the effects of previous viral inoculations (including the possible occult infection) on eclipse time are also minimal (Fig. 1B). Our data showed that the median eclipse time on intermediate dose anal challenge leading to SIVmac251 infection was about 7 days. An early intervention, ideally, would induce recall responses and block the viral transmission before day 7. However, we did observe viral dissemination as rapid as 4 days in some of the animals. More rapid viral dissemination in these animals was associated with high frequency of colorectal Ki67+CCR5+CD4+T cells before viral inoculation. Studies have demonstrated that high levels of chemoattractant, proinflammatory and immune activation genes were induced at the early stage of vaginal19 or rectal SIV infection,20 which can recruit and activate virus-susceptible target cells into the mucosa. We found that the preexisting Ki67+CCR5+CD4+T cells in the colorectal mucosa determined the speed of viral dissemination to the periphery. These viral target cells at the portal of viral entry fueled the initial viral replication even before the recruitment of the viral target cells by virus-induced proinflammatory chemokines/cytokines, and thus accelerated the viral dissemination. Reducing these cells would be beneficial not only for decreasing the acute VLs as we have observed before,7 but also for extending the eclipse time so that the host has more time to mount protective immunity while the virus is localized to the tissue of entry. The latter is especially important in HIV vaccine development, as more time for the host to induce protective recall responses could make a difference for the ultimate infection outcome. However, the delayed correlation of rectal VL at day 14 with plasma VLs at day 21 and 28 suggested that the rectal VL might impact the later plasma VLs. This gave us a hint that any interventions that reduce early rectal VL might still have the chance of reducing systemic VLs. Thus, the priority for developing any early interventions would be limitation of viral target cells at the portal of entry combined with the induction of protective immunity at the mucosal sites.
The authors thank Nancy Miller from the National Institute of Allergy and Infectious Diseases for providing the SIVmac251 challenge stock.
1. Haase AT. Early events in sexual transmission of HIV and SIV and opportunities for interventions. Annu Rev Med. 2011;62:127–139.
2. Miller CJ, Li Q, Abel K, et al.. Propagation and dissemination of infection after vaginal transmission of simian immunodeficiency virus. J Virol. 2005;79:9217–9227.
3. Miyake A, Ibuki K, Enose Y, et al.. Rapid dissemination of a pathogenic simian/human immunodeficiency virus to systemic organs and active replication in lymphoid tissues following intrarectal infection. J Gen Virol. 2006;87:1311–1320.
4. Ribeiro Dos Santos P, Rancez M, Pretet JL, et al.. Rapid dissemination of SIV follows multisite entry after rectal inoculation. PLoS One. 2011;6:e19493.
5. Liu J, Keele BF, Li H, et al.. Low-dose mucosal simian immunodeficiency virus infection restricts early replication kinetics and transmitted virus variants in rhesus monkeys. J Virol. 2010;84:10406–10412.
6. Whitney JB, Hill AL, Sanisetty S, et al.. Rapid seeding of the viral reservoir prior to SIV viraemia in rhesus monkeys. Nature. 2014;512:74–77.
7. Sui Y, Hogg A, Wang Y, et al.. Vaccine-induced myeloid cell population dampens protective immunity to SIV. J Clin Invest. 2014;124:2538–2549.
8. Sui Y, Gagnon S, Dzutsev A, et al.. TLR agonists and/or IL-15 adjuvanted mucosal SIV vaccine reduced gut CD4(+) memory T cell loss in SIVmac251-challenged rhesus macaques. Vaccine. 2011;30:59–68.
9. Sui Y, Zhu Q, Gagnon S, et al.. Innate and adaptive immune correlates of vaccine and adjuvant-induced control of mucosal transmission of SIV in macaques. Proc Natl Acad Sci U S A. 2010;107:9843–9848.
10. Giorgi JV, Hultin LE, McKeating JA, et al.. Shorter survival in advanced human immunodeficiency virus type 1 infection is more closely associated with T lymphocyte activation than with plasma virus burden or virus chemokine coreceptor usage. J Infect Dis. 1999;179:859–870.
11. Lawn SD, Butera ST, Folks TM. Contribution of immune activation to the pathogenesis and transmission of human immunodeficiency virus type 1 infection. Clin Microbiol Rev. 2001;14:753–777; table of contents.
12. Begaud E, Chartier L, Marechal V, et al.. Reduced CD4 T cell activation and in vitro susceptibility to HIV-1 infection in exposed uninfected Central Africans. Retrovirology. 2006;3:35.
13. Koning FA, Otto SA, Hazenberg MD, et al.. Low-level CD4+ T cell activation is associated with low susceptibility to HIV-1 infection. J Immunol. 2005;175:6117–6122.
14. McLaren PJ, Ball TB, Wachihi C, et al.. HIV-exposed seronegative commercial sex workers show a quiescent phenotype in the CD4+ T cell compartment and reduced expression of HIV-dependent host factors. J Infect Dis. 2010;202(suppl 3):S339–S344.
15. Songok EM, Luo M, Liang B, et al.. Microarray analysis of HIV resistant female sex workers reveal a gene expression signature pattern reminiscent of a lowered immune activation state. PLoS One. 2012;7:e30048.
16. Keele BF, Li H, Learn GH, et al.. Low-dose rectal inoculation of rhesus macaques by SIVsmE660 or SIVmac251 recapitulates human mucosal infection by HIV-1. J Exp Med. 2009;206:1117–1134.
17. Keele BF, Giorgi EE, Salazar-Gonzalez JF, et al.. Identification and characterization of transmitted and early founder virus envelopes in primary HIV-1 infection. Proc Natl Acad Sci U S A. 2008;105:7552–7557.
18. Varela M, Landskron L, Lai RP, et al.. Molecular evolution analysis of the human immunodeficiency virus type 1 envelope in simian/human immunodeficiency virus-infected macaques: implications for challenge dose selection. J Virol. 2011;85:10332–10345.
19. Li QS, Estes JD, Schlievert PM, et al.. Glycerol monolaurate prevents mucosal SIV transmission. Nature. 2009;458:1034–1113.
20. Lu W, Ma F, Churbanov A, et al.. Virus-host mucosal interactions during early SIV rectal transmission. Virology. 2014;464–465:406–414.