Prevention of SHIV transmission by topical IFN-ß treatment.

Understanding vaginal and rectal HIV transmission and protective cellular and molecular mechanisms is critical for designing new prevention strategies, including those required for an effective vaccine. The determinants of protection against HIV infection are, however, poorly understood. Increasing evidence suggest that innate immune defenses may help protect mucosal surfaces from HIV transmission in highly exposed, uninfected subjects. More recent studies suggest that systemically administered type 1 interferon protects against simian immunodeficiency virus infection of macaques. Here we hypothesized that topically applied type 1 interferons might stimulate vaginal innate responses that could protect against HIV transmission. We therefore applied a recombinant human type 1 interferon (IFN-ß) to the vagina of rhesus macaques and vaginally challenged them with pathogenic simian/human immunodeficiency virus (SHIV). Vaginal administration of IFN-ß resulted in marked local changes in immune cell phenotype, increasing immune activation and HIV co-receptor expression, yet provided significant protection from SHIV acquisition as interferon response genes were also upregulated. These data suggest that protection from vaginal HIV acquisition may be achieved by activating innate mucosal defenses.

IL-17-producing innate lymphoid cells are restricted to mucosal tissues and are depleted in SIV-infected macaques.

Innate lymphoid cells (ILCs) are an emerging subset of lymphocytes involved in surveillance against virally infected cells. Here, we show CD3(-)CD8(high) lymphocytes in macaque blood include major subsets of ILCs including natural killer (NK) cells expressing CD16, NKp46, and NKG2A, but also populations of ILCs in mucosal tissues having different properties. One ILC subset secreted interleukin (IL)-17 (ILC17), but these were restricted to mucosal tissues. Some mucosal ILC17 cells expressed classical NK-cell markers, but little NKG2A or NKG2D. Some ILC17 cells secreted IL-22 and tumor necrosis factor-α, but few produced interferon (IFN)-γ or contained granzyme B. IL-17 production by ILCs was induced by IL-6, transforming growth factor-ß, and IL-23. Further, simian immunodeficiency virus (SIV) infection resulted in a significant loss of ILC17 cells, especially in the jejunum, which persisted throughout SIV infection. These findings indicate that ILC17 cells may be involved in innate mucosal immune responses, and their loss may contribute to loss of intestinal mucosal integrity and disease progression in human immunodeficiency virus (HIV)/SIV infection.

Monitoring alpha4beta7 integrin expression on circulating CD4+ T cells as a surrogate marker for tracking intestinal CD4+ T-cell loss in SIV infection.

Intestinal CD4+ T cells are rapidly and profoundly depleted in human immunodeficiency virus (HIV)-infected patients and simian immunodeficiency virus (SIV)-infected macaques. However, monitoring intestinal cells in humans is difficult, and identifying surrogate markers in the blood, which correlate with loss or restoration of intestinal CD4+ T cells could be helpful in monitoring the success of therapeutic strategies and vaccine candidates. Recent studies indicate HIV utilizes the intestinal homing molecule alpha4beta7 for attachment and signaling of CD4+ T cells, suggesting this molecule may have a central role in HIV pathogenesis. Here, we compared beta7(HIGH) integrin expression on CD4+ T cells in blood with loss of CD4+ T cells in the intestine of macaques throughout SIV infection. The loss of beta7(HIGH) CD4+ T cells in blood closely paralleled the loss of intestinal CD4+ T cells, and proved to be a more reliable marker of intestinal CD4+ T-cell loss than monitoring CCR5+ memory CD4+ T cells. These data are consistent with a recent hypothesis that alpha4beta7 has a role in the selective depletion of intestinal CD4+ T cells, and indicate that monitoring beta7(HIGH) expression on CD4+ T cells in the blood may be a useful surrogate for estimating intestinal CD4+ T cell loss and restoration in HIV-infected patients.

Cyclic changes in the vaginal epithelium of normal rhesus macaques.

Studies in nonhuman primates indicate that changes in the thickness and integrity of the vaginal epithelium affect the transmission rates of HIV-1, but few studies have examined the normal variations that may occur in the vagina of normal macaques as a result of aging or changes in the menstrual cycle. This study was conducted to determine if differences occur in the thickness of the vaginal mucosa with age or menses. Vaginal mucosal thickness was compared in 46 rhesus macaques grouped as juvenile (1-3 years old), mature cycling (3-21 years old), and geriatric (> 21 years old). Epithelia of mature cycling macaques were also compared at different stages of the menstrual cycle. Older females (> 21 years) had the thinnest and least keratinized epithelium of all groups, followed by the youngest females (< 3 years). The vaginal epithelium was also thinner in cycling macaques during menses compared to the follicular stage. In addition, young, geriatric, or cycling macaques during menses had minimal keratinization. We hypothesize that normal physiologic changes in the vaginal epithelium of women occur with age and menses, which may affect a woman's susceptibility to HIV-1 transmission and other sexually transmitted diseases. Also, age and menstrual cycle should be considered when designing vaginal transmission experiments in rhesus macaques.

Immune responses induced by intranasal imiquimod and implications for therapeutics in rhinovirus infections.

Notwithstanding the progress recently made in immunology and virology, there is yet no effective, specific treatment for the common cold. Symptomatic treatment is minimally effective. An anecdotal report of rapid clearing of the common cold of recent onset after intranasal application of imiquimod in several subjects by one of the authors, made us test the hypothesis that this treatment works through the secretion of interferon by the nasal mucosa. We decided to do an animal study in primates (Indian Macaca Mulata): 5 treatment and 3 control animals were used. Imiquimod or placebo was massaged into the nares of the animals and periodic samples of post-nasal fluid were taken and measurements for Interferon alpha (IFNalpha) and Tumor Necrosis Factor alpha (TNFalpha) were made by ELISA methods, and kinetic studies. mRNA IFNalpha was also isolated and analyzed by quantitative competitive RT-PCR. The internal standard was constructed to be complementary to and compete with oligonucleotide primers and for amplification of target sequences. One intranasal application of imiquimod rapidly (1-4 Hours) induced high levels of mRNA for IFNalpha, and minimal levels in the control animals. Rapid induction of INFalpha, and proportional increase of TNFalpha sustained for 4 and 6 hours respectively were noted. No adverse reactions to treatment were found in macaques during this short period of intranasal imiquimod usage (except in one macaque with a short period of lacrimation). No animal had cytotoxic effects when examined at 6 hr, 12 hr, 24 hr or 48 hr, except one animal, which had an episode of lacrimation for 6 hr post treatment. Thus both safety and efficacy of short treatment with imiquimod is proven in this animal model. Proof of principle for intranasal treatment of the common cold with imiquimod is shown. We think that this work will encourage a number of double blind clinical trials to confirm the effectiveness of the intranasal treatment of the common cold with imiquimod.

Transient early post-inoculation anti-retroviral treatment facilitates controlled infection with sparing of CD4+ T cells in gut-associated lymphoid tissues in SIVmac239-infected rhesus macaques, but not resistance to rechallenge.

Like human immunodeficiency virus infection of humans, infection of rhesus macaques with pathogenic simian immunodeficiency virus (SIV) strains typically results in persistent progressive infection, leading to clinically significant immunosuppression. In previous studies, we administered short term anti-retroviral treatment, shortly after intravenous inoculation with SIVsmE660, in an effort to allow immunologic sensitization under conditions not characterized by overwhelming cytopathic infection compromising the developing immune response. We showed that such treatment allowed control of off treatment viremia and was associated with resistance to rechallenge. Control of off treatment viremia was associated, at least in part, with CD8+ lymphocytes, based on in vivo CD8 depletion studies. In the present study, six rhesus macaques were infected intravenously with 100 MID50 of SIVmac239; four then received 30 days of treatment with tenofovir 9-[2-(R)-(phosphonomethoxy)propyl]adenine (PMPA); 20-30 mg/kg, subcutaneously) starting 24 hours post-inoculation. Tenofovir-treated animals showed low (<500 copy Eq/ml) or undetectable (<100 copy Eq/ml) plasma SIV RNA levels during treatment, with undetectable plasma viremia following discontinuation of treatment. Plasma SIV RNA remained <100 copy Eq/ml, even after depletion of CD8+ lymphocytes, 6 weeks after discontinuation of tenofovir treatment. In contrast to untreated infected control animals that showed substantial depletion of CD4+ T cells from gut-associated lymphoid tissues (GALT), tenofovir-treated animals showed sparing of GALT CD4+ T cells both during the treatment period and in the off treatment follow-up period. However, in contrast to earlier results with animals infected with SIVsmE660, in the present study, the animals did not develop readily measurable cellular anti-SIV immune responses, and did not resist homologous rechallenge with SIVmac239, administered 44 weeks after the initial infection. Differences in the animals and virus strains employed may in part account for the differences in results observed. Comparative analysis of virologic and immunologic parameters in this model system may provide important insights for understanding the basis of effective immunologic control of SIV infection.

Longitudinal follow up of SIVmac pathogenesis in rhesus macaques of Chinese origin: emergence of B cell lymphoma.

Two subspecies of rhesus (Rh) macaques, the Chinese (Ch) and Indian (Ind) subspecies were infected intravenously with 100TCID50 SIVmac239. CD4+, CD8+ T cells, plasma viral loads, depletion of intestinal lymphocytes with memory phenotype, humoral immune responses and clinical courses were monitored for 600 days. The pathogenesis of SIVmac was also compared with primary human immunodeficiency virus (HIV) infection of humans. Plasma viral loads in Ch Rh were lower in the acute and chronic phases compared with Ind Rh. SIVmac pathogenesis in Ch Rh was closer to virus loads in untreated HIV infected humans. Ch Rh had higher CD4/CD8 ratios, stronger antibody responses and interestingly, less depletion of intestinal memory CCR5+ CD4+ T lymphocytes compared with Ind Rh. One Ch Rh developed B cell origin lymphoma at 570 days post-infection, the first such report in this subspecies. Three of four Ind Rh developed AIDS within 6 months. The findings indicate that Ch Rh are more resistant to SIVmac pathogenesis compared with Ind Rh and that Ch Rh paralleled HIV-1 infections in untreated adult humans. The SIVmac infected Ch Rh subspecies are an acceptable model for HIV/AIDS.

Simian immunodeficiency virus (SIV)-specific cytotoxic T lymphocytes in gastrointestinal tissues of chronically SIV-infected rhesus monkeys.

Although systemic virus-specific cytotoxic T lymphocyte (CTL) responses are of critical importance in controlling virus replication in individuals infected with human immunodeficiency virus 1 (HIV-1), little is known about this immune response in the gastrointestinal (GI) tract. This study investigated the GI tract CTL response in a nonhuman primate model for HIV-1 infection, simian immunodeficiency virus (SIV)-infected rhesus monkeys. Lymphocytes from duodenal pinch biopsy specimens were obtained from 9 chronically SIVmac-infected rhesus monkeys and GI tract lymphocytes were harvested from the jejunum and ileum of 4 euthanized SIVmac-infected rhesus monkeys. Lymphocytes were also assessed in GI mucosal tissues by in situ staining in histologic specimens. SIVmac Gag-specific CTLs were assessed in the monkeys using the tetramer technology. These GI mucosal tissues of chronically SIVmac-infected rhesus monkeys contained levels of CTLs comparable to those found in the peripheral blood and lymph nodes. The present studies suggest that the CD8(+) CTL response in GI mucosal sites is comparable to that seen systemically in SIVmac-infected rhesus monkeys.

The mucosal immune system: primary target for HIV infection and AIDS.

Despite intensive research, several questions remain regarding the pathogenesis of infection with HIV-1. Recently, it has been shown that simian immunodeficiency virus (SIV) selectively targets and destroys specific subsets of CD4+ T cells that are abundant in mucosal tissues but rare in peripheral lymphoid tissues. This finding could be highly relevant in explaining a major paradox in the infection and elimination of CD4+ T cells during HIV infection: the progressive decline in the number of CD4+ T cells in the blood, despite the paucity of HIV-infected cells in this tissue. This article discusses the hypothesis that infection with HIV and SIV, and the resulting disease, is governed by the state of cellular activation and the expression of chemokine receptors by specific subsets of CD4+ T cells residing in mucosal lymphoid tissues, rather than those found in the peripheral blood or lymph nodes.

Emergence and kinetics of simian immunodeficiency virus-specific CD8(+) T cells in the intestines of macaques during primary infection.

In this report, three Mamu-A*01(+) rhesus macaques were examined to compare the emergence of simian immunodeficiency virus (SIV)-specific CD8(+) T cells in the intestines and blood in early SIV infection using a major histocompatibility complex class I tetramer complexed with the Gag(181-189) peptide. Fourteen days after intravenous inoculation with SIVmac251, large numbers of SIV Gag(181-189)-specific CD8(+) T cells were detected in the intestinal mucosa (3.1 to 11.5% of CD3(+) CD8(+) lymphocytes) as well as in the blood (3.1 to 13.4%) of all three macaques. By 21 days postinoculation, levels of tetramer-binding cells had dropped in both the intestines and blood. At day 63, however, levels of SIV Gag(181-189)-specific CD8(+) T cells in the intestines had rebounded in all three macaques to levels that were higher (8.6 to 18.7%) than those at day 21. In contrast, percentages of tetramer-binding cells in the peripheral blood remained comparatively stable (2.5 to 4.5%) at this time point. In summary, SIV Gag(181-189)-specific CD8(+) T cells appeared in both the intestinal mucosa and peripheral blood at a comparable rate and magnitude in primary SIV infection. Given that the intestine is a major site of early viral replication as well as the site where most of the total body lymphocyte pool resides, these data indicate that it is also an early and important site of development of antiviral immune responses.

Induction of disseminated Mycobacterium avium in simian AIDS is dependent upon simian immunodeficiency virus strain and defective granuloma formation.

Mycobacterium avium complex (MAC) is the most common disseminated bacterial disease in patients infected by the human immunodeficiency virus. Although murine models of disseminated MAC exist, they are primarily based on underlying genetic susceptibilities and cannot adequately address the complex interactions that occur between host, mycobacteria, and immunosuppressive lentivirus. To address this problem we have developed an experimental system to co-inoculate rhesus macaques with the simian immunodeficiency virus (SIV) and a clinical M. avium isolate that results in a disease virtually identical to that observed in human cases. Using this experimental system we have found that the development of disseminated MAC is dependent on viral strain. Animals co-infected with SIVmac251 and M. avium developed progressive disease, whereas control animals and animals inoculated with closely related viruses (SIVmac239 and SIVmac239MER) developed self-limiting infections. The ability of animals infected with SIVmac239 or SIVmac239MER to eliminate mycobacterial disease was independent of viral load and CD4 T-cell number but was correlated with the size and composition of microgranulomas. This work establishes a novel primate model of disseminated MAC in the context of immunosuppressive lentiviral infection and advances our understanding of why human immunodeficiency virus-infected patients are remarkably sensitive to the development of mycobacterial disease.

Dynamics of CCR5 expression by CD4(+) T cells in lymphoid tissues during simian immunodeficiency virus infection.

Early viral replication and profound CD4(+) T-cell depletion occur preferentially in intestinal tissues of macaques infected with simian immunodeficiency virus (SIV). Here we show that a much higher percentage of CD4(+) T cells in the intestine express CCR5 compared with those found in the peripheral blood, spleen, or lymph nodes. In addition, the selectivity and extent of the CD4(+) T-cell loss in SIV infection may depend upon these cells coexpressing CCR5 and having a "memory" phenotype (CD45RA(-)). Following intravenous infection with SIVmac251, memory CD4(+) CCR5(+) T cells were selectively eliminated within 14 days in all major lymphoid tissues (intestine, spleen, and lymph nodes). However, the effect on CD4(+) T-cell numbers was most profound in the intestine, where cells of this phenotype predominate. The CD4(+) T cells that remain after 14 days of infection lacked CCR5 and/or were naive (CD45RA(+)). Furthermore, when animals in the terminal stages of SIV infection (with AIDS) were examined, virtually no CCR5-expressing CD4(+) T cells were found in lymphoid tissues, and all of the remaining CD4(+) T cells were naive and coexpressed CXCR4. These findings suggest that chemokine receptor usage determines which cells are targeted for SIV infection and elimination in vivo.

Induction of mucosal homing virus-specific CD8(+) T lymphocytes by attenuated simian immunodeficiency virus.

Induction of virus-specific T-cell responses in mucosal as well as systemic compartments of the immune system is likely to be a critical feature of an effective AIDS vaccine. We investigated whether virus-specific CD8(+) lymphocytes induced in rhesus macaques by immunization with attenuated simian immunodeficiency virus (SIV), an approach that is highly effective in eliciting protection against mucosal challenge, express the mucosa-homing receptor alpha4beta7 and traffic to the intestinal mucosa. SIV-specific CD8(+) T cells expressing alpha4beta7 were detected in peripheral blood and intestine of macaques infected with attenuated SIV. In contrast, virus-specific T cells in blood of animals immunized cutaneously by a combined DNA-modified vaccinia virus Ankara regimen did not express alpha4beta7. These results demonstrate the selective induction of SIV-specific CD8(+) T lymphocytes expressing alpha4beta7 by a vaccine approach that replicates in mucosal tissue and suggest that induction of virus-specific lymphocytes that are able to home to mucosal sites may be an important characteristic of a successful AIDS vaccine.

Simian immunodeficiency virus-specific cytotoxic T lymphocytes and cell-associated viral RNA levels in distinct lymphoid compartments of SIVmac-infected rhesus monkeys.

Major histocompatibility class I-peptide tetramer technology and simian immunodeficiency virus of macaques (SIVmac)-infected rhesus monkeys were used to clarify the distribution of acquired immunodeficiency syndrome virus-specific cytotoxic T lymphocytes (CTL) in secondary lymphoid organs and to assess the relationship between these CTL and the extent of viral replication in the various anatomic compartments. SIVmac Gag epitope-specific CD8(+) T cells were evaluated in the spleen, bone marrow, tonsils, thymus, and 5 different lymph node compartments of 4 SIVmac-infected rhesus monkeys. The average percentage of CD8(+) T lymphocytes that bound this tetramer in all the different lymph node compartments was similar to that in peripheral blood lymphocytes in individual monkeys. The percentage of CD8(+) T cells that bound the tetramer in the thymus was uniformly low in the monkeys. However, the percentage of CD8(+) T cells that bound the tetramer in bone marrow and spleen was consistently higher than that seen in lymph nodes and peripheral blood. The phenotypic profile of the tetramer-binding CD8(+) T lymphocytes in the different lymphoid compartments was similar, showing a high expression of activation-associated adhesion molecules and a low level expression of naive T-cell-associated molecules. Surprisingly, no correlation was evident between the percentage of tetramer-binding CD8(+) T lymphocytes and the magnitude of the cell-associated SIV RNA level in each lymphoid compartment of individual monkeys. These studies suggest that a dynamic process of trafficking may obscure the tendency of CTL to localize in particular regional lymph nodes or that some lymphoid organs may provide milieus that are particularly conducive to CTL expansion. (Blood. 2000;96:1474-1479)

Differential effects of simian immunodeficiency virus infection on immune inductive and effector sites in the rectal mucosa of rhesus macaques.

The rectal mucosa, a region involved in human immunodeficiency virus/simian immunodeficiency virus (SIV) infection and transmission, contains immune inductive sites, rectal lymphoid nodules (RLN), and effector sites, the lamina propria (LP). This study was designed to evaluate cell populations involved in rectal mucosal immune function in both RLN and LP, by immunocytochemical analysis of rectal mucosa from 11 SIV-infected (2 to 21 months postinfection) and five naive rhesus macaques. In the rectum, as previously observed in other intestinal regions, CD4(+) cells were dramatically reduced in the LP of SIV-infected macaques, but high numbers of CD4(+) cells remained in RLN indicating maintenance of T cell help in inductive sites. Cells expressing the mucosal homing receptor alpha4beta7 were dramatically decreased in the RLN and LP of most SIV-infected macaques. The RLN of both naive and SIV-infected macaques contained high numbers of CD68 + MHC-II+ macrophages and cells expressing the co-stimulatory molecules B7-2 and CD40, as well as IgM + MHCII+ and IgM + CD40+ B cells, indicating maintenance of antigen presentation capacity. The LP of all three macaques SIV-infected for 2 months contained many B7-2+ cells, suggesting increased activation of antigen-presenting cells. LP of SIV-infected rectal mucosa contained increased numbers of IgM+ cells, confirming previous observations in small intestine and colon. The data suggest that antigen-presentation capacity is maintained in inductive sites of SIV-infected rectal mucosa, but immune effector functions may be altered.

Simian immunodeficiency virus (SIV)-specific CTL are present in large numbers in livers of SIV-infected rhesus monkeys.

The immunopathogenesis of AIDS-associated hepatitis was explored in the SIV/rhesus monkey model. The livers of SIV-infected monkeys showed a mild hepatitis, with a predominantly CD8+ T lymphocyte infiltration in the periportal fields and sinusoids. These liver-associated CD8+ T cells were comprised of a high percentage of SIV-specific CTL as defined by MHC class I/Gag peptide tetramer binding and Gag peptide epitope-specific lytic activity. There was insufficient viral replication in these livers to account for attracting this large number of functional virus-specific CTL to the liver. There was also no evidence that the predominant population of CTL were functionally end-stage cells trapped in the liver and destined to undergo apoptotic cell death in that organ. Interestingly, we noted that liver tetramer-binding cells showed an increased expression of CD62L, an adhesion molecule usually only rarely expressed on tetramer-binding cells. This observation suggests that the expression of specific adhesion molecules by CTL might facilitate the capture of these cells in the liver. These results demonstrate that functional SIV-specific CD8+ T cells are present in large numbers in the liver of chronically SIV-infected monkeys. Thus, the liver may be a trap for virus-specific cytotoxic T cells.

Identifying the target cell in primary simian immunodeficiency virus (SIV) infection: highly activated memory CD4(+) T cells are rapidly eliminated in early SIV infection in vivo.

It has recently been shown that rapid and profound CD4(+) T-cell depletion occurs almost exclusively within the intestinal tract of simian immunodeficiency virus (SIV)-infected macaques within days of infection. Here we demonstrate (by three- and four-color flow cytometry) that this depletion is specific to a definable subset of CD4(+) T cells, namely, those having both a highly and/or acutely activated (CD69(+) CD38(+) HLA-DR(+)) and memory (CD45RA(-) Leu8(-)) phenotype. Moreover, we demonstrate that this subset of helper T cells is found primarily within the intestinal lamina propria. Viral tropism for this particular cell type (which has been previously suggested by various studies in vitro) could explain why profound CD4(+) T-cell depletion occurs in the intestine and not in peripheral lymphoid tissues in early SIV infection. Furthermore, we demonstrate that an acute loss of this specific subset of activated memory CD4(+) T cells may also be detected in peripheral blood and lymph nodes in early SIV infection. However, since this particular cell type is present in such small numbers in circulation, its loss does not significantly affect total CD4(+) T cell counts. This finding suggests that SIV and, presumably, human immunodeficiency virus specifically infect, replicate in, and eliminate definable subsets of CD4(+) T cells in vivo.

Sexual transmission and propagation of SIV and HIV in resting and activated CD4+ T cells.

In sexual transmission of simian immunodeficiency virus, and early and later stages of human immunodeficiency virus-type 1 (HIV-1) infection, both viruses were found to replicate predominantly in CD4(+) T cells at the portal of entry and in lymphoid tissues. Infection was propagated not only in activated and proliferating T cells but also, surprisingly, in resting T cells. The infected proliferating cells correspond to the short-lived population that produces the bulk of HIV-1. Most of the HIV-1-infected resting T cells persisted after antiretroviral therapy. Latently and chronically infected cells that may be derived from this population pose challenges to eradicating infection and developing an effective vaccine.