Expression of DC-SIGN by Dendritic Cells of Intestinal and Genital Mucosae in Humans and Rhesus Macaques

To better understand the role of dendritic cells (DCs) in human immunodeficiency virus (HIV) transmission at mucosal surfaces, we examined the expressions of the HIV adhesion molecule, dendritic-cell-specific ICAM-3 grabbing nonintegrin (DC-SIGN), its closely related homologue DC-SIGNR, and HIV coreceptors by distinct DC populations in the intestinal and genital tracts of humans and rhesus macaques. We also developed monoclonal antibodies (MAbs) specific for DC-SIGN or DC-SIGNR. In the Peyer's patches, DC-SIGN expression was detected in the interfollicular regions and in clusters of cells in the subepithelial dome regions. DC-SIGN expression was not found on plasmacytoid DCs. DC-SIGNR expression was restricted to endothelial cells in approximately one-third of the capillaries in the terminal ileum. In the vaginal epithelium, Langerhans' cells did not express DC-SIGN, whereas subepithelial DCs in the lamina propria expressed moderate levels of DC-SIGN. Finally, the rectum contained cells that expressed high levels of DC-SIGN throughout the entire thickness of the mucosa, while solitary lymphoid nodules within the rectum showed very little staining for DC-SIGN. Triple-color analysis of rectal tissue indicated that CCR5(+) CD4(+) DC-SIGN(+) DCs were localized just beneath the luminal epithelium. These findings suggest that DC-SIGN(+) DCs could play a role in the transmission of primate lentiviruses in the ileum and the rectum whereas accessibility to DC-SIGN(+) cells is limited in an intact vaginal mucosa. Finally, we identified a MAb that blocked simian immunodeficiency virus interactions with rhesus macaque DC-SIGN. This and other specific MAbs may be used to assess the relevance of DC-SIGN in virus transmission in vivo.     

Distribution of SIV infection in the gastrointestinal tract of rhesus macaques at early and terminal stages of AIDS

Intestinal tissues from SIV-infected rhesus macaques with subclinical as well as with terminal SIV disease were evaluated for SIV infection. SIV-infected mononuclear cells were detected throughout the gastrointestinal tract in all animals. In early infection, SIV-infected cells were diffusely distributed in lymphoid tissue and lamina propria, whereas in late stages of the disease, the viral infection was more severe and widely disseminated. Lymphoid cells of the lamina propria and gut-associated lymphoid tissue were the primary targets of SIV.     

Simian immunodeficiency virus infection in neonatal macaques

Children with human immunodeficiency virus infection often have higher viral loads and progress to AIDS more rapidly than adults. Since the intestinal tract is a major site of early viral replication and CD4(+) T-cell depletion in adults, we examined the effects of simian immunodeficiency virus (SIV) on both peripheral and intestinal lymphocytes from 13 neonatal macaques infected with SIVmac239. Normal neonates had more CD4(+) T cells and fewer CD8(+) T cells in all tissues than adults. Surprisingly, neonates had substantial percentages of CD4(+) T cells with an activated, memory phenotype (effector CD4(+) T cells) in the lamina propria of the intestine compared to peripheral lymphoid tissues, even when examined on the day of birth. Moreover, profound and selective depletion of jejunum lamina propria CD4(+) T cells occurred in neonatal macaques within 21 days of infection, which was preceded by large numbers of SIV-infected cells in this compartment. Furthermore, neonates with less CD4(+) T-cell depletion in tissues tended to have higher viral loads. The persistence of intestinal lamina propria CD4(+) T cells in some neonates with high viral loads suggests that increased turnover and/or resistance to CD4(+) T-cell loss may contribute to the higher viral loads and increased severity of disease in neonatal hosts.     

Both mucosal and systemic routes of immunization with the live,attenuated NYVAC/simian immunodeficiency virus SIV(gpe) recombinant vaccine result in gag-specific CD8(+) T-cell responses in mucosal tissues of macaques

As most human immunodeficiency virus (HIV) infection occurs via mucosal surfaces, an important goal of vaccination may be the induction of virus-specific immune responses at mucosal sites to contain viral infection early on. Here we designed a study in macaques carrying the major histocompatibility complex class I Mamu-A(*)01 molecule to assess the capacity of the highly attenuated poxvirus NYVAC/simian immunodeficiency virus (SIV) SIV(gpe) vaccine candidate administered by the intranasal, intramuscular, or intrarectal route to induce mucosal immunity. All macaques, including one naive macaque, were exposed to SIV(mac251) by the intrarectal route and sacrificed 48 h after infection. The kinetics of immune response at various time points following immunization with NYVAC/SIV(gpe) and the anamnestic response to SIV(mac251) at 48 h after challenge were assessed in blood, in serial rectal and vaginal biopsy samples, and in tissues at euthanasia with an SIV(mac) Gag-specific tetramer. In addition, at euthanasia, antigen-specific cells producing gamma interferon or tumor necrosis factor alpha from the jejunum lamina propria were quantified in all macaques. Surprisingly, antigen-specific CD8(+) T cells were found in the mucosal tissues of all immunized macaques regardless of whether the vaccine was administered by a mucosal route (intranasal or intrarectal) or systemically. In addition, following mucosal SIV(mac251) challenge, antigen-specific responses were mainly confined to mucosal tissues, again regardless of the route of immunization. We conclude that immunization with a live vector vaccine results in the appearance of CD8(+) T-cell responses at mucosal sites even when the vaccine is delivered by nonmucosal routes.     

Heterogeneity of the simian immunodeficiency virus (SIV) specific CD8(+) T-cell response in mucosal tissues during SIV primary infection

Virus-specific CD8(+) T cells play an important role in controlling viral replication during acute primary infection. At this early stage, mucosal tissues represent a major site of viral replication. Therefore, the presence of functional virus-specific CD8(+) effector T cells in the mucosa during primary infection is a key issue in the pathogenesis of infection. In order to evaluate the extent of this response, six rhesus macaques were infected with simian immunodeficiency virus (SIV)mac251 and sacrificed on day 28 following infection. The functional activity of SIV-effector CD8(+) T cells was evaluated by means of a gamma-IFN ELISpot assay with autologous cells expressing SIV env, gag, pol and nef genes as antigen-presenting cells. This evaluation was performed on PBMCs, spleen, peripheral lymph node, gut-associated lymph node and lamina propria lymphocytes isolated from different mucosal sites. In parallel, the cell-associated viral load was quantified in all these tissues. Five macaques had gamma-IFN SIV-specific CD8(+) T cells in PBMCs and/or lymph nodes. However, in these macaques, these CD8(+) T cells were only present in seven mucosal sites out of 24 tested (the lamina propria lymphocytes of the duodenum, jejunum, ileum and colon were evaluated separately for each animal), whereas they were detected in all corresponding gut-associated lymph nodes. In addition, the mean frequency of SIV-specific gamma-IFN-secreting CD8(+) T cells was 117 +/- 228 per 10(6) cells in the lamina propria vs. 958 +/- 1184 in gut associated lymph nodes (P = 0.001). No overall correlation was observed between the CD8(+) T-cell activity and the viral load: among the 17 mucosal sites in which the virus was isolated, no specific activity was detected in 13 sites. In conclusion, these data indicate that the frequencies of SIV-specific gamma-IFN-secreting CD8(+) T cells are low in the mucosa during early primary infection. This may be of importance with regard to the intense viral replication observed in the mucosa at this stage.     

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.     

Gastrointestinal epithelium is an early extrathymic site for increased prevalence of CD34(+) progenitor cells in contrast to the thymus during primary simian immunodeficiency virus infection

The objective of this study was to determine the effects of primary simian immunodeficiency virus (SIV) infection on the prevalence and phenotype of progenitor cells present in the gastrointestinal epithelia of SIV-infected rhesus macaques, a primate model for human immunodeficiency virus pathogenesis. The gastrointestinal epithelium was residence to progenitor cells expressing CD34 antigen, a subset of which also coexpressed Thy-1 and c-kit receptors, suggesting that the CD34(+) population in the intestine comprised a subpopulation of primitive precursors. Following experimental SIVmac251 infection, an early increase in the proportions of CD34(+) Thy-1(+) and CD34(+) c-kit+ progenitor cells was observed in the gastrointestinal epithelium. In contrast, the proportion of CD34(+) cells in the thymus declined during primary SIV infection, which was characterized by a decrease in the frequency of CD34(+) Thy-1(+) progenitor cells. A severe depletion in the frequency of CD4-committed CD34(+) progenitors was observed in the gastrointestinal epithelium 2 weeks after SIV infection which persisted even 4 weeks after infection. A coincident increase in the frequency of CD8- committed CD34(+) progenitor cells was observed during primary SIV infection. These results indicate that in contrast to the primary lymphoid organs such as the thymus, the gastrointestinal epithelium may be an early extrathymic site for the increased prevalence of both primitive and committed CD34(+) progenitor cells. The gastrointestinal epithelium may potentially play an important role in maintaining T-cell homeostasis in the intestinal mucosa during primary SIV infection.     

Capture and transfer of simian immunodeficiency virus by macaque dendritic cells is enhanced by DC-SIGN

Dendritic cells (DCs) are among the first cells encountered by human and simian immunodeficiency virus (HIV and SIV) following mucosal infection. Because these cells efficiently capture and transmit virus to T cells, they may play a major role in mediating HIV and SIV infection. Recently, a C-type lectin protein present on DCs, DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN), was shown to efficiently bind and present HIV and SIV to CD4(+), coreceptor-positive cells in trans. However, the significance of DC-SIGN for virus transmission and pathogenesis in vivo remains unclear. Because SIV infection of macaques may represent the best model to study the importance of DC-SIGN in HIV infection, we cloned and characterized pig-tailed macaque DC-SIGN and generated monoclonal antibodies (MAbs) against it. We demonstrate that, like human DC-SIGN, pig-tailed macaque DC-SIGN (ptDC-SIGN) is expressed on DCs and macrophages but not on monocytes, T cells, or B cells. Moderate levels of ptDC-SIGN expression were detected on the surface of DCs, and low-level expression was found on macrophages. Additionally, we show that ptDC-SIGN efficiently binds and transmits replication-competent SIVmne variants to CD4(+), coreceptor-positive cells. Moreover, transmission of virus between pig-tailed macaque DCs and CD4(+) T cells is largely ptDC-SIGN dependent. Interestingly, MAbs directed against ptDC-SIGN vary in the capacity to block transmission of different SIVmne variants. These data demonstrate that ptDC-SIGN plays a central role in transmitting virus from macaque DCs to T cells, and they suggest that SIVmne variants may differ in their interactions with ptDC-SIGN. Thus, SIVmne infection of pig-tailed macaques may provide an opportunity to investigate the significance of DC-SIGN in primate lentiviral infections.     

Increased macrophage infection upon subcutaneous inoculation of rhesus macaques with simian immunodeficiency virus-loaded dendritic cells or T cells but not with cell-free virus

Information on the establishment of immunodeficiency virus infection through transmission of infected cells is sparse. Dendritic cells (DCs) and T cells may be central to the onset and subsequent spread of infection following mucosal exposure. To directly investigate the consequences of virus being introduced by DCs or T cells, we reinjected ex vivo simian immunodeficiency virus (SIV)-loaded autologous immature DCs and T cells subcutaneously (s.c.) into healthy macaques. s.c. injection of cell-bound virus was used to mirror what may happen if virus-loaded cells pass through an epithelium or perhaps DCs and T cells that immediately entrap cell-free virus, having just crossed an epithelial barrier. Virus load in the plasma was monitored along with combined in situ hybridization and immunohistochemistry to identify the cells replicating virus in the lymphoid tissues. Both DCs and T cells transmitted infection after being pulsed with either wild-type or nef-defective (delta nef) SIVmac239. As seen in animals infected intravenously, replication of delta nef was attenuated compared to that of wild-type virus when introduced in either cell-bound form. Upon examination of the draining lymph nodes (LNs) during the first days of infection, virus-producing CD4(+) T cells predominated in control animals that received s.c. cell-free virus. In dramatic contrast, both SIV-positive macrophages and T cells were detected in the LNs of monkeys infected with cell-associated SIV. Therefore, although both cell-free and cell-associated viruses are infectious, the initial cells amplifying the virus differ. This may have important implications for the subsequent dissemination of infection and/or induction of antiretroviral immunity.     

Mucosal infection and vaccination against feline immunodeficiency virus.

Feline immunodeficiency virus (FIV) infection is a naturally occurring lentiviral infection of cats which progresses to immunodeficiency in a manner strikingly similar to that observed in HIV infection in man. The rectal and cervico-vaginal mucosae are common routes of transmission of HIV and it has been shown that the gastrointestinal tract is an important site of HIV infection and primary pathology. Although biting is the principle mode of transmission for FIV, we have shown that it is possible to reliably infect cats via both the rectal and vaginal routes. Using a biotin-streptavidin linked immunoperoxidase technique we have detected FIV core and envelope proteins in the colonic follicle associated epithelial cells, cells within the lymphoid follice and occasional cells in the lamina propria. Further, in the intestine we have detected FIV RNA and proviral DNA in epithelial cells, colonic lymphoid aggregates and isolated lamina propria cells. We have studied a group of asymptotic cats which have been rectally infected with FIV for 1 year or longer and shown an increase in the number of lamina propria CD8+ cells and greater levels of IL-2, IL-6, IL-10 and gamma-IFN mRNA. Since these cats remained clinically healthy these results might suggest that both local antibody and class I restricted cytotoxic lymphocytes (CTLs) may play a role in control of viral replication. We have investigated a range of vaccination regimes for their ability to generate responses which would protect from rectal challenge with virulent virus. Cats have been immunized with whole virus (FIV-pet, FIV-GLA-8), V3, V3MAP or C2 with cholera toxin (CT), or Quil A based adjuvants via rectal, intra-nasal, parenteral or targeted lymph node routes, and challenged rectally with ten mucosal cat infectious doses (MCID) of FIV-GLA-8. We have shown that the adjuvant effects of cholera toxin and Quil A are not influenced by the route of delivery (intraperitoneal (i.p.) versus rectal) with CT more effective in stimulating humoral and Quil A more effective in stimulating cellular responses to FIV antigens. However we have shown that, quantitatively, CT is more effective when used as an adjuvant via the intra-nasal than the rectal route. Recently, we have begun to investigate if the promising results obtained with targeted lymph node (TLN) vaccination in monkeys could be reproduced in the cat. We have shown that TLN was more effective than rectal immunisation in stimulating both humoral and proliferative responses. In a preliminary study we have also been able to detect FIV specific CTLs and have observed protection from rectal challenge in four out of four cats.     

Distinct expression patterns of CD69 in mucosal and systemic lymphoid tissues in primary SIV infection of rhesus macaques

Although the intestinal tract plays a major role in early human immunodeficiency virus (HIV) infection, the role of immune activation and viral replication in intestinal tissues is not completely understood. Further, increasing evidence suggests the early leukocyte activation antigen CD69 may be involved in the development or regulation of important T cell subsets, as well as a major regulatory molecule of immune responses. Using the simian immunodeficiency virus (SIV) rhesus macaque model, we compared expression of CD69 on T cells from the intestine, spleen, lymph nodes, and blood of normal and SIV-infected macaques throughout infection. In uninfected macaques, the majority of intestinal lamina propria CD4+ T cells had a memory (CD95+) phenotype and co-expressed CD69, and essentially all intestinal CCR5+ cells co-expressed CD69. In contrast, systemic lymphoid tissues had far fewer CD69+ T cells, and many had a naïve phenotype. Further, marked, selective depletion of intestinal CD4+CD69+ T cells occurred in early SIV infection, and this depletion persisted throughout infection. Markedly increased levels of CD8+CD69+ T cells were detected after SIV infection in virtually all tissues, including the intestine. Further, confocal microscopy demonstrated selective, productive infection of CD3+CD69+ T cells in the intestine in early infection. Combined, these results indicate CD69+CD4+ T cells are a major early target for viral infection, and their rapid loss by direct infection may have profound effects on intestinal immune regulation in HIV infected patients.     

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.     

Glycosylation of Simian Immunodeficiency Virus Influences Immune-Tissue Targeting during Primary Infection, Leading to Immunodeficiency or Viral Control

Glycans of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) play pivotal roles in modulating virus-target cell interactions. We have previously reported that, whereas SIVmac239 is pathogenic, its deglycosylated essentially nonpathogenic mutant (Δ5G) serves as a live-attenuated vaccine, although both replicate similarly during primary infection. These findings prompted us to determine whether such a polarized clinical outcome was due to differences in the immune tissues targeted by these viruses, where functionally and phenotypically different memory CD4(+) T cells reside. The results showed that Δ5G replicates in secondary lymphoid tissue (SLT) at 1- to 2-log-lower levels than SIVmac239, whereas SIVmac239-infected but not Δ5G-infected animals deplete CXCR3(+) CCR5(+) transitional memory (TrM) CD4(+) T cells. An early robust Δ5G replication was localized to small intestinal tissue, especially the lamina propria (effector site) rather than isolated lymphoid follicles (inductive site) and was associated with the induction and depletion of CCR6(+) CXCR3(-) CCR5(+) effector memory CD4(+) T cells. These results suggest that differential glycosylation of Env dictates the type of tissue-resident CD4(+) T cells that are targeted, which leads to pathogenic infection of TrM-Th1 cells in SLT and nonpathogenic infection of Th17 cells in the small intestine, respectively.     

Spatial alterations between CD4(+) T follicular helper, B, and CD8(+) T cells during simian immunodeficiency virus infection: T/B cell homeostasis, activation, and potential mechanism for viral escape

HIV/SIV infections induce chronic immune activation with remodeling of lymphoid architecture and hypergammaglobulinemia, although the mechanisms leading to such symptoms remain to be fully elucidated. Moreover, lymph nodes have been highlighted as a predilection site for SIV escape in vivo. Following 20 rhesus macaques infected with SIVmac239 as they progress from pre-infection to acute and chronic infection, we document for the first time, to our knowledge, the local dynamics of T follicular helper (T(FH)) cells and B cells in situ. Progression of SIV infection was accompanied by increased numbers of well-delineated follicles containing germinal centers (GCs) and T(FH) cells with a progressive increase in the density of programmed death-1 (PD-1) expression in lymph nodes. The rise in PD-1(+) T(FH) cells was followed by a substantial accumulation of Ki67(+) B cells within GCs. However, unlike in blood, major increases in the frequency of CD27(+) memory B cells were observed in lymph nodes, indicating increased turnover of these cells, correlated with increases in total and SIV specific Ab levels. Of importance, compared with T cell zones, GCs seemed to exclude CD8(+) T cells while harboring increasing numbers of CD4(+) T cells, many of which are positive for SIVgag, providing an environment particularly beneficial for virus replication and reservoirs. Our data highlight for the first time, to our knowledge, important spatial interactions of GC cell subsets during SIV infection, the capacity of lymphoid tissues to maintain stable relative levels of circulating B cell subsets, and a potential mechanism for viral reservoirs within GCs during SIV infection.     

Unique pathology in simian immunodeficiency virus-infected rapid progressor macaques is consistent with a pathogenesis distinct from that of classical AIDS

Simian immunodeficiency virus (SIV) infection of macaques and human immunodeficiency virus type 1 (HIV-1) infection of humans result in variable but generally fatal disease outcomes. Most SIV-infected macaques progress to AIDS over a period of 1 to 3 years, in the face of robust SIV-specific immune responses (conventional progressors [CP]). A small number of SIV-inoculated macaques mount transient immune responses and progress rapidly to AIDS (rapid progressors [RP]). We speculated that the underlying pathogenic mechanisms may differ between RP and CP macaques. We compared the pathological lesions, virus loads, and distribution of virus and target cells in SIVsmE660- or SIVsmE543-infected RP and CP rhesus macaques at terminal disease. RP macaques developed a wasting syndrome characterized by severe SIV enteropathy in the absence of opportunistic infections. In contrast, opportunistic infections were commonly observed in CP macaques. RP and CP macaques showed distinct patterns of CD4(+) T-cell depletion, with a selective loss of memory cells in RP macaques and a generalized (naive and memory) CD4 depletion in CP macaques. In situ hybridization demonstrated higher levels of virus expression in lymphoid tissues (P < 0.001) of RP macaques and a broader distribution to include many nonlymphoid tissues. Finally, SIV was preferentially expressed in macrophages in RP macaques whereas the primary target cells in CP macaques were T lymphocytes at end stage disease. These data suggest distinct pathogenic mechanisms leading to the deaths of these two groups of animals, with CP macaques being more representative of HIV-induced AIDS in humans.     

Simian immunodeficiency virus infection induces severe loss of intestinal central memory T cells which impairs CD4+ T-cell restoration during antiretroviral therapy

BACKGROUND: Simian immunodeficiency virus (SIV) infection leads to severe loss of intestinal CD4(+) T cells and, as compared to peripheral blood, restoration of these cells is slow during antiretroviral therapy (ART). Mechanisms for this delay have not been examined in context of which specific CD4(+) memory subsets or lost and fail to regenerate during ART. METHODS: Fifteen rhesus macaques were infected with SIV, five of which received ART (FTC/PMPA) for 30 weeks. Viral loads were measured by real-time PCR. Flow cytometric analysis determined changes in T-cell subsets and their proliferative state. RESULTS: Changes in proliferative CD4(+) memory subsets during infection accelerated their depletion. This reduced the central memory CD4(+) T-cell pool and contributed to slow CD4(+) T-cell restoration during ART. CONCLUSION: There was a lack of restoration of the CD4(+) central memory and effector memory T-cell subsets in gut-associated lymphoid tissue during ART, which may contribute to the altered intestinal T-cell homeostasis in SIV infection.     

Immunodeficiency in the absence of high viral load in pig-tailed macaques infected with Simian immunodeficiency virus SIVsun or SIVlhoest

Simian immunodeficiency virus (SIV) is known to result in an asymptomatic infection of its natural African monkey host. However, some SIV strains are capable of inducing AIDS-like symptoms and death upon experimental infection of Asian macaques. To further investigate the virulence of natural SIV isolates from African monkeys, pig-tailed (PT) macaques were inoculated intravenously with either of two recently discovered novel lentiviruses, SIVlhoest and SIVsun. Both viruses were apparently apathogenic in their natural hosts but caused immunodeficiency in PT macaques. Infection was characterized by a progressive loss of CD4(+) lymphocytes in the peripheral blood and lymph nodes, generalized lymphoid depletion, a wasting syndrome, and opportunistic infections, such as Mycobacterium avium or Pneumocystis carinii infections. However, unlike SIVsm/mac infection of macaques, SIVlhoest and SIVsun infections in PT macaques were not accompanied by high viral loads during the chronic disease stage. In addition, no significant correlation between the viral load at set point (12 weeks postinfection) and survival could be found. Five out of eight SIVlhoest-infected and three out of four SIVsun-infected macaques succumbed to AIDS during the first 5 years of infection. Thus, the survival of SIVsun- and SIVlhoest-infected animals was significantly longer than that of SIVagm- or SIVsm-infected macaques. All PT macaques maintained strong SIV antibody responses despite progression to SIV-induced AIDS. The development of immunodeficiency in the face of low viremia suggests that SIVlhoest and SIVsun infections of macaques may model unique aspects of the pathogenesis of human immunodeficiency virus infection in humans.     

nef gene is required for robust productive infection by simian immunodeficiency virus of T-cell-rich paracortex in lymph nodes

The pathogenesis of AIDS virus infection in a nonhuman primate AIDS model was studied by comparing plasma viral loads, CD4(+) T-cell subpopulations in peripheral blood mononuclear cells, and simian immunodeficiency virus (SIV) infection in lymph nodes for rhesus macaques infected with a pathogenic molecularly cloned SIVmac239 strain and those infected with its nef deletion mutant (Deltanef). In agreement with many reports, whereas SIVmac239 infection induced AIDS and depletion of memory CD4(+) T cells in 2 to 3 years postinfection (p.i.), Deltanef infection did not induce any manifestation associated with AIDS up to 6.5 years p.i. To explore the difference in SIV infection in lymphoid tissues, we biopsied lymph nodes at 2, 8, 72, and 82 weeks p.i. and analyzed them by pathological techniques. Maximal numbers of SIV-infected cells (SIV Gag(+), Env(+), and RNA(+)) were detected at 2 weeks p.i. in both the SIVmac239-infected animals and the Deltanef-infected animals. In the SIVmac239-infected animals, most of the infected cells were localized in the T-cell-rich paracortex, whereas in the Deltanef-infected animals, most were localized in B-cell-rich follicles and in the border region between the paracortex and the follicles. Analyses by double staining of CD68(+) macrophages and SIV Gag(+) cells and by double staining of CD3(+) T cells and SIV Env(+) cells revealed that SIV-infected cells were identified as CD4(+) T cells in either the SIVmac239 or the Deltanef infection. Whereas the many functions of Nef protein were reported from in vitro studies, our finding of SIVmac239 replication in the T-cell-rich paracortex in the lymph nodes supports the reported roles of Nef protein in T-cell activation and enhancement of viral infectivity. Furthermore, the abundance of SIVmac239 infection and the paucity of Deltanef infection in the T-cell-rich paracortex accounted for the differences in viral replication and pathogenicity between SIVmac239 and the Deltanef mutant. Thus, our in vivo study indicated that the nef gene enhances SIV replication by robust productive infection in memory CD4(+) T cells in the T-cell-rich region in lymphoid tissues.