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Although IL-15 has been implicated in the pathogenic hyperimmune activation that drives progressive HIV and SIV infection, as well as in the generation of HIV/SIV target cells, it also supports NK and T cell homeostasis and effector activity, potentially benefiting the host. To understand the role of IL-15 in SIV infection and pathogenesis, we treated two cohorts of SIVmac239-infected rhesus macaques (RM; Macaca mulatta), one with chronic infection, the other with primary infection, with a rhesusized, IL-15–neutralizing mAb (versus an IgG isotype control) for up to 10 wk (n = 7–9 RM per group). In both cohorts, anti–IL-15 was highly efficient at blocking IL-15 signaling in vivo, causing 1) profound depletion of NK cells in blood and tissues throughout the treatment period; 2) substantial, albeit transient, depletion of CD8+ effector memory T cells (TEM) (but not the naive and central memory subsets); and 3) CD4+ and CD8+ TEM hyperproliferation. In primary infection, reduced frequencies of SIV-specific effector T cells in an extralymphoid tissue site were also observed. Despite these effects, the kinetics and extent of SIV replication, CD4+ T cell depletion, and the onset of AIDS were comparable between anti–IL-15– and control-treated groups in both cohorts. However, RM treated with anti–IL-15 during primary infection manifested accelerated reactivation of RM rhadinovirus. Thus, IL-15 support of NK cell and TEM homeostasis does not play a demonstrable, nonredundant role in SIV replication or CD4+ T cell deletion dynamics but may contribute to immune control of oncogenic γ-herpesviruses.

Prophylactic vaccination of rhesus macaques with rhesus cytomegalovirus (RhCMV) vectors expressing simian immunodeficiency virus (SIV) antigens (RhCMV/SIV) elicits immune responses that stringently control highly pathogenic SIV infection, with subsequent apparent clearance of the infection, in ~50% of vaccinees. In contrast, here, we show that therapeutic RhCMV/SIV vaccination of rhesus macaques previously infected with SIV and given continuous combination antiretroviral therapy (cART) beginning 4–9 d post-SIV infection does not mediate measurable SIV reservoir clearance during over 600 d of follow-up on cART relative to RhCMV/control vaccination. However, none of the six animals started on cART on day four or five, across both RhCMV/SIV- and RhCMV/control-vaccinated groups, those rhesus macaques with SIV reservoirs most closely resembling those of prophylactically RhCMV/SIV-vaccinated and protected animals early in their course, showed post-cART viral rebound with up to nine months of follow-up. Moreover, at necropsy, these rhesus macaques showed little to no evidence of replication-competent SIV. These results suggest that the early SIV reservoir is limited in durability and that effective blockade of viral replication and spread in this critical time window by either pharmacologic or immunologic suppression may result in reduction, and potentially loss, of rebound-competent virus over a period of ~two years. Early and prolonged administration of antiretroviral therapy to SIV-infected and post-exposure-vaccinated rhesus macaques was associated with absence or delay of detectable virus after therapy interruption.

Chronic-phase HIV and simian immunodeficiency virus (SIV) replication is reduced by as much as 10,000-fold in elite controllers (ECs) compared with typical progressors (TPs), but sufficient viral replication persists in EC tissues to allow viral sequence evolution and induce excess immune activation. Here we show that productive SIV infection in rhesus monkey ECs, but not TPs, is markedly restricted to CD4(+) follicular helper T (TFH) cells, suggesting that these EC monkeys' highly effective SIV-specific CD8(+) T cells can effectively clear productive SIV infection from extrafollicular sites, but their relative exclusion from B cell follicles prevents their elimination of productively infected TFH cells. CD8(+) lymphocyte depletion in EC monkeys resulted in a dramatic re-distribution of productive SIV infection to non-TFH cells, with restriction of productive infection to TFH cells resuming upon CD8(+) T cell recovery. Thus, B cell follicles constitute 'sanctuaries' for persistent SIV replication in the presence of potent anti-viral CD8(+) T cell responses, potentially complicating efforts to cure HIV infection with therapeutic vaccination or T cell immunotherapy.

Memory CD4+ T cell homeostasis and AIDS progression are independent of naive CD4+ T cells in SIV infection of nonhuman primates., The development of AIDS in chronic HIV/simian immunodeficiency virus (SIV) infection has been closely linked to progressive failure of CD4+ memory T cell (TM) homeostasis. CD4+ naive T cells (TN) also decline in these infections, but their contribution to disease progression is less clear. We assessed the role of CD4+ TN in SIV pathogenesis using rhesus macaques (RMs) selectively and permanently depleted of CD4+ TN before SIV infection. CD4+ TN-depleted and CD4+ TN-repleted RMs were created by subjecting juvenile RMs to thymectomy versus sham surgery, respectively, followed by total CD4+ T cell depletion and recovery from this depletion. Although thymectomized and sham-treated RMs manifested comparable CD4+ TM recovery, only sham-treated RMs reconstituted CD4+ TN. CD4+ TN-depleted RMs responded to SIVmac239 infection with markedly attenuated SIV-specific CD4+ T cell responses, delayed SIVenv-specific Ab responses, and reduced SIV-specific CD8+ T cell responses. However, CD4+ TN-depleted and -repleted groups showed similar levels of SIV replication. Moreover, CD4+ TN deficiency had no significant effect on CD4+ TM homeostasis (either on or off anti-retroviral therapy) or disease progression. These data demonstrate that the CD4+ TN compartment is dispensable for CD4+ TM homeostasis in progressive SIV infection, and they confirm that CD4+ TM comprise a homeostatically independent compartment that is intrinsically capable of self-renewal.

Depletion of CD8(+) lymphocytes during acute simian immunodeficiency virus (SIV) infection of rhesus macaques (RMs) results in irreversible prolongation of peak-level viral replication and rapid disease progression, consistent with a major role for CD8(+) lymphocytes in determining postacute-phase viral replication set points. However, we report that CD8(+) lymphocyte depletion is also associated with a dramatic induction of proliferation among CD4(+) effector memory T (T(EM)) cells and, to a lesser extent, transitional memory T (T(TrM)) cells, raising the question of whether an increased availability of optimal (activated/proliferating), CD4(+)/CCR5(+) SIV "target" cells contributes to this accelerated pathogenesis. In keeping with this, depletion of CD8(+) lymphocytes in SIV(-) RMs led to a sustained increase in the number of potential CD4(+) SIV targets, whereas such depletion in acute SIV infection led to increased target cell consumption. However, we found that the excess CD4(+) T(EM) cell proliferation of CD8(+) lymphocyte-depleted, acutely SIV-infected RMs was completely inhibited by interleukin (IL)-15 neutralization, and that this inhibition did not abrogate the rapidly progressive infection in these RMs. Moreover, although administration of IL-15 during acute infection induced robust CD4(+) T(EM) and T(TrM) cell proliferation, it did not recapitulate the viral dynamics of CD8(+) lymphocyte depletion. These data suggest that CD8(+) lymphocyte function has a larger impact on the outcome of acute SIV infection than the number and/or activation status of target cells available for infection and viral production.

Primary simian immunodeficiency virus (SIV) infections of rhesus macaques result in the dramatic depletion of CD4(+) CCR5(+) effector-memory T (T(EM)) cells from extra-lymphoid effector sites, but in most infections, an increased rate of CD4(+) memory T cell proliferation appears to prevent collapse of effector site CD4(+) T(EM) cell populations and acute-phase AIDS. Eventually, persistent SIV replication results in chronic-phase AIDS, but the responsible mechanisms remain controversial. Here, we demonstrate that in the chronic phase of progressive SIV infection, effector site CD4(+) T(EM) cell populations manifest a slow, continuous decline, and that the degree of this depletion remains a highly significant correlate of late-onset AIDS. We further show that due to persistent immune activation, effector site CD4(+) T(EM) cells are predominantly short-lived, and that their homeostasis is strikingly dependent on the production of new CD4(+) T(EM) cells from central-memory T (T(CM)) cell precursors. The instability of effector site CD4(+) T(EM) cell populations over time was not explained by increasing destruction of these cells, but rather was attributable to progressive reduction in their production, secondary to decreasing numbers of CCR5(-) CD4(+) T(CM) cells. These data suggest that although CD4(+) T(EM) cell depletion is a proximate mechanism of immunodeficiency, the tempo of this depletion and the timing of disease onset are largely determined by destruction, failing production, and gradual decline of CD4(+) T(CM) cells.

The mechanisms linking human immunodeficiency virus replication to the progressive immunodeficiency of acquired immune deficiency syndrome are controversial, particularly the relative contribution of CD4+ T cell destruction. Here, we used the simian immunodeficiency virus (SIV) model to investigate the relationship between systemic CD4+ T cell dynamics and rapid disease progression. Of 18 rhesus macaques (RMs) infected with CCR5-tropic SIVmac239 (n = 14) or CXCR4-tropic SIVmac155T3 (n = 4), 4 of the former group manifested end-stage SIV disease by 200 d after infection. In SIVmac155T3 infections, naive CD4+ T cells were dramatically depleted, but this population was spared by SIVmac239, even in rapid progressors. In contrast, all SIVmac239-infected RMs demonstrated substantial systemic depletion of CD4+ memory T cells by day 28 after infection. Surprisingly, the extent of CD4+ memory T cell depletion was not, by itself, a strong predictor of rapid progression. However, in all RMs destined for stable infection, this depletion was countered by a striking increase in production of short-lived CD4+ memory T cells, many of which rapidly migrated to tissue. In all rapid progressors (P < 0.0001), production of these cells initiated but failed by day 42 of infection, and tissue delivery of new CD4+ memory T cells ceased. Thus, although profound depletion of tissue CD4+ memory T cells appeared to be a prerequisite for early pathogenesis, it was the inability to respond to this depletion with sustained production of tissue-homing CD4+ memory T cells that best distinguished rapid progressors, suggesting that mechanisms of the CD4+ memory T cell generation play a crucial role in maintaining immune homeostasis in stable SIV infection.

Retroviruses produced from the quail packaging cell line SE21Q1b predominantly contain cellular RNAs instead of viral RNAs. These RNAs can be reverse transcribed and integrated into the genomes of newly infected cells and are thereafter referred to as newly formed retrogenes. We investigated whether retrogene formation can occur within SE21Q1b cells themselves and whether this occurs intracellularly or via extracellular reinfection. By using packaging cell line mutants derived from the SE21Q1b provirus and selectable reporter constructs, we found that the process requires envelope glycoproteins and a retroviral packaging signal. Our results suggest that extracellular reinfection is the primary route of retrotransposition of nonviral RNAs.

Live attenuated simian immunodeficiency virus (SIV) vaccines (LAVs) remain the most efficacious of all vaccines in nonhuman primate models of HIV and AIDS, yet the basis of their robust protection remains poorly understood. Here we show that the degree of LAV-mediated protection against intravenous wild-type SIVmac239 challenge strongly correlates with the magnitude and function of SIV-specific, effector-differentiated T cells in the lymph node but not with the responses of such T cells in the blood or with other cellular, humoral and innate immune parameters. We found that maintenance of protective T cell responses is associated with persistent LAV replication in the lymph node, which occurs almost exclusively in follicular helper T cells. Thus, effective LAVs maintain lymphoid tissue-based, effector-differentiated, SIV-specific T cells that intercept and suppress early wild-type SIV amplification and, if present in sufficient frequencies, can completely control and perhaps clear infection, an observation that provides a rationale for the development of safe, persistent vectors that can elicit and maintain such responses.

The rhesus macaque (RM) is a critical animal model for studies of viral pathogenesis and immunity, yet fundamental aspects of their cellular immune response remain poorly defined. One such deficiency is the lack of validated phenotypic signatures for their naive and memory T cell subsets, and the resultant unavailability of accurate information on their memory T cell development, homeostasis, and function. In this study, we report a phenotypic paradigm allowing definitive characterization of these subsets and their comprehensive functional analysis. Naive T cells are optimally delineated by their homogeneous CD95lowCD28highβ7 integrinint (CD4+) or CD95lowCD28intCD11alow (CD8+) phenotypes. This subset 1) was present in blood and secondary lymph tissues, but not effector sites; 2) vastly predominated in the fetal/neonatal immune system, but rapidly diminished with postnatal age; 3) lacked IFN-γ production capability, and specific responses to RM CMV; and 4) demonstrated low in vivo proliferative activity. CD4+ and CD8+ memory subsets were CD95high, but otherwise phenotypically heterogeneous and included all IFN-γ production, RM CMV-specific responses, effector site T cells, and demonstrated high in vivo proliferative activity (∼10 times the naive subset). These analyses also revealed the RM “effector memory” subset within the overall memory population. This population, best defined by lack of CD28 expression, contained the majority of RM CMV-specific cells, was highly enriched in extralymphoid effector sites, and comprised an increasing proportion of total memory cells with age. The effector memory subset demonstrated similar in vivo proliferative activity and survival as CD28+ “central memory” T cells, consistent with independent homeostatic regulation.

A chicken glyceraldehyde 3-phosphate dehydrogenase (GAPDH) processed pseudogene was identified by inverse PCR using oligonucleotide primers specific for the 5′ region of the GAPDH mRNA. Molecular cloning and sequence analysis of this genomic sequence shows that the processed pseudogene is incomplete and arranged in a permuted tail-to-head order. We propose that the tail-to-head organization is the result of circularization and breakage of a GAPDH retrogene prior to chromosomal integration. PCR analysis of DNAs from quail, pheasant, and various jungle fowl, shows that the processed pseudogene was formed after the three genera diverged but prior to Gallus speciation. This is the first report of a chicken GAPDH processed pseudogene sequence. This is also the first published report of a processed pseudogene with a tail-to-head organization.

The avian packaging cell line SE21Q1b produces particles which encapsidate cellular RNAs. Such RNAs can be reverse transcribed by endogenous polymerase and integrated into the genomes of newly infected cells (M. Linial, Cell 49:93-102, 1987). Genomic RNA is not packaged because the packaging (psi) region of the provirus is deleted. The provirus also lacks the negative-strand primer binding site, which prevents efficient reverse transcription of randomly packaged genomic RNA. Previous work from our laboratory suggested that the trans-acting defect which allows packaging of cellular mRNA mapped to the provirus but did not map to the nucleocapsid region of the gag gene (D.J. Anderson, P. Lee, K. L. Levine, J. Sang, S. A. Shah, O. O. Yang, P. R. Shank, and M. L. Linial, J. Virol. 66:204-216, 1992). We have found, using proviral recombinants between SE21Q1b and wild-type Rous sarcoma virus, that packaging of cellular RNAs does not map to the gag gene. Rather, the propensity of SE21Q1b particles to package cellular mRNA is a function of the high level of particle production in these cells and not of any specific viral structural proteins.

Cloning and sequencing of Tripneustes gratilla genomic DNA and cDNA encoding a developmentally regulated, embryonic messenger RNA, referred to as Tg616, revealed an actin-encoding gene orthologous to the CyI actin gene described from Strongylocentrotus purpuratus. Tg616 and SpCyI share: (1) 150 nucleotides of highly conserved sequence 5′ of the transcription start site, (2) 95% nucleotide sequence identity in the protein encoding regions, which specify identical amino acid residues in 375 of 377 positions, and (3) extensive nucleotide sequence identity in the 3′ untranslated region of their messenger RNAs. Tg616 was therefore designated TgCyI. In situ hybridization shows sequential activation of TgCyI in various cells of the embryo. TgCyI mRNA becomes abundant in primary and secondary mesenchyme cells as they prepare to enter the blastocoel, in prospective aboral ectoderm cells at blastula stage, in gut cells during gut differentiation, and in oral ectoderm at pluteus stage. This pattern of embryonic gene expression is more complex than any of the major patterns of developmentally upregulated genes observed in S. purpuratus embryos and is distinct from SpCyI expression which is progressively restricted to the gut and oral ectoderm.

Using a previously cloned, developmentally regulated mRNA sequence expressed predominantly in the endoderm of sea urchin pluteus larvae, we isolated genomic clones and additional cDNA clones to define the gene and the protein it encodes. Nucleic acid sequencing revealed that the gene consists of four exons interrupted by three introns and spans approximately 3600 bp. It encodes a low-molecular-weight protein with polar ends. A stretch of Glu and Asp residues at its carboxyl terminus suggests that it is a nucleic acid-binding protein and a stretch of four Lys residues near the amino terminus suggests a nuclear localization signal.