Recombinant Yellow Fever Vaccine Virus 17D Expressing Simian Immunodeficiency Virus SIVmac239 Gag Induces SIV-Specific CD8+ T-Cell Responses in Rhesus Macaques

Here we describe a novel vaccine vector for expressing human immunodeficiency virus (HIV) antigens. We show that recombinant attenuated yellow fever vaccine virus 17D expressing simian immunodeficiency virus SIVmac239 Gag sequences can be used as a vector to generate SIV-specific CD8⁺ T-cell responses in the rhesus macaque. Priming with recombinant BCG expressing SIV antigens increased the frequency of these SIV-specific CD8⁺ T-cell responses after recombinant YF17D boosting. These recombinant YF17D-induced SIV-specific CD8⁺ T cells secreted several cytokines, were largely effector memory T cells, and suppressed viral replication in CD4⁺ T cells.None of the vaccine regimens tested in human immunodeficiency virus (HIV) vaccine efficacy trials to date have either reduced the rate of HIV infection or reduced the level of HIV replication. Structural features and the enormous variability of the envelope glycoprotein have frustrated efforts to induce broadly reactive neutralizing antibodies against HIV (10). Investigators have therefore focused their attention on T-cell-based vaccines (40). Simian immunodeficiency virus (SIV) challenge of rhesus macaques vaccinated with T-cell-based vaccines has shown that it is possible to control virus replication after SIV infection (22, 41, 42). The recent STEP trial of a recombinant Ad5-vectored vaccine was widely seen as an important test of this concept (http://www.hvtn.org/media/pr/step111307.html) (9, 25). Unfortunately, vaccinees became infected at higher rates than the controls (9). While it is still not clear what caused the enhanced infection rate in the vaccinated group, future Ad5-based human vaccine trials may be difficult to justify. We therefore need to develop new vaccine vectors for delivering SIV and HIV genes. Several other viral vectors currently under consideration include nonreplicating adenovirus (Ad)-based vectors (1, 21, 22), Venezuelan equine encephalitis (VEE) virus (12, 20), adeno-associated virus (AAV) (19), modified vaccinia virus Ankara (MVA) (3, 4, 13, 15, 18, 38), NYVAC (6), cytomegalovirus (CMV) (16), and replicating Ad (30). However, only a few of these have shown promise in monkey trials using rigorous SIV challenges.We explored whether the small (11-kb) yellow fever vaccine flavivirus 17D (YF17D) might be a suitable vector for HIV vaccines. The YF17D vaccine is inexpensive, production and quality control protocols already exist, and it disseminates widely in vivo after a single dose (27). Importantly, methods for the manipulation of the YF17D genome were recently established (7, 8, 24, 28). This effective vaccine has been safely used on >400 million people in the last 70 years (27). Additionally, the YF17D strain elicits robust CD8⁺ T-cell responses in humans (26). Chimeric YF17D is presently being developed as a vaccine for other flaviviruses, such as Japanese encephalitis virus (28), dengue virus (14), and West Nile virus (29). Inserts expressing a malaria B-cell epitope have been engineered into the E protein of YF17D (7). In murine models, recombinant YF17D viruses have generated robust and specific responses to engineered antigens inserted between the 2B and NS3 proteins in vivo (24, 35).We first used the YF17D vaccine virus to infect four Mamu-A*01-positive macaques. The vaccine virus replicated in these four animals and induced neutralizing antibodies in all four macaques by 2 weeks postvaccination (Fig. 1A and B). To monitor the CD8⁺ T-cell immune response against YF17D, we scanned its proteome for peptides that might bind to Mamu-A*01 using the major histocompatibility complex (MHC) pathway algorithm (31). We synthesized the 52 YF17D-derived peptides most likely to bind to Mamu-A*01 based on their predicted affinity for this MHC class I molecule. We then used a gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) assay to screen these peptides in YF17D-immunized animals at several time points after vaccination and discovered that four Mamu-A*01-binding peptides, LTPVTMAEV (LV9_1285-1293), VSPGNGWMI (VI9_3250-3258), MSPKGISRM (MM9_2179-2187), and TTPFGQQRVF (TF10_2853-2862), were recognized in vivo (Fig. ​(Fig.1C).1C). Using a previously reported protocol (26), we also observed CD8⁺ T-cell activation in all four animals (Fig. 1D and E). Thus, as was observed previously, the YF17D vaccine virus replicates in Indian rhesus monkeys (36) and induces neutralizing antibodies, yellow fever 17D-specific Mamu-A*01-restricted CD8⁺ T-cell responses, and CD8⁺ T-cell activation.Open in a separate windowFIG. 1.YF17D replicates and induces neutralizing antibodies, virus-specific CD8⁺ T cells, and the activation of CD8⁺ T cells in rhesus macaques. (A) Replication of YF17D during the first 10 days after vaccination with two different doses, as measured by quantitative PCR (Q-PCR) using the following primers: forward primer YF-17D 10188 (5′-GCGGATCACTGATTGGAATGAC-3′), reverse primer YF-17D 10264 (5′-CGTTCGGATACGATGGATGACTA-3′), and probe 6-carboxyfluorescein (6Fam)-5′-AATAGGGCCACCTGGGCCTCCC-3′-6-carboxytetramethylrhodamine (TamraQ). (B) Titer of neutralizing antibodies determined at 2 and 5 weeks after YF17D vaccination. (C) Fresh PBMC from vaccinees (100,000 cells/well) were used in IFN-γ ELISPOT assays (41) to assess T-cell responses against YF17D. We used 4 epitopes (LTPVTMAEV [LV9_1285-1293], VSPGNGWMI [VI9_3250-3258], MSPKGISRM [MM9_2179-2187], and TTPFGQQRVF [TF10_2853-2862]) predicted to bind to Mamu-A*01 as defined by the MHC pathway algorithm (31). All IFN-γ ELISPOT results were considered positive if they were ≥50 SFC/10⁶ PBMC and ≥2 standard deviations over the background. (D) Identification of activated CD8⁺ T cells after vaccination with YF17D based on the expression of the proliferation and proapoptotic markers Ki-67 and Bcl-2, respectively (26). We stained whole blood cells with antibodies against CD3 and CD8. We then permeabilized and subsequently labeled these cells with Bcl-2- and Ki-67-specific antibodies. The flow graphs were gated on CD3⁺ CD8⁺ lymphocytes. (E) Expression kinetics of Ki-67 and Bcl-2 in CD8⁺ T cells after vaccination with YF17D.We next engineered the YF17D vaccine virus to express amino acids 45 to 269 of SIVmac239 Gag (rYF17D/SIVGag_45-269) by inserting a yellow fever codon-optimized sequence between the genes encoding the viral proteins E and NS1. This recombinant virus replicated and induced neutralizing antibodies in mice (data not shown). We then tested the rYF17D/SIVGag_45-269 construct in six Mamu-A*01-positive Indian rhesus macaques. We found evidence for the viral replication of rYF17D/SIVGag_45-269 for five of these six macaques (Fig. ​(Fig.2A).2A). However, neutralizing antibodies were evident for all six animals at 2 weeks postvaccination (Fig. ​(Fig.2B).2B). Furthermore, all animals developed SIV-specific CD8⁺ T cells after a single immunization with rYF17D/SIVGag_45-269 (Fig. ​(Fig.2C).2C). To test whether a second dose of this vaccine could boost virus-specific T-cell responses, we administered rYF17D/SIVGag_45-269 (2.0 × 10⁵ PFU) to four macaques on day 28 after the first immunization and monitored cellular immune responses. With the exception of animal r04091, the rYF17D/SIVGag_45-269 boost did not increase the frequency of the vaccine-induced T-cell responses. This recombinant vaccine virus also induced CD8⁺ T-cell activation in the majority of the vaccinated animals (Fig. ​(Fig.2D2D).Open in a separate windowFIG. 2.rYF17D/SIVGag_45-269 replicates and induces neutralizing antibodies, virus-specific CD8⁺ T cells, and the activation of CD8⁺ T cells in rhesus macaques. (A) Replication of rYF17D/SIVGag_45-269 during the first 10 days after vaccination with two different doses as measured by Q-PCR using the YF17D-specific primers described in the legend of Fig. ​Fig.1.1. (B) Titer of neutralizing antibodies determined at 2 and 5 weeks after rYF17D/SIVGag_45-269 vaccination. The low levels of neutralization for animal r02013 were observed in three separate assays. (C) Fresh PBMC from vaccinees (100,000 cells/well) were used in IFN-γ ELISPOT assays to assess T-cell responses against the YF17D vector (red) and the SIV Gag(45-269) insert (black) at several time points postvaccination. We measured YF17D-specific responses using the same epitopes described in the legend of Fig. ​Fig.1.1. For SIV Gag-specific responses, we used 6 pools of 15-mers overlapping by 11 amino acids spanning the entire length of the SIVmac239 Gag insert. In addition, we measured Mamu-A*01-restricted responses against the dominant Gag_181-189CM9 and subdominant Gag_254-262QI9 epitopes. Four animals received a second dose of rYF17D/SIVGag_45-269 on day 28 after the first vaccination (dashed line). (D) Expression kinetics of Ki-67 and Bcl-2 in CD8⁺ T cells after vaccination with rYF17D/SIVGag_45-269. This assay was performed as described in the legend of Fig. ​Fig.11.We could not detect differences in vaccine-induced immune responses between the group of animals vaccinated with YF17D and the group vaccinated with rYF17D/SIVGag_45-269. There was, however, considerable animal-to-animal variability. Animal r02034, which was vaccinated with YF17D, exhibited massive CD8⁺ T-cell activation (a peak of 35% at day 14) (Fig. ​(Fig.1E),1E), which was probably induced by the high levels of viral replication (16,800 copies/ml at day 5) (Fig. ​(Fig.1A).1A). It was difficult to see differences between the neutralizing antibody responses induced by YF17D and those induced by rYF17D/SIVGag_45-269 (Fig. ​(Fig.1B1B and ​and2B).2B). However, neutralizing antibodies in animal r02013 decreased by 5 weeks postvaccination. It was also difficult to detect differences in the YF17D-specific CD8⁺ T-cell responses induced by these two vaccines. Peak Mamu-A*01-restricted CD8⁺ T-cell responses against YF17D ranged from barely detectable (animal r02110 at day 11) (Fig. ​(Fig.1C)1C) to 265 spot-forming cells (SFCs)/10⁶ peripheral blood mononuclear cells (PBMC) (animal r02034 at day 28) (Fig. ​(Fig.1C).1C). Similarly, three of the rYF17D/SIVGag_45-269-vaccinated animals (animals r04091, r04051, and r02013) made low-frequency CD8⁺ T-cell responses against the Mamu-A*01-bound YF17D peptides, whereas the other three animals (animals r03130, r02049, and r02042) recognized these epitopes with responses ranging from 50 to 200 SFCs/10⁶ PBMC (Fig. ​(Fig.2C).2C). For almost every rYF17D/SIVGag_45-269-vaccinated animal, the Gag_181-189CM9-specific responses (range, 50 to 750 SFCs/10⁶ PBMC) were higher than those generated against the Mamu-A*01-restricted YF17D epitopes (range, 0 to 175 SFCs/10⁶ PBMC), suggesting that the recombinant virus replicated stably in vivo (Fig. ​(Fig.2C).2C). Thus, the recombinant YF17D virus replicated and induced both virus-specific neutralizing antibodies and CD8⁺ T cells that were not demonstrably different from those induced by YF17D alone.Most viral vectors are usually more efficient after a prime with DNA or recombinant BCG (rBCG) (4, 11, 15, 18). We therefore used rYF17D/SIVGag_45-269 to boost two macaques that had been primed with rBCG expressing SIV proteins (Fig. ​(Fig.3A).3A). We detected no SIV-specific responses after either of the two priming rBCG vaccinations. Unfortunately, while the recombinant YF17D virus replicated well in animal r01056, we found evidence for only low levels of replication of rYF17D/SIVGag_45-269 on day 5 postvaccination for animal r01108 (7 copies/ml) (Fig. ​(Fig.3B).3B). Both animals, however, generated neutralizing antibodies at 2 weeks postvaccination (Fig. ​(Fig.3C).3C). Encouragingly, we detected high-frequency CD8⁺ T-cell responses in the Mamu-A*01-positive macaque (animal r01056) after boosting with rYF17D/SIVGag_45-269 (Fig. 3D to F). These responses were directed mainly against the Mamu-A*01-restricted Gag_181-189CM9 epitope, which is contained in the peptide pool Gag E (Fig. ​(Fig.3D).3D). Furthermore, the boost induced a massive activation of animal r01056''s CD8⁺ T cells, peaking at 35% at 17 days postvaccination (Fig. ​(Fig.3E).3E). Of these activated CD8⁺ T cells, approximately 10% were directed against the Gag_181-189CM9 epitope, with a frequency of 3.5% of CD8⁺ T cells (Fig. ​(Fig.3E).3E). These epitope-specific CD8⁺ T cells made IFN-γ, tumor necrosis factor alpha (TNF-α), macrophage inflammatory protein 1β (MIP-1β), and degranulated (Fig. ​(Fig.3F3F and data not shown). Thus, an rBCG prime followed by a recombinant yellow fever 17D boost induced polyfunctional antigen-specific CD8⁺ T cells.Open in a separate windowFIG. 3.rYF17D/SIVGag_45-269 vaccination induced a robust expansion of Gag-specific responses in an rBCG-primed macaque. (A) Vaccination scheme. We immunized two rhesus macaques with rBCG intradermally (i.d.) (2.0 × 10⁵ CFU), rBCG orally (10⁷ CFU), and rYF17D/SIVGag_45-269 subcutaneously (2.0 × 10⁵ PFU) at 6-month intervals. rBCG was engineered to express 18 minigenes containing sequences of Gag, Vif, Nef, Rev, and Tat from SIVmac239. (B) Replication of rYF17D/SIVGag_45-269 during the first 10 days after vaccination as measured by Q-PCR using the YF17D-specific primers described in the legend of Fig. ​Fig.1.1. (C) Titer of neutralizing antibodies determined at 2 and 5 weeks after rYF17D/SIVGag_45-269 vaccination. (D) Fresh PBMC from animal r01056 (100,000 cells/well) were used in IFN-γ ELISPOT assays to assess T-cell responses against the YF17D vector (red) and the SIV Gag(45-269) insert (black) at several time points postvaccination. (E) Kinetics of CD8⁺ T-cell activation (as described in the legend of Fig. ​Fig.1)1) and expansion of Gag_181-189CM9-specific CD8⁺ T cells in animal r01056 after vaccination with rYF17D/SIVGag_45-269. (F) Vaccination with rYF17D/SIVGag_45-269 induced robust CD8⁺ T-cell responses against Gag_181-189CM9 in r01056. CD8⁺ T-cell activation (Ki-67⁺/Bcl-2⁻) for baseline and day 13 are shown. Gag_181-189CM9-specific responses were measured by tetramer staining and intracellular cytokine staining (ICS) with antibodies against MIP-1β and IFN-γ.Vaccine-induced CD8⁺ T cells are usually central memory T cells (T_CM) or effector memory T cells (T_EM). These two subsets of CD8⁺ T cells differ in function and surface markers (23). Repeated boosting drives CD8⁺ T cells toward the T_EM subset (23). We therefore determined whether a rBCG prime followed by a rYF17D/SIVGag_45-269 boost induced T_CM or T_EM CD8⁺ T cells. Staining of PBMC obtained on day 30 postvaccination revealed that the SIV-specific CD8⁺ T cells were largely T_EM cells since the majority of them were CD28 negative (Fig. ​(Fig.4A).4A). Furthermore, these cells persisted with the same phenotype until day 60 after vaccination (Fig. ​(Fig.4B).4B). It was recently suggested that T_EM cells residing in the mucosae can effectively control infection after a low-dose challenge with SIVmac239 (16).Open in a separate windowFIG. 4.rYF17D/SIVGag_45-269 vaccination of animal r01056 induced effector memory Gag_181-189CM9-specific CD8⁺ T cells that suppressed viral replication in CD4⁺ targets. (A and B) Frequency and memory phenotype of tetramer-positive Gag_181-189-specific CD8⁺ T cells in animal r01056 on day 30 (A) and day 60 (B) after rYF17D/SIVGag_45-269 vaccination. CD28 and CD95 expression profiles of tetramer-positive cells show a polarized effector memory phenotype. Cells were gated on CD3⁺ CD8⁺ lymphocytes. (C) Ex vivo Gag_181-189CM9-specific CD8⁺ T cells from animal r01056 inhibit viral replication from SIVmac239-infected CD4⁺ T cells. Gag_181-189CM9-specific CD8⁺ T cells from three SIV-infected Mamu-A*01-positive animals and rYF17D/SIVGag_45-269-vaccinated animal r01056 were tested for their ability to suppress viral replication from SIV-infected CD4⁺ T cells (39). Forty-eight hours after the incubation of various ratios of SIV-infected CD4⁺ T cells and Gag_181-189CM9-specific CD8⁺ T cells, the supernatant was removed and measured for viral RNA (vRNA) copies per ml by Q-PCR. We observed no suppression when effectors were incubated with CD4⁺ targets from Mamu-A*01-negative animals (data not shown). Animal rh2029 was infected with SIVmac239 (viral load, ∼10⁵ vRNA copies/ml) containing mutations in 8 Mamu-B*08-restricted epitopes as part of another study (37). Animal r01080 was vaccinated with a DNA/Ad5 regimen expressing Gag, Rev, Tat, and Nef and later infected with SIVmac239 (viral load, ∼10³ vRNA copies/ml) (42). Animal r95061 was vaccinated with a DNA/MVA regimen containing Gag_181-189CM9 and was later challenged with SIVmac239 (undetectable viral load) (2).We then assessed whether rYF17D/SIVGag_45-269-induced CD8⁺ T cells could recognize virally infected CD4⁺ T cells. We have shown that these vaccine-induced CD8⁺ T cells stain for tetramers and produce cytokines after stimulation with synthetic peptides (Fig. ​(Fig.3).3). None of these assays, however, tested whether these SIV-specific CD8⁺ T cells recognize SIV-infected cells and reduce viral replication. We therefore used a newly developed assay (39) to determine whether vaccine-induced CD8⁺ T cells can reduce viral replication in CD4⁺ T cells. We sorted tetramer-positive (Gag_181-189CM9-specific) lymphocytes directly from fresh PBMC and incubated them for 48 h with SIVmac239-infected CD4⁺ T cells expressing Mamu-A*01. We assessed the percentage of CD4⁺ T cells that expressed SIV Gag p27 (data not shown) and the quantity of virus in the culture supernatant (Fig. ​(Fig.4C).4C). Vaccine-induced CD8⁺ T cells reduced viral replication to the same extent as that seen with Gag_181-189CM9-specific CD8⁺ T cells purified from three SIVmac239-infected rhesus macaques, including an elite controller rhesus macaque, animal r95061 (Fig. ​(Fig.4C4C).The most encouraging aspect of this study is that rBCG primed a high-frequency CD8⁺ T-cell response after boosting with rYF17D/SIVGag_45-269. These CD8⁺ T cells reached frequencies that were similar to those induced by an rBCG prime followed by an Ad5 boost (11). Even without the benefit of the rBCG prime, the levels of CD8⁺ T cells induced by a single rYF17D/SIVGag_45-269 vaccination were equivalent to those induced by our best SIV vaccine, SIVmac239ΔNef. Recombinant YF17D generated an average of 195 SFCs/10⁶ PBMC (range, 100 to 750 SFCs/10⁶ PBMC) (n = 6), whereas SIVmac239ΔNef induced an average of 238 SFCs/10⁶ PBMC (range, 150 to 320 SFCs/10⁶ PBMC) (n = 3) (32). It is also possible that any YF17D/HIV recombinants would likely replicate better in humans than they have in rhesus macaques and thus induce more robust immune responses. Also, rBCG was shown previously to be effective in humans (5, 17, 33, 34) and may be more useful at priming T-cell responses in humans than it has been in our limited study with rhesus macaques. These two vectors have long-distinguished safety and efficacy histories in humans and may therefore be well suited for HIV vaccine development.     

TLR4 Ligands Augment Antigen-Specific CD8+ T Lymphocyte Responses Elicited by a Viral Vaccine Vector

Toll-like receptor (TLR) ligands are critical activators of innate immunity and are being developed as vaccine adjuvants. However, their utility in conjunction with viral vector-based vaccines remains unclear. In this study, we evaluated the impact of a variety of TLR ligands on antigen-specific CD8⁺ T lymphocyte responses elicited by a recombinant adenovirus serotype 26 (rAd26) vector expressing simian immunodeficiency virus Gag in mice. The TLR3 ligand poly(I:C) suppressed Gag-specific cellular immune responses, whereas the TLR4 ligands lipopolysaccharide and monophosphoryl lipid A substantially augmented the magnitude and functionality of these responses by a MyD88- and TRIF-dependent mechanism. These data demonstrate that TLR ligands can modulate the immunogenicity of viral vaccine vectors both positively and negatively. Moreover, these findings suggest the potential utility of TLR4 ligands as adjuvants for rAd vector-based vaccines.Toll-like receptors (TLRs) are critical sensors of infection with a fundamental role in the activation of innate immune responses and the subsequent modulation of pathogen-specific adaptive immunity (2). TLR ligands have therefore emerged as potential vaccine adjuvants, particularly in the context of peptide, protein, and DNA vaccines (17). In particular, TLR agonists are widely reported to modulate antibody and T helper lymphocyte responses, and in some cases CD8⁺ T lymphocyte responses, elicited by protein-based vaccines (5, 19, 33, 41). However, far less is known about the impact of TLR ligands on the immunogenicity of viral vector-based vaccines.Compared with DNA vaccines, viral vectors are typically more immunogenic, presumably as a result of the activation of innate immunity via multiple TLRs or other pattern recognition receptors (29). Viral vectors elicit robust T lymphocyte responses and thus are attractive vaccine candidates for pathogens such as human immunodeficiency virus type 1 (HIV-1) and malaria (10). Whether the addition of exogenous TLR agonists might further enhance the immunogenicity of viral vectors, however, remains unclear. The few studies that have explored the utility of TLR adjuvants with viral vectors have typically shown no or mild enhancement of antibody and T lymphocyte responses (7, 26). We therefore sought to determine systematically whether TLR ligands can modulate cellular immune responses elicited by a recombinant adenovirus serotype 26 (rAd26) vector in mice.C57BL/6 mice (n = 7 to 8/group) were immunized with a single injection of 3 × 10⁸ viral particles (vp) rAd26-Gag alone or combined with various TLR ligands (1). Vectors were mixed with soluble TLR agonists 1 h prior to intramuscular (i.m.) injection into both quadriceps muscles. Cellular immune responses were assessed by D^b/AL11 tetramer binding assays (3, 6), gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) assays (6), and multiparameter intracellular cytokine staining (ICS) assays (14). As shown in Fig. ​Fig.11 A, immunization with rAd26-Gag plus either 20 μg Pam3CSK (TLR1/2 ligand) (25), 20 μg Pam2CSK (TLR2/6 ligand) (9, 20), 10 μg flagellin (TLR5 ligand) (5, 8), 100 μg CLO97 (TLR7 ligand) (41), or 40 μg CpG (TLR9 ligand) (40) (all obtained from InvivoGen, San Diego, CA) elicited AL11-specific tetramer-positive responses (3, 6) that were similar to those detected in the unadjuvanted groups.Open in a separate windowFIG. 1.Antigen-specific CD8⁺ T cell responses elicited by rAd26-Gag are modulated by soluble TLR ligands. (A) C57BL/6 mice (n = 7 to 8 mice/group) were immunized once with 3 × 10⁸ vp rAd26-Gag alone or 3 × 10⁸ vp rAd26-Gag combined with the following TLR ligands: 20 μg synthetic triacylated lipoprotein (Pam3CSK; TLR1/2 ligand), 20 μg synthetic diacylated lipoprotein (Pam2CSK; TLR 2/6 ligand), 100 μg poly(I:C) (TLR3 ligand), 10 μg LPS (TLR4 ligand), 10 μg flagellin (TLR5 ligand), 100 μg CLO97 (TLR7 ligand), or 40 μg unmethylated CpG-oligodeoxynucleotides (CpG; TLR9 ligand). Gag-specific cellular immune responses were assayed by D^b/AL11 tetramer binding assays at multiple time points following injection. (B) At week 4 following immunization, functional immune responses from mice immunized with rAd26 vaccine alone or with 10 μg LPS or 100 μg poly(I:C) were assessed by IFN-γ ELISPOT assays in response to pooled Gag peptides, the CD8⁺ T lymphocyte epitopes AL11 and KV9, and the CD4⁺ T lymphocyte epitope DD13. (C) Assessment of the dose response of LPS (10 μg, 2 μg, 0.4 μg) and poly(I:C) (100 μg, 20 μg, 4 μg) with rAd26-Gag (n = 4 mice/group) by D^b/AL11 tetramer binding assays. (D) Mice were immunized once i.m. with 3 × 10⁸ vp rAd26-Gag alone, rAd26-Gag with 2 μg LPS, or rAd26-Gag with 20 μg poly(I:C) (n = 4 to 8 mice/group), and Gag-specific CD8⁺ T cell responses in splenocytes were assessed 4 weeks after vaccination by intracellular cytokine assays for IFN-γ, TNF-α, IL-2, and CD107. Responses to pooled Gag peptides are presented for each individual combination of functions and collated as the number of functions elaborated as a percent of total CD8⁺ T lymphocytes (insert; bar graph) and as the fraction of Gag-specific CD8⁺ T lymphocytes (insert; pie charts). Mean responses with standard errors are shown (*, P < 0.001; **, P < 0.05; two-tailed t test).The TLR3 ligand poly(I:C) (InvivoGen, San Diego, CA), however, markedly suppressed responses to the rAd26-Gag vaccine (Fig. ​(Fig.1A).1A). This finding contrasts with prior reports demonstrating its adjuvanticity for protein antigen vaccines (22, 34, 37). By day 28, mice that received the vaccine plus 100 μg poly(I:C) developed Gag-specific CD8⁺ T lymphocyte responses that were significantly lower (1.7%) than those of mice that received the vaccine alone (5.4%; P < 0.001; two-tailed t test). Similarly, IFN-γ ELISPOT responses in mice that received poly(I:C) were lower than those observed in the unadjuvanted group (Fig. ​(Fig.1B)1B) (6). In a dose response study (Fig. ​(Fig.1C),1C), 100-μg, 20-μg, and 4-μg doses of poly(I:C) all resulted in diminished tetramer-positive responses.In contrast, the TLR4 ligand lipopolysaccharide (LPS) (Ultrapure LPS from Escherichia coli 0111:B4; InvivoGen, San Diego, CA) substantially enhanced Gag-specific CD8⁺ T lymphocyte responses elicited by the rAd26-Gag vaccine (Fig. ​(Fig.1A).1A). At day 28, tetramer-positive responses in mice that received the vaccine plus 10 μg LPS (9.6%) were significantly higher than those in the unadjuvanted group (5.4%; P = 0.04). Moreover, IFN-γ ELISPOT responses (6, 21) to pooled Gag peptides, the CD8⁺ T lymphocyte epitopes AL11 and KV9, and the CD4⁺ T lymphocyte epitope DD13 were greater in mice that received the vaccine with LPS than in mice that received the vaccine alone at week 4 after immunization (P = 0.02) (Fig. ​(Fig.1B).1B). To further quantify this effect, mice were immunized once i.m. (n = 4 mice/group) with rAd26-Gag with various doses of LPS (10 μg, 2 μg, 0.4 μg). Tetramer-positive responses were enhanced by 10 μg and 2 μg LPS but not by 0.4 μg LPS (Fig. ​(Fig.1C),1C), indicating that this LPS effect was dose dependent. No overt clinical toxicities were observed by using these doses of LPS in mice.We next evaluated the functionality of CD8⁺ T lymphocyte responses by multiparameter ICS assays that assessed IFN-γ, tumor necrosis factor alpha (TNF-α), interleukin-2 (IL-2), and the cytotoxic degranulation marker CD107 expression at week 4 following immunization with rAd26-Gag alone, rAd26-Gag with 2 μg LPS, or rAd26-Gag with 20 μg poly(I:C) (n = 4 to 8 mice/group) (15). As shown in Fig. ​Fig.1D,1D, the addition of LPS significantly enhanced not only the overall magnitude of Gag-specific CD8⁺ T lymphocyte responses (P = 0.04) but also the fraction of Gag-specific CD8⁺ T lymphocytes that expressed two or more effector functions (P = 0.04). In particular, the LPS-adjuvanted group induced higher levels of single-function CD107⁺, 2-function TNF-α⁺ CD107⁺, as well as 3-function IFN-γ⁺ TNF-α⁺ CD107⁺ CD8⁺ T lymphocytes than mice that received rAd26-Gag alone. These data show that LPS enhanced both the magnitude and functionality of antigen-specific cellular responses elicited by rAd26-Gag. In contrast, the addition of poly(I:C) diminished both the overall magnitude of Gag-specific responses and the fraction of these responses that were multifunctional.We further characterized the opposing effects of poly(I:C) and LPS by administering the rAd26-Gag vaccine with both poly(I:C) and LPS. C57BL/6 mice (n = 4 mice/group) were immunized with a single injection of rAd26-Gag alone or with 10 μg LPS, 60 μg poly(I:C), or both TLR ligands. As shown in Fig. ​Fig.22 A, administration of both TLR ligands resulted in reduced Gag-specific responses, suggesting that the suppressive effect of poly(I:C) was dominant over the enhancing effect of LPS. To determine the durability of the effects of poly(I:C) and LPS, C57BL/6 mice were primed with rAd26-Gag alone or with 2 μg LPS or 20 μg poly(I:C) (n = 4 mice/group) and were boosted on day 35 with a single i.m. injection of the heterologous vector rAd5HVR48(1-7) also expressing simian immunodeficiency virus (SIV) Gag (32). As shown in Fig. ​Fig.2B,2B, the mice that received poly(I:C) with the priming immunization responded to the boosting immunization with Gag-specific responses that were comparable to those observed in the mice that received rAd26-Gag alone. In contrast, mice that received LPS with the priming immunization exhibited sustained enhanced Gag-specific tetramer and ELISPOT responses, demonstrating the proliferative potential of antigen-specific CD8⁺ T lymphocytes elicited by the LPS-adjuvanted rAd26-Gag vaccine.Open in a separate windowFIG. 2.Dominant suppressive effect of poly(I:C) over LPS with the rAd26-Gag vaccine. (A) Mice were immunized once i.m. with 3 × 10⁸ vp rAd26-Gag alone or with 20 μg poly(I:C), 2 μg LPS, or both poly(I:C) and LPS (n = 4 mice/group). Gag-specific CD8⁺ T lymphocyte responses were assessed by D^b/AL11 tetramer binding assays and IFN-γ ELISPOT assays 4 weeks after immunization. (B) Mice were primed once with 3 × 10⁸ vp rAd26-Gag alone or with 2 μg LPS or 20 μg poly(I:C) and then boosted (↓) with 3 × 10⁸ vp rAd5HVR48(1-7) at week 5. Gag-specific cellular immune responses were assessed by D^b/AL11 tetramer binding assays and by IFN-γ ELISPOT responses at week 4 postboost. Mean responses with standard errors are shown.We next investigated whether the mechanism underlying the immunomodulatory effects of LPS and poly(I:C) involved the expected TLR signaling pathways. Although LPS and poly(I:C) are chiefly considered TLR ligands, poly(I:C) can also signal through the intracellular sensor MDA-5 (14), and both LPS and poly(I:C) may activate inflammasomes through Nalp3 (12, 28). To explore whether the effects of LPS and poly(I:C) involved TLR signaling, we utilized C57BL/6 mice lacking TRIF (Jackson Laboratory, Bar Harbor, ME), which is utilized by TLR3, or C57BL/6 mice lacking MyD88 (provided by S. Akira and B. Pulendran), which is utilized by the majority of TLRs. In particular, TLR4 signals through both TRIF and MyD88. Wild-type, MyD88^−/−, and TRIF^−/− mice (n = 4 mice/group) were immunized with rAd26-Gag vaccine alone or with 2 μg LPS or 20 μg poly(I:C). As shown in Fig. ​Fig.3,3, the adjuvant activity of LPS was abrogated in both MyD88^−/− and TRIF^−/− mice (Fig. 3A and B), suggesting that the adjuvanticity of the TLR4 ligand LPS was dependent on both MyD88 and TRIF, as expected. In contrast, the suppressive effect of poly(I:C) was observed in MyD88^−/− mice but not in TRIF^−/− mice (Fig. 3A and B), indicating that the suppressive effect of the TLR3 ligand poly(I:C) was dependent on TRIF, rather than MDA-5 or nonspecific effects (14, 39). These data confirm that the immunomodulatory effects of LPS and poly(I:C) were dependent on the expected TLR signaling pathways.Open in a separate windowFIG. 3.The immunomodulatory effects of poly(I:C) and LPS are TLR dependent. MyD88−/− and TRIF−/− mice (n = 4 mice/group) were immunized once i.m. with 3 × 10⁸ vp rAd26-Gag alone or with 2 μg LPS or 20 μg poly(I:C). (A) D^b/AL11 tetramer binding assays were performed at multiple time points following injection, and (B) IFN-γ ELISPOT responses were assessed 4 weeks after immunization. Mean responses with standard errors are shown.LPS is not a likely adjuvant for clinical development as a result of its toxicities, and alternative TLR4 ligands have been developed for potential clinical use. In particular, monophosphoryl lipid A (MPLA) is an LPS derivative that retains the immunologically active lipid A portion of the parent molecule (23, 27). The reduced toxicity of MPLA is attributed to the preferential recruitment of TRIF upon TLR4 activation, resulting in decreased induction of inflammatory cytokines (18). To determine if MPLA can similarly adjuvant cellular immune responses elicited by rAd26-Gag, C57BL/6 mice were immunized with 3 × 10⁷, 3 × 10⁸, or 3 × 10⁹ vp rAd26-Gag alone or with 5 μg MPLA (derived from Salmonella enterica serovar Minnesota R595 LPS; InvivoGen, San Diego, CA) (n = 4 mice/group). This optimal dose of MPLA was selected by dose response studies (data not shown). As shown in Fig. ​Fig.44 A, Gag-specific IFN-γ ELISPOT responses to the lowest dose of vector were essentially undetectable in the unadjuvanted group, consistent with prior observations (1). In contrast, clear responses were observed in the mice that received 3 × 10⁷ vp rAd26-Gag with MPLA (P < 0.01; two-tailed t test). Mice that received the 3 × 10⁸ vp and 3 × 10⁹ vp doses of rAd26-Gag with MPLA also exhibited higher Gag-specific cellular immune responses than the unadjuvanted groups (P < 0.01). Functionality of these Gag-specific CD8⁺ T lymphocyte responses, as measured by multiparameter ICS assays assessing IFN-γ, TNF-α, IL-2, and CD107 expression, was also greater in mice that received rAd26-Gag with MPLA compared with rAd26-Gag (P < 0.05 for the lowest dose group) (Fig. ​(Fig.4B).4B). Thus, the TLR4 ligand MPLA also augmented antigen-specific CD8⁺ T lymphocyte responses elicited by rAd26-Gag.Open in a separate windowFIG. 4.The TLR4 ligand MPLA augments the immunogenicity of rAd26-Gag. C57BL/6 mice (n = 4 mice/group) were immunized once i.m. with 3 × 10⁷, 3 × 10⁸, or 3 × 10⁹ vp rAd26-Gag with or without 5 μg MPLA. Gag-specific cellular immune responses were assessed 4 weeks after immunization by IFN-γ ELISPOT responses (*, P < 0.01 for responses to pooled Gag peptides; two-tailed t test) (A) and by ICS for IFN-γ, TNF-α, IL-2, and CD107 (B). Responses to pooled Gag peptides in mice immunized with 3 × 10⁷ vp rAd26-Gag with or without 5 μg MPLA are presented for each individual combination of functions and collated as the number of functions as a fraction of the total Gag-specific CD8⁺ T lymphocyte response (insert; pie charts) (**, P < 0.05). (C) Cytokine levels were measured in sera of mice 8 h after immunization with 3 × 10⁸ vp rAd26-Gag alone or 3 × 10⁸ vp rAd26-Gag with 5 μg MPLA or 2 μg LPS (n = 4 mice/group). Mean responses with standard errors are shown.To explore differences in acute inflammatory responses following MPLA and LPS administration, serum levels of IL-1α, IL-6, granulocyte colony-stimulating factor (G-CSF), and IP-10 were assessed 8 h after vaccination in duplicate using multiplexed fluorescent bead-based immunoassays (Millipore, Billerica, MA) and analyzed on the Luminex 100 IS (Luminex, Austin, TX). As shown in Fig. ​Fig.4C,4C, mice that received MPLA had lower levels of the MyD88-associated acute proinflammatory cytokines IL-1α and IL-6 than mice that received LPS, as expected. Levels of IP-10 and G-CSF, which are associated with TRIF activation (18), were comparable (Fig. ​(Fig.4B).4B). These data confirm that MPLA resulted in lower levels of systemic inflammatory cytokine secretion than LPS.Optimization of the immunogenicity of viral vectors is an important research priority. However, there have been few reports addressing the potential use of adjuvants together with viral vectors. Combining alum with rAd35 elicited improved antibody responses to a malaria antigen (24), and the addition of TLR9 agonists (CpGs) resulted in paradoxically diminished immune responses elicited by a rAd5 vector but improved protection against a cancer antigen (13). Most recently, Appledorn et al. reported enhanced antigen-specific T lymphocyte responses with the coadministration of a rAd vector engineered to express a novel TLR5 agonist (4). Our study extends these findings and represents the first systematic investigation of the capacity of a panel of soluble TLR ligands to modulate rAd-elicited CD8⁺ T lymphocyte responses.The TLR agonists that modulated vaccine-elicited immune responses in this study included poly(I:C), LPS, and MPLA. These ligands have all been reported to augment CD8⁺ T lymphocyte responses elicited by peptide or protein vaccines (11, 22, 31, 33, 42), presumably through enhanced cross-presentation (34, 35). TLR signaling has been shown to be important for virus-elicited CD8⁺ T lymphocyte responses (38), often through activation of multiple TLRs or other pattern recognition receptors (30). The activation of TLR4 by LPS or MPLA with a viral vector most likely provides an additive or synergistic signal, probably resulting in enhanced APC maturation in the appropriate cytokine milieu. Moreover, immunization of the viral vector and LPS at different sites abrogated the observed adjuvanticity (data not shown), indicating that TLR4 adjuvanticity involves a local mechanism of action. However, the mechanism by which a TLR3 agonist suppresses immunogenicity of a viral vector remains unclear. It is possible that the high levels of type I interferon elicited by poly(I:C) (data not shown) may limit expression from the rAd26 vector. Alternatively, poly(I:C) has been reported to elicit IL-10 secretion, and this suppressive cytokine may limit CD8⁺ T cell proliferation (22, 36). The unexpected suppressive activity of poly(I:C) illustrates the inherent complexity of viral vectors compared to protein-based vaccines (16, 37).Our data demonstrate that antigen-specific CD8⁺ T lymphocyte responses elicited by a rAd26-Gag vaccine vector can be both positively and negatively modulated by soluble TLR ligands, and the mechanism underlying these observations involves the expected TRIF and MyD88 signaling pathways. In particular, the TLR4 ligands LPS and MPLA substantially augmented the magnitude and functionality of antigen-specific cellular immune responses elicited by this vaccine vector. These findings suggest that TLR ligands, particularly MPLA, deserve further exploration as potential adjuvants to improve the immunogenicity and protective efficacy of viral vaccine vectors.     

Effective Downregulation of HLA-A*2 and HLA-B*57 by Primary Human Immunodeficiency Virus Type 1 Isolates Cultured from Elite Suppressors

Elite controllers or suppressors (ES) are human immunodeficiency virus type 1 (HIV-1)-infected patients who control viral replication to <50 copies/ml without antiretroviral therapy. Downregulation of HLA class I molecules is an important mechanism used by HIV-1 to evade the immune system. In this study, we showed that primary isolates from ES are as effective as isolates obtained from patients with progressive HIV-1 disease at downregulating HLA-A*2 and HLA-B*57 molecules on primary CD4⁺ T cells. Thus, a diminished ability of viral isolates from ES to evade HIV-specific immune responses probably does not contribute to the control of viral replication in these patients.Long-term nonprogressors (LTNP) are human immunodeficiency virus type 1 (HIV-1)-infected individuals who maintain normal CD4⁺ T-cell counts and remain asymptomatic for longer than 10 years without therapy (7). Although many LTNP have detectable levels of HIV-1 RNA in their plasma, patients known as elite suppressors (ES) have viral loads of <50 RNA copies/ml. Understanding the factors involved in the maintenance of LTNP and ES statuses may be critical for the development of effective vaccines and immunotherapeutic treatments. One such factor under investigation is the role of cytotoxic-T-lymphocyte (CTL) responses. Several studies have shown that the HLA-B*27 and -B*57 alleles are overrepresented in cohorts of ES (13, 16, 19, 28, 29, 34). These findings suggest important roles for major histocompatibility complex class I (MHC-I) restriction and CD8⁺ T-cell responses in the control of viremia. Indeed, multiple studies have documented qualitatively superior CD8⁺ T cell function in ES compared to that in chronic HIV progressors (CP) (2, 5, 12, 27, 28, 37, 47).Other studies suggest that some ES and LTNP are infected with attenuated viruses. One illustrative example comes from studies done on the Sydney Blood Bank Cohort, in which an LTNP donor transmitted an HIV-1 isolate with a large deletion in nef and the U3 region of the long terminal repeat to multiple recipients, all of whom became LTNP (11, 21). As in the Sydney Blood Bank Cohort studies, several other investigators have detected viruses with defective nef genes in LTNP and ES (1, 8, 18, 23, 25, 35, 36, 38, 43). In contrast, other studies showed that CD4⁺ T cells from ES could produce Gag when they were stimulated in vitro (20, 26), and full-length sequence analyses of plasma and proviral genomes revealed no evidence of significant deletions (30). Recent studies have suggested that plasma isolates (31) and replication-competent viruses (32) from HLA-B*57/B58*01 ES and LTNP, respectively, are less fit than isolates from B*57/B*5801 CP, but the difference in fitness observed is unlikely to fully explain the control of viral replication in these patients. Furthermore, we recently performed detailed genotypic and phenotypic analyses of replication-competent viruses isolated from ES and showed that these viruses were fully replication competent (6) Although nef is not required for viral replication in vitro, it has been strongly associated with pathogenesis in vivo (reviewed in reference 14). It is thus possible that some ES isolates are replication competent but have mutations in nef that result in diminished pathogenesis.nef has been shown to be involved in the downregulation of both CD4 (15) and MHC-I (41). Several studies have shown that nef-induced MHC-I downregulation has a major impact on CTL function. In a seminal study, a dramatic reduction in HLA-A*2 expression by CD4⁺ T cells infected with wild-type virus but not by those infected with a virus carrying a defective nef gene was demonstrated. This downregulation resulted in diminished killing of HIV-1-infected cells by CTL clones specific for an HLA-A*2-restricted HIV-1 Gag epitope (10). Similarly, nef-mediated MHC-I downregulation was shown to impair the ability of HIV-1-specific CTL clones to suppress viral replication (42, 44). While these findings strongly suggest that HIV-1 partially evades the immune response by inducing MHC-I downregulation, other studies have demonstrated that primary CD8⁺ T cells from some ES and CP could effectively respond to autologous viral replication in autologous CD4⁺ T cells (26).We tested the hypothesis that ES are infected with HIV-1 isolates that are less capable of downregulating MHC-I molecules. This could potentially cause the isolates to be more susceptible to CD8⁺ T-cell suppression of replication and may explain the superior CD8⁺ T-cell responses reported in prior ES studies (2, 5, 12, 27, 28, 37, 47). To date, fully characterized replication-competent isolates have been reported from just six ES subjects (1, 3, 6). We compared the MHC-I downregulation capacity of isolates from five of these ES to that of isolates obtained from resting CD4⁺ T cells of eight patients with progressive disease (viral load, >10,000 copies/ml). In order to develop a physiological model for HIV-1-induced MHC-I downregulation, we enriched primary CD4⁺ T cells from peripheral blood mononuclear cells (PBMC) from donors who were HLA-A*2 and/or HLA-B*57 positive by CD8⁺ T cell depletion with magnetic beads (Dynal), followed by activation in vitro with phytohemagglutinin for 3 days. For evaluation of HLA-A*2 downregulation, CD4⁺ T cells were obtained from HIV-seronegative donors. CD4⁺ T cells from ES were used for the evaluation of HLA-B*57 downregulation. This allele was as effectively downregulated in these ES as it was in multiple HLA-B*57 CP (data not shown). Following activation, the cells were infected with primary HIV-1 isolates from ES or CP by spinoculation (33). The primary isolates were obtained as previously described from latently infected CD4⁺ T cells (9). The median peak viral load and CD4⁺ T-cell nadir of the CP from whom viral isolates were obtained was 81,000 copies/ml and 279 cells/μl, respectively, and thus these isolates should be effective at HLA downregulation (22).At different time points, the cells were harvested and stained with either fluorescein isothiocyanate (FITC)-conjugated anti-HLA-A*2 (Becton Dickinson) and tricolor-conjugated anti-CD4 antibodies (Caltag) or biotinylated anti-HLA-B*57 antibody (One Lambda) followed by FITC-conjugated streptavidin, peridinin chlorophyll protein-Cy5.5-conjugated anti-CD4 antibody (Becton Dickenson), and allophycocyanin-conjugated anti-CD3 antibody. The cells were fixed and permeabilized with Cytofix/Cytoperm solution (Becton Dickenson). Intracellular staining was then performed with the phycoerythrin-conjugated Gag-specific monoclonal antibody Kc57 or an immunoglobulin G1 mouse isotype control (Beckman Coulter). A total of 100,000 to 500,000 events were analyzed for each sample. HLA typing of ES was performed as previously described (4). The HLA-specific antibodies were tested on cells from a panel of ES with known HLA types to confirm specificity.MHC-I downregulation was measured by comparing the mean fluorescence intensities (MFI) of HLA-A*2 and HLA-B*57 on HIV-1-infected versus noninfected CD4⁺ T cells. Infected cells were defined as cells that stained positive for intracellular Gag and had downregulated CD4 (Fig. ​(Fig.1).1). Uninfected CD4⁺ T cells were defined as cells that expressed high levels of CD4 and were negative for intracellular Gag protein. In order to standardize values, we determined relative MFI by dividing the MFI of the infected population by that of the CD4-positive, uninfected population. The Wilcoxon Mann-Whitney test was used to analyze the data.Open in a separate windowFIG. 1.Analysis of HLA-B*57 downregulation on HIV-1-infected cells. (A) CD8⁺ T-cell-depleted PBMC were stained with anti-HLA-B*57 and anti-CD4 monoclonal antibodies 3 days after infection with primary isolates from an ES (ES8) or a CP (CP2). Cells in quadrant 1 are uninfected CD4⁺ T cells, and cells in quadrant 4 (Gag-positive, low levels of CD4) are infected cells that have downregulated CD4. (PE, phycoerythrin; IgG, immunoglobulin G.) (B) The MFI of HLA-B*57 were compared for uninfected (quadrant 1) and infected (quadrant 4) cells from each sample.To determine if there was a difference in the ability of HIV-1 isolates cultured from ES versus CP to downregulate HLA-A*2, we measured the MFI of this molecule on infected CD4⁺ T cells that had downregulated CD4. On average, primary CD4⁺ T cells infected by ES viruses had levels of MHC-I downregulation of about two- to threefold, with relative MFI of 0.51, 0.37, and 0.30 on days 2, 3, and 4, respectively. Similarly, cells infected by isolates cultured from CP had relative MFI of 0.46, 0.36, and 0.33 on days 2, 3, and 4, respectively (Fig. ​(Fig.2B).2B). These differences were not significantly different at any time point.Open in a separate windowFIG. 2.(A) Relative MFIs of HLA-A*02 on cells infected with isolates from five ES (triangles) and eight CP (squares) on days 2 to 4 postinfection. The relative MFI is defined as the MFI of the infected cells divided by the MFI of the uninfected CD4⁺ T cells in each sample. The horizontal bars represent the median for each group. (B) Average relative MFI of HLA-A*02 for cells infected with isolates from ES and CP on each day. (C) Average relative MFI of HLA-A*02 for cells infected with the wild-type NL4-3-green fluorescent protein virus (diamonds) or the Nef⁻ Vpr⁻ mutant virus (circles).In order to rule out nonspecific downregulation of MHC-I on infected cells, we determined the MFI of HLA-DR and CD45 RO on cells infected with isolates from two subjects. The average relative MFI of the two proteins were 1.28 and 1.48, respectively, indicating that the MHC-I was in fact specifically downregulated. Since mutations in Nef have been shown to abrograte HLA downregulation, we also compared HLA-A2 downregulation by the HIV-1-based reporter construct NL4-3-green fluorescent protein and a Nef⁻ Vpr⁻ mutant vector (45, 46). As shown in Fig. ​Fig.2C,2C, no downregulation of HLA-A2 was seen at any point after infection with the Nef⁻ Vpr⁻ mutant virus, whereas infection with wild-type virus caused a degree of downregulation that was similar to that seen with primary isolates from ES and CP. Finally, we also looked at CD3 downregulation, as this molecule has been shown to be downregulated by Nef from HIV-2 and many simian immunodeficiency virus (SIV) isolates but not from HIV-1 (39). Furthermore, since SIVsmm nef isolated from sooty mangabeys with preserved CD4⁺ T-cell counts causes significantly more downregulation than SIVsmm nef from sooty mangabeys with CD4⁺ T-cell depletion (40), we determined whether isolates from ES also selectively downregulated this molecule. As shown in Fig. ​Fig.3A,3A, there was no significant downmodulation of CD3 after infection of cells with isolates from ES or CP.Open in a separate windowFIG. 3.(A) Relative MFI of CD3 on cells infected with isolates from five ES (triangles) and five CP (squares) on day 3 postinfection. The horizontal bars represent the median for each group. (B) Relative MFI of HLA-B*57 on cells infected with isolates from ES and CP.Epidemiologic studies have suggested that HLA-B alleles play a larger role than HLA-A alleles in determining the outcome of infection (17). Furthermore, while HLA-B*57 is the most overrepresented allele seen in ES, there have not been any studies looking at downregulation of this MHC-I protein. Activated CD4⁺ T cells from an HLA-B*5703-positive ES were infected with isolates from five ES and five CP, and the degree of HLA-B*57 downregulation was measured on day 3. As shown in Fig. ​Fig.3B,3B, the average relative MFI of cells infected with isolates from five ES was 0.53, which was not significantly different from the average relative MFI of 0.64 that was seen in cells infected with isolates from five progressors.While it appeared that there was generally more downregulation of HLA-A*2 than HLA-B*57, the studies were performed in cells from different donors, and this precluded a direct comparison of the MFI of the two MHC-I alleles. Two ES in our cohort were positive for both HLA alleles, and the degrees of downregulation of these proteins could thus be compared. CD4⁺ T cells from ES8 were infected with autologous virus (6), and cells from ES9 were infected with a primary isolate from the CP who transmitted virus to her (3). For patient ES8, HLA-A2 showed a greater degree of downregulation than HLA-B57 at day 3 (a relative MFI of 0.36 versus 0.62) (Fig. ​(Fig.4).4). In contrast, in ES9 the degrees of downregulation of the two proteins were nearly identical (a relative MFI of 0.35 for HLA-A2 versus 0.31 for HLA-B*57).Open in a separate windowFIG. 4.Comparison of the relative MFI of HLA-A*02 and HLA-B*57 on CD8⁺ T-cell-depleted PBMC from ES8 and ES9 that were infected with autologous virus (ES8) or with the primary isolate from the CP who transmitted the virus to ES9. The MFI of HLA-A*2 or HLA-B*57 on uninfected CD4⁺ T cells (top panels) and infected cells that had downregulated CD4 (bottom panels) are shown.This is the first study to look at downregulation of MHC-I proteins on CD4⁺ T cells infected with HIV-1 isolates cultured from ES CD4⁺ T cells. We used a physiological model where primary CD4⁺ T cells were infected with primary HIV-1 isolates. One advantage of this approach is that it accounts for HLA downregulation mediated by viral proteins such as Tat (24), as well as Nef. Similar amounts of MHC-I downregulation were seen for cells infected with replication-competent isolates cultured from ES and progressors. These results demonstrate that most ES are not infected by HIV-1 virions that are deficient in downregulating MHC-I compared to those of CP. Thus, it is likely that other factors enable ES to control viremia. The identification of these factors will have implications for the design of HIV-1 vaccines.     

Murine Norovirus Infection Has No Significant Effect on Adaptive Immunity to Vaccinia Virus or Influenza A Virus

Murine norovirus (MNV) is endemic in many research mouse colonies. Although MNV infections are typically asymptomatic in immunocompetent mice, the effects of MNV infection on subsequent experimental viral infections are poorly documented. Here, we infected C57BL/6 mice with MNV and then with either vaccinia virus or influenza A virus. MNV infection had no effect on CD8⁺ T-cell or antibody responses to secondary viruses or to secondary virus-induced morbidity or mortality. While our findings suggest that MNV has little influence on host immunity in immunocompetent mice, we would urge caution regarding the potential effects of MNV on immune responses to viruses and other pathogens, which must be determined on a system-by-system basis.Human norovirus (NoV) infections cause greater than 90% of nonbacterial gastroenteritis cases (4, 5) and are an important public health concern. Murine noroviruses (MNV) were recently identified (7) as highly pathogenic agents in immunocompromised mice, and serological studies indicate that over 20% of mice in research colonies are exposed to MNV (6). As with NoV, MNV is spread through the fecal-oral route. While NoV rapidly causes gastrointestinal symptoms and fever in healthy individuals, MNV is typically asymptomatic in immunocompetent mice.MNV isolates are both genetically and biologically diverse (13). In wild-type (wt) mice, some strains of MNV are rapidly cleared, while others persist (13). Controlling MNV infections requires elements of both innate and adaptive immunity. Mice with defects in interferon (IFN) signaling pathways demonstrate increased MNV lethality (7, 9). CD4⁺ and CD8⁺ T cells and B cells are all needed for complete MNV clearance (1, 2). Natural exposure of immunocompromised mice to MNV leads to inflammation of the liver, lungs, and peritoneal and pleural cavities (14).It is well established that infection with natural mouse viruses can greatly impact immune responses to infections with other viruses. The prevalence of MNV in research mouse colonies might therefore lead to irreproducible and variable results that significantly impact research efforts. Indeed, MNV was recently reported to alter disease progression in a mouse model of bacterium-induced inflammatory bowel disease (8). Concern over the potential effects of MNV on viral immunology research prompted a dedicated workshop at the 2008 Keystone Viral Immunity meeting (http://www.keystonesymposia.org). In the present study, we examined the effect of MNV infection on adaptive immune responses in wt mice to influenza A virus (IAV) and vaccinia virus (VV).We infected C57BL/6 mice perorally with a high dose (3 × 10⁷ PFU/mouse) of a plaque-purified MNV stock derived from MNV-CR6p2 (13). The capacity of this plaque-purified virus to persist in wt mice has been confirmed by quantitative PCR analysis and a plaque assay (D. Strong, L. Thackray, and H. Virgin, unpublished observation). We confirmed that the mice were infected by measuring anti-MNV antibodies (Abs) by using an enzyme-linked immunosorbent assay (ELISA) (data not shown). For all experiments, mice were infected with MNV at Washington University and shipped 4 to 5 days later to NIAID for further study. To contain MNV, infected mice were housed in microisolator cages in a quarantine room. In some experiments, control mice were housed in the same room as MNV-infected mice. Sera collected from control mice did not contain anti-MNV Abs as determined by ELISA (data not shown), confirming that transmission of MNV between mice housed in microisolator cages can be prevented by proper cage changing and aseptic handling of samples from infected mice.Upon intraperitoneal (i.p.) infection with either VV or IAV, mice mount robust CD8⁺ T-cell responses that peak, respectively, on day 6 or 7. Anti-VV and anti-IAV CD8⁺ T-cell responses in C57BL/6 mice conform to a well-established immunodominance hierarchy (3, 10). To determine to what extent MNV infection alters the magnitude and/or immunodominance hierarchy of CD8⁺ T-cell responses, we infected C57BL/6 mice i.p. with either VV or IAV 19 days following MNV infection. As controls, naïve mice (MNV negative) were infected with either virus. Lymphocytes were isolated from mice 6 days postinfection with VV and 7 days postinfection with IAV. The fraction of antigen-specific CD8⁺ T cells present in spleen and peritoneal exudate cells (PEC) was determined by intracellular IFN-γ staining after stimulation with synthetic peptides. MNV infection had little effect on the magnitude of splenic or PEC CD8⁺ T cells responding to VV (Fig. 1A and B) or IAV (Fig. 1C and D) infection. Regardless of MNV exposure history, splenic and PEC responses were dominated by B8R- and A8R-specific CD8⁺ T cells following VV infection (Fig. 1A and B) and by PA-specific and NP-specific CD8⁺ T cells following IAV infection (Fig. 1C and D).Open in a separate windowFIG. 1.MNV exposure does not alter CD8⁺ T-cell responses to VV or IAV. MNV-infected and naïve C57BL/6 mice were infected i.p. with ∼1 × 10⁶ PFU of VV (A and B) or ∼1 × 10⁷ 50% tissue culture infective dose units of IAV (C and D), and specific CD8⁺ T cells were determined by intracellular IFN-γ staining after restimulating lymphocytes with peptides. Lymphocytes isolated from the spleen (A and C) and peritoneal cavity (B and D) were tested. MNV infections were completed 19 days prior to VV or IAV infections. Means and SEM are shown in panels A and C. A two-way analysis of variance and Bonferroni statistical analysis were completed for these experiments. Cells were pooled for peritoneal lavage samples as shown in panels B and D. Four to five mice/group were used for each experiment; data are representative of two independent experiments.To examine the effect of MNV infection on antiviral Ab responses, MNV-infected and control C57BL/6 mice were infected intranasally (i.n.) with a sublethal dose of either VV or IAV. Three weeks later, levels of anti-VV and anti-IAV Abs were determined by ELISA and hemagglutination inhibition assays, respectively. MNV infection did not significantly modify the magnitude of Ab responses to VV (Fig. ​(Fig.2A)2A) or IAV (Fig. ​(Fig.2B).2B). Next, we determined the effect of MNV infection on heavy chain class switching of anti-VV or anti-IAV Ab responses. Anti-VV and anti-IAV Ab responses exhibited similar heavy chain profiles dominated by immunoglobulin G2b (IgG2b) Abs regardless of MNV status (Fig. 2C and D). Thus, the CD8⁺ T-cell and Ab response to both VV and IAV appears to be essentially unaffected by chronic MNV infection. Since IgG anti-VV or anti-IAV Ab responses are entirely dependent on CD4⁺ T-cell help (11, 12), we can also infer that MNV also does not significantly affect CD4⁺ T-cell responses to VV or IAV.Open in a separate windowFIG. 2.MNV exposure does not alter Ab responses to VV or IAV. MNV-infected and naïve C57BL/6 mice were infected i.n. with ∼1 × 10³ PFU of VV (A and C) or ∼50 50% tissue culture infective dose units of IAV (B and D), and virus-specific Abs were determined by ELISA (A, C, and D) or hemagglutination inhibition (B). The ELISA results shown in panel A measured the total IgG, while the ELISA results shown in panels C and D measured the individual isotype indicated. MNV infections were completed 19 days prior to VV or IAV infections. Means and standard errors of the means are shown in panels A, C, and D. Means are shown as lines in panel B. A two-way analysis of variance and Bonferroni statistical analysis were completed for experiments shown in panels A, C, and D, and t tests were completed for the experiment shown in panel B. Four to five mice/group were used for each experiment. O.D., optical density; HAI, hemagglutination inhibition.T-cell and Ab responses, together with innate immune mechanisms, collaborate to control viral replication and limit pathogenesis. To examine the effect of chronic MNV infection on VV-induced or IAV-induced pathogenesis, we infected C57BL/6 mice i.n. with a lethal or sublethal dose of VV or IAV and monitored body weight over a 16-day period. MNV-CR6p2 infection had no significant effect on morbidity or mortality from either virus (Fig. ​(Fig.33 and ​and4).4). Since MNV isolates are highly diverse, we decided to examine the effects of a second strain of MNV (MNV-CW3) which is fully cleared in immunocompetent mice. Mice that cleared MNV-CW3 (19 days post-MNV infection) were infected i.n. with VV or IAV. Once again, this strain of MNV had no effect on VV-induced or IAV-induced morbidity or mortality (Fig. ​(Fig.33 and ​and4).4). Future studies should address the extent to which other MNV strains affect the generation of adaptive immune responses to secondary viral infections.Open in a separate windowFIG. 3.MNV does not increase morbidity following subsequent i.n. infection with VV or IAV. MNV-infected and naïve C57BL/6 mice were infected i.n. with a sublethal dose of VV (∼1 × 10³ PFU) (A) or IAV (∼50 50% tissue culture infective dose units) (B), and weight loss was recorded for 16 days postinfection. MNV infections were completed 19 days prior to VV or IAV infections. A two-way analysis of variance and Bonferroni statistical analysis were completed. Four to five mice/group were used for each experiment.Open in a separate windowFIG. 4.MNV does not increase mortality following subsequent i.n. infection with VV or IAV. MNV-infected and naïve C57BL/6 mice were infected i.n. with VV (∼1 × 10⁴ PFU) (A) or IAV (∼500 50% tissue culture infective dose units) (B), and survival was monitored for 16 days postinfection. MNV infections were completed 19 days prior to VV or IAV infections. Eight to 10 mice/group were used for each experiment.Taken together, these data demonstrate that MNV infection has no significant effects on the measured immune response to VV or IAV. Our results cannot, however, be simply extrapolated to other viruses or microorganisms. Rather, the effect of MNV infection on host immunity in mouse model disease systems needs to be established on a system-by-system basis. Without this knowledge, the possible confounding effects of MNV infection will continue to undermine the confidence in results obtained using mice in colonies in which MNV infections are endemic.     

Mononucleosis and Antigen-Driven T Cell Responses Have Different Requirements for Interleukin-2 Signaling in Murine Gammaherpesvirus Infection

Interleukin-2 (IL-2) has been implicated as being necessary for the optimal formation of primary CD8⁺ T cell responses against various pathogens. Here we have examined the role that IL-2 signaling plays in several aspects of a CD8⁺ T cell response against murine gammaherpesvirus 68 (MHV-68). Exposure to MHV-68 causes a persistent infection, along with infectious mononucleosis, providing a model for studying these processes in mice. Our study indicates that CD25 is necessary for optimal expansion of the antigen-specific CD8⁺ T cell response but not for the long-term memory response. Contrastingly, IL-2 signaling through CD25 is absolutely required for CD8⁺ T cell mononucleosis.Members of the gammaherpesvirus family are associated with significant diseases, such as nasopharyngeal carcinoma, lymphoid malignancies, and infectious mononucleosis (16). Murine gammaherpesvirus 68 (MHV-68) is a γ2-herpesvirus related to the human pathogens Epstein-Barr virus (EBV) and Kaposi''s sarcoma virus (19, 21). Intranasal (i.n.) infection of mice with MHV-68 results in acute infection of the lung epithelium, which is eventually controlled; however, the virus also establishes a latent infection in B cells, dendritic cells, and macrophages that is maintained throughout the life of the host (8, 9). Infection with MHV-68 generates a broad array of antigen-specific CD8⁺ T cells that can control the virus without eliminating persistent infection (5, 12, 13). Additionally, CD4⁺ T cells and neutralizing antibodies are thought to be critical for the prevention of virus reactivation (3, 6).A major complication of EBV infection is infectious mononucleosis (16), which occurs when infection is delayed until puberty. Signs of disease include dramatic lymph node enlargement and the presence of large numbers of activated CD8⁺ T cells in the peripheral blood. Similarly to EBV infection, MHV-68 induces a polyclonal activation of B cells upon establishment of latency. Concurrently, a CD8⁺ T cell-dominated lymphocytosis of the peripheral blood occurs, as seen with EBV. However, there are distinct differences between the two types of infectious mononucleosis. CD8⁺ T cell lymphocytosis seen with EBV consists of a broad array of T cell receptor specificities, a large proportion of which are specific for EBV epitopes. In contrast, MHV-68-induced mononucleosis is dominated by oligoclonal Vβ4⁺ CD8⁺ T cells that are not reactive to MHV-68 epitopes. With MHV-68, the expansion of this population is dramatic, with levels reaching upwards of 60% of the peripheral blood CD8⁺ T cell population (20). This occurs in different mouse strains, across at least five different major histocompatibility complex (MHC) class I haplotypes. However, it is important to note that infection of wood mice (Apodemus sylvaticus) does not induce splenomegaly, as seen with laboratory strains of mice, indicating a potential lack of Vβ4 expansion that may be species related (14). Interestingly, evidence suggests that Vβ4⁺ CD8⁺ T cell expansion does not require classical MHC class Ia antigen presentation (4). Recent studies instead implicate a secreted viral protein, M1, capable of stimulating the Vβ4+ T cell population in a novel manner, and the authors propose a role for Vβ4+ T cells in control of MHV-68 infection (7).We and others have recently shown that IL-2 signaling during the early stages of a response to acute viral and bacterial pathogens is required for optimal expansion and differentiation of CD8⁺ T cells (15, 17, 18). However, reports with other viruses have shown IL-2-independent primary CD8⁺ T cell responses (1, 22). Therefore, we wished to determine whether IL-2 signals are necessary for the expansion, maintenance, and/or recall of CD8⁺ T cell responses during murine gammaherpesvirus infection.We generated chimeric mice through lethal irradiation of C57BL/6 mice followed by adoptive transfer of mixed bone marrow from C57BL/6 wild-type (WT) and CD25^−/− donors, as previously described (17). Following previous described protocols, mice were given bone marrow in a 2:1 ratio of CD25^−/−/WT to generate equally proportioned congenic populations in recipient mice (see Fig. S1 in the supplemental material) (1, 17). The resultant mice contained CD8⁺ T cells of both WT and CD25^−/− origin, which could be distinguished by congenic markers. Chimeric mice were infected intranasally with 400 PFU of MHV-68, and the kinetics of the CD8⁺ T cell response were followed by antibody and tetramer staining of peripheral blood for CD8⁺ T cells specific for the epitopes ORF6₄₈₇ (p56) and ORF61₅₂₄ (p79), as previously described (13). While antigen-specific CD25^−/− CD8⁺ T cells were initially able to proliferate in response to infection, the peak response was significantly lower than that of the wild-type cells (Fig. ​(Fig.11 A and B). This indicates that while CD25 is dispensable for early activation of CD8⁺ T cells, IL-2 signaling is required for full expansion of the antigen-specific response to MHV-68. Despite this deficit in the acute antiviral response, the resultant memory populations were not statistically different between the groups (Fig. 1A and B). In our previous report, CD25^−/− CD8⁺ T cells were unable to fully differentiate into short-lived effector cells (SLECs), defined as KLRG1^high CD127^low (17). To determine if MHV-68-specific responses were also unable to fully differentiate, we infected chimeric mice and stained p79⁺ CD8⁺ T cells for the cell surface markers KLRG1 and CD127. At the peak of the response (14 days postinfection [p.i.]), p79⁺ WT cells had differentiated into SLEC (KLRG1^high CD127^low), memory precursor (MPEC) (KLRG1^low CD127^high), and doubly positive populations. However, the p79⁺ CD25^−/− cells failed to form the SLEC population and instead had a corresponding increase in the MPEC population, indicating that CD25 is necessary for full effector differentiation of gammaherpesvirus-specific CD8⁺ T cell responses (Fig. 1C and D).Open in a separate windowFIG. 1.IL-2 signals are necessary for the optimal expansion of MHV-68-specific CD8⁺ T cells. WT/CD25^−/− chimeric mice were infected with MHV-68 intranasally and bled at set time points. The antigen-specific responses against two dominant epitopes, p79 (A) and p56 (B), were determined via tetramer staining of peripheral blood. p79-specific CD8⁺ T cells from the WT and CD25^−/− populations were stained at the peak of the response (day 14 p.i.) for KLRG1 and CD127 to determine their ability to differentiate into short-lived and memory precursor effector cells (C and D). *, P < 0.05; **, P < 0.01; ***, P < 0.001. Error bars represent standard deviations from the means. Four mice were used per group, and data are representative of at least two experiments.To determine whether antigen-specific CD25^−/− CD8⁺ T cells were capable of optimally responding to a secondary challenge, we infected chimeric mice with MHV-68 and waited 60 days before challenging with recombinant vaccinia virus (rVV) expressing the ORF61₅₂₄ epitope (2 × 10⁶ PFU, intraperitoneal). It is necessary to use a heterologous virus to induce a recall CD8⁺ T cell response since MHV-68 generates a robust neutralizing antibody response, preventing secondary infection. Previous studies with rVV indicate that the recall response of MHV-68-specific CD8⁺ T cells is antigen dependent, since administration of rVV expressing an irrelevant epitope had no effect upon the MHV-68-specific populations (2). WT and CD25^−/− cells were able to respond to the secondary challenge with similar kinetics (Fig. ​(Fig.22 A and B), indicating that MHV-68 memory CD8⁺ T cells are capable of a generating a recall response in the absence of IL-2 signaling. These data, together with our previous report (17), show that the dependence on CD25 for formation of the SLEC population is conserved between both persistent and acute virus infections.Open in a separate windowFIG. 2.CD25^−/− CD8⁺ T cells can respond to secondary challenge. WT/CD25^−/− chimeric mice were infected with MHV-68 i.n. After 60 days, the percentage of peripheral blood CD8⁺ T cells specific for p79 was determined. Mice were then challenged with rVV p79, and the p79⁺ CD8⁺ population was determined 5 days postchallenge (A). The numbers in the box represent the averages ± standard deviations. The average fold increase was calculated to determine the ability of WT and CD25^−/− CD8⁺ T cells to respond to a secondary challenge (B). Error bars represent standard deviations from the means. Four mice were used per group, and data are representative of at least two experiments.WT CD8⁺ T cells underwent a dramatic expansion between days 15 and 21 p.i. (Fig. ​(Fig.3A),3A), consistent with infectious mononucleosis (10). Interestingly, we did not observe a similar expansion of CD25^−/− CD8⁺ T cells, indicating a role for IL-2 signaling in the expansion of CD8⁺ T cells during mononucleosis (Fig. ​(Fig.3A).3A). Since mononucleosis is dominated by Vβ4+ CD8⁺ T cells, we analyzed these T cells from both naive and infected mice (17 days p.i.) for expression of CD25 by flow cytometry. While Vβ4⁺ CD8⁺ T cells from the spleen and peripheral blood of naive mice did not express detectable levels of CD25, mice infected with MHV-68 expressed intermediate levels of CD25 during the time period when dramatic expansion of Vβ4⁺ T cells occurs (Fig. 3B and C). Consistent with a role for IL-2 signaling in Vβ4 expansion, we observed a severe deficit in expansion in the CD25^−/− population of chimeric mice, since the percentage of WT Vβ4⁺ cells increased dramatically between days 14 and 36 p.i., accompanied by only a small expansion of the CD25^−/− Vβ4⁺ population over the same period (Fig. ​(Fig.44 A and B).Open in a separate windowFIG. 3.Vβ4⁺ CD8⁺ T cells express CD25 upon infection with MHV-68. WT/CD25^−/− chimeric mice were infected with MHV-68 i.n., and the percentage of peripheral blood cells that were CD8⁺ was determined over time for each congenic population (A). Vβ4⁺ CD8⁺ T cells from naive and MHV-68-infected mice (day 17 p.i.) were analyzed for expression of CD25 (B and C). Isotype control, filled histogram; naive mice, dashed line; infected mice, solid line (**, P < 0.01). Error bars represent standard deviations from the means. Four mice were used per group, and data are representative of at least two experiments.Open in a separate windowFIG. 4.CD8⁺ T cell-based infectious mononucleosis does not occur in the absence of IL-2 signaling in MHV-68-infected mice. WT/CD25^−/− chimeric mice were infected with MHV-68 i.n., and the percentage of Vβ4⁺ CD8⁺ T cells was determined over time for each congenic population. Representative plots from day 36 p.i. (A) or the averages over time (B) are shown. WT and CD25^−/− CD8⁺ T cells from chimeric mice were analyzed for expression of CD62L over time. Representative plots from day 24 p.i. (C) or the averages over time (D) are shown. *, P < 0.05; **, P < 0.01). Error bars represent standard deviations from the means. Four mice were used per group, and data are representative of at least two experiments.During infectious mononucleosis, CD8⁺ T cells are in a highly activated state and thus express low levels of CD62L (20). Therefore, we analyzed CD8⁺ T cells from chimeric mice for expression of CD62L. After MHV-68 infection, the majority of WT CD8⁺ T cells in the peripheral blood were CD62L^low, as previously reported (Fig. 4C and D) (20). Interestingly, CD25^−/− CD8⁺ T cells failed to develop this dominant CD62L^low population, indicating that CD25 is necessary for the activation of the CD8⁺ T cell compartment in addition to cell expansion during mononucleosis (Fig. 4C and D). When we analyzed the Vβ4⁺ CD8⁺ T cell compartment, we observed that WT cells downregulated expression of CD62L. While Vβ4⁺ cells from the CD25^−/− compartment also decreased expression of CD62L, they did so to a lesser extent both as a percentage and on a per-cell basis (see Fig. S2 in the supplemental material).In these studies, we have shown that signaling through CD25 is necessary for the generation of an optimal primary CD8⁺ T cell response against a gammaherpesvirus, since virus-specific CD8⁺ T cells were unable to expand as robustly as WT cells and did not fully differentiate into short-lived effector cells. These observations are consistent with previous results from our lab and findings of others using a variety of acute infection models (17, 18). However, not all persistent infections appear to require CD25, since the m45-specific response to murine cytomegalovirus (MCMV) infection occurs normally in the absence of IL-2 signals (1). What allows for some responses to be independent of IL-2 remains unknown. Potential explanations could involve differences in tropism, the route of infection, or the amount of proinflammatory cytokines induced by each infection. Despite the dependence on CD25 for the short-term effector response, the memory CD8⁺ T cell response remained intact in the absence of IL-2 signaling. In contrast, Vβ4 expansion and mononucleosis never attained normal levels. Unlike the antigen-specific response, which relies upon peptide/MHC interactions for induction, mononucleosis does not rely upon conventional antigen presentation (4). Instead, the M1 protein of MHV-68, expressed during the establishment and expansion of latency in the spleen, appears to drive Vβ4 expansion (7). Interestingly, our evidence shows that both antigen-dependent and -independent CD8⁺ T cell expansion require CD25. Antigen-specific T cells also undergo an apoptotic contraction phase, followed by a lower frequency of cells surviving as relatively quiescent memory cells. In contrast, during mononucleosis caused by MHV-68, CD8⁺ T cells remain in an activated state and do not undergo a marked contraction, providing a potential explanation as to why the WT and CD25^−/− Vβ4 populations continue to differ in both number and phenotype later in the response.Earlier studies have also identified CD4⁺ T cells as being critical for the development of MHV-68-induced infectious mononucleosis (11, 20). We have previously shown that CD4⁺ T cell help was critical for robust expression of CD25 on activated antigen-specific CD8⁺ T cells. Interestingly, when we measured CD25 expression on Vβ4⁺ cells from mice lacking CD4⁺ T cells, we saw a moderate decrease in the level of CD25 expressed (data not shown), indicating one potential reason why CD4-deficient mice do not experience infectious mononucleosis. However, it is likely that other factors involving CD4⁺ T cells and activation of B cells are also involved (10).In conclusion, the significance of these studies is twofold. First, they shed light on the requirements for MHV-68-induced mononucleosis. Second, our data illustrate that CD25 is required for both antigen-specific and non-antigen-specific activation of CD8⁺ T cell responses, while being dispensable for memory cell formation. This knowledge may be useful in developing new T cell-based immune therapies to enhance control of persistent gammaherpesvirus infections.      

Simian Immunodeficiency Virus Infection Induces Expansion of α4β7+ and Cytotoxic CD56+ NK Cells

Herein we demonstrate that chronic simian immunodeficiency virus (SIV) infection induces significant upregulation of the gut-homing marker α4β7 on macaque NK cells, coupled with downregulation of the lymph node-trafficking marker, CCR7. Interestingly, in naïve animals, α4β7 expression was associated with increased NK cell activation and, on CD16⁺ NK cells, delineated a unique dual-function cytotoxic-CD107a⁺/gamma interferon (IFN-γ)-secreting population. However, while SIV infection increased CD107a expression on stimulated CD56⁺ NK cells, α4β7⁺ and α4β7⁻ NK cells were affected similarly. These findings suggest that SIV infection redirects NK cells away from the lymph nodes to the gut mucosae but alters NK cell function independent of trafficking repertoires.Human peripheral blood contains two primary subsets of natural killer (NK) cells—a major CD16⁺ CD56^dim subset and a minor CD16⁻ CD56^bright subset. We have defined subsets of rhesus macaque (Macaca mulatta) NK cells and found that, similarly, macaque peripheral blood is dominated by a CD16⁺ CD56⁻ subset but contains two minor CD16⁻ CD56⁺ and CD16⁻ CD56⁻ subpopulations (34). As in humans, macaque CD16⁻ CD56⁺ NK cells are the primary producers of gamma interferon (IFN-γ) and express cell surface markers such as CCR7 and CD62L that mediate homing to lymph nodes, whereas CD16⁺ CD56⁻ NK cells do not express CCR7 or CD62L and primarily mediate cytolytic functions (7, 12, 30, 34). In both humans and macaques, the distribution of NK subsets in peripheral blood is distinct from that observed in lymph nodes and mucosal tissues, where NK cells are primarily CD56⁺ (9, 12, 30, 35).NK cells are important for the control of multiple viral infections, and increasing evidence suggests that NK cells play a significant role in controlling human immunodeficiency virus (HIV) infection (3, 5, 13, 14, 19, 21, 22, 24, 33), as well as simian immunodeficiency virus (SIV) infection of rhesus macaques and sooty mangabeys (6, 16, 26). HIV and SIV primarily replicate in the gut mucosa (18), and although we and others have demonstrated the presence of NK cells in the gastrointestinal tracts of both humans and rhesus macaques (8, 9, 25, 30), the nature of these NK cells is still poorly understood. Interestingly, the integrin α4β7, which directs lymphocyte trafficking to the gut (4), has been shown to be expressed on peripheral blood NK cells in humans and rhesus macaques (11, 27). This finding suggests that the gut mucosa is a site of active NK cell trafficking.Despite the importance of gut-associated lymphoid tissue in HIV/SIV pathogenesis, little is known about the effects of infection on NK cell homing to these tissues. In order to address this deficit, a total of 47 Indian rhesus macaques were studied, 27 of which were SIV naïve and 20 infected with either SIVmac239 (5) or SIVmac251 (15) for more than 150 days (mean duration of infection, 293 days). All animals were housed at the New England Primate Research Center and maintained in accordance with the guidelines of the Committee on Animals of the Harvard Medical School and the Guide for the Care and Use of Laboratory Animals (23a).PBMC isolation and polychromatic flow cytometry staining were carried out using protocols described previously for our laboratory (29, 31); the antibodies used are listed in Table ​Table1.1. NK cells were defined as CD3⁻ CD8α⁺ NKG2A⁺ (30, 34), and CD16 and CD56 expression were used to delineate three primary subsets: CD56⁻ CD16⁺ (CD16⁺), the dominant subset; CD56⁺ CD16⁻ (CD56⁺); and CD56⁻ CD16⁻ (double negative [DN]) (Fig. ​(Fig.11 A). The results of polychromatic flow cytometry analyses demonstrated that α4β7 was expressed at the highest levels on CD16⁺ NK cells and that, while expression on this subset was not altered during SIV infection, α4β7 was significantly upregulated on both CD56⁺ and DN NK cells in SIV-infected animals (Fig. 1B and C). Interestingly, CCR7, which is expressed only on the CD56⁺ and DN NK cell subsets in macaques (30, 34), was concomitantly downregulated on these subsets of NK cells during chronic SIV infection (Fig. ​(Fig.1B).1B). The relationship between the two markers delineated a dichotomous expression pattern between naïve and SIV-infected macaques (Fig. ​(Fig.1D).1D). This dramatic shift in CD56⁺ and DN NK cell trafficking repertoires is likely indicative of increased homing of these NK subsets to the gut coupled to decreased homing to lymph nodes. Also, as shown in Fig. ​Fig.1E,1E, the absolute numbers of both CD16⁺ and DN NK cells increased during chronic SIV infection, resulting in increased absolute numbers of gut-homing α4β7⁺ cells in both subsets. Interestingly, while the absolute numbers of all CD56⁺ NK cells tended to decrease during chronic SIV infection, the absolute numbers of the α4β7⁺ CD56⁺ NK cell subset increased slightly (Fig. ​(Fig.1E,1E, middle panel), further suggesting that multiple subsets of α4β7⁺ NK cells increase during chronic SIV infection.Open in a separate windowFIG. 1.Comparison of α4β7 expression on NK cell subsets in naïve and SIV-infected macaques. (A) Macaque NK cell subsets were defined as CD3⁻ CD8α⁺ NKG2A⁺ (30, 34) and then further delineated into CD56⁺, CD16⁺, and DN subsets. (B) Representative flow cytometry plots of α4β7 and CCR7 expression on NK cell subsets in naïve and SIV-infected macaques. (C) Percentages of α4β7⁺ cells above the background level were compared between naïve and SIV-infected macaques for CD56⁺, CD16⁺, and DN NK subsets. (D) Relationships between α4β7 and CCR7 expression on CD56⁺ and DN NK cells in naïve and SIV-infected macaques. (E) Absolute numbers of total circulating NK cells were determined by using a bead-based flow cytometric assay as described previously (29, 30), and α4β7⁺ NK cell subset counts were extrapolated using these data combined with NK cell frequency data determined by polychromatic flow cytometry (panel A). Horizontal bars indicate median values for 20 to 27 animals. Student''s t tests were used to compare naive and SIV-infected animal groups; P values of >0.05 are considered statistically significant.

TABLE 1.

Antibodies used in polychromatic flow cytometry analyses

Important Role for the Murid Herpesvirus 4 Ribonucleotide Reductase Large Subunit in Host Colonization via the Respiratory Tract

Viral enzymes that process small molecules provide potential chemotherapeutic targets. A key constraint—the replicative potential of spontaneous enzyme mutants—has been hard to define with human gammaherpesviruses because of their narrow species tropisms. Here, we disrupted the murid herpesvirus 4 (MuHV-4) ORF61, which encodes its ribonucleotide reductase (RNR) large subunit. Mutant viruses showed delayed in vitro lytic replication, failed to establish infection via the upper respiratory tract, and replicated to only a very limited extent in the lower respiratory tract without reaching lymphoid tissue. RNR could therefore provide a good target for gammaherpesvirus chemotherapy.Cellular deoxyribonucleotide synthesis is strongly cell cycle dependent. DNA viruses replicating in noncycling cells must therefore either induce cellular enzymes or supply their own. Most herpesviruses encode multiple homologs of nucleotide metabolism enzymes, including both subunits of the cellular ribonucleotide reductase (RNR) (4). While most in vivo cells are resting, most in vitro cell lines divide continuously (29). The importance of viral RNRs may therefore only be apparent in vivo (14). In contrast to alpha- and betaherpesviruses, gammaherpesviruses cause disease mainly through latency-associated cell proliferation. However, gamma-2 herpesviruses show lytic gene expression in sites of latency (9, 17), and lytic reactivation could potentially alleviate some gammaherpesvirus-infected cancers (7, 8). Therefore, it is important also to understand the pathogenetic roles of gammaherpesvirus lytic cycle enzymes, such as RNR.The known human gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi''s sarcoma-associated herpesvirus (KSHV) have narrow species tropisms that preclude most pathogenesis studies. In contrast, murid herpesvirus 4 (MuHV-4) (21, 26) allows gammaherpesvirus host colonization to be studied in vivo. After intranasal (i.n.) inoculation, MuHV-4 replicates lytically in lung epithelial cells before seeding to lymphoid tissue (27). Long-term virus loads are independent of extensive primary lytic spread (25). However, whether persistence requires some lytic gene expression remains unclear. Replication-deficient viral DNA reached the spleen after intraperitoneal (i.p.) but not i.n. virus inoculation (15, 20, 28), suggesting that virus dissemination from the lung to lymphoid tissue requires lytic replication. In addition, less invasive inoculations may increase further the viral functions required to establish a persistent infection. Thymidine kinase (TK)-deficient MuHV-4 given i.n. without general anesthesia, in which method the wild-type virus infects the upper respiratory tract and reaches lymphoid tissue without infecting the lungs (18), fails to colonize in mice at all (12). The implication is that virions using a likely physiological route of host entry must replicate in terminally differentiated cells to establish a significant infection. However, some unusual features of gammaherpesvirus TKs (11) suggest that they have functions besides thymidine phosphorylation. We therefore targeted here another enzyme linked to viral DNA replication, the MuHV-4 RNR. We aimed to define the in vivo importance of a potential therapeutic target and to advance generally our understanding of gammaherpesvirus pathogenesis.Transposon insertions in the MuHV-4 RNR small (ORF60) and large (ORF61) RNR subunit genes have been described as either attenuating or not for lytic replication in vitro (19, 23). We disrupted ORF61 (RNR⁻) by inserting stop codons close to its 5′ end (Fig. ​(Fig.11 a). An EcoRI-L genomic clone (coordinates 80644 to 84996) in pUC19 (6) was digested with AleI to remove nucleotides 82320 to 82534 of ORF61 (82865 to 80514). An oligonucleotide encoding multiple stop codons and an EcoRI restriction site (5′-CTAGCATGCTAGAATTCTAGCATGCATG-3′) was ligated in place. Nucleotides 81365 to 83883 were then PCR amplified, including a BamHI site in the 81365 primer, cloned as a BglII/BamHI fragment into the BamHI site of pST76K-SR, and recombined into a MuHV-4 bacterial artificial chromosome (BAC) (1). A revertant virus was made by reconstituting the corresponding, unmutated genomic fragment. Southern blots (5) of viral DNA (Fig. ​(Fig.1b)1b) confirmed the expected genomic structures, and immunoblots (5) of infected cell lysates (Fig. ​(Fig.1c)1c) established that mutant viruses no longer expressed the RNR large subunit.Open in a separate windowFIG. 1.Disruption of the MuHV-4 ORF61. (a) Schematic diagram of the ORF61 (RNR large) locus, showing the mutation introduced and relevant restriction sites. (b) Viral DNA was digested with EcoRI and probed for ORF61. Oligonucleotide insertion into ORF61 changes a 4,352-bp wild-type band to 2,462 bp plus 1,676 bp. The 2,462-bp fragment is not visible because it overlaps the probe by only 331 nucleotides (nt) and comigrates with a background band of unknown origin. WT, wild type; REV, revertant; RNR⁻, mutant; RNR⁻ ind, independent mutant. WT luc⁺ is MuHV-4 expressing luciferase from an ORF57/ORF58 intergenic cassette. RNR⁻ luc⁺ and RNR⁻ luc⁺ind have ORF61 disrupted on this background. (c) Infected cell lysates were immunoblotted for gp150 (virion envelope glycoprotein, monoclonal antibody [MAb] T1A1), ORF17 (capsid component, MAb 150-7D1), TK (tegument component, MAb CS-4A5), and ORF61 (MAb PS-8A7). (d) BHK-21 cells were infected with RNR⁺ or RNR⁻ viruses (0.01 eGFP units/cell, 2 h, 37°C), washed two times with phosphate-buffered saline (PBS) to remove unbound virions, and cultured at 37°C to allow virus spread. Infectivity (in eGFP units) at each time point was determined on fresh BHK-21 cells in the presence of phosphonoacetic acid to prevent further viral spread, with the number of eGFP-postive cells counted 18 h later by flow cytometry. (e) BHK-21 cells were infected with RNR⁺ or RNR⁻ viruses (2 eGFP units/cell, 2 h, 37°C), washed in medium (pH 3) to inactivate nonendocytosed virions, and cultured at 37°C to allow virus replication. The infectivity of replicate cultures was then assayed as described in the legend of panel d. (f) BHK-21 cells were incubated with RNR⁺ or RNR⁻ viruses (0.3 eGFP units/cell, 37°C) for the times indicated, and the numbers of eGFP-positive cells in the cultures were then determined by flow cytometry.RNR⁻ viruses were noticeably slower than RNR⁺ viruses when spreading through BHK-21 cell monolayers after BAC DNA transfection. Normalizing by immunoblot signal, RNR⁻ virus stocks had titers similar to that of the wild type by viral enhanced green fluorescent protein (eGFP) expression but 10- to 100-fold lower plaque titers. Using eGFP expression as a readout, RNR⁻ virion production after a low multiplicity of infection lagged 1 day behind that of the wild type (Fig. ​(Fig.1d).1d). Maximum infectivity yields were also reduced, but once BHK-21 cells become confluent, they support MuHV-4 lytic infection poorly, so this was probably a consequence of the slower lytic spread. After a high multiplicity of infection (Fig. ​(Fig.1e),1e), RNR⁻ mutants showed a 10-h lag in virion production and no difference in the final yield. They showed no defect in single-cycle eGFP expression (Fig. ​(Fig.1f),1f), implying normal virion entry. Therefore, the main RNR⁻ defect lay in infectious virion production.For in vivo experiments, the loxP-flanked viral BAC-eGFP cassette must be removed (1). Therefore, to monitor infection in vivo without having to rely on new virion production as a readout, we transferred the RNR mutation onto a luciferase-positive (luc⁺) background (18). Viral luciferase expression (from an early lytic promoter) by in vitro luminometry (18) was independent of either viral DNA replication or RNR expression (Fig. ​(Fig.22 a). After i.n. inoculation of anesthetized mice, RNR⁻ luciferase signals measured in vivo by i.p. luciferin injection and IVIS Lumina charge-coupled-device (CCD) camera scanning (18) were visible in lungs (Fig. ​(Fig.2b)2b) but were 100-fold lower than those of the RNR⁺ controls (Fig. ​(Fig.2c).2c). A severe impairment of RNR⁻ lytic replication was confirmed by plaque assay (18) (Fig. ​(Fig.2d);2d); the difference between RNR⁻ and RNR⁺ plaque titers greatly exceeded any difference in plaquing efficiency.Open in a separate windowFIG. 2.Host colonization by RNR⁻ MuHV-4 mutants. (a) BHK-21 cells were left uninfected or infected overnight with RNR⁺ or RNR⁻ luc⁺ MuHV-4 and then assayed for luciferase expression by luminometry. Phosphonoacetic acid (PAA; 100 μg/ml) was either added or not to cultures to block viral late gene expression. Each point shows the mean ± standard deviation from triplicate cultures. (b) BALB/c mice were infected i.n. under general anesthesia with RNR⁻ or RNR⁺ luc⁺ MuHV-4 (5 × 10³ PFU) and then assayed for luciferase expression by luciferin injection and CCD camera scanning. The images are from 5 days postinfection. Note that the RNR⁺ and RNR⁻ images have different sensitivity scales. (c) For quantitation, dorsal and ventral luciferase signals were summed. Each point shows 1 mouse. The dashed lines show detection thresholds. The RNR⁺ signal was significantly greater than the RNR⁻ signal for all sites and time points (P < 0.001 by Student''s t test). (d) C57BL/6 mice were infected i.n. under anesthesia with RNR⁻ or RNR⁺ MuHV-4 (5 × 10³ PFU). Five days later, infectious virus loads in noses and lungs were measured by plaque assay. Each point shows 1 mouse. RNR⁻ infections yielded no plaques and therefore are shown at the sensitivity limits of each assay. (e) BALB/c mice were infected i.n. with RNR⁻ or RNR⁺ MuHV-4 without anesthesia and then monitored by luciferin injection and CCD camera scanning. Each point shows the summed ventral and dorsal signals of the relevant region for 1 mouse. Neck signals correspond to the superficial cervical lymph nodes (SCLN). The dashed lines show detection thresholds. RNR⁻ luciferase signals were undetectable at all time points.No RNR⁻ luciferase signals were visible in noses, nor did RNR⁻ MuHV-4 give signals in the superficial cervical lymph nodes (SCLN), which drain the nose (Fig. ​(Fig.2c).2c). This lack of live imaging signals from the upper respiratory tract was confirmed by ex vivo imaging of SCLN at day 14 postinfection. We examined upper respiratory tract infection further with an independently derived luc⁺ RNR⁻ mutant, inoculating i.n. without anesthesia so as to avoid virus aspiration into the lungs. No RNR⁻ luciferase signals were detected, while wild-type signals were readily observed in the nose and superficial cervical lymph nodes (Fig. ​(Fig.2e2e).Like RNR⁻ MuHV-4, TK⁻ mutants are severely attenuated for lytic replication in the lower respiratory tract. However, they eventually establish a reactivatable latent infection and induce virus-specific antibody (3). Latent virus titers in spleens peak at 1 month postinoculation. Infectious center assays showed no RNR⁻ infection of spleens at that time (Fig. ​(Fig.33 a). We also looked for viral DNA in spleens by quantitative PCR (Fig. ​(Fig.3b).3b). Genomic coordinates 4166 to 4252 were amplified and hybridized to a probe with coordinates 4218 to 4189. Viral genome copies, relative to the cellular adenosine phosphoribosyl transferase copy number, were calculated from standard curves of cloned plasmid DNA (10). No RNR⁻ viral DNA was detected. ELISA for MuHV-4-specific serum IgG (24) detected an antibody response after lung infection but not upper respiratory tract infection of BALB/c mice with RNR⁻ MuHV-4 (Fig. ​(Fig.3c).3c). There was a similar lack of antibody 1 month after upper respiratory tract infection of C57BL/6 mice with independently derived RNR⁻ mutants (Fig. ​(Fig.3d)3d) and 3 months after exposure of 6 BALB/c mice to RNR⁻ luc⁺ MuHV-4. In contrast, i.p. RNR⁻ luc⁺ MuHV-4 gave lower luciferase signals than RNR⁺ luc⁺ MuHV-4 (Fig. ​(Fig.44 a), but RNR⁻ infectious centers (Fig. ​(Fig.4b)4b) and viral genomes (Fig. ​(Fig.4c)4c) were detected in spleens, and enzyme-linked immunosorbent assays (ELISAs) (Fig. ​(Fig.4d)4d) showed MuHV-4-specific serum IgG.Open in a separate windowFIG. 3.Spleen colonization by RNR⁻ MuHV-4. (a) BALB/c or C57BL/6 mice were infected i.n. either with general anesthesia (lung infection) or without (nose infection). One month later, spleens were assayed for recoverable latent virus by infectious center assay. Lower detection limit, 10 infectious centers per spleen. (b) The spleens described in the legend of panel a were further analyzed for viral DNA by quantitative PCR. Copy numbers are expressed relative to the cellular adenosine phosphoribosyl transferase copy number in each sample. The dashed lines show lower detection limits (1 viral copy/10,000 cellular copies). (c) Sera from BALB/c mice after i.n. infection either with (lung infection) or without (nose infection) general anesthesia were assayed for MuHV-4-specific IgG by ELISA. Each line shows the absorbance curve for 1 mouse. The dashed lines show naive serum. (d) Sera from C57BL/6 mice 1 month after infection with independent RNR mutants were analyzed for MuHV-4-specific IgG, as described in the legend to panel c.Open in a separate windowFIG. 4.Intraperitoneal infection with RNR⁺ and RNR⁻ MuHV-4. (a) Mice were infected i.p. with RNR⁻ luc⁺ or RNR⁺ luc⁺ MuHV-4 and then monitored for luciferase expression. Each point shows the total abdominal signal of 1 mouse. The x axis is at the lower limit of signal detection above the background. (b) Spleens were assayed for recoverable virus by infectious center assay 10 days after i.p. infection with RNR⁻ luc⁺ or RNR⁺ luc⁺ MuHV-4. Each point shows the titer of 1 mouse. One log₁₀ infectious center per mouse corresponds to the lower limit of detection. (c) Spleen DNA was analyzed for viral genome content by quantitative PCR. Each point shows viral copy/cellular copy for the mean of triplicate reactions for 1 mouse. (d) Sera taken 10 days after i.p. infection with RNR⁻ luc⁺ or RNR⁺ luc⁺ MuHV-4 were assayed for MuHV-4-specific IgG by ELISA. Each line shows the absorbance values for the serum of 1 mouse. “Naive” represents age-matched, uninfected controls.The failure of both the RNR large subunit (ORF61) and TK⁻ MuHV-4 mutants to infect via the upper respiratory tract argues that this requires viral replication in a nucleotide-poor cell. The additional lack of lymphoid RNR⁻ infection after inoculation into the lungs seemed likely to reflect a defect in virus transport, as RNR⁻ MuHV-4 did colonize the spleen after i.p. inoculation. It is also possible that the first cells infected simply produced no infectious virions, although this seemed a more likely explanation for upper respiratory tract infection being undetectable; lung infection progressed sufficiently to give detectable luciferase expression and to induce an antiviral antibody response. How transport from lung to lymphoid tissue occurs is unknown, but likely scenarios include latently infected dendritic cells (22) carrying MuHV-4 along afferent lymphatics to germinal centers and cell-free virions being captured in lymph nodes by subcapsular sinus macrophages (13). Therefore, RNR may be important for MuHV-4 to spread from myeloid cells to B cells.The difference between RNR⁻ and TK⁻ mutants in host colonization via the lung—TK⁻ mutants reached lymphoid tissue whereas RNR⁻ mutants did not—could reflect additional ORF61 functions, as precedent exists for functional drift (2, 16). Alternatively, RNR may be needed more than TK for MuHV-4 replication in some cell types. Formidable hurdles to RNR-based therapies remain: human gammaherpesvirus infections rarely present until latency is well established, so blocking virus spread to lymphoid tissue may have a limited impact, and no drugs sufficiently selective to target viral RNRs in a clinical setting have yet emerged. Nevertheless, the severe in vivo attenuation of RNR⁻ MuHV-4 suggested that RNR may be a viable target for limiting gammaherpesvirus lytic spread.     

HIV Infection Is Associated with a Preferential Decline in Less-Differentiated CD56dim CD16+ NK Cells

HIV-1 infection is characterized by loss of CD56^dim CD16⁺ NK cells and increased terminal differentiation on various lymphocyte subsets. We identified a decrease of CD57⁻ and CD57^dim cells but not of CD57^bright cells on CD56^dim CD16⁺ NK cells in chronic HIV infection. Increasing CD57 expression was strongly associated with increasing frequencies of killer immunoglobulin-like receptors (KIRs) and granzyme B-expressing cells but decreasing percentages of cells expressing CD27⁺, HLA-DR⁺, Ki-67⁺, and CD107a. Our data indicate that HIV leads to a decline of less-differentiated cells and suggest that CD57 is a useful marker for terminal differentiation on NK cells.NK cells are effector cells of innate immunity which are pivotal as first-line defense against viral infections, such as HIV infection (14). Large genotypic studies demonstrated a delayed onset of AIDS in HIV-seropositive individuals carrying the activating receptor KIR3DS1 and/or alleles of the inhibiting receptor KIR3DL1 in conjunction with HLA-Bw4-80I (18, 19). Development of NK cells mainly takes place in the bone marrow, from which mature NK cells move out to reside and circulate in peripheral sites (13). Mature NK cells are characterized by granules which harbor granzymes and perforin. These NK cells are fully armed, “ready-to-go” effector cells (17).A number of NK cell abnormalities have been reported in HIV infection (9), including high activation status (2, 10), increased turnover (16), differential expression of activating and inhibitory receptors (20), impaired interaction with dendritic cells (12), and loss of CD56^dim CD16⁺ NK cells (23). CD56^dim CD16⁺ NK cells represent the largest NK cell subset in peripheral blood in healthy individuals. The expression of killer immunoglobulin-like receptors (KIRs) and CD57 are predominant features of this subpopulation (8, 15). CD57 expression on NK cells has been previously associated with replicative senescence on T and NK cells (4), raising the question of how HIV-1 infection alters CD57 expression on CD56^dim CD16⁺ NK cells.To the best of our knowledge, no one has addressed the phenotypic and functional properties of CD56^dim CD16⁺ NK cells that are preferentially lost during HIV infection. Here, we provide evidence that increasing CD57 expression indicates terminal differentiation in healthy individuals, as well in as HIV-infected subjects. We furthermore show that HIV infection is associated with preferential loss of less-differentiated cells, which are characterized by high activation status and turnover.In this study, blood samples from 37 HIV-seropositive individuals and 15 healthy subjects were analyzed; all HIV-infected patients were either antiretroviral therapy naïve or untreated for more than one year. The HIV-positive study cohort comprised 10 patients with a viral load of less than 2,000 copies/ml, 14 patients with a viral load ranging from 2,000/ml to 20,000 copies/ml, and 13 patients with a viral load above 20,000 copies/ml. CD4 T cell counts ranged from 180/μl to 1,355/μl, the average being 457.3/μl.The study was approved by the local ethics commission (Ethikkommission der Medizinischen Hochschule Hannover, Votum No. 3150), and all study participants gave informed written consent for their participation.Flow cytometric analysis was performed on cryopreserved peripheral blood mononuclear cells (PBMCs) as previously described (21, 22). A list of monoclonal antibodies employed in this study is available upon request. For intracellular analysis of granzyme B, perforin, and Ki-67, we used a fixation and permeabilization kit (Invitrogen). At least 1 million events were acquired for each sample, using either a FACSAria or LSR II flow cytometer (BD Biosciences). Data were analyzed with FlowJo (TreeStar). Lymphocytes were defined by forward and side scatter. CD3⁺, CD14⁺, CD19⁺, dead cells, and cell aggregates were removed from analysis based on peridinin chlorophyll protein and Viaprobe staining and gating on a plot of forward-scatter area versus forward-scatter height (Fig. ​(Fig.1A).1A). NK cells and their distinctive subpopulations were defined based on their CD56 and/or CD16 expression. Fluorescence-minus-one (FMO) staining was used to determine threshold values for the expression of specific markers.Open in a separate windowFIG. 1.HIV infection is associated with loss of CD57⁻ and CD57^dim but not CD57^bright CD56^dim CD16⁺ NK cells. (A) Representative gating scheme for identification of NK cells. NK cells were defined as CD3⁻ CD14⁻ CD19⁻ lymphocytes expressing either CD56 or CD16 or both. We divided CD56^dim CD16⁺ NK cells into three subsets based on their level of CD57 expression: CD57⁻, CD57^dim, and CD57^bright cells. Numbers on FACS plots indicate frequency of gated population. SSC-A, side scatter area; FSC-A, forward scatter area; FSC-W, forward scatter width. (B) Comparison of percentages of the CD57⁻, CD57^dim, and CD57^bright subpopulations in control subjects (n = 14) and HIV-seropositive individuals (n = 34) on CD56^dim CD16⁺ NK cells. ns, not significant (P > 0.05); **, P < 0.01; ***, P < 0.001. (C) Frequencies of CD57⁻, CD57^dim, and CD57^bright expressing CD56^dim CD16⁺ NK cells in relation to total NK cells in control subjects (n = 14) and HIV-seropositive individuals (n = 34). (D) Mean frequency of CD56^dim CD16⁺ NK cells in 14 control individuals and in 34 HIV-infected people and the distribution of CD57⁻, CD57^dim, and CD57^bright cells within CD56^dim CD16⁺ NK cells is shown. (E) Relationship between percentage of CD57^dim CD56^dim CD16⁺NK cells and percentage of CD56^neg CD16⁺ NK cells on total NK cells. Horizontal bars in dot plots show the means.NK cells as defined above were sorted from cryopreserved PBMCs on a FACSAria (purities ranged from 91% to 99%). An amount of 10⁵ NK cells was plated per well and stimulated with 10 ng/ml interleukin-15 (IL-15), 100 ng/ml IL-12, and 5 × 10⁴ K562 cells. A CD107a degranulation assay was performed as described previously (1, 12). GraphPad Prism (version 5.0) software was used for statistical evaluation of data. Correlation analysis was performed using the Pearson test. The unpaired t test was performed when two groups were compared, and all t tests were two tailed. Comparison of more than two groups was performed using one-way analysis of variance followed by Tukey''s post-hoc test. P values of less than 0.05 were considered significant.We found that CD57 on NK cells was predominantly expressed on the CD56^dim CD16⁺ population (Fig. ​(Fig.1A).1A). The expression patterns of CD57 allowed us to differentiate between three subfractions within CD56^dim CD16⁺ NK cells, namely, CD57⁻, CD57^dim, and CD57^bright cells. The frequency of the CD57^bright subpopulation on CD56^dim CD16⁺ NK cells was increased compared to the frequency of the CD57^dim subpopulation on CD56^dim CD16⁺ NK cells in HIV-seropositive patients but not in HIV-seronegative control subjects (Fig. ​(Fig.1B).1B). This relative increase was associated with substantial reductions of the CD57⁻ CD56^dim and the CD57^dim CD56^dim NK cell subpopulations of total NK cells in our HIV-seropositive cohort compared to these subpopulations in healthy control subjects (means, 36.6% versus 24.8% [P = 0.0002] and 22.4% versus 15.4% [P = 0.0001]), but the frequencies of CD57^bright CD56^dim NK cells within total NK cells were similar between HIV-infected patients and HIV-seronegative individuals (Fig. ​(Fig.1C).1C). In accordance with previously published data (3, 23), we could confirm that there is a relative loss of CD56^dim CD16⁺ NK cells in HIV infection (mean, 84.3% versus 67.0%, P = 0.0004) (Fig. ​(Fig.1D).1D). Our data indicate that this loss is predominantly due to decreased numbers of CD57⁻ CD56^dim and CD57^dim CD56^dim NK cells, leading to a relative overrepresentation of CD57^bright cells within CD56^dim CD16⁺ NK cells in HIV infection (Fig. ​(Fig.1C).1C). There was no significant correlation between the relative loss of CD57⁻ and CD57^dim NK cells and absolute numbers of CD56^dim CD16⁺ NK cells, but there was a significant inverse correlation between loss of CD57^dim NK cells and increasing percentages of CD56⁻ CD16⁺ cells (Pearson r = −0.54, P = 0.001) (Fig. ​(Fig.1E1E).To determine whether the relative decrease of CD57⁻ and CD57^dim NK cells was associated with parameters of HIV disease progression, we performed correlation analysis of the percentages of CD57⁻ or CD57^dim cells with viral load and CD4 T cell counts. We found no such correlations (Pearson r < 0.2 and P > 0.05 for all) (data not shown). A recent cross-sectional and longitudinal study demonstrated that changes in the NK cell compartment, as shown by down-modulation of Siglec-7 on CD56^dim NK cells, are associated with HIV viremia (5). The longitudinal data in the study indicated that the full restoration of NK cell pathologies required 24 months of antiviral treatment. This suggests that alterations in the NK cell compartment can be driven by HIV viral load but that these changes seem to require a significant amount of time.We next investigated the phenotypic and functional properties of the CD57⁻, CD57^dim, and CD57^bright subpopulations on CD56^dim CD16⁺ NK cells. For KIR2DL2/DL3/DS2, we detected increasing prevalences of KIR-expressing NK cells with increasing expression of CD57 in both healthy control subjects and HIV-infected blood donors (Fig. ​(Fig.2A).2A). As for KIR3DS1/DL1, we found an increase of KIR⁺-expressing NK cells between CD57⁻ and CD57^bright cells in control individuals and significant differences in percentages of KIR3DS1/DL1-expressing NK cells between CD57⁻ and CD57^dim, as well as between CD57⁻ and CD57^bright, NK cells in our HIV-positive cohort (Fig. ​(Fig.2A).2A). These results suggest that increasing CD57 expression is associated with higher numbers of KIR-expressing NK cells in control subjects and HIV-infected subjects.Open in a separate windowFIG. 2.Phenotypic characterization of the CD57⁻, CD57^dim, and CD57^bright subpopulations of CD56^dim CD16⁺ NK cells. Representative flow cytometry plots for one control and one HIV-infected subject and summary data for all individuals whose PBMCs were analyzed are shown. CD57⁻, CD57^dim, and CD57^bright NK cells are concatenated to visualize them in a single dot plot. Numbers in contour plots indicate percentages of gated events of the respective subset. (A) Percentages of KIR2DL2/DL3/DS2 and KIR3DS1/DL1-expressing CD57⁻, CD57^dim, and CD57^bright cells were analyzed in control individuals (n = 15) and HIV-infected subjects (n = 37). (B) Numbers of HLA-DR-expressing and CD27-expressing CD57⁻, CD57^dim, and CD57^bright cells in control individuals'' (n = 15) and HIV-infected subjects'' (n = 37) PBMCs were analyzed. Horizontal bars in dot plots show the means. ns, not significant (P > 0.05); *, P < 0.05; **, P < 0.01; ***, P < 0.001.We next addressed the question of whether increasing CD57 expression is linked to differential phenotypic properties of NK cells and analyzed the HLA-DR and CD27 expression of the CD57⁻, CD57^dim, and CD57^bright subpopulations on CD56^dim CD16⁺ NK cells. A significantly higher fraction of NK cells expressed HLA-DR in the CD57⁻ than in the CD57^bright subset in both healthy control individuals and HIV-infected subjects (Fig. ​(Fig.2B).2B). A considerably higher portion of NK cells was positive for HLA-DR in HIV-infected individuals than in control subjects (means, 3.2% versus 13.2% [P < 0.0001], 1.8% versus 10.4% [P = 0.001], and 0.9% versus 6.5% [P = 0.005] for CD57⁻, CD57^dim, and CD57^bright subpopulations, respectively). We furthermore detected marked differences in frequencies of cells expressing CD27, a member of the tumor necrosis factor (TNF) receptor family (24). CD57⁻ NK cells displayed the highest percentages of CD27⁺ cells, whereas CD57^bright cells were almost all negative for CD27, in both control individuals and HIV-seropositive subjects (Fig. ​(Fig.2B).2B). We thus show that increasing expression of CD57 is associated with differential activation status and differential phenotype.Next, we sought to determine whether CD57 is linked to differential functional phenotypes by assessing the intracellular expression of granzyme B, perforin, and Ki-67. The frequencies of perforin-expressing NK cells did not vary within the different CD57 subsets of CD56^dim CD16⁺ NK cells (Fig. ​(Fig.3A).3A). However, we found that CD57^bright cells displayed the highest frequencies of granzyme B⁺ in both control and HIV-seropositive subjects, whereas CD57⁻ cells exhibited the lowest percentages for granzyme B⁺ cells (Fig. ​(Fig.3A).3A). Conversely, when we studied the expression of Ki-67, we identified the opposite trend: less than 5% of CD57^bright cells in control individuals and less than 10% of CD57^bright cells in HIV-infected study subjects expressed Ki-67 (Fig. ​(Fig.3B).3B). The highest numbers of Ki-67⁺ cells were found in the CD57⁻ population.Open in a separate windowFIG. 3.Functional characterization of CD57⁻, CD57^dim, and CD57^bright cells within the CD56^dim CD16⁺ NK cell population. (A) Representative staining results for granzyme B and perforin and summary data for control (n = 14) and HIV-seropositive subjects (n = 36). Numbers in the concatenated contour plots indicate percentages of gated events of the respective subset. B cells were defined as the negative control for granzyme and perforin staining. (B) Percentages of Ki-67⁺ and CD107a⁺ cells on CD57⁻, CD57^dim, and CD57^bright cells within the CD56^dim NK cell population in control (n = 14 and n = 9, respectively) and HIV-seropositive (n = 36 and n = 21, respectively) subjects'' PBMCs were analyzed. Horizontal bars in dot plots show the means. NC, negative control; ns, not significant (P > 0.05); *, P < 0.05; **, P < 0.01; ***, P < 0.001.We also assessed the presence of the degranulation marker CD107a on CD57⁻, CD57^dim, and CD57^bright subpopulations of CD56^dim CD16⁺ NK cells after stimulation with IL-12 and IL-15 and exposure to K562 cells. Similarly to what we had observed for Ki-67 expression, CD57⁻ cells were the most efficient at degranulation when compared with CD57^dim and CD57^bright cells in HIV-infected individuals. Comparison to healthy controls revealed that there was a higher expression of CD107a in HIV-seropositive subjects for each CD57 subset. However, the most effective degranulation occurred in the CD57⁻ and CD57^dim subsets, which are preferentially depleted in HIV infection.We focused our analysis on CD56^dim CD16⁺ NK cells because they constitute the largest NK cell subset in peripheral blood, they are the major NK cell subset expressing CD57 and KIRs, and they are the most prominent subpopulation for cytolytic activity. CD56^dim CD16⁺ cells but not CD56^bright CD16⁻ NK cells were reported to be decreased in HIV-infected subjects (23), which we could confirm in our experiments (data not shown). We did not find CD57 on CD56^bright CD16⁻ NK cells either in healthy or in HIV-infected individuals. CD57 has been described as a marker for replicative senescence, and its expression has been associated with shorter telomeres and diminished proliferative capacities on T and NK cells (4). The presence of this marker on CD56^dim CD16⁺ but not on CD56^bright CD16⁺ NK cells might explain why the latter subset was shown to proliferate more efficiently upon cytokine stimulation (6). We demonstrated that increasing CD57 expression on NK cells was associated with lower numbers of CD27-expressing cells, a marker which is mainly expressed by CD56^bright CD16⁻ NK cells (24). CD56^bright CD16⁻ cells were suggested to be early NK cells, which differentiate from CD34^dim CD45RA⁺ hematopoietic precursor cells with high expression of integrin α₄β₇ (11). These cells can furthermore give rise to CD56^dim CD16⁺ NK cells (7). Our data support this hypothesis, as we show that CD57 can be found on CD56^dim CD16⁺ NK cells but not on CD56^bright NK cells, whereas the opposite is observed for CD27.We demonstrate that differential CD57 expression is associated with distinct functional characteristics. We show for the first time that increasing expression of CD57 on CD56^dim CD16⁺ NK cells is associated with increasing prevalence of KIR⁺ and granzyme B⁺ cells. These cells appear to be more mature and differentiated in terms of KIR and granzyme B expression but less functionally active, as shown by decreased expression of Ki-67 and CD107a. We therefore propose that CD57 is not only a marker for replicative senescence but, in addition, a marker for terminal differentiation on NK cells, which is characterized by increased expression of KIR and higher granzyme B content and “counterbalanced” by decreased degranulation (CD107a) and decreased proliferation (Ki-67).Notably, we observed consistently higher frequencies of granzyme B⁺ cells in all three subsets within CD56^dim CD16⁺ NK cells from HIV-seropositive individuals than in healthy control subjects (means, 52.9% versus 78.7% [P < 0.0001], 65.3% versus 89.6% [P < 0.0001], and 76.5% versus 95.0% [P < 0.0001]for CD57⁻, CD57^dim, and CD57^bright subpopulations, respectively) (Fig. ​(Fig.1C).1C). Furthermore, HIV infection was associated with higher numbers of Ki-67-expressing NK cells (means, 8.4% versus 16.1% [P = 0.0005], 5.3% versus 11.6% [P = 0.0016], and 4.1% versus 6.2% [P = 0.04]) (Fig. ​(Fig.1C).1C). These changes, including the strong increase in HLA-DR-expressing NK cells, probably reflect the systemic immune activation in HIV-infected individuals.In summary, these findings support a view of a differential regulation of NK function and are in concordance with maturation of NK cells with high expression of CD57 on NK cells with a more terminally differentiated phenotype. Our data indicate that high turnover; activation status; and active degranulation as characterized by the expression of Ki-67, HLA-DR, and CD107a are mainly features of CD57⁻ and much less of CD57^dim NK cells. HIV infection is associated with increased activation, proliferation, and cytotoxicity during “early” stages of CD56^dim CD16⁺ NK cell differentiation compared to their occurrence in healthy controls, but those are the very cells that are significantly decreased in chronic HIV infection. A loss of these functionally more active NK cells may be a yet-unappreciated factor in overall NK cell pathology and a further possible explanation for the impairment of NK cells in their contribution to viral control in HIV infection.     

Efficacious Early Antiviral Activity of HIV Gag- and Pol-Specific HLA-B*2705-Restricted CD8+ T Cells

The association between HLA-B*2705 and the immune control of human immunodeficiency virus type 1 (HIV-1) has previously been linked to the targeting of the HLA-B*2705-restricted Gag epitope KRWIILGLNK (KK10) by CD8⁺ T cells. In order to better define the mechanisms of the HLA-B*2705 immune control of HIV, we first characterized the CD8⁺ T-cell responses of nine highly active antiretroviral therapy (HAART)-naïve B*2705-positive subjects. Unexpectedly, we observed a strong response to an HLA-B*2705-restricted Pol epitope, KRKGGIGGY (KY9), in 8/9 subjects. The magnitude of the KY9 response was only marginally lower than that of the KK10-specific response (median, 695 versus 867 spot-forming cells [SFC]/million peripheral blood mononuclear cells [PBMCs]; not significant [NS]), and viral escape mutants were observed in both KY9 and KK10, resulting from selection pressure driven by the respective CD8⁺ T-cell response. By comparing inhibitions of viral replication by CD8⁺ T cells specific for the Gag KK10, Pol KY9, and Vpr VL9 HLA-B*2705-restricted epitopes, we observed a consistent hierarchy of antiviral efficacy (Gag KK10 > Pol KY9 > Vpr VL9). This hierarchy was associated with early recognition of HIV-1-infected cells, within 6 h of infection, by KK10- and KY9-specific CD8⁺ T cells but not until 18 h postinfection by VL9-specific CD8⁺ T cells. There was no association between antiviral efficacy and proliferative capacity, cytotoxicity, polyfunctionality, or T-cell receptor (TCR) avidity. These data are consistent with previous studies indicating an important role for the B*2705-Gag KK10 response in the control of HIV but also suggest a previously unrecognized role played by the subdominant Pol-specific KY9 response in HLA-B*2705-mediated control of HIV and that the recognition of HIV-infected cells by CD8⁺ T cells early in the viral life cycle may be important for viral containment in HIV-infected individuals.Current human immunodeficiency virus (HIV) vaccine strategies are focused on emulating the protective effect observed for HIV-infected individuals carrying alleles such as B*2705 by inducing the virus-specific CD8⁺ T-cell responses that are thought to be responsible for delaying or preventing disease progression. Understanding why such alleles confer protection facilitates a rational approach to vaccine design. It has been hypothesized that the slow progression to AIDS exhibited by HLA-B*2705-positive (HLA-B*2705⁺) HIV-infected individuals is due to the immunodominant B*27-restricted CD8⁺ T-cell response toward the p24 Gag epitope KRWIILGLNK (KK10) (Gag residues 263 to 272). Escape from this epitope typically occurs late in infection and is associated with rapid progression to AIDS (14, 16). The commonly selected mutation R264K abrogates CD8⁺ T-cell recognition but also confers a substantial fitness cost to the virus, and the selection of compensatory mutations is required to restore viral replicative capacity (19, 29, 30). This has prompted the hypothesis that CD8⁺ T-cell responses that can drive escape mutations that reduce viral fitness are a contributing factor in the immune control of HIV, either by promoting the outgrowth of a viral quasispecies with a lower replicative capacity or by delaying the selection of escape mutations, both of which may slow the onset of AIDS (11, 21, 25).To better understand how CD8⁺ T cells can be most effective against HIV, recent studies have directly assessed the antiviral activity of CD8⁺ T cells via the viral suppression of HIV-infected CD4⁺ T cells during coculture. Such studies indicated that Gag-specific CD8⁺ T cells have a higher potency for viral suppression than Env-specific CD8⁺ T cells (10), supporting previous data indicating that broad CD8⁺ T-cell targeting of Gag epitopes was associated a with lower viral set point and, hence, slower progression to AIDS (20). A recent study of simian immunodeficiency virus (SIV) suggested that the protective effect of Gag-specific CD8⁺ T cells is mediated by the early presentation of Gag epitopes, processed from the viral Gag protein from incoming virions during infection, which can sensitize target cells for lysis by Gag-specific CD8⁺ T cells within 6 h of infection (26, 27). In addition, it was proposed previously that the ability of CD8⁺ T cells to secrete multiple cytokines may also be an important correlate of immune protection (6), and a further recent study demonstrated a more polyfunctional cytokine profile of Gag-specific B*2705-KK10 CD8⁺ T-cell responses than those of other HIV-specific CD8⁺ T-cell responses (1). The ability of CD8⁺ T cells to proliferate in response to the cognate epitope peptide has also been associated with immune control (1, 12). Other studies demonstrated the importance of lytic granule loading of CD8⁺ T cells for the effective elimination of HIV-infected cells (6, 22). However, the induction of a Gag KK10-specific CD8⁺ T-cell vaccine response in a B*2705-positive vaccinee did not protect against rapid progression following subsequent HIV-1 infection (5). This anecdotal case suggests the possibility that HLA-B*2705-associated immune control of HIV-1 may not be dependent on the Gag KK10-specific CD8⁺ T-cell response alone.Since current vaccine strategies hope to induce a protective effect, such as that observed for HLA-B*2705⁺ HIV-infected individuals, the study of the functional and phenotypic characteristics of B*2705-specific CD8⁺ T cells provides an opportunity to redefine the proposed correlates of immune protection essential for rational vaccine design. In this study we analyze three different specificities of HLA-B*2705-restricted CD8⁺ T cells from chronically HIV-infected individuals in order to directly compare antiviral activity with potential correlates of immune protection, including the kinetics of viral inhibition, cytokine profile, granzyme production, proliferative capacity, and cytotoxicity.     

Interleukin-7 enhances proliferation responses to T-cell receptor stimulation in naïve CD4+ T cells from human immunodeficiency virus-infected persons

Proliferation responses of naïve CD4⁺ T cells to T-cell receptor and interleukin-7 (IL-7) stimulation were evaluated by using cells from human immunodeficiency virus-positive (HIV⁺) donors. IL-7 enhanced responses to T-cell receptor stimulation, and the magnitude of this enhancement was similar in cells from healthy controls and from HIV⁺ subjects. The overall response to T-cell receptor stimulation alone or in combination with IL-7, however, was diminished among viremic HIV⁺ donors and occurred independent of antigen-presenting cells. Frequencies of CD127⁺ cells were related to the magnitudes of proliferation enhancement that were mediated by IL-7. Thus, IL-7 enhances but does not fully restore the function of naïve CD4⁺ T cells from HIV-infected persons.Interleukin-7 (IL-7) plays an important role in T-cell homeostasis by modulating thymic output (1, 16, 22) and by enhancing the peripheral expansion and survival of both naïve and memory T-cell subsets (12, 18, 20, 25, 26, 31, 32). Under normal circumstances, the homeostatic maintenance of naïve CD4⁺ T cells is regulated by at least two types of signals that include T-cell receptor (TCR) engagement and IL-7 (10, 26, 30). In addition, IL-7 may play an important role in the conversion of effector T cells into long-term memory cells (12, 14).Homeostasis of T cells is dysregulated in human immunodeficiency virus (HIV) infection such that there is a marked depletion of CD4⁺ cells and a progressive loss of naïve CD4 and CD8⁺ T cells (24). Although the mechanisms for these deficiencies are not fully understood, it is possible that impairments in T-cell proliferation and responsiveness to immunomodulatory cytokines could play a role. In HIV disease, IL-7 is increased in plasma (2, 5, 11, 15, 19, 21, 23) and the alpha chain of the IL-7 receptor, CD127, is less frequently expressed among T lymphocytes (2, 5, 11, 21, 23). The ability of patient T cells to respond to IL-7 stimulation may be diminished in HIV disease but may not be related to the density of CD127 expression as it is in T cells from healthy controls (4). Moreover, the responsiveness of T cells, including naïve CD4⁺ lymphocytes, to TCR stimulation is diminished in HIV disease (27-29). Thus, defects in responsiveness to cytokines or TCR stimulation could contribute to the perturbations in T-cell proliferation and survival in HIV disease.In these studies, we examined the responsiveness of naïve CD4⁺ T cells from viremic HIV-positive (HIV⁺) donors (median plasma HIV RNA level, 25,200 copies/ml [range, 1,015 to 1,000,000 copies/ml]; median CD4 cell count, 429 cells/μl [range, 41 to 950 cells/μl]; median age, 38 years [range, 22 to 64 years]; n = 25) and aviremic, highly active antiretroviral therapy (HAART)-treated HIV⁺ donors (plasma HIV RNA level, <400 copies/ml; median CD4 cell count, 309 cells/μl [range, 74 to 918 cells/μl]; median age, 48 years [range, 37 to 55 years]; n = 12) to the combined stimulus of recombinant IL-7 (Cytheris) plus agonistic anti-CD3 antibody. Peripheral blood mononuclear cells (PBMC) were depleted of CD45RO⁺ cells by magnetic bead depletion (>90% purity) and were incubated in medium alone or were stimulated with anti-CD3 antibody, IL-7, or anti-CD3 antibody plus IL-7. CD4⁺CD45RO⁻CD28⁺CD27⁺ cells were assessed for the expression of Ki67 2 days poststimulation by flow cytometric analyses. The addition of IL-7 to anti-CD3 antibody enhanced the induction of Ki67 expression in cells from both HIV⁺ and HIV-negative (HIV⁻) donors (Fig. ​(Fig.11 and Fig. ​Fig.2).2). A diminished response to anti-CD3 antibody was observed among naïve CD4⁺ T cells from viremic HIV⁺ donors. In contrast, cells from aviremic HIV⁺ donors (all receiving antiretroviral therapy) had normal responses to anti-CD3 antibody compared to cells from healthy donors (Fig. ​(Fig.2).2). Importantly, the addition of IL-7 to the cultures significantly improved the responses to above those observed with anti-CD3 alone in HIV⁻ and HIV⁺ donors, regardless of viremia (Wilcoxon signed ranks test; for each comparison, P was <0.04), and the magnitude of that enhancement, although slightly diminished in cells from HIV⁺ donors, was not significantly different between groups of subjects when measured as either the enhancement (n-fold; not shown) or as the change in percent Ki67⁺ cells above the background observed for cells stimulated with anti-CD3 alone (Fig. ​(Fig.3).3). Although IL-7 enhanced responses to TCR stimulation in HIV⁻ subjects, the overall magnitude of the responses among cells from HIV viremic subjects did not reach the levels seen with cells from healthy donors, even in the presence of IL-7 (Fig. ​(Fig.2).2). It should be noted, however, that these functional readouts were not related to clinical indices of plasma HIV RNA level, CD4 cell count, or age when considered as continuous variables, suggesting that the functional perturbations in naïve CD4⁺ T cells are probably undermined by complexities extending beyond HIV replication (not shown). Together, these results suggest that TCR responsiveness is diminished in naïve CD4⁺ T cells from viremic HIV⁺ subjects, whereas responsiveness to IL-7 stimulation is relatively preserved.Open in a separate windowFIG. 1.IL-7 enhances the induction of Ki67 expression in naïve CD4⁺ T cells from healthy controls and HIV⁺ donors. CD45RO-depleted PBMC were incubated with anti-CD3 antibody (100 ng/ml), IL-7 (50 ng/ml), anti-CD3 antibody plus IL-7, or medium alone (RPMI with 10% fetal bovine serum). Cells were gated on CD4⁺CD27⁺CD28⁺ lymphocytes and examined for Ki67 expression by intracellular flow cytometry.Open in a separate windowFIG. 2.IL-7 responsiveness in cells from viremic and aviremic HIV⁺ donors. Plotted values represent the percentages of CD4⁺CD27⁺CD28⁺CD45RO⁻ T cells that expressed Ki67 after a 2-day incubation with anti-CD3 or with anti-CD3 plus IL-7. Percentages of Ki67⁺ cells in cultures without stimulation or with IL-7 only were subtracted from the values shown. Responses of cells from healthy controls (n = 9), HIV⁺ subjects with plasma HIV RNA levels of >400 copies/ml (n = 25), and HIV⁺ subjects on HAART with suppressed viral replication (<400 copies/ml; n = 12) are shown. Statistically significant differences between cells from controls and HIV⁺ donors are indicated. Analyses included Kruskal-Wallis test (P = 0.002) for multigroup comparisons and Mann-Whitney U test for comparison of two groups (*, P < 0.05).Open in a separate windowFIG. 3.IL-7 enhances responses to anti-CD3 antibody stimulation to a similar degree in cells from HIV⁺ and HIV⁻ donors. Naïve CD4⁺ T cells were incubated with IL-7, anti-CD3, anti-CD3 plus IL-7, or medium alone for 2 days. Background division (percent Ki67⁺ cells) in medium alone or IL-7 alone was first subtracted from the responses observed with cells stimulated with anti-CD3 alone or with anti-CD3 plus IL-7, respectively. The magnitude of IL-7 enhancement was then calculated by subtracting the percentage of naïve CD4⁺ cells that expressed Ki67⁺ after anti-CD3 antibody stimulation from the percentage of naïve CD4⁺ cells that expressed Ki67 after stimulation with anti-CD3 plus IL-7. n = 9, 25, and 12 for healthy controls, viremic subjects, and aviremic subjects, respectively.Previous studies indicate that the frequency of CD127⁺ T cells, particularly memory T-cell subsets, is reduced in patients with HIV disease (5, 11, 21, 23). This could, in part, result from the modulation of receptor expression through increased exposure to IL-7 in vivo and also may reflect accumulation of CD127⁻ effector memory cells (21). We assessed the expression of CD127 in naïve CD4⁺CD45RA⁺CD28⁺CD27⁺ and memory CD4⁺CD45RO⁺ T cells in a subset of patients and asked if the frequencies of CD127⁺ cells were related to the induction of Ki67 expression by anti-CD3 or by anti-CD3 plus IL-7 among naïve CD4⁺ T cells. We reasoned that the ability of IL-7 to enhance responses to TCR stimulation might be limited if CD127 expression was diminished among naïve CD4⁺ T cells from HIV⁺ donors. Alternatively, a defect in functional responses also could be related to increased exposure to IL-7 in vivo, as may be reflected by the absence of CD127 receptor expression on memory T-cell subsets.In agreement with previous studies, our results suggest that CD127 expression is relatively preserved in naïve CD4⁺ T cells from HIV⁺ donors (representative histograms in Fig. ​Fig.4)4) (mean percentage of CD127⁺ cells, 87 and 83 for HIV⁻ donors [n = 5] and HIV⁺ donors [n = 17], respectively; P = 0.96) but is diminished in memory CD4⁺ T cells from HIV⁺ donors (mean percentage of CD127⁺ cells, 83 and 59 for HIV⁻ and HIV⁺ donors, respectively; P = 0.01). The frequencies of CD127⁺ naïve T cells were directly related to the frequencies of CD127⁺ memory T cells (Spearman''s correlations; r = 0.711, P = 0.001; n = 18) in HIV⁺ subjects. This result suggests that a similar mechanism modulates the expression of CD127 in these T-cell subsets, even though the loss of CD127 expression is clearly greater among the memory T cells in HIV disease. Neither CD127 expression among naïve CD4⁺ T cells nor CD127 expression among memory CD4⁺ T cells was related to the functional response of naïve CD4⁺ T cells to anti-CD3 (r = 0.238 and P = 0.36 for naïve CD127 expression; r = 0.293 and P = 0.25 for memory CD127 expression) or to anti-CD3 plus IL-7 (r = 0.32 and P = 0.21 for naïve CD127 expression; r = 0.31 and P = 0.22 for memory CD127 expression). There was a relationship between the percentage of CD127⁺ naïve T cells and the delta Ki67 expression that resulted from the addition of IL-7 to anti-CD3-treated cultures (percentage of Ki67⁺ cells in cultures treated with anti-CD3 plus IL-7 minus the percentage of Ki67⁺ cells in cultures treated with anti-CD3 alone) (Fig. ​(Fig.4).4). This relationship was statistically significant by Pearson''s correlation (r = 0.5, P = 0.041), the use of which was justified based on the normal distribution of the data. Spearman''s analysis, which is independent of data distribution, indicated a similar trend that was not statistically significant (r = 0.41, P = 0.1). The mean fluorescence intensity of CD127 expression on CD4⁺CD45RA⁺CD27⁺CD28⁺ T cells was not significantly related to the delta Ki67 expression induced by IL-7 but also suggested a trend consistent with a direct relationship between these indices (r = 0.45 and P = 0.07 by Pearson''s correlation; r = 0.34 and P = 0.18 by Spearman''s correlation). Despite the relative preservation of IL-7 receptor in naïve CD4⁺ T cells from HIV⁺ donors, the association between the frequencies of CD127⁺ cells and CD4⁺ T-cell proliferation responses to TCR plus IL-7 suggests that subtle IL-7 receptor perturbations might contribute to functional defects of naïve CD4⁺ T cells in HIV-infected persons.Open in a separate windowFIG. 4.CD127 receptor expression is related to enhancement of proliferation by IL-7. (A) Whole blood from a healthy control and an HIV-infected person was examined by flow cytometry for expression of CD127 on CD4⁺CD45RA⁺CD27⁺CD28⁺ (naïve) T cells. The gating strategy for identifying naïve cells involved an initial gate for lymphocyte forward and side scatter (SSC) characteristics (not shown) and then sequential gates for CD4 positive, CD45RA positive and, finally, CD28⁺CD27⁺ double-positive cells. (B) Plotted values indicating the relationship between the delta Ki67 expression in naïve CD4⁺ T cells and the percentage of CD127⁺ naïve T cells that was determined by using freshly isolated whole blood. The delta Ki67 expression was calculated by subtracting the percentage of naïve CD4⁺ cells that expressed Ki67⁺ after anti-CD3 antibody stimulation from the percentage of naïve CD4⁺ cells that expressed Ki67 after stimulation with anti-CD3 plus IL-7.To consider the possibility that antigen-presenting cells could contribute to the diminished response of T-cells to stimulation with TCR plus IL-7, we next asked if defects in TCR-plus-IL-7 stimulation could be detected in purified naïve CD4⁺ T-cell populations. CD4⁺CD45RO⁻ cells were negatively selected by magnetic bead depletion, achieving a purity of >90% as determined by flow cytometric analyses. Purified naïve CD4⁺ T cells were labeled with carboxy fluorescein succinimidyl ester (CFSE) tracking dye and incubated with IL-7, anti-CD3 antibody that was immobilized on a plate, anti-CD3 plus IL-7, or medium alone. The induction of proliferation was measured 7 days later by the dilution of CFSE tracking dye among CD4⁺CD27⁺ cells by calculating the division index (average number of cell divisions of all CD4⁺CD27⁺ cells) and the proliferation index (average number of divisions of CD4⁺CD27⁺ cells that had diluted tracking dye; Flow-Jo analysis software). These purified CD4⁺ T cells proliferated poorly in response to anti-CD3 antibody stimulation alone, providing functional evidence that the samples were free of antigen-presenting cell contamination (Fig. ​(Fig.5A).5A). The combined treatment of anti-CD3 and IL-7 induced cellular expansion, whereas alone, neither stimulus induced cellular proliferation during the 7-day period (Fig. ​(Fig.5A).5A). Responses of cells from HIV⁺ donors were reduced compared to those of cells from healthy donors, confirming that the defects in naïve CD4⁺ T-cell expansion are independent of antigen-presenting cells and not fully corrected by IL-7 (Fig. ​(Fig.5B5B).Open in a separate windowFIG. 5.Diminished responses to TCR plus IL-7 in purified naïve CD4⁺ T cells from HIV⁺ donors. CD4⁺CD45RO⁻ cells were purified from PBMC by negative selection. Cells from HIV⁺ donors (n = 7) and healthy controls (n = 7) were labeled with CSFE and incubated with anti-CD3 immobilized on a plate (5 μg/ml, overnight at 4°C) plus IL-7 (10 ng/ml). CFSE dye dilution was measured among the CD4⁺CD27⁺ cells. (A) Representative histograms showing the dilution of CFSE and CD27 expression among cells incubated with anti-CD3 antibody alone, IL-7 alone, or the combination of anti-CD3 plus IL-7. Placements of quadrant gates were based on an isotype control antibody stain (for CD27 expression) and on cells that had been incubated in medium alone (for CFSE dye dilution). (B) Division indices (average number of cell divisions among CD4⁺CD27⁺ cells) and proliferation indices (average number of cell divisions among CD4⁺CD27⁺ cells that had diluted tracking dye) are shown.IL-7 is a promising candidate for therapeutic and vaccine adjuvant applications in HIV disease. This cytokine may be especially beneficial in circumstances of immune reconstitution, since it normally plays an essential role in T-cell proliferation and survival. Here, we demonstrate that IL-7 efficiently enhances TCR-triggered naïve CD4⁺ T-cell expansion in cells from healthy individuals and from HIV⁺ donors. The mechanism of IL-7 activity is not discerned in these experiments but may involve effects on survival, such as the induction of Bcl-2 (9), or may involve the enhancement of IL-2 or IL-2 receptor expression (6, 8). In any case, our studies provide evidence that IL-7 should provide an effective therapy for the regulation of naïve CD4⁺ T-cell homeostasis and may be useful for vaccine adjuvant applications in HIV disease. The potential of this approach has been illustrated by recent human trials of IL-7 that demonstrated the expansion of naïve T cells in vivo after IL-7 administration to HIV-infected persons (13) and by animal studies, wherein IL-7 administration enhanced T-cell responses to immunization in mice (17).Notably, the depletion studies and purification methods employed here did not necessarily eliminate terminally differentiated effector memory CD4⁺ T cells from our cultures; however, studies of CMV-specific terminally differentiated cells suggested that these cells are primarily CD27⁻ (3), and the use of three markers to identify naïve CD4⁺ T cells, including the ones used here (CD27, CD28, and CD45RO) is estimated to provide 98% assurance that the cells being examined are truly naïve (7). Thus, it is likely that terminally differentiated cells were largely removed from our analyses.Our observations provide confirmation of a significant defect in the responses of naïve CD4⁺ T cells to TCR triggering in HIV disease, and this defect is not fully corrected by IL-7, as shown here, or by IL-2, as we demonstrated previously (27). These deficiencies are reproduced even among naïve CD4⁺ T cells that are purified from professional antigen-presenting cells, indicating that the defects are intrinsic to the T cells and not a consequence of dysfunctional antigen-presenting cells. We propose that functional defects in naïve CD4⁺ T cells from HIV⁺ donors stem primarily from deficiencies in TCR signaling. Further studies that define the nature of naïve CD4⁺ T-cell defects in HIV disease will be required to address the underlying mechanisms.     

Functional Analysis of the Nicotinate Mononucleotide:5,6-Dimethylbenzimidazole Phosphoribosyltransferase (CobT) Enzyme,Involved in the Late Steps of Coenzyme B12 Biosynthesis in Salmonella enterica

In Salmonella enterica, the CobT enzyme activates the lower ligand base during the assembly of the nucleotide loop of adenosylcobalamin (AdoCbl) and other cobamides. Previously, mutational analysis identified a class of alleles (class M) that failed to restore AdoCbl biosynthesis during intragenic complementation studies. To learn why class M cobT mutations were deleterious, we determined the nature of three class M cobT alleles and performed in vivo and in vitro functional analyses guided by available structural data on the wild-type CobT (CobT^WT) enzyme. We analyzed the effects of the variants CobT(G257D), CobT(G171D), CobT(G320D), and CobT(C160A). The latter was not a class M variant but was of interest because of the potential role of a disulfide bond between residues C160 and C256 in CobT activity. Substitutions G171D, G257D, and G320D had profound negative effects on the catalytic efficiency of the enzyme. The C160A substitution rendered the enzyme fivefold less efficient than CobT^WT. The CobT(G320D) protein was unstable, and results of structure-guided site-directed mutagenesis suggest that either variants CobT(G257D) and CobT(G171D) have less affinity for 5,6-dimethylbenzimidazole (DMB) or access of DMB to the active site is restricted in these variant proteins. The reported lack of intragenic complementation among class M cobT alleles is caused in some cases by unstable proteins, and in others it may be caused by the formation of dimers between two mutant CobT proteins with residual activity that is so low that the resulting CobT dimer cannot synthesize sufficient product to keep up with even the lowest demand for AdoCbl.Cobalamin (Cbl, also known as B₁₂) is a structurally complex cyclic tetrapyrrole with a cobalt ion coordinated by equatorial bonds with pyrrolic nitrogen atoms and is unique among cyclic tetrapyrroles (e.g., heme, chlorophylls, coenzyme F₄₃₀) in that it has an upper and a lower axial ligand (Fig. ​(Fig.1).1). The coenzymic form of Cbl is known as adenosylcobalamin (AdoCbl) or coenzyme B₁₂.Open in a separate windowFIG. 1.Role of CobT in the late steps of AdoCbl biosynthesis. This branch of the AdoCbl biosynthetic pathway is known as the NLA pathway. The black box in the inner membrane represents the corrinoid-specific ABC transporter BtuCD. The inset shows the chemical structure of AdoCbl; the coring ring in the scheme is represented by the rhomboid cartoon with the Co ion in the middle.Some bacteria and archaea synthesize AdoCbl de novo or from preformed precursors such as cobyric acid (Cby) or cobinamide (Cbi) (Fig. ​(Fig.1)1) (11, 32). The enterobacterium Salmonella enterica serovar Typhimurium LT2 (hereafter referred to as S. Typhimurium) synthesizes the corrin ring of AdoCbl de novo only under anoxic conditions (15). Although oxygen blocks de novo corrin ring biosynthesis in this bacterium, it does not block the assembly of adenosylcobalamin (AdoCbl, coenzyme B₁₂) if cells are provided with preformed, incomplete corrinoids such as Cbi or Cby (11, 13-15).The late steps in AdoCbl biosynthesis can be divided into two different branches that comprise the nucleotide loop assembly (NLA) pathway (19). One of the branches of the NLA pathway activates the lower ligand base, while the other one activates adenosylcobinamide (AdoCbi) to AdoCbi-GDP (Fig. ​(Fig.11).In this paper, we focus on the activation of 5,6-dimethylbenzimidazole (DMB), the lower ligand base of AdoCbl. There are two ways in which DMB can be activated. In both cases, the CobT enzyme (EC 2.4.2.21) catalyzes the reaction, but the product of the reaction varies, depending on whether the cosubstrate of CobT is NAD⁺ or its precursor nicotinate mononucleotide (NaMN). If NaMN is the substrate, CobT synthesizes α-ribazole-phosphate (α-RP) (reaction: DMB + NaMN → α-RP + Na) (30). If NAD⁺ substitutes for NaMN, CobT synthesizes α-DMB adenine dinucleotide (α-DAD) (reaction: DMB + NAD⁺ → α-DAD + Nm) (18). α-RP is a good substrate for the AdoCbl-5′-phosphate (AdoCbl-P) synthase (CobS; EC 2.7.8.26) enzyme (19, 34), suggesting that an unidentified enzyme cleaves α-DAD into α-RP and AMP (reaction: α-DAD → α-RP + AMP). Although it is possible that CobS may use α-DAD as a substrate, to date, data are not available to support this idea. The AdoCbl-P phosphatase (CobC; EC 3.1.3.73) enzyme catalyzes the last step of the NLA pathway to yield AdoCbl (Fig. ​(Fig.1)1) (34).Early genetic studies identified four classes of cobT alleles, namely, classes J, K, L, and M (12); class M was an intriguing class of mutations because they did not display intragenic complementation (12). Here we identify the nature of class M cobT mutations, report the initial in vitro and in vivo characterization of class M CobT variant proteins, and propose structural explanations for the observed deficiencies in CobT activity caused by class M mutations.     

Uncoupling Human Immunodeficiency Virus Type 1 gag and pol Reading Frames: Role of the Transframe Protein p6* in Viral Replication

Apart from its regulatory role in protease (PR) activation, little is known about the function of the human immunodeficiency virus type 1 transframe protein p6* in the virus life cycle. p6* is located between the nucleocapsid and PR domains in the Gag-Pol polyprotein precursor and is cleaved by PR during viral maturation. We have recently reported that the central region of p6* can be extensively mutated without abolishing viral infectivity and replication in vitro. However, mutagenesis of the entire p6*-coding sequence in the proviral context is not feasible without affecting the superimposed frameshift signal or the overlapping p1-p6^gag sequences. To overcome these limitations, we created a novel NL4-3-derived provirus by displacing the original frameshift signal to the 3′ end of the gag gene, thereby uncoupling the p6* gene sequence from the p1-p6^gag reading frame. The resulting virus (AL) proved to be replication competent in different cell cultures and thus represents an elegant tool for detailed analysis of p6* function. Hence, extensive deletions or substitutions were introduced into the p6* gene sequence of the AL provirus, and effects on particle release, protein processing, and viral infectivity were evaluated. Interestingly, neither the deletion of 63% of all p6* residues nor the partial substitution by a heterologous sequence affected virus growth and infectivity, suggesting that p6* is widely dispensable for viral in vitro replication. However, the insertion of a larger reporter sequence interfered with virus production and maturation, implying that the length or conformation of this spacer region might be critical for p6* function.The transframe protein p6*, also referred to as TFP-p6^pol, is one of the least-characterized gene products in human immunodeficiency virus type 1 (HIV-1), consistently raising controversial discussions on its main function in the viral life cycle. p6* is located at the amino terminus of the Pol moiety within the Gag-Pol precursor, which is synthesized following a programmed ribosomal −1 frameshift during translation (Fig. ​(Fig.1).1). In contrast, the Gag precursor is generated by conventional translation at a 20-fold-higher rate (summarized in reference 13). The characterization of recombinant p6* protein by nuclear magnetic resonance analysis revealed a highly flexible structure (3), suggesting that p6* might serve as an adjustable hinge within the large Gag-Pol precursor to facilitate folding of the bulky protein domains during viral assembly. Proper folding of the embedded homodimeric protease (PR) is an essential prerequisite for full activity following sequential autocleavage from the full-length Gag-Pol polyprotein (summarized in reference 13). Indeed, the p6* residues have been demonstrated to stabilize the PR dimer and dictate the folding propensities of PR precursors, thereby influencing the rate of PR maturation (6, 10, 21). Interestingly, the autoprocessing of a truncated Gag-PR precursor was clearly accelerated when the p6* sequence was deleted, leading to the proposal that the active site of the PR might be less accessible in the presence of p6* (26). Further observations that p6* itself is sequentially cleaved by the PR (1, 7, 22, 23, 24, 30, 31, 32, 41) strengthened the hypothesis that the transframe protein contributes to spatiotemporal activation of the PR. Indeed, we and others have previously shown that the PR needs to be released from the flanking p6* residues to be capable of completing all virus-associated processing steps (24, 27, 36). The potential of p6* to regulate PR activity has been further corroborated by our finding that recombinant p6* protein comprising a free accessible carboxyl terminus is a potent inhibitor of the mature PR in vitro (28). Apart from the carboxyl-terminal tetrapeptide mainly contributing to the observed inhibition, the amino-terminal octapeptide of p6* (TFP) has been reported to inhibit PR activity in vitro (21).Open in a separate windowFIG. 1.Gag and Gag-Pol polyproteins encoded by NL4-3 (NL) and AL proviruses. (A) Schematic representation of the Gag and Gag-Pol polyprotein precursors. The p6* domain within Gag-Pol is highlighted in black, and the p1-p6^gag region inserted in the Gag-Pol precursor of AL is shaded gray; the five residues appended to p6^gag in Gag are indicated by a black vertical line. MA, matrix protein; NC, nucleocapsid protein; IN, integrase. (B) The Gag (gray) and Pol (black) products translated in the presence of the original frameshift site in NL are indicated on top, and the corresponding passage synthesized in AL after destruction of the slippery site is shown below. The active (NL) and inactive (AL) slippery sites are underlined, and the 3′ nucleotides contributing to the stem-loop structure are in parentheses. (C) An artificial frameshift site was introduced at the 3′ end of the gag gene in AL, resulting in the synthesis of a Gag-Pol polyprotein with p6* directly fused to p6^gag. The gag reading frame in AL is terminated by an artificial stop codon (boxed) and is extended by the residues ANFLG. The E₄→V₄ substitution in p6* is marked by a circle, and numbers at the left indicate amino acid positions within Gag and Gag-Pol with respect to the Gag start codon.The fact that p6* has a substantial size of 55 to 72 amino acids, depending on the isolate, raises the question of whether the transframe protein exerts other functions apart from PR regulation. In this regard, p6* has also been proposed to be a possible binding partner of the viral Nef protein, the major pathogenicity factor during HIV infection (9). However, the target site within p6* for Nef interaction has not been mapped so far.Although it is completely overlapped by the p1-p6^gag domains, the p6* sequence offers much room for natural polymorphisms (5). Furthermore, amino acid insertions or duplications, as well as deletions of up to 13 residues, in p6* sequences of infectious isolates have been reported, some of which are associated with viral drug resistance (4, 29, 35, 38). We have recently shown that nonconservative substitutions of up to 70% of the p6* residues in a provirus clone did not abolish viral growth or infectivity in various cell lines, suggesting that the central p6* region is widely dispensable for viral in vitro replication (19, 27).Unlike the variable center, the p6* amino terminus is highly conserved owing to the overlapping p1^gag sequence (14) and the RNA frameshift signal composed of a heptanucleotide slippery site (UUUUUUA) and a 3′ pseudoknot structure (11). This complex sequence context imposes major restrictions on mutational analysis of the p6* amino terminus in the viral background (24). To allow for a more in-depth functional analysis of the conserved p6* residues in the viral context, we have established a novel NL4-3-based provirus with p1-p6^gag and p6* reading frames uncoupled via a dislocated frameshift site. Thereby, we could demonstrate that neither the deletion of the bulk of p6* nor the insertion of a short unrelated sequence significantly affected replication and infectivity of the virus in different cell cultures. However, the insertion of a five times larger green fluorescent protein (GFP)-encoding sequence into the p6* reading frame was associated with a clear loss of particle production and infectivity. Therefore, we conclude that p6* is a spacer protein of limited length which is widely dispensable for viral replication in vitro.