Effector Cell Recruitment with Novel Fv-based Dual-affinity Re-targeting Protein Leads to Potent Tumor Cytolysis and in Vivo B-cell Depletion

Bispecific antibodies capable of redirecting the lytic potential of immune effector cells to kill tumor targets have long been recognized as a potentially potent biological therapeutic intervention. Unfortunately, efforts to produce such molecules have been limited owing to inefficient production and poor stability properties. Here, we describe a novel Fv-derived strategy based on a covalently linked bispecific diabody structure that we term dual-affinity re-targeting (DART). As a model system, we linked an Fv specific for human CD16 (FcγRIII) on effector cells to an Fv specific for mouse or human CD32B (FcγRIIB), a normal B-cell and tumor target antigen. DART proteins were produced at high levels in mammalian cells, retained the binding activity of the respective parental Fv domains as well as bispecific binding, and showed extended storage and serum stability. Functionally, the DART molecules demonstrated extremely potent, dose-dependent cytotoxicity in retargeting human PBMC against B-lymphoma cell lines as well as in mediating autologous B-cell depletion in culture. In vivo studies in mice demonstrated effective B-cell depletion that was dependent on the transgenic expression of both CD16A on the effector cells and CD32B on the B-cell targets. Furthermore, DART proteins showed potent in vivo protective activity in a human Burkitt's lymphoma cell xenograft model. Thus, DART represents a biologically potent format that provides a versatile platform for generating bispecific antibody fragments for redirected killing and, with the selection of appropriate binding partners, applications outside of tumor cell cytotoxicity.     

Brain-derived neurotrophic factor (BDNF) induces polarized signaling of small GTPase (Rac1) protein at the onset of Schwann cell myelination through partitioning-defective 3 (Par3) protein

Brain-derived neurotrophic factor (BDNF) was shown to play a role in Schwann cell myelination by recruiting Par3 to the axon-glial interface, but the underlying mechanism has remained unclear. Here we report that Par3 regulates Rac1 activation by BDNF but not by NRG1-Type III in Schwann cells, although both ligands activate Rac1 in vivo. During development, active Rac1 signaling is localized to the axon-glial interface in Schwann cells by a Par3-dependent polarization mechanism. Knockdown of p75 and Par3 individually inhibits Rac1 activation, whereas constitutive activation of Rac1 disturbs the polarized activation of Rac1 in vivo. Polarized Rac1 activation is necessary for myelination as Par3 knockdown attenuates myelination in mouse sciatic nerves as well as in zebrafish. Specifically, Par3 knockdown in zebrafish disrupts proper alignment between the axon and Schwann cells without perturbing Schwann cell migration, suggesting that localized Rac1 activation at the axon-glial interface helps identify the initial wrapping sites. We therefore conclude that polarization of Rac1 activation is critical for myelination.     

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.     

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.     

A quantitative method for inferring locomotory shifts in amniotes during ontogeny,its application to dinosaurs and its bearing on the evolution of posture

Evolutionary transitions between quadrupedal and bipedal postures are pivotal to the diversification of amniotes on land, including in our own lineage (Hominini). Heterochrony is suggested as a macroevolutionary mechanism for postural transitions but understanding postural evolution in deep time is hindered by a lack of methods for inferring posture in extinct species. Dinosaurs are an excellent natural laboratory for understanding postural transitions because they demonstrate at least four instances of quadrupedality evolving from bipedality, and heterochronic processes have been put forward as an explanatory model for these transitions. We extend a quantitative method for reliably inferring posture in tetrapods to the study of ontogenetic postural transitions using measurements of proportional limb robusticity. We apply this to ontogenetic series of living and extinct amniotes, focusing on dinosaurs. Our method correctly predicts the general pattern of ontogenetic conservation of quadrupedal and bipedal postures in many living amniote species and infers the same pattern in some dinosaurs. Furthermore, it correctly predicts the ontogenetic postural shift from quadrupedal crawling to bipedal walking in humans. We also infer a transition from early ontogenetic quadrupedality to late-ontogenetic bipedality in the transitional sauropodomorph dinosaur Mussaurus patagonicus and possibly in the early branching ceratopsian Psittacosaurus lujiatunensis but not in the sauropodomorph Massospondylus carinatus. The phylogenetic positions of these ontogenetic shifts suggest that heterochrony may play a role in the macroevolution of posture, at least in dinosaurs. Our method has substantial potential for testing evolutionary transitions between locomotor modes, especially in elucidating the role of evolutionary mechanisms like heterochrony.     

Neutralizing Polyclonal IgG Present during Acute Infection Prevents Rapid Disease Onset in Simian-Human Immunodeficiency Virus SHIVSF162P3-Infected Infant Rhesus Macaques

Simian-human immunodeficiency virus (SHIV) models for human immunodeficiency virus (HIV) infection have been widely used in passive studies with HIV neutralizing antibodies (NAbs) to test for protection against infection. However, because SHIV-infected adult macaques often rapidly control plasma viremia and any resulting pathogenesis is minor, the model has been unsuitable for studying the impact of antibodies on pathogenesis in infected animals. We found that SHIV_SF162P3 infection in 1-month-old rhesus macaques not only results in high persistent plasma viremia but also leads to very rapid disease progression within 12 to 16 weeks. In this model, passive transfer of high doses of neutralizing IgG (SHIVIG) prevents infection. Here, we show that at lower doses, SHIVIG reduces both plasma and peripheral blood mononuclear cell (PBMC)-associated viremia and mitigates pathogenesis in infected animals. Moreover, production of endogenous NAbs correlated with lower set-point viremia and 100% survival of infected animals. New SHIV models are needed to investigate whether passively transferred antibodies or antibodies elicited by vaccination that fall short of providing sterilizing immunity impact disease progression or influence immune responses. The 1-month-old rhesus macaque SHIV model of infection provides a new tool to investigate the effects of antibodies on viral replication and clearance, mechanisms of B cell maintenance, and the induction of adaptive immunity in disease progression.     

The effect of chronic seaweed subsidies on herbivory: plant-mediated fertilization pathway overshadows lizard-mediated predator pathways

Flows of energy and materials link ecosystems worldwide and have important consequences for the structure of ecological communities. While these resource subsidies typically enter recipient food webs through multiple channels, most previous studies focussed on a single pathway of resource input. We used path analysis to evaluate multiple pathways connecting chronic marine resource inputs (in the form of seaweed deposits) and herbivory in a shoreline terrestrial ecosystem. We found statistical support for a fertilization effect (seaweed increased foliar nitrogen content, leading to greater herbivory) and a lizard numerical response effect (seaweed increased lizard densities, leading to reduced herbivory), but not for a lizard diet-shift effect (seaweed increased the proportion of marine-derived prey in lizard diets, but lizard diet was not strongly associated with herbivory). Greater seaweed abundance was associated with greater herbivory, and the fertilization effect was larger than the combined lizard effects. Thus, the bottom-up, plant-mediated effect of fertilization on herbivory overshadowed the top-down effects of lizard predators. These results, from unmanipulated shoreline plots with persistent differences in chronic seaweed deposition, differ from those of a previous experimental study of the short-term effects of a pulse of seaweed deposition: while the increase in herbivory in response to chronic seaweed deposition was due to the fertilization effect, the short-term increase in herbivory in response to a pulse of seaweed deposition was due to the lizard diet-shift effect. This contrast highlights the importance of the temporal pattern of resource inputs in determining the mechanism of community response to resource subsidies.     

Pulses of marine subsidies amplify reproductive potential of lizards by increasing individual growth rate

Pulsed resource subsidies can have profound effects on recipient communities. The effects of resource pulses are often mediated by increases in the density of consumer populations. Here we investigate several mechanisms linking experimental pulses of seaweed deposition to population‐level responses in the brown anole Anolis sagrei. Subsidized lizards grew approximately 30% faster than lizards in seaweed‐removal plots, but there was no effect of seaweed subsidies on survival or body condition. Breeding is strongly seasonal in A. sagrei, resulting in a limited reproductive window of opportunity. Accelerated growth allows subsidized females to reach sexual maturity earlier and thereby exploit more of this window, which is projected to double fecundity in their first year of life. These results show how changes in an individual trait can translate pulses of resource input into reproductive output. Further, they highlight the importance of seasonal timing in mechanistically linking individual‐, population‐ and community‐level responses to pulsed resource subsidies.     

The effect of disturbance on an ant–plant mutualism

Protective ant–plant mutualisms—where plants provide food or shelter to ants and ants protect the plants from herbivores—are a common feature in many ecological communities, but few studies have examined the effect of disturbance on these interactions. Disturbance may affect the relationship between plants and their associated ant mutualists by increasing the plants’ susceptibility to herbivores, changing the amount of reward provided for the ants, and altering the abundance of ants and other predators. Pruning was used to simulate the damage to buttonwood mangrove (Conocarpus erectus) caused by hurricanes. Pruned plants grew faster than unpruned plants, produced lower levels of physical anti-herbivore defenses (trichomes, toughness), and higher levels of chemical defenses (tannins) and extrafloral nectaries. Thus, simulated hurricane damage increased plant growth and the amount of reward provided to ant mutualists, but did not have consistent effects on other anti-herbivore defenses. Both herbivores and ants increased in abundance on pruned plants, indicating that the effects of simulated hurricane damage on plant traits were propagated to higher trophic levels. Ant-exclusion led to higher leaf damage on both pruned and upruned plants. The effect of ant-exclusion did not differ between pruned and unpruned plants, despite the fact that pruned plants had higher ant and herbivore densities, produced more extrafloral nectaries, and had fewer physical defenses. Another common predator, clubionid spiders, increased in abundance on pruned plants from which ants had been excluded. I suggest that compensatory predation by these spiders diminished the effect of ant-exclusion on pruned plants.     

Gathering Local Knowledge in Madagascar Results in a Major Increase in the Known Range and Number of Sites for Critically Endangered Greater Bamboo Lemurs (Prolemur simus)

Greater bamboo lemurs (Prolemur simus) are endemic to Madagascar and are the only recognized species within their genus. The IUCN lists the species as critically endangered, with very few confirmed distribution records and <200 individuals known in the wild. With the aim of contributing to its conservation, we attempted to find previously unknown sites containing the species. Working closely with local communities, and basing our methodology on the gathering of local knowledge, we surveyed 44 sites in and around the Ankeniheny-Zahamena Corridor. We found evidence of the presence of Prolemur simus at 18 sites. We made direct sightings at 6 sites, of a total of 65 confirmed individuals, and identified their characteristic feeding remains at the other sites. Twelve of the sites are located in midaltitude rain forest within the corridor, and 6 lie in isolated and degraded lowland areas outside the corridor. These discoveries more than double the number of sites where the species is known to occur, and extend its known range 85 km further north. We identified numerous threats to the newly discovered sites, including hunting pressure, habitat destruction, habitat disturbance, and habitat fragmentation. Demographic factors related to small population sizes and population isolation may also impact the viability of the populations. Our results illustrate the benefits of systematic gathering of local knowledge when searching for rare or secretive primates.