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The new entry inhibitor eCD4-Ig, consisting of the immunoadhesin form of CD4 (CD4-Ig) fused to a small CCR5-mimetic sulfopeptide, avidly binds two highly conserved sites of the HIV-1 Env protein; the inhibitor has high potency and breadth and can neutralize 100% of a diverse panel of neutralization-resistant HIV-1 viruses, and when delivered to macaques using an adeno-associated virus vector, it can provide effective long-term protection from multiple challenges with simian/human immunodeficiency virus. HIV-1 entry inhibitors with vaccine-like action This study describes a novel class of highly potent HIV-1 entry inhibitors that can be delivered with a gene-therapy vector to provide an effective alternative to conventional vaccines for HIV-1. To enter cells, HIV-1 first binds its cellular receptor CD4, then the co-receptor CCR5 or CXCR4 The new entry inhibitor consists of the immunoadhesin CD4-Ig fused to a sulfopeptide mimicking CCR5. This fusion, called eCD4-Ig, avidly binds the Env protein of HIV-1 and irreversibly inactivates it. Michael Farzan and colleagues show that this inhibitor has exceptional potency and breadth and can neutralize 100% of a diverse panel of neutralization-resistant HIV-1. When delivered to macaques using an adeno-associated virus, it can protect them from multiple challenges with virus. Long-term in vivo expression of a broad and potent entry inhibitor could circumvent the need for a conventional vaccine for HIV-1. Adeno-associated virus (AAV) vectors can stably express HIV-1 broadly neutralizing antibodies (bNAbs) 1 , 2 . However, even the best bNAbs neutralize 10–50% of HIV-1 isolates inefficiently (80% inhibitory concentration (IC 80 ) > 5 μg ml −1 ), suggesting that high concentrations of these antibodies would be necessary to achieve general protection 3 , 4 , 5 , 6 . Here we show that eCD4-Ig, a fusion of CD4-Ig with a small CCR5-mimetic sulfopeptide, binds avidly and cooperatively to the HIV-1 envelope glycoprotein (Env) and is more potent than the best bNAbs (geometric mean half-maximum inhibitory concentration (IC 50 ) < 0.05 μg ml −1 ). Because eCD4-Ig binds only conserved regions of Env, it is also much broader than any bNAb. For example, eCD4-Ig efficiently neutralized 100% of a diverse panel of neutralization-resistant HIV-1, HIV-2 and simian immunodeficiency virus isolates, including a comprehensive set of isolates resistant to the CD4-binding site bNAbs VRC01, NIH45-46 and 3BNC117. Rhesus macaques inoculated with an AAV vector stably expressed 17–77 μg ml −1 of fully functional rhesus eCD4-Ig for more than 40 weeks, and these macaques were protected from several infectious challenges with SHIV-AD8. Rhesus eCD4-Ig was also markedly less immunogenic than rhesus forms of four well-characterized bNAbs. Our data suggest that AAV-delivered eCD4-Ig can function like an effective HIV-1 vaccine.

The interaction between the HIV-1 envelope glycoprotein, Env, and the host receptor, CD4, marks the first step of viral entry. This event leads to coreceptor association and subsequent conformational rearrangements of Env that promote fusion of the viral and target-cell membranes. Env is crucial to viral replication and, as the sole viral protein on the surface of HIV-1, is under constant pressure by the host’s immune response. Rhesus macaques have been developed as a model organism to study HIV-1 transmission and pathogenesis. However, rhesus macaques differ from humans in aspects that prevent replication of HIV-1. Therefore, simian immunodeficiency viruses, SIVs, have been utilized to model human HIV-1 infection. In Chapter 2 we show that SIV utilizes rhesus CD4 less efficiently than HIV-1 does human CD4. We identified a single residue in CD4, isoleucine 39, that largely accounted for this difference. We further identified two residue changes in the CD4-binding site of Env that improved SIVmac239 use of rhesus CD4. We propose that rhesus macaques infected with an SIVmac239 variant bearing these mutations might better reflect some aspects of human HIV-1 disease, for example infection of cells that express lower levels of CD4 in the brain. In Chapter 3 we study the inhibitor eCD4-Ig, which bears CD4 domains 1 and 2 and a coreceptor mimetic peptide. We show that virus escapes this inhibitor more slowly than the potent broadly neutralizing antibody NIH45-46 and that escape likely associates with a fitness loss. We analyzed viruses from in vitro and in vivo studies and identified escape mutations in the apex region, V3 loop and CD4-binding site. In one in vivo case, SIVmac239 exploited a single residue difference between cellular rhesus CD4 and eCD4-Ig. It did so by acquiring one of the two CD4-binding site mutations described in Chapter 2, linking the two chapters in an unexpected way. Collectively these studies add to our understanding of the CD4-Env interaction, which is especially important for the development of eCD4-Ig as an antiretroviral agent in a clinical setting. In Chapter 4, we compare eCD4-Ig with its major competitors, broadly neutralizing antibodies and conventional vaccine strategies.

Applications of mode-locked fiber lasers benefit from robust and self-starting mode-locking, spectral tuning, high pulse energy and high average power. All-polarization-maintaining (PM) fiber lasers mode-locked with a phase-biased nonlinear amplifying loop mirror (NALM) have been shown to be very robust and reliably self-starting, and provide either spectral tuning or high pulse energy, but not both. We report on a simple method for concurrent spectral tuning and nanojoule-level pulse energy scaling of an all-PM phase-biased NALM mode-locked Yb:fiber laser, which we demonstrate over a 54 nm tuning range, reaching up to 1.67 nJ pulse energy and 126 mW average power. Unlike other laser configurations, our results show that net normal dispersion is not necessary or optimal for scaling the pulse energy of this type of mode-locked fiber laser.

We report a simple and compact design of a dispersion compensated mode-locked Yb:fiber oscillator based on a nonlinear amplifying loop mirror (NALM). The fully polarization maintaining (PM) fiber integrated laser features a chirped fiber Bragg grating (CFBG) for dispersion compensation and a fiber integrated compact non-reciprocal phase bias device, which is alignment-free. The main design parameters were determined by numerically simulating the pulse evolution in the oscillator and by analyzing their impact on the laser performance. Experimentally, we achieved an 88 fs compressed pulse duration with sub-fs timing jitter at 54 MHz repetition rate and 51 mW of output power with 5.5 * 10-5 [20 Hz, 1 MHz] integrated relative intensity noise (RIN). Furthermore, we demonstrate tight phase-locking of the laser's carrier-envelope offset frequency (fceo) to a stable radio frequency (RF) reference and of one frequency comb tooth to a stable optical reference at 291 THz.

T-cell antigen receptors (TCRs) exhibit inherent cross-reactivity which broadens the spectrum of epitopes that are recognizable by a finite TCR-repertoire but also carries the risk of autoimmunity. However, TCRs support also a high level of antigen specificity as they allow T-cells to discriminate single antigenic peptide/MHC complexes (pMHCs) against millions of structurally related self-pMHCs, in some cases based on the absence or presence of a single methyl-group. How TCRs manage to convey such seemingly contrary properties and why some T-cells become over time autoreactive despite negative thymic selection, has remained elusive. Here, we devised a non-invasive molecular live cell imaging platform to investigate the biophysical parameters governing stimulatory TCR:pMHC interactions in settings of autoreactivity and anti-viral responses - two extremes in T-cell antigen recognition. We show that CMV-specific CD8+ RA14-T-cells respond effectively to even a single HLA-A2/CMV (A2/CMV) antigen, with synaptic TCR:pMHC lifetimes lasting seconds. In contrast, cross-reactivity of type 1 diabetes (T1D)-associated CD8+ 1E6 T-cells towards HLA-A2/preproinsulin (A2/PPI) self-epitopes involved ten-fold less stable synaptic TCR interactions resulting in severely attenuated ZAP70 recruitment and downstream signaling. Compared to A2/CMV-engaged RA14 T-cells, 1E6-T-cells required for activation 4000 or more A2/PPI and at least 100-times as many simultaneously pMHC-engaged TCRs. In support of antigen discrimination, CD8 co-engagement of MHC class I (MHCI) strengthened both settings of TCR:pMHC interactions equally but was essential only for sensitized virus detection but not autorecognition (1000-versus 5-fold enhancement). We conclude that the binding dynamics of TCRs and CD8 with pMHC shape the boundaries of central tolerance in the physiological context of the phenomenal yet also differential T-cell antigen detection capacity, TCR-cross-reactivity and self-antigen abundance. Gained insights are integral to a molecular and quantitative understanding of CD8+ T-cell mediated autoimmunity and protective immunity against infections and cancer. With the use of newly devised molecular live-cell imaging modalities we measured with unprecedented precision T-cell antigen recognition dynamics in human T-cells in settings of anti-viral immunity and autoimmunity-causing cross-reactivity. These two extremes within the spectrum of T-cell antigen detection differed substantially with regard to synaptic TCR: antigen-engagement, the level of sensitization through the CD8-coreceptor and the overall efficiency of ensuing downstream signaling. Our results demarcate limits of central tolerance and protective immunity and set quantitative boundaries on the occurrence of autoimmunity with direct implications for T-cell-based designs of immunotherapies.

We present a flexible all-polarization-maintaining (PM) mode-locked ytterbium (Yb):fiber laser based on a nonlinear amplifying loop mirror (NALM). In addition to providing detailed design considerations, we discuss the different operation regimes accessible by this versatile laser architecture and experimentally analyze five representative mode-locking states. These five states were obtained in a 78-MHz configuration at different intracavity group delay dispersion (GDD) values ranging from anomalous (-0.035 ps\\(^2\\)) to normal (+0.015 ps\\(^2\\)). We put a particular focus on the characterization of the intensity noise as well as the free-running linewidth of the carrier-envelope-offset (CEO) frequency as a function of the different operation regimes. We observe that operation points far from the spontaneous emission peak of Yb (~1030 nm) and close to zero intracavity dispersion can be found, where the influence of pump noise is strongly suppressed. For such an operation point, we show that a CEO linewidth of less than 10-kHz at 1 s integration can be obtained without any active stabilization.

We demonstrate dual-comb generation from an all-polarization-maintaining dual-color ytterbium (Yb) fiber laser. Two pulse trains with center wavelengths at 1030 nm and 1060 nm respectively are generated within the same laser cavity with a repetition rate around 77 MHz. Dual-color operation is induced using a tunable mechanical spectral filter, which cuts the gain spectrum into two spectral regions that can be independently mode-locked. Spectral overlap of the two pulse trains is achieved outside the laser cavity by amplifying the 1030-nm pulses and broadening them in a nonlinear fiber. Spatially overlapping the two arms on a simple photodiode then generates a down-converted radio frequency comb. The difference in repetition rates between the two pulse trains and hence the line spacing of the down-converted comb can easily be tuned in this setup. This feature allows for a flexible adjustment of the tradeoff between non-aliasing bandwidth vs. measurement time in spectroscopy applications. Furthermore, we show that by fine-tuning the center-wavelengths of the two pulse trains, we are able to shift the down-converted frequency comb along the radio-frequency axis. The usability of this dual-comb setup is demonstrated by measuring the transmission of two different etalons while the laser is completely free-running.