Biology subjects

Hahn, B. H.

Publications and source records attributed to Hahn, B. H..

30 records · Page 2Linked to original sources

bioRxiv2022

A germline-targeting chimpanzee SIV envelope glycoprotein elicits a new class of V2-apex directed cross-neutralizing antibodies

HIV-1 and its SIV precursors share a broadly neutralizing antibody (bNAb) epitope in variable loop 2 (V2) at the envelope glycoprotein (Env) trimer apex. Here, we tested the immunogenicity of germline-targeting versions of a chimpanzee SIV (SIVcpz) Env in human V2-apex bNAb heavy-chain precursor-expressing knock-in mice and as chimeric simian-chimpanzee immunodeficiency viruses (SCIVs) in rhesus macaques (RMs). Trimer immunization of knock-in mice induced V2-directed NAbs, indicating activation of V2-apex bNAb precursor-expressing mouse B cells. SCIV infection of RMs elicited high-titer viremia, potent autologous tier 2 neutralizing antibodies, and rapid sequence escape in the canonical V2-apex epitope. Six of seven animals also developed low-titer heterologous plasma breadth that mapped to the V2-apex. Antibody cloning from two of these identified multiple expanded lineages with long heavy chain third complementarity determining regions that cross-neutralized as many as 7 of 19 primary HIV-1 strains, but with low potency. Negative stain electron microscopy (NSEM) of members of the two most cross-reactive lineages confirmed V2 targeting but identified an angle of approach distinct from prototypical V2-apex bNAbs, with antibody binding either requiring or inducing an occluded-open trimer. Probing with conformation-sensitive, non-neutralizing antibodies revealed that SCIV-expressed Envs as well as some primary HIV-1 Envs adopted a more open conformation, thereby exposing a conserved V2 epitope that is occluded in closed SIVcpz and HIV-1 Env trimers. These results expand the spectrum of V2-apex targeted antibodies that can contribute to neutralization breadth and identify novel SIV Env platforms for further development as germline-targeting and immunofocusing immunogens. One sentence summaryA cryptic V2 epitope in occluded-open HIV and SIV Env trimers is the target of a new class of V2-directed cross-neutralizing antibodies.

immunology↗

bioRxiv2022

Small CD4 mimetics sensitize HIV-1-infected macrophages to antibody-dependent cellular cytotoxicity

HIV-1 Envelope (Env) conformation determines the susceptibility of infected CD4+ T cells to Antibody Dependent Cellular Cytotoxicity (ADCC). Upon interaction with CD4, Env adopts more "open" conformations, exposing ADCC epitopes. HIV-1 limits Env-CD4 interaction and protects infected cells against ADCC by downregulating CD4 via Nef, Vpu and Env. Limited data exists however of the role of these proteins in downmodulating CD4 on infected macrophages and how this impacts Env conformation. While Nef, Vpu and Env are all required to efficiently downregulate CD4 on infected CD4+T cells, we show here that any one of these proteins is sufficient to downmodulate most CD4 from the surface of infected macrophages. Consistent with this finding, Nef and Vpu have a lesser impact on Env conformation and ADCC sensitivity in infected macrophages compared to CD4+T cells. However, treatment of infected macrophages with small CD4-mimetics expose vulnerable CD4-induced Env epitopes and sensitize them to ADCC.

microbiology↗

bioRxiv2022

HIV-1 Vpu restricts Fc-mediated effector functions in vivo

Non-neutralizing antibodies (nnAbs) can eliminate HIV-1-infected cells via antibody-dependent cellular cytotoxicity (ADCC) and were identified as a correlate of protection in the RV144 vaccine trial. Fc-mediated effector functions of nnAbs were recently shown to alter the course of HIV-1 infection in vivo using a vpu-defective virus. Since Vpu is known to downregulate cell surface CD4, which triggers conformational changes in the viral envelope glycoprotein (Env), we ask whether the lack of Vpu expression was linked to the observed nnAbs activity. We found that restoring Vpu expression greatly reduces nnAb recognition of infected cells, rendering them resistant to ADCC responses. Moreover, administration of a nnAb in humanized mice reduces viral loads only in animals infected with a vpu-defective but not with a wildtype virus. Finally, nnAb Fc-effector functions are observed only on cells expressing Env in the "open" conformation. This work highlights the importance of Vpu-mediated evasion of humoral responses.

microbiology↗

bioRxiv2022

Macrophage activation of cGAS and TRIM5 distinguish pandemic and non-pandemic HIV

Pandemic viruses remain a global threat to health and economics but how they adapt to become pandemic remains poorly understood. Here we compare pandemic HIV-1(M) and non-pandemic HIV-(O) and HIV-2 strains finding that non-pandemic HIV replicate poorly in myeloid cell models due to activation of cGAS and TRIM5, and ensuing antiviral responses. We use phylogenetics and viral capsid structural biology to define specific differences between pandemic and non-pandemic HIV capsids and demonstrate that their genetic reversal in HIV-1(M) mutants causes TRIM5, cGAS and innate immune activation. We propose a model in which the parental lineage of pandemic HIV-1(M) has uniquely evolved a dynamic capsid that avoids activation of cGAS and TRIM5 to establish cloaked replication in myeloid cells. The unique adaptations of the pandemic virus lineage suggests a role in effective human-to-human transmissibility and highlight the importance of avoiding innate immune activation during pandemic human-to-human viral transmission.

immunology↗

bioRxiv2021

Detection of the HIV-1 accessory proteins Nef and Vpu by flow cytometry represents a new tool to study their functional interplay within a single infected CD4+ T cell

The HIV-1 Nef and Vpu accessory proteins are known to protect infected cells from antibody-dependent cellular cytotoxicity (ADCC) responses by limiting exposure of CD4-induced (CD4i) envelope (Env) epitopes at the cell surface. Although both proteins target the host receptor CD4 for degradation, the extent of their functional redundancy is unknown. Here, we developed an intracellular staining technique that permits the intracellular detection of both Nef and Vpu in primary CD4+ T cells by flow cytometry. Using this method, we show that the combined expression of Nef and Vpu predicts the susceptibility of HIV-1-infected primary CD4+ T cells to ADCC by HIV+ plasma. We also show that Vpu cannot compensate for the absence of Nef, thus providing an explanation for why some infectious molecular clones that carry a LucR reporter gene upstream of Nef render infected cells more susceptible to ADCC responses. Our method thus represents a new tool to dissect the biological activity of Nef and Vpu in the context of other host and viral proteins within single infected CD4+ T cells. IMPORTANCEHIV-1 Nef and Vpu exert several biological functions that are important for viral immune evasion, release and replication. Here, we developed a new method allowing simultaneous detection of these accessory proteins in their native form together with some of their cellular substrates. This allowed us to show that Vpu cannot compensate the lack of a functional Nef, which has implication for studies that use Nef-defective viruses to study ADCC responses.

microbiology↗

bioRxiv2021

HIV-1 envelope glycoproteins proteolytic cleavage protects infected cells from ADCC mediated by plasma from infected individuals

The HIV-1 envelope glycoprotein (Env) is synthesized in the endoplasmic reticulum as a trimeric gp160 precursor, which requires proteolytic cleavage by a cellular furin protease to mediate virus-cell fusion. Env is conformationally flexible, but controls its transition from the unbound "closed" conformation (State 1) to downstream CD4-bound conformations (States 2/3), which are required for fusion. In particular, HIV-1 has evolved several mechanisms that reduce the premature "opening" of Env which exposes highly conserved epitopes recognized by non-neutralizing antibodies (nnAbs) capable of mediating antibody-dependent cellular cytotoxicity (ADCC). Env cleavage decreases its conformational transitions favoring the adoption of the "closed" conformation. Here we altered the gp160 furin cleavage site to impair Env cleavage and to examine its impact on ADCC responses mediated by plasma from HIV-1-infected individuals. We found that infected primary CD4+ T cells expressing uncleaved, but not wildtype, Env are efficiently recognized by nnAbs and become highly susceptible to ADCC responses mediated by plasma from HIV-1-infected individuals. Thus, HIV-1 limits the exposure of uncleaved Env at the surface of HIV-1-infected cells at least in part to escape ADCC responses.

microbiology↗

bioRxiv2021

Red Blood Cells Function as DNA Sensors

Erythrocytes have long been mistaken as exclusively inert oxygen carriers lacking immune function. Here we show that red blood cells (RBCs) serve as immune sensors through surface expression of the nucleic acid-sensing toll-like receptor 9 (TLR9), a classically endosomal receptor that initiates immune responses following the detection of unmethylated CpG motifs present in pathogen and mitochondrial DNA. Mammalian RBCs express TLR9 on their surface and bind CpG-containing bacterial, malarial, and mitochondrial DNA. Erythrocyte-bound CpG DNA increases during infection, and CpG-carrying RBCs trigger accelerated erythrophagocytosis and innate immune activation characterized by RBC-TLR9 dependent local and systemic cytokine production. Thus, RBC nucleic acid detection and capture regulates red cell clearance and immune responses and provides evidence for RBCs as innate immune sentinels during pathologic states. One Sentence SummaryThe ability of RBCs to detect and bind cell-free nucleic acids contributes to immunity during acute inflammatory states.

immunology↗

bioRxiv2021

New SHIVs and Improved Design Strategy for Modeling HIV-1 Transmission, Immunopathogenesis, Prevention and Cure

Simian-human immunodeficiency virus (SHIV) chimeras contain the HIV-1 envelope (env) gene embedded within an SIVmac proviral backbone. Previously, we showed that substitution of Env residue 375-Ser by bulky aromatic residues enhances Env binding to rhesus CD4 and enables primary or transmitted/founder (T/F) HIV-1 Envs to support efficient SHIV replication in rhesus macaques (RMs). Here, we test this design strategy more broadly by constructing and analyzing SHIVs containing ten strategically selected primary or T/F HIV-1 Envs corresponding to subtypes A, B, C, AE and AG, each with six allelic variants at position 375. All ten SHIVs bearing wildtype Env375 residues replicated efficiently in human CD4+ T cells, but only one of these replicated efficiently in rhesus CD4+ T cells. This was a SHIV whose subtype AE Env naturally contained a bulky aromatic His residue at position 375. Replacement of wildtype Env375 residues by Trp, Tyr, Phe or His in the other nine SHIVs uniformly led to efficient replication in rhesus CD4+ T in vitro and in RMs in vivo. Env375-Trp - the residue found most frequently among SIV strains infecting Old World monkeys - was favored for SHIV replication in RMs, although some SHIVs preferred Env375-Tyr, -His or -Phe. Nine SHIVs containing optimized Env375 alleles were grown large scale in primary activated rhesus CD4+ T cells to serve as challenge stocks in preclinical prevention trials. These virus stocks were genetically homogeneous, native-like in Env antigenicity and tier-2 neutralization sensitivity, transmissible by rectal, vaginal, penile, oral or intravenous inoculation routes, and exhibited acute and early replication kinetics that were indistinguishable from HIV-1 infection in humans. Finally, to expedite future SHIV constructions and eliminate short redundant elements in tat1 and env gp41 that were spontaneously deleted in chronically infected monkeys, we engineered a simplified second-generation SHIV design scheme and validated it in RMs. Overall, our findings demonstrate that SHIVs bearing primary or T/F Envs with bulky aromatic amino acid substitutions at position Env375 consistently replicate in RMs, recapitulating many features of HIV-1 infection in humans. We further show that SHIV challenge stocks grown in primary rhesus CD4+ T cells are efficiently transmitted by mucosal routes common to HIV-1 infection and can be used effectively to test for vaccine efficacy in preclinical monkey trials.

microbiology↗

bioRxiv2020

Natural Cystatin C fragments inhibit GPR15-mediated HIV and SIV infection without interfering with GPR15L signaling

GPR15 is a G protein-coupled receptor proposed to play a role in mucosal immunity that also serves as entry cofactor for HIV and SIV. To discover novel endogenous GPR15 ligands, we screened a hemofiltrate-derived peptide library for inhibitors of GPR15-mediated SIV infection. Our approach identified a C-terminal fragment of Cystatin C (CysC95-146) that specifically inhibits GPR15-dependent HIV-1, HIV-2 and SIV infection. In contrast, GPR15L, the chemokine ligand of GPR15, failed to inhibit virus infection. We found that Cystatin C fragments preventing GPR15-mediated viral entry do not interfere with GPR15L signaling and are generated by proteases activated at sites of inflammation. The antiretroviral activity of CysC95-146 was confirmed in primary CD4+ T cells and is conserved in simian hosts of SIV infection. Thus, we identified a potent endogenous inhibitor of GPR15-mediated HIV and SIV infection that does not interfere with the physiological function of this G protein-coupled receptor.

microbiology↗

bioRxiv2020

Species-specific differences in antagonism of APOBEC3 proteins by HIV-2 and SIVsmm Vif proteins

SIVsmm infecting sooty mangabeys has been transmitted to humans on at least nine independent occasions, giving rise to HIV-2 groups A to I. SIVsmm isolates replicate in human T cells and seem capable of overcoming major human restriction factors without adaptation. However, only groups A and B are responsible for the HIV-2 epidemic in Sub-Saharan Africa and it is largely unclear whether adaptive changes were associated with significant spread in humans. To address this, we examined the sensitivity of infectious molecular clones (IMCs) of five HIV-2 strains (4 group A and one AB recombinant) and representatives of five different SIVsmm lineages to inhibition by type I interferon (IFN) and various APOBEC3 proteins. We confirmed that SIVsmm strains replicate in primary human CD4+ T cells. However, SIVsmm replication was highly variable, typically lower relative to HIV-2 isolates and almost entirely prevented by type I IFN treatment. Viral propagation was generally dependent on intact vif genes, highlighting the need for efficient counteraction of APOBEC3 proteins. On average, SIVsmm strains were significantly more susceptible to inhibition by human APOBEC3D, F, G and H than HIV-2 IMCs. For example, human APOBEC3F reduced infectious virus yield of SIVsmm by [~]80% but achieved only [~]40% in the case of HIV-2. Functional and mutational analyses of human, sooty mangabey and rhesus macaque derived alleles revealed that an R128T polymorphism in APOBEC3F is important for species-specific counteraction by HIV-2 and SIVsmm Vif proteins. In addition, we found that changes of Y45H and T84S in SIVsmm Vif increase its ability to antagonize human APOBEC3F. Altogether, our results show that SIVsmm Vifs show some intrinsic activity against human ABOBEC3 proteins, but HIV-2 Vifs acquired adaptive changes to efficiently clear this barrier in the human host. AUTHOR SUMMARYSIVs infecting African monkey species do not infect humans, with one notable exception. SIVsmm from sooty mangabeys managed to cross the species barrier to humans on at least nine independent occasions. This is because SIVsmm strains seem capable of overcoming many innate defense mechanisms without adaptation and that their Vif proteins are active against human APOBEC3 proteins. Here, we show that replication of SIVsmm is highly variable in human CD4 T cells and more sensitive to interferon inhibition compared to HIV-2. While different lineages of SIVsmm were capable of counteracting human APOBEC3 proteins in a Vif-dependent manner, they were significantly more susceptible to inhibition by APOBEC3D/F/G/H compared to HIV-2. Mutational analyses revealed an R128T substitution in APOBEC3F and a T84S change in Vif are relevant for species-specific counteraction by HIV-2 and SIVsmm. Altogether, our results support that HIV-2 group A adapted to humans prior to or during epidemic spread.

immunology↗

bioRxiv2020

Urine as a high-quality source of host genomic DNA from wild populations

The ability to generate genomic data from wild animal populations has the potential to give unprecedented insight into the population history and dynamics of species in their natural habitats. However, in the case of many species, it is impossible legally, ethically, or logistically to obtain tissues samples of high-quality necessary for genomic analyses. In this study we evaluate the success of multiple sources of genetic material (feces, urine, dentin, and dental calculus) and several capture methods (shotgun, whole-genome, exome) in generating genome-scale data in wild eastern chimpanzees (Pan troglodytes schweinfurthii) from Gombe National Park, Tanzania. We found that urine harbors significantly more host DNA than other sources, leading to broader and deeper coverage across the genome. Urine also exhibited a lower rate of allelic dropout. We found exome sequencing to be far more successful than both shotgun sequencing and whole-genome capture at generating usable data from low-quality samples such as feces and dental calculus. These results highlight urine as a promising and untapped source of DNA that can be noninvasively collected from wild populations of many species.

genomics↗

bioRxiv2018

Chimpanzee SIV Envelope trimer: structure and deployment as an HIV vaccine template

Epitope-targeted HIV vaccine design seeks to focus antibody responses to broadly neutralizing antibody (bnAb) sites by sequential immunization. Chimpanzee SIV Envelope (Env) shares a single bnAb site, the V2-apex, with HIV, suggesting its possible utility in an HIV immunization strategy. Accordingly, we generated a chimpanzee SIV Env trimer, MT145K, which displays selective binding to HIV V2-apex bnAbs and precursor versions, but no binding to other HIV specificities. We determined the structure of the MT145K trimer by cryo-EM and showed its architecture was remarkably similar to HIV Env. Immunization of an HIV V2-apex bnAb precursor Ab-expressing knock-in mouse with chimpanzee MT145K trimer induced HIV V2-specific neutralizing responses. Subsequent boosting with an HIV trimer cocktail induced responses exhibiting some virus cross-neutralization. Overall, the chimpanzee MT145K trimer behaves as expected from design both in vitro and in vivo and is an attractive potential component of a sequential immunization regimen to induce V2-apex bnAbs.

immunology↗