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HIV-1 sequence diversity and the presence of archived epitope mutations in antibody binding sites are a major obstacle for the clinical application of broadly neutralizing antibodies (bNAbs) against HIV-1. Specifically, it is unclear to what degree the viral reservoir is compartmentalized and more importantly if virus susceptibility to antibody neutralization differs across tissues. The Last Gift cohort enrolled 7 people with HIV diagnosed with a terminal illness and collected antemortem blood and postmortem tissues across 33 anatomical compartments for near-full-length env HIV-genome sequencing. Using these data, we applied a new Bayesian machine-learning model (MCMC-SVM) that uses HIV-1 envelope sequences and approximated glycan-occupancy information as variables to quantitatively predict the half-maximal-inhibitory concentrations (IC50) of bNAbs. This allowed us to map the landscape of neutralization resistance across each person's tissue reservoirs. Predicted mean susceptibilities across tissues within a participant were relatively homogenous and the susceptibility pattern observed in blood often matched what was predicted for tissues. Selected tissues, like the brain, however, showed in some participants evidence of compartmentalized viral populations with distinct neutralization susceptibilities. Additionally, we found substantial heterogeneity of the range of neutralization susceptibilities across tissues even within an individual, between individuals and also between bNAbs within the same individual (standard deviation of log2(IC50) > 3.4). Blood-based screening methods to determine viral susceptibility to bNAbs might underestimate the presence of resistant viral variants in tissues. To what extent these resistant viruses are clinically relevant, ie. leading to bNAb therapeutic failure, needs to be further explored.

BackgroundHuman immunodeficiency virus type 1 (HIV-1) sequence diversity and the presence of archived epitope muta-tions in antibody binding sites are a major obstacle for the clinical application of broadly neutralizing antibodies (bNAbs) against HIV-1. Specifically, it is unclear to what degree the viral reservoir is compartmentalized and if virus susceptibility to antibody neutralization differs across tissues.MethodsThe Last Gift cohort enrolled 7 people with HIV diagnosed with a terminal illness and collected antemortem blood and postmortem tissues across 33 anatomical compartments for near full-length env HIV genome sequencing. Using these data, we applied a Bayesian machine-learning model (Markov chain Monte Carlo-support vector machine) that uses HIV-1 envelope sequences and approximated glycan-occupancy information to quantitatively predict the half-maximal inhib-itory concentrations (IC50) of bNAbs, allowing us to map neutralization resistance pattern across tissue reservoirs.ResultsPredicted mean susceptibilities across tissues within participants were relatively homogenous, and the susceptibility pattern observed in blood often matched what was predicted for tissues. However, selected tissues, such as the brain, showed ev-idence of compartmentalized viral populations with distinct neutralization susceptibilities in some participants. Additionally, we found substantial heterogeneity in the range of neutralization susceptibilities across tissues within and between indi-viduals, and between bNAbs within individuals (standard deviation of log2(IC50) >3.4).ConclusionsBlood-based screening methods to determine viral susceptibility to bNAbs might underestimate the presence of resistant viral variants in tissues. The extent to which these resistant viruses are clinically relevant, that is, lead to bNAb therapeutic failure, needs to be further explored.

Two main challenges have impeded the development of an effective HIV-1 envelope (Env) vaccine, with antibodies eliciting neutralisation of virions as well as Fc-effector functions, such as antibody-dependent cytotoxicity (ADCC), phagocytosis (ADP) or complement deposition (ADCD). On one hand, designing the right Env vaccine to elicit humoral or cellular protection has been challenging and, to date, SOSIP-Env trimers which are covalently constrained in the closed, pre-fusion conformation are the best vaccine candidate over uncleaved (Unc), open-structured trimers. On the other hand, eliciting heterologous neutralising antibodies in several animal models (including humans) has been difficult. Cows nevertheless produce unique antibodies with long CDRH3 regions, capable of accessing neutralising epitopes beneath the glycan shield, inaccessible for other animals. We tested how differences in clade and/or structure of HIV-1 Env vaccines affect the neutralising activity and Fc-effector functions of antibodies elicited, using recombinant trimers of clades A (KNH1,BG505), B (AD8, PSC89) and C (MW), which exposed either an open structure (Unc gp140) or a closed structure (SOSIP gp140). KNH1/BG505 SOSIP gp140 vaccine elicited the best neutralising IgGs against heterologous tier-2 pseudoviruses with high potency and breadth. While AD8 Unc gp140 also induced neutralisation, it was against only tier-1 pseudoviruses. Nevertheless, it was the only vaccine able to elicit IgGs that engaged CD32 (FcgRIIa), induced phagocytosis and complement-activation. The different antibody profile observed with both vaccines was explained by the Env immunogen structure, as KNH1/BG505 SOSIP gp140 induced mostly IgGs targeting the V1/V2 loop, whereas AD8 Unc gp140 induced antibodies targeting CD4-binding site and CD4-induced epitopes. In addition, analysis of IgG repertoires from animals of KNH1/BG505 SOSIP 100 and AD8 Unc 500 groups showed that KNH1/BG505 SOSIP gp140 induced higher rates of somatic

Natural environments can present diverse challenges, but some genotypes remain fit across many environments. Such "generalists" can be hard to evolve, outcompeted by specialists fitter in any particular environment. Here, inspired by the search for broadly neutralizing antibodies during B cell affinity maturation, we demonstrate that environmental changes on an intermediate timescale can reliably evolve generalists, even when faster or slower environmental changes are unable to do so. We find that changing environments on timescales comparable with evolutionary transients in a population enhance the rate of evolving generalists from specialists, without enhancing the reverse process. The yield of generalists is further increased in more complex dynamic environments, such as a "chirp" of increasing frequency. Our work offers design principles for how nonequilibrium fitness "seascapes" can dynamically funnel populations to genotypes unobtainable in static environments.

The HIV Research for Prevention (HIVR4P) conference is dedicated to advancing HIV prevention research, responding to a growing consensus that effective and durable prevention will require a combination of approaches as well as unprecedented collaboration among scientists, practitioners, and community workers from different fields and geographic areas. The conference theme in 2018, "From Research to Impact," acknowledged an increasing focus on translation of promising research findings into practical, accessible, and affordable HIV prevention options for those who need them worldwide. HIVR4P 2018 was held in Madrid, Spain, on 21-25 October, with >1,400 participants from 52 countries around the globe, representing all aspects of HIV prevention research and implementation. The program included 137 oral and 610 poster presentations. This article presents a brief summary of highlights from the conference. More detailed information, complete abstracts as well as webcasts and daily Rapporteur summaries may be found on the conference website.

The HIV Research for Prevention (HIVR4P) conference is dedicated to advancing HIV prevention research, responding to a growing consensus that effective and durable prevention will require a combination of approaches as well as unprecedented collaboration among scientists, practitioners, and community workers from different fields and geographic areas. The conference theme in 2018, "From Research to Impact," acknowledged an increasing focus on translation of promising research findings into practical, accessible, and affordable HIV prevention options for those who need them worldwide. HIVR4P 2018 was held in Madrid, Spain, on 21-25 October, with >1,400 participants from 52 countries around the globe, representing all aspects of HIV prevention research and implementation. The program included 137 oral and 610 poster presentations. This article presents a brief summary of highlights from the conference. More detailed information, complete abstracts as well as webcasts and daily Rapporteur summaries may be found on the conference website.

HIV-1 Envelope (Env) mediates viral-host membrane fusion after binding host-receptor CD4 and coreceptor. Soluble envelopes (SOSIPs), designed to mimic prefusion conformational states of virion-bound envelopes, are proposed immunogens for eliciting neutralizing antibodies, yet only static structures are available. To evaluate conformational landscapes of ligand-free, CD4-bound, inhibitor-bound, and antibody-bound SOSIPs, we measured inter-subunit distances throughout spin-labeled SOSIPs using double electron-electron resonance (DEER) spectroscopy and compared results to soluble and virion-bound Env structures, and single-molecule fluorescence resonance energy transfer (smFRET)-derived dynamics of virion-bound Envs. Unliganded SOSIP measurements were consistent with closed, neutralizing antibody-bound structures and shielding of non-neutralizing epitopes, demonstrating homogeneity at Env apex, increased flexibility near Env base, and no evidence for the intra-subunit flexibility near Env apex suggested by smFRET. CD4 binding increased inter-subunit distances and heterogeneity, consistent with rearrangements required for coreceptor binding. Results suggest similarities between SOSIPs and virion-bound Envs and demonstrate DEER's relevance for immunogen design.

HIV-1 Envelope (Env) mediates viral-host membrane fusion after binding host-receptor CD4 and coreceptor. Soluble envelopes (SOSIPs), designed to mimic prefusion conformational states of virion-bound envelopes, are proposed immunogens for eliciting neutralizing antibodies, yet only static structures are available. To evaluate conformational landscapes of ligand-free, CD4-bound, inhibitor-bound, and antibody-bound SOSIPs, we measured inter-subunit distances throughout spin-labeled SOSIPs using double electron-electron resonance (DEER) spectroscopy and compared results to soluble and virion-bound Env structures, and single-molecule fluorescence resonance energy transfer (smFRET)-derived dynamics of virion-bound Envs. Unliganded SOSIP measurements were consistent with closed, neutralizing antibody-bound structures and shielding of non-neutralizing epitopes, demonstrating homogeneity at Env apex, increased flexibility near Env base, and no evidence for the intra-subunit flexibility near Env apex suggested by smFRET. CD4 binding increased inter-subunit distances and heterogeneity, consistent with rearrangements required for coreceptor binding. Results suggest similarities between SOSIPs and virion-bound Envs and demonstrate DEER's relevance for immunogen design.

Seit der Entdeckung des humanen Immundefizienz-Virus (HIV) als Auslöser für das erworbenes Immundefektsyndrom (AIDS) hat es großen Fortschritt in den Behandlungsmöglichkeiten gegeben. Ein Impfstoff wird jedoch noch benötigt. Vielversprechende Impfstoffkandidaten sind virale Vektoren, da diese ein umfangreiche, robuste und langanhaltende Immunantwort hervorrufen. Wir arbeiten mit einem chimären Vesikularen stomatitis Virus (VSV-GP), welches das Glycoprotein (GP) des Lymphozytäre-Choriomeningitis-Virus besitzt. VSV-GP ist ein potenter Impfstoffvektor, der keine der Nachteile eines wildtyp VSV aufweist. In dieser Arbeit bewerten wird das Potential von VSV-GP als Impfstoff gegen HIV. Verschiedene HIV-1 Hüllproteingene wurden in das Genom von VSV-GP kloniert, wie bspw. sekretierte oder Membran-verankerte, intakte oder mutierte Furinschnittstelle oder C-Termini mit unterschiedlicher Länge. Unsere Ergebnisse zeigen, dass das Einfügen eines zusätzlichen Antigens sowie die Anwesenheit von zwei Glycoproteinen die Replikation von VSV-GP nicht beeinträchtigen. Alle HIV-1 Env varianten werden von Zellen expremiert und einige werden sehr effizient in die Membran von VSV-GP eingebaut. Wichtige Epitope für das Binden von breit neutralisierenden Antikörpern gegen HIV-1, wie MPER (membrane proximal external region), CD4 binding site, V1V2 und V3 loop werden auf den viralen Partikeln präsentiert. Das Binden von quartären Antikörpern, lässt eine trimäre Strukturv von HIV Env eingebaut in VSV-GP vermuten. Vektoren mit Membran-verankerten Env induzierten höhere Antikörpertiter als sekretierte Env Proteine. In Kaninchen wurden Antikörper hervorgerufen, die Epitop über das gesamte Env verteilt, erkennen. Des weiteren wurde eine Tier 1A neutralisierende Antikörperantwort indiziert, die mit einer Boost-Immunisierung gesteigert werden konnte. Das Friend Virus Model ist ein retrovirales Model für Infektionskrankheiten, bei dem eine Challenge im Maus-Model möglich ist. Wir zeigten, dass VSV-GP, Since the human immunodeficiency virus (HIV) has been discovered as the cause of the acquired immunodeficiency syndrome (AIDS), great progress has been made towards the treatment of HIV. However, a protective vaccine is still urgently needed. Promising vaccine candidates are viral vectors, since they induce a diverse, robust and long-lasting immune response. We are working with the chimeric vesicular stomatitis virus (VSV-GP) containing the glycoprotein GP of the lymphocytic choriomeningitis virus. VSV-GP is a potent viral vaccine vector that overcomes several of the limitations of wild-type VSV. Here, we evaluated the potential of VSV-GP as an HIV vaccine vector. We introduced genes for different variants of the HIV-1 Envelope protein Env, i.e., secreted or membrane-anchored, intact or mutated furin cleavage site or different C-termini, into the genome of VSV-GP. We found that the addition of the Env antigen and the presence of two glycoproteins did not attenuate VSV-GP replication. All HIV-1 Env variants were expressed in VSV-GP infected cells and some were incorporated very efficiently into VSV-GP particles. Crucial epitopes for binding of broadly neutralizing antibodies against HIV-1 such as MPER (membrane proximal external region), CD4 binding site, V1V2 and V3 loop were present on the surface of VSV-GP-Env particles. Binding of quaternary antibodies indicated a trimeric structure of VSV-GP incorporated Env. We detected high HIV-1 antibody titers in mice and showed that vectors expressing membrane-anchored Env elicited higher antibody titers than vectors that express secreted Envs. In rabbits, HIV Env-binding antibodies recognizing different epitopes spanning the whole HIV Env gp140 were induced. Furthermore, tier 1A HIV-1 neutralizing antibodies were detectable after prime immunization and titers further increased after boosting with a second immunization. Using Friend virus as model for retroviral infections where a challenge is possible in a mouse model, we, by Christiane Anika Bresk, Dissertation Medical University of Innsbruck 2019

Seit der Entdeckung des humanen Immundefizienz-Virus (HIV) als Auslöser für das erworbenes Immundefektsyndrom (AIDS) hat es großen Fortschritt in den Behandlungsmöglichkeiten gegeben. Ein Impfstoff wird jedoch noch benötigt. Vielversprechende Impfstoffkandidaten sind virale Vektoren, da diese ein umfangreiche, robuste und langanhaltende Immunantwort hervorrufen. Wir arbeiten mit einem chimären Vesikularen stomatitis Virus (VSV-GP), welches das Glycoprotein (GP) des Lymphozytäre-Choriomeningitis-Virus besitzt. VSV-GP ist ein potenter Impfstoffvektor, der keine der Nachteile eines wildtyp VSV aufweist. In dieser Arbeit bewerten wird das Potential von VSV-GP als Impfstoff gegen HIV. Verschiedene HIV-1 Hüllproteingene wurden in das Genom von VSV-GP kloniert, wie bspw. sekretierte oder Membran-verankerte, intakte oder mutierte Furinschnittstelle oder C-Termini mit unterschiedlicher Länge. Unsere Ergebnisse zeigen, dass das Einfügen eines zusätzlichen Antigens sowie die Anwesenheit von zwei Glycoproteinen die Replikation von VSV-GP nicht beeinträchtigen. Alle HIV-1 Env varianten werden von Zellen expremiert und einige werden sehr effizient in die Membran von VSV-GP eingebaut. Wichtige Epitope für das Binden von breit neutralisierenden Antikörpern gegen HIV-1, wie MPER (membrane proximal external region), CD4 binding site, V1V2 und V3 loop werden auf den viralen Partikeln präsentiert. Das Binden von quartären Antikörpern, lässt eine trimäre Strukturv von HIV Env eingebaut in VSV-GP vermuten. Vektoren mit Membran-verankerten Env induzierten höhere Antikörpertiter als sekretierte Env Proteine. In Kaninchen wurden Antikörper hervorgerufen, die Epitop über das gesamte Env verteilt, erkennen. Des weiteren wurde eine Tier 1A neutralisierende Antikörperantwort indiziert, die mit einer Boost-Immunisierung gesteigert werden konnte. Das Friend Virus Model ist ein retrovirales Model für Infektionskrankheiten, bei dem eine Challenge im Maus-Model möglich ist. Wir zeigten, dass VSV-GP, Since the human immunodeficiency virus (HIV) has been discovered as the cause of the acquired immunodeficiency syndrome (AIDS), great progress has been made towards the treatment of HIV. However, a protective vaccine is still urgently needed. Promising vaccine candidates are viral vectors, since they induce a diverse, robust and long-lasting immune response. We are working with the chimeric vesicular stomatitis virus (VSV-GP) containing the glycoprotein GP of the lymphocytic choriomeningitis virus. VSV-GP is a potent viral vaccine vector that overcomes several of the limitations of wild-type VSV. Here, we evaluated the potential of VSV-GP as an HIV vaccine vector. We introduced genes for different variants of the HIV-1 Envelope protein Env, i.e., secreted or membrane-anchored, intact or mutated furin cleavage site or different C-termini, into the genome of VSV-GP. We found that the addition of the Env antigen and the presence of two glycoproteins did not attenuate VSV-GP replication. All HIV-1 Env variants were expressed in VSV-GP infected cells and some were incorporated very efficiently into VSV-GP particles. Crucial epitopes for binding of broadly neutralizing antibodies against HIV-1 such as MPER (membrane proximal external region), CD4 binding site, V1V2 and V3 loop were present on the surface of VSV-GP-Env particles. Binding of quaternary antibodies indicated a trimeric structure of VSV-GP incorporated Env. We detected high HIV-1 antibody titers in mice and showed that vectors expressing membrane-anchored Env elicited higher antibody titers than vectors that express secreted Envs. In rabbits, HIV Env-binding antibodies recognizing different epitopes spanning the whole HIV Env gp140 were induced. Furthermore, tier 1A HIV-1 neutralizing antibodies were detectable after prime immunization and titers further increased after boosting with a second immunization. Using Friend virus as model for retroviral infections where a challenge is possible in a mouse model, we, by Christiane Anika Bresk, Dissertation Medical University of Innsbruck 2019