Biology subjects

Overbaugh, J.

Publications and source records attributed to Overbaugh, J..

31 records · Page 2Linked to original sources

bioRxiv2020

High-resolution mapping of the neutralizing and binding specificities of polyclonal rabbit serum elicited by HIV Env trimer immunization

Mapping the epitope specificities of polyclonal serum is critical to rational vaccine design. However, most high-resolution mapping approaches involve isolating and characterizing individual monoclonal antibodies, which incompletely defines the full polyclonal response. Here we use two complementary approaches to directly map the specificities of the neutralizing and binding antibodies of polyclonal anti-HIV-1 sera from rabbits immunized with BG505 Env SOSIP trimers. To map the neutralizing specificity, we used mutational antigenic profiling to determine how all amino-acid mutations in Env affected viral neutralization. To map the binding specificity, we used electron microscopy polyclonal epitope mapping (EMPEM) to directly visualize the Fabs in serum bound to Env trimers. Mutational antigenic profiling showed that the dominant neutralizing specificities were the C3/V5 and/or 241/289 glycan hole epitopes, which were generally only a subset of the more diverse binding specificities mapped with EMPEM. Additional differences between binding and neutralization reflected antigenicity differences between virus and soluble Env trimer. Further, mutational antigenic profiling was able to refine epitope specificity in residue-level detail directly from sera, revealing subtle differences across rabbits. Together, mutational antigenic profiling and EMPEM allow for a holistic view of the binding and neutralizing specificity of polyclonal sera and could be used to finely evaluate and guide vaccine design.

microbiology↗

bioRxiv2020

Development of antibody-dependent cell cytotoxicity function in HIV-1 antibodies

A prerequisite for the design of an HIV vaccine that elicits protective antibodies is understanding the developmental pathways that result in desirable antibody features. The development of antibodies that mediate antibody-dependent cellular cytotoxicity (ADCC) is particularly relevant because such antibodies have been associated with HIV protection in humans. We reconstructed the developmental pathways of six human HIV-specific ADCC antibodies using longitudinal antibody sequencing data. Most of the inferred naive antibodies did not mediate detectable ADCC. Gain of antigen binding and ADCC function typically required mutations in complementarity determining regions of one or both chains. Enhancement of ADCC potency often required additional mutations in framework regions. Antigen binding affinity and ADCC activity were correlated, but affinity alone was not sufficient to predict ADCC potency. Thus, elicitation of human ADCC antibodies may require mutations that first enable high affinity antigen recognition, followed by mutations that optimize factors contributing to functional ADCC activity.

immunology↗

bioRxiv2020

Phage-DMS: a comprehensive method for fine mapping of antibody epitopes

Understanding the antibody response is critical to developing vaccine and antibody-based therapies and has inspired the recent development of new methods to isolate antibodies. However, methods to define the antibody-antigen interactions that determine specificity or allow escape have not kept pace. We developed Phage-DMS, a method which combines two powerful approaches - immunoprecipitation of phage peptide libraries and deep mutational scanning (DMS) - to enable high-throughput fine mapping of antibody epitopes. As an example, we designed sequences encoding all possible amino acid variants of HIV Envelope to create phage display libraries. Using Phage-DMS, we identified sites of escape predicted using other approaches for four well characterized HIV monoclonal antibodies with known linear epitopes. In some cases, the results of Phage-DMS refined the epitope beyond what was determined in previous studies. This method has the potential to rapidly and comprehensively screen many antibodies in a single experiment to define sites essential for binding to antigen.

immunology↗

bioRxiv2019

Identification of HIV-1 Envelope Mutations that Enhance Entry Using Macaque CD4 and CCR5

Although Rhesus macaques are an important animal model for HIV-1 vaccine development research, most transmitted HIV-1 strains replicate poorly in macaque cells. A major genetic determinant of this species-specific restriction is a non-synonymous mutation in macaque CD4 that results in reduced HIV-1 Envelope (Env)-mediated viral entry compared to human CD4. Recent research efforts employing either laboratory evolution or structure-guided design strategies have uncovered several mutations in Envs gp120 subunit that enhance binding of macaque CD4 by transmitted/founder HIV-1 viruses. In order to identify additional Env mutations that promote infection of macaque cells, we utilized deep mutational scanning to screen thousands of Env point mutants for those that enhance HIV-1 entry via macaque receptors. We identified many uncharacterized amino acid mutations in the N-terminal heptad repeat (NHR) and C-terminal heptad repeat (CHR) regions of gp41 that increased entry into cells bearing macaque receptors by up to 38-fold. Many of these mutations also modestly increased infection of cells bearing human CD4 and CCR5 (up to 13-fold). NHR/CHR mutations identified by deep mutational scanning that enhanced entry also increased sensitivity to neutralizing antibodies targeting the MPER epitope, and to inactivation by cold-incubation, suggesting that they promote sampling of an intermediate trimer conformation between closed and receptor bound states. Identification of this set of mutations can inform future macaque model studies, and also further our understanding of the relationship between Env structure and function. ImportanceAlthough Rhesus macaques are the favored non-human primate animal model used in HIV-1 research, most circulating HIV-1 strains poorly infect macaque cells. Studies using macaques to model HIV-1 infection often use evolved, or mutant HIV-1 variants that are able to utilize macaque CD4, but these HIV-1 variants poorly model infection by circulating strains. In this work, we sought to identity HIV-1 mutations that would allow entry into macaque cells, but that would maintain critical characteristics of circulating HIV-1 strains. We employed a powerful experimental method to simultaneously assess the effects of thousands of individual HIV-1 mutations on infection of cells bearing macaque receptors. We identified many previously uncharacterized mutations that enhance infection of circulating HIV-1 strains into cells bearing macaque receptors by up to 38-fold. Identification of these mutations may be of use in future macaque model studies.

microbiology↗

bioRxiv2019

Diversity and function of maternal HIV-1-specific antibodies at the time of vertical transmission

Infants of HIV positive mothers can acquire HIV infection by various routes, but even in the absence of antiviral treatment, the majority of these infants do not become infected. There is evidence that maternal antibodies may provide some protection from infection, but gestational maternal antibodies have not yet been characterized in detail. One of the most studied vertically-infected infants is BG505, as the virus from this infant yielded an Envelope protein that was successfully developed as a stable trimer. Here, we isolated and characterized 39 HIV-specific neutralizing monoclonal antibodies (nAbs) from MG505, the mother of BG505, at a time point just prior to vertical transmission. These nAbs belonged to 21 clonal families, employed a variety of VH genes, many were specific for the HIV-1 Env V3 loop, and this V3 specificity correlated with measurable antibody-dependent cellular cytotoxicity (ADCC) activity. The isolated nAbs did not recapitulate the full breadth of heterologous nor autologous virus neutralization by contemporaneous plasma. Notably, we found that the V3-targeting nAb families neutralized one particular maternal Env variant even though all tested variants had low V3 sequence diversity and were measurably bound by these nAbs. None of the nAbs neutralized the BG505 transmitted virus. Furthermore, the MG505 nAb families were found at relatively low frequencies within the maternal B cell repertoire: all less than 0.25% of total IgG sequences. Our findings demonstrate the diversity of HIV-1 nAbs that exist within a single mother, resulting in a collection of antibody specificities that can shape the transmission bottleneck.\n\nImportanceMother-to-child-transmission of HIV-1 offers a unique setting in which maternal antibodies both within the mother and passively-transferred to the infant are present at the time of viral exposure. Untreated HIV-exposed human infants are infected at a rate of 30-40%, meaning that some infants do not get infected despite continued exposure to virus. Since the potential of HIV-specific immune responses to provide protection against HIV is a central goal of HIV vaccine design, understanding the nature of maternal antibodies may provide insights into immune mechanisms of protection. In this study, we isolated and characterized HIV-specific antibodies from the mother of an infant whose transmitted virus has been well studied.

immunology↗

bioRxiv2018

Massively parallel profiling of HIV-1 resistance to the fusion inhibitor enfuvirtide

Resistance to enfuvirtide, the only clinically approved HIV-1 entry inhibitor, has primarily been mapped to the binding site in the N-terminal heptad repeat (NHR) of the Env transmembrane domain and a limited number of allosteric sites. To better delineate the genotypic determinants of resistance, we used deep mutational scanning to quantify how all mutations to HIV-1 Env affect enfuvirtide sensitivity. We identified numerous additional resistance mutations in the NHR and other regions of Env, including the co-receptor binding site. This complete map of resistance sheds light on the diverse mechanisms of enfuvirtide resistance and can inform clinical monitoring of patients.

microbiology↗

bioRxiv2018

Lineage-dependent differences and the role of IFITM3 in the type-I interferon-induced restriction of Zika virus

Type-I interferon (IFN-I) is an important aspect of host innate antiviral response. Recent studies have shown that IFN-I can inhibit Zika virus (ZIKV) replication and that this is mediated in part by Interferon-induced transmembrane protein 3 (IFITM3). ZIKV infections in South America have led to severe congenital syndrome in a subset of infected infants. ZIKV was first identified in Africa, where there is limited evidence for the pathogenic effects associated with the American outbreak, which is fueled by infection with Asian-lineage strains, raising the possibility that the African and Asian ZIKV lineages have distinct pathogenic properties. Given the observation that IFN-I can inhibit ZIKV replication in cell culture, we asked whether ZIKV strains differed in their susceptibility to IFN-I. There was a range of susceptibilities to IFN-I inhibition across virus strains. Virus production in A549 cells was reduced from 3-42-fold for IFN and 63-807-fold for IFN{beta} across a panel of nine viruses, five from the African-lineage and four from the Asian-lineage. African-lineage ZIKV strains were more resistant to IFN-I than Asian-lineage strains, but this difference was only significant for IFN-mediated restriction (p = 0.049). Notably, over-expression of IFITM3 at similar levels induced by IFN-I did not significantly restrict either a prototype African lineage (MR 766) or Asian lineage (PRVABC59) isolate. Moreover, knocking out IFITM3 expression did not result in a significant increase in viral replication or a diminishment of the inhibition by IFN-I. Overall, our findings show that while diverse ZIKV strains are susceptible to the antiviral effects of IFN-I, African-lineage strains are more resistant to IFN. In addition, the majority of the IFN-I-induced inhibition of ZIKV strains cannot be explained by IFITM3, suggesting that other unknown ISGs may be the driving force of the type I IFN response against ZIKV.\n\nAuthor summaryThe innate immune system, and specifically the type-I interferon response, is a critical component of the host response against viral infections. The recent unprecedented spread and severe pathogenic features of Zika virus in the Americas have led to significant interest in characterizing features of Zika virus strains that have fueled the American outbreak. Zika virus was first identified in Africa, where there is limited evidence for the pathogenic effects associated with the American outbreak. Here, we demonstrate that African-lineage Zika virus strains are significantly more resistant to the effects of type-I interferon, and that type-I interferon-mediated restriction of Zika virus strains is not explained by the host factor Interferon-induced transmembrane protein 3. This improved understanding of Zika virus-host interactions may explain certain pathogenic features of Asian-lineage Zika virus strains that have fueled the American Zika virus epidemic, and supports the search for as-yet-unidentified actors in the interferon response against Zika virus.

microbiology↗

bioRxiv2018

Rapid development of an infant-derived HIV-1 broadly neutralizing antibody lineage

HIV-infected infants develop broadly neutralizing plasma responses with more rapid kinetics than adults, suggesting the ontogeny of infant responses could better inform a path to achievable vaccine targets. We developed computational methods to reconstruct the developmental lineage of BF520.1, the first example of a HIV-specific broadly neutralizing antibody (bnAb) from an infant. The BF520.1 inferred naive precursor binds HIV Env and a bnAb evolved within six months of infection and required only 3% mutation. Mutagenesis and structural analyses revealed that for this infant bnAb, substitutions in the kappa chain were critical for activity, particularly in CDRL1. Overall, the developmental pathway of this infant antibody includes features distinct from adult antibodies, including several that may be amenable to better vaccine responses.

immunology↗

bioRxiv2018

An antigenic atlas of HIV-1 escape from broadly neutralizing antibodies

Anti-HIV broadly neutralizing antibodies (bnAbs) have revealed vaccine targets on the viruss Env protein and are themselves promising immunotherapeutics. The efficacy of bnAb-based therapies and vaccines depends in part on how readily the virus can escape neutralization. While structural studies can define contacts between bnAbs and Env, only functional studies can define mutations that confer escape. Here we map how all single amino-acid mutations to Env affect neutralization of HIV by nine bnAbs targeting five epitopes. For most bnAbs, mutations at only a small fraction of structurally defined contact sites mediated escape, and most escape occurred at sites that are near but do not directly contact the antibody. The mutations selected by two pooled bnAbs were similar to those expected from the combination of the bnAbs independent action. Overall, our mutation-level antigenic atlas provides a comprehensive dataset for understanding viral immune escape and refining therapies and vaccines.

immunology↗

bioRxiv2018

A Virus-Packageable CRISPR Screen Identifies Host Factors Mediating Interferon Inhibition of HIV

Interferon (IFN) inhibits HIV replication by inducing an array of antiviral effectors. Here we describe a novel CRISPR knockout screening approach to identify the ensemble of these HIV restriction factors. We assembled a CRISPR sgRNA library specific for Interferon Stimulated Genes (ISGs) into a modified lentiviral vector that allows for packaging of sgRNA-encoding genomes in trans into budding HIV-1 particles. We observed that knockout of Zinc Antiviral Protein (ZAP) improved the performance of the screen due to ZAP-mediated inhibition of the vector. We identify a small panel of IFN-induced HIV restriction factors, including MxB, IFITM1, Tetherin/BST2 and TRIM5 which together explain the inhibitory effects of IFN on the HIV-1 LAI strain in THP-1 cells. Further, we identify novel HIV dependency factors, including SEC62 and TLR2. The ability of IFN-induced restriction factors to inhibit an HIV strain to replicate in human cells suggests that these human restriction factors are incompletely antagonized.

microbiology↗

bioRxiv2018

Complete functional mapping of infection- and vaccine-elicited antibodies against the fusion peptide of HIV

Eliciting broadly neutralizing antibodies (bnAbs) targeting envelope (Env) is a major goal of HIV vaccine development, but cross-clade breadth from immunization has only sporadically been observed. Recently, Xu et al (2018) elicited cross-reactive neutralizing antibody responses in a variety of animal models using immunogens based on the epitope of bnAb VRC34.01. The VRC34.01 antibody, which was elicited by natural human infection, targets the N terminus of the Env fusion peptide, a critical component of the virus entry machinery. Here we precisely characterize the functional epitopes of VRC34.01 and two vaccine-elicited murine antibodies by mapping all single amino-acid mutations to the BG505 Env that affect viral neutralization. While escape from VRC34.01 occurred via mutations in both fusion peptide and distal interacting sites of the Env trimer, escape from the vaccine-elicited antibodies was mediated predominantly by mutations in the fusion peptide. Cryo-electron microscopy of four vaccine-elicited antibodies in complex with Env trimer revealed focused recognition of the fusion peptide and provided a structural basis for development of neutralization breadth. Together, these functional and structural data suggest that the breadth of vaccine-elicited antibodies targeting the fusion peptide can be enhanced by specific interactions with additional portions of Env. Thus, our complete maps of viral escape provide a template to improve the breadth or potency of future vaccine-induced antibodies against Envs fusion peptide.\n\nAuthor summaryA major goal of HIV-1 vaccine design is to elicit antibodies that neutralize diverse strains of HIV-1. Recently, some of us elicited such antibodies in animal models using immunogens based on the epitope of a broad antibody (VRC34.01) isolated from an infected individual. Further improving these vaccine-elicited antibody responses will require a detailed understanding of how the resulting antibodies target HIVs envelope protein (Env). Here, we used mutational antigenic profiling to precisely map the epitope of two vaccine-elicited antibodies and the template VRC34.01 antibody. We did this by quantifying the effect of all possible amino acid mutations to Env on antibody neutralization. Although all antibodies target a similar region of Env, we found clear differences in the functional interaction of Env with the vaccine- and infection-elicited antibodies. We combined these functional data with structural analyses to identify antibody-Env interactions that could improve the breadth of vaccine-elicited antibodies, and thereby help to refine vaccination schemes to achieve broader responses.

immunology↗

bioRxiv2017

Mapping mutational effects along the evolutionary landscape of HIV envelope

The immediate evolutionary space accessible to HIV is largely determined by how single amino-acid mutations affect fitness. These mutational effects can shift as the virus evolves. However, the prevalence of such shifts in mutational effects remains unclear. Here we quantify the effects on viral growth of all amino-acid mutations to two HIV envelope (Env) proteins that differ at >100 residues. Most mutations similarly affect both Envs, but the amino-acid preferences of a minority of sites have clearly shifted. These shifted sites usually prefer a specific amino acid in one Env, but tolerate many amino acids in the other. Surprisingly, shifts are only slightly enriched at sites that have substituted between the Envs -- and many occur at residues that do not even contact substitutions. Therefore, long-range epistasis can unpredictably shift Env's mutational tolerance during HIV evolution, although the amino-acid preferences of most sites are conserved between moderately diverged viral strains.

microbiology↗

bioRxiv2017

Comprehensive mapping of HIV-1 escape from a broadly neutralizing antibody

Precisely defining how viral mutations affect HIVs sensitivity to antibodies is vital to the development and evaluation of vaccines and antibody immunotherapeutics. But despite great effort, a full map of escape mutants has not yet been delineated for even a single anti-HIV antibody. Here we describe a massively parallel experimental approach to quantify how all single amino-acid mutations to Envelope (Env) affect HIVs sensitivity to a neutralizing antibody. We applied this approach to PGT151 and identified novel sites of escape in addition to those previously defined by structural and functional studies, such as glycans at sites 611 and 637, residue 647, and sites in the fusion peptide. Evaluating the effect of each amino acid at each site lends insight into the biochemical basis of escape throughout the epitope. Thus, comprehensive mapping of HIV antibody escape gives a quantitative, mutation-level view of the ways that Env can evade neutralization.

microbiology↗