Cynthia A. Derdeyn - US grants

DESCRIPTION (provided by applicant): Although crystallographic studies have provided a detailed description of the conserved core structure of the HIV-1 surface glycoprotein (gp120), much less is known about the structure and function of the first two hyper-variable regions (VIV2), which protrude from the core surface and often undergo extensive insertions and deletions over the course of disease. Elimination of glycosylation sites or deletion of amino acid residues from the VIV2 domain can influence a number of molecular interactions that are critical to virus entry. However, such studies may not fully describe the natural functions of V1V2, and few studies have comprehensively examined the consequences of natural variability within the V1V2 region during virus transmission or disease progression. Here we will define the structure-function relationships of naturally occurring V1V2 regions as they pertain to heterosexual transmission and virus evolution in Env molecules derived from a large heterosexual couple cohort in Zambia. Our preliminary studies demonstrated that a homogeneous virus population with compact length and restricted N-linked glycosylation in VIV2 were found in the recipient partners of eight heterosexual transmission pairs; however, the viral quasispecies of each donor partner contained up to nine different VIV2 variants, ranging as much as 3-fold in size and 9-fold in the number of glycosylation sites. These data suggest that the molecular determinants of heterosexual transmission are related to the structure of V1V2, and V1V2-chimeric Envs will be constructed from five !transmission pairs and utilized to define this relationship. To better understand the extensive variation that emerges in VIV2 following the acute infection period, the course of sequence evolution in VIV2 will be followed in longitudinal samples collected over a minimum of three years from ten newly identified seroconvertors in the same cohort. We will construct V1V2-chimeric Env molecules to investigate the ,biological consequences of temporal changes within the VIV2 region during infection. Through these studies, we will test the hypotheses that viruses with compact V1V2 regions establish infection and that progressive increases in length and glycosylation arise over the course of infection that contribute to immune evasion and replication fitness, but interfere with virus transmission.

DESCRIPTION: (provided by applicant) Sooty mangabey monkeys (SM) naturally infected with SIV maintain normal levels of CD4+ lymphocytes and do not develop AIDS despite chronic high level viral replication, short longevity of infected CD4+ T cells, and increased rates of CD4+ T cell turnover. Interestingly, despite otherwise intact immune functions, SIV-infected SMs manifest limited or absent anti-SIV specific cytotoxic T cell (CTL) responses. We have further shown that SIV-infected SMs possess preserved bone marrow, thymic and peripheral lymphoid sources for T lymphocyte production, and manifest levels of immune activation and apoptosis far lower than those seen in pathogenic infections with SIV in rhesus macaques (RMs) and with HIV in humans. These data suggest that the direct consequences of high level virus replication alone cannot account for the progressive CD4+ T cell depletion leading to AIDS. Rather, SIV-infected SMs may be spared, by their failure to mount significant antiviral immune responses, much of the bystander damage seen in pathogenic primate lentivirus infections that contributes to both accelerated CD4+ depletion and compromised host immune regenerative capacity. We propose to test the hypothesis that the type and magnitude of the host immune response to virus infections determines whether of not disease occurs by (1) detailed characterization of primary SIV infection in RMs and SMs by virologic, immunologic and genetic methods, (2) induction of active cellular anti-SIV immunity in acutely-infected SMs and evaluation of whether disease develops in an otherwise refractory host, and (3) blockade or deviation of host cellular immune responses to SIV in acutely-infected RMs, and evaluation of whether protection from disease progression is achieved in an otherwise susceptible host.

AIDS Virus; ATGN; Antibody Formation; Antibody Production; Antibody Response; Antigens; CRISP; Cell Surface Glycoproteins; Chronic Disease; Chronic Illness; Computer Retrieval of Information on Scientific Projects Database; ENV; Enrollment; Envelope Glycoprotein gp120, HIV; Envelope Protein; Funding; Gene Products, env; Genes, env; Glycoproteins, env; Grant; HIV Envelope Protein gp120; HIV-1; HIV-I; HIV1; HTLV-III gp120; Heterosexuals; Human immunodeficiency virus 1; Immunodeficiency Virus Type 1, Human; Infection; Institution; Investigators; Membrane Glycoproteins; NIH; National Institutes of Health; National Institutes of Health (U.S.); Nature; Northern Rhodesia; Pathogenesis; Research; Research Personnel; Research Resources; Researchers; Resources; Role; Source; Surface Glycoproteins; Transmission; United States National Institutes of Health; Vaccines; Variant; Variation; Viral; Zambia; antibody biosynthesis; base; chronic disease/disorder; chronic disorder; cohort; design; designing; enroll; env Antigens; env Gene Products; env Genes; env Glycoproteins; env Polyproteins; env Protein; env Protein gp120, HIV; gp120; gp120 ENV Glycoprotein; gp120(HIV); human T cell leukemia virus III; human T lymphotropic virus III; immunogen; immunoglobulin biosynthesis; neutralizing antibody; social role; transmission process; virus characteristic

Project Summary/Abstract Genetically diverse subtypes and recombinant forms of HIV-1 circulate in different regions of the globe and are the basis of the current AIDS pandemic. The biological consequences of this viral diversity are poorly understood and pose s substantial challenge for vaccination. In HIV-1 infected patients, the immune system makes neutralizing antibodies against the HIV-1 envelope glycoproteins, but these proteins are highly variable in sequence, especially between subtypes, and they utilize sophisticated mechanisms to avoid immune recognition. The study of virus neutralization and escape in patients that harbor globally prevalent genetic subtypes of HIV-1 is therefore of substantial interest. The goal of the proposed studies is to better understand how antibodies neutralize subtype C HIV-1, which predominates in sub-Saharan Africa, and to determine how the virus escapes from immune recognition. The studies will be performed using viral envelope clones derived from longitudinally collected material (blood cell DNA and plasma) from recently infected, treatment-naive subjects in a discordant couple cohort in Zambia, where subtype C HIV-1 predominates. The ability of antibodies in patient plasma to neutralize autologous viral envelope clones will be evaluated for a panel of 15 seroconvertors using a single round infection assay, and molecular approaches will be used to isolate patient-derived antibodies, identify neutralization targets, and reconstruct pathways of viral escape. Mutational patterns that occur in a helical domain of the envelope and are peripherally associated with neutralization escape will also be investigated. The hypotheses are that (i) the major escape pathways operative in early subtype C infection differ from those reported to dominate in subtype B infection, (ii) these escape pathways carry a fitness cost and are dependent on ancillary changes in the a2 helix, and (iii) B cell responses in subtype C infection target strain-specific and shared regions of Env that are distinct from those defined for subtype B infection.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Neutralizing antibodies in HIV-1 infection are directed against the envelope (Env) glycoproteins gp120 and gp41. However, HIV-1 Env utilizes highly effective, but poorly defined, mechanisms to evade antibody-mediated neutralization. The objective of these studies is to indentify the initial B cell targets and define mechanisms of viral escape in subjects that are newly infected with subtype A and C HIV-1 in Rwanda and Zambia, respectively. In year 03 of this R01, through collaboration with Dr. Guido Silvestri, we compared neutralizing antibody (Nab) responses against the autologous virus in HIV-1 infection vs. SIV infection of a natural host species, the sooty mangabey (Li et al). We demonstrated that high titer Nab develops in early HIV-1 infection and persists into the chronic stage of infection. However, SIV infection of sooty mangabeys does not elicit high titer Nab responses against the autologous virus. This finding suggests fundamental differences in the B cell response in pathogenic vs. nonpathogenic infection, and we are continuing to investigate the biology underlying this observation. We have demonstrated that B cells in chronic HIV-1 infection express increased levels of PD-1 and decreased levels of BTLA, compared to healthy human subjects (Boliar et al.). Expression of these markers was linked to increased immune activation throughout the B cells subsets and to the aberrant production of immunoglobulin. We are continuing to investigate how these abnormalities contribute to disease progression and failure of the humoral immune response in HIV-1 infection. FUNDING SOURCE: NIH

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Progress towards an HIV-1 vaccine has been stymied by the inability to induce a protective humoral response. Well-characterized neutralization epitopes are either poorly immunogenic or effectively masked on the majority of patient isolates. Newer evidence suggests that even highly masked primary isolates possess sensitive neutralization targets that are recognized by autologous patient sera and occasionally by heterologous sera. This suggests that mapping the epitopes involved may identify novel targets that are capable of inducing broadly neutralizing activities. In this project, we are identifying subtype B and C infected patients that possess broadly neutralizing activities for primary isolates, and localize the target epitopes. We are interested in antibody activities that mediate potent neutralization of heterologous viral envelope (Env) glycoproteins from various subtypes. We screened more than 100 plasma samples from subtype C HIV-1 for cross-neutralizing activities against heterologous Envs in order to discover novel targets that are exposed on typical neutralization-resistant primary isolates. In the past funding period, we identified a subtype C infected subject whose plasma possesses exceptional breadth and potency. We are in the process of characterizing the autologous viral Env and antibody neutralizing activity in this patient, as well as generating monoclonal antibodies from B cells.

In the DNA prime, MVA boost model to be used in Project One of this application, the only immune correlate of protection against mucosal challenge identified thus far is the GM-CSF mediated avidity of vaccine-elicited antibody binding to the envelope ofthe challenge virus. The mechanism for protection is unknown but suggests an important role for the humoral immune response. The goals of the Virology and Antibody Assay Core are therefore to investigate (i) a mechanism for GM-CSF-based antibody protection (Project One), (ii) novel strategies for promoting humoral immunity within the gut mucosa (Project Two), and (iii) vaccine and challenge induced antibody specificities (NHP Monoclonal Antibody Core). The Virology Core is central to these studies and will assess whether neutralizing and Fc-mediated antibody activities correlate with vaccine-induced protection. We will focus on antibodies within mucosal secretions and plasma against viral proteins Env and Gag. The neutralizing antibody component mediates antiviral activity by binding to the virion associated form of Env and preventing infection of a target cell. While neutralizing antibodies are likely to be a critical component of a vaccine, to date no one has elicited Nabs that can mediate neutralization of genetically diverse viruses, a feature that is critical for protection of HlV-1 infected humans. Other nonneutralizing antibody activities have been associated with protection against acquisition and disease progression. These involve recognition ofthe Env expressed on the surface of an infected cell, leading to lysis of that cell (ADCC) in addition to augmenting other non-lytic mechanisms that may contribute to viral suppression (ADCVI). Finally, transmission of HIV-1 and SIV across the genital mucosa is associated with a genetic bottleneck, and we will therefore characterize the variants that establish infection in the vaccinated and control monkeys to determine whether vaccine-induced antibodies imposed selective pressure on Env. Specifically, the Virology Core will determine whether peripheral and mucosal IgG and IgA-mediated antibody activities correlate with immune protection against intravaginal challenge following vaccination and investigate whether a sieve effect is evident.

DESCRIPTION (provided by applicant): The ability to induce antibodies that neutralize (nAb) circulating HIV-1 strains will be critical for a vaccine to exhibit optimal protective efficacy aganst HIV-1. To define the epitopes that can stimulate these responses, monoclonal antibodies (mAb) with broad and potent neutralization capacity have been recovered from a few chronically infected individuals that exhibited superior plasma nAb, but the viral and host B cell events that preceded this broad and potent neutralization phenotype have not been defined. Samples from early infection have not been available from these broadly neutralizing individuals, so questions about how nAb breadth developed cannot yet be answered. Here we identified 5 subtype A and C HIV-1 infected seroconvertors, out of 17, who developed relatively potent cross-clade nAb breadth at ~3 years post-infection. This proposal will investigate how and why nAb breadth developed in these individuals, but not in others who had low or undetectable levels of breadth. Our Preliminary Data suggest that there are fundamental virologic and immune differences between these two patient populations that could explain why early nAb are strain-specific, but later nAb go on to develop heterologous breadth in a subset of subjects. This focus on early infection is consistent with the observation that nAb breadth is generally either present or absent by ~3 years post-infection. Furthermore, our Preliminary Data show that autologous nAb from broad neutralizers in our panel targeted V1V2 and the CD4 binding site, both targets of mAbs with 'elite' heterologous neutralizing activity. We will utilize stored plasma and viable PBMC samples to define the initial nAb targets and viral escape pathways in 5 subjects with nAb breadth and 5 subjects without. We will recover mAbs from single B cell sorts using envelope (Env) gp140 B cell probes and PBMC samples collected at two early time points, and ~3 years post-infection, from these 10 subjects. The mAbs will be characterized genetically and functionally, and a representative subset, including those with breadth, will also be crystallized. To our knowledge, this type of comparative investigation into the early viral and immune determinants of neutralization breadth has not been performed. The proposed aims also provide a strong likelihood of recovering and characterizing broadly neutralizing mAbs that could represent early versions of those isolated from chronic infection. Our hypothesis is that the initil targeting of certain nAb epitopes, such as the CD4 binding site or V1V2, combined with the subsequent influence of escape pathways, leads to the development of nAb breadth in a subset of HIV-1 infected individuals. Specifically, the aims are to (i) Identify the initial neutralizing antibody target and define the viral escape pathways in 5 recently infected subjects who developed heterologous neutralization breadth and 5 subjects that lack breadth and (ii) Determine the genotypic, functional, and structural characteristics of early mAbs that are strain-specific and those that have acquired autologous or heterologous neutralization breadth.

Recent advances in the HIV vaccine research field have led to renewed optimism that vaccine-elicited antibody responses can contribute to protection against HIV-1 infection. These advances include the discovery of more than 40 antibodies with broad neutralization activity against genetically diverse HIV-1 strains, and the correlation of antibody responses with protection in the RV144 phase III vaccine trial. In preclinical studies in nonhuman primates (NHP), using SIV immunogens and challenge virus, we have demonstrated that inclusion of an immunostimulatory ligand, CD40L, and deletion of immunomodulatory genes from the MVA vaccine vector, both augment vaccine-elicited antibody responses. In the case of . CD40L, antibody avidity, neutralizing activity, and specificity were enhanced and correlated with protection against acquisition. Here in Project 3 we will investigate the effects of CD40L on antibody responses elicited against clade C HIV-1 immunogens in NHP in Project 2, and in human subjects enrolled in a phase I clinical trial in Project 1. Furthermore, we will look for potential synergy between CD40L and the modified MVA vaccine vector when used in combination. Project 3 will specifically characterize the antibody responses elicited by these immunization regimens, and compare responses in NHP and humans that received analagous vaccines. The hypotheses to be tested in Project 3 are (i) that inclusion of the CD40L adjuvant will focus the vaccine-elicited antibody response on potentially protective epitopes, (ii) this focused response will be associated with enhanced protection against challenge in NHP, (iii) the MVAA4 will further augment the protective antibody response elicited by the CD40L adjuvanted vaccine by increasing the magnitude of the response, and (iv) similar antibody profiles will be observed in humans and NHP that received analogous CD40L adjuvanted vaccines. The use of an innovative and broad set of experimental tools, combined with the comparison of antibody responses and specificities elicited in human subjects and NHP, will inform us about important similarities and differences that bridge the translational gap between pre-clinical and Clinical vaccine studies.

Abstract (Project Summary/Abstract) Despite a strong and lengthy effort focused on developing an HIV vaccine, immune correlates necessary to achieve robust and sustained protection remain unknown. The discovery and characterization of numerous broadly neutralizing antibodies (bnAbs) that neutralize genetically diverse viruses, and the observation that bnAbs can passively protect against infection in nonhuman primates has generated optimism that strategically selected envelope (Env) immunogens will induce neutralizing antibodies (nAbs) that are broadly protective. Recent studies suggest that Env immunogens that (i) preserve features of the native trimer and (ii) are based on variants from individuals that developed bnAbs are worthy pursuits. We propose to utilize patient-informed Env immunogens delivered via DNA/modified vaccinia Ankara (MVA) immunization followed by protein boost to determine how the natural history and presentation of the Envs shape the genetic and functional antibody landscape in rhesus macaques (RM). The Env immunogen sets are derived from two HIV-1 infected individuals, chosen due to the disparate nature of their early antibody responses, which can be mimicked by vaccination, and subsequent development of ?elite? or ?poor? nAb breadth. Monoclonal antibodies derived from these two individuals 7 months after infection display striking differences in germline usage, clonality, binding affinity, and autologous neutralization. Thus, a detailed understanding of how antibody responses are influenced and altered by the immunogen choice and presentation form will ensure that vaccine efforts can be driven towards bnAbs, while simultaneously avoiding pathways that produce antibodies with limited function. DNA/MVA, a vaccine platform that produces robust and durable Env-specific antibody responses in RM and humans, and has provided protection against SIV challenge, will be used to deliver sequential patient-derived Env immunogens. These immunizations will be followed by a protein boost consisting of either gp120 or ?native flexibly linked? (NFL) gp140 stabilized trimers. Our ensuing multi-parametric analysis will include antibody germline usage, somatic hypermutation, development and longevity of clonal lineages, binding affinity (KD), and capacity to neutralize virus or facilitate Fc-mediated signaling, as well as post-immunization activation of germinal center (GC) B cells and T follicular helper cells (Tfh). To achieve an unprecedented level of resolution, our analysis will take place at the single B cell and monoclonal antibody level, examining plasmablasts, memory B cells, and long lived plasma cells residing in the bone marrow. Based on the results of these analyses, we will select individual B cells from immunized monkeys for RNA-Seq transcriptome analysis to connect B cell biology with antibody functional capacity. Our overall hypothesis is that the immunogens from the ?elite? neutralizer will elicit functional neutralizing antibody responses, and the trimer protein will further augment these. By contrast, immunogens from the ?poor? neutralizer will reveal mechanistic roadblocks to the desired antibody responses.

Project Summary/Abstract HIV research efforts are strongly focused on developing vaccines that can elicit highly protective antibody responses. There is evidence from human and nonhuman vaccine studies that both neutralizing and non- neutralizing antibody activities contribute to protection against acquisition. Importantly, the only phase III vaccine efficacy trial to show a signal of protection in human volunteers demonstrated that antibody dependent cell- mediated cytotoxicity (ADCC) was associated with reduced acquisition, compelling us to know more about this Fc-mediated antiviral function. We previously recovered a unique collection of more than 300 monoclonal antibodies from 6 recently HIV-1 infected individuals from Zambia and Rwanda and found that a subset of these antibodies mediated ADCC against target cells coated with the envelope gp120 protein from the autologous, transmitted/founder (T/F) variant. This activity was associated with antibodies that had shorter CDRH3 regions and lower neutralization activity against the T/F envelope pseudovirus than antibodies lacking ADCC activity. Modeling and competition studies suggested that some of the ADCC-mediating antibodies recognize novel epitopes. Here, we will test these monoclonal antibodies for ADCC against target cells infected with the authentic T/F variant created by molecular cloning, and compare the results with ADCC against target cells coated with the T/F gp120 protein and neutralization of the T/F Env, the latter being the hallmark of antibody binding to the functional, virion-associated envelope spike. By using a large number of autologous T/F envelope-antibody combinations, where the envelope presents the exact epitope or a very close approximation to that which the antibody was selected against, and two widely used ADCC assays, our studies have the potential to reveal previously unappreciated mechanistic features of ADCC and identify novel epitopes that can be further developed and explored for vaccines.

SUMMARY Clinical sequelae of COVID-19 patients include not only acute respiratory distress syndrome (ARDS), but also often acute kidney injury and heart failure. These pathologies share endothelial activation as a common underlying early response to injury, and endothelial cells express high levels of angiotensin converting enzyme 2 (ACE2), a functional receptor for SARS-CoV-2. Activated endothelium not only attracts and promotes leukocyte infiltration into tissues and contributes to the cytokine storm resulting in capillary leakage and edema, but is also prothrombotic. Together, these mechanisms result in tissue inflammation and ischemia, leading to organ failure. Our laboratory discovered that polymerase delta interacting protein 2 (Poldip2) is a novel and important regulator of inflammation, endothelial permeability and potentially coagulation in mice. Mice heterozygous for Poldip2 are largely protected from lipopolysaccharide (LPS)- or P. aeruginosa-induced ARDS. Here, we hypothesize that Poldip2 may be a novel target for treatment of SARS-CoV-2-infected individuals, as downregulation of Poldip2 not only reduces ARDS complications such as edema and the cytokine storm, but also potentially may inhibit thrombosis. To test this hypothesis, we propose to treat SARS-CoV-2-infected mice with an anti-cancer agent undergoing clinical trials that we have recently shown to reduce Poldip2 levels and restore endothelial barrier function. In the first Aim, we will use this agent to downregulate Poldip2 and test its ability to preserve endothelial barrier function and reduce inflammation in response to SARS-CoV-2 infection in mice. The second aim will focus on determining the effect of the anticancer agent and genetic ablation of Poldip2 on basal coagulation and that induced by SARS-CoV-2 infection. We anticipate that pharmacological downregulation of Poldip2 will represent a promising new treatment for COVID-19 patients that can be rapidly translated to the clinic.