Contrast-Enhanced In-Phase Dixon Sequence: Impact on Biopsy Clip Detection on Breast MRI.

BACKGROUND. Identification of breast biopsy clips using conventional MRI sequences may be challenging. A contrast-enhanced in-phase Dixon sequence may have greater conspicuity for areas of susceptibility compared with standard clinical sequences. OBJECTIVE. The purpose of this article is to compare detection of breast biopsy clips on MRI between the contrast-enhanced in-phase Dixon sequence and three routine clinical sequences. METHODS. This retrospective study included 164 patients (mean age, 50.3 years) with a total of 281 breast biopsy clips who underwent contrast-enhanced breast MRI between January 2, 2019, and April 16, 2020. Three radiologists, blinded to the clip location and sequence used, independently annotated biopsy clip locations on three clinical sequences (T1-weighted non-fat-suppressed [NFS], STIR, and first phase from dynamic contrast-enhanced T1-weighted fat-suppressed [FS]) and on a contrast-enhanced in-phase Dixon sequence and then recorded confidence scores (1-4 scale). A study coordinator used all available imaging and reports to localize clips on MRI, which served as the reference standard. A physicist measured clip CNR. Sequences were compared using the McNemar test and two-tailed Wilcoxon signed rank tests. RESULTS. Among the three readers, pooled sensitivity and PPV were 78.2% and 96.2% for T1-weighted NFS, 26.6% and 92.7% for STIR, 61.7% and 95.9% for contrast-enhanced T1-weighted FS, and 85.1% and 95.1% for contrast-enhanced in-phase Dixon sequence. Pooled sensitivity was higher for contrast-enhanced in-phase Dixon sequence than for the other sequences (all p < .05); pooled PPV was not significantly different between contrast-enhanced in-phase Dixon and the other sequences (all p > .05). Mean confidence scores (pooled across readers for true-positive assessments) and mean CNR were 3.0 ± 0.9 (SD) and 1.21 ± 0.61 for T1-weighted NFS, 1.7 ± 0.9 and 0.57 ± 0.69 for STIR, 2.5 ± 1.0 and 0.54 ± 0.61 for contrast-enhanced T1-weighted FS, and 3.5 ± 0.8 and 4.05 ± 2.6 for the contrast-enhanced in-phase Dixon sequence. Pooled mean confidence scores and CNR were higher for contrast-enhanced in-phase Dixon than for the other sequences (all p < .001). CONCLUSION. Compared with clinical sequences, the contrast-enhanced in-phase Dixon sequence had higher sensitivity for detecting breast biopsy clips on MRI and higher reader confidence and CNR, without change in PPV. CLINICAL IMPACT. The contrast-enhanced in-phase Dixon sequence may help address a current challenge in clinical breast MRI interpretation.

Increased Epitope Complexity Correlated with Antibody Affinity Maturation and a Novel Binding Mode Revealed by Structures of Rabbit Antibodies against the Third Variable Loop (V3) of HIV-1 gp120.

The third variable (V3) loop of HIV-1 gp120 is an immunodominant region targeted by neutralizing antibodies (nAbs). Despite limited breadth, better characterization of the structural details of the interactions between these nAbs and their target epitopes would enhance our understanding of the mechanism of neutralization and facilitate designing better immunogens to induce nAbs with greater breadth. Recently, we isolated two anti-V3 neutralizing monoclonal antibodies (MAbs), 10A3 and 10A37, from a rabbit immunized with gp120 of the M group consensus sequence. In this study, crystal structures of these MAbs bound to target epitopes were determined. 10A3 binds to the V3 crown (303TRKSIHIGPGRAF317) using the cradle binding mode, similar to human V3 MAbs encoded by IGHV5-51 germ line genes, and its epitope structure resembles that bound to the human antibodies. In contrast, 10A37, which exhibits greater breadth and potency than 10A3, binds the V3 crown and the succeeding stem region (308HIGPGRAFYTTGEI323). Unexpectedly, the 315RAFYTT320 portion of the epitope existed as helical turns, a V3 structure that has not been observed previously. Its main chain-dominated antigen-antibody interactions not only explain the broad neutralization of 10A37 but also show that its epitope is a potential vaccine target to be further evaluated. In conclusion, our study provides novel insights about neutralization-susceptible epitope structures of the V3 loop of HIV-1 gp120 and demonstrates that, despite low amino acid sequence similarity to human antibody germ line genes, rabbits can serve as a useful animal model to evaluate human vaccine candidates.IMPORTANCE The apex crown of V3 of HIV-1 gp120 is the most immunogenic region of the surface glycoprotein, and many MAbs targeting this region have been developed. Structural understanding of V3 crown MAbs not only can help understand how antibody responses target this unique region but also contribute to immunogen design for vaccine development. We present here crystal structures of two neutralizing V3 MAbs, 10A3 and 10A37, developed from a rabbit immunized with gp120. Our analysis of 10A3 in complex with V3 provided a detailed example of how epitope complexity can evolve with affinity maturation, while that of 10A37 revealed a novel V3 binding mode targeting the C-terminal side of the V3 crown and showed that this region can form a helical structure. Our study provides novel insights about neutralization-susceptible V3 epitope structures and demonstrates that rabbits can serve as a useful animal model to evaluate human vaccine candidates.

Comparison of the vaginal environment in rhesus and cynomolgus macaques pre- and post-lactobacillus colonization.

BACKGROUND: Rhesus and cynomologus macaques are valuable animal models for the study of human immunodeficiency virus (HIV) prevention strategies. However, for such studies focused on the vaginal route of infection, differences in vaginal environment may have deterministic impact on the outcome of such prevention, providing the rationale for this study. METHODS: We tested the vaginal environment of rhesus and cynomolgus macaques longitudinally to characterize the normal microflora based on Nugent scores and pH. This evaluation was extended after colonization of the vaginal space with Lactobacilli in an effort to recreate NHP models representing the healthy human vaginal environment. RESULTS AND CONCLUSION: Nugent scores and pH differed significantly between species, although data from both species were suggestive of stable bacterial vaginosis. Colonization with Lactobacilli was successful in both species leading to lower Nugent score and pH, although rhesus macaques appeared better able to sustain Lactobacillus spp over time.

Detailed characterization of antibody responses against HIV-1 group M consensus gp120 in rabbits.

BACKGROUND: We recently reported induction of broadly neutralizing antibodies (bnAbs) against multiple HIV-1 (human immunodeficiency virus type 1) isolates in rabbits, albeit weak against tier 2 viruses, using a monomeric gp120 derived from an M group consensus sequence (MCON6). To better understand the nature of the neutralizing activity, detailed characterization of immunological properties of the protein was performed. Immunogenic linear epitopes were identified during the course of immunization, and spatial distribution of these epitopes was determined. Subdomain antibody target analyses were done using the gp120 outer domain (gp120-OD) and eOD-GT6, a protein based on a heterologous sequence. In addition, refined epitope mapping analyses were done by competition assays using several nAbs with known epitopes. RESULTS: Based on linear epitope mapping analyses, the V3 loop was most immunogenic, followed by C1 and C5 regions. The V1/V2 loop was surprisingly non-immunogenic. Many immunogenic epitopes were clustered together even when they were distantly separated in primary sequence, suggesting the presence of immunogenic hotspots on the protein surface. Although substantial antibody responses were directed against the outer domain, only about 0.1% of the antibodies bound eOD-GT6. Albeit weak, antibodies against peptides that corresponded to a part of the bnAb VRC01 binding site were detected. Although gp120-induced antibodies could not block VRC01 binding to eOD-GT6, they were able to inhibit VRC01 binding to both gp120 and trimeric BG505 SOSIP gp140. The immune sera also efficiently competed with CD4-IgG2, as well as nAbs 447-52D, PGT121 and PGT126, in binding to gp120. CONCLUSIONS: The results suggest that some antibodies that bind at or near known bnAb epitopes could be partly responsible for the breadth of neutralizing activity induced by gp120 in our study. Immunization strategies that enhance induction of these antibodies relative to others (e.g. V3 loop), and increase their affinity, could improve protective efficacy of an HIV-1 vaccine.

Racial Disparities in Breast Imaging Wait Times Before and After the Implementation of a Same-Day Biopsy Program.

OBJECTIVE: To examine time from screening to diagnostic workup, biopsy, and surgery for non-Hispanic White (NHW) and Black women following implementation of a same-day biopsy program. METHODS: All NHW and Black women with BI-RADS category 0 screening mammogram at Duke University Hospital were identified between August 1, 2020, and August 1, 2021. Patient characteristics were recorded. Time between screening mammogram, diagnostic workup, breast biopsy, surgical consultation, and surgery were recorded. Comparisons were made between NHW and Black women using a multivariable regression model. Diagnostic imaging to biopsy time interval was compared to historical averages before same-day biopsy implementation. RESULTS: There were 2156 women: 69.9% NHW (1508/2156) and 30.1% Black (648/2156). Mean ± standard deviation time from screening to diagnostic imaging overall was 13.5 ±â€…32.5 days but longer for Black (18.0 ±â€…48.3 days) than for NHW women (11.5 ±â€…22.2 days) (P < 0.001). The mean time from diagnostic mammogram to biopsy was 5.9 ±â€…18.9 days, longer for Black (9.0 ±â€…27.9 days) than for NHW women (4.4 ±â€…11.8 days) (P = 0.017). The same-day biopsy program shortened the time from diagnostic imaging to biopsy overall (12.5 ±â€…12.4 days vs 5.9 ±â€…18.9 days; P < 0.001), with a significant reduction for NHW women (12.4 ±â€…11.7 days vs 4.4 ±â€…11.8 days) (P < 0.001) but not Black women (11.5 ±â€…9.9 days vs 9.0 ±â€…27.9 days) (P = 0.527). CONCLUSION: Disparities exist along the breast imaging pathway. A same-day biopsy program benefited NHW women more than Black women.

Role of digital breast tomosynthesis in the evaluation of focal breast pain.

RATIONAL AND OBJECTIVE: To investigate the utility of digital breast tomosynthesis (DBT) in the evaluation of focal breast pain, considering breast density and breast cancer risk. METHODS: Ninety-one cases of focal breast pain evaluated with DBT and ultrasound (US) from 12/30/2014 to 11/9/2017 with 2-year follow-up were identified. Exclusion criteria were non-focal, axillary, or radiating pain; palpable or skin changes; pregnancy or lactation; and history of ipsilateral cancer, trauma, or infection. Demographic data, Tyrer-Cuzick Score (TCS), medical history, breast density, imaging results, and pathology were recorded. Descriptive statistics were reported. RESULTS: Eighteen percent (16/91) of cases demonstrated findings, all benign. Of these, 6% (1/16) were detected by DBT only, 88% (14/16) by US only, and 6% (1/16) by DBT and US. US resulted in 3 benign biopsies. Ninety-nine percent (75/76) of cases with no findings at the site of pain on US also had no findings on DBT. Ninety-eight percent (89/91) of cases with no cancer detected at the site of pain on US also did not have cancer on DBT. DBT detected 2 incidental cancers not associated with pain. DBT and US agreed that there was no finding at the site of pain in 82% (75/91) of cases. A high degree of agreement between DBT and US was seen when stratified by breast density and TCS. CONCLUSION: DBT may be appropriate for the evaluation of focal pain. Low breast cancer incidence was observed at the site of focal pain across all mammographic breast densities and breast cancer risks.

Structural characterization of HIV gp41 with the membrane-proximal external region.

Human immunodeficiency virus, type 1 (HIV-1) envelope glycoprotein (gp120/gp41) plays a critical role in virus infection and pathogenesis. Three of the six monoclonal antibodies considered to have broadly neutralizing activities (2F5, 4E10, and Z13e1) bind to the membrane-proximal external region (MPER) of gp41. This makes the MPER a desirable template for developing immunogens that can elicit antibodies with properties similar to these monoclonal antibodies, with a long term goal of developing antigens that could serve as novel HIV vaccines. In order to provide a structural basis for rational antigen design, an MPER construct, HR1-54Q, was generated for x-ray crystallographic and x-ray footprinting studies to provide both high resolution atomic coordinates and verification of the solution state of the antigen, respectively. The crystal structure of HR1-54Q reveals a trimeric, coiled-coil six-helical bundle, which probably represents a postfusion form of gp41. The MPER portion extends from HR2 in continuation of a slightly bent long helix and is relatively flexible. The structures observed for the 2F5 and 4E10 epitopes agree well with existing structural data, and enzyme-linked immunosorbent assays indicate that the antigen binds well to antibodies that recognize the above epitopes. Hydroxyl radical-mediated protein footprinting of the antigen in solution reveals specifically protected and accessible regions consistent with the predictions based on the trimeric structure from the crystallographic data. Overall, the HR1-54Q antigen, as characterized by crystallography and footprinting, represents a postfusion, trimeric form of HIV gp41, and its structure provides a rational basis for gp41 antigen design suitable for HIV vaccine development.

Induction of Potent and Durable Neutralizing Antibodies Against SARS-CoV-2 Using a Receptor Binding Domain-Based Immunogen.

A novel betacoronavirus (SARS-CoV-2) that causes severe pneumonia emerged through zoonosis in late 2019. The disease, referred to as COVID-19, has an alarming mortality rate and it is having a devastating effect on the global economy and public health systems. A safe, effective vaccine is urgently needed to halt this pandemic. In this study, immunogenicity of the receptor binding domain (RBD) of spike (S) glycoprotein was examined in mice. Animals were immunized with recombinant RBD antigen intraperitoneally using three different adjuvants (Zn-chitosan, Alhydrogel, and Adju-Phos), and antibody responses were followed for over 5 months. Results showed that potent neutralizing antibodies (nAbs) can be induced with 70% neutralization titer (NT70) of ~14,580 against live, infectious viruses. Although antigen-binding antibody titers decreased gradually over time, sufficiently protective levels of nAbs persisted (NT80 >2,430) over the 5-month observation period. Results also showed that adjuvants have profound effects on kinetics of nAb induction, total antibody titers, antibody avidity, antibody longevity, and B-cell epitopes targeted by the immune system. In conclusion, a recombinant subunit protein immunogen based on the RBD is a highly promising vaccine candidate. Continued evaluation of RBD immunogenicity using different adjuvants and vaccine regimens could further improve vaccine efficacy.

A Structural Landscape of Neutralizing Antibodies Against SARS-CoV-2 Receptor Binding Domain.

SARS-CoV-2, the novel coronavirus responsible for the ongoing COVID-19 pandemic, has been spreading rampantly. The global scientific community has responded rapidly to understand immune correlates of protection to develop vaccines and immunotherapeutics against the virus. The major goal of this mini review is to summarize current understanding of the structural landscape of neutralizing antibodies (nAbs) that target the receptor binding domain (RBD) of viral spike (S) glycoprotein. The RBD plays a critical role in the very first step of the virus life cycle. Better understanding of where and how nAbs bind the RBD should enable identification of sites of vulnerability and facilitate better vaccine design and formulation of immunotherapeutics. Towards this goal, we compiled 38 RBD-binding nAbs with known structures. Review of these nAb structures showed that (1) nAbs can be divided into five general clusters, (2) there are distinct non-neutralizing faces on the RBD, and (3) maximum of potentially four nAbs could bind the RBD simultaneously. Since most of these nAbs were isolated from virus-infected patients, additional analyses of vaccine-induced nAbs could facilitate development of improved vaccines.

Structural analysis of a highly glycosylated and unliganded gp120-based antigen using mass spectrometry.

Structural characterization of the HIV-1 envelope protein gp120 is very important for providing an understanding of the protein's immunogenicity and its binding to cell receptors. So far, the crystallographic structure of gp120 with an intact V3 loop (in the absence of a CD4 coreceptor or antibody) has not been determined. The third variable region (V3) of the gp120 is immunodominant and contains glycosylation signatures that are essential for coreceptor binding and entry of the virus into T-cells. In this study, we characterized the structure of the outer domain of gp120 with an intact V3 loop (gp120-OD8) purified from Drosophila S2 cells utilizing mass spectrometry-based approaches. We mapped the glycosylation sites and calculated the glycosylation occupancy of gp120-OD8; 11 sites from 15 glycosylation motifs were determined as having high-mannose or hybrid glycosylation structures. The specific glycan moieties of nine glycosylation sites from eight unique glycopeptides were determined by a combination of ECD and CID MS approaches. Hydroxyl radical-mediated protein footprinting coupled with mass spectrometry analysis was employed to provide detailed information about protein structure of gp120-OD8 by directly identifying accessible and hydroxyl radical-reactive side chain residues. Comparison of gp120-OD8 experimental footprinting data with a homology model derived from the ligated CD4-gp120-OD8 crystal structure revealed a flexible V3 loop structure in which the V3 tip may provide contacts with the rest of the protein while residues in the V3 base remain solvent accessible. In addition, the data illustrate interactions between specific sugar moieties and amino acid side chains potentially important to the gp120-OD8 structure.

Human immunodeficiency virus type 1 envelope gp120-induced partial T-cell receptor signaling creates an F-actin-depleted zone in the virological synapse.

Cell-to-cell transmission of human immunodeficiency virus type 1 (HIV-1) occurs via a virological synapse (VS), a tight cell-cell junction formed between HIV-infected cells and target cells in which the HIV-1-infected cell polarizes and releases virions toward the noninfected target cell in a gp120- and intercellular adhesion molecule 1 (ICAM-1)-dependent process. The response of the target cell has been less studied. We utilized supported planar bilayers presenting gp120 and ICAM-1 as a reductionist model for the infected-cell membrane and investigated its effect on the target CD4 T cell. This study shows that HIV-1 gp120 interaction with its receptors is initially organized into microclusters that undergo F-actin-dependent consolidation into a central supramolecular activation complex (cSMAC). Src kinases are active in both gp120 microclusters and in the VS cSMAC. The early T-cell receptor (TCR) signaling machinery is partially activated at the VS, and signaling does not propagate to trigger Ca(2+) elevation or increase CD69 expression. However, these partial TCR signals act locally to create an F-actin-depleted zone. We propose a model in which the F-actin-depleted zone formed within the target CD4 T cell enhances the reception of virions by releasing the physical barrier for HIV-1 entry and facilitating postentry events.

Optimal efficient imaging time for identification of sentinel lymph node with Tc-99m Tilmanocept in breast cancer patients.

We determined the optimal imaging time for axillary lymph node (LN) visualization following Tc-99m Tilmanocept in breast cancer patients to establish imaging guidelines that can allow for a reliable and efficient yet high yield study prior to surgery. Retrospective analysis in 651 patients who underwent lymphoscintigraphy, comparing LN visualization on immediate, 15-minute, and 90-minute delayed imaging after injection of Tc-99m Tilmanocept. Statistical analysis was performed using McNemar's test, kappa coefficient, and Pearson Chi-square test. Five hundred and six patients had either immediate or immediate and 90-minute delayed imaging. Of these patients, 203 (40.1%) had both immediate and 90-minute delayed images. Of these 203 patients, 54 (26.6%) had ≥1 lymph node(s) identified immediately and 196 (96.6%) had ≥1 lymph node(s) identified at 90 minutes (P<0.0001). A kappa coefficient of .0256 was observed (95% CI: .0058-.0453). One hundred and forty-five additional patients had 15-minute delayed imaging. Of these patients, 117 (80.7%) had ≥1 lymph node(s) identified, which was significantly fewer compared to the number of patients with ≥1 lymph node(s) detected at 90 minutes (P<0.0001). Ninety-minute delayed imaging is optimal for identifying sentinel lymph node(s) following Tc-99m Tilmanocept injection in breast cancer patients.

Human immunodeficiency virus type 1 envelope gp120 induces a stop signal and virological synapse formation in noninfected CD4+ T cells.

Human immunodeficiency virus type 1 (HIV-1)-infected T cells form a virological synapse with noninfected CD4(+) T cells in order to efficiently transfer HIV-1 virions from cell to cell. The virological synapse is a specialized cellular junction that is similar in some respects to the immunological synapse involved in T-cell activation and effector functions mediated by the T-cell antigen receptor. The immunological synapse stops T-cell migration to allow a sustained interaction between T-cells and antigen-presenting cells. Here, we have asked whether HIV-1 envelope gp120 presented on a surface to mimic an HIV-1-infected cell also delivers a stop signal and if this is sufficient to induce a virological synapse. We demonstrate that HIV-1 gp120-presenting surfaces arrested the migration of primary activated CD4 T cells that occurs spontaneously in the presence of ICAM-1 and induced the formation of a virological synapse, which was characterized by segregated supramolecular structures with a central cluster of envelope surrounded by a ring of ICAM-1. The virological synapse was formed transiently, with the initiation of migration within 30 min. Thus, HIV-1 gp120-presenting surfaces induce a transient stop signal and supramolecular segregation in noninfected CD4(+) T cells.

18F-FDG PET/CT findings in hepatosplenic Gamma-Delta T-cell lymphoma: case reports and review of the literature.

Hepatosplenic Gamma Delta T cell lymphoma (γδHSTL) is a rare, highly aggressive, and rapidly lethal T cell lymphoma which manifests 18F-FDG PET/CT findings that can mimic benign conditions. Patients with γδHSTL present with unexplained symptoms of a hematologic malignancy like the B symptoms of lymphoma including weight loss, fevers, and night sweats, as well as, splenomegaly and hepatomegaly. Thrombocytopenia, anemia, or neutropenia are also common due to spleen, liver and bone marrow involvement. The peripheral blood, however, typically does not show abnormal T cells. The clinical and 18F-FDG PET/CT findings are presented for 3 patients with γδHSTL. Patients with γδHSTL may have a normal 18F-FDG PET/CT or an 18F-FDG PET/CT with any combination of the three findings: splenomegaly with intense FDG uptake; hepatomegaly with increased FDG uptake; and diffuse, increased FDG uptake in the bone marrow. Importantly, lymphadenopathy is usually absent, and most patients show morphologically normal lymph nodes with normal FDG uptake. Due to the aggressive nature of the disease, γδHSTL is a critical diagnosis to consider in patients who present with clinical signs of suspected hematologic malignancy and variable 18F-FDG PET/CT findings. The absence of lymphadenopathy and normal FDG uptake in lymph nodes are typical pertinent negative findings that differentiate γδHSTL from other lymphomas. A bone or liver biopsy is frequently necessary to establish the diagnosis and should be recommended.

Focal Breast Pain: Does Breast Density Affect the Need for Ultrasound?

RATIONALE AND OBJECTIVES: This study aimed to determine the utility of directed ultrasound and digital mammogram for evaluating focal breast pain in women with different mammographic breast densities. MATERIALS AND METHODS: This institutional review board-approved and Health Insurance Portability and Accountability Act-compliant retrospective study included 413 cases of focal breast pain in 369 women (mean age 53 years). All cases were evaluated with both mammogram and ultrasound and had at least 2 years of imaging follow-up. Exclusion criteria were non-focal, axillary, or radiating pain; palpable or skin changes; pregnancy or lactation; and history of trauma or infection. Breast density, imaging findings, and biopsy results were recorded. Specificity, positive predictive values, and negative predictive values were calculated. RESULTS: Eighteen percent (76 of 413) of cases demonstrated an imaging correlate. Of these, 74% (56 of 76) occurred in dense breasts and 26% (20 of 76) in nondense breasts. Seventy percent (14 of 20) of lesions in nondense breasts were seen with mammography and ultrasound, whereas 30% (6 of 20) were detected only with ultrasound. Of lesions detected in dense breasts, 29% (16 of 56) were seen with mammography and ultrasound, whereas 71% (40 of 56) were detected only with ultrasound. Thirty-one percent (24 of 76) of cases were biopsied, 42% (10 of 24) of which were detected by ultrasound only. No cancer was detected in initial workup. At 2-year follow-up, three women, all with dense breasts, developed cancer in the same quadrant as the initial pain. CONCLUSIONS: Directed ultrasound, when performed in conjunction with digital mammography for the evaluation of focal breast pain in women with nondense breasts, is of low utility and may contribute to unnecessary intervention as a result of incidental findings.

Evaluation of a novel multi-immunogen vaccine strategy for targeting 4E10/10E8 neutralizing epitopes on HIV-1 gp41 membrane proximal external region.

The membrane proximal external region (MPER) of HIV-1 gp41 is targeted by broadly neutralizing antibodies (bnAbs) 4E10 and 10E8. In this proof-of-concept study, we evaluated a novel multi-immunogen vaccine strategy referred to as Incremental, Phased Antigenic Stimulation for Rapid Antibody Maturation (IPAS-RAM) to induce 4E10/10E8-like bnAbs. Rabbits were immunized sequentially, but in a phased manner, with three immunogens that are progressively more native (gp41-28×3, gp41-54CT, and rVV-gp160DH12). Although nAbs were not induced, epitope-mapping analyses indicated that IPAS-RAM vaccination was better able to target antibodies towards the 4E10/10E8 epitopes than homologous prime-boost immunization using gp41-28×3 alone. MPER-specific rabbit monoclonal antibodies were generated, including 9F6. Although it lacked neutralizing activity, the target epitope profile of 9F6 closely resembled those of 4E10 and 10E8 (671NWFDITNWLWYIK683). B-cell repertoire analyses suggested the importance of co-immunizations for maturation of 9F6, which warrants further evaluation of our IPAS-RAM vaccine strategy using an improved priming immunogen.

Differences in serum IgA responses to HIV-1 gp41 in elite controllers compared to viral suppressors on highly active antiretroviral therapy.

Mechanisms responsible for natural control of human immunodeficiency type 1 (HIV) replication in elite controllers (EC) remain incompletely defined. To determine if EC generate high quality HIV-specific IgA responses, we used Western blotting to compare the specificities and frequencies of IgA to HIV antigens in serum of gender-, age- and race-matched EC and aviremic controllers (HC) and viremic noncontrollers (HN) on highly active antiretroviral therapy (HAART). Concentrations and avidity of IgA to HIV antigens were measured using ELISA or multiplex assays. Measurements for IgG were performed in parallel. EC were found to have stronger p24- and V1V2-specific IgG responses than HN, but there were no IgG differences for EC and HC. In contrast, IgA in EC serum bound more frequently to gp160 and gag proteins than IgA in HC or HN. The avidity of anti-gp41 IgA was also greater in EC, and these subjects had stronger IgA responses to the gp41 heptad repeat region 1 (HR1), a reported target of anti-bacterial RNA polymerase antibodies that cross react with gp41. However, EC did not demonstrate greater IgA responses to E. coli RNA polymerase or to peptides containing the shared LRAI sequence, suggesting that most of their HR1-specific IgA antibodies were not induced by intestinal microbiota. In both EC and HAART recipients, the concentrations of HIV-specific IgG were greater than HIV-specific IgA, but their avidities were comparable, implying that they could compete for antigen. Exceptions were C1 peptides and V1V2 loops. IgG and IgA responses to these antigens were discordant, with IgG reacting to V1V2, and IgA reacting to C1, especially in EC. Interestingly, EC with IgG hypergammaglobulinemia had greater HIV-specific IgA and IgG responses than EC with normal total IgG levels. Heterogeneity in EC antibody responses may therefore be due to a more focused HIV-specific B cell response in some of these individuals. Overall, these data suggest that development of HIV-specific IgA responses and affinity maturation of anti-gp41 IgA antibodies occurs to a greater extent in EC than in subjects on HAART. Future studies will be required to determine if IgA antibodies in EC may contribute in control of viral replication.

Modulating immunogenic properties of HIV-1 gp41 membrane-proximal external region by destabilizing six-helix bundle structure.

The C-terminal alpha-helix of gp41 membrane-proximal external region (MPER; (671)NWFDITNWLWYIK(683)) encompassing 4E10/10E8 epitopes is an attractive target for HIV-1 vaccine development. We previously reported that gp41-HR1-54Q, a trimeric protein comprised of the MPER in the context of a stable six-helix bundle (6HB), induced strong immune responses against the helix, but antibodies were directed primarily against the non-neutralizing face of the helix. To better target 4E10/10E8 epitopes, we generated four putative fusion intermediates by introducing double point mutations or deletions in the heptad repeat region 1 (HR1) that destabilize 6HB in varying degrees. One variant, HR1-∆10-54K, elicited antibodies in rabbits that targeted W672, I675 and L679, which are critical for 4E10/10E8 recognition. Overall, the results demonstrated that altering structural parameters of 6HB can influence immunogenic properties of the MPER and antibody targeting. Further exploration of this strategy could allow development of immunogens that could lead to induction of 4E10/10E8-like antibodies.