Autologous neutralizing antibody responses to an HIV envelope glycan hole are not easily broadened in rabbits

Extensive studies with subtype A BG505-derived HIV Env immunogens have revealed that the dominant autologous neutralizing epitope in rabbits is located in an exposed region of the heavily glycosylated trimer that lacks potential N-linked glycosylation sites at positions 230, 241, and 289. The Env derived from B41, a subtype B virus, shares a glycan hole centered on positions 230 and 289. To test whether broader neutralization to the common glycan hole can be achieved, we immunized rabbits with B41 SOSIP alone, as well as B41 and BG505 co-immunization. We isolated autologous neutralizing antibodies (nAbs) and described their structure in complex with the B41 Env. Our data suggest that distinct autologous nAb lineages are induced by BG505 and B41 immunogens, even when both were administered together. In contrast to previously described BG505 glycan hole antibodies, the B41-specific nAbs accommodate the >97% conserved N241 glycan, which is present in B41. Single particle cryo-electron microscopy studies confirmed that B41 and BG505-specific nAbs bind to overlapping glycan hole epitopes. We then used our high-resolution data to guide mutations in the BG505 glycan hole epitope in an attempt to broaden the reactivity of a B41-specific nAb, but only recovered partial binding. Our data demonstrate that lack of cross-reactivity in glycan hole antibodies is due to amino acid differences within the epitope and our attempts to rationally design cross-reactive trimers resulted in only limited success. Thus, even for the immunodominant glycan hole shared between BG505 and B41 the prospect of designing prime-boost immunogens remains difficult

HIV envelope trimer-elicited autologous neutralizing antibodies bind a region overlapping the N332 glycan supersite

To date, immunization studies of rabbits with the BG505 SOSIP.664 HIV envelope glycoprotein trimers have revealed the 241/289 glycan hole as the dominant neutralizing antibody epitope. Here, we isolated monoclonal antibodies from a rabbit that did not exhibit glycan hole–dependent autologous serum neutralization. The antibodies did not compete with a previously isolated glycan hole–specific antibody but did compete with N332 glycan supersite broadly neutralizing antibodies. A 3.5-Å cryoEM structure of one of the antibodies in complex with the BG505 SOSIP.v5.2 trimer demonstrated that while the epitope recognized overlapped the N332 glycan supersite by contacting the GDIR motif at the base of V3, primary contacts were located in the variable V1 loop. These data suggest that strain-specific responses to V1 may interfere with broadly neutralizing responses to the N332 glycan supersite and vaccine immunogens may require engineering to minimize these off-target responses or steer them toward a more desirable pathway

Intraguild Predation and Native Lady Beetle Decline

Coccinellid communities across North America have experienced significant changes in recent decades, with declines in several native species reported. One potential mechanism for these declines is interference competition via intraguild predation; specifically, increased predation of native coccinellid eggs and larvae following the introduction of exotic coccinellids. Our previous studies have shown that agricultural fields in Michigan support a higher diversity and abundance of exotic coccinellids than similar fields in Iowa, and that the landscape surrounding agricultural fields across the north central U.S. influences the abundance and activity of coccinellid species. The goal of this study was to quantify the amount of egg predation experienced by a native coccinellid within Michigan and Iowa soybean fields and explore the influence of local and large-scale landscape structure. Using the native lady beetle Coleomegilla maculata as a model, we found that sentinel egg masses were subject to intense predation within both Michigan and Iowa soybean fields, with 60.7% of egg masses attacked and 43.0% of available eggs consumed within 48 h. In Michigan, the exotic coccinellids Coccinella septempunctata and Harmonia axyridis were the most abundant predators found in soybean fields whereas in Iowa, native species including C. maculata, Hippodamia parenthesis and the soft-winged flower beetle Collops nigriceps dominated the predator community. Predator abundance was greater in soybean fields within diverse landscapes, yet variation in predator numbers did not influence the intensity of egg predation observed. In contrast, the strongest predictor of native coccinellid egg predation was the composition of edge habitats bordering specific fields. Field sites surrounded by semi-natural habitats including forests, restored prairies, old fields, and pasturelands experienced greater egg predation than fields surrounded by other croplands. This study shows that intraguild predation by both native and exotic predators may contribute to native coccinellid decline, and that landscape structure interacts with local predator communities to shape the specific outcomes of predator-predator interactions

Holes in the Glycan Shield of the Native HIV Envelope Are a Target of Trimer-Elicited Neutralizing Antibodies

A major advance in the search for an HIV vaccine has been the development of a near-native Envelope trimer (BG505 SOSIP.664) that can induce robust autologous Tier 2 neutralization. Here, potently neutralizing monoclonal antibodies (nAbs) from rabbits immunized with BG505 SOSIP.664 are shown to recognize an immunodominant region of gp120 centered on residue 241. Residue 241 occupies a hole in the glycan defenses of the BG505 isolate, with fewer than 3% of global isolates lacking a glycan site at this position. However, at least one conserved glycan site is missing in 89% of viruses, suggesting the presence of glycan holes in most HIV isolates. Serum evidence is consistent with targeting of holes in natural infection. The immunogenic nature of breaches in the glycan shield has been under-appreciated in previous attempts to understand autologous neutralizing antibody responses and has important potential consequences for HIV vaccine design

Antibodies from rabbits immunized with HIV-1 clade B SOSIP trimers can neutralize multiple clade B viruses by destabilizing the envelope glycoprotein

The high HIV-1 viral diversity is a formidable hurdle for the development of an HIV-1 vaccine. Elicitation of broadly neutralizing antibodies (bNAbs) would offer a solution, but so far immunization strategies have failed to elicit bNAbs efficiently. To overcome the obstacles, it is important to understand the immune responses elicited by current HIV-1 envelope glycoprotein (Env) immunogens. To gain more insight, we characterized monoclonal antibodies (mAbs) isolated from rabbits immunized with Env SOSIP trimers based on the clade B isolate AMC008. Four rabbits that were immunized three times with AMC008 trimer developed robust autologous and sporadic low-titer heterologous neutralizing responses. Seventeen AMC008 trimer-reactive mAbs were isolated using antigen-specific single B cell sorting. Four of these mAbs neutralized the autologous AMC008 virus and several other clade B viruses. When visualized by electron microscopy, the complex of the neutralizing mAbs with the AMC008 trimer showed binding to the gp41 subunit with unusual approach angles and we observed that their neutralization ability depended on their capacity to induce Env trimer dissociation. Thus, AMC008 SOSIP trimer immunization induced clade B neutralizing mAbs with unusual approach angles with neutralizing effects that involve trimer destabilization. Optimizing these responses might provide an avenue to the induction of trimer dissociating bNAbs. IMPORTANCE Roughly 32 million people have died as a consequence of HIV-1 infection since the start of the epidemic and still 1.7 million people get infected with HIV-1 annually. Therefore, a vaccine to prevent HIV-1 infection is urgently needed. Current HIV-1 immunogens are not able to elicit the broad immune responses needed to provide protection against the large variation of HIV-1 strains circulating globally. A better understanding of the humoral immune responses elicited by immunization with state-of-the-art HIV-1 immunogens should facilitate the design of improved HIV-1 vaccine candidates. We identified antibodies with the ability to neutralize multiple HIV-1 viruses by destabilization of the envelope glycoprotein. Their weak but consistent cross-neutralization ability indicates the potential of this epitope to elicit broad responses. The trimer-destabilizing effect of the neutralizing mAbs combined with detailed characterization of the neutralization epitope can be used to shape the next generation of HIV-1 immunogens to elicit improved humoral responses after vaccination

A combined HM-PCR/SNuPE method for high sensitive detection of rare DNA methylation

<p>Abstract</p> <p>Background</p> <p>DNA methylation changes are widely used as early molecular markers in cancer detection. Sensitive detection and classification of rare methylation changes in DNA extracted from circulating body fluids or complex tissue samples is crucial for the understanding of tumor etiology, clinical diagnosis and treatment. In this paper, we describe a combined method to monitor the presence of methylated tumor DNA in an excess of unmethylated background DNA of non-tumorous cells. The method combines heavy methyl-PCR, which favors preferential amplification of methylated marker sequence from bisulfite-treated DNA with a methylation-specific single nucleotide primer extension monitored by ion-pair, reversed-phase, high-performance liquid chromatography separation.</p> <p>Results</p> <p>This combined method allows detection of 14 pg (that is, four to five genomic copies) of methylated chromosomal DNA in a 2000-fold excess (that is, 50 ng) of unmethylated chromosomal background, with an analytical sensitivity of > 90%. We outline a detailed protocol for the combined assay on two examples of known cancer markers (SEPT9 and TMEFF2) and discuss general aspects of assay design and data interpretation. Finally, we provide an application example for rapid testing on tumor methylation in plasma DNA derived from a small cohort of patients with colorectal cancer.</p> <p>Conclusion</p> <p>The method allows unambiguous detection of rare DNA methylation, for example in body fluid or DNA isolates from cells or tissues, with very high sensitivity and accuracy. The application combines standard technologies and can easily be adapted to any target region of interest. It does not require costly reagents and can be used for routine screening of many samples.</p

Antibody responses induced by SHIV infection are more focused than those induced by soluble native HIV-1 envelope trimers in non-human primates

The development of an effective human immunodeficiency virus (HIV-1) vaccine is a high global health priority. Soluble native-like HIV-1 envelope glycoprotein trimers (Env), including those based on the SOSIP design, have shown promise as vaccine candidates by inducing neutralizing antibody responses against the autologous virus in animal models. However, to overcome HIV-1’s extreme diversity a vaccine needs to induce broadly neutralizing antibodies (bNAbs). Such bNAbs can protect non-human primates (NHPs) and humans from infection. The prototypic BG505 SOSIP.664 immunogen is based on the BG505 env sequence isolated from an HIV-1-infected infant from Kenya who developed a bNAb response. Studying bNAb development during natural HIV-1 infection can inform vaccine design, however, it is unclear to what extent vaccine-induced antibody responses to Env are comparable to those induced by natural infection. Here, we compared Env antibody responses in BG505 SOSIP-immunized NHPs with those in BG505 SHIV-infected NHPs, by analyzing monoclonal antibodies (mAbs). We observed three major differences between BG505 SOSIP immunization and BG505 SHIV infection. First, SHIV infection resulted in more clonal expansion and less antibody diversity compared to SOSIP immunization, likely because of higher and/or prolonged antigenic stimulation and increased antigen diversity during infection. Second, while we retrieved comparatively fewer neutralizing mAbs (NAbs) from SOSIP-immunized animals, these NAbs targeted more diverse epitopes compared to NAbs from SHIV-infected animals. However, none of the NAbs, either elicited by vaccination or infection, showed any breadth. Finally, SOSIP immunization elicited antibodies against the base of the trimer, while infection did not, consistent with the base being placed onto the virus membrane in the latter setting. Together these data provide new insights into the antibody response against BG505 Env during infection and immunization and limitations that need to be overcome to induce better responses after vaccination

Electron-Microscopy-Based Epitope Mapping Defines Specificities of Polyclonal Antibodies Elicited during HIV-1 BG505 Envelope Trimer Immunization

Characterizing polyclonal antibody responses via currently available methods is inherently complex and difficult. Mapping epitopes in an immune response is typically incomplete, which creates a barrier to fully understanding the humoral response to antigens and hinders rational vaccine design efforts. Here, we describe a method of characterizing polyclonal responses by using electron microscopy, and we applied this method to the immunization of rabbits with an HIV-1 envelope glycoprotein vaccine candidate, BG505 SOSIP.664. We detected known epitopes within the polyclonal sera and revealed how antibody responses evolved during the prime-boosting strategy to ultimately result in a neutralizing antibody response. We uncovered previously unidentified epitopes, including an epitope proximal to one recognized by human broadly neutralizing antibodies as well as potentially distracting non-neutralizing epitopes. Our method provides an efficient and semiquantitative map of epitopes that are targeted in a polyclonal antibody response and should be of widespread utility in vaccine and infection studies