The HIV-1 broad neutralizing antibody (bnAb) 2F5 has been shown to be poly/self-reactive in vitro, and we previously demonstrated that targeted expression of its VDJ rearrangement alone was sufficient to trigger a profound B cell developmental blockade in 2F5 VH knockin (KI) mice, consistent with central deletion of 2F5 H chain-expressing B cells. Here, we generate a strain expressing the entire 2F5 bnAb specificity, 2F5 VHxVL KI mice, and find an even higher degree of tolerance control than observed in the 2F5 VH KI strain. Although B-cell development was severely impaired in 2F5 VHxVL KI animals, we demonstrate rescue of their B-cells when cultured in IL-7/BAFF. Intriguingly, even under these conditions, most rescued B-cell hybridomas produced mAbs that lacked HIV-1 Envelope (Env) reactivity due to editing of the 2F5 L chain, and the majority of rescued B-cells retained an anergic phenotype. Thus, when clonal deletion is circumvented, κ editing and anergy are additional safeguards preventing 2F5 VH/VL expression by immature/transitional B-cells. Importantly, 7% of rescued B-cells retained 2F5 VH/VL-expression and secreted Env-specific mAbs with HIV-1 neutralizing activity. This “partial” rescue was further corroborated in vivo, as reflected by the anergic phenotype of most rescued B-cells in 2F5 VHxVL KI × Eμ-bcl2 tg mice, and significant (yet modest) enrichment of Env-specific B-cells and serum Igs. The rescued 2F5 mAb-producing B-cell clones in this study are the first examples of in vivo-derived bone marrow precursors specifying HIV-1 bnAbs, and provide a starting point for design of strategies aimed at rescuing such B-cells.

The membrane proximal external region (MPER) of HIV-1 gp41 has several features that make it an attractive antibody-based vaccine target, but eliciting an effective gp41 MPER-specific protective antibody response remains elusive. One fundamental issue is whether the failure to make gp41 MPER-specific broadly neutralizing antibodies like 2F5 and 4E10 is due to structural constraints with the gp41 MPER, or alternatively, if gp41 MPER epitope-specific B cells are lost to immunological tolerance. An equally important question is how B cells interact with, and respond to, the gp41 MPER epitope, including whether they engage this epitope in a non-canonical manner i.e., by non-paratopic recognition via B cell receptors (BCR). To begin understanding how B cells engage the gp41 MPER, we characterized B cell-gp41 MPER interactions in BALB/c and C57BL/6 mice. Surprisingly, we found that a significant (∼7%) fraction of splenic B cells from BALB/c, but not C57BL/6 mice, bound the gp41 MPER via their BCRs. This strain-specific binding was concentrated in IgMhi subsets, including marginal zone and peritoneal B1 B cells, and correlated with enriched fractions (∼15%) of gp41 MPER-specific IgM secreted by in vitro-activated splenic B cells. Analysis of Igha (BALB/c) and Ighb (C57BL/6) congenic mice demonstrated that gp41 MPER binding was controlled by determinants of the Igha locus. Mapping of MPER gp41 interactions with IgMa identified MPER residues distinct from those to which mAb 2F5 binds and demonstrated the requirement of Fc CH regions. Importantly, gp41 MPER ligation produced detectable BCR-proximal signaling events, suggesting that interactions between gp41 MPER and IgMa determinants may elicit partial B cell activation. These data suggest that low avidity, non-paratopic interactions between the gp41 MPER and membrane Ig on naïve B cells may interfere with or divert bnAb responses.

Varicella-zoster virus (VZV) is renowned for its very low titer when grown in cultured cells. There remains no single explanation for the low infectivity. In this study, viral particles on the surfaces of infected cells were examined by several imaging technologies. Few surface particles were detected at 48 h postinfection (hpi), but numerous particles were observed at 72 and 96 hpi. At 72 hpi, 75% of the particles resembled light (L) particles, i.e., envelopes without capsids. By 96 hpi, 85% of all particles resembled L particles. Subsequently, the envelopes of complete virions and L particles were investigated to determine their glycoprotein constituents. Glycoproteins gE, gI, and gB were detected in the envelopes of both types of particles in similar numbers; i.e., there appeared to be no difference in the glycoprotein content of the L particles. The viral particles emerged onto the cell surface amid actin-based filopodia, which were present in abundance within viral highways. Viral particles were easily detected at the base of and along the exterior surfaces of the filopodia. VZV particles were not detected within filopodia. In short, these results demonstrate that VZV infection of cultured cells produces a larger proportion of aberrant coreless particles than has been seen with any other previously examined alphaherpesvirus. Further, these results suggested a major disassociation between capsid formation and envelopment as an explanation for the invariably low VZV titer in cultured cells.Varicella-zoster virus (VZV) is renowned for its extremely low titer when grown in cultured cells. Explanations for this low titer have been pursued for decades. In this investigation, we hypothesized that one of the earliest explanations, an aberrant structure, was at least partly responsible for the markedly reduced infectivity in cell culture.In 1968, Cook and Stevens reported that extranuclear VZ virions were distinguishable from herpes simplex virions by the presence of pleomorphism: VZV had incomplete coats and loss of central dense cores (2). Those authors suggested that aberrant assembly was a potential reason for the lack of cellfree virus. In their work, Cook and Stevens did not describe in detail the characteristics of extracellular VZV. The cell-associated nature of VZV has limited the repertoire of techniques available to those wishing to study VZV outside the cellular environment. Earlier studies (41) using scanning electron microscopy (SEM) illustrated the presence of viral particles arranged in clusters on the surface of infected human melanoma cells (MeWo). In 1995, Harson and Grose (18) reported a very distinguishing characteristic of VZV-infected cells. Thousands of extracellular viral particles were seen on the surfaces of fused cell monolayers. Instead of being randomly distributed over the cell surface, egressed viral particles were arranged in an elongated pattern termed "viral highways." This viral egress phenotype was seen with all VZV strains examined in MeWo cells, including the vaccine strain VZV...

In the course of examining the trafficking pathways of varicella-zoster virus (VZV) glycoproteins gE, gI, gH, and gB, we discovered that all four are synthesized within 4 to 6 h postinfection (hpi) in cultured cells. Thereafter, they travel via the trans-Golgi network to the outer cell membrane. When we carried out a similar analysis on VZV gC, we observed little gC biosynthesis in the first 72 hpi. Further examination disclosed that gC was present in the inocula of infected cells, but no new gC biosynthesis occurred during the first 24 to 48 h thereafter, during which time new synthesis of gE, gH, and major capsid protein was easily detectable. Similarly, delayed gC biosynthesis was confirmed with three different VZV strains and two different cell lines. Bioinformatics analyses disclosed the presence of PBX/HOX consensus binding domains in the promoter/ enhancer regions of the genes for VZV gC and ORF4 protein (whose orthologs transactivate gC in other herpesviruses). Bioinformatics analysis also identified two HOXA9 activation regions on ORF4 protein. Treatment of infected cultures with chemicals known to induce the production of PBX/HOX transcription proteins, namely, hexamethylene bisacetamide (HMBA) and retinoic acid, led to more rapid gC biosynthesis. Immunoblotting demonstrated a fivefold increase in the HOXA9 protein after HMBA treatment. In summary, these results documented that gC was not produced during early VZV replication cycles, presumably related to a deficiency in the PBX/HOX transcription factors. Furthermore, these results explain the apparent spontaneous loss of VZV gC in some passaged viruses, as well as other anomalous gC results.Varicella-zoster virus (VZV) is a very cell-associated virus in cell culture. In addition, the infectivity titers are extremely low, usually less than 1,000,000 U per 25-cm 2 monolayer. Further, the virions produced in cell culture have an aberrant appearance. Explanations for these observations are a subject of continuing research. To this end, we have been investigating the biosynthesis and maturation of several VZV structural glycoproteins found in the envelope of the virion, especially the predominant gE/gI complex. These two glycoproteins are synthesized in the Golgi and then traffic through the trans-Golgi network en route to the outer plasma membrane within the expected 12-h replication cycle of an alphaherpesvirus. After endocytosis via their tyrosine-and dileucine-based motifs, they travel back to the trans-Golgi network (12). At this location, the glycoproteins appear to be incorporated into the virion assembly vacuoles. Two other major VZV glycoproteins, gH and gB, have similar endocytosis motifs and similar trafficking profiles.During the glycoprotein trafficking studies, VZV gC was used as a control because there are no obvious trafficking motifs in its short cytoplasmic tail (20). There have been clues that gC is important for virion production. The severe combined immunodeficient (SCID) mouse has become an important animal model for the investigation...

Patients with metastatic triple-negative breast cancer have a poor prognosis. Sacituzumab govitecan (IMMU-132) is an antibody-drug conjugate that contains the irinotecan active metabolite, SN-38, linked to a humanized monoclonal antibody targeting trophoblast cell surface antigen 2, which is overexpressed in many solid tumors. In a basket design phase I/II study, sacituzumab govitecan demonstrated promising single-agent therapeutic activity in multiple cancer cohorts, leading to accelerated approval by the U.S. Food and Drug Administration of sacituzumab govitecan-hziy (TRODELVY) for the treatment of patients with metastatic triple-negative breast cancer who had received at least two prior therapies in