Abstract Introduction: Despite the theoretic potential of immunostimulatory therapy in the treatment of cancer, the disruption of negative checkpoints in the tumor microenvironment remains the dominant clinical strategy. The failure of immunostimulation, utilizing agonistic cytokines or activating antibodies, can be attributed to off target side effects, the failure to preferentially activate cytotoxic lymphocytes (CTLs) over regulatory T cells (Tregs), and the development of T cell exhaustion resulting from Jak/STAT/PI3K signaling in T cells. Methods: To address these issues we designed a first in class immunostimulatory fusion protein (RB101) that utilizes NKG2D-targeting (without activation of the NKG2D signaling) to exclusively activate CTLs (CD8+ T, NK, γδ T cells). Previous approaches to target CD8+ T cells relied upon activation of the Jak/STAT/PI3K signal transduction pathway. In contrast, RB101 treatment results in selective activation of the nuclear factor of activated T cell (NFAT) signaling pathway. NFAT activation results in increased mitochondrial biogenesis, increased T cell receptor recognition of tumor antigens, accelerated proliferation, and augmented generation of memory T cells when compared to canonical cytokines and alternative methods of immunostimulation. Results: We observed that the unique combination of NFAT signaling and NKG2D-targeting led to CTL priming and expansion without systematic toxicity in a non-human primate (NHP) model. Subcutaneous RB101 administration to healthy cynomolgus macaques at or below the No Observed Adverse Event Level (NOAEL) increases the number of interferon gamma and granzyme B positive CD8+ T and NK cells 10-100-fold over vehicle treated animals. In addition, such administration resulted in >6-fold increase in CD8+ T/Treg and NK/Treg ratios, two leading indicators of clinical efficacy. Similar results were evident in human PBMC cultures in vitro. Subcutaneous RB101 administration results in partial and/or complete responses in mice bearing multiple syngeneic murine tumors. RB101 treatment also drives improved tumor control when combined with checkpoint inhibitors or radiation. Conclusion: The powerful immune response generated by signaling through the NFAT pathway has long been evident in organ allograft rejection. Clinical organ transplantation only became possible with clinical therapies targeting this signaling pathway (such as with tacrolimus). To our knowledge, RB101 is the first therapeutic designed to stimulate the NFAT pathway of T cell activation. The precise targeting of this stimulus to CTLs allows for the safe, subcutaneous administration of RB101 and results in a compelling preclinical profile of safety and efficacy in human cells and across multiple animal models. Citation Format: Eric Lazear, Dan Watkins, Alexander Krupnick, John Westwick. A first in class immunotherapy that selectively activates the NFAT pathway in CD8+ T cells [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr A112.

As data and protocols are increasingly generated, accessed, and manipulated electronically, many labs are shifting to storing data in electronic laboratory notebooks (ELNs). ELNs provide clear advantages in readability, shareability, searchability, accessibility, and data security over their paper counterparts. Our interdisciplinary program implemented ELNs across several sections of our gateway molecular biology laboratory course and analyzed the impact on 384 students (13 sections total from Fall 2016 to Spring 2018) using surveys, focus groups, and grade data. We found that students prefer the electronic format, citing the ease of transitioning to lab reports and the longevity of their data. Course grades overall were not affected by the change. Based on our experience, we offer suggestions for smoothing the transition to an ELN system in undergraduate and graduate laboratory courses, including use during remote teaching.

BACKGROUND: Long-term survival of lung transplants lags behind other solid organs due to early onset of a fibrotic form of chronic rejection known as chronic lung allograft dysfunction (CLAD). Preventing CLAD is difficult as multiple immunologic and physiologic insults contribute to its development. Targeting fibroblast activation, which is the final common pathway leading to CLAD, offers the opportunity to ameliorate fibrosis irrespective of the initiating insult. Thy-1 is a surface glycoprotein that controls fibroblast differentiation and activation. METHODS: To study the role of Thy-1 in CLAD, we utilized the minor antigen mismatched C57BL/6 (B6wild-type) or B6Thy-1(-/--)> C57BL/10 (B10) model of murine orthotopic lung transplantation with postoperative bacterial infection modeled by intratracheal lipopolysaccharide (LPS) administration. The effects of LPS on Thy-1 expression, proliferation, and gene expression were assessed in fibroblasts in vitro and the therapeutic potential of Thy-1 replacement was assessed in vivo. RESULTS: More severe CLAD was evident in B6Thy-1(-/--)-> B10grafts compared to B6wild-type-> B10 grafts. LPS further accentuated fibrosis in B6wild-type-> B10 grafts with some, but limited, effects on B6Thy-1(-/--)-> B10grafts. LPS contributed to Thy-1 loss from Thy-1(+) fibroblasts in vitro due to a decrease in mRNA expression. In addition, LPS promoted proliferation and upregulation of multiple inflammatory pathways in Thy-1(-) fibroblasts by gene expression analysis. Most importantly, replacement of Thy-1 through exogenous administration ameliorated the fibrotic phenotype post-LPS mediated modeling of infection. CONCLUSIONS: Our findings suggest that the loss of Thy-1 on fibroblasts is a previously unrecognized cause of CLAD and its replacement may offer therapeutic applications for amelioration of this disease post-transplantation in the setting of infectious stress responses. J Heart Lung Transplant 2022;41:1044-1054 (c) Published by Elsevier Inc.

Cytokine therapy is limited by undesirable off-target side effects as well as terminal differentiation and exhaustion of chronically stimulated T cells. Here, we describe the signaling properties of a potentially unique cytokine by design, where T cell surface binding and signaling are separated between 2 different families of receptors. This fusion protein cytokine, called OMCPmutIL-2, bound with high affinity to the cytotoxic lymphocyte-defining immunoreceptor NKG2D but signaled through the common γ chain cytokine receptor. In addition to precise activation of cytotoxic T cells due to redirected binding, OMCPmutIL-2 resulted in superior activation of both human and murine CD8+ T cells by improving their survival and memory cell generation and decreasing exhaustion. This functional improvement was the direct result of altered signal transduction based on the reorganization of surface membrane lipid rafts that led to Janus kinase-3-mediated phosphorylation of the T cell receptor rather than STAT/AKT signaling intermediates. This potentially novel signaling pathway increased CD8+ T cell response to low-affinity antigens, activated nuclear factor of activated T cells transcription factors, and promoted mitochondrial biogenesis. OMCPmutIL-2 thus outperformed other common γ chain cytokines as a catalyst for in vitro CD8+ T cell expansion and in vivo CD8+ T cell-based immunotherapy.

Key Points Retargeting IL-2 delivery to CTLs improves immunotherapy. Regulatory lymphocytes hinder ex vivo expansion of CTLs. Tumor-infiltrating NK cells and γδ T cells facilitate adoptive transfer immunotherapy. Ex vivo expansion followed by reinfusion of tumor-infiltrating leukocytes (TILs) has been used successfully for the treatment of multiple malignancies. Most protocols rely on the use of the cytokine IL-2 to expand TILs prior to reinfusion. In addition, TIL administration relies on systemic administration of IL-2 after reinfusion to support transferred cell survival. The use of IL-2, however, can be problematic because of its preferential expansion of regulatory T and myeloid cells as well as its systemic side effects. In this study, we describe the use of a novel IL-2 mutant retargeted to NKG2D rather than the high-affinity IL-2R for TIL-mediated immunotherapy in a murine model of malignant melanoma. We demonstrate that the NKG2D-retargeted IL-2 (called OMCPmutIL-2) preferentially expands TIL-resident CTLs, such as CD8+ T cells, NK cells, and γδT cells, whereas wild-type IL-2 provides a growth advantage for CD4+Foxp3+ T cells as well as myeloid cells. OMCPmutIL-2–expanded CTLs express higher levels of tumor-homing receptors, such as LFA-1, CD49a, and CXCR3, which correlate with TIL localization to the tumor bed after i.v. injection. Consistent with this, OMCPmutIL-2–expanded TILs provided superior tumor control compared with those expanded in wild-type IL-2. Our data demonstrate that adoptive transfer immunotherapy can be improved by rational retargeting of cytokine signaling to NKG2D-expressing CTLs rather than indiscriminate expansion of all TILs.

The rapid development of molecular biotechnology presents a curricular challenge for educators trying to provide students with relevant coursework. A comprehensive biology education should also include opportunities for students to develop intellectual and technical skills through authentic research experiences. Integrating relevant and interesting research projects into their classes, however, can be a challenging task for instructors. To address these varied demands, we redesigned our existing molecular cloning course to incorporate an independent research project assessing calcium signaling. In the revised course, students use traditional and recombination-based cloning strategies to generate bacterial and mammalian expression vectors encoding CaMPARI, a novel fluorescent calcium indicator. Bacterially-expressed CaMPARI is used in protein quantification and purification assays. Students must also design their own research project evaluating the effect of chemotherapeutic agents on calcium signaling in a mammalian system. Revised and novel labs were designed to be modular, facilitating their integration into the course over 2 years. End-of-semester student evaluations were compared between years revealing a significant difference in students' perception of the course's difficulty between years. This change in attitude highlights potential pedagogical considerations that must be examined when introducing new material and activities into existing courses. Since calcium signaling is important for cellular process across diverse species, instructors may be able to develop research projects within their respective areas of interest. Integration of authentic research experiences into the curriculum is challenging; however, the framework described here provides a versatile structure that can be adapted to merge diverse instructor interests with evolving educational needs.

Abstract A growing arm of adoptive immunotherapy for cancer involves the isolation, expansion and reinfusion of autologous tumor infiltrating leukocytes or TILs. TILs are enriched for tumor-reactive cytotoxic cells that are rendered inactive or anergic by multiple immunosuppressive mechanisms operating in solid tumors. Their separation from the tumor microenvironment followed by ex vivo activation and expansion in the presence of T cell receptor stimulation and interleukin-2 (IL-2) has been described to reverse this dysfunction and control tumor growth in some patients. Unfortunately, a large number of patients achieve no response or clinical benefit suggesting that TIL immunotherapy could benefit from refinement and improvement. Part of the limitation involves the reliance on IL-2 to support T cell expansion both in vitro and in vivo as this cytokine can result in substantial expansion and activation of regulatory T cells (Tregs) that limit the efficacy of immunotherapy. We have recently described a novel re-targeted form of IL-2, which utilizes NKG2D rather than α chain of IL-2R to form the high affinity receptor complex for β and γ chain signal transduction. This redirected cytokine fusion protein consists of a cowpox virus encoded NKG2D ligand called orthopoxvirus major histocompatibility complex class I-like protein or OMCP along with non-IL-2R-α binding mutant form of IL-2 (OMCPmutIL-2). To evaluate the effect of NKG2D-redirected cytokine delivery on TIL expansion, we isolated TILs from progressively growing B16ova melanoma and expanded them with transient anti-CD3/CD28 stimulation in the presence of continuously replenished wild-type IL-2 or OMCPmutIL-2. Compared to wild-type IL-2 the use of OMCPmutIL-2 resulted in specific and preferential expansion of NK cells, CD8+ T cells as well as γδ T cells after 2 weeks of culture. Significantly higher expansion was observed in CD8+ T (p=0.037), antigen-specific CD8+ tetramer+ (p=0.0014), NK cells (p<0.001) and T cells (p=0.002), with decreased expansion of Tregs (p=0.045) and MDSCs (p=0.0122) indicating preferential expansion of more cytolytic subtypes of TILs by OMCPmut.IL-2. Human melanoma TILs demonstrated a similar trend with NK, NKT, CD8+T and T cells expanding significantly more in OMCPmut.IL-2 whereas MDSCs and Tregs expanded more in IL-2. We next evaluated tumor growth in C57BL/6 mice bearing established melanoma reconstituted with 5 × 106 TILs expanded in either wild-type IL-2 or OMCPmutIL-2. We found significant tumor control in the group which received OMCPmutIL-2 expanded TIL compared to IL-2 expanded ones (178.83±140.01vs 970.15±330.47mm3 on day 22 of growth). We conclude that the use of the NKG2D retargeted common γ-chain cytokine, called OMCPmutIL-2, facilitates expansion of multiple lineages of TIL-resident cytotoxic lymphocytes and improves adoptive transfer immunotherapy over wild-type IL-2. Our data thus suggests that retargeting stimulation away from the traditional IL-2R-α chain represents a rational approach to TIL immunotherapy. Citation Format: Anirban Banerjee, Yizhan Guo, Lea Paragas, Jacqueline Slobin, Bayan Mahgoub, Dongge Li, Sarah Hein, John Westwick, Eric Lazear, Alexander S. Krupnick. Targeted expansion of NKG2D-expressing tumor infiltrating leukocytes improves adoptive transfer immunotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 878.

NKG2D is a key component of cytotoxic antitumor and antiviral responses. Multiple viruses evade NKG2D recognition by blocking NKG2D ligand expression on infected cells. In contrast, cowpox virus targets NKG2D directly by encoding a secreted antagonist, Orthopoxvirus MHC Class I-like Protein (OMCP). We have previously reported that OMCP also binds to the orphan receptor FcRL5 on innate B cells. Here, we demonstrate that mammalian-derived, glycosylated OMCP binds NKG2D but not FcRL5. Cowpox viruses either lacking OMCP, or expressing an NKG2D-binding deficient mutant, are significantly attenuated in wild type and FcRL5-deficient mice but not NKG2D-deficient mice, demonstrating that OMCP is critical in subverting NKG2D-mediated immunity . Next we determined the structure of OMCP bound to human NKG2D. Despite a structure similar to that of host NKG2D ligands, OMCP uses a drastically different orientation for NKG2D binding. The re-orientation of OMCP is associated with dramatically higher affinity for human NKG2D and the targeted interface is highly conserved in mammalian NKG2Ds, increasing the zoonotic potential of cowpox virus. We also show that cell surface presented OMCP can trigger NKG2D effector functions equivalently to host NKG2D ligands, demonstrating that NKG2D-mediated signaling requires clustering but is insensitive to binding orientation. Thus, in contrast to TCR/MHC interactions, the docking topology of NKG2D with its ligands does not appear to regulate its activation.

HSV virus-cell and cell-cell fusion requires multiple interactions between four essential virion envelope glycoproteins, gD, gB, gH, and gL, and between gD and a cellular receptor, nectin-1 or herpesvirus entry mediator (HVEM). Current models suggest that binding of gD to receptors induces a conformational change that leads to activation of gH/gL and consequent triggering of the prefusion form of gB to promote membrane fusion. Since protein-protein interactions guide each step of fusion, identifying the sites of interaction may lead to the identification of potential therapeutic targets that block this process. We have previously identified two "faces" on gD: one for receptor binding and the other for its presumed interaction with gH/gL. We previously separated the gD monoclonal antibodies (MAbs) into five competition communities. MAbs from two communities (MC2 and MC5) neutralize virus infection and block cell-cell fusion but do not block receptor binding, suggesting that they block binding of gD to gH/gL. Using a combination of classical epitope mapping of gD mutants with fusion and entry assays, we identified two residues (R67 and P54) on the presumed gH/gL interaction face of gD that allowed for fusion and viral entry but were no longer sensitive to inhibition by MC2 or MC5, yet both were blocked by other MAbs. As neutralizing antibodies interfere with essential steps in the fusion pathway, our studies strongly suggest that these key residues block the interaction of gD with gH/gL. IMPORTANCE Virus entry and cell-cell fusion mediated by HSV require gD, gH/gL, gB, and a gD receptor. Neutralizing antibodies directed against any of these proteins bind to residues within key functional sites and interfere with an essential step in the fusion pathway. Thus, the epitopes of these MAbs identify critical, functional sites on their target proteins. Unlike many anti-gD MAbs, which block binding of gD to a cellular receptor, two, MC2 and MC5, block a separate, downstream step in the fusion pathway which is presumed to be the activation of the modulator of fusion, gH/gL. By combining epitope mapping of a panel of gD mutants with fusion and virus entry assays, we have identified residues that are critical in the binding and function of these two MAbs. This new information helps to define the site of the presumptive interaction of gD with gH/gL, of which we have limited knowledge.

A recurrent theme in viral immune evasion is the sabotage of MHC-I antigen presentation, which brings virus the concomitant issue of ‘missing-self’ recognition by NK cells that use inhibitory receptors to detect surface MHC-I proteins. Here, we report that rodent herpesvirus Peru (RHVP) encodes a Qa-1 like protein (pQa-1) via RNA splicing to counteract NK activation. While pQa-1 surface expression is stabilized by the same canonical peptides presented by murine Qa-1, pQa-1 is GPI-anchored and resistant to the activity of RHVP pK3, a ubiquitin ligase that targets MHC-I for degradation. pQa-1 tetramer staining indicates that it recognizes CD94/NKG2A receptors. Consistently, pQa-1 selectively inhibits NKG2A+ NK cells and expression of pQa-1 can protect tumor cells from NK control in vivo. Collectively, these findings reveal an innovative NK evasion strategy wherein RHVP encodes a modified Qa-1 mimic refractory to MHC-I sabotage and capable of specifically engaging inhibitory receptors to circumvent NK activation.

Human cytomegalovirus (HCMV) is a significant cause of disease in immune-compromised adults and immune naïve newborns. No vaccine exists to prevent HCMV infection, and current antiviral therapies have toxic side effects that limit the duration and intensity of their use. There is thus an urgent need for new strategies to treat HCMV infection. Repurposing existing drugs as antivirals is an attractive approach to limit the time and cost of new antiviral drug development. Virus-induced changes in infected cells are often driven by changes in cellular kinase activity, which led us to hypothesize that defining the complement of kinases (the kinome), whose abundance or expression is altered during infection would identify existing kinase inhibitors that could be repurposed as new antivirals. To this end, we applied a kinase capture technique, multiplexed kinase inhibitor bead-mass spectrometry (MIB-MS) kinome, to quantitatively measure perturbations in >240 cellular kinases simultaneously in cells infected with a laboratory-adapted (AD169) or clinical (TB40E) HCMV strain. MIB-MS profiling identified time-dependent increases and decreases in MIB binding of multiple kinases including cell cycle kinases, receptor tyrosine kinases, and mitotic kinases. Based on the kinome data, we tested the antiviral effects of kinase inhibitors and other compounds, several of which are in clinical use or development. Using a novel flow cytometry-based assay and a fluorescent reporter virus we identified three compounds that inhibited HCMV replication with IC50 values of <1 μm, and at doses that were not toxic to uninfected cells. The most potent inhibitor of HCMV replication was OTSSP167 (IC50 <1.2 nm), a MELK inhibitor, blocked HCMV early gene expression and viral DNA accumulation, resulting in a >3 log decrease in virus replication. These results show the utility of MIB-MS kinome profiling for identifying existing kinase inhibitors that can potentially be repurposed as novel antiviral drugs.

The use of high-dose interleukin-2 (IL-2) has fallen out of favor due to severe life-threatening side effects. We have recently described a unique way of directly targeting IL-2 to cytotoxic lymphocytes using a virally encoded immune evasion protein and an IL-2 mutant that avoids off-target side effects such as activation of regulatory T cells and vascular endothelium.

Despite over 20 years of clinical use, IL-2 has not fulfilled expectations as a safe and effective form of tumour immunotherapy. Expression of the high affinity IL-2Rα chain on regulatory T cells mitigates the anti-tumour immune response and its expression on vascular endothelium is responsible for life threatening complications such as diffuse capillary leak and pulmonary oedema. Here we describe the development of a recombinant fusion protein comprised of a cowpox virus encoded NKG2D binding protein (OMCP) and a mutated form of IL-2 with poor affinity for IL-2Rα. This fusion protein (OMCP-mutIL-2) potently and selectively activates IL-2 signalling only on NKG2D-bearing cells, such as natural killer (NK) cells, without broadly activating IL-2Rα-bearing cells. OMCP-mutIL-2 provides superior tumour control in several mouse models of malignancy and is not limited by mouse strain-specific variability of NK function. In addition, OMCP-mutIL-2 lacks the toxicity and vascular complications associated with parental wild-type IL-2.

Herpes simplex virus entry is initiated by glycoprotein D (gD) binding to a cellular receptor, such as HVEM or nectin-1. gD is activated by receptor-induced displacement of the C-terminus from the core of the glycoprotein. Binding of HVEM requires the formation of an N-terminal hairpin loop of gD; once formed this loop masks the nectin-1 binding site on the core of gD. We found that HVEM and nectin-1 exhibit non-reciprocal competition for binding to gD. The N-terminus of gD does not spontaneously form a stable hairpin in the absence of receptor and HVEM does not appear to rely on a pre-existing hairpin for binding to gD(3C-38C) mutants. However, HVEM function is affected by mutations that impair optimal hairpin formation. Furthermore, nectin-1 induces a new conformation of the N-terminus of gD. We conclude that the conformation of the N-terminus of gD is actively modified by the direct action of both receptors.

ABSTRACT The NKG2D receptor is expressed on the surface of NK, T, and macrophage lineage cells and plays an important role in antiviral and antitumor immunity. To evade NKG2D recognition, herpesviruses block the expression of NKG2D ligands on the surface of infected cells using a diverse repertoire of sabotage methods. Cowpox and monkeypox viruses have taken an alternate approach by encoding a soluble NKG2D ligand, the orthopoxvirus major histocompatibility complex (MHC) class I-like protein (OMCP), which can block NKG2D-mediated cytotoxicity. This approach has the advantage of targeting a single conserved receptor instead of numerous host ligands that exhibit significant sequence diversity. Here, we show that OMCP binds the NKG2D homodimer as a monomer and competitively blocks host ligand engagement. We have also determined the 2.25-Å-resolution crystal structure of OMCP from the cowpox virus Brighton Red strain, revealing a truncated MHC class I-like platform domain consisting of a beta sheet flanked with two antiparallel alpha helices. OMCP is generally similar in structure to known host NKG2D ligands but has notable variations in regions typically used to engage NKG2D. Additionally, the determinants responsible for the 14-fold-higher affinity of OMCP for human than for murine NKG2D were mapped to a single loop in the NKG2D ligand-binding pocket.

ABSTRACT As the receptor-binding protein of herpes simplex virus (HSV), gD plays an essential role in virus entry. In its native state, the last 56 amino acids of the ectodomain C terminus (C-term) occlude binding to its receptors, herpesvirus entry mediator (HVEM) and nectin-1. Although it is clear that movement of the C-term must occur to permit receptor binding, we believe that this conformational change is also a key event for triggering later steps leading to fusion. Specifically, gD mutants containing disulfide bonds that constrain the C-term are deficient in their ability to trigger fusion following receptor binding. In this report, we show that two newly made monoclonal antibodies (MAbs), MC2 and MC5, have virus-neutralizing activity but do not block binding of gD to either receptor. In contrast, all previously characterized neutralizing anti-gD MAbs block binding of gD to a receptor(s). Interestingly, instead of blocking receptor binding, MC2 significantly enhances the affinity of gD for both receptors. Several nonneutralizing MAbs (MC4, MC10, and MC14) also enhanced gD-receptor binding. While MC2 and MC5 recognized different epitopes on the core of gD, these nonneutralizing MAbs recognized the gD C-term. Both the neutralizing capacity and rate of neutralization of virus by MC2 are uniquely enhanced when MC2 is combined with MAb MC4, MC10, or MC14. We suggest that MC2 and MC5 prevent gD from performing a function that triggers later steps leading to fusion and that the epitope for MC2 is normally occluded by the C-term of the gD ectodomain.

32 As the receptor-binding protein of HSV, gD plays an essential role in virus entry. In its 33 native state, the last 56 amino acids of the ectodomain C-terminus (C-term) occlude 34 binding to its receptors, HVEM and nectin-1. Although it is clear that movement of the C35 term must occur to permit receptor binding, we believe that this conformational change is 36 also a key event for triggering later steps leading to fusion. Specifically, gD mutants 37 containing disulfide bonds that constrain the C-term are deficient in their ability to trigger 38 fusion following receptor binding. In this report, we show that two newly made MAbs, MC2 39 and MC5, have virus-neutralizing activity but do not block binding of gD to either receptor. 40 In contrast, all previously characterized neutralizing anti-gD MAbs block binding of gD to 41 receptor(s). Interestingly, instead of blocking receptor binding, MC2 significantly enhances 42 the affinity of gD for both receptors. Several non-neutralizing MAbs also enhanced gD43 receptor binding (MC4, MC10, MC14). While MC2 and MC5 recognized different epitopes 44 on the core of gD, these non-neutralizing MAbs recognized the gD C-term. Both the 45 neutralizing capacity and rate of neutralization of virus by MC2 is uniquely enhanced when 46 combined with MAbs MC4, MC10, or MC14. We suggest that MC2 and MC5 prevent gD from 47 performing a function that triggers later steps leading to fusion, and that the epitope for 48 MC2 is normally occluded by the C-term of the gD ectodomain. 49 50 on O cber 3, 2017 by gest http/jvi.asm .rg/ D ow nladed fom