Peripheral Blood CD34 Donor Chimerism has Greater Clinical Utility Than CD3 for Detecting Relapse after Allogeneic Stem Cell Transplantation for Acute Myeloid Leukemia or Myelodysplastic Syndrome.

Monitoring of donor chimerism (DC) may detect early relapse following allogeneic hematopoietic stem cell transplantation (allo-SCT) for acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). Most centers use unfractionated peripheral blood or T-cells to monitor DC, although CD34+ DC may be more predictive. The limited adoption of CD34+ DC may be due to the lack of detailed, comparative studies. To address this knowledge gap, we compared peripheral blood CD34+ and CD3+ DC in 134 patients who underwent allo-SCT for AML or MDS. In July 2011, the Alfred Hospital Bone Marrow Transplantation Service adopted routine monitoring of DC in the lineage-specific CD34+ and CD3+ cell subsets from peripheral blood at 1, 2, 3, 4, 6, 9, and 12 months post-transplantation for AML or MDS. Immunologic interventions, including rapid withdrawal of immunosuppression, azacitidine, and donor lymphocyte infusion, were prespecified for CD34+ DC ≤80%. Overall, CD34+ DC ≤80% detected 32 of 40 relapses (positive predictive value [PPV], 68%; negative predictive value [NPV], 91%), compared with 13 of 40 relapses for CD3+ DC ≤80% (PPV, 52%; NPV, 75%). Receiver operating characteristic analysis showed the superiority of CD34+ DC, with the greatest value at day 120 post-transplantation. CD3+ DC provided additional value in only 3 cases, preceding CD34+ DC ≤80% by 1 month. We further show that the CD34+ DC sample can be used to detect NPM1mut, with the combination of CD34+ DC ≤80% and NPM1mut identifying the highest risk of relapse. Among the 24 patients in morphologic remission at the time of CD34+ DC ≤80%, 15 (62.5%) responded to immunologic interventions (rapid withdrawal of immunosuppression, azacitidine, or donor lymphocyte infusion) with recovery of CD34+ DC >80%, and 11 of these patients remained in complete remission for a median of 34 months (range, 28 to 97 months). In contrast, the other 9 patients did not respond to the clinical intervention and relapsed within a median of 59 days after detecting CD34+ DC ≤80%. The CD34+ DC was significantly higher in responders than in nonresponders (median, 72% versus 56%; P = .015, Mann-Whitney U test). Overall, monitoring of CD34+ DC was considered clinically useful (early diagnosis of relapse enabling preemptive therapy or predicting low risk of relapse) in 107 of 125 evaluable patients (86%). Our findings show that peripheral blood CD34+ DC is feasible and superior to CD3+ DC for predicting relapse. It also provides a source of DNA for measurable residual disease testing, which may further stratify the risk of relapse. If validated by an independent cohort, our results suggest that CD34+ should be used in preference to CD3+ DC for detecting early relapse and guiding immunologic interventions following allo-SCT for AML or MDS.

Predictors and outcomes of dose reduction of methotrexate and cyclosporin graft-versus-host disease prophylaxis following allogeneic haemopoietic cell transplantation.

BACKGROUND: Concern regarding dose-related toxicities of methotrexate (MTX) and cyclosporin (CYA) graft-versus-host disease (GVHD) prophylaxis occasionally leads to dose alterations post allogeneic haemopoietic cell transplant (alloHCT). AIMS: To clarify causes of MTX and CYA dose alteration and assess impact on patient outcomes, including GVHD, relapse, non-relapse mortality (NRM) and overall survival (OS). METHODS: Analysis of retrospective data was performed in a single tertiary centre of patients who underwent alloHCT for any indication and who received GVHD prophylaxis with CYA and MTX between the years 2011 and 2015. Univariate analysis was conducted using the log-rank test for OS and using competing risk regression for NRM, relapse and GVHD. Fisher exact tests were used to determine if an association existed between each of the pre-transplant variables and MTX alteration. Multivariate models for OS and NRM were constructed using Cox proportional hazards modelling and competing risk regression respectively. RESULTS: Fifty-four (28%) of 196 had MTX alterations and 61/187 (33%) had CYA alterations. Reasons for MTX alteration included mucositis, renal or liver impairment, fluid overload and sepsis. Causes of CYA alteration were numerous, but most commonly due to acute kidney impairment. MTX alteration was associated with inferior OS (hazard ratio 2.4; P = <0.001) and higher NRM (odds ratio (OR) 4.6; P < 0.001) at 6 years post-landmark. CYA alteration was associated with greater NRM (OR 2.7; P = 0.0137) at 6 years. GVHD rates were unaffected by dose alteration. CONCLUSIONS: Our findings suggest dose alteration in MTX and CYA GVHD prophylaxis is associated with adverse survival outcomes in alloHCT, without a significant impact on GVHD rates.

Antibody affinity versus dengue morphology influences neutralization.

Different strains within a dengue serotype (DENV1-4) can have smooth, or "bumpy" surface morphologies with different antigenic characteristics at average body temperature (37°C). We determined the neutralizing properties of a serotype cross-reactive human monoclonal antibody (HMAb) 1C19 for strains with differing morphologies within the DENV1 and DENV2 serotypes. We mapped the 1C19 epitope to E protein domain II by hydrogen deuterium exchange mass spectrometry, cryoEM and molecular dynamics simulations, revealing that this epitope is likely partially hidden on the virus surface. We showed the antibody has high affinity for binding to recombinant DENV1 E proteins compared to those of DENV2, consistent with its strong neutralizing activities for all DENV1 strains tested regardless of their morphologies. This finding suggests that the antibody could out-compete E-to-E interaction for binding to its epitope. In contrast, for DENV2, HMAb 1C19 can only neutralize when the epitope becomes exposed on the bumpy-surfaced particle. Although HMAb 1C19 is not a suitable therapeutic candidate, this study with HMAb 1C19 shows the importance of choosing a high-affinity antibody that could neutralize diverse dengue virus morphologies for therapeutic purposes.

Evaluation of EuroFlow minimal residual disease measurement and donor chimerism monitoring following tandem auto-allogeneic transplantation for multiple myeloma.

Prognostic factors for multiple myeloma (MM) after allogeneic haemopoietic stem cell transplantation (alloHSCT) are poorly characterised. Two potential factors include minimal residual disease (MRD) and CD3+ donor-specific chimerism. We retrospectively examined 93 consecutive patients who received upfront or deferred tandem auto-alloHSCT. Bone marrow (Euroflow) MRD was assessed pre-alloHSCT and 3-monthly post-alloHSCT. CD3+ donor chimerism was assessed at D30, D60, D90, 6 m and 12 m post-alloHSCT. There was no statistical difference between upfront and deferred transplants in progression free survival (PFS) (34 m vs. 15 m respectively, p = 0.20) and overall survival (OS) (75.5 m vs. 62.7 m respectively, p = 0.56). Patients who were MRD-positive post-alloHSCT had inferior PFS to MRD-negative patients from 6 m (6 m HR 3.32, p = 0.02; 9 m HR 4.08, p = 0.003; 12 m HR 4.47, p = 0.008). Attainment or maintenance of MRD-negativity predicted reduced relapse risk (23.5% vs. 62.5%, p = 0.04). However, there was no significant difference in OS between the MRD-negative and positive groups. Full CD3+ donor chimerism at early time points (D30 and D90) was associated with increased risk of acute GVHD (D30 p < 0.001, D90 p = 0.006) and extensive chronic GVHD (D90 p = 0.04), but not PFS or OS. These data support the use of sequential MRD evaluation post-alloHSCT to inform intervention to eradicate persistent or emergent MRD-positive disease.

High flavivirus structural plasticity demonstrated by a non-spherical morphological variant.

Previous flavivirus (dengue and Zika viruses) studies showed largely spherical particles either with smooth or bumpy surfaces. Here, we demonstrate flavivirus particles have high structural plasticity by the induction of a non-spherical morphology at elevated temperatures: the club-shaped particle (clubSP), which contains a cylindrical tail and a disc-like head. Complex formation of DENV and ZIKV with Fab C10 stabilize the viruses allowing cryoEM structural determination to ~10 Å resolution. The caterpillar-shaped (catSP) Fab C10:ZIKV complex shows Fabs locking the E protein raft structure containing three E dimers. However, compared to the original spherical structure, the rafts have rotated relative to each other. The helical tail structure of Fab C10:DENV3 clubSP showed although the Fab locked an E protein dimer, the dimers have shifted laterally. Morphological diversity, including clubSP and the previously identified bumpy and smooth-surfaced spherical particles, may help flavivirus survival and immune evasion.

DENGUE VIRUS. Cryo-EM structure of an antibody that neutralizes dengue virus type 2 by locking E protein dimers.

There are four closely-related dengue virus (DENV) serotypes. Infection with one serotype generates antibodies that may cross-react and enhance infection with other serotypes in a secondary infection. We demonstrated that DENV serotype 2 (DENV2)-specific human monoclonal antibody (HMAb) 2D22 is therapeutic in a mouse model of antibody-enhanced severe dengue disease. We determined the cryo-electron microscopy (cryo-EM) structures of HMAb 2D22 complexed with two different DENV2 strains. HMAb 2D22 binds across viral envelope (E) proteins in the dimeric structure, which probably blocks the E protein reorganization required for virus fusion. HMAb 2D22 "locks" two-thirds of or all dimers on the virus surface, depending on the strain, but neutralizes these DENV2 strains with equal potency. The epitope defined by HMAb 2D22 is a potential target for vaccines and therapeutics.

A highly potent human antibody neutralizes dengue virus serotype 3 by binding across three surface proteins.

Dengue virus (DENV) infects ~400 million people annually. There is no licensed vaccine or therapeutic drug. Only a small fraction of the total DENV-specific antibodies in a naturally occurring dengue infection consists of highly neutralizing antibodies. Here we show that the DENV-specific human monoclonal antibody 5J7 is exceptionally potent, neutralizing 50% of virus at nanogram-range antibody concentration. The 9 Å resolution cryo-electron microscopy structure of the Fab 5J7-DENV complex shows that a single Fab molecule binds across three envelope proteins and engages three functionally important domains, each from a different envelope protein. These domains are critical for receptor binding and fusion to the endosomal membrane. The ability to bind to multiple domains allows the antibody to fully coat the virus surface with only 60 copies of Fab, that is, half the amount compared with other potent antibodies. Our study reveals a highly efficient and unusual mechanism of molecular recognition by an antibody.

Dengue virus purification and sample preparation for cryo-electron microscopy.

Cryo-electron microscopy (cryo-EM) is a valuable tool used to study the structures of icosahedral viruses without having to resort to crystallization. During the last few decades, significant progress has been made where virus structures previously resolved only to low resolution have now breached the sub-nanometer threshold. Critical to such excellent results are the acquisition of highly purified virus samples and well-frozen samples in vitreous ice. With the virus particles locked in their native conformations, cryo-EM together with single-particle analysis can then be deployed to study the structures of the viruses in their fully hydrated states.

A potent anti-dengue human antibody preferentially recognizes the conformation of E protein monomers assembled on the virus surface.

Dengue virus (DENV), which consists of four serotypes (DENV1-4), infects over 400 million people annually. Previous studies have indicated most human monoclonal antibodies (HMAbs) from dengue patients are cross-reactive and poorly neutralizing. Rare neutralizing HMAbs are usually serotype-specific and bind to quaternary structure-dependent epitopes. We determined the structure of DENV1 complexed with Fab fragments of a highly potent HMAb 1F4 to 6 Å resolution by cryo-EM. Although HMAb 1F4 appeared to bind to virus and not E proteins in ELISAs in the previous study, our structure showed that the epitope is located within an envelope (E) protein monomer, and not across neighboring E proteins. The Fab molecules bind to domain I (DI), and DI-DII hinge of the E protein. We also showed that HMAb 1F4 can neutralize DENV at different stages of viral entry in a cell type and receptor dependent manner. The structure reveals the mechanism by which this potent and specific antibody blocks viral infection.

Immature and mature dengue serotype 1 virus structures provide insight into the maturation process.

Dengue virus is a major human pathogen that has four serotypes (DENV1 to -4). Here we report the cryoelectron microscopy (cryo-EM) structures of immature and mature DENV1 at 6- and 4.5-Å resolution, respectively. The subnanometer-resolution maps allow accurate placement of all of the surface proteins. Although the immature and mature viruses showed vastly different surface protein organizations, the envelope protein transmembrane (E-TM) regions remain in similar positions. The pivotal role of the E-TM regions leads to the identification of the start and end positions of all surface proteins during maturation.

Development of orf virus as a bifunctional recombinant vaccine: surface display of Echinococcus granulosus antigen EG95 by fusion to membrane structural proteins.

The parapoxvirus, orf virus (ORFV) causes superficial skin lesions in infected sheep. Unattenuated ORFV is used globally to vaccinate against orf. Recombinant poxviruses are proven delivery systems and we investigated strategies to express the immunogenic Echinococcus granulosus peptide EG95 from ORFV with the aim of developing a recombinant bivalent vaccine. EG95 is an oncosphere protein of the cestode E. granulosus, a parasite responsible for causing cystic hydatid disease in a wide range of hosts including humans and grazing animals such as sheep. Recombinant viruses were produced in which EG95 was expressed by itself or fused to ORFV envelope-associated structural proteins 10 kDa and F1L. Infection studies in sheep showed that specific antibodies were produced against ORFV and EG95 and that the antibody levels against EG95 were comparable to that of animals immunized with purified EG95 in Quil A adjuvant, an immunization regime that is known to afford protection. A single exposure to the dual vaccine has potential for protecting lambs against orf and for priming against EG95 so as to respond strongly to a later injection of EG95 protein.

Investigation of orf virus structure and morphogenesis using recombinants expressing FLAG-tagged envelope structural proteins: evidence for wrapped virus particles and egress from infected cells.

Orf virus (ORFV) is the type species of the genus Parapoxvirus, but little is known about the structure or morphogenesis of the virus. In contrast, the structure and morphogenesis of vaccinia virus (VACV) has been extensively studied. VACV has two main infectious forms, mature virion (MV) and extracellular virion (EV). The MV is wrapped by two additional membranes derived from the trans-Golgi to produce a wrapped virion (WV), the outermost of which is lost by cellular membrane fusion during viral egress to form the EV. Genome sequencing of ORFV has revealed that it has homologues of almost all of the VACV structural genes. Notable exceptions are A36R, K2L, A56R and B5R, which are associated with WV and EV envelopes. This study investigated the morphogenesis and structure of ORFV by fusing FLAG peptide to the structural proteins 10 kDa, F1L and ORF-110 to form recombinant viruses. 10 kDa and F1L are homologues of VACV A27L and H3L MV membrane proteins, whilst ORF-110 is homologous to VACV A34R, an EV membrane protein. Immunogold labelling of FLAG proteins on virus particles isolated from lysed cells showed that FLAG-F1L and FLAG-10 kDa were displayed on the surface of infectious particles, whereas ORF-110-FLAG could not be detected. Western blot analysis of solubilized recombinant ORF-110-FLAG particles revealed that ORF-110-FLAG was abundant and undergoes post-translational modification indicative of endoplasmic reticulum trafficking. Fluorescent microscopy confirmed the prediction that ORF-110-FLAG localized to the Golgi in virus-infected cells. Finally, immunogold labelling of EVs showed that ORF-110-FLAG became exposed on the surface of EV-like particles as a result of egress from the cell.