Anus preservation in low rectal adenocarcinoma based on MMR/MSI status (APRAM): a study protocol for a randomised, controlled, open-label, multicentre phase III trial.

BACKGROUND: Anus preservation has been a challenge in the treatment of patients with low rectal adenocarcinoma (within 5 cm from the anal verge) because it is difficult to spare the anus with its functioning sphincter complex under the safe margin of tumour resection. Patients with dMMR/MSI-H can achieve a favourable complete response (CR) rate by using a single immune checkpoint inhibitor. For patients with pMMR/MSS/MSI-L, intensified neoadjuvant three-drug chemotherapy may be the preferred option for anal preservation. In addition, the watch and wait (W&W) strategy has been proven safe and feasible for patients with rectal cancer who achieve a clinical complete response (cCR). Therefore, we initiated this clinical trial to explore the optimal neoadjuvant treatment pattern for patients with low locally advanced rectal cancer (LARC) with different MMR/MSI statuses, aiming to achieve a higher cCR rate with the W&W strategy and ultimately provide more patients with a chance of anus preservation. METHODS: This is a randomised, controlled, open-label, multicentre phase III trial. Patients with clinical stage T2-4 and/or N + tumours located within 5 cm from the anal verge are considered eligible. Based on the results of pathological biopsy, the patients are divided into two groups: dMMR/MSI-H and pMMR/MSS. Patients in the dMMR/MSI-H group will be randomly allocated in a 1:1 ratio to either arm A (monoimmunotherapy) or arm B (short-course radiotherapy followed by monoimmunotherapy). Patients in the pMMR/MSS group will be initially treated with long-term pelvic radiation with concurrent capecitabine combined with irinotecan. Two weeks after the completion of chemoradiotherapy (CRT), the patients will be randomly allocated in a 1:1 ratio to arm C (XELIRI six cycle regime) or arm D (FOLFIRINOX nine cycle regime). The irinotecan dose will be adjusted according to the UGT1A1-genotype. After treatment, a comprehensive assessment will be performed to determine whether a cCR has been achieved. If achieved, the W&W strategy will be adopted; otherwise, total mesorectal excision (TME) will be performed. The primary endpoint is cCR with the maintenance of 12 months at least, determined using digital rectal examination, endoscopy, and rectal MRI or PET/CT as a supplementary method. DISCUSSION: APRAM will explore the best anus preservation model for low LARC, combining the strategies of consolidation chemotherapy, immunotherapy, and short-course radiotherapy, and aims to preserve the anus of more patients using W&W. Our study provides an accurate individual treatment mode based on the MMR/MSI status for patients with low LARC, and more patients will receive the opportunity for anus preservation under our therapeutic strategy, which would transform into long-term benefits. TRIAL REGISTRATION: Clinicaltrials.gov NCT05669092 (Registered 28th Nov 2022).

Glutamine as a Potential Noninvasive Biomarker for Human Embryo Selection.

To determine whether glutamine consumption is associated with embryo quality and aneuploidy, a retrospective study was conducted in an in vitro fertilization center. Spent embryo culture media from patients undergoing assisted reproduction treatment and preimplantation genetic testing (PGT) were obtained on day 3 of in vitro culture. Embryo quality was assessed for cell number and fragmentation rate. PGT for aneuploidy was performed using whole genome amplification and DNA sequencing. Glutamine levels in spent embryo culture media were analyzed by gas chromatography-mass spectrometry. The results demonstrated that glutamine was a primary contributor to the classification of the good-quality and poor-quality embryos based on the orthogonal partial least-squares discriminant analysis model. Glutamine consumption in the poor-quality embryos was significantly higher than that in the good-quality embryos (P < 0.05). A significant increase in glutamine consumption was observed from aneuploid embryos compared with that from euploid embryos (P < 0.01). The Pearson correlation coefficients between embryo quality and glutamine consumption, and between aneuploidy and glutamine consumption, were 0.430 and 0.757, respectively. The area under the ROC curve was 0.938 (95% CI: 0.902-0.975) for identifying aneuploidy. Animal experiments demonstrate that increased glutamine consumption may be a compensatory mechanism to mitigate oxidative stress. Our data suggest that glutamine consumption is associated with embryo quality and aneuploidy. Glutamine may serve as a molecular indicator for embryo assessment and aneuploidy testing.

Postoperative Chemoradiotherapy With Capecitabine and Oxaliplatin vs Capecitabine for Stage II to III Rectal Cancer: A Randomized Clinical Trial.

Importance: Several studies have explored the efficacy and toxic effects of concurrent 5-fluorouracil (5-FU)- or capecitabine-based chemoradiotherapy (CRT) with or without oxaliplatin in the neoadjuvant setting. Addition of oxaliplatin to 5-FU or capecitabine elicited similar outcomes but with significantly increased toxic effects; however, there is a need for randomized clinical trials comparing 2 CRT regimens for patients receiving CRT in the adjuvant setting. Objective: To explore the efficacy and toxic effects of oxaliplatin combined with postoperative concurrent capecitabine and radiotherapy (RT) for pathological stage II and III rectal cancer. Design, Setting, and Participants: This multicenter randomized clinical trial enrolled patients from 7 centers in China between April 1, 2008, and December 30, 2015. Patients with pathologically confirmed stage II and III rectal cancer were randomized (1:1) to receive concurrent CRT with capecitabine or capecitabine plus oxaliplatin. Analysis was conducted from December 31, 2019, to March 15, 2020. Interventions: RT comprised 45 to 50 Gy in 25 fractions of 1.8 to 2.0 Gy over 5 weeks. In the capecitabine with RT group, concurrent chemotherapy included 2 cycles of capecitabine (1600 mg/m2) on days 1 to 14 and 22 to 35. The capecitabine and oxaliplatin with RT group received identical postoperative RT to that in the capecitabine with RT group combined with capecitabine (1300 mg/m2) on days 1 to 14 and 22 to 35 and a 2-hour infusion of oxaliplatin (60 mg/m2) on weeks 1, 2, 4, and 5. Patients in both groups received adjuvant chemotherapy (capecitabine or fluorouracil and oxaliplatin) after CRT. Main Outcomes and Measures: The primary end point was 3-year disease-free survival (DFS). Results: A total of 589 patients (median [IQR] age, 55 [47-52] years; 375 [63.7%] men and 214 [36.3%] women) were enrolled, including 294 patients randomized to the capecitabine with RT group and 295 patients randomized to the capecitabine and oxaliplatin with RT group. Median (IQR) follow-up was 68 (45-96) months. Most patients had stage III disease (574 patients [75.9%]). Three-year DFS was 76.3% for the capecitabine with RT group and 74.1% for the capecitabine and oxaliplatin with RT group, and 5-year DFS was 72.0% for the capecitabine with RT group and 71.1% for the capecitabine and oxaliplatin with RT group (hazard ratio [HR], 1.07; 95% CI, 0.79-1.44; P = .68). There was no significant difference between groups in overall survival (HR, 0.93; 95% CI, 0.64-1.34; P = .70) or local recurrence (HR, 0.61; 95% CI, 0.31-1.22; P = .16). More grade 3 and 4 acute toxic effects were observed in the capecitabine and oxaliplatin with RT group than in the capecitabine with RT group (114 patients [38.6%] vs 84 patients [28.6%]; P = .01). Conclusions and Relevance: This randomized clinical trial found that addition of oxaliplatin to capecitabine-based postoperative CRT did not improve the efficacy of treatment but increased the risk of severe acute toxic effects. This finding highlights the basic role of postoperative capecitabine with RT for patients with locally advanced rectal cancer. Trial Registration: ClinicalTrials.gov Identifier: NCT00714077.

Determination of higenamine in multi-matrix by gas chromatography-mass spectrometry combined with derivatization technology.

Higenamine (HG), a cardioactive component of some foods and medicines, has been listed in the doping category by the International Olympic Committee, which may lead to misuse by athletes. We report the development of a gas chromatography-mass spectrometry (GC-MS) method for determination of HG in various matrix samples (biological samples, different forms of Chinese patent medicine, Chinese herbal medicine) based on acylation derivatization of HG by heptafluorobutyric anhydride. Under optimal conditions, the linearity of HG in the range of 5-200 ng mL-1 was acceptable (R2 > 0.999), and the limit of detection (LOD) and limit of quantitation (LOQ) for HG was 1.52 ng mL-1 and 5 ng mL-1, respectively. Low, medium, and high concentrations (25, 100 and 160 ng mL-1) of HG were added to plasma, urine, oral liquid, capsule, watered bolus, honeyed bolus and Chinese herbal medicine samples, with recovery ranging from 82.70 to 109.80%, intra-day and inter-day precisions were both less than 3.39%. The results indicated that the method had sufficient sensitivity for analysis of biological samples, and Chinese patent and herbal medicine.

An antibody against the F glycoprotein inhibits Nipah and Hendra virus infections.

Nipah virus (NiV) and Hendra virus (HeV) are zoonotic henipaviruses (HNVs) responsible for outbreaks of encephalitis and respiratory illness with fatality rates of 50-100%. No vaccines or licensed therapeutics currently exist to protect humans against NiV or HeV. HNVs enter host cells by fusing the viral and cellular membranes via the concerted action of the attachment (G) and fusion (F) glycoproteins, the main targets of the humoral immune response. Here, we describe the isolation and humanization of a potent monoclonal antibody cross-neutralizing NiV and HeV. Cryo-electron microscopy, triggering and fusion studies show the antibody binds to a prefusion-specific quaternary epitope, conserved in NiV F and HeV F glycoproteins, and prevents membrane fusion and viral entry. This work supports the importance of the HNV prefusion F conformation for eliciting a robust immune response and paves the way for using this antibody for prophylaxis and post-exposure therapy with NiV- and HeV-infected individuals.

Rescue and characterization of recombinant cedar virus, a non-pathogenic Henipavirus species.

BACKGROUND: Hendra virus and Nipah virus are zoonotic viruses that have caused severe to fatal disease in livestock and human populations. The isolation of Cedar virus, a non-pathogenic virus species in the genus Henipavirus, closely-related to the highly pathogenic Hendra virus and Nipah virus offers an opportunity to investigate differences in pathogenesis and receptor tropism among these viruses. METHODS: We constructed full-length cDNA clones of Cedar virus from synthetic oligonucleotides and rescued two replication-competent, recombinant Cedar virus variants: a recombinant wild-type Cedar virus and a recombinant Cedar virus that expresses a green fluorescent protein from an open reading frame inserted between the phosphoprotein and matrix genes. Replication kinetics of both viruses and stimulation of the interferon pathway were characterized in vitro. Cellular tropism for ephrin-B type ligands was qualitatively investigated by microscopy and quantitatively by a split-luciferase fusion assay. RESULTS: Successful rescue of recombinant Cedar virus expressing a green fluorescent protein did not significantly affect virus replication compared to the recombinant wild-type Cedar virus. We demonstrated that recombinant Cedar virus stimulated the interferon pathway and utilized the established Hendra virus and Nipah virus receptor, ephrin-B2, but not ephrin-B3 to mediate virus entry. We further characterized virus-mediated membrane fusion kinetics of Cedar virus with the known henipavirus receptors ephrin-B2 and ephrin-B3. CONCLUSIONS: The recombinant Cedar virus platform may be utilized to characterize the determinants of pathogenesis across the henipaviruses, investigate their receptor tropisms, and identify novel pan-henipavirus antivirals. Moreover, these experiments can be conducted safely under BSL-2 conditions.

Patients with pathological stage N2 rectal cancer treated with early adjuvant chemotherapy have a lower treatment failure rate.

BACKGROUND: In this era of oxaliplatin-based adjuvant therapy, the optimal sequence in which chemoradiotherapy should be administered for pathological stage N2 rectal cancer is unknown. The aim of this study was to investigate this sequence. METHODS: In the primary adjuvant concurrent chemoradiotherapy (A-CRT) group (n = 71), postoperative concurrent chemoradiotherapy was administered before adjuvant chemotherapy. In the primary adjuvant chemotherapy (A-CT) group (n = 43), postoperative concurrent chemoradiotherapy was administered during or after adjuvant chemotherapy. Postoperative radiotherapy comprised 45-50.4 Gy in 25-28 fractions. Concurrent chemotherapy comprised two cycles of oral capecitabine (1,600 mg/m2) on days 1-14 and 22-35. Patients receiving adjuvant chemotherapy with four or more cycles of XELOX (oxaliplatin plus capecitabine) or eight or more cycles of FOLFOX (fluorouracil, leucovorin, and oxaliplatin) were included. RESULTS: Between June 2005 and December 2013, data for 114 qualified rectal cancer patients were analyzed. The percentages of patients in whom treatment failed in the A-CRT and A-CT groups were 33.8% and 16.3%, respectively (p = 0.042). More patients had distant metastases in the A-CRT group than in the A-CT group (32.4% vs. 14.3%, p = 0.028). Multivariate analysis indicated that the sequence in which chemoradiotherapy was administered (A-CT vs. A-CRT) was an independent prognostic factor for both estimated disease-free survival [hazard ratio (HR) 0.345, 95% confidence interval (CI) 0.137-0.868, p = 0.024] and estimated distant metastasis-free survival (HR 0.366, 95% CI 0.143-0.938, p = 0.036). CONCLUSIONS: In pathological stage N2 rectal cancer patients, administering adjuvant chemotherapy before chemoradiotherapy led to a lower rate of treatment failure, especially with respect to distant metastasis. Adjuvant chemotherapy prescribed as early as possible might benefit this cohort of patients in this era of oxaliplatin-based adjuvant therapy.

Adjuvant treatment may benefit patients with high-risk upper rectal cancer: A nomogram and recursive partitioning analysis of 547 patients.

PURPOSE: The role of adjuvant chemoradiotherapy (ACRT) or adjuvant chemotherapy (ACT) in treating patients with locally advanced upper rectal cancer (URC) after total mesorectal excision (TME) surgery remains unclear. We developed a clinical nomogram and a recursive partitioning analysis (RPA)-based risk stratification system for predicting 5-year cancer-specific survival (CSS) to determine whether these individuals require ACRT or ACT. MATERIALS AND METHODS: This retrospective analysis included 547 patients with primary URC. A nomogram was developed based on the Cox regression model. The performance of the model was assessed by concordance index (C-index) and calibration curve in internal validation with bootstrapping. RPA stratified patients into risk groups based on their tumor characteristics. RESULTS: Five independent prognostic factors (age, preoperative increased carcinoembryonic antigen and carcinoma antigen 19-9, positive lymph node [PLN] number, tumor deposit [TD], pathological T classification) were identified and entered into the predictive nomogram. The bootstrap-corrected C-index was 0.757. RPA stratification of the three prognostic groups showed obviously different prognosis. Only the high-risk group (patients with PLN ≤ 6 and TD, or PLN > 6) benefited from ACRT plus ACT when compared with surgery followed by ACRT or ACT, and surgery alone (5-year CSS: 70.8% vs. 57.8% vs. 15.6%, P < 0.001). CONCLUSIONS: Our nomogram predicts 5-year CSS after TME surgery for locally advanced rectal cancer and RPA-based stratification indicates that ACRT plus ACT post-surgery may be an important treatment plan with potentially ignificant survival advantages in high-risk URC. This may help to select candidates of adjuvant treatment in prospective studies.

Possible contribution of IMRT in postoperative radiochemotherapy for rectal cancer: analysis on 1798 patients by prediction model.

The evidence for adjuvant therapy in locally advanced rectal cancer after TME surgery is sparse. The aim of this study was to identify predicting factors of overall survival (OS) in these patients and combine them into a nomogram for individualized treatment. 1798 patients with pathologically staged II/III rectal adenocarcinoma treated by radical TME surgery from a single center's database were reviewed. The nomogram was derived by Cox proportional hazards regression. Its performance was assessed by concordance index and calibration curve in internal validation with bootstrapping. Pooled Cox model analysis identified age, sex, grade of histology, pathological T and N stage, residual tumor, concurrent radiochemotherapy (RTCT), adjuvant chemotherapy cycles (CT), radiotherapy (RT) unexpected interruption days and intensity-modulated radiation therapy (IMRT) as significant covariates for 5-year OS (P<0.05). Postoperative RTCT, CT and IMRT all improved OS. The proposed model can predict 5-year OS with a C-index of 0.7105. IMRT significantly benefited OS in multivariate analysis (p=0.0441).In conclusion, our nomogram can predict 5-year OS after TME surgery for locally advanced rectal cancer with simple and effective advantage. This model may provide not only baseline OS estimate but also a tool for candidates selecting of adjuvant treatment in prospective studies.

Interim analysis of postoperative chemoradiotherapy with capecitabine and oxaliplatin versus capecitabine alone for pathological stage II and III rectal cancer: a randomized multicenter phase III trial.

The aim of this study is to present an interim analysis of a phase III trial (NCT00714077) of postoperative concurrent capecitabine and radiotherapy with or without oxaliplatin for pathological stage II and III rectal cancer. Patients with pathologically confirmed stage II and III rectal cancer were randomized to either radiotherapy with concurrent capecitabine (Cap-RT group) or with capecitabine and oxaliplatin (Capox-RT group). The primary endpoint was 3-year disease-free survival rate (DFS). The 3-year DFS rate was 73.9% in the Capox-RT group and 71.6% in the Cap-RT group (HR 0.92, p = 0.647), respectively. No significant difference was observed in overall survival, cumulative incidence of local recurrence and distant metastasis between the two groups (p > 0.05). More grade 3-4 acute toxicity was observed in the Capox-RT group than in the Cap-RT group (38.1% vs. 29.2%, p = 0.041). Inclusion of oxaliplatin in the capecitabine-based postoperative regimen did not improve DFS but increased toxicities for pathological stage II and III rectal cancer in this interim analysis.

A recombinant Hendra virus G glycoprotein subunit vaccine protects nonhuman primates against Hendra virus challenge.

UNLABELLED: Hendra virus (HeV) is a zoonotic emerging virus belonging to the family Paramyxoviridae. HeV causes severe and often fatal respiratory and/or neurologic disease in both animals and humans. Currently, there are no licensed vaccines or antiviral drugs approved for human use. A number of animal models have been developed for studying HeV infection, with the African green monkey (AGM) appearing to most faithfully reproduce the human disease. Here, we assessed the utility of a newly developed recombinant subunit vaccine based on the HeV attachment (G) glycoprotein in the AGM model. Four AGMs were vaccinated with two doses of the HeV vaccine (sGHeV) containing Alhydrogel, four AGMs received the sGHeV with Alhydrogel and CpG, and four control animals did not receive the sGHeV vaccine. Animals were challenged with a high dose of infectious HeV 21 days after the boost vaccination. None of the eight specifically vaccinated animals showed any evidence of clinical illness and survived the challenge. All four controls became severely ill with symptoms consistent with HeV infection, and three of the four animals succumbed 8 days after exposure. Success of the recombinant subunit vaccine in AGMs provides pivotal data in supporting its further preclinical development for potential human use. IMPORTANCE: A Hendra virus attachment (G) glycoprotein subunit vaccine was tested in nonhuman primates to assess its ability to protect them from a lethal infection with Hendra virus. It was found that all vaccinated African green monkeys were completely protected against subsequent Hendra virus infection and disease. The success of this new subunit vaccine in nonhuman primates provides critical data in support of its further development for future human use.

Hendra virus vaccine, a one health approach to protecting horse, human, and environmental health.

In recent years, the emergence of several highly pathogenic zoonotic diseases in humans has led to a renewed emphasis on the interconnectedness of human, animal, and environmental health, otherwise known as One Health. For example, Hendra virus (HeV), a zoonotic paramyxovirus, was discovered in 1994, and since then, infections have occurred in 7 humans, each of whom had a strong epidemiologic link to similarly affected horses. As a consequence of these outbreaks, eradication of bat populations was discussed, despite their crucial environmental roles in pollination and reduction of the insect population. We describe the development and evaluation of a vaccine for horses with the potential for breaking the chain of HeV transmission from bats to horses to humans, thereby protecting horse, human, and environmental health. The HeV vaccine for horses is a key example of a One Health approach to the control of human disease.

Use of cross-reactive serological assays for detecting novel pathogens in wildlife: assessing an appropriate cutoff for henipavirus assays in African bats.

Reservoir hosts of novel pathogens are often identified or suspected as such on the basis of serological assay results, prior to the isolation of the pathogen itself. Serological assays might therefore be used outside of their original, validated scope in order to infer seroprevalences in reservoir host populations, until such time that specific diagnostic assays can be developed. This is particularly the case in wildlife disease research. The absence of positive and negative control samples and gold standard diagnostic assays presents challenges in determining an appropriate threshold, or 'cutoff', for the assay that enables differentiation between seronegative and seropositive individuals. Here, multiple methods were explored to determine an appropriate cutoff for a multiplexed microsphere assay that is used to detect henipavirus antibody binding in fruit bat plasma. These methods included calculating multiples of 'negative' control assay values, receiver operating characteristic curve analyses, and Bayesian mixture models to assess the distribution of assay outputs for classifying seropositive and seronegative individuals within different age classes. As for any diagnostic assay, the most appropriate cutoff determination method and value selected must be made according to the aims of the study. This study is presented as an example for others where reference samples, and assays that have been characterised previously, are absent.

Vaccination of ferrets with a recombinant G glycoprotein subunit vaccine provides protection against Nipah virus disease for over 12 months.

BACKGROUND: Nipah virus (NiV) is a zoonotic virus belonging to the henipavirus genus in the family Paramyxoviridae. Since NiV was first identified in 1999, outbreaks have continued to occur in humans in Bangladesh and India on an almost annual basis with case fatality rates reported between 40% and 100%. METHODS: Ferrets were vaccinated with 4, 20 or 100 µg HeVsG formulated with the human use approved adjuvant, CpG, in a prime-boost regime. One half of the ferrets were exposed to NiV at 20 days post boost vaccination and the other at 434 days post vaccination. The presence of virus or viral genome was assessed in ferret fluids and tissues using real-time PCR, virus isolation, histopathology, and immunohistochemistry; serology was also carried out. Non-immunised ferrets were also exposed to virus to confirm the pathogenicity of the inoculum. RESULTS: Ferrets exposed to Nipah virus 20 days post vaccination remained clinically healthy. Virus or viral genome was not detected in any tissues or fluids of the vaccinated ferrets; lesions and antigen were not identified on immunohistological examination of tissues; and there was no increase in antibody titre during the observation period, consistent with failure of virus replication. Of the ferrets challenged 434 days post vaccination, all five remained well throughout the study period; viral genome - but not virus - was recovered from nasal secretions of one ferret given 20 µg HeVsG and bronchial lymph nodes of the other. There was no increase in antibody titre during the observation period, consistent with lack of stimulation of a humoral memory response. CONCLUSIONS: We have previously shown that ferrets vaccinated with 4, 20 or 100 µg HeVsG formulated with CpG adjuvant, which is currently in several human clinical trials, were protected from HeV disease. Here we show, under similar conditions of use, that the vaccine also provides protection against NiV-induced disease. Such protection persists for at least 12 months post-vaccination, with data supporting only localised and self-limiting virus replication in 2 of 5 animals. These results augur well for acceptability of the vaccine to industry.

Neutralizing antibody responses in macaques induced by human immunodeficiency virus type 1 monovalent or trivalent envelope glycoproteins.

A major goal of efforts to develop a vaccine to prevent HIV-1 infection is induction of broadly cross-reactive neutralizing antibodies (bcnAb). In previous studies we have demonstrated induction of neutralizing antibodies that did cross-react among multiple primary and laboratory strains of HIV-1, but neutralized with limited potency. In the present study we tested the hypothesis that immunization with multiple HIV-1 envelope glycoproteins (Envs) would result in a more potent and cross-reactive neutralizing response. One Env, CM243(N610Q), was selected on the basis of studies of the effects of single and multiple mutations of the four gp41 glycosylation sites. The other two Envs included R2 (subtype B) and 14/00/4 (subtype F), both of which were obtained from donors with bcnAb. Rhesus monkeys were immunized using a prime boost regimen as in previous studies. Individual groups of monkeys were immunized with either one of the three Envs or all three. The single N610Q and N615Q mutations of CM243 Env did not disrupt protein secretion, processing into, or reactivity with mAbs, unlike other single or multiple deglycosylation mutations. In rabbit studies the N610Q mutation alone or in combination was associated with an enhanced neutralizing response against homologous and heterologous subtype E viruses. In the subsequent monkey study the response induced by the R2 Env regimen was equivalent to the trivalent regimen and superior to the other monovalent regimens against the virus panel used for testing. The 14/00/4 Env induced responses superior to CM243(N610Q). The results indicate that elimination of the glycosylation site near the gp41 loop results in enhanced immunogenicity, but that immunization of monkeys with these three distinct Envs was not more immunogenic than with one.

A Hendra virus G glycoprotein subunit vaccine protects African green monkeys from Nipah virus challenge.

In the 1990s, Hendra virus and Nipah virus (NiV), two closely related and previously unrecognized paramyxoviruses that cause severe disease and death in humans and a variety of animals, were discovered in Australia and Malaysia, respectively. Outbreaks of disease have occurred nearly every year since NiV was first discovered, with case fatality ranging from 10 to 100%. In the African green monkey (AGM), NiV causes a severe lethal respiratory and/or neurological disease that essentially mirrors fatal human disease. Thus, the AGM represents a reliable disease model for vaccine and therapeutic efficacy testing. We show that vaccination of AGMs with a recombinant subunit vaccine based on the henipavirus attachment G glycoprotein affords complete protection against subsequent NiV infection with no evidence of clinical disease, virus replication, or pathology observed in any challenged subjects. Success of the recombinant subunit vaccine in nonhuman primates provides crucial data in supporting its further preclinical development for potential human use.

Biochemical, conformational, and immunogenic analysis of soluble trimeric forms of henipavirus fusion glycoproteins.

The henipaviruses, Hendra virus (HeV) and Nipah virus (NiV), are paramyxoviruses discovered in the mid- to late 1990s that possess a broad host tropism and are known to cause severe and often fatal disease in both humans and animals. HeV and NiV infect cells by a pH-independent membrane fusion mechanism facilitated by their attachment (G) and fusion (F) glycoproteins. Here, several soluble forms of henipavirus F (sF) were engineered and characterized. Recombinant sF was produced by deleting the transmembrane (TM) and cytoplasmic tail (CT) domains and appending a glycosylphosphatidylinositol (GPI) anchor signal sequence followed by GPI-phospholipase D digestion, appending a trimeric coiled-coil (GCNt) domain (sF(GCNt)), or deleting the TM, CT, and fusion peptide domain. These sF glycoproteins were produced as F(0) precursors, and all were apparent stable trimers recognized by NiV-specific antisera. Surprisingly, however, only the GCNt-appended constructs (sF(GCNt)) could elicit cross-reactive henipavirus-neutralizing antibody in mice. In addition, sF(GCNt) constructs could be triggered in vitro by protease cleavage and heat to transition from an apparent prefusion to postfusion conformation, transitioning through an intermediate that could be captured by a peptide corresponding to the C-terminal heptad repeat domain of F. The pre- and postfusion structures of sF(GCNt) and non-GCNt-appended sF could be revealed by electron microscopy and were distinguishable by F-specific monoclonal antibodies. These data suggest that only certain sF constructs could serve as potential subunit vaccine immunogens against henipaviruses and also establish important tools for further structural, functional, and diagnostic studies on these important emerging viruses.

Site occupancy and glycan compositional analysis of two soluble recombinant forms of the attachment glycoprotein of Hendra virus.

Hendra virus (HeV) continues to cause morbidity and mortality in both humans and horses with a number of sporadic outbreaks. HeV has two structural membrane glycoproteins that mediate the infection of host cells: the attachment (G) and the fusion (F) glycoproteins that are essential for receptor binding and virion-host cell membrane fusion, respectively. N-linked glycosylation of viral envelope proteins are critical post-translation modifications that have been implicated in roles of structural integrity, virus replication and evasion of the host immune response. Deciphering the glycan composition and structure on these glycoproteins may assist in the development of glycan-targeted therapeutic intervention strategies. We examined the site occupancy and glycan composition of recombinant soluble G (sG) glycoproteins expressed in two different mammalian cell systems, transient human embryonic kidney 293 (HEK293) cells and vaccinia virus (VV)-HeLa cells, using a suite of biochemical and biophysical tools: electrophoresis, lectin binding and tandem mass spectrometry. The N-linked glycans of both VV and HEK293-derived sG glycoproteins carried predominantly mono- and disialylated complex-type N-glycans and a smaller population of high mannose-type glycans. All seven consensus sequences for N-linked glycosylation were definitively found to be occupied in the VV-derived protein, whereas only four sites were found and characterized in the HEK293-derived protein. We also report, for the first time, the existence of O-linked glycosylation sites in both proteins. The striking characteristic of both proteins was glycan heterogeneity in both N- and O-linked sites. The structural features of G protein glycosylation were also determined by X-ray crystallography and interactions with the ephrin-B2 receptor are discussed.

A neutralizing human monoclonal antibody protects african green monkeys from hendra virus challenge.

Hendra virus (HeV) is a recently emerged zoonotic paramyxovirus that can cause a severe and often fatal disease in horses and humans. HeV is categorized as a biosafety level 4 agent, which has made the development of animal models and testing of potential therapeutics and vaccines challenging. Infection of African green monkeys (AGMs) with HeV was recently demonstrated, and disease mirrored fatal HeV infection in humans, manifesting as a multisystemic vasculitis with widespread virus replication in vascular tissues and severe pathologic manifestations in the lung, spleen, and brain. Here, we demonstrate that m102.4, a potent HeV-neutralizing human monoclonal antibody (hmAb), can protect AGMs from disease after infection with HeV. Fourteen AGMs were challenged intratracheally with a lethal dose of HeV, and 12 subjects were infused twice with a 100-mg dose of m102.4 beginning at either 10, 24, or 72 hours after infection and again about 48 hours later. The presence of viral RNA, infectious virus, and HeV-specific immune responses demonstrated that all subjects were infected after challenge. All 12 AGMs that received m102.4 survived infection, whereas the untreated control subjects succumbed to disease on day 8 after infection. Animals in the 72-hour treatment group exhibited neurological signs of disease, but all animals started to recover by day 16 after infection. These results represent successful post-exposure in vivo efficacy by an investigational drug against HeV and highlight the potential impact a hmAb can have on human disease.

A recombinant Hendra virus G glycoprotein-based subunit vaccine protects ferrets from lethal Hendra virus challenge.

The henipaviruses, Hendra virus (HeV) and Nipah virus (NiV), are two deadly zoonotic viruses for which no vaccines or therapeutics have yet been approved for human or livestock use. In 14 outbreaks since 1994 HeV has been responsible for multiple fatalities in horses and humans, with all known human infections resulting from close contact with infected horses. A vaccine that prevents virus shedding in infected horses could interrupt the chain of transmission to humans and therefore prevent HeV disease in both. Here we characterise HeV infection in a ferret model and show that it closely mirrors the disease seen in humans and horses with induction of systemic vasculitis, including involvement of the pulmonary and central nervous systems. This model of HeV infection in the ferret was used to assess the immunogenicity and protective efficacy of a subunit vaccine based on a recombinant soluble version of the HeV attachment glycoprotein G (HeVsG), adjuvanted with CpG. We report that ferrets vaccinated with a 100 µg, 20 µg or 4 µg dose of HeVsG remained free of clinical signs of HeV infection following a challenge with 5000 TCID50 of HeV. In addition, and of considerable importance, no evidence of virus or viral genome was detected in any tissues or body fluids in any ferret in the 100 and 20 µg groups, while genome was detected in the nasal washes only of one animal in the 4 µg group. Together, our findings indicate that 100 µg or 20 µg doses of HeVsG vaccine can completely prevent a productive HeV infection in the ferret, suggesting that vaccination to prevent the infection and shedding of HeV is possible.