Lyophilisation of influenza, rabies and Marburg lentiviral pseudotype viruses for the development and distribution of a neutralisation-assay based diagnostic kit

Pseudotype viruses (PVs) are chimeric, replication-deficient virions that mimic wild-type virus entry mechanisms and can be safely employed in neutralisation assays, bypassing the need for high biosafety requirements and performing comparably to established serological assays. However, PV supernatant necessitates -80°C long-term storage and cold-chain maintenance during transport, which limits the scope of dissemination and application throughout resource-limited laboratories. We therefore investigated the effects of lyophilisation on influenza, rabies and Marburg PV stability, with a view to developing a pseudotype virus neutralisation assay (PVNA) based kit suitable for affordable global distribution. Infectivity of each PV was calculated after lyophilisation and immediate reconstitution, as well as subsequent to incubation of freeze-dried pellets at varying temperatures, humidities and timepoints. Integrity of glycoprotein structure following treatment was also assessed by employing lyophilised PVs in downstream PVNAs. In the presence of 0.5M sucrose-PBS cryoprotectant, each freeze-dried pseudotype was stably stored for 4 weeks at up to 37°C and could be neutralised to the same potency as unlyophilised PVs when employed in PVNAs. These results confirm the viability of a freeze-dried PVNA-based kit, which could significantly facilitate low-cost serology for a wide portfolio of emerging infectious viruses.

Development of broad-spectrum human monoclonal antibodies for rabies post-exposure prophylaxis

Currently available rabies post-exposure prophylaxis (PEP) for use in humans includes equine or human rabies immunoglobulins (RIG). The replacement of RIG with an equally or more potent and safer product is strongly encouraged due to the high costs and limited availability of existing RIG. In this study, we identified two broadly neutralizing human monoclonal antibodies that represent a valid and affordable alternative to RIG in rabies PEP. Memory B cells from four selected vaccinated donors were immortalized and monoclonal antibodies were tested for neutralizing activity and epitope specificity. Two antibodies, identified as RVC20 and RVC58 (binding to antigenic site I and III, respectively), were selected for their potency and broad-spectrum reactivity. In vitro, RVC20 and RVC58 were able to neutralize all 35 rabies virus (RABV) and 25 non-RABV lyssaviruses. They showed higher potency and breath compared to antibodies under clinical development (namely CR57, CR4098, and RAB1) and commercially available human RIG. In vivo, the RVC20–RVC58 cocktail protected Syrian hamsters from a lethal RABV challenge and did not affect the endogenous hamster post-vaccination antibody response.

Targeting protein tyrosine phosphatase SHP2 for therapeutic intervention

Protein tyrosine phosphatases have been the focus of considerable research efforts aimed at developing novel therapeutics; however, these targets are often characterized as being ‘undruggable’ due to the challenge of achieving selectivity, potency and cell permeability. More recently, there has been renewed interest in developing inhibitors of the tyrosine phosphatase SHP2 (PTPN11) in the light of its broad role in cancer, specifically juvenile myelomonocytic leukemia, and recent studies that implicate SHP2 as a key factor in breast cancer progression. Recent significant advances in the field of SHP2 inhibitor development raise the question: are we on the verge of a new era of protein tyrosine phosphatase-directed therapeutics? This article critically appraises recent developments, assesses ongoing challenges and presents a perspective on possible future directions.

A monovalent chimpanzee adenovirus Ebola vaccine boosted with MVA

BACKGROUND The West African outbreak of Ebola virus disease that peaked in 2014 has caused more than 11,000 deaths. The development of an effective Ebola vaccine is a priority for control of a future outbreak. METHODS In this phase 1 study, we administered a single dose of the chimpanzee adenovirus 3 (ChAd3) vaccine encoding the surface glycoprotein of Zaire ebolavirus (ZEBOV) to 60 healthy adult volunteers in Oxford, United Kingdom. The vaccine was administered in three dose levels — 1×1010 viral particles, 2.5×1010 viral particles, and 5×1010 viral particles — with 20 participants in each group. We then assessed the effect of adding a booster dose of a modified vaccinia Ankara (MVA) strain, encoding the same Ebola virus glyco- protein, in 30 of the 60 participants and evaluated a reduced prime–boost interval in another 16 participants. We also compared antibody responses to inactivated whole Ebola virus virions and neutralizing antibody activity with those observed in phase 1 studies of a recombinant vesicular stomatitis virus–based vaccine expressing a ZEBOV glycoprotein (rVSV-ZEBOV) to determine relative potency and assess durability. RESULTS No safety concerns were identified at any of the dose levels studied. Four weeks after immunization with the ChAd3 vaccine, ZEBOV-specific antibody responses were similar to those induced by rVSV-ZEBOV vaccination, with a geometric mean titer of 752 and 921, respectively. ZEBOV neutralization activity was also similar with the two vaccines (geo- metric mean titer, 14.9 and 22.2, respectively). Boosting with the MVA vector increased virus-specific antibodies by a factor of 12 (geometric mean titer, 9007) and increased glycoprotein-specific CD8+ T cells by a factor of 5. Significant increases in neutralizing antibodies were seen after boosting in all 30 participants (geometric mean titer, 139; P<0.001). Virus-specific antibody responses in participants primed with ChAd3 remained positive 6 months after vaccination (geometric mean titer, 758) but were significantly higher in those who had received the MVA booster (geometric mean titer, 1750; P<0.001). CONCLUSIONS The ChAd3 vaccine boosted with MVA elicited B-cell and T-cell immune responses to ZEBOV that were superior to those induced by the ChAd3 vaccine alone. (Funded by the Wellcome Trust and others; ClinicalTrials.gov number, NCT02240875.)