Mutations and transient conformational movements of receptor binding domain (RBD) that make neutralizing epitopes momentarily unavailable, present immune escape routes to SARS-CoV-2. To mitigate viral escape, we developed a cocktail of neutralizing antibodies (NAbs) targeting epitopes located on different domains of spike (S) protein. Screening of a library of monoclonal antibodies generated from peripheral blood mononuclear cells of COVID-19 convalescent patients yielded potent NAbs, targeting N-terminal domain (NTD) and RBD domain of S, effective at nM concentrations. Remarkably, combination of RBD-targeting NAbs and NTD-binding NAb, FC05, enhanced the neutralization potency in cell-based assays and animal model. Results of competitive SPR assays and cryo-EM structures of Fabs bound to S unveil determinants of immunogenicity. Combinations of immunogens, identified in NTD and RBD of S, when immunized in rabbits and macaques elicited potent protective immune responses against SARS-CoV-2. More importantly, two immunizations of this combination of NTD and RBD immunogens provided complete protection in macaques against SARS-CoV-2 challenge, without observable antibody-dependent enhancement of infection. These results provide a proof-of-concept for neutralization-based immunogen design targeting SARS-CoV-2 NTD and RBD.

To investigate whether healthy animals are potential carriers of rabies virus in China, 153 domestic dogs were collected from a rabies enzootic area, Anlong county in Guizhou Province, and monitored for 6 months. Initially, findings of rabies virus antigen in the saliva of 15 dogs by an enzyme-linked immunosorbent assay (ELISA) test suggested they might be carriers. These 15 dogs were kept under observation for 6 months. None of the dogs showed any clinical signs of rabies during the observation period. Moreover, using the ELISA test alone, detection of rabies virus antigen in saliva of some animals was not consistent during the observation period. However, none of the saliva samples collected either at the time of acquisition or during the observation period was found to be positive for rabies virus RNA by reverse transcriptase-polymerase chain reaction (RT-PCR). Furthermore, neither viral antigen nor viral RNA was detected in the brain samples collected at the time of euthanasia. These results do not provide support for the contention that healthy dogs act as carriers in rabies. Caution is urged when preliminary and nondefinitive tests, such as ELISA, are used to infer clinical status related to rabies.

Advanced mRNA vaccines play vital roles against SARS-CoV-2. However, most current mRNA delivery platforms need to be stored at −20 °C or −70 °C due to their poor stability, which severely restricts their availability. Herein, we develop a lyophilization technique to prepare SARS-CoV-2 mRNA-lipid nanoparticle vaccines with long-term thermostability. The physiochemical properties and bioactivities of lyophilized vaccines showed no change at 25 °C over 6 months, and the lyophilized SARS-CoV-2 mRNA vaccines could elicit potent humoral and cellular immunity whether in mice, rabbits, or rhesus macaques. Furthermore, in the human trial, administration of lyophilized Omicron mRNA vaccine as a booster shot also engendered strong immunity without severe adverse events, where the titers of neutralizing antibodies against Omicron BA.1/BA.2/BA.4 were increased by at least 253-fold after a booster shot following two doses of the commercial inactivated vaccine, CoronaVac. This lyophilization platform overcomes the instability of mRNA vaccines without affecting their bioactivity and significantly improves their accessibility, particularly in remote regions.

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The therapeutic recombinant human keratinocyte growth factor 1 (rhKGF-1) was approved by the FDA for oral mucositis resulting from hematopoietic stem cell transplantation for hematological malignancies in 2004. However, no recommended bioassay for rhKGF-1 bioactivity has been recorded in the U.S. Pharmacopoeia. In this study, we developed an rhKGF-1-dependent bioassay for determining rhKGF-1 bioactivity based on HEK293 and HaCat cell lines that stably expressed the luciferase reporter driven by the serum response element (SRE) and human fibroblast growth factor receptor (FGFR2) IIIb. A good responsiveness to rhKGF-1 and rhKGF-2 shared by target HEK293/HaCat cell lines was demonstrated. Our stringent validation was completely focused on specificity, linearity, accuracy, precision, and robustness according to the International Council for Harmonization (ICH) Q2 (R1) guidelines, AAPS/FDA Bioanalytical Workshop and the Chinese Pharmacopoeia. We confirmed the reliability of the method in determining rhKGF bioactivity. The validated method is highly timesaving, sensitive, and simple, and is especially valuable for providing information for quality control during the manufacture, research, and development of therapeutic rhKGF.