The 2022 mpox outbreak led the World Health Organization (WHO) to declare it a public health emergency of international concern (PHEIC). There is a need to develop more effective and safer mpox virus (MPXV)-specific vaccines in response to the mpox epidemic. The mRNA vaccine is a promising platform to protect against MPXV infection. In this study, we construct two bivalent MPXV mRNA vaccines, designated LBA (B6R-A29L) and LAM (A35R-M1R), and a quadrivalent mRNA vaccine, LBAAM (B6R-A35R-A29L-M1R). The immunogenicity and protective efficacy of these vaccines alone or in combination were evaluated in a lethal mouse model. All mRNA vaccine candidates could elicit potential antigen-specific humoral and cellular immune responses and provide protection against vaccinia virus (VACV) infection. The protective effect of the combination of two bivalent mRNA vaccines and the quadrivalent vaccine was superior to that of the individual bivalent mRNA vaccine. Our study provides valuable insights for the development of more efficient and safer mRNA vaccines against mpox.

BACKGROUNDAs Omicron is prompted to replicate in the upper airway, neutralizing antibodies (NAbs) delivered through inhalation might inhibit early-stage infection in the respiratory tract. Thus, elucidating the prophylactic efficacy of NAbs via nasal spray addresses an important clinical need.METHODSThe applicable potential of a nasal spray cocktail containing 2 NAbs was characterized by testing its neutralizing potency, synergetic neutralizing mechanism, emergency protective and therapeutic efficacy in a hamster model, and pharmacokinetics/pharmacodynamic (PK/PD) in human nasal cavity.RESULTSThe 2 NAbs displayed broad neutralizing efficacy against Omicron, and they could structurally compensate each other in blocking the Spike-ACE2 interaction. When administrated through the intranasal mucosal route, this cocktail demonstrated profound efficacy in the emergency prevention in hamsters challenged with authentic Omicron BA.1. The investigator-initiated trial in healthy volunteers confirmed the safety and the PK/PD of the NAb cocktail delivered via nasal spray. Nasal samples from the participants receiving 4 administrations over a course of 16 hours demonstrated potent neutralization against Omicron BA.5 in an ex vivo pseudovirus neutralization assay.CONCLUSIONThese results demonstrate that the NAb cocktail nasal spray provides a good basis for clinical prophylactic efficacy against Omicron infections.TRIAL REGISTRATIONwww.chictr.org.cn, ChiCTR2200066525.FUNDINGThe National Science and Technology Major Project (2017ZX10202203), the National Key Research and Development Program of China (2018YFA0507100), Guangzhou National Laboratory (SRPG22-015), Lingang Laboratory (LG202101-01-07), Science and Technology Commission of Shanghai Municipality (YDZX20213100001556), and the Emergency Project from the Science & Technology Commission of Chongqing (cstc2021jscx-fyzxX0001).

Monkey B virus (BV) infection in humans and other macaque species has a mortality rate of approximately 80%. Because BV infects humans through bites, scratches, and other injuries inflicted by macaques, the simple and rapid diagnosis of BV in field laboratories is of great importance to protect veterinarians, laboratory researchers, and support personnels from the threat of infection. Two recombinase polymerase amplification (RPA) assays with a closed vertical flow (VF) visualization strip (RPA-VF-UL27 and RPA-VF-US6) were developed that target two conserved genes combined with a one-off, closed visualization strip device. We compared the sensitivities and specificities of the two assays after optimization of the reaction conditions. The performance of RPA-VF-US6 at room temperature was determined to evaluate its potential in point-of-care (POC) testing. RPA-VF-US6 specifically detected the positive plasmid control (rather than nucleic acids of herpesviruses) with a detection limit of 28 copies, while RPA-VF-UL27 had cross-reactivity with HSV-1, but even 3.4 copies of plasmid standards were readout by this assay. Moreover, RPA-VF-US6 had excellent performance at room temperature (the detection limit was 2,800 plasmid copies), indicating the potential of RPA-VF-US6 in POC testing. We developed two RPA assays for BV visualization diagnosis. RPA-VF-US6 is a simple, rapid, and specific detection method for BV. The entire reaction can be performed at a constant temperature within 30 min, suggesting the potential of RPA-VF-US6 for POC testing in field laboratories without sophisticated instruments.

Neutralizing monoclonal antibodies (NAbs) with prophylactic and therapeutic efficacy have demonstrated fundamental importance in the control of SARS-CoV-2 transmission. However, their wide application has been largely limited by high cost and inconvenience in administration. Here, we developed an intranasal spray containing two synergetic human NAbs that could broadly neutralize the emerging Omicron variants in vitro. A unique synergetic neutralizing mechanism was identified that the two NAbs bound to exclusive epitopes on the RBD and structurally compensate each other in blocking the Spike-ACE2 interaction. Importantly, when given at low dosages for three consecutive days through the intranasal mucosal route, this cocktail showed significant improvement in the emergency preventive and therapeutic effects in hamsters challenged with authentic Omicron BA.1. Further, we performed an investigator-initiated trail in healthy volunteers (ChiCTR2200066525) to study the safety and pharmacokinetics of the antibody cocktail administrated as nasal spray. The nasal spray is generally safe and well tolerated without treatment related severe abnormal effects. The antibody cocktail nasal spray demonstrated nasal concentrations higher than the IC90 of neutralization activity against Omicron BA.4/5 even at 24 hours post dosing. Furthermore, nasal samples from the study subjects demonstrated potent neutralization activity against Omicron BA.4/5 in an ex vivo pseudovirus neutralization assay. Together, we provide a novel approach for NAb regimens, a potentially highly effective product with broad applicable perspective in depressing the infection risk of new epidemic variant and ameliorating the heavy medical burden of hospital.One Sentence Summary An intranasal spray of two synergetic antibodies cocktail neutralizing Omicron BA.4/5 and an initial clinical evaluation in healthy volunteers.### Competing Interest StatementAilong Huang and Aishun Jin declare the following competing interests: Patent has been filed for some of the antibodies presented here (patent application number: PCT/CN2020/115480, PCT/CN2021/078150, PCT/CN2021/113261; patent applicants: Chongqing Medical University). All other authors declare no competing interests.### Clinical TrialChiCTR2200066525### Funding StatementThis study was funded by the Natural Science Foundation of Hubei Province of China (2019CFA076) The National Natural Science Foundation of China (32170949, 81871639, 92169109, 81871656 and 8181101099); the National Science and Technology Major Project (2017ZX10202203) The National Key Research and Development Program of China (2018YFA0507100 and 2016YFD0500300); Guangzhou National Laboratory (SRPG22-015) Lingang Laboratory (LG202101-01-07); Science and Technology Commission of Shanghai Municipality YDZX20213100001556 and 20XD1422900). The first-in-human Investigator Initiated Study was funded by the Emergency Project from the Science & Technology Commission of Chongqing (cstc2021jscx-fyzxX0001)### Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:Ethics committee of the Second Affiliated Hospital of Chongqing Medical University (Chongqing, China) gave ethical approval for this work (Study # AY-62-8001).I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.YesAll data produced in the present study are available upon reasonable request to the authors

During the two-year pandemic of coronavirus disease 2019（COVID-19）, its causative agent, severe acute respiratory syndrome coronavirus 2（SARS-CoV-2）, has been evolving. SARS-CoV-2 Delta, a variant of concern, has become the dominant circulating strain worldwide within just a few months. Here, we performed a comprehensive analysis of a new B.1.617.2 Delta strain（Delta630） compared with the early WIV04 strain（WIV04） in vitro and in vivo, in terms of replication, infectivity, pathogenicity, and transmission in hamsters. When inoculated intranasally, Delta630 led to more pronounced weight loss and more severe disease in hamsters. Moreover, 40%mortality occurred about one week after infection with 104PFU of Delta630, whereas no deaths occurred even after infection with 105PFU of WIV04 or other strains belonging to the Delta variant. Moreover, Delta630outgrew over WIV04 in the competitive aerosol transmission experiment. Taken together, the Delta630 strain showed increased replication ability, pathogenicity, and transmissibility over WIV04 in hamsters. To our knowledge, this is the first SARS-CoV-2 strain that causes death in a hamster model, which could be an asset for the efficacy evaluation of vaccines and antivirals against infections of SARS-CoV-2 Delta strains. The underlying molecular mechanisms of increased virulence and transmission await further analysis.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still rapidly spreading worldwide. Many drugs and vaccines have been approved for clinical use show efficacy in the treatment and prevention of SARS-CoV-2 infections. However, the emergence of SARS-CoV-2 variants of concern (VOCs), such as Delta (B.1.617.2) and the recently emerged Omicron (B.1.1.529), has seriously challenged the application of current therapeutics. Therefore, there is still a pressing need for identification of new broad-spectrum antivirals. Here, we further characterized a human antibody (58G6), which we previously isolated from a patient, with a broadly authentic virus-neutralizing activity that inhibits the Delta and Omicron variants with half-maximal inhibitory concentrations (IC50) of 1.69 ng/ml and 54.31 ng/ml, respectively. 58G6 shows prophylactic and therapeutic efficacy in hamsters challenged with the Delta and Omicron variants through nasal delivery. Notably, a very low dosage (2 mg/kg daily) of 58G6 efficiently prevented Omicron variant replication in the lungs. These advantages may overcome the efficacy limitation of currently approved neutralizing antibodies that can be administered only by intravenous injection. In general, 58G6 is a promising prophylactic and therapeutic candidate against current circulating VOCs and even future emerging mutants. To the best of our knowledge, 58G6 is one of the most potent neutralizing antibodies against Omicron, with a broader spectrum than those approved for clinical use. 58G6 could be developed as a nebulized therapy, which would be more cost effective and user friendly and enhance the clinical outcome compared to that obtained with direct nasal delivery.

Current SARS-CoV-2 Omicron subvariants impose a heavy burden on global health systems by evading immunity from most developed neutralizing antibodies and vaccines. Here, we identified a nanobody (aSA3) that strongly cross-reacts with the receptor binding domain (RBD) of both SARS-CoV-1 and wild-type (WT) SARS-CoV-2. The dimeric construct of aSA3 (aSA3-Fc) tightly binds and potently neutralizes both SARS-CoV-1 and WT SARS-CoV-2. Based on X-ray crystallography, we engineered a bispecific nanobody dimer (2-3-Fc) by fusing aSA3-Fc to aRBD-2, a previously identified broad-spectrum nanobody targeting an RBD epitope distinct from aSA3. 2-3-Fc exhibits single-digit ng/mL neutralizing potency against all major variants of concerns including BA.5. In hamsters, a single systemic dose of 2-3-Fc at 10 mg/kg conferred substantial efficacy against Omicron infection. More importantly, even at three low doses of 0.5 mg/kg, 2-3-Fc prophylactically administered through the intranasal route drastically reduced viral RNA loads and completely eliminated infectious Omicron particles in the trachea and lungs. Finally, we discovered that 2(Y29G)-3-Fc containing a Y29G substitution in aRBD-2 showed better activity than 2-3-Fc in neutralizing BA.2.75, a recent Omicron subvariant that emerged in India. This study expands the arsenal against SARS-CoV-1, provides potential therapeutic and prophylactic candidates that fully cover major SARS-CoV-2 variants, and may offer a simple preventive approach against Omicron and its subvariants.

Neutralizing antibodies (NAbs) can prevent and treat infections caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, continuously emerging variants, such as Omicron, have significantly reduced the potency of most known NAbs. The selection of NAbs with broad neutralizing activities and the identification of conserved critical epitopes are still urgently needed. Here, we identified an extremely potent antibody (55A8) by single B-cell sorting from convalescent SARS-CoV-2-infected patients that recognized the receptor-binding domain (RBD) in the SARS-CoV-2 spike (S) protein. 55A8 could bind to wild-type SARS-CoV-2, Omicron BA.1 and Omicron BA.2 simultaneously with 58G6, a NAb previously identified by our group. Importantly, an antibody cocktail containing 55A8 and 58G6 (2-cocktail) showed synergetic neutralizing activity with a half-maximal inhibitory concentration (IC 50 ) in the picomolar range in vitro and prophylactic efficacy in hamsters challenged with Omicron (BA.1) through intranasal delivery at an extraordinarily low dosage (25 μg of each antibody daily) at 3 days post-infection. Structural analysis by cryo-electron microscopy (cryo-EM) revealed that 55A8 is a Class III NAb that recognizes a highly conserved epitope. It could block angiotensin-converting enzyme 2 (ACE2) binding to the RBD in the S protein trimer via steric hindrance. The epitopes in the RBD recognized by 55A8 and 58G6 were found to be different and complementary, which could explain the synergetic mechanism of these two NAbs. Our findings not only provide a potential antibody cocktail for clinical use against infection with current SARS-CoV-2 strains and future variants but also identify critical epitope information for the development of better antiviral agents.

ABSTRACT SARS-CoV-2 has infected more than 400 million people around the globe and caused millions of deaths. Since its identification in November 2021, Omicron, a highly transmissible variant, has become the dominant variant in most countries. Omicron’s highly mutated spike protein, the main target of vaccine development, significantly compromises the immune protection from current vaccination. We develop an mRNA vaccine (S Omicron -6P) based on an Omicron-specific sequence. In mice, S Omicron -6P shows superior neutralizing antibodies inducing abilities to a clinically approved inactivated virus vaccine, a clinically approved protein subunit vaccine, and an mRNA vaccine (S WT -2P) with the same sequence of BNT162b2 RNA. Significantly, S Omicron -6P induces a 14.4∼27.7-fold and a 28.3∼50.3-fold increase of neutralizing activity against the pseudovirus of Omicron and authentic Omicron compared to S WT -2P, respectively. In addition, two doses S Omicron -6P significantly protects Syrian hamsters against challenge with SARS-CoV-2 Omicron variant and elicits high titers of nAbs in a dose-dependent manner in macaques. Our results suggest that S Omicron -6P offers advantages over current vaccines, and it will be helpful for those with weak immunity.