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Mumps virus (MuV) is a highly contagious paramyxovirus that is endemic in most regions of the world and continues to cause outbreaks even in highly immunized populations. Outbreaks of mumps in countries with high measles, mumps, and rubella vaccination coverage have been attributed to waning immunity and antigenic differences between the Jeryl Lynn vaccine strain (genotype A) and circulating wild-type viruses. To obtain a subunit vaccine, we used structure-based design to engineer the mumps fusion (F) glycoprotein stabilized in its prefusion conformation (Pre-F) as well as a chimeric immunogen comprising Pre-F linked to mumps hemagglutinin neuraminidase (HN); in mice, both Pre-F antigen and the chimeric antigen elicited potent cross-reactive plaque reducing neutralizing titers to genotypes A, G, and H mumps. A crystal structure of mumps Pre-F at 2.16 Å resolution validated the stabilization strategy, while a post-fusion form of F was engineered as a comparator. Monoclonal antibodies to mumps Pre-F and HN were isolated from immunized mice; 7 of 14 Pre-F-specific antibodies and 9 of 15 HN-specific antibodies were capable of neutralizing genotype G MuV with a range of potencies. Additionally, 7 of 14 Pre-F-specific antibodies neutralized genotype A mumps. Structural and binding analyses of Pre-F-specific antibodies revealed binding to four discrete neutralizing antigenic sites and binding analyses of HN-specific antibodies revealed binding to five discrete neutralizing antigenic sites. Overall, the PreF and the chimeric Pre-F/HN immunogens are promising candidates to boost MMR-elicited immunity to mumps or as a next-generation vaccine., Competing Interests: Competing interests statement:G.B.E.S.-J. is an shareholder of Mevox, Ltd. R.J.L, B.S.G., and G.B.E.S.-J. are co-inventors on US Provisional 62/946,902.

Flavivirus assembly at the endoplasmic reticulum is driven by the structural proteins envelope (E) and premembrane (prM). Here, contrary to the established paradigm for flavivirus assembly, we demonstrate that the biogenesis of flavivirus particles does not require an intact prM nor proteolytic activation. The expression of E preceded by a truncated version of prM (M-E) was sufficient for the formation of non-infectious Zika virus subviral particles and pseudo-infectious reporter virions. Subviral particles encoded by a ZIKV M-E DNA vaccine elicited a neutralizing antibody response that was insensitive to the virion maturation state, a feature of flavivirus humoral immunity shown to correlate with protection. M-E vaccines that uniformly present structural features shared with mature virions offer a higher quality and broadly applicable approach to flavivirus vaccination., Competing Interests: Competing interests: The authors declare that they have no competing interests.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a plasma protein that controls cholesterol homeostasis. Here, we design a human PCSK9 mimic, named HIT01, with no consecutive 9-residue stretch in common with any human protein as a potential heart attack vaccine. Murine immunizations with HIT01 reduce low-density lipoprotein (LDL) and cholesterol levels by 40% and 30%, respectively. Immunization of cynomolgus macaques with HIT01-K21Q-R218E, a cleavage-resistant variant, elicits high-titer PCSK9-directed antibody responses and significantly reduces serum levels of cholesterol 2 weeks after each immunization. However, HIT01-K21Q-R218E immunizations also increase serum PCSK9 levels by up to 5-fold, likely due to PCSK9-binding antibodies altering the half-life of PCSK9. While vaccination with a PCSK9 mimic can induce antibodies that block interactions of PCSK9 with the LDL receptor, PCSK9-binding antibodies appear to alter homeostatic levels of PCSK9, thereby confounding its vaccine impact. Our results nevertheless suggest a mechanism for increasing the half-life of soluble regulatory factors by vaccination., Competing Interests: Declaration of interests B.Z., G.-Y.C., W.-P.K., Y.W., C.W.C., J.R.M., L.O., T.Z., Y.Y., T.L., and P.D.K. are inventors on a United States provisional patent application submitted by the NIH describing PCSK9-mimicking vaccine immunogens., (Published by Elsevier Inc.)

The receptor-binding domain (RBD) of the SARS-CoV-2 spike is a primary target of neutralizing antibodies and a key component of licensed vaccines. Substantial mutations in RBD, however, enable current variants to escape immunogenicity generated by vaccination with the ancestral (WA1) strain. Here, we produce and assess self-assembling nanoparticles displaying RBDs from WA1 and BA.5 strains by using the SpyTag:SpyCatcher system for coupling. We observed both WA1- and BA.5-RBD nanoparticles to degrade substantially after a few days at 37 °C. Incorporation of nine RBD-stabilizing mutations, however, increased yield ~five-fold and stability such that more than 50% of either the WA1- or BA.5-RBD nanoparticle was retained after one week at 37 °C. Murine immunizations revealed that the stabilized RBD-nanoparticles induced ~100-fold higher autologous neutralization titers than the prefusion-stabilized (S2P) spike at a 2 μg dose. Even at a 25-fold lower dose where S2P-induced neutralization titers were below the detection limit, the stabilized BA.5-RBD nanoparticle induced homologous titers of 12,795 ID 50 and heterologous titers against WA1 of 1767 ID 50 . Assessment against a panel of β-coronavirus variants revealed both the stabilized BA.5-RBD nanoparticle and the stabilized WA1-BA.5-(mosaic)-RBD nanoparticle to elicit much higher neutralization breadth than the stabilized WA1-RBD nanoparticle. The extraordinary titer and high neutralization breadth elicited by stabilized RBD nanoparticles from strain BA.5 make them strong candidates for next-generation COVID-19 vaccines.

The Pneumoviridae family of viruses includes human metapneumovirus (HMPV) and respiratory syncytial virus (RSV). The closely related Paramyxoviridae family includes parainfluenza viruses (PIVs). These three viral pathogens cause acute respiratory tract infections with substantial disease burden in the young, the elderly, and the immune-compromised. While promising subunit vaccines are being developed with prefusion-stabilized forms of the fusion glycoproteins (Fs) of RSV and PIVs, for which neutralizing titers elicited by the prefusion (pre-F) conformation of F are much higher than for the postfusion (post-F) conformation, with HMPV, pre-F and post-F immunogens described thus far elicit similar neutralizing responses, and it has been unclear which conformation, pre-F or post-F, would be the most effective HMPV F-vaccine immunogen. Here, we investigate the impact of further stabilizing HMPV F in the pre-F state. We replaced the furin-cleavage site with a flexible linker, creating a single chain F that yielded increased amounts of pre-F stabilized trimers, enabling the generation and assessment of F trimers stabilized by multiple disulfide bonds. Introduced prolines could increase both expression yields and antigenic recognition by the pre-F specific antibody, MPE8. The cryo-EM structure of a triple disulfide-stabilized pre-F trimer with the variable region of antibody MPE8 at 3.25-Å resolution confirmed the formation of designed disulfides and provided structural details on the MPE8 interface. Immunogenicity assessments in naïve mice showed the triple disulfide-stabilized pre-F trimer could elicit high titer neutralization, >10-fold higher than elicited by post-F. Immunogenicity assessments in pre-exposed rhesus macaques showed the triple disulfide-stabilized pre-F could recall high neutralizing titers after a single immunization, with little discrimination in the recall response between pre-F and post-F immunogens. However, the triple disulfide-stabilized pre-F adsorbed HMPV-directed responses from commercially available pooled human immunoglobulin more fully than post-F. Collectively, these results suggest single-chain triple disulfide-stabilized pre-F trimers to be promising HMPV-vaccine antigens., Competing Interests: LO, BZ, TZ, WPK, YT, JRM, UJB, and PDK are inventors on a U.S. Patent Application filed on their behalf by the National Institutes of Health. The other authors declare no competing interests., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

New vaccine delivery technologies, such as mRNA, have played a critical role in the rapid and efficient control of SARS-CoV-2, helping to end the COVID-19 pandemic. Enveloped virus-like particles (eVLPs) are often more immunogenic than protein subunit immunogens and could be an effective vaccine platform. Here, we investigated whether the genetic delivery of eVLPs could achieve strong immune responses in mice as previously reported with the immunization of in vitro purified eVLPs. We utilized Newcastle disease virus-like particles (NDVLPs) to display SARS-CoV-2 prefusion-stabilized spikes from the WA-1 or Beta variant (S-2P or S-2Pᵦ, respectively) and evaluated neutralizing murine immune responses achieved by a single-gene-transcript DNA construct for the WA-1 or Beta variant (which we named S-2P-NDVLP-1T and S-2Pᵦ-NDVLP-1T, respectively), by multiple-gene-transcript DNA constructs for the Beta variant (S-2Pᵦ-NDVLP-3T), and by a protein subunit-DNA construct for the WA-1 or Beta variant (S-2P-TM or S-2Pᵦ-TM, respectively). The genetic delivery of S-2P-NDVLP-1T or S-2Pᵦ-NDVLP-1T yielded modest neutralizing responses after a single immunization and high neutralizing responses after a second immunization, comparable to previously reported results in mice immunized with in vitro purified S-2P-NDVLPs. Notably, genetic delivery of S-2Pᵦ-NDVLP-3T yielded significantly higher neutralizing responses in mice after a second immunization than S-2Pᵦ-NDVLP-1T or S-2Pᵦ-TM. Genetic delivery also elicited high spike-specific T-cell responses. Collectively, these results indicate that genetic delivery can provide an effective means to immunize eVLPs and that a multiple-gene transcript eVLP platform may be especially efficacious and inform the design of improved vaccines.

While several COVID-19 vaccines have been in use, more effective and durable vaccines are needed to combat the ongoing COVID-19 pandemic. Here, we report highly immunogenic self-assembling SARS-CoV-2 spike-HBsAg nanoparticles displaying a six-proline-stabilized WA1 (wild type, WT) spike S6P on a HBsAg core. These S6P-HBsAgs bound diverse domain-specific SARS-CoV-2 monoclonal antibodies. In mice with and without a HBV pre-vaccination, DNA immunization with S6P-HBsAgs elicited significantly more potent and durable neutralizing antibody (nAb) responses against diverse SARS-CoV-2 strains than that of soluble S2P or S6P, or full-length S2P with its coding sequence matching mRNA-1273. The nAb responses elicited by S6P-HBsAgs persisted substantially longer than by soluble S2P or S6P and appeared to be enhanced by HBsAg pre-exposure. These data show that genetic delivery of SARS-CoV-2 S6P-HBsAg nanoparticles can elicit greater and more durable nAb responses than non-nanoparticle forms of stabilized spike. Our findings highlight the potential of S6P-HBsAgs as next generation genetic vaccine candidates against SARS-CoV-2., (© 2023. Springer Nature Limited.)

Soluble HIV-1-envelope (Env) trimers elicit immune responses that target their solvent-exposed protein bases, the result of removing these trimers from their native membrane-bound context. To assess whether glycosylation could limit these base responses, we introduced sequons encoding potential N -linked glycosylation sites (PNGSs) into base-proximal regions. Expression and antigenic analyses indicated trimers bearing six-introduced PNGSs to have reduced base recognition. Cryo-EM analysis revealed trimers with introduced PNGSs to be prone to disassembly and introduced PNGS to be disordered. Protein-base and glycan-base trimers induced reciprocally symmetric ELISA responses, in which only a small fraction of the antibody response to glycan-base trimers recognized protein-base trimers and vice versa. EM polyclonal epitope mapping revealed glycan-base trimers -even those that were stable biochemically- to elicit antibodies that recognized disassembled trimers. Introduced glycans can thus mask the protein base but their introduction may yield neo-epitopes that dominate the immune response., Competing Interests: NIH has submitted a PCG patent application (PCT/US2023/065009) for Env trimers described in this manuscript on which A.S.O., C.C., T.Z., D.H.R., A.C., R.R., Y.Y., and P.D.K. are co-inventors. The other authors declare no competing interests.

Enterovirus D68 (EV-D68) causes severe respiratory illness in children and can result in a debilitating paralytic disease known as acute flaccid myelitis. No treatment or vaccine for EV-D68 infection is available. Here, we demonstrate that virus-like particle (VLP) vaccines elicit a protective neutralizing antibody against homologous and heterologous EV-D68 subclades. VLP based on a B1 subclade 2014 outbreak strain elicited comparable B1 EV-D68 neutralizing activity as an inactivated viral particle vaccine in mice. Both immunogens elicited weaker cross-neutralization against heterologous viruses. A B3 VLP vaccine elicited more robust neutralization of B3 subclade viruses with improved cross-neutralization. A balanced CD4 + T helper response was achieved using a carbomer-based adjuvant, Adjuplex. Nonhuman primates immunized with this B3 VLP Adjuplex formulation generated robust neutralizing antibodies against homologous and heterologous subclade viruses. Our results suggest that both vaccine strain and adjuvant selection are critical elements for improving the breadth of protective immunity against EV-D68.

Zika virus (ZIKV), an arbovirus transmitted by mosquitoes, was identified as a cause of congenital disease during a major outbreak in the Americas in 2016. Vaccine design strategies relied on limited available isolate sequence information due to the rapid response necessary. The first-generation ZIKV mRNA vaccine, mRNA-1325, was initially generated and, as additional strain sequences became available, a second mRNA vaccine, mRNA-1893, was developed. Herein, we compared the immune responses following mRNA-1325 and mRNA-1893 vaccination and reported that mRNA-1893 generated comparable neutralizing antibody titers to mRNA-1325 at 1/20th of the dose and provided complete protection from ZIKV challenge in non-human primates. In-depth characterization of these vaccines indicated that the observed immunologic differences could be attributed to a single amino acid residue difference that compromised mRNA-1325 virus-like particle formation., (© 2023. The Author(s).)

The FtsQBL is an essential molecular complex sitting midway through bacterial divisome assembly. To visualize and understand its structure, and the consequences of its membrane anchorage, we produced a model of the E. coli complex using the deep-learning prediction utility, AlphaFold 2. The heterotrimeric model was inserted into a 3-lipid model membrane and subjected to a 500-ns atomistic molecular dynamics simulation. The model is superb in quality and captures most experimentally derived structural features, at both the secondary structure and the side-chain levels. The model consists of a uniquely interlocking module contributed by the C-terminal regions of all three proteins. The functionally important constriction control domain residues of FtsB and FtsL are located at a fixed vertical position of ∼43-49 Å from the membrane surface. While the periplasmic domains of all three proteins are well-defined and rigid, the single transmembrane helices of each are flexible and their collective twisting and bending contribute to most structural variations, according to principal component analysis. Considering FtsQ only, the protein is more flexible in its free state relative to its complexed state-with the biggest structural changes located at the elbow between the transmembrane helix and the α-domain. The disordered N-terminal domains of FtsQ and FtsL associate with the cytoplasmic surface of the inner membrane instead of freely venturing into the solvent. Contact network analysis highlighted the formation of the interlocking trimeric module in FtsQBL as playing a central role in mediating the overall structure of the complex., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

In 2019, there were about 100,000 kidney transplants globally, with more than a quarter of them performed in the United States. Unfortunately, some engrafted organs are lost to polyomavirus-associated nephropathy (PyVAN) caused by BK and JC viruses (BKPyV and JCPyV). Both viruses cause brain disease and possibly bladder cancer in immunosuppressed individuals. Transplant patients are routinely monitored for BKPyV viremia, which is an accepted hallmark of nascent nephropathy. If viremia is detected, a reduction in immunosuppressive therapy is standard care, but the intervention comes with increased risk of immune rejection of the engrafted organ. Recent reports have suggested that transplant recipients with high levels of polyomavirus-neutralizing antibodies are protected against PyVAN. Virus-like particle (VLP) vaccines, similar to approved human papillomavirus vaccines, have an excellent safety record and are known to induce high levels of neutralizing antibodies and long-lasting protection from infection. In this study, we demonstrate that VLPs representing BKPyV genotypes I, II, and IV, as well as JCPyV genotype 2 produced in insect cells elicit robust antibody titers. In rhesus macaques, all monkeys developed neutralizing antibody titers above a previously proposed protective threshold of 10,000. A second inoculation, administered 19 weeks after priming, boosted titers to a plateau of ≥ 25,000 that was maintained for almost two years. No vaccine-related adverse events were observed in any macaques. A multivalent BK/JC VLP immunogen did not show inferiority compared to the single-genotype VLP immunogens. Considering these encouraging results, we believe a clinical trial administering the multivalent VLP vaccine in patients waiting to receive a kidney transplant is warranted to evaluate its ability to reduce or eliminate PyVAN., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Dr. Christopher B. Buck and Dr. Diana V. Pastrana are both inventors in the following: Patent # 9764022 Methods and compositions for inhibiting polyomavirus-associated pathology; and patent # 9931393 Immunogenic JC polyomavirus compositions and methods for use., (Published by Elsevier Ltd.)

New Delhi metallo-β-lactamase 1 (NDM-1) is an important causative factor of antimicrobial resistance due to its efficient hydrolysis of a broad range of β-lactam compounds. The two zinc ions at the active site play essential roles in the NDM-1 catalytic activities. In a previous work, H116, one of the three ligands at the Zn1 site, was mutated in order to investigate the nature of zinc ion chelation. We report here the crystal structure of the NDM-1 H116Q mutant, that was designed to convert a B1 di-zinc enzyme into a B3 type, which either still binds two zinc ions or binds only one at the Zn2 site. The effect of mutation on the overall structure is minimal. Unexpectedly, no zinc ion was observed in the crystal structure. The Zn2-site ligating residue C221 forms a covalent bond with the nearby K121, a residue important in maintaining the active-site structure. The largest conformational changes were found at main-chain and side-chain atoms at residues 232-236 (loop 10), the proper configuration of which is known to be essential for substrate binding. The catalytic-site mutation caused little local changes, yet the effects were amplified and propagated to the substrate binding residues. There were big changes in the ψ angles of residues G232 and L234, which resulted in the side chain of N233 being displaced away from the substrate-binding site. In summary, we failed in turning a B1 enzyme into a B3 enzyme, yet we produced a zinc-less NDM-1 with residual activities., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Conjugate-vaccine immunogens require three components: a carrier protein, an antigen, and a crosslinker, capable of coupling antigen to carrier protein, while preserving both T-cell responses from carrier protein and B-cell responses from antigen. We previously showed that the N-terminal eight residues of the HIV-1 fusion peptide (FP8) as an antigen could prime for broad cross-clade neutralizing responses, that recombinant heavy chain of tetanus toxin (rTTHC) as a carrier protein provided optimal responses, and that choice of crosslinker could impact both antigenicity and immunogenicity. Here, we delve more deeply into the impact of varying the linker between FP8 and rTTHC. In specific, we assessed the physical properties, the antigenicity, and the immunogenicity of conjugates for crosslinkers ranging in spacer-arm length from 1.5 to 95.2 Å, with varying hydrophobicity and crosslinking-functional groups. Conjugates coupled with different degrees of multimerization and peptide-to-rTTHC stoichiometry, but all were well recognized by HIV-fusion-peptide-directed antibodies VRC34.01, VRC34.05, PGT151, and ACS202 except for the conjugate with the longest linker (24-PEGylated SMCC; SM(PEG)24), which had lower affinity for ACS202, as did the conjugate with the shortest linker (succinimidyl iodoacetate; SIA), which also had the lowest peptide-to-rTTHC stoichiometry. Murine immunizations testing seven FP8-rTTHC conjugates elicited fusion-peptide-directed antibody responses, with SIA- and SM(PEG)24-linked conjugates eliciting lower responses than the other five conjugates. After boosting with prefusion-closed envelope trimers from strains BG505 clade A and consensus clade C, trimer-directed antibody-binding responses were lower for the SIA-linked conjugate; elicited neutralizing responses were similar, however, though statistically lower for the SM(PEG)24-linked conjugate, when tested against a strain especially sensitive to fusion-peptide-directed responses. Overall, correlation analyses revealed the immunogenicity of FP8-rTTHC conjugates to be negatively impacted by hydrophilicity and extremes of length or low peptide-carrier stoichiometry, but robust to other linker parameters, with several commonly used crosslinkers yielding statistically indistinguishable serological results.

Bacteria expressing NDM-1 have been labeled as superbugs because it confers upon them resistance to a broad range of β-lactam antibiotics. The enzyme has a di‑zinc active centre, with the Zn2 site extensively studied. The roles of active-site Zn1 ligand residues are, however, still not fully understood. We carried out structure-function studies using the mutants, H116A, H116N, and H116Q. Zinc content analysis showed that Zn1 binding was weakened by 40 to 60% in the H116 mutants. The enzymatic-activity studies showed that the lower hydrolysis rates were mainly caused by their weaker substrate binding. The catalytic efficiency (k cat /K m ) of the mutants followed the order: WT > > H116Q (decreased by 4-20 fold) > H116A (decreased by 20-700 fold) ≥ H116N (decreased by 6-800 fold). The maximum effect was observed on H116N against penicillin G, whereas ampicillin was not hydrolyzed at all. The fold-increase of K m values, which informs the weakening of substrate binding, were: H116A by 5-45 fold; H116N by 6-100 fold; H116Q by 2-10 fold. Molecular dynamics simulations suggested that the Zn1 site mutations affected the positions of Zn2 and the bridging hydroxide, by 0.8 to 1.2 Å, with the largest changes of ~1.5 Å observed on Zn2 ligand C221. A native hydrogen bond between H118 and D236 was disrupted in the H116N and H116Q mutants, which led to increased flexibility of loop 10. Consequently, residue N233 was no longer maintained at an optimal position for substrate binding. H116 connected loop 7 across Zn1 to loop 10, thereby contributed to the overall integrity. This work revealed that the H116-Zn1 interaction plays a critical role in defining the substrate-binding site. From these results, it can be inferred that inhibition strategies targeting the zinc ions may be a new direction for drug development., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: New approaches for the prevention and elimination of malaria, a leading cause of illness and death among infants and young children globally, are needed., Methods: We conducted a phase 1 clinical trial to assess the safety and pharmacokinetics of L9LS, a next-generation antimalarial monoclonal antibody, and its protective efficacy against controlled human malaria infection in healthy adults who had never had malaria or received a vaccine for malaria. The participants received L9LS either intravenously or subcutaneously at a dose of 1 mg, 5 mg, or 20 mg per kilogram of body weight. Within 2 to 6 weeks after the administration of L9LS, both the participants who received L9LS and the control participants underwent controlled human malaria infection in which they were exposed to mosquitoes carrying Plasmodium falciparum (3D7 strain)., Results: No safety concerns were identified. L9LS had an estimated half-life of 56 days, and it had dose linearity, with the highest mean (±SD) maximum serum concentration (C max ) of 914.2±146.5 μg per milliliter observed in participants who had received 20 mg per kilogram intravenously and the lowest mean C max of 41.5±4.7 μg per milliliter observed in those who had received 1 mg per kilogram intravenously; the mean C max was 164.8±31.1 in the participants who had received 5 mg per kilogram intravenously and 68.9±22.3 in those who had received 5 mg per kilogram subcutaneously. A total of 17 L9LS recipients and 6 control participants underwent controlled human malaria infection. Of the 17 participants who received a single dose of L9LS, 15 (88%) were protected after controlled human malaria infection. Parasitemia did not develop in any of the participants who received 5 or 20 mg per kilogram of intravenous L9LS. Parasitemia developed in 1 of 5 participants who received 1 mg per kilogram intravenously, 1 of 5 participants who received 5 mg per kilogram subcutaneously, and all 6 control participants through 21 days after the controlled human malaria infection. Protection conferred by L9LS was seen at serum concentrations as low as 9.2 μg per milliliter., Conclusions: In this small trial, L9LS administered intravenously or subcutaneously protected recipients against malaria after controlled infection, without evident safety concerns. (Funded by the National Institute of Allergy and Infectious Diseases; VRC 614 ClinicalTrials.gov number, NCT05019729.)., (Copyright © 2022 Massachusetts Medical Society.)

FtsQBL is a transmembrane protein complex in the divisome of Escherichia coli that plays a critical role in regulating cell division. Although extensive efforts have been made to investigate the interactions between the three involved proteins, FtsQ, FtsB, and FtsL, the detailed interaction mechanism is still poorly understood. In this study, we used hydrogen-deuterium exchange mass spectrometry to investigate these full-length proteins and their complexes. We also dissected the structural dynamic changes and the related binding interfaces within the complexes. Our data revealed that FtsB and FtsL interact at both the periplasmic and transmembrane regions to form a stable complex. Furthermore, the periplasmic region of FtsB underwent significant conformational changes. With the help of computational modeling, our results suggest that FtsBL complexation may bring the respective constriction control domains (CCDs) in close proximity. We show that when FtsBL adopts a coiled-coil structure, the CCDs are fixed at a vertical position relative to the membrane surface; thus, this conformational change may be essential for FtsBL's interaction with other divisome proteins. In the FtsQBL complex, intriguingly, we show only FtsB interacts with FtsQ at its C-terminal region, which stiffens a large area of the β-domain of FtsQ. Consistent with this, we found the connection between the α- and β-domains in FtsQ is also strengthened in the complex. Overall, the present study provides important experimental evidence detailing the local interactions between the full-length FtsB, FtsL, and FtsQ protein, as well as valuable insights into the roles of FtsQBL complexation in regulating divisome activity., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Currently, licensed seasonal influenza vaccines display variable vaccine effectiveness, and there remains a need for novel vaccine platforms capable of inducing broader responses against viral protein domains conserved among influenza subtypes. We conducted a first-in-human, randomized, open-label, phase 1 clinical trial ( NCT03186781 ) to evaluate a novel ferritin (H2HA-Ferritin) nanoparticle influenza vaccine platform. The H2 subtype has not circulated in humans since 1968. Adults born after 1968 have been exposed to only the H1 subtype of group 1 influenza viruses, which shares a conserved stem with H2. Including both H2-naive and H2-exposed adults in the trial allowed us to evaluate memory responses against the conserved stem domain in the presence or absence of pre-existing responses against the immunodominant HA head domain. Fifty healthy participants 18-70 years of age received H2HA-Ferritin intramuscularly as a single 20-μg dose (n = 5) or a 60-μg dose either twice in a homologous (n = 25) prime-boost regimen or once in a heterologous (n = 20) prime-boost regimen after a matched H2 DNA vaccine prime. The primary objective of this trial was to evaluate the safety and tolerability of H2HA-Ferritin either alone or in prime-boost regimens. The secondary objective was to evaluate antibody responses after vaccination. Both vaccines were safe and well tolerated, with the most common solicited symptom being mild headache after both H2HA-Ferritin (n = 15, 22%) and H2 DNA (n = 5, 25%). Exploratory analyses identified neutralizing antibody responses elicited by the H2HA-Ferritin vaccine in both H2-naive and H2-exposed populations. Furthermore, broadly neutralizing antibody responses against group 1 influenza viruses, including both seasonal H1 and avian H5 subtypes, were induced in the H2-naive population through targeting the HA stem. This ferritin nanoparticle vaccine technology represents a novel, safe and immunogenic platform with potential application for pandemic preparedness and universal influenza vaccine development., (© 2022. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

The SARS-CoV-2 spike is the primary target of virus-neutralizing antibodies and critical to the development of effective vaccines against COVID-19. Here, we demonstrate that the prefusion-stabilized two-proline "S2P" spike-widely employed for laboratory work and clinical studies-unfolds when stored at 4 °C, physiological pH, as observed by electron microscopy (EM) and differential scanning calorimetry, but that its trimeric, native-like conformation can be reacquired by low pH treatment. When stored for approximately 1 week, this unfolding does not significantly alter antigenic characteristics; however, longer storage diminishes antibody binding, and month-old spike elicits virtually no neutralization in mice despite inducing high ELISA-binding titers. Cryo-EM structures reveal the folded fraction of spike to decrease with aging; however, its structure remains largely similar, although with varying mobility of the receptor-binding domain. Thus, the SARS-CoV-2 spike is susceptible to unfolding, which affects immunogenicity, highlighting the need to monitor its integrity., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Published by Elsevier Inc.)

Human metapneumovirus (HMPV) is a major cause of respiratory disease worldwide, particularly among children and the elderly. Although there is no licensed HMPV vaccine, promising candidates have been identified for related pneumoviruses based on the structure-based stabilization of the fusion (F) glycoprotein trimer, with prefusion-stabilized F glycoprotein trimers eliciting significantly higher neutralizing responses than their postfusion F counterparts. However, immunization with HMPV F trimers in either prefusion or postfusion conformations has been reported to elicit equivalent neutralization responses. Here we investigate the impact of stabilizing disulfides, especially interprotomer disulfides (IP-DSs) linking protomers of the F trimer, on the elicitation of HMPV-neutralizing responses. We designed F trimer disulfides, screened for their expression, and used electron microscopy (EM) to confirm their formation, including that of an unexpected postfusion variant. In mice, IP-DS-stabilized prefusion and postfusion HMPV F elicited significantly higher neutralizing responses than non-IP-DS-stabilized HMPV Fs. In macaques, the impact of IP-DS stabilization was more measured, although IP-DS-stabilized variants of either prefusion or postfusion HMPV F induced neutralizing responses many times the average titers observed in a healthy human cohort. Serological and absorption-based analyses of macaque responses revealed elicited HMPV-neutralizing responses to be absorbed differently by IP-DS-containing and by non-IP-DS-containing postfusion Fs, suggesting IP-DS stabilization to alter not only the immunogenicity of select epitopes but their antigenicity as well. We speculate the observed increase in immunogenicity by IP-DS trimers to be related to reduced interprotomer flexibility within the HMPV F trimer., Competing Interests: The authors declare no competing interest.

Background: Vaccines to prevent coronavirus disease 2019 (Covid-19) are urgently needed. The effect of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines on viral replication in both upper and lower airways is important to evaluate in nonhuman primates., Methods: Nonhuman primates received 10 or 100 μg of mRNA-1273, a vaccine encoding the prefusion-stabilized spike protein of SARS-CoV-2, or no vaccine. Antibody and T-cell responses were assessed before upper- and lower-airway challenge with SARS-CoV-2. Active viral replication and viral genomes in bronchoalveolar-lavage (BAL) fluid and nasal swab specimens were assessed by polymerase chain reaction, and histopathological analysis and viral quantification were performed on lung-tissue specimens., Results: The mRNA-1273 vaccine candidate induced antibody levels exceeding those in human convalescent-phase serum, with live-virus reciprocal 50% inhibitory dilution (ID 50 ) geometric mean titers of 501 in the 10-μg dose group and 3481 in the 100-μg dose group. Vaccination induced type 1 helper T-cell (Th1)-biased CD4 T-cell responses and low or undetectable Th2 or CD8 T-cell responses. Viral replication was not detectable in BAL fluid by day 2 after challenge in seven of eight animals in both vaccinated groups. No viral replication was detectable in the nose of any of the eight animals in the 100-μg dose group by day 2 after challenge, and limited inflammation or detectable viral genome or antigen was noted in lungs of animals in either vaccine group., Conclusions: Vaccination of nonhuman primates with mRNA-1273 induced robust SARS-CoV-2 neutralizing activity, rapid protection in the upper and lower airways, and no pathologic changes in the lung. (Funded by the National Institutes of Health and others.)., (Copyright © 2020 Massachusetts Medical Society.)

A vaccine for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is needed to control the coronavirus disease 2019 (COVID-19) global pandemic. Structural studies have led to the development of mutations that stabilize Betacoronavirus spike proteins in the prefusion state, improving their expression and increasing immunogenicity 1 . This principle has been applied to design mRNA-1273, an mRNA vaccine that encodes a SARS-CoV-2 spike protein that is stabilized in the prefusion conformation. Here we show that mRNA-1273 induces potent neutralizing antibody responses to both wild-type (D614) and D614G mutant 2 SARS-CoV-2 as well as CD8 + T cell responses, and protects against SARS-CoV-2 infection in the lungs and noses of mice without evidence of immunopathology. mRNA-1273 is currently in a phase III trial to evaluate its efficacy.

Hydroponic produce is gaining popularity due to its suitability for urban agriculture. The general public also considers that hydroponic produce is free from microbiological contamination. In this study, we compared the frequency and abundance of tetracycline-resistant and sulphadiazine-resistant bacteria and the minimal inhibitory concentration (MIC) of these isolates in conventional, organic, and hydroponic lettuce sold in retail. We also determined the frequency of samples carrying tetB , tetX , sul1 , sul2 , and int1 genes by PCR and further quantified the copy number of tetX , sul1 , and int1 genes in samples positive for these genes using qPCR. As expected, the number of resistant bacteria and the MICs of these isolates were lowest in hydroponic lettuce and highest in organic lettuce. All tested resistant genes, except int1 , were detected in samples of all three production methods, but no significant difference was observed between the three groups in the frequency of samples carrying the resistance genes examined or in their copy number. To the best of our knowledge, it is the first study directly reporting the existence of antibiotic-resistant bacteria and resistance genes in hydroponic vegetables sold in retail. The result highlights that the risk of antibiotic-resistant bacteria contamination in hydroponic produce should be further investigated.

Zika virus (ZIKV) is the cause of a pandemic associated with microcephaly in newborns and Guillain-Barre syndrome in adults. Currently, there are no available treatments or vaccines for ZIKV, and the development of a safe and effective vaccine is a high priority for many global health organizations. We describe the development of ZIKV vaccine candidates using the self-amplifying messenger RNA (SAM) platform technology delivered by cationic nanoemulsion (CNE) that allows bedside mixing and is particularly useful for rapid responses to pandemic outbreaks. Two immunizations of either of the two lead SAM (CNE) vaccine candidates elicited potent neutralizing antibody responses to ZIKV in mice and nonhuman primates. Both SAM (CNE) vaccines protected these animals from ZIKV challenge, with one candidate providing complete protection against ZIKV infection in nonhuman primates. The data provide a preclinical proof of concept that a SAM (CNE) vaccine candidate can rapidly elicit protective immunity against ZIKV., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

The emergence of Zika virus (ZIKV) in the Americas stimulated the development of multiple ZIKV vaccine candidates. We previously developed two related DNA vaccine candidates encoding ZIKV structural proteins that were immunogenic in animal models and humans. We sought to identify neutralizing antibody (NAb) properties induced by each vaccine that correlated with protection in nonhuman primates (NHPs). Despite eliciting equivalent NAb titers in NHPs, these vaccines were not equally protective. The transfer of equivalent titers of vaccine-elicited NAb into AG129 mice also revealed nonequivalent protection, indicating qualitative differences among antibodies (Abs) elicited by these vaccines. Both vaccines elicited Abs with similar binding titers against envelope protein monomers and those incorporated into virus-like particles, as well as a comparable capacity to orchestrate phagocytosis. Functional analysis of vaccine-elicited NAbs from NHPs and humans revealed a capacity to neutralize the structurally mature form of the ZIKV virion that varied in magnitude among vaccine candidates. Conversely, sensitivity to the virion maturation state was not a characteristic of NAbs induced by natural or experimental infection. Passive transfer experiments in mice revealed that neutralization of mature ZIKV virions more accurately predicts protection from ZIKV infection. These findings demonstrate that NAb correlates of protection may differ among vaccine antigens when assayed using standard neutralization platforms and suggest that measurements of Ab quality, including the capacity to neutralize mature virions, will be critical for defining correlates of ZIKV vaccine-induced immunity., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The vaccine elicitation of broadly neutralizing antibodies against HIV-1 is a long-sought goal. We previously reported the amino-terminal eight residues of the HIV-1-fusion peptide (FP8) - when conjugated to the carrier protein, keyhole limpet hemocyanin (KLH) - to be capable of inducing broadly neutralizing responses against HIV-1 in animal models. However, KLH is a multi-subunit particle derived from a natural source, and its manufacture as a clinical product remains a challenge. Here we report the preclinical development of recombinant tetanus toxoid heavy chain fragment (rTTHC) linked to FP8 (FP8-rTTHC) as a suitable FP-conjugate vaccine immunogen. We assessed 16 conjugates, made by coupling the 4 most prevalent FP8 sequences with 4 carrier proteins: the aforementioned KLH and rTTHC; the H. influenzae protein D (HiD); and the cross-reactive material from diphtheria toxin (CRM197). While each of the 16 FP8-carrier conjugates could elicit HIV-1-neutralizing responses, rTTHC conjugates induced higher FP-directed responses overall. A Sulfo-SIAB linker yielded superior results over an SM(PEG)2 linker but combinations of carriers, conjugation ratio of peptide to carrier, or choice of adjuvant (Adjuplex or Alum) did not significantly impact elicited FP-directed neutralizing responses in mice. Overall, SIAB-linked FP8-rTTHC appears to be a promising vaccine candidate for advancing to clinical assessment.

The genus Cucumis contains 52 species, including two economically significant crops, cucumber and melon, as well as other important species. Cucumis anguria var. anguria is a wild relative of C. melon , native to Africa. Cucumis anguria is rich in vitamins and minerals in gherkin fruits and carries broad-spectrum resistance to multiplex biotic and abiotic stress, such as powdery mildew, fusarium wilt, and meloidogyn incognita. Cucumis anguria provides a valuable gene pool for crop improvement of Cucumis crops. In this study, the complete chloroplast (cp) genome sequence of C. anguria was determined using next-generation sequencing. The entire cp genome was determined to be 156,577 bp in length. It contained large single-copy (LSC) and small single-copy (SSC) regions of 85,971 and 18,100 bp, respectively, which were separated by a pair of 26,253 bp inverted repeat (IR) regions. The genome contained 134 genes, including 88 protein-coding genes, 37 tRNA genes, 8 rRNA genes and 1 pseudogene infA. The overall GC content of the genome is 37.0%. A phylogenetic tree reconstructed by 48 chloroplast genomes reveals that C. anguria is a separate branch in Cucumis ., Competing Interests: No potential conflict of interest was reported by the authors., (© 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)

Background: Respiratory syncytial virus (RSV) subtypes, A and B, co-circulate in annual epidemics and alternate in dominance. We have shown that a subtype A RSV fusion (F) glycoprotein, stabilized in its prefusion conformation by DS-Cav1 mutations, is a promising RSV-vaccine immunogen, capable of boosting RSV-neutralizing titers in healthy adults. In both humans and vaccine-tested animals, neutralizing titers elicited by this subtype A DS-Cav1 immunogen were ~ 2- to 3-fold higher against the homologous subtype A virus than against the heterologous subtype B virus., Methods: To understand the molecular basis for this subtype difference, we introduced DS-Cav1 mutations into RSV strain B18537 F, determined the trimeric crystal structure, and carried out immunogenicity studies., Results: The B18537 DS-Cav1 F structure at 2-Å resolution afforded a precise delineation of prefusion F characteristics, including those of antigenic site Ø, a key trimer-apex site. Structural comparison with the subtype A prefusion F indicated 11% of surface residues to be different, with an alpha-carbon root-mean-square deviation (RMSD) of 1.2 Å; antigenic site Ø, however, differed in 23% of its surface residues and had an alpha-carbon RMSD of 2.2 Å. Immunization of vaccine-tested animals with DS-Cav1-stabilized B18537 F induced neutralizing responses ~100-fold higher than with postfusion B18537 F. Notably, elicited responses neutralized RSV subtypes A and B at similar levels and were directed towards both conserved equatorial and diverse apical regions., Conclusion: We propose that structural differences in apical and equatorial sites-coupled to differently focused immune responses-provide a molecular explanation for observed differences in elicited subtype A and B neutralizing responses., Competing Interests: The authors declare no conflict of interests., (© Pathogens and Immunity 2019.)

Zika virus (ZIKV) caused an epidemic of congenital malformations in 2015-2016. Although many vaccine candidates have been generated, few have demonstrated efficacy against congenital ZIKV infection. Here, we evaluated lipid-encapsulated messenger RNA (mRNA) vaccines and a DNA plasmid vaccine encoding the prM-E genes of ZIKV in mouse models of congenital infection. Although the DNA vaccine provided comparable efficacy against vertical transmission of ZIKV, the mRNA vaccines, including one that minimizes antibody-dependent enhancement of infection, elicited higher levels of antigen-specific long-lived plasma cells and memory B cells. Despite the induction of robust neutralizing antibody titers by all vaccines, breakthrough seeding of the placenta and fetal head was observed in a small subset of type I interferon signaling-deficient immunocompromised dams. In comparison, evaluation of one of the mRNA vaccines in a human STAT2-knockin transgenic immunocompetent mouse showed complete protection against congenital ZIKV transmission. These data will inform ongoing human ZIKV vaccine development efforts and enhance our understanding of the correlates of vaccine-induced protection., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Western, Eastern, and Venezuelan equine encephalitis viruses (WEEV, EEEV, and VEEV, respectively) are important mosquito-borne agents that pose public health and bioterrorism threats. Despite considerable advances in understanding alphavirus replication, there are currently no available effective vaccines or antiviral treatments against these highly lethal pathogens. To develop a potential countermeasure for viral encephalitis, we generated a trivalent, or three-component, EEV vaccine composed of virus-like particles (VLPs). Monovalent VLPs elicited neutralizing antibody responses and protected mice and nonhuman primates (NHPs) against homologous challenges, but they were not cross-protective. In contrast, NHPs immunized with trivalent VLPs were completely protected against aerosol challenge by each of these three EEVs. Passive transfer of IgG from immunized NHPs protected mice against aerosolized EEV challenge, demonstrating that the mechanism of protection was humoral. Because they are replication incompetent, these trivalent VLPs represent a potentially safe and effective vaccine that can protect against diverse encephalitis viruses., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

MERS-CoV is present in dromedary camels throughout the Middle East and Africa. Dromedary camels are the primary zoonotic reservoir for human infections. Interruption of the zoonotic transmission chain from camels to humans, therefore, may be an effective strategy to control the ongoing MERS-CoV outbreak. Here we show that vaccination with an adjuvanted MERS-CoV Spike protein subunit vaccine confers complete protection from MERS-CoV disease in alpaca and results in reduced and delayed viral shedding in the upper airways of dromedary camels. Protection in alpaca correlates with high serum neutralizing antibody titers. Lower titers of serum neutralizing antibodies correlate with delayed and significantly reduced shedding in the nasal turbinates of dromedary camels. Together, these data indicate that induction of robust neutralizing humoral immune responses by vaccination of naïve animals reduces shedding that potentially could diminish the risk of zoonotic transmission.

The present vaccine against influenza virus has the inevitable risk of antigenic discordance between the vaccine and the circulating strains, which diminishes vaccine efficacy. This necessitates new approaches that provide broader protection against influenza. Here we designed a vaccine using the hypervariable receptor-binding domain (RBD) of viral hemagglutinin displayed on a nanoparticle (np) able to elicit antibody responses that neutralize H1N1 influenza viruses spanning over 90 years. Co-display of RBDs from multiple strains across time, so that the adjacent RBDs are heterotypic, provides an avidity advantage to cross-reactive B cells. Immunization with the mosaic RBD-np elicited broader antibody responses than those induced by an admixture of nanoparticles encompassing the same set of RBDs as separate homotypic arrays. Furthermore, we identified a broadly neutralizing monoclonal antibody in a mouse immunized with mosaic RBD-np. The mosaic antigen array signifies a unique approach that subverts monotypic immunodominance and allows otherwise subdominant cross-reactive B cell responses to emerge.

The emergence of new pathogens, such as Middle East respiratory syndrome coronavirus (MERS-CoV), poses serious challenges to global public health and highlights the urgent need for methods to rapidly identify and characterize potential therapeutic or prevention options, such as neutralizing antibodies. Spike (S) proteins are present on the surface of MERS-CoV virions and mediate viral entry. S is the primary target for MERS-CoV vaccine and antibody development, and it has become increasingly important to understand MERS-CoV antibody binding specificity and function. Commonly used serological methods like ELISA, biolayer interferometry, and flow cytometry are informative, but limited. Here, we demonstrate a high-throughput protein binding inhibition assay using image cytometry. The image cytometry-based high-throughput screening method was developed by selecting a cell type with high DPP4 expression and defining optimal seeding density and protein binding conditions. The ability of monoclonal antibodies to inhibit MERS-CoV S binding was then tested. Binding inhibition results were comparable with those described in previous literature for MERS-CoV spike monomer and showed similar patterns as neutralization results. The coefficient of variation (CV) of our cell-based assay was <10%. The proposed image cytometry method provides an efficient approach for characterizing potential therapeutic antibodies for combating MERS-CoV that compares favorably with current methods. The ability to rapidly determine direct antibody binding to host cells in a high-throughput manner can be applied to study other pathogen-antibody interactions and thus can impact future research on viral pathogens., (Published by Elsevier B.V.)

Parainfluenza virus types 1-4 (PIV1-4) are highly infectious human pathogens, of which PIV3 is most commonly responsible for severe respiratory illness in newborns, elderly, and immunocompromised individuals. To obtain a vaccine effective against all four PIV types, we engineered mutations in each of the four PIV fusion (F) glycoproteins to stabilize their metastable prefusion states, as such stabilization had previously enabled the elicitation of high-titer neutralizing antibodies against the related respiratory syncytial virus. A cryoelectron microscopy structure of an engineered PIV3 F prefusion-stabilized trimer, bound to the prefusion-specific antibody PIA174, revealed atomic-level details for how introduced mutations improved stability as well as how a single PIA174 antibody recognized the trimeric apex of prefusion PIV3 F. Nine combinations of six newly identified disulfides and two cavity-filling mutations stabilized the prefusion PIV3 F immunogens and induced 200- to 500-fold higher neutralizing titers in mice than were elicited by PIV3 F in the postfusion conformation. For PIV1, PIV2, and PIV4, we also obtained stabilized prefusion Fs, for which prefusion versus postfusion titers were 2- to 20-fold higher. Elicited murine responses were PIV type-specific, with little cross-neutralization of other PIVs. In nonhuman primates (NHPs), quadrivalent immunization with prefusion-stabilized Fs from PIV1-4 consistently induced potent neutralizing responses against all four PIVs. For PIV3, the average elicited NHP titer from the quadrivalent immunization was more than fivefold higher than any titer observed in a cohort of over 100 human adults, highlighting the ability of a prefusion-stabilized immunogen to elicit especially potent neutralization., Competing Interests: The authors declare no conflict of interest.

Middle East respiratory syndrome coronavirus (MERS-CoV) causes a highly lethal pulmonary infection with ∼35% mortality. The potential for a future pandemic originating from animal reservoirs or health care-associated events is a major public health concern. There are no vaccines or therapeutic agents currently available for MERS-CoV. Using a probe-based single B cell cloning strategy, we have identified and characterized multiple neutralizing monoclonal antibodies (MAbs) specifically binding to the receptor-binding domain (RBD) or S1 (non-RBD) regions from a convalescent MERS-CoV-infected patient and from immunized rhesus macaques. RBD-specific MAbs tended to have greater neutralizing potency than non-RBD S1-specific MAbs. Six RBD-specific and five S1-specific MAbs could be sorted into four RBD and three non-RBD distinct binding patterns, based on competition assays, mapping neutralization escape variants, and structural analysis. We determined cocrystal structures for two MAbs targeting the RBD from different angles and show they can bind the RBD only in the "out" position. We then showed that selected RBD-specific, non-RBD S1-specific, and S2-specific MAbs given prophylactically prevented MERS-CoV replication in lungs and protected mice from lethal challenge. Importantly, combining RBD- and non-RBD MAbs delayed the emergence of escape mutations in a cell-based virus escape assay. These studies identify MAbs targeting different antigenic sites on S that will be useful for defining mechanisms of MERS-CoV neutralization and for developing more effective interventions to prevent or treat MERS-CoV infections. IMPORTANCE MERS-CoV causes a highly lethal respiratory infection for which no vaccines or antiviral therapeutic options are currently available. Based on continuing exposure from established reservoirs in dromedary camels and bats, transmission of MERS-CoV into humans and future outbreaks are expected. Using structurally defined probes for the MERS-CoV spike glycoprotein (S), the target for neutralizing antibodies, single B cells were sorted from a convalescent human and immunized nonhuman primates (NHPs). MAbs produced from paired immunoglobulin gene sequences were mapped to multiple epitopes within and outside the receptor-binding domain (RBD) and protected against lethal MERS infection in a murine model following passive immunization. Importantly, combining MAbs targeting distinct epitopes prevented viral neutralization escape from RBD-directed MAbs. These data suggest that antibody responses to multiple domains on CoV spike protein may improve immunity and will guide future vaccine and therapeutic development efforts., (Copyright © 2018 American Society for Microbiology.)

We present a technology to screen millions of B cells for natively paired human antibody repertoires. Libraries of natively paired, variable region heavy and light (V H :V L ) amplicons are expressed in a yeast display platform that is optimized for human Fab surface expression. Using our method we identify HIV-1 broadly neutralizing antibodies (bNAbs) from an HIV-1 slow progressor and high-affinity neutralizing antibodies against Ebola virus glycoprotein and influenza hemagglutinin.

Appropriate adjuvant selection may be essential to optimize the potency and to tailor the immune response of subunit vaccines. To induce protective responses against respiratory syncytial virus (RSV)-a highly prevalent childhood pathogen without a licensed vaccine-we previously engineered a pre-fusion-stabilized trimeric RSV F (pre-F) "DS-Cav1" immunogen, which induced high titer RSV-neutralizing antibodies, in mice and non-human primates, when formulated with adjuvants Poly (I:C) and Poly (IC:LC), respectively. To assess the impact of different adjuvants, here we formulated RSV F DS-Cav1 with multiple adjuvants and assessed immune responses. Very high RSV-neutralizing antibody responses (19,006 EC50) were observed in naïve mice immunized with 2 doses of DS-Cav1 adjuvanted with Sigma adjuvant system (SAS), an oil-in-water adjuvant, plus Carbopol; high responses (3658-7108) were observed with DS-Cav1 adjuvanted with Alum, SAS alone, Adjuplex, Poly (I:C) and Poly (IC:LC); and moderate responses (1251-2129) were observed with DS-Cav1 adjuvanted with the TLR4 agonist MPLA, Alum plus MPLA or AddaVax. In contrast, DS-Cav1 without adjuvant induced low-level responses (6). A balanced IgG1 and IgG2a (Th2/Th1) immune response was elicited in most of the high to very high response groups (all but Alum and Adjuplex). We also tested the immune response induced by DS-Cav1 in elderly mice with pre-existing DS-Cav1 immunity; we observed that DS-Cav1 adjuvanted with SAS plus Carbopol boosted the response 2-3-fold, whereas DS-Cav1 adjuvanted with alum boosted the response 5-fold. Finally, we tested whether a mixture of ISA 71 VG and Carbopol would enhanced the antibody response in DS-Cav1 immunized calves. While pre-F-stabilized bovine RSV F induced very high titers in mice when adjuvanted with SAS plus Carbopol, the addition of Carbopol to ISA 71 VG did not enhance immune responses in calves. The vaccine response to pre-F-stabilized RSV F is augmented by adjuvant, but the degree of adjuvant-induced enhancement appears to be both context-dependent and species-specific.

Background: Total glucosides of paeony (TGP) is commonly used to treat rheumatoid arthritis (RA) in China. However, clinical practice hasn't been well informed by evidence from appropriately conducted systematic reviews. This PRISMA-compliant systematic review aims at examining the effectiveness and safety of TGP for RA., Methods: Randomized controlled trials (RCTs) comparing TGP with placebo, no treatment, or disease-modifying antirheumatic drugs (DMARDs) for patients with RA were retrieved by searching seven databases. Primary outcomes included disease improvement and disease remission. Secondary outcomes included adverse effects, pain, health-related quality of life, C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR). Data extraction and analyses were conducted according to the Cochrane standards. We assessed risk of bias for each included studies and quality of evidence on pre-specified outcomes., Results: Eight studies enrolling 1209 patients with active RA were included in this systematic review. On the basis of traditional DMARD(s), TGP might be beneficial for patients with RA in improvement of American College of Rheumatology (ACR) 20 response rate, ACR 50 response rate, ACR70 response rate, and in reduction of adverse effects, compared with no treatment. The overall methodological quality of included studies and the quality of evidence for each outcome were limited., Conclusions: Current trials suggested potential benefits of TGP for RA on the basis of traditional DMARD(s). Therefore, TGP may be a good choice for RA as an adjuvant therapy. However, considering the limited methodological quality and strength of evidence, high-quality RCTs are warranted to support the use of TGP for RA., (Copyright © 2017. Published by Elsevier Ltd.)

Zika virus infection during pregnancy can cause congenital abnormities or fetal demise. The persistence of Zika virus in the male reproductive system poses a risk of sexual transmission. Here we demonstrate that live-attenuated Zika virus vaccine candidates containing deletions in the 3' untranslated region of the Zika virus genome (ZIKV-3'UTR-LAV) prevent viral transmission during pregnancy and testis damage in mice, as well as infection of nonhuman primates. After a single-dose vaccination, pregnant mice challenged with Zika virus at embryonic day 6 and evaluated at embryonic day 13 show markedly diminished levels of viral RNA in maternal, placental, and fetal tissues. Vaccinated male mice challenged with Zika virus were protected against testis infection, injury, and oligospermia. A single immunization of rhesus macaques elicited a rapid and robust antibody response, conferring complete protection upon challenge. Furthermore, the ZIKV-3'UTR-LAV vaccine candidates have a desirable safety profile. These results suggest that further development of ZIKV-3'UTR-LAV is warranted for humans.Zika virus infection can result in congenital disorders and cause disease in adults, and there is currently no approved vaccine. Here Shan et al. show that a single dose of a live-attenuated Zika vaccine prevents infection, testis damage and transmission to the fetus during pregnancy in different animal models.

Middle East respiratory syndrome coronavirus (MERS-CoV) is a lineage C betacoronavirus that since its emergence in 2012 has caused outbreaks in human populations with case-fatality rates of ∼36%. As in other coronaviruses, the spike (S) glycoprotein of MERS-CoV mediates receptor recognition and membrane fusion and is the primary target of the humoral immune response during infection. Here we use structure-based design to develop a generalizable strategy for retaining coronavirus S proteins in the antigenically optimal prefusion conformation and demonstrate that our engineered immunogen is able to elicit high neutralizing antibody titers against MERS-CoV. We also determined high-resolution structures of the trimeric MERS-CoV S ectodomain in complex with G4, a stem-directed neutralizing antibody. The structures reveal that G4 recognizes a glycosylated loop that is variable among coronaviruses and they define four conformational states of the trimer wherein each receptor-binding domain is either tightly packed at the membrane-distal apex or rotated into a receptor-accessible conformation. Our studies suggest a potential mechanism for fusion initiation through sequential receptor-binding events and provide a foundation for the structure-based design of coronavirus vaccines., Competing Interests: Conflict of interest statement: J.P., N.W., K.S.C., R.N.K., H.L.T., C.A.C., B.S.G., A.B.W., and J.S.M. are inventors on US patent application no. 62/412,703, entitled “Prefusion Coronavirus Spike Proteins and Their Use.” L.W., W.S., W.-P.K., and B.S.G. are inventors on US patent application no. PCT/US2016/019395, entitled “Middle East Respiratory Syndrome Coronavirus Immunogens, Antibodies and Their Use.”

Bovine respiratory syncytial virus, a major cause of respiratory disease in calves, is closely related to human RSV, a leading cause of respiratory disease in infants. Recently, promising human RSV-vaccine candidates have been engineered that stabilize the metastable fusion (F) glycoprotein in its prefusion state; however, the absence of a relevant animal model for human RSV has complicated assessment of these vaccine candidates. Here, we use a combination of structure-based design, antigenic characterization, and X-ray crystallography to translate human RSV F stabilization into the bovine context. A "DS2" version of bovine respiratory syncytial virus F with subunits covalently fused, fusion peptide removed, and pre-fusion conformation stabilized by cavity-filling mutations and intra- and inter-protomer disulfides was recognized by pre-fusion-specific antibodies, AM14, D25, and MPE8, and elicited bovine respiratory syncytial virus-neutralizing titers in calves >100-fold higher than those elicited by post-fusion F. When challenged with a heterologous bovine respiratory syncytial virus, virus was not detected in nasal secretions nor in respiratory tract samples of DS2-immunized calves; by contrast bovine respiratory syncytial virus was detected in all post-fusion- and placebo-immunized calves. Our results demonstrate proof-of-concept that DS2-stabilized RSV F immunogens can induce highly protective immunity from RSV in a native host with implications for the efficacy of prefusion-stabilized F vaccines in humans and for the prevention of bovine respiratory syncytial virus in calves., Competing Interests: Competing Interests The authors declare that an intellectual property application has been filed.

Objective To observe the effect of Bushen Qiangdu Recipe (BQR) on the entheses ossification histomorphology of articular ligament of DBA/1 mice with spontaneous ankylosing spondylitis (AS) , and to study its mechanism for prevention and treatment of AS. Methods Thirty 12-week old male DBA/1 mice were randomly divided into the model group, the positive drug group, low, medium, high dose BQR groups, 6 in each group. Another 6 C57BLE mice of the same age were recruited as a blank control group. BQR containing 11. 25, 22. 50, 45.00 g/kg crude drugs was respectively adminis- tered to mice in low, medium, high dose BQR groups by gastrogavage, 0. 2 mL for each mouse, once per day. Celecoxib Capsule (0. 2 mL/0. 8 mg for each mouse, once per day) was administered to mice in the positive drug group by gastrogavage. Equal volume of normal saline was administered to mice in the model group and the blank control group by gastrogavage. All mice were fed and intragastically adminis- tered for 12 successive weeks. Body weight, diet, stools, and hair were routinely observed. Signs of ar- thritis were evaluated once per two weeks. Mice were sacrifice, and then general observation of achilles tendon was performed. The achilles tendon tissue was HE stained. Protein expressions of alkaline phos- phatase (ALP) , bone gamma-carboxyglutamic-acid-containing proteins (BGP) , Dickkopfl (DKK1) , and Wnt5a in the achilles tendon were detected using immunohistochemical method. Results Compared with the blank control group, the scoring of arthritis obviously increased in the model group (P <0. 05). But the scoring of arthritis was obviously lower in the 3 BQR groups and the positive drug group than in the model group (P <0. 05). Histopathological results of achilles tendon tissue showed that no infiltration of inflammatory cells or fibroblasts occurred in the normal group. Their histomorphological structures were normal. Cartilage formation and bone formation at various degrees occurred in the model group. Filtration of fibroblast-like cells occurred in inflammatory cells and attachment points. Scattered lymphocyte infiltra- tion was often seen in the achilles tendon tissue of each medicated group. Cartilage formation and bone formation were rarely seen. Compared with the blank control group, the scoring of arthritis increased in the model group (P <0. 05). Compared with the model group, the scoring of arthritis was decreased in the 3 BQR groups and the positive drug group (P <0. 05). Compared with the blank control group, protein expression of DKK1 decreased and protein expression of Wnt5a increased in the model group (P <0. 05). Compared with the model group, protein expression of DKK1 increased and protein expression of Wnt5a decreased in middle and high dose BQR groups (P <0. 05). Conclusion BQR could delay the occur- rence and development of arthritis and ossification in DBA/1 mice of spontaneous AS model possibly by inhibiting classical Wnt pathway.

Molecular understanding of serological immunity to influenza has been confounded by the complexity of the polyclonal antibody response in humans. Here we used high-resolution proteomics analysis of immunoglobulin (referred to as Ig-seq) coupled with high-throughput sequencing of transcripts encoding B cell receptors (BCR-seq) to quantitatively determine the antibody repertoire at the individual clonotype level in the sera of young adults before and after vaccination with trivalent seasonal influenza vaccine. The serum repertoire comprised between 40 and 147 clonotypes that were specific to each of the three monovalent components of the trivalent influenza vaccine, with boosted pre-existing clonotypes accounting for ∼60% of the response. An unexpectedly high fraction of serum antibodies recognized both the H1 and H3 monovalent vaccines. Recombinant versions of these H1 + H3 cross-reactive antibodies showed broad binding to hemagglutinins (HAs) from previously circulating virus strains; several of these antibodies, which were prevalent in the serum of multiple donors, recognized the same conserved epitope in the HA head domain. Although the HA-head-specific H1 + H3 antibodies did not show neutralization activity in vitro, they protected mice against infection with the H1N1 and H3N2 virus strains when administered before or after challenge. Collectively, our data reveal unanticipated insights regarding the serological response to influenza vaccination and raise questions about the added benefits of using a quadrivalent vaccine instead of a trivalent vaccine., Competing Interests: The authors declare no competing financial interests.

Zika virus (ZIKV) was identified as a cause of congenital disease during the explosive outbreak in the Americas and Caribbean that began in 2015. Because of the ongoing fetal risk from endemic disease and travel-related exposures, a vaccine to prevent viremia in women of childbearing age and their partners is imperative. We found that vaccination with DNA expressing the premembrane and envelope proteins of ZIKV was immunogenic in mice and nonhuman primates, and protection against viremia after ZIKV challenge correlated with serum neutralizing activity. These data not only indicate that DNA vaccination could be a successful approach to protect against ZIKV infection, but also suggest a protective threshold of vaccine-induced neutralizing activity that prevents viremia after acute infection., (Copyright © 2016, American Association for the Advancement of Science.)

The design of immunogens that elicit broadly reactive neutralizing antibodies (bnAbs) has been a major obstacle to HIV-1 vaccine development. One approach to assess potential immunogens is to use mice expressing precursors of human bnAbs as vaccination models. The bnAbs of the VRC01-class derive from the IGHV1-2 immunoglobulin heavy chain and neutralize a wide spectrum of HIV-1 strains via targeting the CD4 binding site of the envelope glycoprotein gp120. We now describe a mouse vaccination model that allows a germline human IGHV1-2(∗)02 segment to undergo normal V(D)J recombination and, thereby, leads to the generation of peripheral B cells that express a highly diverse repertoire of VRC01-related receptors. When sequentially immunized with modified gp120 glycoproteins designed to engage VRC01 germline and intermediate antibodies, IGHV1-2(∗)02-rearranging mice, which also express a VRC01-antibody precursor light chain, can support the affinity maturation of VRC01 precursor antibodies into HIV-neutralizing antibody lineages., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Structure-based design of vaccines, particularly the iterative optimization used so successfully in the structure-based design of drugs, has been a long-sought goal. We previously developed a first-generation vaccine antigen called DS-Cav1, comprising a prefusion-stabilized form of the fusion (F) glycoprotein, which elicits high-titer protective responses against respiratory syncytial virus (RSV) in mice and macaques. Here we report the improvement of DS-Cav1 through iterative cycles of structure-based design that significantly increased the titer of RSV-protective responses. The resultant second-generation 'DS2'-stabilized immunogens have their F subunits genetically linked, their fusion peptides deleted and their interprotomer movements stabilized by an additional disulfide bond. These DS2 immunogens are promising vaccine candidates with superior attributes, such as their lack of a requirement for furin cleavage and their increased antigenic stability against heat inactivation. The iterative structure-based improvement described here may have utility in the optimization of other vaccine antigens.

Antibodies capable of neutralizing divergent influenza A viruses could form the basis of a universal vaccine. Here, from subjects enrolled in an H5N1 DNA/MIV-prime-boost influenza vaccine trial, we sorted hemagglutinin cross-reactive memory B cells and identified three antibody classes, each capable of neutralizing diverse subtypes of group 1 and group 2 influenza A viruses. Co-crystal structures with hemagglutinin revealed that each class utilized characteristic germline genes and convergent sequence motifs to recognize overlapping epitopes in the hemagglutinin stem. All six analyzed subjects had sequences from at least one multidonor class, and-in half the subjects-multidonor-class sequences were recovered from >40% of cross-reactive B cells. By contrast, these multidonor-class sequences were rare in published antibody datasets. Vaccination with a divergent hemagglutinin can thus increase the frequency of B cells encoding broad influenza A-neutralizing antibodies. We propose the sequence signature-quantified prevalence of these B cells as a metric to guide universal influenza A immunization strategies., (Published by Elsevier Inc.)