The SARS-CoV-2 virus has caused a global crisis, resulting in 0.5 billion infections and over 6 million deaths as of March 2022. Fortunately, infection and hospitalization rates were curbed due to ...the rollout of DNA and mRNA vaccines. However, the efficacy of these vaccines significantly drops a few months post immunization, from 88% down to 47% in the case of the Pfizer BNT162 vaccine. The emergence of variant strains, especially delta and omicron, have also significantly reduced vaccine efficacy. We propose peptide vaccines as a potential solution to address the inadequacies of the current vaccines. Peptide vaccines can be easily modified to target emerging strains, have greater stability, and do not require cold-chain storage. We screened five peptide fragments (B1-B5) derived from the SARS-CoV-2 spike protein to identify neutralizing B-cell peptide antigens. We then investigated adjuvant systems for efficient stimulation of immune responses against the most promising peptide antigens, including liposomal formulations of polyleucine (L10) and polymethylacrylate (PMA), as well as classical adjuvants (CFA and MF59). Immune efficacy of formulations was evaluated using competitive ELISA, pseudovirion neutralization, and live virus neutralization assays. Unfortunately, peptide conjugation to L10 and PMA dramatically altered the secondary structure, resulting in low antibody neutralization efficacy. Of the peptides tested, only B3 administered with CFA or MF59 was highly immunogenic. Thus, a peptide vaccine relying on B3 may provide an attractive alternative to currently marketed vaccines.
The use of dengue virus (DENV) vaccines has been hindered by the complexities of antibody dependent enhancement (ADE). Current late-stage vaccine candidates utilize attenuated and chimeric DENVs that ...produce particles of varying maturities. Antibodies that are elicited by preferentially exposed epitopes on immature virions have been linked to increased ADE. We aimed to further understand the humoral immunity promoted by DENV particles of varying maturities in an AG129 mouse model using a chimeric insect specific vaccine candidate, bDENV-2. We immunized mice with mature, partially mature, and immature bDENV-2 and found that immunization with partially mature bDENV-2 produced more robust and cross-neutralizing immune responses than immunization with immature or mature bDENV-2. Upon challenge with mouse adapted DENV-2 (D220), we observed 80% protection for mature bDENV-2 vaccinated mice and 100% for immature and partially mature vaccinated mice, suggesting that protection to homotypic challenge is not dependent on maturation. Finally, we found reduced in vitro ADE at subneutralising serum concentrations for mice immunized with mature bDENV-2. These results suggest that both immature and mature DENV particles play a role in homotypic protection; however, the increased risk of in vitro ADE from immature particles indicates potential safety benefits from mature DENV-based vaccines.
Arboviruses cycle between, and replicate in, both invertebrate and vertebrate hosts, which for Zika virus (ZIKV) involves Aedes mosquitoes and primates
. The viral determinants required for ...replication in such obligate hosts are under strong purifying selection during natural virus evolution, making it challenging to resolve which determinants are optimal for viral fitness in each host. Herein we describe a deep mutational scanning (DMS) strategy
whereby a viral cDNA library was constructed containing all codon substitutions in the C-terminal 204 amino acids of ZIKV envelope protein (E). The cDNA library was transfected into C6/36 (Aedes) and Vero (primate) cells, with subsequent deep sequencing and computational analyses of recovered viruses showing that substitutions K316Q and S461G, or Q350L and T397S, conferred substantial replicative advantages in mosquito and primate cells, respectively. A 316Q/461G virus was constructed and shown to be replication-defective in mammalian cells due to severely compromised virus particle formation and secretion. The 316Q/461G virus was also highly attenuated in human brain organoids, and illustrated utility as a vaccine in mice. This approach can thus imitate evolutionary selection in a matter of days and identify amino acids key to the regulation of virus replication in specific host environments.
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) Omicron variant sub-lineages spread rapidly worldwide, mostly due to their immune-evasive properties. This has put a significant part ...of the population at risk for severe disease and underscores the need for effective anti-SARS-CoV-2 agents against emergent strains in vulnerable patients. Camelid nanobodies are attractive therapeutic candidates due to their high stability, ease of large-scale production, and potential for delivery via inhalation. Here, we characterize the receptor binding domain (RBD)-specific nanobody W25 and show superior neutralization activity toward Omicron sub-lineages in comparison to all other SARS-CoV2 variants. Structure analysis of W25 in complex with the SARS-CoV2 spike glycoprotein shows that W25 engages an RBD epitope not covered by any of the antibodies previously approved for emergency use. In vivo evaluation of W25 prophylactic and therapeutic treatments across multiple SARS-CoV-2 variant infection models, together with W25 biodistribution analysis in mice, demonstrates favorable pre-clinical properties. Together, these data endorse W25 for further clinical development.
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•The nanobody W25 efficiently neutralizes SARS-CoV2 omicron sub-lineages•A dimeric W25-Fc fusion shows enhanced neutralization activity•W25 binds a unique conserved SARS-CoV2 spike RBD epitope•W25 protects K18-huACE2 mice from Wuhan and Omicron SARS-CoV-2 infections
Public health; Information system model; Decision science
Subunit vaccines exhibit favorable safety and immunogenicity profiles and can be designed to mimic native antigen structures. However, pairing with an appropriate adjuvant is imperative in order to ...elicit effective humoral and cellular immune responses. In this study, we aimed to determine an optimal adjuvant pairing with the prefusion form of influenza haemagglutinin (HA) or respiratory syncytial virus (RSV) fusion (F) subunit vaccines in BALB/c mice in order to inform future subunit vaccine adjuvant selection. We tested a panel of adjuvants, including aluminum hydroxide (alhydrogel), QS21, Addavax, Addavax with QS21 (AdQS21), and Army Liposome Formulation 55 with monophosphoryl lipid A and QS21 (ALF55). We found that all adjuvants elicited robust humoral responses in comparison to placebo, with the induction of potent neutralizing antibodies observed in all adjuvanted groups against influenza and in AdQS21, alhydrogel, and ALF55 against RSV. Upon HA vaccination, we observed that none of the adjuvants were able to significantly increase the frequency of CD4
and CD8
IFN-γ
cells when compared to unadjuvanted antigen. The varying responses to antigens with each adjuvant highlights that those adjuvants most suited for pairing purposes can vary depending on the antigen used and/or the desired immune response. We therefore suggest that an adjuvant trial for different subunit vaccines in development would likely be necessary in preclinical studies.
Abstract In 2022, a genotype IV (GIV) strain of Japanese encephalitis virus (JEV) caused an unprecedented and widespread outbreak of disease in pigs and humans in Australia. As no veterinary vaccines ...against JEV are approved in Australia and all current approved human and veterinary vaccines are derived from genotype (G) III JEV strains, we used the recently described insect-specific Binjari virus (BinJV) chimeric flavivirus vaccine technology to produce a JEV GIV vaccine candidate. Herein we describe the production of a chimeric virus displaying the structural prM and E proteins of a JEV GIV isolate obtained from a stillborn piglet (JEV NSW/22 ) in the genomic backbone of BinJV (BinJ/JEV NSW/22- prME). BinJ/JEV NSW/22- prME was shown to be antigenically indistinguishable from the JEV NSW/22 parental virus by K D analysis and a panel of JEV-reactive monoclonal antibodies in ELISA. BinJ/JEV NSW/22- prME replicated efficiently in C6/36 cells, reaching titres of >10 7 infectious units/mL - an important attribute for vaccine manufacture. As expected, BinJ/JEV NSW/22- prME failed to replicate in a variety of vertebrate cells lines. When used to immunise mice, the vaccine induced a potent virus neutralising response against JEV NSW/22 and to GII and GIII JEV strains. The BinJ/JEV NSW/22- prME vaccine provided complete protection against lethal challenge with JEV NSW/22 , whilst also providing partial protection against viraemia and disease for the related Murray Valley encephalitis virus. Our results demonstrate that BinJ/JEV NSW/22- prME is a promising vaccine candidate against JEV.
This protocol describes an ELISA-based procedure for accurate measurement of SARS-CoV-2 spike protein-receptor binding domain (RBD) neutralization efficacy by murine immune serum. The procedure ...requires a small amount of S-protein/RBD and angiotensin converting enzyme-2 (ACE2). A high-throughput, simple ELISA technique is employed. Plate-coated-RBDs are allowed to interact with the serum, then soluble ACE2 is added, followed by secondary antibodies and substrate. The key steps in this procedure include (1) serum heat treatment to prevent non-specific interactions, (2) proper use of blank controls to detect side reactions and eliminate secondary antibody cross-reactivity, (3) the addition of an optimal amount of saturating ACE2 to maximize sensitivity and prevent non-competitive co-occurrence of RBD-ACE2 binding and neutralization, and (4) mechanistically derived neutralization calculation using a calibration curve. Even manually, the protocol can be completed in 16 h for >30 serum samples; this includes the 7.5 h of incubation time. This automatable, high-throughput, competitive ELISA assay can screen a large number of sera, and does not require sterile conditions or special containment measures, as live viruses are not employed. In comparison to the 'gold standard' assays (virus neutralization titers (VNT) or plaque reduction neutralization titers (PRNT)), which are laborious and time consuming and require special containment measures due to their use of live viruses. This simple, alternative neutralization efficacy assay can be a great asset for initial vaccine development stages. The assay successfully passed conventional validation parameters (sensitivity, specificity, precision, and accuracy) and results with moderately neutralizing murine sera correlated with VNT assay results (R
= 0.975,
= 25), demonstrating high sensitivity.
The ongoing coronavirus disease 2019 (COVID-19) pandemic continues to disrupt essential health services in 90 percent of countries today. The spike (S) protein found on the surface of the causative ...agent, the SARS-CoV-2 virus, has been the prime target for current vaccine research since antibodies directed against the S protein were found to neutralize the virus. However, as new variants emerge, mutations within the spike protein have given rise to potential immune evasion of the response generated by the current generation of SARS-CoV-2 vaccines. In this study, a modified, HexaPro S protein subunit vaccine, delivered using a needle-free high-density microarray patch (HD-MAP), was investigated for its immunogenicity and virus-neutralizing abilities. Mice given two doses of the vaccine candidate generated potent antibody responses capable of neutralizing the parental SARS-CoV-2 virus as well as the variants of concern, Alpha and Delta. These results demonstrate that this alternative vaccination strategy has the potential to mitigate the effect of emerging viral variants.
Complications arising from dengue virus infection include potentially fatal vascular leak, and severe disease has been linked with excessive immune cell activation. An understanding of the triggers ...of this activation is critical for the development of appropriately targeted disease control strategies. We show here that the secreted form of the dengue virus nonstructural protein 1 (NS1) is a pathogen-associated molecular pattern (PAMP). Highly purified NS1 devoid of bacterial endotoxin activity directly activated mouse macrophages and human peripheral blood mononuclear cells (PBMCs) via Toll-like receptor 4 (TLR4), leading to the induction and release of proinflammatory cytokines and chemokines. In an in vitro model of vascular leak, treatment with NS1 alone resulted in the disruption of endothelial cell monolayer integrity. Both NS1-mediated activation of PBMCs and NS1-induced vascular leak in vitro were inhibited by a TLR4 antagonist and by anti-TLR4 antibody treatment. The importance of TLR4 activation in vivo was confirmed by the reduction in capillary leak by a TLR4 antagonist in a mouse model of dengue virus infection. These results pinpoint NS1 as a viral toxin counterpart of the bacterial endotoxin lipopolysaccharide (LPS). Similar to the role of LPS in septic shock, NS1 might contribute to vascular leak in dengue patients, which highlights TLR4 antagonists as a possible therapeutic option.
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