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Neutralizing antibodies correlate with protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Recent studies, however, show that binding antibody titers, in the absence of robust neutralizing activity, also correlate with protection against disease progression. Non-neutralizing antibodies cannot directly protect against infection but may recruit effector cells and thus contribute to the clearance of infected cells. Additionally, they often bind conserved epitopes across multiple variants. Here, we characterize 42 human monoclonal antibodies (mAbs) from coronavirus disease 2019 (COVID-19)-vaccinated individuals. Most of these antibodies exhibit no neutralizing activity in vitro, but several non-neutralizing antibodies provide protection against lethal challenge with SARS-CoV-2 in different animal models. A subset of those mAbs shows a clear dependence on Fc-mediated effector functions. We have determined the structures of three non-neutralizing antibodies, with two targeting the receptor-binding domain and one that binds the subdomain 1 region. Our data confirm the real-world observation in humans that non-neutralizing antibodies to SARS-CoV-2 can be protective., Competing Interests: Declaration of interests The Icahn School of Medicine at Mount Sinai has filed patent applications relating to SARS-CoV-2 serological assays, NDV-based SARS-CoV-2 vaccines, influenza virus vaccines, and influenza virus therapeutics, which list F.K. as co-inventor. V.S. is also listed as inventor on the SARS-CoV-2 serological assays patent, and W.S. is listed as inventor on the NDV-based SARS-CoV-2 vaccine IP. Mount Sinai has spun out a company, Kantaro, to market serological tests for SARS-CoV-2 and another company, Castlevax, to develop SARS-CoV-2 vaccines. F.K. and W.S. are co-founders and scientific advisory board members of Castlevax. F.K. has consulted for Merck, Curevac, Seqirus, GSK, and Pfizer and is currently consulting for Third Rock Ventures, Sanofi, Gritstone, and Avimex. F.K. is a recipient of royalties from a licensing agreement with Leyden Laboratories B.V. The Krammer laboratory is also collaborating with Dynavax on influenza vaccine development and VIR on influenza therapeutics development. The Ellebedy laboratory has received funding under sponsored research agreements from Moderna, Emergent BioSolutions, and AbbVie. A.H.E. has received consulting and speaking fees from InBios International, Inc., Fimbrion Therapeutics, RGAX, Mubadala Investment Company, AstraZeneca, Moderna, Pfizer, GSK, Danaher, Third Rock Ventures, Goldman Sachs, and Morgan Stanley, is the founder of ImmuneBio Consulting, and is a recipient of royalties from licensing agreements with Abbvie and Leyden Laboratories B.V., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

We have developed a new universal influenza B vaccination strategy based on inactivated influenza B viruses displaying mosaic hemagglutinins (mHAs). Recombinant mHA viruses were constructed by replacing the four major antigenic sites of influenza B virus HAs, with those from exotic avian influenza A virus HAs. Sequential vaccination of naïve mice with mHA-based vaccines elicited higher immune responses towards the immuno-subdominant conserved epitopes of the HA than vaccination with wildtype viruses. Among the different preparations tested, mHA split vaccines were less immunogenic than their whole inactivated virus counterparts. This lower immunogenicity was overcome by the combination with adjuvants. mHA split vaccines adjuvanted with a Toll-like receptor-9 agonist (CpG 1018) increased Th1 immunity and in vivo cross-protection, whereas adjuvanting with an MF59-like oil-in-water nano-emulsion (AddaVax) enhanced and broadened humoral immune responses and antibody-mediated cross-protection. The mHA vaccines with or without adjuvant were subsequently evaluated in mice that were previously immunized to closely mimic human pre-existing immunity to influenza B viruses and the contribution of innate and cellular immunity was evaluated in this model. We believe these preclinical studies using the mHA strategy represent a major step toward the evaluation of a universal influenza B virus vaccine in clinical trials., Competing Interests: Competing interests The Icahn School of Medicine at Mount Sinai and Dynavax have filed a patent application in which P.P., W.S., I.G.D. and F.K. are listed as co-inventors. The Icahn School of Medicine at Mount Sinai has filed patent applications relating to SARS-CoV-2 serological assays, NDV-based SARS-CoV-2 vaccines, influenza virus vaccines and influenza virus therapeutics which list F.K. as co-inventor. Mount Sinai has spun out a company, Kantaro, to market serological tests for SARS-CoV-2 and another company, Castlevax, to develop SARS-CoV-2 vaccines. F.K., P.P. and W.S. are co-founder of Castlevax. F.K. has consulted for Merck, Curevac, Seqirus and Pfizer and is currently consulting for 3rd Rock Ventures, GSK, Gritstone and Avimex. The Krammer laboratory is also collaborating with Dynavax on influenza A virus vaccine development. All other authors declare no competing interests., (© 2024. The Author(s).)

Background: In order to prevent the emergence and spread of future variants of concern of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), developing vaccines capable of stopping transmission is crucial. The SARS-CoV-2 vaccine NDV-HXP-S can be administered live intranasally (IN) and thus induce protective immunity in the upper respiratory tract. The vaccine is based on Newcastle disease virus (NDV) expressing a stabilised SARS-CoV-2 spike protein. NDV-HXP-S can be produced as influenza virus vaccine at low cost in embryonated chicken eggs., Methods: The NDV-HXP-S vaccine was genetically engineered to match the Omicron variants of concern (VOC) BA.1 and BA.5 and tested as an IN two or three dose vaccination regimen in female mice. Furthermore, female mice intramuscularly (IM) vaccinated with mRNA-lipid nanoparticles (LNPs) were IN boosted with NDV-HXP-S. Systemic humoral immunity, memory T cell responses in the lungs and spleens as well as immunoglobulin A (IgA) responses in distinct mucosal tissues were characterised., Findings: NDV-HXP-S Omicron variant vaccines elicited high mucosal IgA and serum IgG titers against respective SARS-CoV-2 VOC in female mice following IN administration and protected against challenge from matched variants. Additionally, antigen-specific memory B cells and local T cell responses in the lungs were induced. Host immunity against the NDV vector did not interfere with boosting. Intramuscular vaccination with mRNA-LNPs was enhanced by IN NDV-HXP-S boosting resulting in improvement of serum neutralization titers and induction of mucosal immunity., Interpretation: We demonstrate that NDV-HXP-S Omicron variant vaccines utilised for primary immunizations or boosting efficiently elicit humoral and cellular immunity. The described induction of systemic and mucosal immunity has the potential to reduce infection and transmission., Funding: This work was partially funded by the NIAIDCenters of Excellence for Influenza Research and Response (CEIRR) and by the NIAID Collaborative Vaccine Innovation Centers and by institutional funding from the Icahn School of Medicine at Mount Sinai. See under Acknowledgements for details., Competing Interests: Declaration of interests The Icahn School of Medicine at Mount Sinai has filed patent applications entitled “RECOMBINANT NEWCASTLE DISEASE VIRUS EXPRESSING SARS-COV-2 SPIKE PROTEIN AND USES THEREOF” which names P.P., A.G.S, F.K. and W.S. as inventors. Mount Sinai is seeking to commercialise this vaccine; therefore, the institution and its faculty inventors could benefit financially. I.G.D. has co-chaired at the ninth ESWI Influenza conference, which has no competing interest with this work. The M.S. laboratory has received unrelated research funding in sponsored research agreements from 7Hills Pharma, ArgenX N.V., Moderna and Phio Pharmaceuticals, which has no competing interest with this work. F.K. has consulted for Merck, Seqirus, Curevac and Pfizer, and is currently consulting for Pfizer, Third Rock Ventures, GSK and Avimex. The FK laboratory is also collaborating with Pfizer on animal models of SARS-CoV-2. The A.G.-S. laboratory has received research support from GSK, Pfizer, Senhwa Biosciences, Kenall Manufacturing, Blade Therapeutics, Avimex, Johnson & Johnson, Dynavax, 7Hills Pharma, Pharmamar, ImmunityBio, Accurius, Nanocomposix, Hexamer, N-fold LLC, Model Medicines, Atea Pharma, Applied Biological Laboratories and Merck. A.G.S. has consulting agreements for the following companies involving cash and/or stock: Castlevax, Amovir, Vivaldi Biosciences, Contrafect, 7Hills Pharma, Avimex, Pagoda, Accurius, Esperovax, Farmak, Applied Biological Laboratories, Pharmamar, CureLab Oncology, CureLab Veterinary, Synairgen, Paratus and Pfizer. A.G.S. has been an invited speaker in meeting events organised by Seqirus, Janssen, Abbott and Astrazeneca. A.G.S. is inventor on patents and patent applications on the use of antivirals and vaccines for the treatment and prevention of virus infections and cancer, owned by the Icahn School of Medicine at Mount Sinai, New York. Specifically, A.G.S., a member of the faculty of the Icahn School of Medicine at Mount Sinai (Mount Sinai) is an inventor of a novel COVID-19 vaccine currently being investigated in clinical trials. Mount Sinai is advancing the development of this vaccine and related technologies for potential commercial use. Mount Sinai has created CastleVax Inc., a Mount Sinai company, and has licensed the applicable IP to it. Mount Sinai will receive financial compensation from CastleVax Inc. pursuant to that license if vaccine development proceeds and as an owner of the company subject to the sale of its ownership interest in the future. Subject to Mount Sinai receiving such financial consideration, A.G.S. will receive a portion of that consideration pursuant to the terms of the Mount Sinai Intellectual Property Policy. All other authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Neutralizing antibodies correlate with protection against SARS-CoV-2. Recent studies, however, show that binding antibody titers, in the absence of robust neutralizing activity, also correlate with protection from disease progression. Non-neutralizing antibodies cannot directly protect from infection but may recruit effector cells thus contribute to the clearance of infected cells. Also, they often bind conserved epitopes across multiple variants. We characterized 42 human mAbs from COVID-19 vaccinated individuals. Most of these antibodies exhibited no neutralizing activity in vitro but several non-neutralizing antibodies protected against lethal challenge with SARS-CoV-2 in different animal models. A subset of those mAbs showed a clear dependence on Fc-mediated effector functions. We determined the structures of three non-neutralizing antibodies with two targeting the RBD, and one that targeting the SD1 region. Our data confirms the real-world observation in humans that non-neutralizing antibodies to SARS-CoV-2 can be protective., Competing Interests: Conflict of interest statement The Icahn School of Medicine at Mount Sinai has filed patent applications relating to SARS-CoV-2 serological assays, NDV-based SARS-CoV-2 vaccines influenza virus vaccines and influenza virus therapeutics which list Florian Krammer as co-inventor. Dr. Simon is also listed on the SARS-CoV-2 serological assays patent. Mount Sinai has spun out a company, Kantaro, to market serological tests for SARS-CoV-2 and another company, Castlevax, to develop SARS-CoV-2 vaccines. Florian Krammer is co-founder and scientific advisory board member of Castlevax. Florian Krammer has consulted for Merck, Curevac, Seqirus and Pfizer and is currently consulting for 3rd Rock Ventures, GSK, Gritstone and Avimex. The Krammer laboratory is also collaborating with Dynavax on influenza vaccine development. The Ellebedy laboratory has received funding under sponsored research agreements from Moderna, Emergent BioSolutions, and AbbVie. A.H.E. has received consulting and speaking fees from InBios International, Inc, Fimbrion Therapeutics, RGAX, Mubadala Investment Company, AstraZeneca, Moderna, Pfizer, GSK, Danaher, Third Rock Ventures, Goldman Sachs, and Morgan Stanley; is the founder of ImmuneBio Consulting and a recipient of royalties from licensing agreements with Abbvie and Leyden Laboratories B.V.

Seasonal influenza virus vaccines are effective when they are well matched to circulating strains. Because of antigenic drift/change in the immunodominant hemagglutinin (HA) head domain, annual vaccine reformulations are necessary to maintain a match with circulating strains. In addition, seasonal vaccines provide little to no protection against newly emerging pandemic strains. Sequential vaccination with chimeric HA (cHA) constructs has been proven to direct the immune response toward the immunosubdominant but more conserved HA stalk domain. In this study, we show that immunization with group 2 cHA split vaccines in combination with the CpG 1018 adjuvant elicits broadly cross-reactive antibodies against all group 2 HAs, as well as systemic and local antigen-specific T cell responses. Antibodies elicited after sequential vaccination are directed to conserved regions of the HA such as the stalk and the trimer interface and also to the N2 neuraminidase (NA). Immunized mice were fully protected from challenge with a broad panel of influenza A viruses.

There is still a need for safe, efficient, and low-cost coronavirus disease 2019 (COVID-19) vaccines that can stop transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here we evaluated a vaccine candidate based on a live recombinant Newcastle disease virus (NDV) that expresses a stable version of the spike protein in infected cells as well as on the surface of the viral particle (AVX/COVID-12-HEXAPRO, also known as NDV-HXP-S). This vaccine candidate can be grown in embryonated eggs at a low cost, similar to influenza virus vaccines, and it can also be administered intranasally, potentially to induce mucosal immunity. We evaluated this vaccine candidate in prime-boost regimens via intramuscular, intranasal, or intranasal followed by intramuscular routes in an open-label non-randomized non-placebo-controlled phase I clinical trial in Mexico in 91 volunteers. The primary objective of the trial was to assess vaccine safety, and the secondary objective was to determine the immunogenicity of the different vaccine regimens. In the interim analysis reported here, the vaccine was found to be safe, and the higher doses tested were found to be immunogenic when given intramuscularly or intranasally followed by intramuscular administration, providing the basis for further clinical development of the vaccine candidate. The study is registered under ClinicalTrials.gov identifier NCT04871737., (© 2023. The Author(s).)

New influenza strains are constantly emerging, causing seasonal epidemics and raising concerns to the risk of a new global pandemic. Since vaccination is an effective method to prevent the spread of the disease and reduce its severity, the development of robust bioprocesses for producing pandemic influenza vaccines is exceptionally important. Herein, a membrane chromatography-based downstream processing platform with a demonstrated industrial application potential was established. Cell culture-derived influenza virus H1N1/A/PR/8/34 was harvested from benchtop bioreactor cultures. For the clarification of the cell culture broth, a depth filtration was selected as an alternative to centrifugation. After inactivation, an anion exchange chromatography membrane was used for viral capture and further processing. Additionally, two pandemic influenza virus strains, the H7N9 subtype of the A/Anhui/1/2013 and H3N2/A/Hong Kong/8/64, were successfully processed through similar downstream process steps establishing optimized process parameters. Overall, 41.3-62.5% viral recovery was achieved, with the removal of 86.3-96.5% host cell DNA and 95.5-99.7% of proteins. The proposed membrane chromatography purification is a scalable and generic method for the processing of different influenza strains and is a promising alternative to the current industrial purification of influenza vaccines based on ultracentrifugation methodologies.

Equitable access to vaccines is necessary to limit the global impact of the coronavirus disease 2019 (COVID-19) pandemic and the emergence of new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. In previous studies, we described the development of a low-cost vaccine based on a Newcastle Disease virus (NDV) expressing the prefusion-stabilized spike protein from SARS-CoV-2, named NDV-HXP-S. Here, we present the development of next-generation NDV-HXP-S variant vaccines, which express the stabilized spike protein of the Beta, Gamma, and Delta variants of concerns (VOC). Combinations of variant vaccines in bivalent, trivalent, and tetravalent formulations were tested for immunogenicity and protection in mice. We show that the trivalent preparation, composed of the ancestral Wuhan, Beta, and Delta vaccines, substantially increases the levels of protection and of cross-neutralizing antibodies against mismatched, phylogenetically distant variants, including the currently circulating Omicron variant. IMPORTANCE This manuscript describes an extended work on the Newcastle disease virus (NDV)-based vaccine focusing on multivalent formulations of NDV vectors expressing different prefusion-stabilized versions of the spike proteins of different SARS-CoV-2 variants of concern (VOC). We demonstrate here that this low-cost NDV platform can be easily adapted to construct vaccines against SARS-CoV-2 variants. Importantly, we show that the trivalent preparation, composed of the ancestral Wuhan, Beta, and Delta vaccines, substantially increases the levels of protection and of cross-neutralizing antibodies against mismatched, phylogenetically distant variants, including the currently circulating Omicron variant. We believe that these findings will help to guide efforts for pandemic preparedness against new variants in the future.

DNA delivery with polyethylenimine (PEI) has been widely used in the last three decades for the transfection of mammalian cells. Advances in novel characterization techniques at the nanometric scale offer new opportunities to revisit the physicochemical properties of DNA/PEI polyplexes that lead to efficient transfection. In this work, these properties are tuned by studying the synergies between simple parameters such as NaCl concentration, pH and incubation time in the DNA/PEI polyplex preparation protocol by means of Design of Experiments (DoE). By doing so, a model is obtained where an optimal NaCl concentration of 125 mM and an incubation time of 11 min provided the highest transfection yields. Correlation analyses between the physicochemical properties of DNA/PEI polyplexes and the predicted model responses revealed the existence of an optimal degree of aggregation in the pre-complexing solution to attain the highest transfection efficiencies. The presence of these micrometric DNA/PEI polyplex aggregates was confirmed by several nanoparticle characterization techniques including cryo-TEM, DLS and flow virometry. The findings provide a better understanding of the role of DNA/PEI aggregates in transient gene expression approaches, in particular considering that similar complexation protocols and saline solutions are widely used for the transfection of mammalian cell cultures., (Copyright © 2022. Published by Elsevier B.V.)

Equitable access to vaccines is necessary to limit the global impact of the coronavirus disease 2019 (COVID-19) pandemic and the emergence of new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. In previous studies, we described the development of a low-cost vaccine based on a Newcastle Disease virus (NDV) expressing the prefusion stabilized spike protein from SARS-CoV-2, named NDV-HXP-S. Here, we present the development of next-generation NDV-HXP-S variant vaccines, which express the stabilized spike protein of the Beta, Gamma and Delta variants of concerns (VOC). Combinations of variant vaccines in bivalent, trivalent and tetravalent formulations were tested for immunogenicity and protection in mice. We show that the trivalent preparation, composed of the ancestral Wuhan, Beta and Delta vaccines, substantially increases the levels of protection and of cross-neutralizing antibodies against mismatched, phylogenetically distant variants, including the currently circulating Omicron variant.

Gag virus-like particles (VLPs) are promising vaccine candidates against infectious diseases. VLPs are generally produced using the insect cell/baculovirus expression vector system (BEVS), or in mammalian cells by plasmid DNA transient gene expression (TGE). However, VLPs produced with the insect cell/BEVS are difficult to purify and might not display the appropriate post-translational modifications, whereas plasmid DNA TGE approaches are expensive and have a limited scale-up capability. In this study, the production of Gag VLPs with the BacMam expression system in a suspension culture of HEK293 cells is addressed. The optimal conditions of multiplicity of infection (MOI), viable cell density (VCD) at infection, and butyric acid (BA) concentration that maximize cell transduction and VLP production are determined. In these conditions, a maximum cell transduction efficiency of 91.5 ± 1.1%, and a VLP titer of 2.8 ± 0.1 × 10 9 VLPs/mL are achieved. Successful VLP generation in transduced HEK293 cells is validated using super-resolution fluorescence microscopy, with VLPs produced resembling immature HIV-1 virions and with an average size comprised in the 100-200 nm range. Additionally, evidence that BacMam transduction occurs via different pathways including dynamin-mediated endocytosis and macropinocytosis is provided. This work puts the basis for future studies aiming at scaling up the BacMam baculovirus system as an alternative strategy for VLP production.

Background: End-stage renal failure of unknown origin (ESRD-UO) is a public health problem in Mexico and many regions of the world. The prevalence of ESRD-UO in Aguascalientes, Mexico, is one of the highest worldwide, particularly in adults between 20 and 40 years of age. Our aim was to screen adolescents for chronic kidney disease (CKD) to identify risk factors and histologically characterize adolescents with persistent albuminuria., Methods: This was a cross-sectional, observational and comparative study of adolescents in whom serum creatinine and the albumin:creatinine ratio (ACR) were determined when screening for CKD. A clinical evaluation and risk factor survey were conducted. Patients with an abnormal ACR (≥30 mg/g) or a low glomerular filtration rate (GFR) (≤75 mL/min/1.73 m 2 ) were re-evaluated and a renal ultrasound (US) was obtained. A kidney biopsy was performed in patients with persistent albuminuria., Results: A total of 513 students were included; 19 had persistent albuminuria and 494 were controls. The prevalence of persistent albuminuria was 3.7% [95% confidence interval (CI) 2.1-5.3]. Only one patient had a decreased GFR. None of the patients with persistent albuminuria had anatomical abnormalities of the urinary tract by renal US. Patients with persistent albuminuria had a decreased total renal volume compared with the control group (150 versus 195 mL/m 2 ; P < 0.01). Eighteen kidney biopsies were performed; 72% had glomerulomegaly and only one patient had mild fibrosis. Podocyte abnormalities were evident on electron microscopy, including partial fusion (100%), microvillous degeneration (80%) and increased organelles (60%). Risk factors for persistent albuminuria were: homestead proximity to maize crops, the use of pesticides at the father's workplace, a family history of CKD and blood pressure abnormalities. The body mass index and breastfeeding were protective factors., Conclusions: The prevalence of persistent albuminuria in adolescents in Aguascalientes is high and histologic compromise is characterized by podocyte injury in the absence of fibrosis. The renal volume of persistent albuminuria patients was decreased, suggesting oligonephronia. Exposure to environmental toxins such as pesticides, even prenatally, may be responsible for this pathological entity. Screening programs in adolescents by determining ACR are necessary in this setting., (© The Author(s) 2022. Published by Oxford University Press on behalf of the ERA.)

Despite global vaccination efforts, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve and spread globally. Relatively high vaccination rates have been achieved in most regions of the United States and several countries worldwide. However, access to vaccines in low- and mid-income countries (LMICs) is still suboptimal. Second generation vaccines that are universally affordable and induce systemic and mucosal immunity are needed. Here we performed an extended safety and immunogenicity analysis of a second-generation SARS-CoV-2 vaccine consisting of a live Newcastle disease virus vector expressing a pre-fusion stabilized version of the spike protein (NDV-HXP-S) administered intranasally (IN), intramuscularly (IM), or IN followed by IM in Sprague Dawley rats. Local reactogenicity, systemic toxicity, and post-mortem histopathology were assessed after the vaccine administration, with no indication of severe local or systemic reactions. Immunogenicity studies showed that the three vaccination regimens tested elicited high antibody titers against the wild type SARS-CoV-2 spike protein and the NDV vector. Moreover, high antibody titers were induced against the spike of B.1.1.7 (alpha), B.1.351 (beta) and B.1.617.2 (delta) variants of concern (VOCs). Importantly, robust levels of serum antibodies with neutralizing activity against the authentic SARS-CoV-2 USA-WA1/2020 isolate were detected after the boost. Overall, our study expands the pre-clinical safety and immunogenicity characterization of NDV-HXP-S and reinforces previous findings in other animal models about its high immunogenicity. Clinical testing of this vaccination approach is ongoing in different countries including Thailand, Vietnam, Brazil and Mexico., Competing Interests: The Icahn School of Medicine at Mount Sinai has filed patent applications relating to NDV-based SARS-CoV-2 vaccines which list WS, FK, AG-S and PP as co-inventors. FK is also listed as inventor on patent applications for SARS-CoV-2 serological assays filed by the Icahn School of Medicine at Mount Sinai. Mount Sinai has spun out a company, Kantaro, to market serological tests for SARS-CoV-2. FK has consulted for Merck and Pfizer (before 2020) as well as Goldman Sachs, and is currently consulting for Pfizer, Seqirus and Avimex. The Krammer laboratory is also collaborating with Pfizer on animal models of SARS-CoV-2. The Adolfo García-Sastre laboratory has received research support from Pfizer, Senhwa Biosciences, Kenall Manufacturing, Avimex, Johnson & Johnson, Dynavax, 7Hills Pharma, Pharmamar, ImmunityBio, Accurius, Nanocomposix, Hexamer, N-fold LLC, Model Medicines and Merck, outside of the reported work. AG-S has consulting agreements for the following companies involving cash and/or stock: Vivaldi Biosciences, Contrafect, 7Hills Pharma, Avimex, Vaxalto, Pagoda, Accurius, Esperovax, Farmak, Applied Biological Laboratories, Pharmamar, and Pfizer. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Tcheou, Raskin, Singh, Kawabata, Bielak, Sun, González-Domínguez, Sather, García-Sastre, Palese, Krammer and Carreño.)

Rapid development of COVID-19 vaccines has helped mitigating SARS-CoV-2 spread, but more equitable allocation of vaccines is necessary to limit the global impact of the COVID-19 pandemic and the emergence of additional variants of concern. We have developed a COVID-19 vaccine candidate based on Newcastle disease virus (NDV) that can be manufactured at high yields in embryonated eggs. Here, we show that the NDV vector expressing an optimized spike antigen (NDV-HXP-S) is a versatile vaccine inducing protective antibody responses. NDV-HXP-S can be administered intramuscularly as inactivated vaccine or intranasally as live vaccine. We show that NDV-HXP-S GMP-produced in Vietnam, Thailand and Brazil is effective in the hamster model. Furthermore, we show that intramuscular vaccination with NDV-HXP-S reduces replication of tested variants of concerns in mice. The immunity conferred by NDV-HXP-S effectively counteracts SARS-CoV-2 infection in mice and hamsters., (© 2021. The Author(s).)

Gag-based virus-like particles (VLPs) have high potential as scaffolds for the development of chimeric vaccines and delivery strategies. The production of purified preparations that can be preserved independently from cold chains is highly desirable to facilitate distribution and access worldwide. In this work, a nimble purification has been developed, facilitating the production of Gag VLPs. Suspension-adapted HEK 293 cells cultured in chemically defined cell culture media were used to produce the VLPs. A four-step downstream process (DSP) consisting of membrane filtration, ion-exchange chromatography, polishing, and lyophilization was developed. The purification of VLPs from other contaminants such as host cell proteins (HCP), double-stranded DNA, or extracellular vesicles (EVs) was confirmed after their DSP. A concentration of 2.2 ± 0.8 × 10 9 VLPs/mL in the lyophilized samples was obtained after its storage at room temperature for two months. Morphology and structural integrity of purified VLPs was assessed by cryo-TEM and NTA. Likewise, the purification methodologies proposed here could be easily scaled up and applied to purify similar enveloped viruses and vesicles.

Influenza viruses undergo antigenic changes in the immuno-dominant hemagglutinin (HA) head domain, necessitating annual re-formulation of and re-vaccination with seasonal influenza virus vaccines for continuing protection. We previously synthesized mosaic HA (mHA) proteins of influenza B viruses which redirect the immune response towards the immuno-subdominant conserved epitopes of the HA via sequential immunization. As ~90% of current influenza virus vaccines are manufactured using the inactivated virus platform, we generated and sequentially vaccinated mice with inactivated influenza B viruses displaying either the homologous (same B HA backbones) or the heterologous (different B HA backbones) mosaic HAs. Both approaches induced long-lasting and cross-protective antibody responses showing strong antibody-dependent cellular cytotoxicity (ADCC) activity. We believe the B virus mHA vaccine candidates represent a major step towards a universal influenza B virus vaccine., Competing Interests: The Icahn School of Medicine at Mount Sinai has filed patent applications entitled “INFLUENZA VIRUS HEMAGGLUTININ PROTEINS AND USES THEREOF” which names PP, FK, AG-S as inventors. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling Editor declared a past co-authorship with one of the authors FK., (Copyright © 2021 Liu, Strohmeier, González-Domínguez, Tan, Simon, Krammer, García-Sastre, Palese and Sun.)

Virus-like particles (VLPs) have emerged as a powerful scaffold for antigen presentation and delivery strategies. Compared to single protein-based therapeutics, quality assessment requires a higher degree of refinement due to the structure of VLPs and their similar properties to extracellular vesicles (EVs). Advances in the field of nanotechnology with single particle and high-resolution analysis techniques provide appealing approaches to VLP characterization. In this study, six different biophysical methods have been assessed for the characterization of HIV-1-based VLPs produced in mammalian and insect cell platforms. Sample preparation and equipment set-up were optimized for the six strategies evaluated. Electron Microscopy (EM) disclosed the presence of several types of EVs within VLP preparations and cryogenic transmission electron microscopy (cryo-TEM) resulted in the best technique to resolve the VLP ultrastructure. The use of super-resolution fluorescence microscopy (SRFM), nanoparticle tracking analysis (NTA) and flow virometry enabled the high throughput quantification of VLPs. Interestingly, differences in the determination of nanoparticle concentration were observed between techniques. Moreover, NTA and flow virometry allowed the quantification of both EVs and VLPs within the same experiment while analyzing particle size distribution (PSD), simultaneously. These results provide new insights into the use of different analytical tools to monitor the production of nanoparticle-based biologicals and their associated contaminants., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Virus-like particles (VLPs) have become a promising platform for vaccine production. VLPs are formed by structural viral proteins that inherently self-assemble when expressed in a host cell. They represent a highly immunogenic and safe vaccine platform, due to the absence of the viral genome and its high protein density. One of the most important parameters in vaccine production is the quality of the product. A related bottleneck in VLP-based products is the presence of cellular vesicles as a major contaminant in the preparations, which will require the set up of techniques allowing for specific discrimination of VLPs from host vesicular bodies. In this work novel, to our knowledge, multifrequency (MF) atomic force microscopy (AFM) has permitted full structural nanophysical characterization by its access to the virus capsid of the HIV-based VLPs. The assessment of these particles by advanced amplitude modulation-frequency modulation (AM-FM) viscoelastic mapping mode has enhanced the imaging resolution of their nanomechanical properties, opening a new window for the study of the biophysical attributes of VLPs. Finally, the identification and differentiation of HIV-based VLPs from cellular vesicles has been performed under ambient conditions, providing, to our knowledge, novel methodology for the monitoring and quality control of VLPs., (Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The manufacturing of biopharmaceuticals in mammalian cells typically relies on the use of stable producer cell lines. However, in recent years, transient gene expression has emerged as a suitable technology for rapid production of biopharmaceuticals. Transient gene expression is particularly well suited for early developmental phases, where several potential therapeutic targets need to be produced and tested in vivo. As a relatively new bioprocessing modality, a number of opportunities exist for improving cell culture productivity upon transient transfection. For instance, several compounds have shown positive effects on transient gene expression. These transfection enhancers either facilitate entry of PEI/DNA transfection complexes into the cell or nucleus or increase levels of gene expression. In this work, the potential of combining transfection enhancers to increase Gag-based virus-like particle production levels upon transfection of suspension-growing HEK 293 cells is evaluated. Using Plackett-Burman design of experiments, it is first tested the effect of eight transfection enhancers: trichostatin A, valproic acid, sodium butyrate, dimethyl sulfoxide (DMSO), lithium acetate, caffeine, hydroxyurea, and nocodazole. An optimal combination of compounds exhibiting the highest effect on gene expression levels was subsequently identified using a surface response experimental design. The optimal consisted on the addition of 20 mM lithium acetate, 3.36 mM valproic acid, and 5.04 mM caffeine which increased VLP production levels 3.8-fold, while maintaining cell culture viability at 94%.