Lyophilized Filovirus Glycoprotein Vaccines: Peroxides in a Vaccine Formulation with Polysorbate 80-Containing Adjuvant are Associated with Reduced Neutralizing Antibody Titers in Both Mice and Non-Human Primates.

Zaire ebolavirus, Sudan ebolavirus, and Marburg marburgvirus are the filoviruses most commonly associated with human disease. Previously, we administered a three-dose regimen of trivalent vaccines comprising glycoprotein antigens from each virus in mice and non-human primates (NHPs). The vaccines, which contained a polysorbate 80-stabilized squalane-in-water emulsion adjuvant and were lyophilized from a solution containing trehalose, produced high antibody levels against all three filovirus antigens. Subsequently, single-vial formulations containing a higher concentration of adjuvant were generated for testing in NHPs, but these vaccines elicited lower neutralizing antibody titers in NHPs than previously tested formulations. In order to explain these results, in the current work we measured the size of adjuvant emulsion droplets and the peroxide levels present in the vaccines after lyophilization and reconstitution and tested the effects of these variables on the immune response in mice. Increases in squalane droplet sizes were observed when the ratio of adjuvant to trehalose was increased beyond a critical value, but antibody and neutralizing antibody titers in mice were independent of the droplet size. Higher levels of peroxides in the vaccines correlated with higher concentrations of adjuvant in the formulations, and higher peroxide levels were associated with increased levels of oxidative damage to glycoprotein antigens. Neutralizing titers in mice were inversely correlated with peroxide levels in the vaccines, but peroxide levels could be reduced by adding free methionine, resulting in retention of high neutralizing antibody titers. Overall, the results suggest that oxidation of glycoprotein antigens by peroxides in the polysorbate 80-stabilized squalane-in-water emulsion adjuvant, but not lyophilization-induced increases in adjuvant emulsion droplet size may have been responsible for the decreased neutralizing titers seen in formulations containing higher amounts of adjuvant.

Recombinant Protein Filovirus Vaccines Protect Cynomolgus Macaques From Ebola, Sudan, and Marburg Viruses.

Ebola (EBOV), Marburg (MARV) and Sudan (SUDV) viruses are the three filoviruses which have caused the most fatalities in humans. Transmission from animals into the human population typically causes outbreaks of limited scale in endemic regions. In contrast, the 2013-16 outbreak in several West African countries claimed more than 11,000 lives revealing the true epidemic potential of filoviruses. This is further emphasized by the difficulty seen with controlling the 2018-2020 outbreak of EBOV in the Democratic Republic of Congo (DRC), despite the availability of two emergency use-approved vaccines and several experimental therapeutics targeting EBOV. Moreover, there are currently no vaccine options to protect against the other epidemic filoviruses. Protection of a monovalent EBOV vaccine against other filoviruses has never been demonstrated in primate challenge studies substantiating a significant void in capability should a MARV or SUDV outbreak of similar magnitude occur. Herein we show progress on developing vaccines based on recombinant filovirus glycoproteins (GP) from EBOV, MARV and SUDV produced using the Drosophila S2 platform. The highly purified recombinant subunit vaccines formulated with CoVaccine HT™ adjuvant have not caused any safety concerns (no adverse reactions or clinical chemistry abnormalities) in preclinical testing. Candidate formulations elicit potent immune responses in mice, guinea pigs and non-human primates (NHPs) and consistently produce high antigen-specific IgG titers. Three doses of an EBOV candidate vaccine elicit full protection against lethal EBOV infection in the cynomolgus challenge model while one of four animals infected after only two doses showed delayed onset of Ebola Virus Disease (EVD) and eventually succumbed to infection while the other three animals survived challenge. The monovalent MARV or SUDV vaccine candidates completely protected cynomolgus macaques from infection with lethal doses of MARV or SUDV. It was further demonstrated that combinations of MARV or SUDV with the EBOV vaccine can be formulated yielding bivalent vaccines retaining full efficacy. The recombinant subunit vaccine platform should therefore allow the development of a safe and efficacious multivalent vaccine candidate for protection against Ebola, Marburg and Sudan Virus Disease.

Single-vial filovirus glycoprotein vaccines: Biophysical characteristics and immunogenicity after co-lyophilization with adjuvant.

Zaire ebolavirus (EBOV), Sudan ebolavirus (SUDV), and Marburg marburgvirus (MARV) are the most prevalent and pathogenic species of filovirus. Previously, we showed that glycoprotein antigens from each virus could be lyophilized to create thermostable monovalent subunit vaccines. However, cross-protection is not expected from the monovalent vaccines and therefore developing a trivalent filovirus vaccine would be desirable. Subunit protein vaccines often require the addition of an adjuvant to sufficiently boost the immunogenicity. Typically, liquid suspensions or emulsions of adjuvants and lyophilized antigens are stored in separate vials to avoid destabilizing interactions and are only mixed immediately before administration. Herein, we describe the development and characterization of monovalent and trivalent filovirus vaccines that are co-lyophilized with a squalane-in-water emulsion adjuvant. We found that the single-vial presentation retained adjuvant particle diameter and zeta potential after lyophilization and reconstitution. Furthermore, the trivalent vaccines elicited high antibody levels against all three antigens in mice and non-human primates. These results advance the prospect of developing a single-vial trivalent filovirus vaccine, which would enable easier distribution and administration of the vaccine to resource-poor areas.

Browning, Starch Gelatinization, Water Sorption, Glass Transition, and Caking Properties of Freeze-dried Maca ( Lepidium meyenii Walpers) Powders.

The browning, gelatinization of starch, water sorption, glass transition, and caking properties of freeze-dried maca ( Lepidium meyenii Walpers) powders were investigated and compared with a commercial maca powder. The freeze-dried maca powders had lower optical density (browning) and higher enthalpy change for starch gelatinization than the commercial maca. This resulted from a difference in thermal history. The equilibrium water contents of the freeze-dried maca powders were higher than those of commercial maca at each water activity ( a w ) because of differences in amorphous part. The glass transition temperature ( T g ) was evaluated by differential scanning calorimetry. There was a negligible difference in the anhydrous T g (79.5-80.2 ºC) among the samples. The T g -depression of freeze-dried maca powders induced by water sorption was more gradual than that of the commercial maca due to a difference in water insoluble material content. From the results, critical water activity ( a wc ) was determined as the a w at which T g becomes 25 ºC. There was negligible caking below a w = 0.328. At higher a w , the degree of caking remarkably increased with a large variation depending on the samples. The degree of caking could be described uniformly as a function of a w / a wc . From these results, we propose an empirical approach to predict the caking of maca powders.

Immunotherapy for the treatment of canine transmissible venereal tumor based in dendritic cells pulsed with tumoral exosomes.

Context: Exosomes secreted by tumor cells are a good source of cellular components that stimulate the immune response, such as alarmins (mRNA, tetraspanins (CD9, CD63, CD81), heat-shock proteins, major histocompatibility complex class I molecules) and tumor-associated antigens. These properties permit to pulsed dendritic cells in the immunotherapy for many cancers types. The aim of this study was to demonstrate the use of exosomes derived from canine transmissible venereal tumor (CTVT) as an antigen to pulsed dendritic cells and its administration in dogs with CTVT as treatment against this disease. Material and methods: From primary culture of CTVT cells the exosomes were isolated and characterized by scanning electron microscopy assay, dot blot and protein quantification. The monocytes of each patient were differentiated to dendritic cells (DC) and pulsed with CTVT exosomes (CTVTE). Phagocytosis, tumor size, populations of lymphocytes and IFN-c levels were evaluated. Results: The CTVTE showed a size around 90 nm. CD81, CD63, CD9 and Hsp70 were expressed. Monocytes showed an expression of 85.71% for CD14+, 12.3% for CD80+, 0.1% for CD83+ and 0.8% for DLA-II. In DC 5.1% for CD14+, 86.7% for CD80+, 90.1% for CD83+ and 92.6% for DLA-II and a phagocytosis of 63% was obtained by FITC Dextran test. No side effects were observed in the experimental groups with our therapy. Tumor regression was of 100% at the seventh week, as well as an increase in the level of IFN-γ (142 pg/ml), and CD4+ (28%) and CD8+ (34%) cell percentage. Discusion and conclusion: These results have shown that DC pulsed with tumor exosomes induce regression of the TVT in dogs.

Immunogenic potential of three transmissible venereal tumor cell lysates to prime canine-dendritic cells for cancer immunotherapy.

Whole tumor cell lysates consist of a mixture of tumor antigens and danger associated molecular patterns (DAMPs) that can be used for dendritic cell maturation and consequently for the activation of a polyclonal T cell-specific tumor response. We evaluated the in vitro efficacy of three different preparations of canine transmissible venereal tumor (TVT) cell lysates: hypochlorous acid-whole tumor cell lysates (HOCl-L), heat shock-whole tumor cell lysates (HS-L), and freeze-thaw cycles-whole tumor cell lysates (FT-L) for the maturation of canine-derived dendritic cells. Our results showed calreticulin, HSP70, and HSP90 release in the three tumor lysates preparations (HOCl-L, HS-L, and FT-L); however, HMGB1 was detected only in HOCl-L and FT-L. Additionally, the uptake by HOCl-L pulsed dendritic cell (DC) increased compared to HS-L and FT-L pulsed DC; and dendritic cell maturation was confirmed by the appropriate cell surface markers (CD11c, CD80, CD83, and MHCII). Furthermore, dendritic cells pulsed with HOCl-L, HS-L or FT-L were cultured with canine lymphocytes. There was an increase of Th1-type cytokines (IL-12, TNF-α, and IFN-γ), in all the tumor cell lysates co-cultures, this correlates with T lymphocyte activation and cytotoxic response. Our data confirm that TVT cell lysates can induce functional canine-DC and that HOCl-L is the most effective one. This preparation of TVT cell lysates with HOCl is an attractive approach that allows the recognition of neoantigens as potential tumor targets and DC priming and therefore could be used for cancer immunotherapy against TVT.

Zika virus protection by a single low-dose nucleoside-modified mRNA vaccination.

Zika virus (ZIKV) has recently emerged as a pandemic associated with severe neuropathology in newborns and adults. There are no ZIKV-specific treatments or preventatives. Therefore, the development of a safe and effective vaccine is a high priority. Messenger RNA (mRNA) has emerged as a versatile and highly effective platform to deliver vaccine antigens and therapeutic proteins. Here we demonstrate that a single low-dose intradermal immunization with lipid-nanoparticle-encapsulated nucleoside-modified mRNA (mRNA-LNP) encoding the pre-membrane and envelope glycoproteins of a strain from the ZIKV outbreak in 2013 elicited potent and durable neutralizing antibody responses in mice and non-human primates. Immunization with 30 µg of nucleoside-modified ZIKV mRNA-LNP protected mice against ZIKV challenges at 2 weeks or 5 months after vaccination, and a single dose of 50 µg was sufficient to protect non-human primates against a challenge at 5 weeks after vaccination. These data demonstrate that nucleoside-modified mRNA-LNP elicits rapid and durable protective immunity and therefore represents a new and promising vaccine candidate for the global fight against ZIKV.