Non-infectious Vaccine Research Articles

Virus-like particles of louping ill virus elicit potent neutralizing antibodies targeting multimers of viral envelope protein

Louping ill virus (LIV) is a tick-borne flavivirus that predominantly causes disease in livestock, especially sheep in the British Isles. A preventive vaccine, previously approved for veterinary use but now discontinued, was based on an inactivated whole virion that likely provided protection by induction of neutralizing antibodies recognizing the viral envelope (E) protein. A major disadvantage of the inactivated vaccine was the need for high containment facilities for the propagation of infectious virus, as mandated by the hazard group 3 status of the virus. This study aimed to develop high-efficacy non-infectious protein-based vaccine candidates. Specifically, soluble envelope protein (sE), and virus-like particles (VLPs), comprised of the precursor of membrane and envelope proteins, were generated, characterized, and studied for their immunogenicity in mice. Results showed that the VLPs induced more potent virus neutralizing response compared to sE, even though the total anti-envelope IgG content induced by the two antigens was similar. Depletion of anti-monomeric E protein antibodies from mouse immune sera suggested that the neutralizing antibodies elicited by the VLPs targeted epitopes spanning the highly organized structure of multimer of the E protein, whereas the antibody response induced by sE focused on E monomers. Thus, our results indicate that VLPs represent a promising LIV vaccine candidate.

Designing a sustainable reverse supply chain network for COVID-19 vaccine waste under uncertainty

The vast nationwide COVID-19 vaccination programs are implemented in many countries worldwide. Mass vaccination is causing a rapid increase in infectious and non-infectious vaccine wastes, potentially posing a severe threat if there is no well-organized management plan. This paper develops a mixed-integer mathematical programming model to design a COVID-19 vaccine waste reverse supply chain (CVWRSC) for the first time. The presented problem is based on minimizing the system's total cost and carbon emission. The uncertainty in the tendency rate of vaccination is considered, and a robust optimization approach is used to deal with it, where an interactive fuzzy approach converts the model into a single objective problem. Additionally, a Lagrangian relaxation (LR) algorithm is utilized to deal with the computational difficulty of the large-scale CVWRSC network. The model's practicality is investigated by solving a real-life case study. The results show the gain of the developed integrated network, where the presented framework performs better than the disintegrated vaccine and waste supply chain models. According to the results, vaccination operations and transportation of non-infectious wastes are responsible for a large portion of total cost and emission, respectively. Autoclaving technology plays a vital role in treating infectious wastes. Moreover, the sensitivity analyses demonstrate that the vaccination tendency rate significantly impacts both objective functions. The case study results prove the model's robustness under different realization scenarios, where the average objective function of the robust model is less than the deterministic model ones’ in all scenarios. Finally, some insights are given based on the obtained results.

Vaccination Accelerates Liver-Intrinsic Expression of Megakaryocyte-Related Genes in Response to Blood-Stage Malaria.

Erythropoiesis and megakaryo-/thrombopoiesis occur in the bone marrow proceeding from common, even bipotent, progenitor cells. Recently, we have shown that protective vaccination accelerates extramedullary hepatic erythroblastosis in response to blood-stage malaria of Plasmodium chabaudi. Here, we investigated whether protective vaccination also accelerates extramedullary hepatic megakaryo-/thrombopoiesis. Female Balb/c mice were twice vaccinated with a non-infectious vaccine before infecting with 106 P. chabaudi-parasitized erythrocytes. Using gene expression microarrays and quantitative real-time PCR, transcripts of genes known to be expressed in the bone marrow by cells of the megakaryo-/thrombocytic lineage were compared in livers of vaccination-protected and unprotected mice on days 0, 1, 4, 8, and 11 p.i. Livers of vaccination-protected mice responded with expression of megakaryo-/thrombocytic genes faster to P. chabaudi than those of unvaccinated mice, evidenced at early patency on day 4 p.i., when livers exhibited significantly higher levels of malaria-induced transcripts of the genes Selp and Pdgfb (p-values < 0.0001), Gp5 (p-value < 0.001), and Fli1, Runx1, Myb, Mpl, Gp1ba, Gp1bb, Gp6, Gp9, Pf4, and Clec1b (p-values < 0.01). Together with additionally analyzed genes known to be related to megakaryopoiesis, our data suggest that protective vaccination accelerates liver-intrinsic megakaryo-/thrombopoiesis in response to blood-stage malaria that presumably contributes to vaccination-induced survival of otherwise lethal blood-stage malaria.

Chikungunya virus produced by a persistently infected mosquito cell line comprises a shorter genome and is non-infectious to mammalian cells.

Although RNA viruses have high mutation rates, host cells and organisms work as selective environments, maintaining the viability of virus populations by eliminating deleterious genotypes. In serial passages of RNA viruses in a single cell line, most of these selective bottlenecks are absent, with no virus circulation and replication in different tissues or host alternation. In this work, Aedes aegypti Aag-2 cells were accidentally infected with Chikungunya virus (CHIKV) and Mayaro virus (MAYV). After numerous passages to achieve infection persistency, the infectivity of these viruses was evaluated in Ae. albopictus C6/36 cells, African green monkey Vero cells and primary-cultured human fibroblasts. While these CHIKV and MAYV isolates were still infectious to mosquito cells, they lost their ability to infect mammalian cells. After genome sequencing, it was observed that CHIKV accumulated many nonsynonymous mutations and a significant deletion in the coding sequence of the hypervariable domain in the nsP3 gene. Since MAYV showed very low titres, it was not sequenced successfully. Persistently infected Aag-2 cells also accumulated high loads of short and recombinant CHIKV RNAs, which seemed to have been originated from virus-derived DNAs. In conclusion, the genome of this CHIKV isolate could guide mutagenesis strategies for the production of attenuated or non-infectious (to mammals) CHIKV vaccine candidates. Our results also reinforce that a paradox is expected during passages of cells persistently infected by RNA viruses: more loosening for the development of more diverse virus genotypes and more pressure for virus specialization to this constant cellular environment.

Cancer attrition immunotherapy

Cancer is a deadly disease wherein the immune system is inefficient, resulting in malignant tumors that metastasize. An active and robust immune system is the body’s natural defense. Research efforts continue exploring alternative immunotherapies to fight cancer. This communication proposes non-infectious vaccines and hyper-allergenic skin creams to starve malignant tumors through immune metabolic interference.

RCSB Protein Data Bank resources for structure-facilitated design of mRNA vaccines for existing and emerging viral pathogens

Structural biologists provide direct insights into the molecular bases of human health and disease. The open-access Protein Data Bank (PDB) stores and delivers three-dimensional (3D) biostructure data that facilitate discovery and development of therapeutic agents and diagnostic tools. We are in the midst of a revolution in vaccinology. Non-infectious mRNA vaccines have been proven during the coronavirus disease 2019 (COVID-19) pandemic. This new technology underpins nimble discovery and clinical development platforms that use knowledge of 3D viral protein structures for societal benefit. The RCSB PDB supports vaccine designers through expert biocuration and rigorous validation of 3D structures; open-access dissemination of structure information; and search, visualization, and analysis tools for structure-guided design efforts. This resource article examines the structural biology underpinning the success of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) mRNA vaccines and enumerates some of the many protein structures in the PDB archive that could guide design of new countermeasures against existing and emerging viral pathogens.

Mammalian expression of virus-like particles as a proof of principle for next generation polio vaccines

Global vaccination programs using live-attenuated oral and inactivated polio vaccine (OPV and IPV) have almost eradicated poliovirus (PV) but these vaccines or their production pose significant risk in a polio-free world. Recombinant PV virus-like particles (VLPs), lacking the viral genome, represent safe next-generation vaccines, however their production requires optimisation. Here we present an efficient mammalian expression strategy producing good yields of wild-type PV VLPs for all three serotypes and a thermostabilised variant for PV3. Whilst the wild-type VLPs were predominantly in the non-native C-antigenic form, the thermostabilised PV3 VLPs adopted the native D-antigenic conformation eliciting neutralising antibody titres equivalent to the current IPV and were indistinguishable from natural empty particles by cryo-electron microscopy with a similar stabilising lipidic pocket-factor in the VP1 β-barrel. This factor may not be available in alternative expression systems, which may require synthetic pocket-binding factors. VLPs equivalent to these mammalian expressed thermostabilized particles, represent safer non-infectious vaccine candidates for the post-eradication era.

Distinguishing Features of High- and Low-Dose Vaccine against Ocular HSV-1 Infection Correlates with Recognition of Specific HSV-1–Encoded Proteins

The protective efficacy of a live-attenuated HSV type 1 (HSV-1) vaccine, HSV-1 0∆ nuclear location signal (NLS), was evaluated in mice prophylactically in response to ocular HSV-1 challenge. Mice vaccinated with the HSV-1 0∆NLS were found to be more resistant to subsequent ocular virus challenge in terms of viral shedding, spread, the inflammatory response, and ocular pathology in a dose-dependent fashion. Specifically, a strong neutralizing Ab profile associated with low virus titers recovered from the cornea and trigeminal ganglia was observed in vaccinated mice in a dose-dependent fashion with doses ranging from 1 × 103 to 1 × 105 PFU HSV-1 0∆NLS. This correlation also existed in terms of viral latency in the trigeminal ganglia, corneal neovascularization, and leukocyte infiltration and expression of inflammatory cytokines and chemokines in infected tissue with the higher doses (1 × 104-1 × 105 PFU) of the HSV-1 0∆NLS-vaccinated mice, displaying reduced viral latency, ocular pathology, or inflammation in comparison with the lowest dose (1 × 103 PFU) or vehicle vaccine employed. Fifteen HSV-1-encoded proteins were uniquely recognized by antisera from high-dose (1 × 105 PFU)-vaccinated mice in comparison with low-dose (1 × 103 PFU)- or vehicle-vaccinated animals. Passive immunization using high-dose-vaccinated, but not low-dose-vaccinated, mouse sera showed significant efficacy against ocular pathology in HSV-1-challenged animals. In summary, we have identified the minimal protective dose of HSV-1 0∆NLS vaccine in mice to prevent HSV-mediated disease and identified candidate proteins that may be useful in the development of a noninfectious prophylactic vaccine against the insidious HSV-1 pathogen.

Vaccination accelerates hepatic erythroblastosis induced by blood-stage malaria

BackgroundVaccination induces survival of otherwise lethal blood-stage infections of the experimental malaria Plasmodium chabaudi. Blood-stage malaria induces extramedullary erythropoiesis in the liver. This study investigates how vaccination affects the course of malaria-induced expression of erythrocytic genes in the liver.MethodsFemale Balb/c mice were vaccinated at week 3 and week 1 before challenging with 106P. chabaudi-parasitized erythrocytes. The non-infectious vaccine consisted of erythrocyte ghosts isolated from P. chabaudi-infected erythrocytes. Gene expression microarrays and quantitative real-time PCR were used to compare mRNA expression of different erythrocytic genes in the liver of vaccination-protected and non-protected mice during infections on days 0, 1, 4, 8, and 11 p.i.ResultsGlobal transcriptomics analyses reveal vaccination-induced modifications of malaria-induced increases in hepatic gene expression on days 4 and 11 p.i. On these days, vaccination also alters hepatic expression of the erythropoiesis-involved genes Ermap, Kel, Rhd, Rhag, Slc4a1, Gypa, Add2, Ank1, Epb4.1, Epb4.2, Epb4.9, Spta1, Sptb, Tmod1, Ahsp, Acyp1, Gata1, Gfi1b, Tal1, Klf1, Epor, and Cldn13. In vaccination-protected mice, expression of these genes, except Epb4.1, is significantly higher on day 4 p.i. than in un-protected non-vaccinated mice, reaches maximal expression at peak parasitaemia on day 8 p.i., and is slowed down or even decreased towards the end of crisis phase on day 11 p.i.. After day 1 p.i., Epor expression takes about the same course as that of the other erythroid genes. Hepatic expression of Epo, however, is delayed in both vaccinated and non-vaccinated mice for the first 4 days p.i. and is maximal at significantly higher levels in vaccinated mice on day 8 p.i., before declining towards the end of crisis phase on day 11 p.i.ConclusionThe present data indicate that vaccination accelerates malaria-induced erythroblastosis in the liver for 1–2 days. This may contribute to earlier replenishment of peripheral red blood cells by liver-derived reticulocytes, which may favour final survival of otherwise lethal blood-stage malaria, since reticulocytes are not preferred as host cells by P. chabaudi.

Gene expression of the liver of vaccination-protected mice in response to early patent infections of Plasmodium chabaudi blood-stage malaria

BackgroundThe role of the liver for survival of blood-stage malaria is only poorly understood. In experimental blood-stage malaria with Plasmodium chabaudi, protective vaccination induces healing and, thus, survival of otherwise lethal infections. This model is appropriate to study the role of the liver in vaccination-induced survival of blood-stage malaria.MethodsFemale Balb/c mice were vaccinated with a non-infectious vaccine consisting of plasma membranes isolated in the form of erythrocyte ghosts from P. chabaudi-infected erythrocytes at week 3 and week 1 before infection with P. chabaudi blood-stage malaria. Gene expression microarrays and quantitative real-time PCR were used to investigate the response of the liver, in terms of expression of mRNA and long intergenic non-coding (linc)RNA, to vaccination-induced healing infections and lethal P. chabaudi malaria at early patency on day 4 post infection, when parasitized erythrocytes begin to appear in peripheral blood.ResultsIn vaccination-induced healing infections, 23 genes were identified to be induced in the liver by > tenfold at p < 0.01. More than one-third were genes known to be involved in erythropoiesis, such as Kel, Rhag, Ahsp, Ermap, Slc4a1, Cldn13 Gata1, and Gfi1b. Another group of > tenfold expressed genes include genes involved in natural cytotoxicity, such as those encoding killer cell lectin-like receptors Klrb1a, Klrc3, Klrd1, the natural cytotoxicity-triggering receptor 1 Ncr1, as well as the granzyme B encoding Gzmb. Additionally, a series of genes involved in the control of cell cycle and mitosis were identified: Ccnb1, Cdc25c, Ckap2l were expressed > tenfold only in vaccination-protected mice, and the expression of 22 genes was at least 100% higher in vaccination-protected mice than in non-vaccinated mice. Furthermore, distinct lincRNA species were changed by > threefold in livers of vaccination-protected mice, whereas lethal malaria induced different lincRNAs.ConclusionThe present data suggest that protective vaccination accelerates the malaria-induced occurrence of extramedullary erythropoiesis, generation of liver-resident cytotoxic cells, and regeneration from malaria-induced injury in the liver at early patency, which may be critical for final survival of otherwise lethal blood-stage malaria of P. chabaudi.

Recombinant Zika Virus Subunits Are Immunogenic and Efficacious in Mice.

Following the 2015 Zika virus (ZIKV) outbreaks in the South Pacific, Caribbean, and Americas, ZIKV has emerged as a serious threat due to its association with infantile microcephaly and other neurologic disorders. Despite an international effort to develop a safe and effective vaccine to combat congenital Zika syndrome and ZIKV infection, only DNA and mRNA vaccines encoding the precursor membrane (prM) and envelope (E) proteins, an inactivated-ZIKV vaccine, and a measles virus-based ZIKV vaccine are currently in phase I or II (prM/E DNA) clinical trials. A ZIKV vaccine based on a nonreplicating, recombinant subunit platform offers a higher safety profile than other ZIKV vaccine candidates but is still highly immunogenic, inducing high virus-neutralizing antibody titers. Here, we describe the production and purification of Drosophila melanogaster S2 insect cell-derived, soluble ZIKV E protein and evaluate its immunogenicity and efficacy in three different mouse strains. As expected, significant virus-specific antibody titers were observed when using formulations containing clinically relevant adjuvants. Immunized mice challenged with live virus demonstrate inhibition of virus replication. Importantly, plaque reduction neutralization tests (PRNTs) indicate the high-titer production of neutralizing antibodies, a correlate of protection in the defense against ZIKV infection. ZIKV challenge of immunocompetent mice led to full protection against viremia with two doses of adjuvanted vaccine candidates. These data demonstrate a proof of concept and establish recombinant subunit immunogens as an effective vaccine candidate against ZIKV infection. IMPORTANCE The recent outbreaks of Zika virus (ZIKV) infection in French Polynesia, the Caribbean, and the Americas have highlighted the severe neuropathological sequelae that such an infection may cause. The development of a safe, effective ZIKV vaccine is critical for several reasons: (i) the difficulty in diagnosing an active infection due to common nonspecific symptoms, (ii) the lack of a specific antiviral therapy, and (iii) the potentially devastating pathological effects of in utero infection. Moreover, a vaccine with an excellent safety profile, such as a nonreplicating, noninfectious vaccine, would be ideal for high-risk people (e.g., pregnant women, immunocompromised patients, and elderly individuals). This report describes the development of a recombinant subunit protein vaccine candidate derived from stably transformed insect cells expressing the ZIKV envelope protein in vitro, the primary antigen to which effective virus-neutralizing antibodies are engendered by immunized animals for several other flaviviruses; the vaccine candidate elicits effective virus-neutralizing antibodies against ZIKV and provides protection against ZIKV infection in mice.

Toward the development of a one-dose classical swine fever subunit vaccine: antigen titration, immunity onset, and duration of immunity.

Highly contagious classical swine fever (CSF) remains a major trade and health problem in the pig industry, resulting in large economic losses worldwide. In CSF-endemic countries, attenuated CSF virus (CSFV) vaccines have been routinely used to control the disease. However, eradication of CSFV in a geographical area would require permanent reduction to zero presence of the virus. It is therefore of paramount importance to develop a safe, potent, and non-infectious CSF vaccine. We have previously reported on a cost-effective CSF E2 subunit vaccine, KNB-E2, which can protect against CSF symptoms in a single dose containing 75 µg of recombinant CSFV glycoprotein E2. In this study, we report on a series of animal studies undertaken to elucidate further the efficacy of KNB-E2. We found that pigs vaccinated with a single KNB-E2 dose containing 25 µg of recombinant CSFV glycoprotein E2 were protected from clinical symptoms of CSF. In addition, KNB-E2-mediated reduction of CSF symptoms was observed at two weeks post-vaccination and the vaccinated pigs continued to exhibit reduced CSF clinical signs when virus challenged at two months and four months post-vaccination. These results suggest that KNB-E2 effectively reduces CSF clinical signs, indicating the potential of this vaccine for safely minimizing CSF-related losses.

Vesicular Stomatitis Virus Pseudotyped with Ebola Virus Glycoprotein Serves as a Protective, Noninfectious Vaccine against Ebola Virus Challenge in Mice.

The recent Ebola virus (EBOV) epidemic in West Africa demonstrates the potential for a significant public health burden caused by filoviral infections. No vaccine or antiviral is currently FDA approved. To expand the vaccine options potentially available, we assessed protection conferred by an EBOV vaccine composed of vesicular stomatitis virus pseudovirions that lack native G glycoprotein (VSVΔG) and bear EBOV glycoprotein (GP). These pseudovirions mediate a single round of infection. Both single-dose and prime/boost vaccination regimens protected mice against lethal challenge with mouse-adapted Ebola virus (ma-EBOV) in a dose-dependent manner. The prime/boost regimen provided significantly better protection than a single dose. As N-linked glycans are thought to shield conserved regions of the EBOV GP receptor-binding domain (RBD), thereby blocking epitopes within the RBD, we also tested whether VSVΔG bearing EBOV GPs that lack GP1 N-linked glycans provided effective immunity against challenge with ma-EBOV or a more distantly related virus, Sudan virus. Using a prime/boost strategy, high doses of GP/VSVΔG partially or fully denuded of N-linked glycans on GP1 protected mice against ma-EBOV challenge, but these mutants were no more effective than wild-type (WT) GP/VSVΔG and did not provide cross protection against Sudan virus. As reported for other EBOV vaccine platforms, the protection conferred correlated with the quantity of EBOV GP-specific Ig produced but not with the production of neutralizing antibodies. Our results show that EBOV GP/VSVΔG pseudovirions serve as a successful vaccination platform in a rodent model of Ebola virus disease and that GP1 N-glycan loss does not influence immunogenicity or vaccination success.IMPORTANCE The West African Ebola virus epidemic was the largest to date, with more than 28,000 people infected. No FDA-approved vaccines are yet available, but in a trial vaccination strategy in West Africa, recombinant, infectious VSV encoding the Ebola virus glycoprotein effectively prevented virus-associated disease. VSVΔG pseudovirion vaccines may prove as efficacious and have better safety, but they have not been tested to date. Thus, we tested the efficacy of VSVΔG pseudovirions bearing Ebola virus glycoprotein as a vaccine platform. We found that wild-type Ebola virus glycoprotein, in the context of this platform, provides robust protection of EBOV-challenged mice. Further, we found that removal of the heavy glycan shield surrounding conserved regions of the glycoprotein does not enhance vaccine efficacy.

Schizogony and gametogony of oocyst-deficient T-263 strain of Toxoplasma gondii

Oocysts are important stage for the spread of Toxoplasma gondii because they are environmentally resistant. Among all hosts of T. gondii, only felids can excrete oocysts. Cats that have excreted T. gondii oocysts after primary infection develop immunity to re-excretion of oocysts, and this immunity appears to be long-lasting. It would be desirable to have a non-infectious vaccine for the prevention of T. gondii infection in cats and to understand mechanism of immunity to excretion of oocysts. An initial step will be to indentify stage/stages of the parasite for induction of immunity. A chemically-induced mutant of T. gondii, T-263, is immunogenic but lacks the capacity to form oocysts in cats. Cats fed live bradyzoites of T-263 do not excrete oocysts after challenge with oocyst producing strains. However, it is not known at what stage of the parasite development the oocyst formation is halted. Here, four cats were fed live tissue cysts of the T-263 strain and examined for enteroepithelial stages and oocyst production. Two cats were administered methyl prednisolone aceatate (20mg/kg) once intramuscularly and these cats were euthanized 5 and 7days post inoculation. No oocysts but immature and mature schizonts (types D and E), male, and female gamonts were detected in two cats euthanized. The remaining two cats did not excrete oocysts examined 3–14days post inoculation, but both seroconverted and developed antibody titers of 1:400 tested by the modification agglutination test, indicating exposure to the inocula. The results demonstrate that the T-263 strain is defective in oocyst formation and the observations should help future studies in identification of genes/factors responsible for oocyst formation in the intestine of cats.

Differential miRNA Expression in the Liver of Balb/c Mice Protected by Vaccination during Crisis of Plasmodium chabaudi Blood-Stage Malaria.

MicroRNAs are increasingly recognized as epigenetic regulators for outcome of diverse infectious diseases and vaccination efficacy, but little information referring to this exists for malaria. This study investigates possible effects of both protective vaccination and P. chabaudi malaria on the miRNome of the liver as an effector against blood-stage malaria using miRNA microarrays and quantitative PCR. Plasmodium chabaudi blood-stage malaria takes a lethal outcome in female Balb/c mice, but a self-healing course after immunization with a non-infectious blood-stage vaccine. The liver robustly expresses 71 miRNA species at varying levels, among which 65 miRNA species respond to malaria evidenced as steadily increasing or decreasing expressions reaching highest or lowest levels toward the end of the crisis phase on day 11 p.i. in lethal malaria. Protective vaccination does not affect constitutive miRNA expression, but leads to significant (p < 0.05) changes in the expression of 41 miRNA species, however evidenced only during crisis. In vaccination-induced self-healing infections, 18 miRNA-species are up- and 14 miRNA-species are down-regulated by more than 50% during crisis in relation to non-vaccinated mice. Vaccination-induced self-healing and survival of otherwise lethal infections of P. chabaudi activate epigenetic miRNA-regulated remodeling processes in the liver manifesting themselves during crisis. Especially, liver regeneration is accelerated as suggested by upregulation of let-7a-5p, let-7b-5p, let-7c-5p, let-7d-5p, let-7f-5p, let-7g-5p, let-7i-5p, miR-26a, miR-122-5p, miR30a, miR27a, and mir-29a, whereas the up-regulated expression of miR-142-3p by more than 100% is compatible with the view of enhanced hepatic erythropoiesis, possibly at expense of megakaryopoiesis, during crisis of P. chabaudi blood-stage malaria.

A thermostable, chromatographically purified Ebola nano-VLP vaccine

BackgroundFilovirus virus-like particles (VLP) are strong immunogens with the potential for development into a safe, non-infectious vaccine. However, the large size and filamentous structure of this virus has heretofore made production of such a vaccine difficult. Herein, we present new assays and a purification procedure to yield a better characterized and more stable product.MethodsSonication of VLP was used to produce smaller “nano-VLP”, which were purified by membrane chromatography. The sizes and lengths of VLP particles were analyzed using electron microscopy and an assay based on transient occlusion of a nanopore. Using conformationally-sensitive antibodies, we developed an in vitro assay for measuring GP conformational integrity in the context of VLP, and used it to profile thermal stability.ResultsWe developed a new procedure for rapid isolation of Ebola VLP using membrane chromatography that yields a filterable and immunogenic product. Disruption of VLP filaments by sonication followed by filtration produced smaller particles of more uniform size, having a mean diameter close to 230 nm. These reduced-size VLP retained GP conformation and were protective against mouse-adapted Ebola challenge in mice. The “nano-VLP” consists of GP-coated particles in a mixture of morphologies including circular, branched, “6”-shaped, and filamentous ones up to ~1,500 nm in length. Lyophilization conferred a high level of thermostability on the nano-VLP. Unlike Ebola VLP in solution, which underwent denaturation of GP upon moderate heating, the lyophilized nano-VLP can withstand at least 1 h at 75°C, while retaining conformational integrity of GP and the ability to confer protective immunity in a mouse model.ConclusionsWe showed that Ebola virus-like particles can be reduced in size to a more amenable range for manipulation, and that these smaller particles retained their temperature stability, the structure of the GP antigen, and the ability to stimulate a protective immune response in mice. We developed a new purification scheme for “nano-VLP” that is more easily scaled up and filterable. The product could also be made thermostable by lyophilization, which is highly significant for vaccines used in tropical countries without a reliable “cold-chain” of refrigeration.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-015-0593-y) contains supplementary material, which is available to authorized users.

Nonviral oncogenic antigens and the inflammatory signals driving early cancer development as targets for cancer immunoprevention.

Cancer immunoprevention is an emerging field that holds much promise. Within the past 20 years, prophylactic vaccines have been implemented on the population level for the immunoprevention of carcinomas induced by viruses, specifically hepatitis B virus (HBV) and human papillomavirus (HPV) infection. Armed with the success of prophylactic vaccines that prevent viral-induced tumors, the field must overcome its next hurdle: to develop robust prophylactic vaccines that prevent the remaining >80% of human cancers not induced by viral infection. In this review, we discuss some of the most promising non-virus-associated prophylactic vaccines that target endogenous neoantigens, including the earliest oncogene products, altered mucin 1 (MUC1) and α-enolase (ENO1), all of which produce new targets in the earliest stages of nonviral-induced tumorigenesis. We also highlight a novel attenuated Listeria monocytogenes-based vaccine expressing mutant oncogene Kras(G12D) (LM-Kras) effective in a pancreatic cancer model. A novel chimeric human/rat HER-2 plasmid vaccine (HuRT-DNA vaccine) effective in a breast cancer model is also discussed. In addition to prophylactic vaccine developments, this review highlights the potential use of classic drugs, such as aspirin and metformin, as chemopreventive agents that can potentially be used as adjuvants to enhance the anticancer immunogenicity and efficacy of noninfectious prophylactic vaccines by modulating the inflammatory pathways within the early tumor microenvironment (TME) that propels tumorigenesis. Finally, timing of prophylactic vaccine administration is critical to its immunopreventive efficacy, providing a necessary role of current and emerging biomarkers for cancer screening and early cancer detection.

Immunoreactivity and trypsin sensitivity of recombinant virus-like particles of foot-and-mouth disease virus.

Foot-and-mouth disease (FMD) is an important infection affecting the health and productivity of cloven-hoofed livestock. Development of improved vaccines and diagnostic reagents is being explored to facilitate the disease control. There is an emerging interest in virus-like particles (VLPs), as their constituent structural proteins are the major immunogens. The VLPs are similar to natural virus particles but lack viral nucleic acid. The objective of the present study was to express the VLPs of FMD virus (FMDV) serotype Asia-1 (IND 63/72), using baculovirus system and characterize them for antigenic structure. The VLPs expressed in insect cells showed immunoreactivity similar to inactivated cell culture FMDV. Further they possess similar sensitivity to trypsin as the inactivated cell culture FMDV, suggesting that trypsin-sensitive antigenic sites could be similarly arranged. Our findings suggest that the FMD VLPs have similar antigenic conformational feature like the wild type virus, thus supporting their utility in development of non-infectious FMD vaccines and/or diagnostic assays.

Tetravalent recombinant dengue virus-like particles as potential vaccine candidates: immunological properties.

BackgroundCurrently, a licensed vaccine for Dengue Virus (DENV) is not yet available. Virus-like particles (VLP) have shown considerable promise for use as vaccines and have many advantages compared to many other types of viral vaccines. VLPs have been found to have high immunogenic potencies, providing protection against various pathogens.ResultsIn the current study, four DENV-VLP serotypes were successfully expressed in Pichia pastoris, based on co-expression of the prM and E proteins. The effects of a tetravalent VLP vaccine were also examined. Immunization with purified, recombinant, tetravalent DENV1-4 VLPs induced specific antibodies against all DENV1-4 antigens in mice. The antibody titers were higher after immunization with the tetravalent VLP vaccine compared to titers after immunization with any of the dengue serotype VLPs alone. Indirect immunofluorescence assay (IFA) results indicated that sera from VLP immunized mice recognized the native viral antigens. TNF-α and IL-10 were significantly higher in mice immunized with tetravalent DENV-VLP compared to those mice received PBS. The tetravalent VLP appeared to stimulate neutralizing antibodies against each viral serotype, as shown by PRNT50 analysis (1:32 against DENV1 and 2, and 1:16 against DENV3 and 4). The highest titers with the tetravalent VLP vaccine were still a little lower than the monovalent VLP against the corresponding serotype. The protection rates of tetravalent DENV-VLP immune sera against challenges with DENV1 to 4 serotypes in suckling mice were 77, 92, 100, and 100%, respectively, indicating greater protective efficacy compared with monovalent immune sera.ConclusionsOur results provide an important basis for the development of the dengue VLP as a promising non-infectious candidate vaccine for dengue infection.

Vaccine-Induced Protection of Rhesus Macaques against Plasma Viremia after Intradermal Infection with a European Lineage 1 Strain of West Nile Virus

The mosquito-borne West Nile virus (WNV) causes human and animal disease with outbreaks in several parts of the world including North America, the Mediterranean countries, Central and East Europe, the Middle East, and Africa. Particularly in elderly people and individuals with an impaired immune system, infection with WNV can progress into a serious neuroinvasive disease. Currently, no treatment or vaccine is available to protect humans against infection or disease. The goal of this study was to develop a WNV-vaccine that is safe to use in these high-risk human target populations. We performed a vaccine efficacy study in non-human primates using the contemporary, pathogenic European WNV genotype 1a challenge strain, WNV-Ita09. Two vaccine strategies were evaluated in rhesus macaques (Macaca mulatta) using recombinant soluble WNV envelope (E) ectodomain adjuvanted with Matrix-M, either with or without DNA priming. The DNA priming immunization was performed with WNV-DermaVir nanoparticles. Both vaccination strategies successfully induced humoral and cellular immune responses that completely protected the macaques against the development of viremia. In addition, the vaccine was well tolerated by all animals. Overall, The WNV E protein adjuvanted with Matrix-M is a promising vaccine candidate for a non-infectious WNV vaccine for use in humans, including at-risk populations.