Heterologous Virus Challenge Research Articles

Low-inflammatory lipid nanoparticle-based mRNA vaccine elicits protective immunity against H5N1 influenza virus with reduced adverse reactions.

Messenger RNA vaccines based on lipid nanoparticles (mRNA-LNPs) are promising vaccine modalities. However, mRNA-LNP vaccines frequently cause adverse reactions such as swelling and fever in humans, partly due to the inflammatory nature of LNP. Modification of the ionizable lipids used in LNPs is one approach to avoid these adverse reactions. Here, we report the development of mRNA-LNP vaccines with better protective immunity and reduced adverse reactions using LNPs, which contain a disulfide (SS)-cleavable bond and pH-activated lipid-like materials with oleic acid (ssPalmO) as anionizable lipid (LNPssPalmO). We used mRNA expressing H5N1 subtype high-pathogenicity avian influenza virus-derived hemagglutinin or neuraminidaseto generate mRNA-LNP vaccines against H5N1 influenza. Compared with conventional LNPs, mRNA-LNPssPalmO induced comparable antigen-specific antibodiesand better interferon-γ (IFN-γ)-producing T helper type 1 responses in mice. Both mRNA-LNPssPalmO and conventional mRNA-LNPs conferred strong protection against homologous H5N1 virus challenge. In addition, mRNA-LNPssPalmO showed better cross-protection against heterologous H5N1 virus challenge compared with conventionalmRNA-LNPs. Furthermore, we observed that mRNA-LNPssPalmO induced less-inflammatory responses (e.g., inflammatory cytokine production, vascular hyperpermeability) and fewer adverse reactions (e.g., weight loss, fever) compared with conventional mRNA-LNPs. These results suggest that mRNA-LNPssPalmO would be a safe alternative toconventional vaccines to overcome mRNA-LNP vaccine hesitancy.

Proteolysis-targeting influenza vaccine strains induce broad-spectrum immunity and in vivo protection.

Generating effective live vaccines from intact viruses remains challenging owing to considerations of safety and immunogenicity. Approaches that can be applied in a systematic manner are needed. Here we created a library of live attenuated influenza vaccines by using diverse cellular E3 ubiquitin ligases to generate proteolysis-targeting (PROTAR) influenza A viruses. PROTAR viruses were engineered to be attenuated by the ubiquitin-proteasome system, which mediates viral protein degradation in conventional host cells, but allows efficient replication in engineered cell lines for large-scale manufacturing. Depending on the degron-E3 ligase pairs, viruses showed varying degrees of attenuation. In animal models, PROTAR viruses were highly attenuated and elicited robust, broad, strain-dependent humoral, mucosal and cellular immunity. In addition, they provided cross-reactive protection against homologous and heterologous viral challenges. This study provides a systematic approach for developing safe and effective vaccines, with potential applications in designing live attenuated vaccines against other pathogens.

PROTAR Vaccine 2.0 generates influenza vaccines by degrading multiple viral proteins.

Manipulating viral protein stability using the cellular ubiquitin-proteasome system (UPS) represents a promising approach for developing live-attenuated vaccines. The first-generation proteolysis-targeting (PROTAR) vaccine had limitations, as it incorporates proteasome-targeting degrons (PTDs) at only the terminal ends of viral proteins, potentially restricting its broad application. Here we developed the next-generation PROTAR vaccine approach, referred to as PROTAR 2.0, which enabled flexible incorporation of PTDs at various genomic loci of influenza viruses, including internal regions and terminal ends. The PROTAR 2.0 influenza viruses maintained efficient replication in UPS-deficient cells for large-scale production but were attenuated by PTD-mediated proteasomal degradation of viral proteins in conventional cells. Incorporation of multiple PTDs into one virus generated optimized PROTAR 2.0 vaccine candidates. In animal models, PROTAR 2.0 vaccine candidates were highly attenuated and a single-dose intranasal immunization induced robust and broad immune responses that provided complete cross-reactive protection against both homologous and heterologous viral challenges.

CXCL13 promotes broad immune responses induced by circular RNA vaccines.

Antibody responses induced by current vaccines for influenza and SARS-CoV-2 often lack robust cross-reactivity. As hubs where diverse immune cells converge and interact, the alterations in the immune microenvironment within lymph nodes (LNs) are intricately linked to immune responses. Herein, we designed a lipid nanoparticle (LNP) loaded with circular RNA (circRNA) and targeted to LNs, in which CXCL13 was directly integrated into antigen-encoding circRNA strands. We demonstrated that CXCL13 alters the transcriptomic profiles of LNs, especially the upregulation of IL-21 and IL-4. Meanwhile, CXCL13 promotes the formation of germinal center and elicits robust antigen-specific T cell responses. With the codelivery of CXCL13 and the antigen, CXCL13 enhances cross-reactive antibodies against influenza virus and SARS-CoV-2, achieving protection against both homologous and heterologous influenza virus challenges in a mouse model. Notably, the targeted modification of LNP surfaces with antibodies helps address some of the challenges associated with lyophilized LNP vaccines, which is crucial for the long-term storage of LNP-circRNA vaccines. Overall, the circRNA-based antigen-CXCL13 coexpression system developed herein provides a simple and robust platform that enhances the magnitude and breadth of antibody responses against multiple viral glycoproteins, highlighting the potential utility of CXCL13 in inducing broad immune responses.

M2e nanovaccines supplemented with recombinant hemagglutinin protect chickens against heterologous HPAI H5N1 challenge

Current poultry vaccines against influenza A viruses target the globular head region of the hemagglutinin (HA1), providing limited protection against antigenically divergent strains. Experimental subunit vaccines based on the conserved ectodomain of the matrix protein 2 (M2e) induce cross-reactive antibody responses, but fail to fully prevent virus shedding after low pathogenic avian influenza (LPAI) virus challenge, and are ineffective against highly pathogenic avian influenza (HPAI) viruses. This study assessed the benefits of combining nanoparticles bearing three tandem M2e repeats (NR-3M2e nanorings or NF-3M2e nanofilaments) with an HA1 subunit vaccine in protecting chickens against a heterologous HPAI H5N1 virus challenge. Chickens vaccinated with the combined formulations developed M2e and HA1-specific antibodies, were fully protected from clinical disease and mortality, and showed no histopathological lesions or virus shedding, unlike those given only HA1, NR-3M2e, or NF-3M2e. Thus, the combined vaccine formulations provided complete cross-protection against HPAI H5N1 virus, and prevented environmental virus shedding, crucial for controlling avian influenza outbreaks.

A Broad Influenza Vaccine Based on a Heat-Activated, Tissue-Restricted Replication-Competent Herpesvirus.

Vaccination with transiently activated replication-competent controlled herpesviruses (RCCVs) expressing influenza A virus hemagglutinins broadly protects mice against lethal influenza virus challenges. The non-replicating RCCVs can be activated to transiently replicate with high efficiency. Activation involves a brief heat treatment to the epidermal administration site in the presence of a drug. The drug co-control is intended as a block to inadvertent reactivation in the nervous system and, secondarily, viremia under adverse conditions. While the broad protective effects observed raise an expectation that RCCVs may be developed as universal flu vaccines, the need for administering a co-activating drug may dampen enthusiasm for such a development. To replace the drug co-control, we isolated keratin gene promoters that were active in skin cells but inactive in nerve cells and other cells in vitro. In a mouse model of lethal central nervous system (CNS) infection, the administration of a recombinant that had the promoter of the infected cell protein 8 (ICP8) gene of a wild-type herpes simplex virus 1 (HSV-1) strain replaced by a keratin promoter did not result in any clinical signs, even at doses of 500 times wild-type virus LD50. Replication of the recombinant was undetectable in brain homogenates. Second-generation RCCVs expressing a subtype H1 hemagglutinin (HA) were generated in which the infected cell protein 4 (ICP4) genes were controlled by a heat switch and the ICP8 gene by the keratin promoter. In mice, these RCCVs replicated efficiently and in a heat-controlled fashion in the epidermal administration site. Immunization with the activated RCCVs induced robust neutralizing antibody responses against influenza viruses and protected against heterologous and cross-group influenza virus challenges.

Boosting DNA vaccine power by lipid nanoparticles surface engineered with amphiphilic bioresorbable copolymer

Successful DNA vaccination generally requires the aid of either a viral vector within vaccine components or electroporation device into muscle or skin of the host. However, these systems come with certain obstacles, including limited transgene capacity, broad pre-existing immunity in humans, and substantial cell death caused by high voltage pulses, respectively. In this study, we repurposed the use of an amphiphilic bioresorbable copolymer (ABC), named PLA-PEG, as surface engineering agent that conciliates lipid nanoparticle (LNP) between stability during preparation and biocompatibility post-vaccination. The LNP carrier can be loaded with SARS-CoV-2 spike-specific DNA; in this form, the DNA-LNP is immunogenic in hamsters and elicits protective immunity following DNA-LNP vaccination against heterologous virus challenge or as a hybrid-type vaccine booster against SARS-CoV-2 variants. The data provide comprehensive information on the relationships between LNP composition, manufacturing process and vaccine efficacy. The outcomes of this study offer new insights into designing next-generation LNP formulations and pave the way for boosting vaccine power to combat existing and possible emerging infectious diseases/pathogens.

Vaccination with antigenically complex hemagglutinin mixtures confers broad protection from influenza disease.

Current seasonal influenza virus vaccines induce responses primarily against immunodominant but highly plastic epitopes in the globular head of the hemagglutinin (HA) glycoprotein. Because of viral antigenic drift at these sites, vaccines need to be updated and readministered annually. To increase the breadth of influenza vaccine-mediated protection, we developed an antigenically complex mixture of recombinant HAs designed to redirect immune responses to more conserved domains of the protein. Vaccine-induced antibodies were disproportionally redistributed to the more conserved stalk of the HA without hindering, and in some cases improving, antibody responses against the head domain. These improved responses led to increased protection against homologous and heterologous viral challenges in both mice and ferrets compared with conventional vaccine approaches. Thus, antigenically complex protein mixtures can at least partially overcome HA head domain antigenic immunodominance and may represent a step toward a more universal influenza vaccine.

An Alternative Serological Measure for Assessing Foot-and-Mouth Disease Vaccine Efficacy against Homologous and Heterologous Viral Challenges in Pigs.

To analyze the relationship between homologous and heterologous serological titers of immunized pigs and their protection statuses against FMD virus challenges, in the present study, the correlation between the virus neutralization titers at 21 and 28 dpv and the protection statuses at 28 dpv against challenge with FMD virus was analyzed using data sets comprising five different combinations of homologous or heterologous challenge experiments in pigs vaccinated with type O (n = 96), A (n = 69), and Asia 1 (n = 74). As a result, the experiments were divided into three groups (21D-1, 21D-2, and 21D-3) in the 21-dpv model and two groups (28D-1 and 28D-2) in the 28-dpv model. Each response curve of groups 21D-1 and 21D-2 in the 21-dpv model was very similar to each curve of groups 28D-1 and 28D-2 in the 28-dpv model, respectively, even though there was an exceptional extra group (21D-3) in the 21-dpv model. The average titers estimating 0.75 probability of protection ranged from 1.06 to 1.62 log10 in the 21-dpv model and from 1.26 to 1.64 log10 in the 28-dpv model. In summary, we demonstrated that the serological method is useful for predicting the homologous and heterologous protection statuses of vaccinated pigs.

Hemagglutinin virus-like particles incorporated with membrane-bound cytokine adjuvants provide protection against homologous and heterologous influenza virus challenge in aged mice

BackgroundCurrent influenza vaccines deliver satisfactory results in young people but are less effective in the elderly. Development of vaccines for an ever-increasing aging population has been an arduous challenge due to immunosenescence that impairs the immune response in the aged, both quantitatively and qualitatively.ResultsTo potentially enhance vaccine efficacy in the elderly, we investigated the immunogenicity and cross-protection of influenza hemagglutinin virus-like particles (HA-VLP) incorporated with glycosylphosphatidylinositol (GPI)-anchored cytokine-adjuvants (GPI-GM-CSF and GPI-IL-12) via protein transfer in aged mice. Lung viral replication against homologous and heterologous influenza viruses was significantly reduced in aged mice after vaccination with cytokine incorporated VLPs (HA-VLP-Cyt) in comparison to HA-VLP alone. Enhanced IFN-γ+CD4+ and IFN-γ+CD8+ T cell responses were also observed in aged mice immunized with HA-VLP-Cyt when compared to HA-VLP alone.ConclusionsCytokine-adjuvanted influenza HA-VLP vaccine induced enhanced protective response against homologous influenza A virus infection in aged mice. Influenza HA-VLP vaccine with GPI-cytokines also induced enhanced T cell responses correlating with better protection against heterologous infection in the absence of neutralizing antibodies. The results suggest that a vaccination strategy using cytokine-adjuvanted influenza HA-VLPs could be used to enhance protection against influenza A virus in the elderly.

Neutralizing and protective murine monoclonal antibodies to the hemagglutinin of influenza H5 clades 2.3.2.1 and 2.3.4.4.

Highly pathogenic avian H5 influenza viruses have spread and diversified genetically and antigenically into multiple clades and subclades. Most isolates of currently circulating H5 viruses are in clade 2.3.2.1 or 2.3.4.4. Panels of murine monoclonal antibodies (mAbs) were generated to the influenza hemagglutinin (HA) of H5 viruses from the clade 2.3.2.1 H5N1 vaccine virus A/duck/Bangladesh/19097/2013 and the clade 2.3.4.4 H5N8 vaccine virus A/gyrfalcon/Washington/41088-6/2014. Antibodies were selected and characterized for binding, neutralization, epitope recognition, cross-reactivity with other H5 viruses, and the ability to provide protection in passive transfer experiments. All mAbs bound homologous HA in an ELISA format; mAbs 5C2 and 6H6 were broadly binding for other H5 HAs. Potently neutralizing mAbs were identified in each panel, and all neutralizing mAbs provided protection in passive transfer experiments in mice challenged with a homologous clade influenza virus. Cross-reacting mAb 5C2 neutralized a wide variety of clade 2.3.2.1 viruses, as well as H5 viruses from other clades, and also provided protection against heterologous H5 clade influenza virus challenge. Epitope analysis indicated that the majority of mAbs recognized epitopes in the globular head of the HA. The mAb 5C2 appeared to recognize an epitope below the globular head but above the stalk region of HA. The results suggested that these H5 mAbs would be useful for virus and vaccine characterization. The results confirmed the functional cross-reactivity of mAb 5C2, which appears to bind a novel epitope, and suggest the therapeutic potential for H5 infections in humans with further development.

Evaluation and Determination of a Suitable Passage Number of Codon Pair Deoptimized PRRSV-1 Vaccine Candidate in Pigs.

Porcine reproductive and respiratory syndrome virus (PRRSV) is major economic problem given its effects on swine health and productivity. Therefore, we evaluated the genetic stability of a codon pair de-optimized (CPD) PRRSV, E38-ORF7 CPD, as well as the master seed passage threshold that elicited an effective immune response in pigs against heterologous virus challenge. The genetic stability and immune response of every 10th passage (out of 40) of E38-ORF7 CPD was analyzed through whole genome sequencing and inoculation in 3-week-old pigs. E38-ORF7 CPD passages were limited to 20 based on the full-length mutation analysis and animal test results. After 20 passages, the virus could not induce antibodies to provide effective immunity and mutations accumulated in the gene, which differed from the CPD gene, presenting a reason for low infectivity. Conclusively, the optimal passage number of E38-ORF7 CPD is 20. As a vaccine, this may help overcome the highly diverse PRRSV infection with substantially enhanced genetic stability.

Antigenic diversity of type 1 polioviruses and its implications for the efficacy of polio vaccines.

Inactivated Polio Vaccines (IPV) and live Oral Polio Vaccine (OPV) were introduced in the mid-20th century, and their coordinated worldwide use led to almost complete elimination of the disease, with only one serotype of poliovirus remaining endemic in just two countries. Polio eradication will lead to discontinuation of OPV use and its replacement with IPV or other vaccines that are currently under development that will need to be tested in clinical trials. Despite decades of research, questions remain about the serological correlates of polio vaccine efficacy, specifically whether the vaccines are equally protective against immunologically different strains of the same serotype. The absence of significant morbidity does not allow use of a protection endpoint in clinical trials, so the answer could be obtained only by using surrogate markers such as immunogenicity. In this study, a panel of wild and vaccine-derived polioviruses of serotype 1 were tested in neutralization assays with sera from vaccine-immunized individuals. The results demonstrated that there was a significant difference in titers of neutralizing antibodies in human sera when measured against different strains. When measured with a homologous strain used for vaccine manufacture all subjects had detectable levels of antibodies, while neutralization tests with some heterologous strains failed to detect neutralizing antibodies in a number of subjects. Administration of a booster dose of IPV led to a significant increase in neutralizing titers against all strains. Results of the experiments using animal sera, performed to obtain more information on protectivity of neutralizing antibodies against heterologous strains, were consistent with the results obtained in the assays using human sera. These results are discussed in the context of serological biomarkers of protection against poliomyelitis, suggesting that potency of vaccines made from serologically different strains should be determined against both homologous and heterologous challenge viruses.

Development of in ovo-compatible NS1-truncated live attenuated influenza vaccines by modulation of hemagglutinin cleavage and polymerase acidic X frameshifting sites

Emerging avian influenza viruses pose a high risk to poultry production, necessitating the need for more broadly protective vaccines. Live attenuated influenza vaccines offer excellent protective efficacies but their use in poultry farms is discouraged due to safety concerns related to emergence of reassortant viruses. Vaccination of chicken embryos inside eggs (in ovo) induces early immunity in young chicks while reduces the safety concerns related to the use of live vaccines on farms. However, in ovo vaccination using influenza viruses severely affects the egg hatchability. We previously engineered a high interferon-inducing live attenuated influenza vaccine candidate with an enhanced protective efficacy in chickens. Here, we asked whether we could further modify this high interferon-inducing vaccine candidate to develop an in ovo-compatible live attenuated influenza vaccine. We first showed that the enhanced interferon responses induced by the vaccine is not enough to attenuate the virus in ovo. To reduce the pathogenicity of the virus for chicken embryos, we replaced the hemagglutinin cleavage site of the H7 vaccine virus (PENPKTR/GL) with that of the H6-subtype viruses (PQIETR/GL) and disrupted the ribosomal frameshifting site responsible for viral polymerase acidic X protein expression. In ovo vaccination of chickens with up to 105 median egg infectious dose of the modified vaccine had minimal effects on hatchability while protecting the chickens against a heterologous challenge virus at two weeks of age. This study demonstrates that targeted genetic mutations can be applied to further attenuate and enhance the safety of live attenuated influenza vaccines to develop future in ovo vaccines for poultry.

Infant rhesus macaques immunized against SARS-CoV-2 are protected against heterologous virus challenge 1 year later.

The U.S. Food and Drug Administration only gave emergency use authorization of the BNT162b2 and mRNA-1273 SARS-CoV-2 vaccines for infants 6 months and older in June 2022. Yet questions regarding the durability of vaccine efficacy, especially against emerging variants, in this age group remain. We demonstrated previously that a two-dose regimen of stabilized prefusion Washington SARS-CoV-2 S-2P spike (S) protein encoded by mRNA encapsulated in lipid nanoparticles (mRNA-LNP) or purified S-2P mixed with 3M-052, a synthetic Toll-like receptor (TLR) 7/8 agonist, in a squalene emulsion (Protein+3M-052-SE) was safe and immunogenic in infant rhesus macaques. Here, we demonstrate that broadly neutralizing and spike-binding antibodies against variants of concern (VOCs), as well as T cell responses, persisted for 12 months. At 1 year, corresponding to human toddler age, we challenged vaccinated rhesus macaques and age-matched nonvaccinated controls intranasally and intratracheally with a high dose of heterologous SARS-CoV-2 B.1.617.2 (Delta). Seven of eight control rhesus macaques exhibited severe interstitial pneumonia and high virus replication in the upper and lower respiratory tract. In contrast, vaccinated rhesus macaques had faster viral clearance with mild to no pneumonia. Neutralizing and binding antibody responses to the B.1.617.2 variant at the day of challenge correlated with lung pathology and reduced virus replication. Overall, the Protein+3M-052-SE vaccine provided superior protection to the mRNA-LNP vaccine, emphasizing opportunities for optimization of current vaccine platforms. The observed efficacy of both vaccines 1 year after vaccination supports the implementation of an early-life SARS-CoV-2 vaccine.

Versatile live-attenuated SARS-CoV-2 vaccine platform applicable to variants induces protective immunity

Live-attenuated vaccines are generally highly effective. Here, we aimed to develop one against SARS-CoV-2, based on the identification of three types of temperature-sensitive (TS) strains with mutations in nonstructural proteins (nsp), impaired proliferation at 37-39ºC, and the capacity to induce protective immunity in Syrian hamsters. To develop a live-attenuated vaccine, we generated a virus that combined all these TS-associated mutations (rTS-all), which showed a robust TS phenotype in vitro and high attenuation in vivo. The vaccine induced an effective cross-reactive immune response and protected hamsters against homologous or heterologous viral challenges. Importantly, rTS-all rarely reverted to the wild-type phenotype. By combining these mutations with an Omicron spike protein to construct a recombinant virus, protection against the Omicron strain was obtained. We show that immediate and effective live-attenuated vaccine candidates against SARS-CoV-2 variants may be developed using rTS-all as a backbone to incorporate the spike protein of the variants.

Respiratory Vaccination with Hemagglutinin Nanoliposomes Protects Mice from Homologous and Heterologous Strains of Influenza Virus.

Intranasal vaccination offers the potential advantage of needle-free prevention of respiratory pathogens such as influenza viruses with induction of mucosal immune responses. Optimal design of adjuvants and antigen delivery vehicles for intranasal delivery has not yet been well established. Here, we report that an adjuvant-containing nanoliposome antigen display system that converts soluble influenza hemagglutinin antigens into nanoparticles is effective for intranasal immunization. Intranasal delivery of nanoliposomes in mice delivers the particles to resident immune cells in the respiratory tract, inducing a mucosal response in the respiratory system as evidenced by nasal and lung localized IgA antibody production, while also producing systemic IgG antibodies. Intranasal vaccination with nanoliposome particles decorated with nanogram doses of hemagglutinin protected mice from homologous and heterologous H3N2 and H1N1 influenza virus challenge. IMPORTANCE A self-assembling influenza virus vaccine platform that seamlessly converts soluble antigens into nanoparticles is demonstrated with various H1N1 and H3N2 influenza antigens to protect mice against influenza virus challenge following intranasal vaccination. Mucosal immune responses following liposome delivery to lung antigen-presenting cells are demonstrated.

Immunogenic Cross-Reactivity between Goose and Muscovy Duck Parvoviruses: Evaluation of Cross-Protection Provided by Mono- or Bivalent Vaccine.

To investigate the immunogenic cross reactivity between goose parvovirus (GPV) and Muscovy duck parvovirus (MDPV), cross-neutralization was carried out with serum samples collected from birds after infection with one of the two waterfowl parvoviruses. The significantly higher virus neutralization titer obtained against the homologous virus than against the heterologous one suggests important differences between the GPV and MDPV antigenic make up that affects the induced protective virus-neutralizing antibody specificity. This was further confirmed by cross-protection studies carried out in waterfowl parvovirus antibody-free Muscovy ducks immunized at one day of age with whole-virus inactivated oil-emulsion vaccines containing either GPV or MDPV as a monovalent vaccine, or both viruses as a bivalent vaccine. Protection against the clinical disease (growth retardation and feathering disorders) provided by the monovalent vaccine was complete against homologous virus challenge at 2 weeks post-vaccination, while the protection against the heterologous virus challenge was significantly lower (p < 0.001). Only the bivalent vaccine containing both goose and Muscovy duck parvoviruses in an inactivated form protected the birds (90–100%) against both waterfowl parvoviruses that can cause disease in Muscovy ducks. Both the cross-neutralization and cross-protection results indicated that adequate protection in Muscovy ducks against the two waterfowl parvoviruses could be achieved only with a vaccine containing both goose and Muscovy duck parvoviruses. Our results showed that the inactivated vaccine applied at one day of age could induce fast immunity (by 2 weeks post-vaccination), providing complete clinical protection in maternal antibody-free birds. It was also demonstrated that day-old vaccination of ducks with maternal antibodies with bivalent vaccine induced active immunity, resulting in 90 to 100% protection by 3 weeks of age, after the decline of maternal antibodies. A booster vaccination administered at 3 weeks of age following the day-old vaccination resulted in a strong and durable immunity against the clinical disease during the susceptible age of the birds.

Generation of a live attenuated influenza A vaccine by proteolysis targeting.

The usefulness of live attenuated virus vaccines has been limited by suboptimal immunogenicity, safety concerns or cumbersome manufacturing processes and techniques. Here we describe the generation of a live attenuated influenza A virus vaccine using proteolysis-targeting chimeric (PROTAC) technology to degrade viral proteins via the endogenous ubiquitin-proteasome system of host cells. We engineered the genome of influenza A viruses in stable cell lines engineered for virus production to introduce a conditionally removable proteasome-targeting domain, generating fully infective PROTAC viruses that were live attenuated by the host protein degradation machinery upon infection. In mouse and ferret models, PROTAC viruses were highly attenuated and able to elicit robust and broad humoral, mucosal and cellular immunity against homologous and heterologous virus challenges. PROTAC-mediated attenuation of viruses may be broadly applicable for generating live attenuated vaccines.

Structural Stability and Antigenicity of Universal Equine H3N8 Hemagglutinin Trimer upon Release from Polyanhydride Nanoparticles and Pentablock Copolymer Hydrogels.

Seasonal influenza A virus infections present substantial costs to both health and economic resources each year. Current seasonal influenza vaccines provide suboptimal protection and require annual reformulation to match circulating strains. In this work, a recombinant equine H3N8 hemagglutinin trimer (rH33) known to generate cross-protective antibodies and protect animals against sublethal, heterologous virus challenge was used as a candidate vaccine antigen. Nanoadjuvants such as polyanhydride nanoparticles and pentablock copolymer hydrogels have been shown to be effective adjuvants, inducing both rapid and long-lived protective immunity against influenza A virus. In this work, polyanhydride nanoparticles and pentablock copolymer hydrogels were used to provide sustained release of the novel rH33 while also facilitating the retention of its structure and antigenicity. These studies lay the groundwork for the development of a novel universal influenza A virus nanovaccine by combining the equine H3N8 rH33 and polymeric nanoadjuvant platforms.