Cross-protective efficacy and safety of an adenovirus-based universal influenza vaccine expressing nucleoprotein, hemagglutinin, and the ectodomain of matrix protein 2.

It is necessary to develop universal vaccines that act broadly and continuously to combat regular seasonal epidemics of influenza and rare pandemics. The aim of this study was to find the optimal dose regimen for the efficacy and safety of a mixture of previously developed recombinant adenovirus-based vaccines that expressed influenza nucleoprotein, hemagglutinin, and ectodomain of matrix protein 2 (rAd/NP and rAd/HA-M2e). The vaccine efficacy and safety were measured in the immunized mice with the mixture of rAd/NP and rAd/HA-M2e intranasally or intramuscularly. The minimum dose that would be efficacious in a single intranasal administration of the vaccine mixture and cross-protective efficacy against various influenza strains were examined. In addition, the immune responses that may affect the cross-protective efficacy were measured. We found that intranasal administration is an optimal route for 107 pfu of vaccine mixture, which is effective against pre-existing immunity against adenovirus. In a study to find the minimum dose with vaccine efficacy, the 106 pfu of vaccine mixture showed higher antibody titers to the nucleoprotein than did the same dose of rAd/NP alone in the serum of immunized mice. The 106 pfu of vaccine mixture overcame the morbidity and mortality of mice against the lethal dose of pH1N1, H3N2, and H5N1 influenza infections. No noticeable side effects were observed in single and repeated toxicity studies. We found that the mucosal administration of adenovirus-based universal influenza vaccine has both efficacy and safety, and can provide cross-protection against various influenza infections even at doses lower than those previously known to be effective.

Whole genome sequencing of recombinant viruses obtained from co-infection and superinfection of Vero cells with modified vaccinia virus ankara vectored influenza vaccine and a naturally occurring cowpox virus.

Modified vaccinia virus Ankara (MVA) has been widely tested in clinical trials as recombinant vector vaccine against infectious diseases and cancers in humans and animals. However, one biosafety concern about the use of MVA vectored vaccine is the potential for MVA to recombine with naturally occurring orthopoxviruses in cells and hosts in which it multiplies poorly and, therefore, producing viruses with mosaic genomes with altered genetic and phenotypic properties. We previously conducted co-infection and superinfection experiments with MVA vectored influenza vaccine (MVA-HANP) and a feline Cowpox virus (CPXV-No-F1) in Vero cells (that were semi-permissive to MVA infection) and showed that recombination occurred in both co-infected and superinfected cells. In this study, we selected the putative recombinant viruses and performed genomic characterization of these viruses. Some putative recombinant viruses displayed plaque morphology distinct of that of the parental viruses. Our analysis demonstrated that they had mosaic genomes of different lengths. The recombinant viruses, with a genome more similar to MVA-HANP (>50%), rescued deleted and/or fragmented genes in MVA and gained new host ranges genes. Our analysis also revealed that some MVA-HANP contained a partially deleted transgene expression cassette and one recombinant virus contained part of the transgene expression cassette similar to that incomplete MVA-HANP. The recombination in co-infected and superinfected Vero cells resulted in recombinant viruses with unpredictable biological and genetic properties as well as recovery of delete/fragmented genes in MVA and transfer of the transgene into replication competent CPXV. These results are relevant to hazard characterization and risk assessment of MVA vectored biologicals.

One HA stalk topping multiple heads as a novel influenza vaccine.

Classic chimeric hemagglutinin (cHA) was designed to induce immune responses against the conserved stalk domain of HA. However, it is unclear whether combining more than one HA head domain onto one stalk domain is immunogenic and further induce immune responses against influenza viruses. Here, we constructed numerous novel cHAs comprising two or three fuzed head domains from different subtypes grafted onto one stalk domain, designated as cH1-H3, cH1-H7, cH1-H3-H7, and cH1-H7-H3. The three-dimensional structures of these novel cHAs were modelled using bioinformatics simulations. Structural analysis showed that the intact neutralizing epitopes were exposed in cH1-H7 and were predicted to be immunogenic. The immunogenicity of the cHAs constructs was evaluated in mice using a chimpanzee adenoviral vector (AdC68) vaccine platform. The results demonstrated that cH1-H7 expressed by AdC68 (AdC68-cH1-H7) induced the production of high levels of binding antibodies, neutralizing antibodies, and hemagglutinin inhibition antibodies against homologous pandemic H1N1, drifted seasonal H1N1, and H7N9 virus. Moreover, vaccinated mice were fully protected from a lethal challenge with the aforementioned influenza viruses. Hence, cH1-H7 cHAs with potent immunogenicity might be a potential novel vaccine to provide protection against different subtypes of influenza virus.

SpyTag/SpyCatcher display of influenza M2e peptide on norovirus-like particle provides stronger immunization than direct genetic fusion.

Introduction: Virus-like particles (VLPs) are similar in size and shape to their respective viruses, but free of viral genetic material. This makes VLP-based vaccines incapable of causing infection, but still effective in mounting immune responses. Noro-VLPs consist of 180 copies of the VP1 capsid protein. The particle tolerates C-terminal fusion partners, and VP1 fused with a C-terminal SpyTag self-assembles into a VLP with SpyTag protruding from its surface, enabling conjugation of antigens via SpyCatcher. Methods: To compare SpyCatcher-mediated coupling and direct peptide fusion in experimental vaccination, we genetically fused the ectodomain of influenza matrix-2 protein (M2e) directly on the C-terminus of norovirus VP1 capsid protein. VLPs decorated with SpyCatcher-M2e and VLPs with direct M2 efusion were used to immunize mice. Results and discussion: We found that direct genetic fusion of M2e on noro-VLP raised few M2e antibodies in the mouse model, presumably because the short linker positions the peptide between the protruding domains of noro-VLP, limiting its accessibility. On the other hand, adding aluminum hydroxide adjuvant to the previously described SpyCatcher-M2e-decorated noro-VLP vaccine gave a strong response against M2e. Surprisingly, simple SpyCatcher-fused M2e without VLP display also functioned as a potent immunogen, which suggests that the commonly used protein linker SpyCatcher-SpyTag may serve a second role as an activator of the immune system in vaccine preparations. Based on the measured anti-M2e antibodies and cellular responses, both SpyCatcher-M2e as well as M2e presented on the noro-VLP via SpyTag/Catcher show potential for the development of universal influenza vaccines.

Development of transgenic algae strain expressing CTB-M2e fusion gene an approach towards the development of a universal edible vaccine in algae.

Avian Influenza, the most studied virus, is of high concern due to its zoonotic pandemic potential. In recent years, several influenza vaccines have been used with the broad goal of managing and in certain cases, eliminating the disease. The matrix 2 extracellular domain (M2e), is one of the key targets of the universal influenza vaccine, a liner peptide that is conserved throughout all influenza A subtypes virus. Many recombinant influenza proteins have been expressed in yeast and plants for vaccine development. A remarkable development has been made in the field of biotechnology to explore the potential of microalga as an expression host. In this study, we designed a fusion gene code for M2e peptide and CTB protein as M2e's natural form has a low level of immunogenicity. The fusion gene was cloned in the Chloroplast transformation vector pSRSapI and expressed in the TN72 mutant strain of Chlamydomonas reinhardii. The expression of the targeted protein was confirmed by ECL western blot analysis. A GM1-ELISA was carried out to detect the affinity of fusion protein for GM1 monosialoganglioside and the significant P-value is lower than 0.05. Immunogenicity assay on chicken detected the anti-M2e bodies in chicken serum. This study gives evidence of therapeutic protein production through algae chloroplast and a stable, selection free and low cost oral delivery for universal vaccine against influenza A virus.

Bivalent vaccines effectively protect mice against influenza A and respiratory syncytial viruses.

Influenza and Respiratory Syncytial virus (RSV) infections together contribute significantly to the burden of acute lower respiratory tract infections. Despite the disease burden, no approved RSV vaccine is available. While approved vaccines are available for influenza, seasonal vaccination is required to maintain protection. In addition to both being respiratory viruses, they follow a common seasonality, which warrants the necessity for a concerted vaccination approach. Here, we designed bivalent vaccines by utilizing highly conserved sequences, targeting both influenza A and RSV, as either a chimeric antigen or individual antigens separated by a ribosome skipping sequence. These vaccines were found to be effective in protecting the animals from challenge by either virus, with mechanisms of protection being substantially interrogated in this communication.

Evaluation of Efficacy of Oil Adjuvanted H5N6 Inactivated Vaccine against Highly Pathogenic H5N6 and H5N1 Influenza Viruses Infected Chickens.

BACKGROUND: Over the last 20 years, circulating highly pathogenic (HP) Asian H5 subtype avian influenza viruses have caused global pandemics in poultry and sporadic infections in humans. Vaccines are a desirable solution to prevent viral infections in poultry and reduce transmission to humans. Herein, we investigated the efficacy of an oil-adjuvanted inactivated H5N6 vaccine against highly pathogenic H5N6 and H5N1 influenza virus infections in chickens. METHODS: The polybasic amino acid cleavage site depleted HA gene and NA gene of A/Waterfowl/Korea/S57/2016 (clade 2.3.4.4) (H5N6) was assembled with the rest of the A/PR/8/34 (H1N1) genes to construct the vaccine virus. The vaccine virus was propagated in fertilized eggs, partially purified using a tangential flow filtration (TFF) system, and inactivated using formalin. The chickens were intramuscularly immunized with 384 HA, 192HA, and 96HA units of oil-adjuvanted inactivated H5N6 vaccine. Antibody titer, survival rate, and lung pathology were evaluated against the homologous H5N6: A/waterfowl/Korea/S57/2016 (clade 2.3.4.4) and heterologous H5N1: A/Hong Kong/213/2003 (clade 1) viruses 12 and 4 weeks post-vaccination (p.v.), respectively. Data were statistically analyzed using the Mann-Whitney U test. RESULTS: The 384HA (n = 10) and 192HA (n = 5) antigen-immunized chickens showed 100% survival after lethal infections with homologous H5N6, and no virus shedding was observed from tracheal and cloacal routes. All chickens that received the 384HA vaccine survived the challenge of heterologous H5N1 after 4 weeks of immunization. The chickens that received the 384HA vaccine showed mean HI titers of 60 and 240 after 12 and 4 weeks of vaccination, respectively, against HP H5N6, whereas a mean HI titer of 80 was observed in sera collected 4 weeks after vaccination against HP H5N1. CONCLUSIONS: Our findings indicate that one dose of 384HA oil-adjuvanted inactivated H5N6 vaccine can induce a long-lasting immune response against both homologous H5N6 and heterologous H5N1 infections in chickens.

Flagellin/Virus-like Particle Hybrid Platform with High Immunogenicity, Safety, and Versatility for Vaccine Development.

Hepatitis B core (HBc) virus-like particles (VLPs) and flagellin are highly immunogenic and widely explored vaccine delivery platforms. Yet, HBc VLPs mainly allow the insertion of relatively short antigenic epitopes into the immunodominant c/e1 loop without affecting VLP assembly, and flagellin-based vaccines carry the risk of inducing systemic adverse reactions. This study explored a hybrid flagellin/HBc VLP (FH VLP) platform to present heterologous antigens by replacing the surface-exposed D3 domain of flagellin. FH VLPs were prepared by the insertion of flagellin gene into the c/e1 loop of HBc, followed by E. coli expression, purification, and self-assembly into VLPs. Using the ectodomain of influenza matrix protein 2 (M2e) and ovalbumin (OVA) as models, we found that the D3 domain of flagellin could be replaced with four tandem copies of M2e or the cytotoxic T lymphocyte (CTL) epitope of OVA without interfering with the FH VLP assembly, while the insertion of four tandem copies of M2e into the c/e1 loop of HBc disrupted the VLP assembly. FH VLP-based M2e vaccine elicited potent anti-M2e antibody responses and conferred significant protection against multiple influenza A viral strains, while FljB- or HBc-based M2e vaccine failed to elicit significant protection. FH VLP-based OVA peptide vaccine elicited more potent CTL responses and protection against OVA-expressing lymphoma or melanoma challenges than FljB- or HBc-based OVA peptide vaccine. FH VLP-based vaccines showed a good systemic safety, while flagellin-based vaccines significantly increased serum interleukin 6 and tumor necrosis factor α levels and also rectal temperature at increased doses. We further found that the incorporation of a clinical CpG 1018 adjuvant could enhance the efficacy of FH VLP-based vaccines. Our data support FH VLPs to be a highly immunogenic, safe, and versatile platform for vaccine development to elicit potent humoral and cellular immune responses.

Generation of an avian influenza DIVA vaccine with a H3-peptide replacement located at HA2 against both highly and low pathogenic H7N9 virus.

A differentiating infected from vaccinated animals (DIVA) vaccine is an ideal strategy for viral eradication in poultry. Here, according to the emerging highly pathogenic H7N9 avian influenza virus (AIV), a DIVA vaccine strain, named rGD4HALo-mH3-TX, was successfully developed, based on a substituted 12 peptide of H3 virus located at HA2. In order to meet with the safety requirement of vaccine production, the multi-basic amino acid located at the HA cleavage site was modified. Meanwhile, six inner viral genes from a H9N2 AIV TX strainwere introduced for increasing viral production. The rGD4HALo-mH3-TX strain displayed a similar reproductive ability with rGD4 and low pathogenicity in chickens, suggesting a good productivity and safety. In immuned chickens, rGD4HALo-mH3-TX induced a similar antibody level with rGD4 and provided 100% clinical protection and 90% shedding protection against highly pathogenic virus challenge. rGD4HALo-mH3-TX strain also produced a good cross-protection against low pathogenic AIV JD/17. Moreover, serological DIVA characteristics were evaluated by a successfully established competitive inhibition ELISA based on a 3G10 monoclonal antibody, and the result showed a strong reactivity with antisera of chickens vaccinated with H7 subtype strains but not rGD4HALo-mH3-TX. Collectedly, rGD4HALo-mH3-TX is a promising DIVA vaccine candidate against both high and low pathogenic H7N9 subtype AIV.

A Single Vaccine Protects against SARS-CoV-2 and Influenza Virus in Mice.

The ongoing SARS-CoV-2 pandemic poses a severe global threat to public health, as do influenza viruses and other coronaviruses. Here, we present chimpanzee adenovirus 68 (AdC68)-based vaccines designed to universally target coronaviruses and influenza. Our design is centered on an immunogen generated by fusing the SARS-CoV-2 receptor-binding domain (RBD) to the conserved stalk of H7N9 hemagglutinin (HA). Remarkably, the constructed vaccine effectively induced both SARS-CoV-2-targeting antibodies and anti-influenza antibodies in mice, consequently affording protection from lethal SARS-CoV-2 and H7N9 challenges as well as effective H3N2 control. We propose our AdC68-vectored coronavirus-influenza vaccine as a universal approach toward curbing respiratory virus-causing pandemics. IMPORTANCE The COVID-19 pandemic exemplifies the severe public health threats of respiratory virus infection and influenza A viruses. The currently envisioned strategy for the prevention of respiratory virus-causing diseases requires the comprehensive administration of vaccines tailored for individual viruses. Here, we present an alternative strategy by designing chimpanzee adenovirus 68-based vaccines which target both the SARS-CoV-2 receptor-binding-domain and the conserved stalk of influenza hemagglutinin. When tested in mice, this strategy attained potent neutralizing antibodies against wild-type SARS-CoV-2 and its emerging variants, enabling an effective protection against lethal SARS-CoV-2 challenge. Notably, it also provided complete protection from lethal H7N9 challenge and efficient control of H3N2-induced morbidity. Our study opens a new avenue to universally curb respiratory virus infection by vaccination.

Altering the Immunogenicity of Hemagglutinin Immunogens by Hyperglycosylation and Disulfide Stabilization.

Influenza virus alters glycosylation patterns on its surface exposed glycoproteins to evade host adaptive immune responses. The viral hemagglutinin (HA), in particular the H3 subtype, has increased its overall surface glycosylation since its introduction in 1968. We previously showed that modulating predicted N-linked glycosylation sites on H3 A/Hong Kong/1/1968 HA identified a conserved epitope at the HA interface. This epitope is occluded on the native HA trimer but is likely exposed during HA "breathing" on the virion surface. Antibodies directed to this site are protective via an ADCC-mediated mechanism. This glycan engineering strategy made an otherwise subdominant epitope dominant in the murine model. Here, we asked whether cysteine stabilization of the hyperglycosylated HA trimer could reverse this immunodominance by preventing access to the interface epitope and focus responses to the HA receptor binding site (RBS). While analysis of serum responses from immunized mice did not show a redirection to the RBS, cysteine stabilization did result in an overall reduction in immunogenicity of the interface epitope. Thus, glycan engineering and cysteine stabilization are two strategies that can be used together to alter immunodominance patterns to HA. These results add to rational immunogen design approaches used to manipulate immune responses for the development of next-generation influenza vaccines.

Effect of an Adenovirus-Vectored Universal Influenza Virus Vaccine on Pulmonary Pathophysiology in a Mouse Model.

Current influenza vaccines, live attenuated or inactivated, do not protect against antigenically novel influenza A viruses (IAVs) of pandemic potential, which has driven interest in the development of universal influenza vaccines. Universal influenza vaccine candidates targeting highly conserved antigens of IAV nucleoprotein (NP) are promising as vaccines that induce T cell immunity, but concerns have been raised about the safety of inducing robust CD8 T cell responses in the lungs. Using a mouse model, we systematically evaluated effects of recombinant adenovirus vectors (rAd) expressing IAV NP (A/NP-rAd) or influenza B virus (IBV) NP (B/NP-rAd) on pulmonary inflammation and function after vaccination and following live IAV challenge. After A/NP-rAd or B/NP-rAd vaccination, female mice exhibited robust systemic and pulmonary vaccine-specific B cell and T cell responses and experienced no morbidity (e.g., body mass loss). Both in vivo pulmonary function testing and lung histopathology scoring revealed minimal adverse effects of intranasal rAd vaccination compared with unvaccinated mice. After IAV challenge, A/NP-rAd-vaccinated mice experienced significantly less morbidity, had lower pulmonary virus titers, and developed less pulmonary inflammation than unvaccinated or B/NP-rAd-vaccinated mice. Based on analysis of pulmonary physiology using detailed testing not previously applied to the question of T cell damage, mice protected by vaccination also had better lung function than controls. Results provide evidence that, in this model, adenoviral universal influenza vaccine does not damage pulmonary tissue. In addition, adaptive immunity, in particular, T cell immunity in the lungs, does not cause damage when restimulated but instead mitigates pulmonary damage following IAV infection.IMPORTANCE Respiratory viruses can emerge and spread rapidly before vaccines are available. It would be a tremendous advance to use vaccines that protect against whole categories of viruses, such as universal influenza vaccines, without the need to predict which virus will emerge. The nucleoprotein (NP) of influenza virus provides a target conserved among strains and is a dominant T cell target. In animals, vaccination to NP generates powerful T cell immunity and long-lasting protection against diverse influenza strains. Concerns have been raised, but not evaluated experimentally, that potent local T cell responses might damage the lungs. We analyzed lung function in detail in the setting of such a vaccination. Despite CD8 T cell responses in the lungs, lungs were not damaged and functioned normally after vaccination alone and were protected upon subsequent infection. This precedent provides important support for vaccines based on T cell-mediated protection, currently being considered for both influenza and SARS-CoV-2 vaccines.

A replication-competent adenovirus-vectored influenza vaccine induces durable systemic and mucosal immunity.

BACKGROUNDTo understand the features of a replicating vaccine that might drive potent and durable immune responses to transgene-encoded antigens, we tested a replication-competent adenovirus type 4 encoding influenza virus H5 HA (Ad4-H5-Vtn) administered as an oral capsule or via tonsillar swab or nasal spray.METHODSViral shedding from the nose, mouth, and rectum was measured by PCR and culturing. H5-specific IgG and IgA antibodies were measured by bead array binding assays. Serum antibodies were measured by a pseudovirus entry inhibition, microneutralization, and HA inhibition assays.RESULTSAd4-H5-Vtn DNA was shed from most upper respiratory tract-immunized (URT-immunized) volunteers for 2 to 4 weeks, but cultured from only 60% of participants, with a median duration of 1 day. Ad4-H5-Vtn vaccination induced increases in H5-specific CD4+ and CD8+ T cells in the peripheral blood as well as increases in IgG and IgA in nasal, cervical, and rectal secretions. URT immunizations induced high levels of serum neutralizing antibodies (NAbs) against H5 that remained stable out to week 26. The duration of viral shedding correlated with the magnitude of the NAb response at week 26. Adverse events (AEs) were mild, and peak NAb titers were associated with overall AE frequency and duration. Serum NAb titers could be boosted to very high levels 2 to 5 years after Ad4-H5-Vtn vaccination with recombinant H5 or inactivated split H5N1 vaccine.CONCLUSIONReplicating Ad4 delivered to the URT caused prolonged exposure to antigen, drove durable systemic and mucosal immunity, and proved to be a promising platform for the induction of immunity against viral surface glycoprotein targets.TRIAL REGISTRATIONClinicalTrials.gov NCT01443936 and NCT01806909.FUNDINGIntramural and Extramural Research Programs of the NIAID, NIH (U19 AI109946) and the Centers of Excellence for Influenza Research and Surveillance (CEIRS), NIAID, NIH (contract HHSN272201400008C).

Heterosubtypic Protection Induced by a Live Attenuated Influenza Virus Vaccine Expressing Galactose-α-1,3-Galactose Epitopes in Infected Cells.

Anti-galactose-α-1,3-galactose (anti-α-Gal) antibody is naturally expressed at a high level in humans. It constitutes about 1% of immunoglobulins found in human blood. Here, we designed a live attenuated influenza virus vaccine that can generate α-Gal epitopes in infected cells in order to facilitate opsonization of infected cells, thereby enhancing vaccine-induced immune responses. In the presence of normal human sera, cells infected with this mutant can enhance phagocytosis of human macrophages and cytotoxicity of NK cells in vitro Using a knockout mouse strain that allows expression of anti-α-Gal antibody in vivo, we showed that this strategy can increase vaccine immunogenicity and the breadth of protection. This vaccine can induce 100% protection against a lethal heterosubtypic group 1 (H5) or group 2 (mouse-adapted H3) influenza virus challenge in the mouse model. In contrast, its heterosubtypic protective effect in wild-type or knockout mice that do not have anti-α-Gal antibody expression is only partial, demonstrating that the enhanced vaccine-induced protection requires anti-α-Gal antibody upon vaccination. Anti-α-Gal-expressing knockout mice immunized with this vaccine produce robust humoral and cell-mediated responses upon a lethal virus challenge. This vaccine can stimulate CD11blo/- pulmonary dendritic cells, which are known to be crucial for clearance of influenza virus. Our approach provides a novel strategy for developing next-generation influenza virus vaccines.IMPORTANCE Influenza A viruses have multiple HA subtypes that are antigenically diverse. Classical influenza virus vaccines are subtype specific, and they cannot induce satisfactory heterosubtypic immunity against multiple influenza virus subtypes. Here, we developed a live attenuated H1N1 influenza virus vaccine that allows the expression of α-Gal epitopes by infected cells. Anti-α-Gal antibody is naturally produced by humans. In the presence of this antibody, human cells infected with this experimental vaccine virus can enhance several antibody-mediated immune responses in vitro Importantly, mice expressing anti-α-Gal antibody in vivo can be fully protected by this H1N1 vaccine against a lethal H5 or H3 virus challenge. Our work demonstrates a new strategy for using a single influenza virus strain to induce broadly cross-reactive immune responses against different influenza virus subtypes.

Adenoviral Vectors as Vaccines for Emerging Avian Influenza Viruses.

It is evident that the emergence of infectious diseases, which have the potential for spillover from animal reservoirs, pose an ongoing threat to global health. Zoonotic transmission events have increased in frequency in recent decades due to changes in human behavior, including increased international travel, the wildlife trade, deforestation, and the intensification of farming practices to meet demand for meat consumption. Influenza A viruses (IAV) possess a number of features which make them a pandemic threat and a major concern for human health. Their segmented genome and error-prone process of replication can lead to the emergence of novel reassortant viruses, for which the human population are immunologically naïve. In addition, the ability for IAVs to infect aquatic birds and domestic animals, as well as humans, increases the likelihood for reassortment and the subsequent emergence of novel viruses. Sporadic spillover events in the past few decades have resulted in human infections with highly pathogenic avian influenza (HPAI) viruses, with high mortality. The application of conventional vaccine platforms used for the prevention of seasonal influenza viruses, such as inactivated influenza vaccines (IIVs) or live-attenuated influenza vaccines (LAIVs), in the development of vaccines for HPAI viruses is fraught with challenges. These issues are associated with manufacturing under enhanced biosafety containment, and difficulties in propagating HPAI viruses in embryonated eggs, due to their propensity for lethality in eggs. Overcoming manufacturing hurdles through the use of safer backbones, such as low pathogenicity avian influenza viruses (LPAI), can also be a challenge if incompatible with master strain viruses. Non-replicating adenoviral (Ad) vectors offer a number of advantages for the development of vaccines against HPAI viruses. Their genome is stable and permits the insertion of HPAI virus antigens (Ag), which are expressed in vivo following vaccination. Therefore, their manufacture does not require enhanced biosafety facilities or procedures and is egg-independent. Importantly, Ad vaccines have an exemplary safety and immunogenicity profile in numerous human clinical trials, and can be thermostabilized for stockpiling and pandemic preparedness. This review will discuss the status of Ad-based vaccines designed to protect against avian influenza viruses with pandemic potential.

Protection of White Leghorn chickens by recombinant fowlpox vector vaccine with an updated H5 insert against Mexican H5N2 avian influenza viruses.

Despite decades of vaccination, surveillance, and biosecurity measures, H5N2 low pathogenicity avian influenza (LPAI) virus infections continue in Mexico and neighboring countries. One explanation for tenacity of H5N2 LPAI in Mexico is the antigenic divergence of circulating field viruses compared to licensed vaccines due to antigenic drift. Our phylogenetic analysis indicates that the H5N2 LPAI viruses circulating in Mexico and neighboring countries since 1994 have undergone antigenic drift away from vaccine seed strains. Here we evaluated the efficacy of a new recombinant fowlpox virus vector containing an updated H5 insert (rFPV-H5/2016), more relevant to the current strains circulating in Mexico. We tested the vaccine efficacy against a closely related subcluster 4 Mexican H5N2 LPAI (2010 H5/LP) virus and the historic H5N2 HPAI (1995 H5/HP) virus in White Leghorn chickens. The rFPV-H5/2016 vaccine provided hemagglutinin inhibition (HI) titers pre-challenge against viral antigens from both challenge viruses in almost 100% of the immunized birds, with no differences in number of birds seroconverting or HI titers among all tested doses (1.5, 2.0, and 3.1 log10 mean tissue culture infectious doses/bird). The vaccine conferred 100% clinical protection and a significant decrease in oral and cloacal virus shedding from 1995 H5/HP virus challenged birds when compared to the sham controls at all tested doses. Virus shedding titers from vaccinated 2010 H5/LP virus challenged birds significantly decreased compared to sham birds especially at earlier time points. Our results confirm the efficacy of the new rFPV-H5/2016 against antigenic drift of LPAI virus in Mexico and suggest that this vaccine would be a good candidate, likely as a primer in a prime-boost vaccination program.

Amino Acid Mutations A286V and T437M in the Nucleoprotein Attenuate H7N9 Viruses in Mice.

The low-pathogenic H7N9 influenza viruses that emerged in 2013 acquired an insertion of four amino acids in their hemagglutinin cleavage site and thereby became highly pathogenic to chickens in 2017. Previous studies indicated that these highly pathogenic H7N9 viruses are virulent in chickens but have distinct pathotypes in mice. A/chicken/Guangdong/SD098/2017 (CK/SD098) is avirulent, with a 50% mouse lethal dose (MLD50) of >7.5 log10 50% egg infectious dose (EID50), whereas A/chicken/Hunan/S1220/2017 (CK/S1220) is virulent in mice, with an MLD50 of 3.2 log10 EID50 In this study, we explored the genetic determinants that contribute to the difference in virulence between these two H7N9 viruses by generating a series of reassortants and mutants in the CK/S1220 virus background and testing their virulence in mice. We found that the reassortant CK/1220-SD098-NP, carrying the nucleoprotein (NP) of CK/SD098, was avirulent in mice, with an MLD50 of >107.5 EID50 The NPs of these two viruses differ by two amino acids, at positions 286 and 437. We further demonstrated that the amino acid mutations A286V and T437M of NP independently slowed the process of NP import to and export from the nucleus and thus jointly impaired the viral life cycle and attenuated the virulence of these H7N9 viruses in mice. Our study identified new virulence determinants in NP and provided novel targets for the development of live attenuated vaccines and antiviral drugs against influenza viruses.IMPORTANCE The H7N9 influenza viruses that emerged in China in 2013 have caused over 1,500 human infections, with a mortality rate of nearly 40%. The viruses were initially low pathogenic but became highly pathogenic in chickens at the beginning of 2017 and caused severe disease outbreaks in poultry. Several studies suggested that the highly pathogenic H7N9 viruses have increased virulence in mammals; however, the genetic basis of the virulence of H7N9 viruses in mammals is not fully understood. Here, we found that two amino acids, 286A and 437T, in NP are prerequisites for the virulence of H7N9 viruses in mice and the mutations A286V and T437M collectively eliminate the virulence of H7N9 viruses in mice. Our study further demonstrated that the virulence of influenza viruses is a polygenic trait, and the newly identified virulence-related residues in NP may provide new targets for attenuated influenza vaccine and antiviral drug development.

Mucosal CD8+ T cell responses induced by an MCMV based vaccine vector confer protection against influenza challenge.

Cytomegalovirus (CMV) is a ubiquitous ß-herpesvirus that establishes life-long latent infection in a high percentage of the population worldwide. CMV induces the strongest and most durable CD8+ T cell response known in human clinical medicine. Due to its unique properties, the virus represents a promising candidate vaccine vector for the induction of persistent cellular immunity. To take advantage of this, we constructed a recombinant murine CMV (MCMV) expressing an MHC-I restricted epitope from influenza A virus (IAV) H1N1 within the immediate early 2 (ie2) gene. Only mice that were immunized intranasally (i.n.) were capable of controlling IAV infection, despite the greater potency of the intraperitoneally (i.p.) vaccination in inducing a systemic IAV-specific CD8+ T cell response. The protective capacity of the i.n. immunization was associated with its ability to induce IAV-specific tissue-resident memory CD8+ T (CD8TRM) cells in the lungs. Our data demonstrate that the protective effect exerted by the i.n. immunization was critically mediated by antigen-specific CD8+ T cells. CD8TRM cells promoted the induction of IFNγ and chemokines that facilitate the recruitment of antigen-specific CD8+ T cells to the lungs. Overall, our results showed that locally applied MCMV vectors could induce mucosal immunity at sites of entry, providing superior immune protection against respiratory infections.

Mannosylation of LNP Results in Improved Potency for Self-Amplifying RNA (SAM) Vaccines.

Mannosylation of Lipid Nanoparticles (LNP) can potentially enhance uptake by Antigen Presenting Cells, which are highly abundant in dermal tissues, to improve the potency of Self Amplifying mRNA (SAM) vaccines in comparison to the established unmodified LNP delivery system. In the current studies, we evaluated mannosylated LNP (MLNP), which were obtained by incorporation of a stable Mannose-cholesterol amine conjugate, for the delivery of an influenza (hemagglutinin) encoded SAM vaccine in mice, by both intramuscular and intradermal routes of administration. SAM MLNP exhibited in vitro enhanced uptake in comparison to unglycosylated LNP from bone marrow-derived dendritic cells, and in vivo more rapid onset of the antibody response, independent of the route. The increased binding antibody levels also translated into higher functional hemagglutinin inhibition titers, particularly following intradermal administration. T cell assay on splenocytes from immunized mice also showed an increase in antigen specific CD8+ T responses, following intradermal administration of MLNP SAM vaccines. Induction of enhanced antigen specific CD4+ T cells, correlating with higher IgG2a antibody responses, was also observed. Hence, the present work illustrates the benefit of mannosylation of LNPs to achieve a faster immune response with SAM vaccines and these observations could contribute to the development of novel skin delivery systems for SAM vaccines.

Mucosal vaccine based on attenuated influenza virus and the group B Streptococcus recombinant peptides protected mice from influenza and S. pneumoniae infections.

Although many influenza-related deaths are attributable to secondary bacterial infection with S. pneumoniae, vaccines that simultaneously protect against influenza and pneumococcal infection are currently not developed. The aim of our study was to evaluate the possibility to prevent post-influenza pneumococcal infection using an associated vaccine based on live influenza vaccine (LAIV) combined with recombinant polypeptides derived from superficial factors of Group B streptococcus (GBS) determining pathogenicity. We demonstrated in a model of post-influenza pneumococcal pneumonia that intranasal pneumococcal super-infection seriously complicated the course of A/Shanghai/2/2013(H7N9) CDC-RG virus infection in mice. Associated immunization using LAIV and GBS vaccine (GBSV) prevented post-influenza pneumococcal pneumonia better than mono-LAIV or GBSV immunization. At the same time, parenteral pneumococcal post-influenza infection of immune mice was more severe in the groups immunized using recombinant GBS peptides which can be explained by antibody-dependent enhancement of infection. In this case, the introduction of blockers of histamine receptors type 1 and 2 reduced the burden of secondary pneumococcal infection.