Live Attenuated Influenza Virus Vaccine Research Articles

Expression of the SARS-CoV-2 receptor-binding domain by live attenuated influenza vaccine virus as a strategy for designing a bivalent vaccine against COVID-19 and influenza

Influenza and SARS-CoV-2 are two major respiratory pathogens that cocirculate in humans and cause serious illness with the potential to exacerbate disease in the event of co-infection. To develop a bivalent vaccine, capable of protecting against both infections, we inserted the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein into hemagglutinin (HA) molecule or into the open reading frame of the truncated nonstructural protein 1 (NS1) of live attenuated influenza vaccine (LAIV) virus and assessed phenotypic characteristics of the rescued LAIV-RBD viruses, as well as their immunogenicity in mouse and Syrian hamster animal models. A panel of 9 recombinant LAIV-RBD viruses was rescued using the A/Leningrad/17 backbone. Notably, only two variants with RBD insertions into the HA molecule could express sufficient quantities of RBD protein in infected MDCK cells. Intranasal immunization of mice induced high levels of anti-influenza antibody responses in all chimeric LAIV-RBD viruses, which was comparable to the LAIV virus vector. The RBD-specific antibody responses were most pronounced in the variant expressing RBD194 fragment as a chimeric HA protein. This candidate was further tested in Syrian hamsters and was shown to be immunogenic and capable of protecting animals against both infections.

Clade 2.3.4.4 H5 chimeric cold-adapted attenuated influenza vaccines induced cross-reactive protection in mice and ferrets.

Clade 2.3.4.4 H5Nx avian influenza viruses (AIVs) have circulated globally and caused substantial economic loss. Increasing numbers of humans have been infected with Clade 2.3.4.4 H5N6 AIVs in recent years. Only a few human influenza vaccines have been licensed to date. However, the licensed live attenuated influenza virus vaccine exhibited the potential of being recombinant with the wild-type influenza A virus (IAV). Therefore, we developed a chimeric cold-adapted attenuated influenza vaccine based on the Clade 2.3.4.4 H5 AIVs. These H5 vaccines demonstrate the advantage of being non-recombinant with circulated IAVs in the future influenza vaccine study. The findings of our current study reveal that these H5 vaccines can induce cross-reactive protective efficacy in mice and ferrets. Our H5 vaccines may provide a novel option for developing human-infected Clade 2.3.4.4 H5 AIV vaccines.

A Bivalent Live Attenuated Influenza Virus Vaccine Protects against Drifted H1N2 and H3N2 Clinical Isolates in Swine

Influenza A viruses (IAVs) can cause a highly contagious respiratory disease for many mammalian species. In pigs, IAVs cause high morbidity and low mortality disease in susceptible populations that can have significant financial and production impacts. They can also present opportunities for mutations and gene reassortment, producing influenza strains with pandemic potential. Therefore, it is very important to prevent and control influenza infection in pigs, and the chief way to do so is through vaccination. The subtypes of IAV most prevalent in swine across the world are H1N1, H1N2, and H3N2; however, genetic diversity of these viruses can vary greatly by region. We previously developed an elastase-dependent bivalent live attenuated vaccine using two Canadian swine influenza A virus (swIAV) isolates, A/Swine/Alberta/SD0191/2016 (H1N2) [SD191] and A/Swine/Saskatchewan/SD0069/2015 (H3N2) [SD69], which provided protection against homologous strains. In this study, we demonstrate that this vaccine extends protection in pigs to more current, drifted non-homologous H1N2 and H3N2 strains, A/Swine/MB/SD0467/2019 (H1N2) [SD467] and A/Swine/AB/SD0435/2019 (H3N2) [SD435]. The vaccine elicited a robust immune response in the serum and the lung and reduced viral replication as well as lung pathology associated with these strains. Therefore, this bivalent vaccine remains a strong candidate that would be beneficial to the swine influenza vaccine market in North America.

Cutting Edge: High-Dose Live Attenuated Influenza Vaccines Elicit Pulmonary Tissue-Resident Memory CD8+ T Cells in the Face of Pre-Existing Humoral Immunity.

In this study, we investigated how pre-existing Ab immunity to influenza virus established from prior immunizations affects the development of CD8+ T cell responses evoked after vaccination with a live attenuated vaccine. Using a mouse model and a panel of live attenuated influenza virus vaccine candidates (cold adapted and single cycle), we show that pre-existing influenza-specific Abs directed against the vaccine backbone attenuate the size and quality of the vaccine-induced CD8+ T cell response. Importantly, we show that increasing the vaccine dose can overcome this impediment, resulting in improved vaccine-induced circulating and tissue-resident memory CD8+ T cell responses, which were protective against heterologous influenza challenge. Thus, the reduced size and quality of the T cell response elicited by a live attenuated influenza virus vaccine imparted by the influenza-specific Ab landscape of the vaccinee can be overcome by increasing vaccine dose.

Evaluating α-galactosylceramide as an adjuvant for live attenuated influenza vaccines in pigs

Natural killer T (NKT) cells activated with the glycolipid ligand α-galactosylceramide (α-GalCer) stimulate a wide variety of immune cells that enhance vaccine-mediated immune responses. Several studies have used this approach to adjuvant inactivated and subunit influenza A virus (IAV) vaccines, including to enhance cross-protective influenza immunity. However, less is known about whether α-GalCer can enhance live attenuated influenza virus (LAIV) vaccines, which usually induce superior heterologous and heterosubtypic immunity compared to non-replicating influenza vaccines. The current study used the swine influenza challenge model to assess whether α-GalCer can enhance cross-protective immune responses elicited by a recombinant H3N2 LAIV vaccine (TX98ΔNS1) encoding a truncated NS1 protein. In one study, weaning pigs were administered the H3N2 TX98ΔNS1 LAIV vaccine with 0, 10, 50, and 100 μg/kg doses of α-GalCer, and subsequently challenged with a heterologous H3N2 virus. All treatment groups were protected from infection. However, the addition of α-GalCer appeared to suppress nasal shedding of the LAIV vaccine. In another experiment, pigs vaccinated with the H3N2 LAIV, with or without 50 μg/kg of α-GalCer, were challenged with the heterosubtypic pandemic H1N1 virus. Pigs vaccinated with the LAIV alone generated cross-reactive humoral and cellular responses which blocked virus replication in the airways, and significantly decreased virus shedding. On the other hand, combining the vaccine with α-GalCer reduced cross-protective cellular and antibody responses, and resulted in higher virus titers in respiratory tissues. These findings suggest that: (i) high doses of α-GalCer impair the replication and nasal shedding of the LAIV vaccine; and (ii) α-GalCer might interfere with heterosubtypic cross-protective immune responses. This research raise concerns that should be considered before trying to use NKT cell agonists as a possible adjuvant approach for LAIV vaccines.

Haemagglutinin substitutions N125D, D127E, D222G and R223Q improve replicative fitness and vaccine effectiveness of an A/H1N1pdm09 live attenuated influenza vaccine virus by enhancing α-2,6 receptor binding.

During 2013–14 and 2015–16, A/H1N1pdm09 live attenuated influenza vaccine (LAIV) viruses replicated inefficiently in primary human nasal epithelial cells (hNEC). This led to reduced vaccine effectiveness (VE) in quadrivalent formulations, mediated by inter-strain competition. By mutating the haemagglutinin (HA) protein, we aimed to enhance hNEC replication of a novel A/H1N1pdm09 vaccine strain to overcome competition and improve VE. Combinations of N125D, D127E, D222G and R223Q substitutions were introduced to the HA protein of A/Slovenia/2903/2015 (A/SLOV15). A/SLOV15 S13, containing all four HA substitutions, produced approximately 1000-fold more virus than parental V1 during hNEC infection. Immunogenicity in ferrets was increased by approximately 10-fold, without compromising yield in eggs or antigenic match to wild-type (wt) reference strains. Despite S13 and V1 being antigenically similar, only S13 protected ferrets from wt virus shedding and fever post-challenge. Crucially, these data suggested that enhanced fitness allowed S13 to overcome inter-strain competition in quadrivalent LAIV (QLAIV). This improved efficacy was later validated by real-world VE data. S13 displayed increased binding avidity to a mammalian-like α-2,6 receptor analogue (6-SLN), relative to V1, while maintaining avian-like 3-SLN avidity. In silico modelling of the HA receptor binding site revealed additional interactions in the S13:6-SLN binding network and a mild increase in 6-SLN binding energy, indicating a possible mechanism for increased α-2,6 receptor-binding avidity. These data confirm that rational HA mutagenesis can be used to optimise hNEC replication and VE for A/H1N1pdm09 LAIV viruses.

Efficacy of the NS1-truncated live attenuated influenza virus vaccine for swine against infection with viruses of major North American and European H3N2 lineages

Control of swine influenza A virus (swIAV) in North America and Europe is complicated because multiple antigenically distinct swIAV strains co-circulate in the field, and no vaccine is available that can provide broad cross-protection against all these swIAVs. In 2017, the first live attenuated influenza vaccine (LAIV) for swine was licensed in the US. The non-structural protein 1 (NS1)-truncated cluster I H3N2 strain A/swine/Texas/4199-2/98 NS1del126 (TX98 LAIV) in this vaccine provides partial cross-protection against heterologous North American cluster II and IV H3N2 swIAV strains. Its efficacy against European or more recent North American H3N2 lineages remains to be investigated. In this study, we evaluated the level of cross-protection against heterologous IAVs representative of the major H3N2 swIAV lineages in Europe and North America. TX98 LAIV prevented both nasal shedding and replication in the lungs of a North American cluster IV H3N2 swIAV for 2/4 pigs, prevented considerable nasal shedding of a North American novel human-like H3N2 swIAV for 2/4 pigs, and reduced replication of a European H3N2 swIAV in the lower respiratory tract to minimal titers for 1/3 pigs. Although TX98 LAIV elicited neutralizing antibodies against the homologous virus in serum and to a lesser extent in nose and lungs, no significant cross-reactive antibody titers against the heterologous swIAVs were detected. Partial cross-protection therefore likely relies on cellular and mucosal immune responses against conserved parts of the swIAV proteins. Since TX98 LAIV can offer partial protection against a broad range of H3N2 swIAVs, it might be a suitable priming vaccine for use in a heterologous prime-boost vaccination strategy.

Construction of the vaccine strain of the influenza B virus with chimeric hemagglutinin to induce a cross-protective immune response

BACKGROUND: Influenza viruses cause worldwide epidemics, and the most effective method to prevent influenza disease is regular vaccinations. The development of new generation vaccines is aimed primarily at the formation of an immune response against a wide range of influenza viruses. One of the promising approaches is sequential vaccination with chimeric influenza viruses with identical stem domains of the hemagglutinin surface protein.
 AIM: The development of an experimental vaccine strain of influenza B virus with chimeric hemagglutinin consisting of head and stem domains of influenza B viruses belonging to different genetic lineages.
 MATERIALS AND METHODS: A chimeric influenza hemagglutinin gene was obtained by genetic engineering from the genetic material of B/Victoria and B/Yamagata influenza strains. The gene was inserted into the vector for the reverse genetics of the influenza virus. The influenza B virus strain with chimeric hemagglutinin was obtained by transfection of Vero cells using an 8-plasmid system. The rest of the genes were obtained from the attenuated influenza B virus with cold-adapted and temperature-sensitive phenotypes. The biological properties of the obtained recombinant strain, its infectious titer in developing chicken embryos and MDCK cell culture were evaluated.
 RESULTS: A recombinant vaccine strain has been successfully rescued. The head domain of the hemagglutinin of the virus is inherited from the B/Victoria influenza virus, and the stem domain from the B/Yamagata virus. The virus actively replicated in eggs and MDCK cells, with temperature-sensitive and cold-adapted phenotypes identical to classical live attenuated influenza vaccine viruses. The thermal stability of the chimeric hemagglutinin did not differ significantly from the thermal stability of the hemagglutinins of the donor viruses.
 CONCLUSIONS: The results obtained indicate the possibility of creating a strain with chimeric hemagglutinin, fragments of which are inherited from different genetic lineages. The growth characteristics and biological properties of the strain make it a promising candidate for the experimental evaluation of the possibility of inducing a cross-protective immune response by sequential vaccination with vaccine strains with identical stem hemagglutinin domains.

Interferon mediated prophylactic protection against respiratory viruses conferred by a prototype live attenuated influenza virus vaccine lacking non-structural protein 1

The influenza A non-structural protein 1 (NS1) is known for its ability to hinder the synthesis of type I interferon (IFN) during viral infection. Influenza viruses lacking NS1 (ΔNS1) are under clinical development as live attenuated human influenza virus vaccines and induce potent influenza virus-specific humoral and cellular adaptive immune responses. Attenuation of ΔNS1 influenza viruses is due to their high IFN inducing properties, that limit their replication in vivo. This study demonstrates that pre-treatment with a ΔNS1 virus results in an antiviral state which prevents subsequent replication of homologous and heterologous viruses, preventing disease from virus respiratory pathogens, including SARS-CoV-2. Our studies suggest that ΔNS1 influenza viruses could be used for the prophylaxis of influenza, SARS-CoV-2 and other human respiratory viral infections, and that an influenza virus vaccine based on ΔNS1 live attenuated viruses would confer broad protection against influenza virus infection from the moment of administration, first by non-specific innate immune induction, followed by specific adaptive immunity.

FluB-RAM and FluB-RANS: Genome Rearrangement as Safe and Efficacious Live Attenuated Influenza B Virus Vaccines.

Influenza B virus (IBV) is considered a major respiratory pathogen responsible for seasonal respiratory disease in humans, particularly severe in children and the elderly. Seasonal influenza vaccination is considered the most efficient strategy to prevent and control IBV infections. Live attenuated influenza virus vaccines (LAIVs) are thought to induce both humoral and cellular immune responses by mimicking a natural infection, but their effectiveness has recently come into question. Thus, the opportunity exists to find alternative approaches to improve overall influenza vaccine effectiveness. Two alternative IBV backbones were developed with rearranged genomes, rearranged M (FluB-RAM) and a rearranged NS (FluB-RANS). Both rearranged viruses showed temperature sensitivity in vitro compared with the WT type B/Bris strain, were genetically stable over multiple passages in embryonated chicken eggs and were attenuated in vivo in mice. In a prime-boost regime in naïve mice, both rearranged viruses induced antibodies against HA with hemagglutination inhibition titers considered of protective value. In addition, antibodies against NA and NP were readily detected with potential protective value. Upon lethal IBV challenge, mice previously vaccinated with either FluB-RAM or FluB-RANS were completely protected against clinical disease and mortality. In conclusion, genome re-arrangement renders efficacious LAIV candidates to protect mice against IBV.

Universal Live-Attenuated Influenza Vaccine Candidates Expressing Multiple M2e Epitopes Protect Ferrets against a High-Dose Heterologous Virus Challenge.

The development of an influenza vaccine with broad protection and durability remains an attractive idea due to the high mutation rate of the influenza virus. An extracellular domain of Matrix 2 protein (M2e) is among the most attractive target for the universal influenza vaccine owing to its high conservancy rate. Here, we generated two recombinant live attenuated influenza vaccine (LAIV) candidates encoding four M2e epitopes representing consensus sequences of human, avian and swine influenza viruses, and studied them in a preclinical ferret model. Both LAIV+4M2e viruses induced higher levels of M2e-specific antibodies compared to the control LAIV strain, with the LAIV/HA+4M2e candidate being significantly more immunogenic than the LAIV/NS+4M2e counterpart. A high-dose heterosubtypic influenza virus challenge revealed the highest degree of protection after immunization with LAIV/HA+4M2e strain, followed by the NS-modified LAIV and the classical LAIV virus. Furthermore, only the immune sera from the LAIV/HA+4M2e-immunized ferrets protected mice from a panel of lethal influenza viruses encoding M genes of various origins. These data suggest that the improved cross-protection of the LAIV/HA+4M2e universal influenza vaccine candidate was mediated by the M2e-targeted antibodies. Taking into account the safety profile and improved cross-protective potential, the LAIV/HA+4M2e vaccine warrants its further evaluation in a phase I clinical trial.

Changes in sialic acid binding associated with egg adaptation decrease live attenuated influenza virus replication in human nasal epithelial cell cultures

Live Attenuated Influenza Virus (LAIV) is administered to and replicates in the sinonasal epithelium. Candidate LAIV vaccine strains are selected based on their ability to replicate to a high titer in embryonated hen’s eggs, a process that can lead to mutations which alter the receptor binding and antigenic structure of the hemagglutinin (HA) protein. In the 2012–2013 northern hemisphere vaccine, the H3N2 HA vaccine strain contained three amino acid changes - H156Q, G186V and S219Y – which altered HA antigenic structure and thus presumably decreased vaccine efficacy. To determine if these mutations also altered LAIV replication, reabcombinant viruses were created that encoded the wild-type (WT) parental HA of A/Victoria/361/2011 (WT HA LAIV), the egg adapted HA (EA HA LAIV) from the A/Victoria/361/2011 vaccine strain and an HA protein with additional amino acid changes to promote α2,3 sialic acid binding (2,3 EA HA LAIV). The WT HA LAIV bound α2,6 sialic compared to the EA HA LAIV and 2,3 EA HA LAIV which both demonstrated an increased preference for α2,3 sialic acid. On MDCKs, the WT HA and EA HA LAIVs showed similar replication at 32 °C but at 37 °C the EA HA LAIV replicated to lower infectious virus titers. The 2,3 EA HA LAIV replicated poorly at both temperatures. This replication phenotype was similar on human nasal epithelial cell (hNEC) cultures, however the WT HA LAIV induced the highest amount of IFN-λ and infected more nasal epithelial cells compared to the other viruses. Together, these data indicate that egg adaption mutations in the HA protein that confer preferential α2,3 sialic acid binding may adversely affect LAIV replication and contribute to reduced vaccine efficacy.

Bat influenza vectored NS1-truncated live vaccine protects pigs against heterologous virus challenge

Swine influenza is an important disease for the swine industry. Currently used whole inactivated virus (WIV) vaccines can induce vaccine-associated enhanced respiratory disease (VAERD) in pigs when the vaccine strains mismatch with the infected viruses. Live attenuated influenza virus vaccine (LAIV) is effective to protect pigs against homologous and heterologous swine influenza virus infections without inducing VAERD but has safety concerns due to potential reassortment with circulating viruses. Herein, we used a chimeric bat influenza Bat09:mH3mN2 virus, which contains both surface HA and NA gene open reading frames of the A/swine/Texas/4199–2/1998 (H3N2) and six internal genes from the novel bat H17N10 virus, to develop modified live-attenuated viruses (MLVs) as vaccine candidates which cannot reassort with canonical influenza A viruses by co-infection. Two attenuated MLV vaccine candidates including the virus that expresses a truncated NS1 (Bat09:mH3mN2-NS1-128, MLV1) or expresses both a truncated NS1 and the swine IL-18 (Bat09:mH3mN2-NS1-128-IL-18, MLV2) were generated and evaluated in pigs against a heterologous H3N2 virus using the WIV vaccine as a control. Compared to the WIV vaccine, both MLV vaccines were able to reduce lesions and virus replication in lungs and limit nasal virus shedding without VAERD, also induced significantly higher levels of mucosal IgA response in lungs and significantly increased numbers of antigen-specific IFN-γ secreting cells against the challenge virus. However, no significant difference was observed in efficacy between the MLV1 and MLV2. These results indicate that bat influenza vectored MLV vaccines can be used as a safe live vaccine to prevent swine influenza.

Shedding and transmission of a live attenuated influenza A virus vaccine in pre-weaned pigs under field conditions.

Influenza A virus (IAV) is one of the most important respiratory viruses affecting pig health and vaccination is the most common strategy to control influenza infections. In this field study we assessed the onset and duration of shedding of a live attenuated influenza virus (LAIV) vaccine, its ability to transmit to non-vaccinated pigs and whether the LAIV could be aerosolized and detected in the environment. Thirty-three litters (n = 33) of a farm using the LAIV vaccine were selected for the study, a subset of them (n = 12) were left unvaccinated and a subset of piglets (n = 3) in vaccinated litters were also left unvaccinated to serve as sentinels. Selected piglets from the litters were sampled multiple days post vaccination (DPV) by collecting nasal swabs and blood, and were tested using a LAIV vaccine specific RT-PCR assay and hemagglutination inhibition assay against the LAIV strains respectively. Environmental specimens consisting of air and surface wipes were also collected. One hundred percent (21/21) of the vaccinated litters tested LAIV positive 1 DPV and until 6 DPV. In contrast, only five (5/33) of the thirty-three non-vaccinated pigs tested positive during the course of the study. Viable LAIV was confirmed in vaccinated pigs by cell culture and whole genome sequencing. In addition, low levels of LAIV RNA (RT-PCR Ct values ranging between 33 and 38) were detected in all air specimens collected on the day of vaccination and until 6 DPV (3/10). Pigs had maternally derived antibodies reactive against the LAIV strains which may have influenced the degree of shedding observed. Under the conditions of this study, shedding of the LAIV from vaccinated pigs was limited in time, resulted in minimal transmission to non-vaccinated pigs and was detected in low levels in aerosols collected in the vaccinated rooms likely influenced by the presence of maternally derived antibodies against the LAIV strains.

Safety, Immunogenicity, and Protective Efficacy of a Chimeric A/B Live Attenuated Influenza Vaccine in a Mouse Model.

Influenza A and B viruses cause significant morbidity and mortality worldwide. Current influenza vaccines are composed of three or four strains: A/H1N1, A/H3N2, and B (Victoria and Yamagata lineages). It is of great interest if immunization against both type A and B influenza viruses can be combined in a single vaccine strain, thus reducing the cost of vaccine production and the possibility of strain interference within the multicomponent vaccine. In the current study, we developed an experimental live cold-adapted influenza intertype reassortant (influenza A and B) vaccine on the live attenuated influenza vaccine (LAIV) A/Leningrad/134/17/57 backbone. Hemagglutinin (HA) and neuraminidase (NA) functional domains were inherited from the influenza B/Brisbane/60/2008 strain, whereas their packaging signals were substituted with appropriate fragments of influenza A virus genes. The recombinant A/B virus efficiently replicated in eggs and Madin–Darby Canine Kidney (MDCK) cells under optimal conditions, temperature-sensitive phenotype was maintained, and its antigenic properties matched the influenza B parental virus. The chimeric vaccine was attenuated in mice: after intranasal immunization, viral replication was seen only in nasal turbinates but not in the lungs. Immunological studies demonstrated the induction of IgG antibody responses against the influenza A and B virus, whereas hemagglutination inhibition (HAI) and neutralizing antibodies were detected only against the influenza B virus, resulting in significant protection of immunized animals against influenza B virus challenge. IFNγ-secreting CD8 effector memory T cells (CD44+CD62L−) were detected in mouse splenocytes after stimulation with the specific influenza A peptide (NP366); however, the T-cell response was not sufficient to protect animals against infection with a high-dose mouse-adapted A/California/07/2009 (H1N1pdm09) virus, most probably due to the mismatch of key T-cell epitopes of the H1N1 virus and the LAIV backbone. Overall, generation of the chimeric A/B LAIV virus on a licensed LAIV backbone demonstrated prospects for the development of safe and efficacious vaccine candidates that afford combined protection against both type A and type B influenza viruses; however, further optimization of the T-cell epitope content within the LAIV backbone may be required.

Chimeric Hemagglutinin-Based Live-Attenuated Vaccines Confer Durable Protective Immunity against Influenza A Viruses in a Preclinical Ferret Model.

Epidemic or pandemic influenza can annually cause significant morbidity and mortality in humans. We developed novel chimeric hemagglutinin (cHA)-based universal influenza virus vaccines, which contain a conserved HA stalk domain from a 2009 pandemic H1N1 (pH1N1) strain combined with globular head domains from avian influenza A viruses. Our previous reports demonstrated that prime-boost sequential immunizations induced robust antibody responses directed toward the conserved HA stalk domain in ferrets. Herein, we further followed vaccinated animals for one year to compare the efficacy and durability of these vaccines in the preclinical ferret model of influenza. Although all cHA-based immunization regimens induced durable HA stalk-specific and heterosubtypic antibody responses in ferrets, sequential immunization with live-attenuated influenza virus vaccines (LAIV-LAIV) conferred the best protection against upper respiratory tract infection by a pH1N1 influenza A virus. The findings from this study suggest that our sequential immunization strategy for a cHA-based universal influenza virus vaccine provides durable protective humoral and cellular immunity against influenza virus infection.

A bivalent live attenuated influenza virus vaccine protects against H1N2 and H3N2 viral infection in swine

Swine Influenza A virus (swIAV) poses a substantial burden to the swine industry due to its highly contagious nature, acute viral disease, and ability to cause up to 100 % morbidity. Currently, North American swine are predominately infected with three subtypes of swIAV: H1N1, H1N2, and H3N2. The ability of influenza viruses to cross both directions between humans and swine means that both human and swine-origin viruses as well as new reassortant viruses can pose a substantial public health or pandemic threat. Since the primary method of protection and control against influenza is through vaccination, more effective, new vaccine platforms need to be developed. This study uses two Canadian swIAV isolates, A/Swine/Alberta/SD0191/2016 (H1N2) [SD191] and A/Swine/Saskatchewan/SD0069/2015 (H3N2) [SD69] to design a bivalent live attenuated influenza virus vaccine (LAIV) through reverse genetics. The hemagglutinin (HA) cleavage site from both SD191-WT and SD69-WT were engineered from a trypsin-sensitive to an elastase-sensitive motif, to generate SD191-R342V and SD69-K345V, respectively. The elastase dependent SD191-R342V virus possesses a mutation from arginine to valine at amino acid (aa) 342 on HA, whereas the elastase dependent SD69-K345V virus possesses a mutation from lysine to valine at aa 345 on HA. Both elastase dependent swIAVs are completely dependent on elastase, display comparable growth properties to the wild type (WT) viruses, are genetically stable in vitro, and entirely non-virulent in pigs. Moreover, when these elastase dependent swIAVs were administered together in pigs, they were found to stimulate antibody responses and IFN-γ secreting cells, as well as prevent viral replication and lung pathology associated with WT H1N2 and H3N2 swIAV challenge. Therefore, this bivalent LAIV demonstrates the strong candidacy to protect swine against the predominant influenza subtypes in North America.

Immunogenicity and protective activity of recombinant influenza viruses expressing fragments of ScaAB lipoprotein of group B streptococci in a mouse model

Group B streptococci (GBS) cause a number of serious diseases in humans. The development of an effective vaccine against GBS requires special approaches. In the present study, three recombinant influenza viruses were constructed on the backbone of H7N9 live attenuated influenza vaccine (LAIV) strain expressing fragments of the ScaAB lipoprotein of Streptococcus agalactiae, fused to the surface protein of the virus, hemagglutinin, using a flexible linker. Recombinant viruses with ScaAB inserts of 85, 141, and 200 amino acids were successfully rescued by the means of reverse genetics. The recombinant strains were able to grow in developing chicken embryos and MDCK cells and retained the temperature-sensitive phenotype attributable to the LAIV viruses. Studies of immunogenicity and protective activity of the vaccine candidates in BALB/c mice revealed that the most promising strain was a strain with an insert of 141 amino acids: this variant had optimal immunogenicity against influenza and GBS and had a protective effect against both pathogens. These data indicate that further studies of the recombinant vectored vaccine H7-ScaAB-141 as a combined viral-bacterial vaccine capable of protection against both influenza virus and bacterial infections caused by group B streptococci are warranted.

Neutralizing epitope of the Fusion Protein of Respiratory Syncytial Virus Embedded in the HA Molecule of LAIV Virus is not Sufficient to Prevent RS Virus Pulmonary Replication but Ameliorates Lung Pathology following RSV Infection in Mice

Aims:To develop experimental bivalent vaccines against influenza and RSV using a cold-adapted LAIV backbone.Background:Respiratory syncytial virus (RSV) is a causative agent of bronchiolitis and pneumonia in young children, elderly and immunocompromised adults. No vaccine against RSV has been licensed to date for various reasons. One of the promising platforms for designing RSV vaccine is the use of live attenuated influenza vaccine (LAIV) viruses to deliver RSV epitopes to the respiratory mucosa.Objective:To generate recombinant LAIV viruses encoding a neutralizing epitope of the RSV fusion protein and assess their protective potential against both influenza and RSV infections in a mouse model.Methods:Reverse genetics methods were used to rescue recombinant LAIV+HA/RSV viruses expressing chimeric hemagglutinins encoding the RSV-F epitope at its N-terminus using two different flexible linkers. BALB/c mice were intranasally immunized with two doses of the recombinant viruses and then challenged with the influenza virus or RSV. The LAIV viral vector and formalin-inactivated RSV (FI-RSV) were included as control vaccines. Protection was assessed by the reduction of virus pulmonary titers. In addition, RSV-induced lung pathology was evaluated by histopathology studies.Results:Two rescued chimeric LAIV+HA/RSV viruses were identical to the LAIV vector in terms of replication capacityin vitroandin vivo. The RSV-F neutralizing epitope was successfully expressed only if inserted into the HA molecule via G-linker, but not A-linker. Both chimeric viruses induced high influenza-specific antibody levels and fully protected mice against a lethal influenza challenge virus. However, they induced weak anti-RSV antibody responses which did not prevent RS virus replication upon challenge, and only LAIV-HA+G-RSV variant protected mice against RSV-induced lung pathology.Conclusion:Although the designed LAIV-RSV chimeric viruses were unable to neutralize the RS virus pulmonary replication, the LAIV-HA+G-RSV reduced RSV-induced lung pathology and can be considered a promising bivalent vaccine against influenza and RSV infections and warrants its further development.

Conserved T-cell epitopes of respiratory syncytial virus (RSV) delivered by recombinant live attenuated influenza vaccine viruses efficiently induce RSV-specific lung-localized memory T cells and augment influenza-specific resident memory T-cell responses

Respiratory syncytial virus (RSV) can cause recurrent infection in people because it does not stimulate a long-lived immunological memory. There is an urgent need to develop a safe and efficacious vaccine against RSV that would induce immunological memory without causing immunopathology following natural RSV infection. We have previously generated two recombinant live attenuated influenza vaccine (LAIV) viruses that encode immunodominant T-cell epitopes of RSV M2 protein in the neuraminidase or NS1 genes. These chimeric vaccines afforded protection against influenza and RSV infection in mice, without causing pulmonary eosinophilia or inflammatory RSV disease. The current study assessed the formation of influenza-specific and RSV-specific CD4 and CD8 T-cell responses in the lungs of mice, with special attention to the lung tissue-resident memory T cell subsets (TRM). The RSV epitopes did not affect influenza-specific CD4 effector memory T cell (Tem) levels in the lungs. The majority of these cells formed by LAIV or LAIV-RSV viruses had CD69+CD103- phenotype. Both LAIV+NA/RSV and LAIV+NS/RSV recombinant viruses induced significant levels of RSV M282 epitope-specific lung-localized CD8 Tem cells expressing both CD69 and CD103 TRM markers. Surprisingly, the CD69+CD103+ influenza-specific CD8 Tem responses were augmented by the addition of RSV epitopes, possibly as a result of the local microenvironment formed by the RSV-specific memory T cells differentiating to TRM in the lungs of mice immunized with LAIV-RSV chimeric viruses. This study provides evidence that LAIV vector-based vaccination can induce robust lung-localized T-cell immunity to the inserted T-cell epitope of a foreign pathogen, without altering the immunogenicity of the viral vector itself.