Use of MDCK cells for production of live attenuated influenza vaccine

To develop a cell-based live attenuated influenza vaccine (LAIV) manufacturing process, several different cell lines were evaluated by comparing the titer of viruses after infection with LAIV strains. While several cell lines have been reported to support influenza virus replication, the degree of replication and the ability to support replication of LAIV strains have not been systematically examined. MDCK cells, which have been considered as potential substrates for influenza vaccine production were evaluated in addition to Vero, MRC-5, WI-38 and FRhL cells. MRC-5, WI-38 and FRhL cells produced low to moderate titers of virus with titers equal or below 5.0 log₁₀ TCID₅₀/mL. Both Vero and MDCK cells could support a higher level of virus replication for certain strains, however, Vero cells only produced high titers when grown in the presence of serum. MDCK cells supported high levels of vaccine virus production for multiple different LAIV subtypes in both serum containing and serum-free media. These results suggest that MDCK cell-based production can be used as an alternative production platform to the currently used egg-based LAIV production system.     

Genetic stability of live attenuated vaccines against potentially pandemic influenza viruses

BackgroundEnsuring genetic stability is a prerequisite for live attenuated influenza vaccine (LAIV). This study describes the results of virus shedding and clinical isolates’ testing of Phase I clinical trials of Russian LAIVs against potentially pandemic influenza viruses in healthy adults.MethodsThree live attenuated vaccines against potentially pandemic influenza viruses, H2N2 LAIV, H5N2 LAIV and H7N3 LAIV, generated by classical reassortment in eggs, were studied. For each vaccine tested, subjects were randomly distributed into two groups to receive two doses of either LAIV or placebo at a 3:1 vaccine/placebo ratio. Nasal swabs were examined for vaccine virus shedding by culturing in eggs and by PCR. Vaccine isolates were tested for temperature sensitivity and cold-adaptation (ts/ca phenotypes) and for nucleotide sequence.ResultsThe majority of nasal wash positive specimens were detected on the first day following vaccination. PCR method demonstrated higher sensitivity than routine virus isolation in eggs. None of the placebo recipients had detectable vaccine virus replication.All viruses isolated from the immunized subjects retained the ts/ca phenotypic characteristics of the master donor virus (MDV) and were shown to preserve all attenuating mutations described for the MDV. These data suggest high level of vaccine virus genetic stability after replication in humans.During manufacture process, no additional mutations occurred in the genome of H2N2 LAIV. In contrast, one amino acid change in the HA of H7N3 LAIV and two additional mutations in the HA of H5N2 LAIV manufactured vaccine lot were detected, however, they did not affect their ts/ca phenotypes.ConclusionsOur clinical trials revealed phenotypic and genetic stability of the LAIV viruses recovered from the immunized volunteers. In addition, no vaccine virus was detected in the placebo groups indicating the lack of person-to-person transmission.LAIV TRIAL REGISTRATION at ClinicalTrials.gov: H7N3-NCT01511419; H5N2-NCT01719783; H2N2-NCT01982331.     

Replication of live attenuated cold-adapted H2N2 influenza virus vaccine candidates in non human primates

The development of an H2N2 vaccine is a priority in pandemic preparedness planning. We previously showed that a single dose of a cold-adapted (ca) H2N2 live attenuated influenza vaccine (LAIV) based on the influenza A/Ann Arbor/6/60 (AA ca) virus was immunogenic and efficacious in mice and ferrets. However, in a Phase I clinical trial, viral replication was restricted and immunogenicity was poor. In this study, we compared the replication of four H2N2 LAIV candidate viruses, AA ca, A/Tecumseh/3/67 (TEC67 ca), and two variants of A/Japan/305/57 (JAP57 ca) in three non-human primate (NHP) species: African green monkeys (AGM), cynomolgus macaques (CM) and rhesus macaques (RM). One JAP57 ca virus had glutamine and glycine at HA amino acid positions 226 and 228 (Q–G) that binds to α2-3 linked sialic acids, and one had leucine and serine that binds to α2-3 and α2-6 linked residues (L–S). The replication of all ca viruses was restricted, with low titers detected in the upper respiratory tract of all NHP species, however replication was detected in significantly more CMs than AGMs. The JAP57 ca Q–G and TEC67 ca viruses replicated in a significantly higher percentage of NHPs than the AA ca virus, with the TEC67 ca virus recovered from the greatest percentage of animals. Altering the receptor specificity of the JAP57 ca virus from α2-3 to both α2-3 and α2-6 linked sialic acid residues did not significantly increase the number of animals infected or the titer to which the virus replicated. Taken together, our data show that in NHPs the AA ca virus more closely reflects the human experience than mice or ferret studies. We suggest that CMs and RMs may be the preferred species for evaluating H2N2 LAIV viruses, and the TEC67 ca virus may be the most promising H2N2 LAIV candidate for further evaluation.     

Restricted replication of the live attenuated influenza A virus vaccine during infection of primary differentiated human nasal epithelial cells

Live Attenuated Influenza Vaccine (LAIV) strains are associated with cold adapted, temperature sensitive and attenuated phenotypes that have been studied in non-human or immortalized cell cultures as well as in animal models. Using a primary, differentiated human nasal epithelial cell (hNEC) culture system we compared the replication kinetics, levels of cell-associated viral proteins and virus particle release during infection with LAIV or the corresponding wild type (WT) influenza viruses. At both 33 °C and 37 °C, seasonal influenza virus and an antigenically matched LAIV replicated to similar titers in MDCK cells but seasonal influenza virus replicated to higher titers than LAIV in hNEC cultures, suggesting a greater restriction of LAIV replication in hNEC cultures. Despite the disparity in infectious virus production, the supernatants from H1N1 and LAIV infected hNEC cultures had equivalent amounts of viral proteins and hemagglutination titers, suggesting the formation of non-infectious virus particles by LAIV in hNEC cultures.     

HA and M2 sequences alter the replication of 2013–16 H1 live attenuated influenza vaccine infection in human nasal epithelial cell cultures

From 2013 to 2016, the H1N1 component of live, attenuated influenza vaccine (LAIV) performed very poorly in contrast to the inactivated influenza vaccine. We utilized a primary, differentiated human nasal epithelial cell (hNEC) culture system to assess the replication differences between isogenic LAIVs containing the HA segment from either A/Bolivia/559/2013 (rBol), which showed poor vaccine efficacy, and A/Slovenia/2903/2015 (rSlov), which had reasonable vaccine efficacy. There were minimal differences in infectious virus production in Madin-Darby Canine Kidney (MDCK) cells, but the rSlov LAIV showed markedly improved replication in hNEC cultures at both 32 °C and 37 °C, demonstrating that the HA segment alone could impact LAIV replication in physiologically relevant systems. The rSlov-infected hNEC cultures showed stronger production of interferon and proinflammatory chemokines which might also be contributing to the increased overall vaccine effectiveness through enhanced recruitment and activation of immune cells. An M2-S86A mutation had no positive effects on H1 LAIV replication in hNEC cultures, in contrast to the increased infectious virus production seen in an H3 LAIV. No obvious defects in viral RNA packaging were detected, suggesting that HA function, rather than defective particle production, may be driving the differential infectious virus production in hNEC cultures. Overall, we have shown that not all H1 HA segments can be successfully used in LAIV, and this phenotype cannot be fully explained by segment incompatibilities. Physiologically relevant temperatures and primary cell cultures should be used to demonstrate that candidate LAIVs can replicate efficiently, which is a necessary property for effective vaccines.     

Evaluation of manufacturing feasibility and safety of an MDCK cell-based live attenuated influenza vaccine (LAIV) platform

Cell culture based live attenuated influenza vaccines (LAIV) as an alternative to egg-based LAIV have been explored because of lack of easy access to SPF eggs for large scale production. In this study, feasibility of MDCK platform was assessed by including multiple LAIV strains covering both type A (H1 and H3) and type B seasonal strains as well as the candidate pandemic potential strains like A/H5 and A/H7 for the growth in MDCK cells. A risk assessment study was conducted on the cell banks to evaluate safety concerns related to tumorigenicity with a regulatory perspective. Tumorigenic potential of the MDCK cells was evaluated in nude mice (10⁷cells/mouse) model system. The 50% tumor producing dose (TPD₅₀) of MDCK cells was studied in SCID mice to determine the amount of cells required for induction of tumors. Further, we conducted an oncogenicity study in three sensitive rodent species as per the requirements specified in the WHO guidelines.We determined TPD₅₀ value of 1.9 X 10⁴ cells/mice through subcutaneous route. Our results suggest that, the intranasal route of administration of the cell culture based LAIV pose minimal to no risk of tumorigenicity associated with the host cells. Also, non-oncogenic nature of MDCK cells was demonstrated. Host cell DNA in the vaccine formulations was < 10 ng/dose which ensures vaccine safety. Production efficiency and consistency were characterized and the observed titer values of the viral harvest and the processed bulk were comparable to the expansion in embryonated eggs.The present study clearly establishes the suitability of MDCK cells as a substrate for the manufacture of a safe and viable LAIV.     

H5N2 vaccine viruses on Russian and US live attenuated influenza virus backbones demonstrate similar infectivity,immunogenicity and protection in ferrets

The continued detection of zoonotic influenza infections, most notably due to the avian influenza A H5N1 and H7N9 subtypes, underscores the need for pandemic preparedness. Decades of experience with live attenuated influenza vaccines (LAIVs) for the control of seasonal influenza support the safety and effectiveness of this vaccine platform. All LAIV candidates are derived from one of two licensed master donor viruses (MDVs), cold-adapted (ca) A/Ann Arbor/6/60 or ca A/Leningrad/134/17/57. A number of LAIV candidates targeting avian H5 influenza viruses derived with each MDV have been evaluated in humans, but have differed in their infectivity and immunogenicity. To understand these differences, we generated four H5N2 candidate pandemic LAIVs (pLAIVs) derived from either MDV and compared their biological characteristics in vitro and in vivo. We demonstrate that all candidate pLAIVs, regardless of gene constellation and derivation, were comparable with respect to infectivity, immunogenicity, and protection from challenge in the ferret model of influenza. These observations suggest that differences in clinical performance of H5 pLAIVs may be due to factors other than inherent biological properties of the two MDVs.     

Possible outcomes of reassortment in vivo between wild type and live attenuated influenza vaccine strains

Reassortment of influenza viruses in nature has been well documented. Genetic reassortment plays a key role in emergence of new influenza A strains, including pandemic viruses. Permissive host can be simultaneously coinfected with multiple influenza viruses. During genetic reassortment gene segments are exchanged between parental viruses that may lead to some enhancement of virulence of reassortant progeny. At present, vaccination with live attenuated cold-adapted (ca) reassortant vaccine (LAIV) is used as an effective public health measure for influenza prophylaxis. However, there are concerns about a potential of simultaneous infection of human host with ca and wild type (wt) influenza viruses which might produce progeny that contain novel, more virulent genotypes. The aim of this study was to investigate potential consequences of reassortment of wt with LAIV strains in vivo.     

Engineering temperature sensitive live attenuated influenza vaccines from emerging viruses

The licensed live attenuated influenza A vaccine (LAIV) in the United States is created by making a reassortant containing six internal genes from a cold-adapted master donor strain (ca A/AA/6/60) and two surface glycoprotein genes from a circulating/emerging strain (e.g., A/CA/7/09 for the 2009/2010 H1N1 pandemic). Technologies to rapidly create recombinant viruses directly from patient specimens were used to engineer alternative LAIV candidates that have genomes composed entirely of vRNAs from pandemic or seasonal strains. Multiple mutations involved in the temperature-sensitive (ts) phenotype of the ca A/AA/6/60 master donor strain were introduced into a 2009 H1N1 pandemic strain rA/New York/1682/2009 (rNY1682-WT) to create rNY1682-TS1, and additional mutations identified in other ts viruses were added to rNY1682-TS1 to create rNY1682-TS2. Both rNY1682-TS1 and rNY1682-TS2 replicated efficiently at 30 °C and 33 °C. However, rNY1682-TS1 was partially restricted, and rNY1682-TS2 was completely restricted at 39 °C. Additionally, engineering the TS1 or TS2 mutations into a distantly related human seasonal H1N1 influenza A virus also resulted pronounced restriction of replication in vitro. Clinical symptoms and virus replication in the lungs of mice showed that although rNY1682-TS2 and the licensed FluMist^®-H1N1pdm LAIV that was used to combat the 2009/2010 pandemic were similarly attenuated, the rNY1682-TS2 was more protective upon challenge with a virulent mutant of pandemic H1N1 virus or a heterologous H1N1 (A/PR/8/1934) virus. This study demonstrates that engineering key temperature sensitive mutations (PB1-K391E, D581G, A661T; PB2-P112S, N265S, N556D, Y658H) into the genomes of influenza A viruses attenuates divergent human virus lineages and provides an alternative strategy for the generation of LAIVs.     

An open label Phase I trial of a live attenuated H6N1 influenza virus vaccine in healthy adults

Background

We describe the results of an open label Phase I trial of a live attenuated H6N1 influenza virus vaccine (ClinicalTrials.gov Identifier: NCT00734175).

Methods and findings

We evaluated the safety, infectivity, and immunogenicity of two doses of 10⁷ TCID₅₀ of the H6N1 Teal HK 97/AA ca vaccine, a cold-adapted and temperature sensitive live, attenuated influenza vaccine (LAIV) in healthy seronegative adults.Twenty-two participants received the first dose of the vaccine, and 18 received the second dose of vaccine 4 weeks later. The vaccine had a safety profile similar to that of other investigational LAIVs bearing avian hemagglutinin (HA) and neuraminidase (NA) genes. The vaccine was highly restricted in replication: two participants had virus detectable by rRT-PCR beyond day 1 after each dose. Antibody responses to the vaccine were also restricted: 43% of participants developed a serum antibody response as measured by any assay: 5% by hemagglutination-inhibition assay, 5% by microneutralization assay, 29% by ELISA for H6 HA-specific IgG and 24% by ELISA for H6 HA specific IgA after either 1 or 2 doses. Following the second dose, vaccine specific IgG and IgA secreting cells as measured by ELISPOT increased from a mean of 0.6 to 9.2/10⁶ PBMCs and from 0.2 to 2.2/10⁶ PBMCs, respectively.

Conclusion

The H6N1 LAIV had a safety profile similar to that of LAIV bearing other HA and NA genes, but was highly restricted in replication in healthy seronegative adults. The H6N1 LAIV was also not as immunogenic as the seasonal LAIV.     

Live attenuated influenza vaccine strains elicit a greater innate immune response than antigenically-matched seasonal influenza viruses during infection of human nasal epithelial cell cultures

Influenza viruses are global pathogens that infect approximately 10–20% of the world's population each year. Vaccines, including the live attenuated influenza vaccine (LAIV), are the best defense against influenza infections. The LAIV is a novel vaccine that actively replicates in the human nasal epithelium and elicits both mucosal and systemic protective immune responses. The differences in replication and innate immune responses following infection of human nasal epithelium with influenza seasonal wild type (WT) and LAIV viruses remain unknown. Using a model of primary differentiated human nasal epithelial cell (hNECs) cultures, we compared influenza WT and antigenically-matched cold adapted (CA) LAIV virus replication and the subsequent innate immune response including host cellular pattern recognition protein expression, host innate immune gene expression, secreted pro-inflammatory cytokine production, and intracellular viral RNA levels. Growth curves comparing virus replication between WT and LAIV strains revealed significantly less infectious virus production during LAIV compared with WT infection. Despite this disparity in infectious virus production the LAIV strains elicited a more robust innate immune response with increased expression of RIG-I, TLR-3, IFNβ, STAT-1, IRF-7, MxA, and IP-10. There were no differences in cytotoxicity between hNEC cultures infected with WT and LAIV strains as measured by basolateral levels of LDH. Elevated levels of intracellular viral RNA during LAIV as compared with WT virus infection of hNEC cultures at 33 °C may explain the augmented innate immune response via the up-regulation of pattern recognition receptors and down-stream type I IFN expression. Taken together our results suggest that the decreased replication of LAIV strains in human nasal epithelial cells is associated with a robust innate immune response that differs from infection with seasonal influenza viruses, limits LAIV shedding and plays a role in the silent clinical phenotype seen in human LAIV inoculation.     

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.     

H7N3 live attenuated influenza vaccine has a potential to protect against new H7N9 avian influenza virus

After recent emergence of new avian influenza A(H7N9) viruses in humans many people and Governments are asking about H7 influenza vaccine which could provide cross-protection against new viruses, until H7N9 vaccine is prepared from a relevant strain. Here we scientifically justify that available H7N3 live attenuated influenza vaccine (LAIV) can be protective against H7N9 viruses due to the presence of conserved immune epitopes in its hemagglutinin. We used Immune Epitope Database analysis resource to predict B-cell and CTL epitopes distributed across H7N3 HA molecule and assessed their identity with new H7N9 viruses at near 70% and 60% of the epitopes, respectively. In addition, we tested serum samples of volunteers participated in phase I clinical trial of H7N3 LAIV for the presence of anti-H7N9 hemagglutination-inhibition and neutralizing antibodies and found seroconversions in 44.8% of vaccinated persons, which suggests the potential of H7N3 LAIV to protect against new H7N9 avian influenza viruses.     

Production and antigenic properties of influenza virus from suspension MDCK-siat7e cells in a bench-scale bioreactor

In efforts to overcome limitations associated with egg-based influenza vaccines, mammalian cell substrates have gradually emerged as potential production platforms. Recently, a suspension Madin Darby canine kidney (MDCK) cell line for influenza virus production was created by expressing the human siat7e gene. To examine the broad susceptibility of this novel cell line, the scalability of the production process, and the antigenic stability of cell-derived progeny viruses, infection experiments using four current influenza vaccine strains (A/California/07/2009 X-179A H1N1, A/Brisbane/59/2007 IVR-148 H1N1, A/Uruguay/716/2007 X-175C H3N2, and B/Brisbane/60/2008) were performed. In small-scale experiments, this cell line was found to support high-titer replication of all four virus strains. Subsequently, production in a bench-scale bioreactor and the antigenic characteristics of progeny viruses were assessed. High titers of hemagglutinin (at least 1:512) were produced in a 2-L bench-scale bioreactor with all four strains. Immunoblot results demonstrated higher yields in the cells than those obtained in chicken embryonated eggs with three of the four tested strains. Progeny viruses collected after serial passages in this cell line exhibited minimal mutations in the HA-encoding gene. Hemagglutination inhibition (HAI) assays using ferret antiserum confirmed the antigenic stability. As a proof-of-concept this work demonstrates that by using a proper strategy, high yields of biologically active hemagglutinin can be produced from scalable cultures of suspension MDCK-siat7e cells.     

Genetic sequence analysis of influenza viruses and illness severity in ill children previously vaccinated with live attenuated or inactivated influenza vaccine

In a large comparative study in 2004–2005, children aged 6–59 months vaccinated with live attenuated influenza vaccine (LAIV) experienced 55% fewer cases of culture-confirmed influenza illness compared with trivalent inactivated influenza vaccine (TIV) recipients. To better understand the characteristics of the breakthrough influenza illnesses, we analyzed the HA1 genetic sequence for all available samples and examined disease severity by strain and treatment group. All 48 A/H1N1 viruses were well-matched to the vaccine, whereas all 276 A/H3N2 viruses and 349 (96%) influenza B viruses were mismatched to the vaccine. The incidence of fever or lower respiratory illness did not differ by strain; however, LAIV recipients had less febrile disease and fewer lower respiratory illnesses than TIV recipients. Viruses of each influenza B lineage caused more illnesses than A/H1N1 viruses; strategies to enhance protection against multiple influenza B lineages should be pursued.     

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.     

Immunogenicity and efficacy comparison of MDCK cell-based and egg-based live attenuated influenza vaccines of H5 and H7 subtypes in ferrets

During a pandemic, the availability of specific pathogen free chicken eggs is a major bottleneck for up-scaling response to the demand for influenza vaccine. This has led us to explore the use of Madin-Darby Canine Kidney (MDCK) cells for the manufacture of live attenuated influenza vaccine (LAIV) that provides production flexibility and speed. The present study reports the comparison of the immunogenicity and efficacy of two MDCK-based LAIVs against two egg-based LAIVs prepared from the same pandemic potential strains of H5 and H7 subtypes after a single dose of the vaccine followed by a challenge with a homologous wild type strain. The vaccine strains have been generated by classical method of reassortment using the A/Leningrad/134/17/57 master donor strain. Additionally, a prime-boost regimen of the MDCK-based vaccine followed by a challenge with a homologous wild type strain for H5 and H7 immunized ferrets and also a heterologous wild type strain for the H5 immunized animals was studied. No difference in the hemagglutination inhibition and virus neutralization antibody titers against the homologous virus was observed following a single dose of either egg-based or MDCK-based H5 and H7 LAIV vaccine. A second dose of MDCK-based vaccine significantly boosted antibody titers in the vaccinated animals. Both a single dose or two doses of LAIV provided complete protection from lower respiratory tract infection and resulted in a significant reduction in the virus titers recovered from the throat, nasal turbinates and lungs after challenge with the homologous wild type strain. Protection from a challenge with a heterologous strain of H5 was also observed after two doses of the MDCK-based LAIVs. This data strongly supports the use of MDCK as a substrate for the manufacture of LAIV which ensures reliable quality, safety, production flexibility, speed and breadth of protection, features that are highly critical during a pandemic.     

Cross-protection against H7N9 influenza strains using a live-attenuated H7N3 virus vaccine

In 2013, avian H7N9 influenza viruses were detected infecting people in China resulting in high mortality. Influenza H7 vaccines that provide cross-protection against these new viruses are needed until specific H7N9 vaccines are ready to market. In this study, an available H7N3 cold-adapted, temperature sensitive, live attenuated influenza vaccine (LAIV) elicited protective immune responses in ferrets against H7N9 viruses. The H7N3 LAIV administered alone (by intranasal or subcutaneous administration) or in a prime-boost strategy using inactivated H7N9 virus resulted in high HAI titers and protected 100% of the animals against H7N9 challenge. Naïve ferrets passively administered immune serum from H7N3 LAIV infected animals were also protected. In contrast, recombinant HA protein or inactivated viruses did not protect ferrets against challenge and elicited lower antibody titers. Thus, the H7N3 LAIV vaccine was immunogenic in healthy seronegative ferrets and protected these ferrets against the newly emerged H7N9 avian influenza virus.