An elastase-dependent attenuated heterologous swine influenza virus protects against pandemic H1N1 2009 influenza challenge in swine

Influenza virus infections continue to cause production losses in the agricultural industry in addition to being a human public health concern. The primary method to control influenza is through vaccination. However, currently used killed influenza virus vaccines must be closely matched to the challenge virus. The ability of an elastase-dependent live attenuated influenza A virus was evaluated to protect pigs against the pandemic H1N1 2009 influenza virus. Pigs vaccinated intranasally or intratracheally with the elastase-dependent swine influenza virus (SIV) vaccine had significantly reduced macroscopic and microscopic lung lesions and lower viral loads in the lung and in nasal swabs. Thus, elastase-dependent SIV mutants can be used as live-virus vaccines against swine influenza in pigs. In addition, low levels of cross-neutralizing antibodies to H1N1 2009 were elicited prior to challenge by the swine adapted H1N1 avian strain vaccine.     

Efficacy of inactivated swine influenza virus vaccines against the 2009 A/H1N1 influenza virus in pigs

The gene constellation of the 2009 pandemic A/H1N1 virus is a unique combination from swine influenza A viruses (SIV) of North American and Eurasian lineages, but prior to April 2009 had never before been identified in swine or other species. Although its hemagglutinin gene is related to North American H1 SIV, it is unknown if vaccines currently used in U.S. swine would cross-protect against infection with the pandemic A/H1N1. The objective of this study was to evaluate the efficacy of inactivated vaccines prepared with North American swine influenza viruses as well as an experimental homologous A/H1N1 vaccine to prevent infection and disease from 2009 pandemic A/H1N1. All vaccines tested provided partial protection ranging from reduction of pneumonia lesions to significant reduction in virus replication in the lung and nose. The multivalent vaccines demonstrated partial protection; however, none was able to prevent all nasal shedding or clinical disease. An experimental homologous 2009 A/H1N1 monovalent vaccine provided optimal protection with no virus detected from nose or lung at any time point in addition to amelioration of clinical disease. Based on cross-protection demonstrated with the vaccines evaluated in this study, the U.S. swine herd likely has significant immunity to the 2009 A/H1N1 from prior vaccination or natural exposure. However, consideration should be given for development of monovalent homologous vaccines to best protect the swine population thus limiting shedding and the potential transmission of 2009 A/H1N1 from pigs to people.     

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.     

Immune responses and protective efficacy of a recombinant swinepox virus expressing HA1 against swine H1N1 influenza virus in mice and pigs

Swine influenza virus (SIV) is not only an important respiratory pathogen in pigs but also a potent threat to human health. Although immunization with recombinant poxviruses expressing protective antigens as vaccines has been widely used for against many infectious diseases, development of recombinant swinepox virus (rSPV) vector for the purpose has been less successful. Here, we report the construction of a recombinant swinepox virus (rSPV-HA1) expressing hemagglutinin (HA1) of H1N1 SIV. Immune responses and protection efficacy of the vaccination vector were evaluated in both the mouse model and the natural host: pig. Prime and boost inoculations of rSPV-HA1 yielded high levels of neutralization antibody against SIV and elicited potent H1N1 SIV-specific IFN-γ response from T-lymphocytes. Complete protection of pigs against H1N1 SIV challenge was observed. No pigs showed evident systemic and local reactions to the vaccine and no SIV shedding was detected from pigs vaccinated with rSPV-HA1 after challenge. Our data demonstrated that the recombinant swinepox virus encoding HA1 of SIV H1N1 may serve as a promising SIV vaccine for protection against SIV infection.     

Poly I:C adjuvanted inactivated swine influenza vaccine induces heterologous protective immunity in pigs

Swine influenza is widely prevalent in swine herds in North America and Europe causing enormous economic losses and a public health threat. Pigs can be infected by both avian and mammalian influenza viruses and are sources of generation of reassortant influenza viruses capable of causing pandemics in humans. Current commercial vaccines provide satisfactory immunity against homologous viruses; however, protection against heterologous viruses is not adequate. In this study, we evaluated the protective efficacy of an intranasal Poly I:C adjuvanted UV inactivated bivalent swine influenza vaccine consisting of Swine/OH/24366/07 H1N1 and Swine/CO/99 H3N2, referred as PAV, in maternal antibody positive pigs against an antigenic variant and a heterologous swine influenza virus challenge. Groups of three-week-old commercial-grade pigs were immunized intranasally with PAV or a commercial vaccine (CV) twice at 2 weeks intervals. Three weeks after the second immunization, pigs were challenged with the antigenic variant Swine/MN/08 H1N1 (MN08) and the heterologous Swine/NC/10 H1N2 (NC10) influenza virus. Antibodies in serum and respiratory tract, lung lesions, virus shedding in nasal secretions and virus load in lungs were assessed. Intranasal administration of PAV induced challenge viruses specific-hemagglutination inhibition- and IgG antibodies in the serum and IgA and IgG antibodies in the respiratory tract. Importantly, intranasal administration of PAV provided protection against the antigenic variant MN08 and the heterologous NC10 swine influenza viruses as evidenced by significant reductions in lung virus load, gross lung lesions and significantly reduced shedding of challenge viruses in nasal secretions. These results indicate that Poly I:C or its homologues may be effective as vaccine adjuvants capable of generating cross-protective immunity against antigenic variants/heterologous swine influenza viruses in pigs.     

Matrix protein 2 vaccination and protection against influenza viruses, including subtype H5N1

Changes in influenza viruses require regular reformulation of strain-specific influenza vaccines. Vaccines based on conserved antigens provide broader protection. Influenza matrix protein 2 (M2) is highly conserved across influenza A subtypes. To evaluate its efficacy as a vaccine candidate, we vaccinated mice with M2 peptide of a widely shared consensus sequence. This vaccination induced antibodies that cross-reacted with divergent M2 peptide from an H5N1 subtype. A DNA vaccine expressing full-length consensus-sequence M2 (M2-DNA) induced M2-specific antibody responses and protected against challenge with lethal influenza. Mice primed with M2-DNA and then boosted with recombinant adenovirus expressing M2 (M2-Ad) had enhanced antibody responses that crossreacted with human and avian M2 sequences and produced T-cell responses. This M2 prime-boost vaccination conferred broad protection against challenge with lethal influenza A, including an H5N1 strain. Vaccination with M2, with key sequences represented, may provide broad protection against influenza A.     

Inactivated and adjuvanted whole-virion clade 2.3.4 H5N1 pre-pandemic influenza vaccine possesses broad protective efficacy against infection by heterologous clades of highly pathogenic H5N1 avian influenza virus in mice

In this study, we evaluated the immunogenicity and protective efficacy of a candidate attenuated H5N1 pre-pandemic influenza vaccine of clade 2.3.4, rgAnhui, which was reverse genetically generated from highly virulent A/Anhui/01/2005 (H5N1) wild-type virus. When a low-dose antigen (0.3 μg HA) vaccine was combined with aluminum hydroxide adjuvant, virus neutralization and anti-HA IgG antibodies induced in the sera of vaccinated mice showed similar levels as those in mice vaccinated with non-adjuvanted high-dose antigen (3 μg HA) vaccine. Serum antibodies had broad reactivity against highly pathogenic H5N1 viruses of both homologous and heterologous clades. All mice vaccinated with adjuvanted and non-adjuvanted rgAnhui vaccines at low and high antigen doses survived, without any significant weight loss, lethal challenge infection with homologous clade 2.3.4 viruses, including antigenic variant virus and heterologous clade 2.1.3. Mice vaccinated with low-dose antigen without adjuvant, however, exhibited 20% and 60% survival rates against clade 1 and clade 2.2 viruses, respectively; but, addition of adjuvant improved these rates to 80% and 100%, respectively. The data strongly suggest that aluminum hydroxide-adjuvanted rgAnhui vaccine can elicit broad cross-reactive and protective immunities against homologous and heterologous clades, and that the rgAnhui vaccine is a useful pre-pandemic H5N1 vaccine.     

Association of vaccine handling conditions with effectiveness of live attenuated influenza vaccine against H1N1pdm09 viruses in the United States

PurposeThis analysis examined potential causes of the lack of vaccine effectiveness (VE) of live attenuated influenza vaccine (LAIV) against A/H1N1pdm09 viruses in the United States (US) during the 2013–2014 season. Laboratory studies have demonstrated reduced thermal stability of A/California/07/2009, the A/H1N1pdm09 strain utilized in LAIV from 2009 through 2013–2014.MethodsPost hoc analyses of a 2013–2014 test-negative case-control (TNCC) effectiveness study investigated associations between vaccine shipping conditions and LAIV lot effectiveness. Investigational sites provided the LAIV lot numbers administered to each LAIV recipient enrolled in the study, and the vaccine distributor used by the site for commercially purchased vaccine. Additionally, a review was conducted of 2009–2014 pediatric observational TNCC effectiveness studies of LAIV, summarizing effectiveness by type/subtype, season, and geographic location.ResultsFrom the 2013 to 2014 TNCC study, the proportion of LAIV recipients who tested positive for H1N1pdm09 was significantly higher among children who received a lot released between August 1 and September 15, 2013, compared with a lot shipped either earlier or later (21% versus 4%; P < 0.01). A linear relationship was observed between the proportion of subjects testing positive for H1N1pdm09 and outdoor temperatures during truck unloading at distributors’ central locations. The review of LAIV VE studies showed that in the 2010–2011 and 2013–2014 influenza seasons, no significant effectiveness of LAIV against H1N1pdm09 was demonstrated for the trivalent or quadrivalent formulations of LAIV in the US, respectively, in contrast to significant effectiveness against A/H3N2 and B strains during 2010–2014.ConclusionsThis study showed that the lack of VE observed with LAIV in the US against H1N1pdm09 viruses was associated with exposure of some LAIV lots to temperatures above recommended storage conditions during US distribution, and is likely explained by the increased susceptibility of the A/California/7/2009 (H1N1pdm09) LAIV strain to thermal degradation.Clinical trial registry: NCT01997450     

Vaccine-mediated protection of pigs against infection with pandemic H1N1 2009 swine influenza A virus requires a close antigenic match between the vaccine antigen and challenge virus

Swine influenza A virus (SwIV) infection has considerable economic and animal welfare consequences and, because of the zoonotic potential, can also have public health implications. The 2009 pandemic H1N1 ‘swine-origin’ infection is now endemic in both pigs and humans. In Europe, avian-like H1_avN1, human-like H1_huN2, human-like swine H3N2 and, since 2009, pandemic H1N1 (pH1N1) lineage viruses and reassortants, constitute the dominant subtypes. In this study, we used a swine pH1N1 challenge virus to investigate the efficacy of whole inactivated virus vaccines homologous or heterologous to the challenge virus as well as a commercial vaccine. We found that vaccine-mediated protection was most effective when vaccine antigen and challenge virus were homologous and correlated with the specific production of neutralising antibodies and a cellular response to the challenge virus. We conclude that a conventional whole inactivated SwIV vaccine must be antigenically matched to the challenge strain to be an effective control measure.     

Avian influenza H5N1 vaccination efficacy in Egyptian backyard poultry

Raising backyard poultry under low biosecurity conditions is a common practice in Egypt. While vaccination is routinely applied in Egypt in commercial settings to curb the spread of avian influenza viruses, it remains less commonly used in backyard settings. We assessed the immunogenicity and protective efficacy of a H5N1 vaccine based on a contemporary Egyptian clade 2.2.1.2 virus among turkeys, ducks, geese, and chickens raised together in a backyard setting. Results showed that this vaccine elicits an immune response in all tested species reaching up to a hemagglutination inhibition titer of 10 log₂ after a booster dose. However, this response varied between species. When challenged, vaccinated birds survived and shed less virus in comparison with unvaccinated birds. However, unvaccinated ducks showed no symptoms of infection and survived the duration of the experiment. Moreover, vaccinated ducks shed more virus as compared to vaccinated birds of other species. Hence, we recommend avoiding mixing various species in the backyards of Egypt. Our data indicates that vaccination can be effective in the backyard setting in Egypt, although planning should consider the species covered.     

Protective efficacy of a polyvalent influenza A DNA vaccine against both homologous (H1N1pdm09) and heterologous (H5N1) challenge in the ferret model

This study describes the protective efficacy of a novel influenza plasmid DNA vaccine in the ferret challenge model. The rationally designed polyvalent influenza DNA vaccine encodes haemagglutinin and neuraminidase proteins derived from less glycosylated pandemic H1N1 (2009) and H3N2 (1968) virus strains as well as the nucleoprotein (NP) and matrix proteins (M1 and M2) from a different pandemic H1N1 (1918) strain. Needle-free intradermal immunisation with the influenza DNA vaccine protected ferrets against homologous challenge with an H1N1pdm09 virus strain, demonstrated by restriction of viral replication to the upper respiratory tract and reduced duration of viral shedding post-challenge. Breadth of protection was demonstrated in two heterologous efficacy experiments in which animals immunised with the influenza DNA vaccine were protected against challenge with a highly pathogenic avian influenza H5N1 virus strain with reproducible survival and clinical outcomes.     

Complete protection against lethal challenge of novel H7N9 virus with heterologous inactivated H7 vaccine in mice

A prototype H7 influenza vaccine constructed based on the H7N7 outbreak in 2003 was tested for the protective efficacy against the novel H7N9 virus in a lethal murine challenge model. Serum samples from vaccinated mice showed significant neutralizing activity against the H7N9 virus and the mice were completely protected with no significant weight loss. The results have direct implications on how to overcome potential vaccine shortage and identify donors for immune sera for passive immunization.     

Influenza H7N9 LAH-HBc virus-like particle vaccine with adjuvant protects mice against homologous and heterologous influenza viruses

The long alpha-helix (LAH) region located in influenza virus hemagglutinin (HA) shows conservation among different influenza A strains, which could be used as a candidate target of influenza vaccines. Moreover, the hepatitis B virus core protein (HBc) is a carrier for heterologous epitopes in eliciting effective immune responses. We inserted the LAH region of H7N9 influenza virus into the HBc and prepared the LAH-HBc protein, which were capable of self-assembly into virus-like particles (VLP), by using E. coli expression system. Intranasal immunization of the LAH-HBc VLP in combination with chitosan adjuvant or CTB¹ adjuvant in mice could induce both humoral and cellular immune responses effectively and provide complete protection against lethal challenge of homologous H7N9 virus or heterologous H3N2 virus, as well as partial protection against lethal challenge of heterologous H1N1 virus. These results provide a proof of concept for LAH-HBc VLP vaccine that would be fast and easy to be produced and might be an ideal candidate as a rapid-response tool against a future influenza pandemic.     

Single dose of oil-adjuvanted inactivated vaccine protects chickens from lethal infections of highly pathogenic H5N1 influenza virus

The highly pathogenic H5N1 influenza viruses are endemic in poultry in many countries, but continuously infect humans and cause human mortality. H5N1 influenza viruses have been regarded as a pandemic candidate. In a pandemic event by this virus, the protection of poultry with an effective vaccine will help to greatly reduce the spread of this virus to humans since it easily infects poultry. Here we showed that immunization with one dose of oil-adjuvanted inactivated H5N1 vaccine could protect chickens from lethal infection by highly pathogenic H5N1 influenza virus until 12 weeks post-immunization. The complete protection of chickens depended on the amount of HA antigens in the vaccine. Complete homologous protection required over 1.25 μg of HA antigens and complete heterologous protection required over 5.0 μg of HA antigens. The bivalent H5N1 inactivated vaccine composed of 1.25 μg of each antigen from clade 1 and clade 2.3.4 H5N1 influenza virus completely protected chickens from the lethal challenge of both viruses. When we determined the induction of antibody subtypes in tissues including nasal cavity, trachea, and lungs, the IgG subtype of antibody was induced more than the IgM or IgA subtype of antibody. Taken together, our results suggest that one dose of oil-adjuvanted inactivated H5N1 vaccine could provide chickens with sterile immunity against the homologous highly pathogenic H5N1 influenza virus.     

Efficacy of live attenuated vaccines against 2009 pandemic H1N1 influenza in ferrets

The advent of the H1N1 influenza pandemic (pH1N1) in 2009 triggered the rapid production of pandemic influenza vaccines, since seasonal influenza vaccines were expected and demonstrated not to provide significant cross-protection against the newly emerged pandemic virus. To increase vaccine production capacity and further evaluate the effectiveness of different candidate pandemic influenza vaccines, the World Health Organization stimulated the evaluation of different vaccination concepts including the use of live attenuated influenza vaccines (LAIVs). Therefore, we have immunized ferrets intranasally with a single dose of pH1N1-LAIV from different manufacturers. They all induced adequate serum HI antibody titers in the ferrets and protected them against intratracheal wild-type pH1N1 virus challenge: pH1N1 virus replication in the upper respiratory tract and lungs was reduced and no disease signs or severe broncho-interstitial pneumonia were observed in any of the vaccinated ferrets. These data together with the relatively efficient production process emphasize the potential of the LAIV concept for pandemic preparedness.     

Human infection from avian-like influenza A (H1N1) viruses in pigs, China

In investigating influenza in an immunodeficient child in China, in December 2010, we found that the influenza virus showed high sequence identity to that of swine. Serologic evidence indicated that viral persistence in pigs was the source of infection. Continued surveillance of pigs and systemic analysis of swine influenza isolates are needed.     

Protection of pregnant mice,fetuses and neonates from lethality of H5N1 influenza viruses by maternal vaccination

The highly pathogenic H5N1 influenza viruses are one of candidates for the next pandemic. Information on protective immunity for pregnant animals by vaccination against the H5N1 influenza virus is limited. Here, we show that the immunization of pregnant mice with inactivated H5N1 influenza vaccine protects them, their fetuses, and their infant mice from H5N1 influenza viruses. Pregnant mice immunized with two doses of H5N1 influenza vaccine were protected from homologous infections of H5N1 influenza viruses with no viruses detected in fetuses, and that they were protected upto 30% from heterologous infections of H5N1 influenza viruses with viruses detected in fetuses. The infant mice born to mothers immunized with H5N1 influenza vaccine were fully protected from infections of H5N1 influenza viruses for upto 4 weeks of age. The protection of infant mice was closely related to the presence of IgG2a antibody in lung, heart, and rectum tissues. Our results suggest that maternal vaccination may be critical for protecting pregnant animals, their fetuses, and their infant mice from lethal infections of H5N1 influenza viruses.     

甲型H1N1流感疫苗接种对其暴发疫情的影响

目的了解甲型H1N1流感(甲流)疫苗接种后对甲流暴发疫情的影响。方法对2011年4—5月发生在学校的1起甲流暴发疫情进行流行病学描述性分析,用回顾性队列研究的方法分析甲流疫苗接种对该起暴发疫情的影响。结果该校542名师生中191人患病,罹患率为35.24%,发病时间主要集中在4月19—25日,发病人群主要为1～6年级的小学生(χ2=9.972,P0.01),住校生发病高于非住校生,112名接种过甲流疫苗的师生发病率为16.96%,明显低于未接种疫苗的师生(40%),差异有统计学意义(χ2=20.661,P0.05),OR=0.306(95%CI∶0.180～0.521)。结论接种甲流疫苗可以有效预防甲型H1N1流感的发生,减少暴发疫情的发病率。     

Vaccination-challenge studies with a Port Chalmers/73 (H3N2)-based swine influenza virus vaccine: Reflections on vaccine strain updates and on the vaccine potency test

The human A/Port Chalmers/1/73 (H3N2) influenza virus strain, the supposed ancestor of European H3N2 swine influenza viruses (SIVs), was used in most commercial SIV vaccines in Europe until recently. If manufacturers want to update vaccine strains, they have to perform laborious intratracheal (IT) challenge experiments and demonstrate reduced virus titres in the lungs of vaccinated pigs. We aimed to examine (a) the ability of a Port Chalmers/73-based commercial vaccine to induce cross-protection against a contemporary European H3N2 SIV and serologic cross-reaction against H3N2 SIVs from Europe and North America and (b) the validity of intranasal (IN) challenge and virus titrations of nasal swabs as alternatives for IT challenge and titrations of lung tissue in vaccine potency tests. Pigs were vaccinated with Suvaxyn Flu^® and challenged by the IT or IN route with sw/Gent/172/08. Post-vaccination sera were examined in haemagglutination-inhibition assays against vaccine and challenge strains and additional H3N2 SIVs from Europe and North America, including an H3N2 variant virus. Tissues of the respiratory tract and nasal swabs were collected 3 days post challenge (DPCh) and from 0–7 DPCh, respectively, and examined by virus titration. Two vaccinations consistently induced cross-reactive antibodies against European H3N2 SIVs from 1998–2012, but minimal or undetectable antibody titres against North American viruses. Challenge virus titres in the lungs, trachea and nasal mucosa of the vaccinated pigs were significantly reduced after both IT and IN challenge. Yet the reduction of virus titres and nasal shedding was greater after IT challenge. The Port Chalmers/73-based vaccine still offered protection against a European H3N2 SIV isolated 35 years later and with only 86.9% amino acid homology in its HA1, but it is unlikely to protect against H3N2 SIVs that are endemic in North America. We use our data to reflect on vaccine strain updates and on the vaccine potency test.     

Generation of a reassortant avian influenza virus H5N2 vaccine strain capable of protecting chickens against infection with Egyptian H5N1 and H9N2 viruses

BackgroundAvian influenza H5N1 viruses have been enzootic in Egyptian poultry since 2006. Avian influenza H9N2 viruses which have been circulating in Egyptian poultry since 2011 showed high replication rates in embryonated chicken eggs and mammalian cells.MethodsTo investigate which gene segment was responsible for increasing replication, we constructed reassortant influenza viruses using the low pathogenic H1N1 PR8 virus as backbone and included individual genes from A/chicken/Egypt/S4456B/2011(H9N2) virus. Then, we invested this finding to improve a PR8-derived H5N1 influenza vaccine strain by incorporation of the NA segment of H9N2 virus instead of the NA of H5N1. The growth properties of this virus and several other forms of reassortant H5 viruses were compared. Finally, we tested the efficacy of this reassortant vaccine strain in chickens.ResultsWe observed an increase in replication for a reassortant virus expressing the neuraminidase gene (N2) of H9N2 virus relative to that of either parental viruses or reassortant PR8 viruses expressing other genes. Then, we generated an H5N2 vaccine strain based on the H5 from an Egyptian H5N1 virus and the N2 from an Egyptian H9N2 virus on a PR8 backbone. This strain had better replication rates than an H5N2 reassortant strain on an H9N2 backbone and an H5N1 reassortant on a PR8 backbone. This virus was then used to develop a killed, oil-emulsion vaccine and tested for efficacy against H5N1 and H9N2 viruses in chickens. Results showed that this vaccine was immunogenic and reduced mortality and shedding.DiscussionOur findings suggest that an inactivated PR8-derived H5N2 influenza vaccine is efficacious in poultry against H5N1 and H9N2 viruses and the vaccine seed replicates at a high rate thus improving vaccine production.