Heterologous prime-boost vaccination with H3N2 influenza viruses of swine favors cross-clade antibody responses and protection

The emergence of multiple novel lineages of H1 and H3 influenza A viruses in swine has confounded control by inactivated vaccines. Because of substantial genetic and geographic heterogeneity among circulating swine influenza viruses, one vaccine strain per subtype cannot be efficacious against all of the current lineages. We have performed vaccination-challenge studies in pigs to examine whether priming and booster vaccinations with antigenically distinct H3N2 swine influenza viruses could broaden antibody responses and protection. We prepared monovalent whole inactivated, adjuvanted vaccines based on a European and a North American H3N2 swine influenza virus, which showed 81.5% aa homology in the HA1 region of the hemagglutinin and 83.4% in the neuraminidase. Our data show that (i) Priming with European and boosting with North American H3N2 swine influenza virus induces antibodies and protection against both vaccine strains, unlike prime-boost vaccination with a single virus or a single administration of bivalent vaccine. (ii) The heterologous prime-boost vaccination enhances hemagglutination inhibiting, virus neutralizing and neuraminidase inhibiting antibody responses against H3N2 viruses that are antigenically distinct from both vaccine strains. Antibody titers to the most divergent viruses were higher than after two administrations of bivalent vaccine. (iii) However, it does not induce antibodies to the conserved hemagglutinin stalk or to other hemagglutinin subtypes. We conclude that heterologous prime-boost vaccination might broaden protection to H3N2 swine influenza viruses and reduce the total amount of vaccine needed. This strategy holds potential for vaccination against influenza viruses from both humans and swine and for a better control of (reverse) zoonotic transmission of influenza viruses

The post-2009 influenza pandemic era : time to revisit antibody immunodominance

The current inactivated influenza vaccines rely on the induction of neutralizing antibodies against the head domain of the viral hemagglutinin (HA). The HA head contains five immunodominant antigenic sites, all of which are subject to antigenic drift, thereby limiting vaccine efficacy. Bypassing the immune system's tendency to focus on the most variable regions of the HA may be a step toward more broadly protective influenza vaccines. However, this requires a better understanding of the biological meaning of immunodominance, and of the hierarchy between different antigenic sites. In this issue of the JCI, Liu et al. determined the immunodominance of the five antigenic sites of the HA head in experimentally infected mice, guinea pigs, and ferrets. All three species exhibited different preferences for the five sites of the 2009 pandemic H1N1 strain. Moreover, human subjects exhibited yet a different pattern of immunodominance following immunization with the standard inactivated influenza vaccine. Together, these results have important implications for influenza vaccine design and interpretation of animal models

Use of Hemagglutinin Stem Probes Demonstrate Prevalence of Broadly Reactive Group 1 Influenza Antibodies in Human Sera.

A better understanding of the seroprevalence and specificity of influenza HA stem-directed broadly neutralizing antibodies (bNAbs) in the human population could significantly inform influenza vaccine design efforts. Here, we utilized probes comprising headless, HA stabilized stem (SS) to determine the prevalence, binding and neutralization breadth of antibodies directed to HA stem-epitope in a cross-sectional analysis of the general population. Five group-1 HA SS probes, representing five subtypes, were chosen for this analyses. Eighty-four percent of samples analyzed had specific reactivity to at least one probe, with approximately 60% of the samples reactive to H1 probes, and up to 45% reactive to each of the non-circulating subtypes. Thirty percent of analyzed sera had cross-reactivity to at least four of five probes and this reactivity could be blocked by competing with F10 bNAb. Binding cross-reactivity in sera samples significantly correlated with frequency of H1H5 cross-reactive B cells. Interestingly, only 33% of the cross-reactive sera neutralized both H1N1 and H5N1 pseudoviruses. Cross-reactive and neutralizing antibodies were more prevalent in individuals >50 years of age. Our data demonstrate the need to use multiple HA-stem probes to assess for broadly reactive antibodies. Further, a universal vaccine could be designed to boost pre-existing B-cells expressing stem-directed bNAbs

Pandemic Paradox: Early Life H2N2 Pandemic Influenza Infection Enhanced Susceptibility to Death during the 2009 H1N1 Pandemic.

Recent outbreaks of H5, H7, and H9 influenza A viruses in humans have served as a vivid reminder of the potentially devastating effects that a novel pandemic could exert on the modern world. Those who have survived infections with influenza viruses in the past have been protected from subsequent antigenically similar pandemics through adaptive immunity. For example, during the 2009 H1N1 "swine flu" pandemic, those exposed to H1N1 viruses that circulated between 1918 and the 1940s were at a decreased risk for mortality as a result of their previous immunity. It is also generally thought that past exposures to antigenically dissimilar strains of influenza virus may also be beneficial due to cross-reactive cellular immunity. However, cohorts born during prior heterosubtypic pandemics have previously experienced elevated risk of death relative to surrounding cohorts of the same population. Indeed, individuals born during the 1890 H3Nx pandemic experienced the highest levels of excess mortality during the 1918 "Spanish flu." Applying Serfling models to monthly mortality and influenza circulation data between October 1997 and July 2014 in the United States and Mexico, we show corresponding peaks in excess mortality during the 2009 H1N1 "swine flu" pandemic and during the resurgent 2013-2014 H1N1 outbreak for those born at the time of the 1957 H2N2 "Asian flu" pandemic. We suggest that the phenomenon observed in 1918 is not unique and points to exposure to pandemic influenza early in life as a risk factor for mortality during subsequent heterosubtypic pandemics.IMPORTANCE The relatively low mortality experienced by older individuals during the 2009 H1N1 influenza virus pandemic has been well documented. However, reported situations in which previous influenza virus exposures have enhanced susceptibility are rare and poorly understood. One such instance occurred in 1918-when those born during the heterosubtypic 1890 H3Nx influenza virus pandemic experienced the highest levels of excess mortality. Here, we demonstrate that this phenomenon was not unique to the 1918 H1N1 pandemic but that it also occurred during the contemporary 2009 H1N1 pandemic and 2013-2014 H1N1-dominated season for those born during the heterosubtypic 1957 H2N2 "Asian flu" pandemic. These data highlight the heretofore underappreciated phenomenon that, in certain instances, prior exposure to pandemic influenza virus strains can enhance susceptibility during subsequent pandemics. These results have important implications for pandemic risk assessment and should inform laboratory studies aimed at uncovering the mechanism responsible for this effect

A universal influenza virus vaccine candidate confers protection against pandemic H1N1 infection in ferrets

Influenza viruses can cause severe disease and mortality in humans. Due to constant change in their immuno-dominant antigenic sites they can evade adaptive immune responses. Current seasonal influenza virus vaccines therefore require annual re-formulation and re-administration to confer protection from circulating viruses. Additionally, these vaccines cannot protect against novel pandemic influenza virus strains. Novel vaccination approaches attempt to refocus antibody responses towards more conserved domains like the hemagglutinin stalk. Antibodies against the stalk domain are broadly-reactive and can neutralize multiple influenza virus subtypes. However, the stalk domain is immuno-subdominant and not preferentially targeted by the immune system. In this study, we tested if a vaccination strategy based on influenza viruses expressing chimeric hemagglutinins (cH) that contain exotic, divergent head domains, but a conserved H1 stalk domain could induce cross-protective antibody responses in ferrets. We compared a heterologous live-attenuated virus (cH8/1N1) prime followed by an inactivated split virus (cH5/1N1) boost combination approach to two doses of split-virus vaccines (cH8/1N1/cH5/1N1) and the impact of adjuvant on the immune response. Additionally, a ‘standard of care’ control group received 2 rounds of a human trivalent influenza virus vaccine. We found that all universal influenza virus vaccination approaches were successful at inducing stalk-reactive antibody responses in serum. Virus replication was limited to the nasopharynx in the live attenuated/split vaccine groups and nasal wash titers were lower than in the \u27standard of care\u27 control group. No virus replication was detected in the lungs of attenuated/split vaccinated ferrets, while the \u27standard of care\u27 group had similarly high titers as an unvaccinated control group. Our findings demonstrate that - using a chimeric hemagglutinin based heterologous attenuated/split combination strategy - our candidate universal influenza virus vaccine can successfully protect ferrets from pandemic H1N1 infection. The data support further development of this vaccination approach and advancement into clinical trials

A universal Influenza B vaccine using mosaic-hemagglutinin vaccine candidates

Influenza viruses undergo antigenic changes in the immuno-dominant hemagglutinin (HA) head domain, necessitating annual re-formulation of and re-vaccination with seasonal influenza virus vaccines for maintaining the protection. We previously synthesized mosaic HA (mHA) proteins of influenza B viruses which redirect the immune response towards the immuno-subdominant conserved epitopes of the B virus HA via sequential immunization. As ~90% of current influenza virus vaccines are manufactured using the inactivated virus platform, we generated and sequentially vaccinated mice with inactivated influenza B viruses displaying either the homologous (same B HA backbones) or the heterologous (different B HA backbones) mosaic HAs. Both approaches induced long-lasting and cross-protective antibody responses showing strong antibody-dependent cellular cytotoxicity (ADCC) activity. Thereafter, we tested different inactivation methods and adjuvants to increase the cross-protection against phylogenetically distant influenza B viruses from both lineages. The use of CpG 1018 or AddaVax boosted the humoral immune response and protection when combined with any inactivation method. Beta-propiolactone (BPL) inactivation was the best method, with high serum HA antibodies levels that correlated with optimal protection in BALB/C mice challenge studies. We believe that these B virus mHA vaccine candidates represent a major step towards a universal influenza B virus vaccine. Please click Download on the upper right corner to see the full abstract

Epidemiology of the influenza A virus H5N1 subtype and memory of immunity to the H2N2 subtype

Comment on: Why do influenza virus subtypes die out? A hypothesis. [MBio. 2011

Infection with 2009 H1N1 influenza virus primes for immunological memory in human nose-associated lymphoid tissue, offering cross-reactive immunity to H1N1 and avian H5N1 viruses

Influenza is a highly contagious mucosal infection in the respiratory tract. 2009 pandemic H1N1 (pH1N1) virus infection resulted in substantial morbidity and mortality in humans. Little is known on whether immunological memory develops following pH1N1 infection and whether it provides protection against other virus subtypes. Enzyme-linked immunosorbent spot assay was used to analyze hemagglutinin (HA)-specific memory B cell responses after virus antigen stimulation in nasal-associated lymphoid tissues (NALT) from children and adults. Individuals with serological evidence of previous exposure to pH1N1 showed significant cross-reactive HA-specific memory B responses to pH1N1, seasonal H1N1(sH1N1) and avian H5N1(aH5N1) viruses upon pH1N1 virus stimulation. pH1N1 virus antigen elicited stronger cross-reactive memory B cell responses than sH1N1 virus. Intriguingly, aH5N1 virus also activated cross-reactive memory responses to sH1N1 and pH1N1 HAs in those who had previous pH1N1 exposure, and that correlated well with the memory response stimulated by pH1N1 virus antigen. These memory B cell responses resulted in cross-reactive neutralizing antibodies against sH1N1, 1918 H1N1 and aH5N1viruses. 2009 pH1N1 infection appeared to have primed human host with B cell memory in NALT that offers cross-protective mucosal immunity against not only H1N1 but also aH5N1 viruses. These findings may have important implications to future vaccination strategies against influenza. It will be important to induce and/or enhance such cross-protective mucosal memory B cells

Pre-existing Hemagglutinin Stalk Antibodies Correlate with Protection of Lower Respiratory Symptoms in Flu-Infected Transplant Patients

Hemagglutination-inhibitory antibodies are usually highly strain specific with little effect on infection with drifted or shifted strains. The significance of broadly cross-reactive non-HAI anti-influenza antibodies against conserved domains of virus glycoproteins, such as the hemagglutinin (HA) stalk, is of great interest. We characterize a cohort of 40 H1N1pmd09 influenza-infected patients and identify lower respiratory symptoms (LRSs) as a predictor for development of pneumonia. A binomial logistic regression of log10 pre-existing antibody values shows that the probability of LRS occurrence decreased with increased anti-HA full-length and stalk antibody ELISA titers. However, a multilevel logistic regression model adjusted by other potential serocorrelates demonstrates that only antibodies directed against the stalk of HA correlate with protection from lower respiratory infection, limiting disease progression. Our predictive model indicates that a threshold of protective immunity based on broadly cross-reactive HA stalk antibodies could be feasible