Heterogeneity of Early Host Response to Infection with Four Low-Pathogenic H7 Viruses with a Different Evolutionary History in the Field.

Once low-pathogenic avian influenza viruses (LPAIVs) of the H5 and H7 subtypes from wild birds enter into poultry species, there is the possibility of them mutating into highly pathogenic avian influenza viruses (HPAIVs), resulting in severe epizootics with up to 100% mortality. This mutation from a LPAIV to HPAIV strain is the main cause of an AIV's major economic impact on poultry production. Although AIVs are inextricably linked to their hosts in their evolutionary history, the contribution of host-related factors in the emergence of HPAI viruses has only been marginally explored so far. In this study, transcriptomic sequencing of tracheal tissue from chickens infected with four distinct LP H7 viruses, characterized by a different history of pathogenicity evolution in the field, was implemented. Despite the inoculation of a normalized infectious dose of viruses belonging to the same subtype (H7) and pathotype (LPAI), the use of animals of the same age, sex and species as well as the identification of a comparable viral load in the target samples, the analyses revealed a heterogeneity in the gene expression profile in response to infection with each of the H7 viruses administered.

Equine Influenza Virus and Vaccines.

Equine influenza virus (EIV) is a constantly evolving viral pathogen that is responsible for yearly outbreaks of respiratory disease in horses termed equine influenza (EI). There is currently no evidence of circulation of the original H7N7 strain of EIV worldwide; however, the EIV H3N8 strain, which was first isolated in the early 1960s, remains a major threat to most of the world's horse populations. It can also infect dogs. The ability of EIV to constantly accumulate mutations in its antibody-binding sites enables it to evade host protective immunity, making it a successful viral pathogen. Clinical and virological protection against EIV is achieved by stimulation of strong cellular and humoral immunity in vaccinated horses. However, despite EI vaccine updates over the years, EIV remains relevant, because the protective effects of vaccines decay and permit subclinical infections that facilitate transmission into susceptible populations. In this review, we describe how the evolution of EIV drives repeated EI outbreaks even in horse populations with supposedly high vaccination coverage. Next, we discuss the approaches employed to develop efficacious EI vaccines for commercial use and the existing system for recommendations on updating vaccines based on available clinical and virological data to improve protective immunity in vaccinated horse populations. Understanding how EIV biology can be better harnessed to improve EI vaccines is central to controlling EI.

H7N7 Avian Influenza Virus Mutation from Low to High Pathogenicity on a Layer Chicken Farm in the UK.

Avian influenza virus (AIV) subtypes H5 and H7 are capable of mutating from low to high pathogenicity strains, causing high mortality in poultry with significant economic losses globally. During 2015, two outbreaks of H7N7 low pathogenicity AIV (LPAIV) in Germany, and one each in the United Kingdom (UK) and The Netherlands occurred, as well as single outbreaks of H7N7 high pathogenicity AIV (HPAIV) in Germany and the UK. Both HPAIV outbreaks were linked to precursor H7N7 LPAIV outbreaks on the same or adjacent premises. Herein, we describe the clinical, epidemiological, and virological investigations for the H7N7 UK HPAIV outbreak on a farm with layer chickens in mixed free-range and caged units. H7N7 HPAIV was identified and isolated from clinical samples, as well as H7N7 LPAIV, which could not be isolated. Using serological and molecular evidence, we postulate how the viruses spread throughout the premises, indicating potential points of incursion and possible locations for the mutation event. Serological and mortality data suggested that the LPAIV infection preceded the HPAIV infection and afforded some clinical protection against the HPAIV. These results document the identification of a LPAIV to HPAIV mutation in nature, providing insights into factors that drive its manifestation during outbreaks.

An evaluation of cytokine and cellular immune responses to heterologous prime-boost vaccination with influenza A/H7N7-A/H7N9 inactivated vaccine.

The immunologic mechanisms underlying the improved serologic responses to heterologous prime-boost avian influenza vaccination are unclear. An exploratory analysis of the immune responses following 1 dose of influenza A/H7N9 inactivated vaccine in subjects who received an influenza A/H7N7 inactivated vaccine (N = 17) 8 years earlier or who were influenza A/H7-naïve (10) was performed. Plasma IL-6 and IL-21 concentrations by ELISA, the frequency of A/H7N7-specific memory B cells and antibody secreting cells by ELISpot, the frequency of circulating T follicular helper cells and the frequency of T cells expressing IL-6 and IL-21 by flow cytometry were assessed at baseline (D1), and 8 days (D9) and 28 days (D29) after vaccination. We assessed the correlation between these measurements and the D29 serologic responses to the boost vaccine. Plasma IL-6 concentration on D9 significantly correlated with the H7N7 and H7N9 hemagglutination inhibition (HAI) antibody levels (P = .03 and 0.02 respectively); and the percentage of T cells expressing IL-21 on D9 significantly correlated with H7N9 HAI antibody seroconversion (P < .001). Significant associations with other immunologic markers were not detected. We detected an association between plasma IL-6 and intracellular IL-21 and serologic responses to heterologous prime-boost avian influenza vaccination. A clarification of the role of these and additional immunologic markers requires larger clinical trials.

Specific memory B cell response in humans upon infection with highly pathogenic H7N7 avian influenza virus.

H7 avian influenza viruses represent a major public health concern, and worldwide outbreaks raise the risk of a potential pandemic. Understanding the memory B cell response to avian (H7) influenza virus infection in humans could provide insights in the potential key to human infection risks. We investigated an epizootic of the highly pathogenic A(H7N7) in the Netherlands, which in 2003 led to infection of 89 persons and one fatal case. Subtype-specificity of antibodies were determined for confirmed H7N7 infected individuals (cases) (n = 19), contacts of these cases (n = 21) and a comparison group controls (n = 16), by microarray, using recombinant hemagglutinin (HA)1 proteins. The frequency and specificity of memory B cells was determined by detecting subtype-specific antibodies in the culture supernatants from in vitro stimulated oligoclonal B cell cultures, from peripheral blood of cases and controls. All cases (100%) had high antibody titers specific for A(H7N7)2003 (GMT > 100), whereas H7-HA1 antigen binding was detected in 29% of contacts and 31% of controls, suggesting that some of the H7 reactivity stems from cross reactive antibodies. To unravel homotypic and heterotypic responses, the frequency and specificity of memory B cells were determined in 2 cases. Ten of 123 HA1 reactive clones isolated from the cases bound to only H7- HA1, whereas 5 bound both H7 and other HA1 antigens. We recovered at least four different epitopal reactivities, though none of the H7 reactive antibodies were able to neutralize H7 infections in vitro. Our study serologically confirms the infection with H7 avian influenza viruses, and shows that H7 infection triggers a mixture of strain -specific and cross-reactive antibodies.

Safety and immunogenicity of an 8 year interval heterologous prime-boost influenza A/H7N7-H7N9 vaccination.

BACKGROUND: Influenza A/H7N9 viruses are undergoing antigenic drift since their emergence in 2013, and vaccination strategies are needed for pandemic preparedness. Two doses of adjuvanted monovalent inactivated influenza A/H7N9 vaccine (IIV1 A/H7N9) are needed for optimal serological responses. However, administering 2 doses in a pandemic setting might be challenging. We evaluated the immunogenicity of "boosting" with IIV1 A/H7N9 in subjects "primed" 8 years previously with IIV1 A/H7N7. METHODS: We administered 1 booster dose containing 45 mcg of IIV1 A/H7N9 hemagglutinin to 17 recipients of 2 prior doses of IIV1 A/H7N7, and to 10 influenza A/H7-naïve subjects. We tested their post-boosting sera for antibodies (Ab) against homologous influenza A/H7N9 using a hemagglutination inhibition assay; and compared their Ab titers to those in stored sera from recipients of AS03-adjuvanted IIV1 A/H7N9 against 9 strains of influenza A/H7N9 viruses. RESULTS: The percentage of subjects with Ab titers ≥40 on Days 9 and 29 post boosting, respectively, was 65% and 41% in primed subjects and 10% and 0% in unprimed subjects. The Ab titers in recipients of AS03-adjuvanted IIV1 A/H7N9 were higher than those in the prime-boost group against a panel of influenza A/H7N9 viruses, except for 2 highly pathogenic strains. CONCLUSIONS: Priming with IIV1 A/H7 results in serological responses following a delayed boost with 1 dose of unadjuvanted IIV1 A/H7N9, despite lack of antibody response after the prime. Optimizing prime-boost approaches would benefit pandemic preparedness. ClinicalTrials.gov identifier: NCT02586792.

Inactivated H7 Influenza Virus Vaccines Protect Mice despite Inducing Only Low Levels of Neutralizing Antibodies.

Avian influenza viruses of the H7 hemagglutinin (HA) subtype present a significant public health threat, as evidenced by the ongoing outbreak of human A(H7N9) infections in China. When evaluated by hemagglutination inhibition (HI) and microneutralization (MN) assays, H7 viruses and vaccines are found to induce lower level of neutralizing antibodies (nAb) than do their seasonal counterparts, making it difficult to develop and evaluate prepandemic vaccines. We have previously shown that purified recombinant H7 HA appear to be poorly immunogenic in that they induce low levels of HI and MN antibodies. In this study, we immunized mice with whole inactivated reverse genetics reassortant (RG) viruses expressing HA and neuraminidase (NA) from 3 different H7 viruses [A/Shanghai/2/2013(H7N9), A/Netherlands/219/2003(H7N7), and A/New York/107/2003(H7N2)] or with human A(H1N1)pdm09 (A/California/07/2009-like) or A(H3N2) (A/Perth16/2009) viruses. Mice produced equivalent titers of antibodies to all viruses as measured by enzyme-linked immunosorbent assay (ELISA). However, the antibody titers induced by H7 viruses were significantly lower when measured by HI and MN assays. Despite inducing very low levels of nAb, H7 vaccines conferred complete protection against homologous virus challenge in mice, and the serum antibodies directed against the HA head region were capable of mediating protection. The apparently low immunogenicity associated with H7 viruses and vaccines may be at least partly related to measuring antibody titers with the traditional HI and MN assays, which may not provide a true measure of protective immunity associated with H7 immunization. This study underscores the need for development of additional correlates of protection for prepandemic vaccines.IMPORTANCE H7 avian influenza viruses present a serious risk to human health. Preparedness efforts include development of prepandemic vaccines. For seasonal influenza viruses, protection is correlated with antibody titers measured by hemagglutination inhibition (HI) and virus microneutralization (MN) assays. Since H7 vaccines typically induce low titers in HI and MN assays, they have been considered to be poorly immunogenic. We show that in mice H7 whole inactivated virus vaccines (WIVs) were as immunogenic as seasonal WIVs, as they induced similar levels of overall serum antibodies. However, a larger fraction of the antibodies induced by H7 WIV was nonneutralizing in vitro Nevertheless, the H7 WIV completely protected mice against homologous viral challenge, and antibodies directed against the HA head were the major contributor toward immune protection. Vaccines against H7 avian influenza viruses may be more effective than HI and virus neutralization assays suggest, and such vaccines may need other methods for evaluation.

Heterosubtypic cross-reactivity of HA1 antibodies to influenza A, with emphasis on nonhuman subtypes (H5N1, H7N7, H9N2).

Epidemics of influenza A vary greatly in size and age distribution of cases, and this variation is attributed to varying levels of pre-existing immunity. Recent studies have shown that antibody-mediated immune responses are more cross-reactive than previously believed, and shape patterns of humoral immunity to influenza A viruses over long periods. Here we quantify antibody responses to the hemagglutinin subunit 1 (HA1) across a range of subtypes using protein microarray analysis of cross-sectional serological surveys carried out in the Netherlands before and after the A/2009 (H1N1) pandemic. We find significant associations of responses, both within and between subtypes. Interestingly, substantial overall reactivity is observed to HA1 of avian H7N7 and H9N2 viruses. Seroprevalence of H7N7 correlates with antibody titers to A/1968 (H3N2), and is highest in persons born between 1954 and 1969. Seroprevalence of H9N2 is high across all ages, and correlates strongly with A/1957 (H2N2). This correlation is most pronounced in A/2009 (H1N1) infected persons born after 1968 who have never encountered A/1957 (H2N2)-like viruses. We conclude that heterosubtypic antibody cross-reactivity, both between human subtypes and between human and nonhuman subtypes, is common in the human population.

In vivo evasion of MxA by avian influenza viruses requires human signature in the viral nucleoprotein.

Zoonotic transmission of influenza A viruses can give rise to devastating pandemics, but currently it is impossible to predict the pandemic potential of circulating avian influenza viruses. Here, we describe a new mouse model suitable for such risk assessment, based on the observation that the innate restriction factor MxA represents an effective species barrier that must be overcome by zoonotic viruses. Our mouse lacks functional endogenous Mx genes but instead carries the human MX1 locus as a transgene. Such transgenic mice were largely resistant to highly pathogenic avian H5 and H7 influenza A viruses, but were almost as susceptible to infection with influenza viruses of human origin as nontransgenic littermates. Influenza A viruses that successfully established stable lineages in humans have acquired adaptive mutations which allow partial MxA escape. Accordingly, an engineered avian H7N7 influenza virus carrying a nucleoprotein with signature mutations typically found in human virus isolates was more virulent in transgenic mice than parental virus, demonstrating that a few amino acid changes in the viral target protein can mediate escape from MxA restriction in vivo. Similar mutations probably need to be acquired by emerging influenza A viruses before they can spread in the human population.

Antigenic Fingerprinting of Antibody Response in Humans following Exposure to Highly Pathogenic H7N7 Avian Influenza Virus: Evidence for Anti-PA-X Antibodies.

UNLABELLED: Infections with H7 highly pathogenic avian influenza (HPAI) viruses remain a major public health concern. Adaptation of low-pathogenic H7N7 to highly pathogenic H7N7 in Europe in 2015 raised further alarm for a potential pandemic. An in-depth understanding of antibody responses to HPAI H7 virus following infection in humans could provide important insight into virus gene expression as well as define key protective and serodiagnostic targets. Here we used whole-genome gene fragment phage display libraries (GFPDLs) expressing peptides of 15 to 350 amino acids across the complete genome of the HPAI H7N7 A/Netherlands/33/03 virus. The hemagglutinin (HA) antibody epitope repertoires of 15 H7N7-exposed humans identified clear differences between individuals with no hemagglutination inhibition (HI) titers (<1:10) and those with HI titers of >1:40. Several potentially protective H7N7 epitopes close to the HA receptor binding domain (RBD) and neuraminidase (NA) catalytic site were identified. Surface plasmon resonance (SPR) analysis identified a strong correlation between HA1 (but not HA2) binding antibodies and H7N7 HI titers. A proportion of HA1 binding in plasma was contributed by IgA antibodies. Antibodies against the N7 neuraminidase were less frequent but targeted sites close to the sialic acid binding site. Importantly, we identified strong antibody reactivity against PA-X, a putative virulence factor, in most H7N7-exposed individuals, providing the first evidence for in vivo expression of PA-X and its recognition by the immune system during human influenza A virus infection. This knowledge can help inform the development and selection of the most effective countermeasures for prophylactic as well as therapeutic treatments of HPAI H7N7 avian influenza virus. IMPORTANCE: An outbreak of pathogenic H7N7 virus occurred in poultry farms in The Netherlands in 2003. Severe outcome included conjunctivitis, influenza-like illness, and one lethal infection. In this study, we investigated convalescent-phase sera from H7N7-exposed individuals by using a whole-genome phage display library (H7N7-GFPDL) to explore the complete repertoire of post-H7N7-exposure antibodies. PA-X is a recently identified influenza virus virulence protein generated by ribosomal frameshifting in segment 3 of influenza virus coding for PA. However, PA-X expression during influenza virus infection in humans is unknown. We identified strong antibody reactivity against PA-X in most H7N7-exposed individuals (but not in unexposed adults), providing the first evidence for in vivo expression of PA-X and its recognition by the immune system during human infection with pathogenic H7N7 avian influenza virus.

Host genetics determine susceptibility to avian influenza infection and transmission dynamics.

Host-genetic control of influenza virus infection has been the object of little attention. In this study we determined that two inbred lines of chicken differing in their genetic background , Lines 0 and C-B12, were respectively relatively resistant and susceptible to infection with the low pathogenicity influenza virus A/Turkey/England/647/77 as defined by substantial differences in viral shedding trajectories. Resistant birds, although infected, were unable to transmit virus to contact birds, as ultimately only the presence of a sustained cloacal shedding (and not oropharyngeal shedding) was critical for transmission. Restriction of within-bird transmission of virus occurred in the resistant line, with intra-nares or cloacal infection resulting in only local shedding and failing to transmit fully through the gastro-intestinal-pulmonary tract. Resistance to infection was independent of adaptive immune responses, including the expansion of specific IFNγ secreting cells or production of influenza-specific antibody. Genetic resistance to a novel H9N2 virus was less robust, though significant differences between host genotypes were still clearly evident. The existence of host-genetic determination of the outcome of influenza infection offers tools for the further dissection of this regulation and also for understanding the mechanisms of influenza transmission within and between birds.

Serological Evidence of Human Infection with Avian Influenza A H7virus in Egyptian Poultry Growers.

Avian influenza viruses circulate widely in birds, with occasional human infections. Poultry-exposed individuals are considered to be at high risk of infection with avian influenza viruses due to frequent exposure to poultry. Some avian H7 viruses have occasionally been found to infect humans. Seroprevalence of neutralizing antibodies against influenza A/H7N7 virus among poultry-exposed and unexposed individuals in Egypt were assessed during a three-years prospective cohort study. The seroprevalence of antibodies (titer, ≥80) among exposed individuals was 0%, 1.9%, and 2.1% annually while the seroprevalence among the control group remained 0% as measured by virus microneutralization assay. We then confirmed our results using western blot and immunofluorescence assays. Although human infection with H7 in Egypt has not been reported yet, our results suggested that Egyptian poultry growers are exposed to avian H7 viruses. These findings highlight the need for surveillance in the people exposed to poultry to monitor the risk of zoonotic transmission of avian influenza viruses.

Glycosylation of Residue 141 of Subtype H7 Influenza A Hemagglutinin (HA) Affects HA-Pseudovirus Infectivity and Sensitivity to Site A Neutralizing Antibodies.

Human infections with H7 subtype influenza virus have been reported, including an H7N7 outbreak in Netherlands in 2003 and H7N9 infections in China in 2013. Previously, we reported murine monoclonal antibodies (mAbs) that recognize the antigenic site A of H7 hemagglutinin (HA). To better understand protective immunity of H7 vaccines and vaccine candidate selection, we used these mAbs to assess the antigenic relatedness among two H7 HA isolated from past human infections and determine residues that affect susceptibility to neutralization. We found that these mAbs neutralize pseudoviruses bearing HA of A/Shanghai/02/2013(H7N9), but not A/Netherlands/219/2003(H7N7). Glycosylation of the asparagine residue at position 141 (N141) (N133, H3 HA numbering) in the HA of A/Netherlands/219/2003 HA is responsible for this resistance, and it affects the infectivity of HA-pseudoviruses. The presence of threonine at position 143 (T135, H3 HA numbering) in the HA of A/Netherlands/219/2003, rather than an alanine found in the HA of A/Shanghai/02/2013(H7N9), accounts for these differences. These results demonstrate a key role for glycosylation of residue N141 in affecting H7 influenza HA-mediated entry and sensitivity to neutralizing antibodies, which have implications for candidate vaccine design.

Cross-reactivity between avian influenza A (H7N9) virus and divergent H7 subtypic- and heterosubtypic influenza A viruses.

The number of human avian H7N9 influenza infections has been increasing in China. Understanding their antigenic and serologic relationships is crucial for developing diagnostic tools and vaccines. Here, we evaluated the cross-reactivities and neutralizing activities among H7 subtype influenza viruses and between H7N9 and heterosubtype influenza A viruses. We found strong cross-reactivities between H7N9 and divergent H7 subtypic viruses, including H7N2, H7N3, and H7N7. Antisera against H7N2, H7N3, and H7N7 could also effectively neutralize two distinct H7N9 strains. Two-way cross-reactivities exist within group 2, including H3 and H4, whereas one-way cross-reactivities were found across other groups, including H1, H10, H9, and H13. Our data indicate that the hemaglutinins from divergent H7 subtypes may facilitate the development of vaccines for distinct H7N9 infections. Moreover, serologic diagnoses for H7N9 infections need to consider possible interference from the cross-reactivity of H7N9 with other subtype influenza viruses.

Protective efficacy of IFN-ω AND IFN-λs against influenza viruses in induced A549 cells.

The interferon system represents one of the components of the first line defence against influenza virus infection. Interferon omega (IFN-ω) is antigenetically different from IFN-α and IFN-ß and can affect patients who are resistant to these IFNs. To improve the biological characterization of IFN-ω, we compared its activity with those of type I and type III IFNs in induced A549 cells. The antiviral effect on IFN-stimulated A549 cells was most apparent after infection with avian influenza virus. IFN-ω had statistically significant antiviral activity although less than IFN-ß1a, IFN-λ1, or IFN-λ2. On the other hand, IFN-ω appeared more efficient than IFN-α2. Our results also indicate that IFN-λs were more suitable against human highly pathogenic virus. In this case, IFN-λ1 and IFN-λ2 were more potent than type I IFNs.

Amino Acid Substitutions Improve the Immunogenicity of H7N7HA Protein and Protect Mice against Lethal H7N7 Viral Challenge.

Avian influenza A H7N7/NL/219/03 virus creates a serious pandemic threat to human health because it can transmit directly from domestic poultry to humans and from human to human. Our previous vaccine study reported that mice when immunized intranasally (i.n) with live Bac-HA were protected from lethal H7N7/NL/219/03 challenge, whereas incomplete protection was obtained when administered subcutaneously (s.c) due to the fact that H7N7 is a poor inducer of neutralizing antibodies. Interestingly, our recent vaccine studies reported that mice when vaccinated subcutaneously with Bac-HA (H7N9) was protected against both H7N9 (A/Sh2/2013) and H7N7 virus challenge. HA1 region of both H7N7 and H7N9 viruses are differ at 15 amino acid positions. Among those, we selected three amino acid positions (T143, T198 and I211) in HA1 region of H7N7. These amino acids are located within or near the receptor binding site. Following the selection, we substituted the amino acid at these three positions with amino acids found on H7N9HA wild-type. In this study, we evaluate the impact of amino acid substitutions in the H7N7 HA-protein on the immunogenicity. We generated six mutant constructs from wild-type influenza H7N7HA cDNA by site directed mutagenesis, and individually expressed mutant HA protein on the surface of baculovirus (Bac-HAm) and compared their protective efficacy of the vaccines with Bac-H7N7HA wild-type (Bac-HA) by lethal H7N7 viral challenge in a mouse model. We found that mice immunized subcutaneously with Bac-HAm constructs T143A or T198A-I211V or I211V-T143A serum showed significantly higher hemagglutination inhibition and neutralization titer against H7N7 and H7N9 viruses when compared to Bac-HA vaccinated mice groups. We also observed low level of lung viral titer, negligible weight loss and complete protection against lethal H7N7 viral challenge. Our results indicated that amino acid substitution at position 143 or 211 improve immunogenicity of H7N7HA vaccine against H7N7/NL/219/03 virus.

Serum strain-specific or cross-reactive neuraminidase inhibiting antibodies against pandemic А/California/07/2009(H1N1) influenza in healthy volunteers.

BACKGROUND: Pre-existing antibodies to influenza virus neuraminidase may provide protection against infection influenza viruses containing novel hemagglutinin (HA). The aim of our study was to evaluate serum neuraminidase-inhibiting (NI) antibodies against А/California/07/2009(H1N1) [H1N1/2009pdm] and А/New Caledonia/20/1999(H1N1) [H1N1/1999] influenza viruses in relation with the age of participants and hemagglutination-inhibition (HI) antibody levels. Anti-H1N1/2009pdm neuraminidase and anti-H1N1/1999 neuraminidase antibody levels were measured in total 219 serum samples from Russian healthy peoples of various ages examined before and a year after pandemic strain appearance. We adjusted peroxidase-linked lectin micro-procedure to measure NI antibody titers using the reassortant A/H7N1 influenza viruses based on A/equine/Prague/1/56(H7N7). Also, HI antibody titers were estimated against H1N1/2009pdm, H1N1/1999 and a panel of seasonal A/H1N1 influenza viruses. RESULTS: In sera samples collected during the fall of 2010, mean titers of specific HI and NI antibodies to H1N1/2009pdm were 2-2.1 times lower than antibody levels against H1N1/1999. Of the 163 individuals examined, 58 (35.6%) had NI anti-H1N1/2009pdm antibody titers > 1:20, compared to 93 (57.1%) who had NI anti-H1N1/1999 antibody titers > 1:20. There were low correlations between HI and NI antibody levels against either H1N1/1999 or H1N1/2009pdm in the same serum samples. The 24 adults born between 1957 and 1977 expressed very low levels of NI antibodies to A/H1N1 influenza viruses. Persons with low HI anti-H1N1/2009pdm titers but positive to seasonal A/H1N1 demonstrated significantly higher NI anti-A/H1N1 antibody titers than unexposed subjects. In 2005 cross-reactive NI anti-H1N1/2009pdm antibody titers > 1:20 were detected among 7.1% of young people. CONCLUSIONS: Our study confirmed that contact with seasonal influenza viruses may have contributed to generating the cross-reacting anti-H1N1/2009pdm NI antibodies which were detected in the sera of 18-20 years old people examined before the pandemic virus active circulation. The lowest levels of antibodies to the neuraminidase of N1 subtype were in the group of participants born during the circulation of influenza A/H2N2 or A/H3N2 viruses. The low correlation between HI and NI antibody titers suggests that NI antibody detection can be used as an additional test to evaluate the immune response after influenza infections or immunizations.

High-Affinity H7 Head and Stalk Domain-Specific Antibody Responses to an Inactivated Influenza H7N7 Vaccine After Priming With Live Attenuated Influenza Vaccine.

Recent studies have shown that live attenuated influenza vaccines (LAIVs) expressing avian influenza virus hemagglutinins (HAs) prime for strong protective antibody responses to an inactivated influenza vaccine (IIV) containing the HA. To better understand this priming effect, we compared H7 HA head and stalk domain-specific B-cell responses in H7N7 LAIV-primed subjects and non-H7-primed controls after a single dose of H7N7 IIV. As previously reported, H7N7 LAIV-primed subjects but not control subjects generated strong hemagglutination-inhibiting and neutralizing antibody responses to the H7N7 IIV. Here, we found that the quantity, epitope diversity, and affinity of H7 head-specific antibodies increased rapidly in only H7N7 LAIV-primed subjects after receipt of the IIV. However, all cohorts generated a vigorous, high-affinity, stalk-specific antibody response. Consistent increases in circulating memory B-cell frequencies after receipt of the IIV reflected the specificity of high-affinity antibody production. Our findings emphasize the value of LAIVs as a vehicle for prepandemic vaccination.

Live attenuated H7N7 influenza vaccine primes for a vigorous antibody response to inactivated H7N7 influenza vaccine.

BACKGROUND: H7 influenza viruses have emerged as potential pandemic threat. We evaluated the safety and immunogenicity of two candidate H7 pandemic live attenuated influenza vaccines (pLAIV) and their ability to prime for responses to an unadjuvanted H7 pandemic inactivated influenza vaccine (pIIV). METHODS: Healthy seronegative adults received two doses of A/Netherlands/219/03 (H7N7) or one dose of A/chicken/British Columbia/CN-6/04 (H7N3) pLAIV all given as 10(7.5) 50% tissue culture infective doses (TCID50) intranasally. A subset of subjects received one 45 µg dose of H7N7 pIIV containing the A/Mallard/Netherlands/12/2000 HA intramuscularly 18-24 months after pLAIV. Viral shedding was assessed by culture and real-time polymerase chain reaction (rRT-PCR), B cell responses following pLAIV were evaluated by ELISPOT and flow cytometry. Serum antibody was assessed by hemagglutination-inhibition (HAI), microneutralization (MN) and ELISA assays after each vaccine. RESULTS: Serum HAI or MN responses were not detected in any subject following one or two doses of either H7 pLAIV, although some subjects had detectable H7 specific B cells after vaccination. However, 10/13 subjects primed with two doses of H7N7 pLAIV responded to a subsequent dose of the homologous H7N7 pIIV with high titer HAI and MN antibody that cross-reacted with both North American and Eurasian lineage H7 viruses, including H7N9. In contrast, naïve subjects and recipients of a single dose of the mismatched H7N3 pLAIV did not develop HAI or MN antibody after pIIV. CONCLUSIONS: While pLAIVs did not elicit detectable serum MN or HAI antibody, strain-specific pLAIV priming established long term immune memory that was cross-reactive with other H7 influenza strains. Understanding the mechanisms underlying priming by pLAIV may aid in pandemic vaccine development.