N-Linked Glycans and K147 Residue on Hemagglutinin Synergize To Elicit Broadly Reactive H1N1 Influenza Virus Antibodies.

Vaccination is the most effective way to prevent influenza virus infections. However, the diversity of antigenically distinct isolates is a challenge for vaccine development. In order to overcome the antigenic variability and improve the protective efficacy of influenza vaccines, our research group has pioneered the development of computationally optimized broadly reactive antigens (COBRA) for hemagglutinin (HA). Two candidate COBRA HA vaccines, P1 and X6, elicited antibodies with differential patterns of hemagglutination inhibition (HAI) activity against a panel of H1N1 influenza viruses. In order to better understand how these HA antigens elicit broadly reactive immune responses, epitopes in the Cb, Sa, or Sb antigenic sites of seasonal-like and pandemic-like wild-type or COBRA HA antigens were exchanged with homologous regions in the COBRA HA proteins to determine which regions and residues were responsible for the elicited antibody profile. Mice were vaccinated with virus-like particles (VLPs) expressing one of the 12 modified HA antigens (designated V1 to V12), COBRA HA antigens, or wild-type HA antigens. The elicited antisera was assessed for hemagglutination inhibition activity against a panel of historical seasonal-like and pandemic-like H1N1 influenza viruses. Primarily, the pattern of glycosylation sites and residues in the Sa antigenic region, around the receptor binding site (RBS), served as signatures for the elicitation of broadly reactive antibodies by these HA immunogens. Mice were vaccinated with VLPs expressing HA antigens that lacked a glycosylation site at residue 144 and a deleted lysine at position 147 residue were more effective at protecting against morbidity and mortality following infection with pandemic-like and seasonal-like H1N1 influenza viruses.IMPORTANCE There is a great need to develop broadly reactive or universal vaccines against influenza viruses. Advanced, next-generation hemagglutinin (HA) head-based vaccines that elicit protective antibodies against H1N1 influenza viruses have been developed. This study focused on understanding the specific amino acids around the receptor binding site (RBS) that were important in elicitation of these broadly reactive antibodies. Specific glycan sites and amino acids located at the tip of the HA molecule enhanced the elicitation of these broadly reactive antibodies. A better understanding of the HA structures around the RBS will lead to more effective HA immunogens.

Computationally optimized broadly reactive vaccine based upon swine H1N1 influenza hemagglutinin sequences protects against both swine and human isolated viruses.

Swine H1 influenza viruses were stable within pigs for nearly 70 years until in 1998 when a classical swine virus reassorted with avian and human influenza viruses to generate the novel triple reassortant H1N1 strain that eventually led to the 2009 influenza pandemic. Previously, our group demonstrated broad protection against a panel of human H1N1 viruses using HA antigens derived by the COBRA methodology. In this report, the effectiveness of COBRA HA antigens (SW1, SW2, SW3 and SW4), which were designed using only HA sequences from swine H1N1 and H1N2 isolates, were tested in BALB/c mice. The effectiveness of these vaccines were compared to HA sequences designed using both human and swine H1 HA sequences or human only sequences. SW2 and SW4 elicited antibodies that detected the pandemic-like virus, A/California/07/2009 (CA/09), had antibodies with HAI activity against almost all the classical swine influenza viruses isolated from 1973-2015 and all of the Eurasian viruses in our panel. However, sera collected from mice vaccinated with SW2 or SW4 had HAI activity against ~25% of the human seasonal-like influenza viruses isolated from 2009-2015. In contrast, the P1 COBRA HA vaccine (derived from both swine and human HA sequences) elicited antibodies that had HAI activity against both swine and human H1 viruses and protected against CA/09 challenge, but not a human seasonal-like swine H1N2 virus challenge. However, the SW1 vaccine protected against this challenge as well as the homologous vaccine. These results support the idea that a pan-swine-human H1 influenza virus vaccine is possible.

Elicitation of Protective Antibodies against a Broad Panel of H1N1 Viruses in Ferrets Preimmune to Historical H1N1 Influenza Viruses.

Most preclinical animal studies test influenza vaccines in immunologically naive animal models, even though the results of vaccination may not accurately reflect the effectiveness of vaccine candidates in humans that have preexisting immunity to influenza. In this study, novel, broadly reactive influenza vaccine candidates were assessed in preimmune ferrets. These animals were infected with different H1N1 isolates before being vaccinated or infected with another influenza virus. Previously, our group has described the design and characterization of computationally optimized broadly reactive hemagglutinin (HA) antigens (COBRA) for H1N1 isolates. Vaccinating ferrets with virus-like particle (VLP) vaccines expressing COBRA HA proteins elicited antibodies with hemagglutination inhibition (HAI) activity against more H1N1 viruses in the panel than VLP vaccines expressing wild-type HA proteins. Specifically, ferrets infected with the 1986 virus and vaccinated with a single dose of the COBRA HA VLP vaccines elicited antibodies with HAI activity against 11 to 14 of the 15 H1N1 viruses isolated between 1934 and 2013. A subset of ferrets was infected with influenza viruses expressing the COBRA HA antigens. These COBRA preimmune ferrets had superior breadth of HAI activity after vaccination with COBRA HA VLP vaccines than COBRA preimmune ferrets vaccinated with VLP vaccines expressing wild-type HA proteins. Overall, priming naive ferrets with COBRA HA based viruses or using COBRA HA based vaccines to boost preexisting antibodies induced by wild-type H1N1 viruses, COBRA HA antigens elicited sera with the broadest HAI reactivity against multiple antigenic H1N1 viral variants. This is the first report demonstrating the effectiveness of a broadly reactive or universal influenza vaccine in a preimmune ferret model.IMPORTANCE Currently, many groups are testing influenza vaccine candidates to meet the challenge of developing a vaccine that elicits broadly reactive and long-lasting protective immune responses. The goal of these vaccines is to stimulate immune responses that react against most, if not all, circulating influenza strains, over a long period of time in all populations of people. Commonly, these experimental vaccines are tested in naive animal models that do not have anti-influenza immune responses; however, humans have preexisting immunity to influenza viral antigens, particularly antibodies to the HA and NA glycoproteins. Therefore, this study investigated how preexisting antibodies to historical influenza viruses influenced HAI-specific antibodies and protective efficacy using a broadly protective vaccine candidate.

Computationally Optimized Broadly Reactive Hemagglutinin Elicits Hemagglutination Inhibition Antibodies against a Panel of H3N2 Influenza Virus Cocirculating Variants.

Each influenza season, a set of wild-type viruses, representing one H1N1, one H3N2, and one to two influenza B isolates, are selected for inclusion in the annual seasonal influenza vaccine. In order to develop broadly reactive subtype-specific influenza vaccines, a methodology called computationally optimized broadly reactive antigens (COBRA) was used to design novel hemagglutinin (HA) vaccine immunogens. COBRA technology was effectively used to design HA immunogens that elicited antibodies that neutralized H5N1 and H1N1 isolates. In this report, the development and characterization of 17 prototype H3N2 COBRA HA proteins were screened in mice and ferrets for the elicitation of antibodies with HA inhibition (HAI) activity against human seasonal H3N2 viruses that were isolated over the last 48 years. The most effective COBRA HA vaccine regimens elicited antibodies with broader HAI activity against a panel of H3N2 viruses than wild-type H3 HA vaccines. The top leading COBRA HA candidates were tested against cocirculating variants. These variants were not efficiently detected by antibodies elicited by the wild-type HA from viruses selected as the vaccine candidates. The T-11 COBRA HA vaccine elicited antibodies with HAI and neutralization activity against all cocirculating variants from 2004 to 2007. This is the first report demonstrating broader breadth of vaccine-induced antibodies against cocirculating H3N2 strains compared to the wild-type HA antigens that were represented in commercial influenza vaccines.IMPORTANCE There is a need for an improved influenza vaccine that elicits immune responses that recognize a broader number of influenza virus strains to prevent infection and transmission. Using the COBRA approach, a set of vaccines against influenza viruses in the H3N2 subtype was tested for the ability to elicit antibodies that neutralize virus infection against not only historical vaccine strains of H3N2 but also a set of cocirculating variants that circulated between 2004 and 2007. Three of the H3N2 COBRA vaccines recognized all of the cocirculating strains during this era, but the chosen wild-type vaccine strains were not able to elicit antibodies with HAI activity against these cocirculating strains. Therefore, the COBRA vaccines have the ability to elicit protective antibodies against not only the dominant vaccine strains but also minor circulating strains that can evolve into the dominant vaccine strains in the future.

Broadened immunity and protective responses with emulsion-adjuvanted H5 COBRA-VLP vaccines.

A number of challenges for developing a protective pre-pandemic influenza A vaccine exists including predicting the target influenza strain and designing the vaccine for an immunologically naïve population. Manufacturing and supply of the vaccine would also require implementing ways to increase coverage for the largest number of people through dose-sparing methods, while not compromising the potency of the vaccine. Previously, our group described a novel hemagglutinin (HA) for H5N1 influenza derived from a methodology termed computationally optimized broadly reactive antigen (COBRA). This report describes a strategy combining a COBRA-based HA vaccine with an oil-in-water emulsion, resulting in a dose-sparing, immunologically broadened, and protective response against multiple H5N1 isolates. Here, we show that an emulsion-based adjuvant enhances the magnitude and breadth of antibody responses with both a wild-type H5HA (H5N1 WT) and the H5N1 COBRA HA VLP vaccines. The H5N1 COBRA HA VLP, combined with an emulsion adjuvant, elicited HAI specific antibodies against a larger panel of H5N1 viruses that resulted in protection against challenge as efficiently as the homologous, matched vaccine.

Design and Characterization of a Computationally Optimized Broadly Reactive Hemagglutinin Vaccine for H1N1 Influenza Viruses.

UNLABELLED: One of the challenges of developing influenza A vaccines is the diversity of antigenically distinct isolates. Previously, a novel hemagglutinin (HA) for H5N1 influenza was derived from a methodology termed computationally optimized broadly reactive antigen (COBRA). This COBRA HA elicited a broad antibody response against H5N1 isolates from different clades. We now report the development and characterization of a COBRA-based vaccine for both seasonal and pandemic H1N1 influenza virus isolates. Nine prototype H1N1 COBRA HA proteins were developed and tested in mice using a virus-like particle (VLP) format for the elicitation of broadly reactive, functional antibody responses and protection against viral challenge. These candidates were designed to recognize H1N1 viruses isolated within the last 30 years. In addition, several COBRA candidates were designed based on sequences of H1N1 viruses spanning the past 100 years, including modern pandemic H1N1 isolates. Four of the 9 H1N1 COBRA HA proteins (X1, X3, X6, and P1) had the broadest hemagglutination inhibition (HAI) activity against a panel of 17 H1N1 viruses. These vaccines were used in cocktails or prime-boost combinations. The most effective regimens that both elicited the broadest HAI response and protected mice against a pandemic H1N1 challenge were vaccines that contained the P1 COBRA VLP and either the X3 or X6 COBRA VLP vaccine. These mice had little or no detectable viral replication, comparable to that observed with a matched licensed vaccine. This is the first report describing a COBRA-based HA vaccine strategy that elicits a universal, broadly reactive, protective response against seasonal and pandemic H1N1 isolates. IMPORTANCE: Universal influenza vaccine approaches have the potential to be paradigm shifting for the influenza vaccine field, with the goal of replacing the current standard of care with broadly cross-protective vaccines. We have used COBRA technology to develop an HA head-based strategy that elicits antibodies against many H1 strains that have undergone genetic drift and has potential as a "subtype universal" vaccine. Nine HA COBRA candidates were developed, and these vaccines were used alone, in cocktails or in prime-boost combinations. The most effective regimens elicited the broadest hemagglutination inhibition (HAI) response against a panel of H1N1 viruses isolated over the past 100 years. This is the first report describing a COBRA-based HA vaccine strategy that elicits a broadly reactive response against seasonal and pandemic H1N1 isolates.

Sequential Infection in Ferrets with Antigenically Distinct Seasonal H1N1 Influenza Viruses Boosts Hemagglutinin Stalk-Specific Antibodies.

UNLABELLED: Broadly reactive antibodies targeting the conserved hemagglutinin (HA) stalk region are elicited following sequential infection or vaccination with influenza viruses belonging to divergent subtypes and/or expressing antigenically distinct HA globular head domains. Here, we demonstrate, through the use of novel chimeric HA proteins and competitive binding assays, that sequential infection of ferrets with antigenically distinct seasonal H1N1 (sH1N1) influenza virus isolates induced an HA stalk-specific antibody response. Additionally, stalk-specific antibody titers were boosted following sequential infection with antigenically distinct sH1N1 isolates in spite of preexisting, cross-reactive, HA-specific antibody titers. Despite a decline in stalk-specific serum antibody titers, sequential sH1N1 influenza virus-infected ferrets were protected from challenge with a novel H1N1 influenza virus (A/California/07/2009), and these ferrets poorly transmitted the virus to naive contacts. Collectively, these findings indicate that HA stalk-specific antibodies are commonly elicited in ferrets following sequential infection with antigenically distinct sH1N1 influenza virus isolates lacking HA receptor-binding site cross-reactivity and can protect ferrets against a pathogenic novel H1N1 virus. IMPORTANCE: The influenza virus hemagglutinin (HA) is a major target of the humoral immune response following infection and/or seasonal vaccination. While antibodies targeting the receptor-binding pocket of HA possess strong neutralization capacities, these antibodies are largely strain specific and do not confer protection against antigenic drift variant or novel HA subtype-expressing viruses. In contrast, antibodies targeting the conserved stalk region of HA exhibit broader reactivity among viruses within and among influenza virus subtypes. Here, we show that sequential infection of ferrets with antigenically distinct seasonal H1N1 influenza viruses boosts the antibody responses directed at the HA stalk region. Moreover, ferrets possessing HA stalk-specific antibody were protected against novel H1N1 virus infection and did not transmit the virus to naive contacts.

Cocktail of H5N1 COBRA HA vaccines elicit protective antibodies against H5N1 viruses from multiple clades.

Pandemic outbreaks of influenza are caused by the emergence of a pathogenic and transmissible virus to which the human population is immunologically naïve. Recent outbreaks of highly pathogenic avian influenza (HPAI) of the H5N1 subtype are of particular concern because of the high mortality rate (60% case fatality rate) and novel subtype. In this study, we have engineered an influenza virus-like particle (VLP) that contains a synthetic, consensus-based HA molecule using a new methodology, computationally optimized broadly reactive antigen (COBRA). Three COBRA H5N1 HA proteins have been engineered based upon (1) human clade 2 H5N1 sequences, (2) human and avian clade 2 sequences, and (3) all H5N1 influenza sequences recorded between 2005-2008. Each hemagglutinin protein retained the ability to bind the appropriate receptors, as well as the ability to mediate particle fusion, following purification from a mammalian expression system. COBRA VLP vaccines were administered to mice and the humoral immune responses were compared to those induced by VLPs containing an HA derived from a primary viral isolate. Using a single vaccination (0.6 ug HA dose with an adjuvant) all animals vaccinated with COBRA clade 2 HA H5N1 VLPs had protective levels of HAI antibodies to a representative isolate from each subclade of clade 2, but lower titers against other clades. The addition of avian sequences from other clades expanded breadth of HAI antibodies to the divergent clades, but still not all of the 25 H5N1 viruses in the panel were recognized by antibodies elicited any one H5N1 COBRA VLP vaccine. Vaccination of mice with a cocktail of all 3 COBRA HA VLP vaccines, in a prime-boost regimen, elicited an average HAI titer greater than 1:40 against all 25 viruses. Collectively, our findings indicate that the elicited antibody response following VLP vaccination with all 3 COBRA HA vaccine simultaneously elicited a broadly-reactive set of antibodies that recognized H5N1 viruses from 11 H5N1 clades/subclades isolated over a 12-year span.

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.

Potential antigenic explanation for atypical H1N1 infections among middle-aged adults during the 2013-2014 influenza season.

Influenza viruses typically cause the most severe disease in children and elderly individuals. However, H1N1 viruses disproportionately affected middle-aged adults during the 2013-2014 influenza season. Although H1N1 viruses recently acquired several mutations in the hemagglutinin (HA) glycoprotein, classic serological tests used by surveillance laboratories indicate that these mutations do not change antigenic properties of the virus. Here, we show that one of these mutations is located in a region of HA targeted by antibodies elicited in many middle-aged adults. We find that over 42% of individuals born between 1965 and 1979 possess antibodies that recognize this region of HA. Our findings offer a possible antigenic explanation of why middle-aged adults were highly susceptible to H1N1 viruses during the 2013-2014 influenza season. Our data further suggest that a drifted H1N1 strain should be included in future influenza vaccines to potentially reduce morbidity and mortality in this age group.

H5 N-terminal ß sheet promotes oligomerization of H7-HA1 that induces better antibody affinity maturation and enhanced protection against H7N7 and H7N9 viruses compared to inactivated influenza vaccine.

Initiation of mass vaccination is critical in response to influenza pandemic. There is an urgent need of a simple, rapid method for production of influenza vaccine that is more effective than current traditional influenza vaccines. Recent H7N9 transmissions to humans in China with high morbidity/mortality initiated extensive vaccine evaluation. We produced the HA1 domains (amino acids 1-320) from H7N9 and H7N7 strains in E. coli. Both were found to contain primarily monomers/trimers with low oligomeric content. However, when residues from the N-terminal ß sheet (first 8 amino acid) of H7 HA1 domains were swapped with the corresponding amino acids from H5N1, functional oligomeric H7 HA1 were produced (HA1-DS), demonstrating strong receptor binding and hemagglutination. In rabbits, the HA1-DS from either H7N9 or H7N7 generated high neutralization titers against both homologous and heterologous H7 strains, superior to the unmodified H7 HA1 proteins. In ferrets, HA1-DS from H7N7 elicited higher (and faster) HI titers, better protected ferrets from lethality, weight loss, and reduced viral loads following challenge with wild-type highly pathogenic H7N7 virus compared with inactivated H7N7 subunit vaccine. HA1-DS vaccinated ferrets were also better protected from weight loss after challenge with the heterologous H7N9 virus compared with inactivated H7N7 subunit vaccine. Importantly, the H7N7 HA1-DS vaccine induced antibody affinity maturation far superior to the inactivated H7N7 subunit vaccine, which strongly correlated with control of viral loads in the nasal washes after challenge with either H7N7 or H7N9 strains. We conclude that N-terminus ß sheet domain-swap can be used to produce stable functional oligomeric forms of better recombinant HA1 vaccines in simple, inexpensive bacterial system for rapid response to emerging pandemic threat for the global population.

H1N1, but not H3N2, influenza A virus infection protects ferrets from H5N1 encephalitis.

UNLABELLED: Seasonal influenza causes substantial morbidity and mortality because of efficient human-to-human spread. Rarely, zoonotic strains of influenza virus spread to humans, where they have the potential to mediate new pandemics with high mortality. We studied systemic viral spread after intranasal infection with highly pathogenic avian influenza virus (H5N1 [A/Viet Nam/1203/2004]) in ferrets with or without prior pandemic H1N1pdm09 (A/Mexico/4108/2009) or H3N2 (A/Victoria/361/2011) infection. After intranasal challenge with H5N1 influenza virus, naive ferrets rapidly succumbed to systemic infection. Animals challenged with H5N1 influenza virus greater than 3 months after recovering from an initial H1N1pdm09 infection survived H5N1 virus challenge and cleared virus from the respiratory tract 4 days after infection. However, a prolonged low-level infection of hematopoietic elements in the small bowel lamina propria, liver, and spleen was present for greater than 2 weeks postinfection, raising the potential for reassortment of influenza genes in a host infected with multiple strains of influenza. Animals previously infected with an H3N2 influenza virus succumbed to systemic disease and encephalitis after H5N1 virus challenge. These results indicate prior infection with different seasonal influenza strains leads to radically different protection from H5N1 challenge and fatal encephalitis. IMPORTANCE: Seasonal influenza is efficiently transmitted from human to human, causing substantial morbidity and mortality. Rarely, zoonotic strains of influenza virus spread to humans, where they have the potential to mediate new pandemics with high mortality. Infection of naive ferrets with H5N1 avian influenza virus causes a rapid and lethal systemic disease. We studied systemic H5N1 viral spread after infection of ferrets with or without prior exposure to either of two seasonal influenza virus strains, H1N1 and H3N2. Ferrets previously infected with H1N1 survive H5N1 challenge while those previously infected with H3N2 die of encephalitis. However ferrets protected from lethal H5N1 infection develop persistent low-level infection of the small intestine, liver, or spleen, providing a nidus for future viral strain recombination. The mechanism by which prior infection with specific strains of seasonal influenza virus protect from lethal H5N1 challenge needs to be elucidated in order to design effective immunization and treatments.

Immune history shapes specificity of pandemic H1N1 influenza antibody responses.

Human antibody responses against the 2009 pandemic H1N1 (pH1N1) virus are predominantly directed against conserved epitopes in the stalk and receptor-binding domain of the hemagglutinin (HA) protein. This is in stark contrast to pH1N1 antibody responses generated in ferrets, which are focused on the variable Sa antigenic site of HA. Here, we show that most humans born between 1983 and 1996 elicited pH1N1 antibody responses that are directed against an epitope near the HA receptor-binding domain. Importantly, most individuals born before 1983 or after 1996 did not elicit pH1N1 antibodies to this HA epitope. The HAs of most seasonal H1N1 (sH1N1) viruses that circulated between 1983 and 1996 possess a critical K133 amino acid in this HA epitope, whereas this amino acid is either mutated or deleted in most sH1N1 viruses circulating before 1983 or after 1996. We sequentially infected ferrets with a 1991 sH1N1 virus and then a pH1N1 virus. Sera isolated from these animals were directed against the HA epitope involving amino acid K133. These data suggest that the specificity of pH1N1 antibody responses can be shifted to epitopes near the HA receptor-binding domain after sequential infections with sH1N1 and pH1N1 viruses that share homology in this region.

Sequential seasonal H1N1 influenza virus infections protect ferrets against novel 2009 H1N1 influenza virus.

Individuals <60 years of age had the lowest incidence of infection, with ~25% of these people having preexisting, cross-reactive antibodies to novel 2009 H1N1 influenza. Many people >60 years old also had preexisting antibodies to novel H1N1. These observations are puzzling because the seasonal H1N1 viruses circulating during the last 60 years were not antigenically similar to novel H1N1. We therefore hypothesized that a sequence of exposures to antigenically different seasonal H1N1 viruses can elicit an antibody response that protects against novel 2009 H1N1. Ferrets were preinfected with seasonal H1N1 viruses and assessed for cross-reactive antibodies to novel H1N1. Serum from infected ferrets was assayed for cross-reactivity to both seasonal and novel 2009 H1N1 strains. These results were compared to those of ferrets that were sequentially infected with H1N1 viruses isolated prior to 1957 or more-recently isolated viruses. Following seroconversion, ferrets were challenged with novel H1N1 influenza virus and assessed for viral titers in the nasal wash, morbidity, and mortality. There was no hemagglutination inhibition (HAI) cross-reactivity in ferrets infected with any single seasonal H1N1 influenza viruses, with limited protection to challenge. However, sequential H1N1 influenza infections reduced the incidence of disease and elicited cross-reactive antibodies to novel H1N1 isolates. The amount and duration of virus shedding and the frequency of transmission following novel H1N1 challenge were reduced. Exposure to multiple seasonal H1N1 influenza viruses, and not to any single H1N1 influenza virus, elicits a breadth of antibodies that neutralize novel H1N1 even though the host was never exposed to the novel H1N1 influenza viruses.

Oligomeric recombinant H5 HA1 vaccine produced in bacteria protects ferrets from homologous and heterologous wild-type H5N1 influenza challenge and controls viral loads better than subunit H5N1 vaccine by eliciting high-affinity antibodies.

Recombinant hemagglutinin from influenza viruses with pandemic potential can be produced rapidly in various cell substrates. In this study, we compared the functionality and immunogenicity of bacterially produced oligomeric or monomeric HA1 proteins from H5N1 (A/Vietnam/1203/04) with those of the egg-based licensed subunit H5N1 (SU-H5N1) vaccine in ferrets challenged with homologous or heterologous H5N1 highly pathogenic influenza strains. Ferrets were vaccinated twice with the oligomeric or monomeric rHA1 or with SU-H5N1 (Sanofi Pasteur) emulsified with Titermax adjuvant and were challenged with wild-type homologous (A/Vietnam/1203/04; clade 1) or heterologous (A/Whooperswan/Mongolia/244/2005; clade 2.2) virus. Only the oligomeric rHA1 (not the monomeric rHA1) immunogen and the SU-H5N1 vaccine provided protection against the lethality and morbidity of homologous and heterologous highly pathogenic H5N1. Oligomeric rHA1 generated more cross-neutralizing antibodies and higher levels of serum antibody binding to HA1, with stronger avidity and a better IgG/IgM ratio, than monomeric HA1 and SU-H5N1 vaccines, as determined by surface plasmon resonance (SPR). Importantly, viral loads after heterologous H5N1 challenge were more efficiently controlled in ferrets vaccinated with the oligomeric rHA1 immunogen than in SU-H5N1-vaccinated ferrets. The reduction of viral loads in the nasal washes correlated strongly with higher-avidity antibodies to oligomeric rHA1 derived from H5N1 clade 1 and clade 2.2 viruses, as measured by SPR. This is the first study to show the role of antibody avidity for the HA1 globular head domain in reduction of viral loads in the upper respiratory tract, which could significantly reduce viral transmission.

Complex patterns of human antisera reactivity to novel 2009 H1N1 and historical H1N1 influenza strains.

BACKGROUND: During the 2009 influenza pandemic, individuals over the age of 60 had the lowest incidence of infection with approximately 25% of these people having pre-existing, cross-reactive antibodies to novel 2009 H1N1 influenza isolates. It was proposed that older people had pre-existing antibodies induced by previous 1918-like virus infection(s) that cross-reacted to novel H1N1 strains. METHODOLOGY/PRINCIPAL FINDINGS: Using antisera collected from a cohort of individuals collected before the second wave of novel H1N1 infections, only a minority of individuals with 1918 influenza specific antibodies also demonstrated hemagglutination-inhibition activity against the novel H1N1 influenza. In this study, we examined human antisera collected from individuals that ranged between the ages of 1 month and 90 years to determine the profile of seropositive influenza immunity to viruses representing H1N1 antigenic eras over the past 100 years. Even though HAI titers to novel 2009 H1N1 and the 1918 H1N1 influenza viruses were positively associated, the association was far from perfect, particularly for the older and younger age groups. CONCLUSIONS/SIGNIFICANCE: Therefore, there may be a complex set of immune responses that are retained in people infected with seasonal H1N1 that can contribute to the reduced rates of H1N1 influenza infection in older populations.

A computationally optimized hemagglutinin virus-like particle vaccine elicits broadly reactive antibodies that protect nonhuman primates from H5N1 infection.

BACKGROUND: Highly pathogenic H5N1 avian influenza viruses continue to spread via waterfowl, causing lethal infections in humans. Vaccines can prevent the morbidity and mortality associated with pandemic influenza isolates. Predicting the specific isolate that may emerge from the 10 different H5N1 clades is a tremendous challenge for vaccine design. METHODS: In this study, we generated a synthetic hemagglutinin (HA) on the basis of a new method, computationally optimized broadly reactive antigen (COBRA), which uses worldwide sequencing and surveillance efforts that are specifically focused on sequences from H5N1 clade 2 human isolates. RESULTS: Cynomolgus macaques vaccinated with COBRA clade 2 HA H5N1 virus-like particles (VLPs) had hemagglutination-inhibition antibody titers that recognized a broader number of representative isolates from divergent clades as compared to nonhuman primates vaccinated with clade 2.2 HA VLPs. Furthermore, all vaccinated animals were protected from A/Whooper Swan/Mongolia/244/2005 (WS/05) clade 2.2 challenge, with no virus detected in the nasal or tracheal washes. However, COBRA VLP-vaccinated nonhuman primates had reduced lung inflammation and pathologic effects as compared to those that received WS/05 VLP vaccines. CONCLUSIONS: The COBRA clade 2 HA H5N1 VLP elicits broad humoral immunity against multiple H5N1 isolates from different clades. In addition, the COBRA VLP vaccine is more effective than a homologous vaccine against a highly pathogenic avian influenza virus challenge.