No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Single- and multiple-clade influenza A H5N1 vaccines induce cross protection in ferrets
Abstract
The rapid evolution, genetic diversity, broad host range, and increasing human infection with avian influenza A (H5N1) viruses highlight the need for an efficacious cross-clade vaccine. Using the ferret model, we compared induction of cross-reactive immunity and protective efficacy of three single-clade H5N1 vaccines and a novel multiple-clade H5N1 vaccine, with and without MF59 adjuvant. Reverse genetics (rg) was used to generate vaccine viruses containing the hemagglutinin (HA) and neuraminidase genes of wild-type H5N1 viruses. Ferrets received two doses of inactivated whole-virus vaccine separated by 3 weeks. Single-clade vaccines (7.5 microg HA per dose) included rg-A/Vietnam/1203/04 (clade 1), rg-A/Hong Kong/213/03 (clade 1), and rg-A/Japanese White Eye/Hong Kong/1038/06 (clade 2.3). The multiple-clade vaccine contained 3.75 microg HA per dose of each single-clade vaccine and of rg-A/Whooper Swan/Mongolia/244/05 (clade 2.2). Two doses of vaccine were required to substantially increase anti-HA and virus neutralizing antibody titers to H5N1 viruses. MF59 adjuvant enhanced induction of clade-specific and cross-clade serum antibody responses, reduced frequency of infection (as determined by upper respiratory tract virus shedding and seroconversion data), and eliminated disease signs. The rg-A/Hong Kong/213/03 vaccine induced the highest antibody titers to homologous and heterologous H5N1 viruses, while rg-A/Japanese White Eye/Hong Kong/1038/06 vaccine induced the lowest. The multiple-clade vaccine was broadly immunogenic against clade 1 and 2 viruses. The rg-A/Vietnam/1203/04 vaccine (the currently stockpiled H5N1 vaccine) most effectively reduced upper respiratory tract virus shedding after challenge with clade 1 and 2 viruses. Importantly, all vaccines protected against lethal challenge with A/Vietnam/1203/04 virus and provided cross-clade protection.
... A possible approach to this may be the use of adjuvants. It has been shown previously that adjuvants, such as MF59, the AS03 adjuvant system and saponin-based adjuvants have an ability to enhance and broaden the immune response elicited by vaccination [16][17][18][19][20][21][22][23][24][25][26]. ...
... It has previously been demonstrated that adjuvants can enhance and broaden immune responses of seasonal and pandemic vaccines in animal models and in humans [16][17][18][19][20][21][22][23][24]. For example, MF59 (squalene oil-in-water emulsion [33] has been shown to enhance vaccine-elicited humoral immune responses in animals [20,34,35] and in humans [35][36][37][38][39] and it is currently licensed for use in combination with a seasonal influenza vaccine (Fluad®). ...
... It has previously been demonstrated that adjuvants can enhance and broaden immune responses of seasonal and pandemic vaccines in animal models and in humans [16][17][18][19][20][21][22][23][24]. For example, MF59 (squalene oil-in-water emulsion [33] has been shown to enhance vaccine-elicited humoral immune responses in animals [20,34,35] and in humans [35][36][37][38][39] and it is currently licensed for use in combination with a seasonal influenza vaccine (Fluad®). Fluad and an MF59 adjuvated H5N1 pandemic vaccine Figure S3, Additional file 5: Figure S4, Additional file 6: Figure S5) candidate are known to induce H1 and H5 cross-reactive HAI responses in ferrets [20] and in humans [16,17,36,39], respectively. ...
Background
Influenza virus infections are responsible for significant morbidity worldwide and therefore it remains a high priority to develop more broadly protective vaccines. Adjuvation of current seasonal influenza vaccines has the potential to achieve this goal.
Methods
To assess the immune potentiating properties of Matrix-M™, mice were immunized with virosomal trivalent seasonal vaccine adjuvated with Matrix-M™. Serum samples were isolated to determine the hemagglutination inhibiting (HAI) antibody titers against vaccine homologous and heterologous strains. Furthermore, we assess whether adjuvation with Matrix-M™ broadens the protective efficacy of the virosomal trivalent seasonal vaccine against vaccine homologous and heterologous influenza viruses.
Results
Matrix-M™ adjuvation enhanced HAI antibody titers and protection against vaccine homologous strains. Interestingly, Matrix-M™ adjuvation also resulted in HAI antibody titers against heterologous influenza B strains, but not against the tested influenza A strains. Even though the protection against heterologous influenza A was induced by the adjuvated vaccine, in the absence of HAI titers the protection was accompanied by severe clinical scores and body weight loss. In contrast, in the presence of heterologous HAI titers full protection against the heterologous influenza B strain without any disease symptoms was obtained.
Conclusion
The results of this study emphasize the promising potential of a Matrix-M™-adjuvated seasonal trivalent virosomal influenza vaccine. Adjuvation of trivalent virosomal vaccine does not only enhance homologous protection, but in addition induces protection against heterologous strains and thus provides overall more potent and broad protective immunity.
Electronic supplementary material
The online version of this article (doi:10.1186/s12985-015-0435-9) contains supplementary material, which is available to authorized users.
... [6]. Previous work demonstrated that the multiple-clade vaccine with MF59 adjuvant increased clade-specific and cross-clade antibody responses against lethal challenge with clade 1 and 2 viruses [7]. Although clade 0 was the least frequently seen, during the summer and early fall of 1996, an outbreak of disease with 40% morbidity occurred on a goose farm in Guangdong province, China. ...
... A few results showed after receiving a cross-reactive H5N1 influenza vaccine, individuals may require some immunity to protect from lethal challenge of other clade HPAI H5N1 virus. And after immunizing with a cross-reactive H5N1 influenza vaccine, individuals may require only a single dose of strainspecific pandemic vaccine [7,10,11]. ...
... MF59, the first oil-in-water emulsion licensed as an adjuvant for human use, can enhance vaccine immune responses through multiple mechanisms [17,18]. It is worthy of note that the addition of MF59 adjuvant induced a stronger cross-reactive immunity as compared to non-adjuvanted multiple-clade vaccines [7]. A trivalent MF59-adjuvanted seasonal influenza vaccine has shown to induce significantly higher immune responses to influenza vaccination in the elderly, compared with non-adjuvanted vaccines, and to provide cross-reactive immunity against divergent influenza strains. ...
The increase in recent outbreaks and unpredictable changes of highly pathogenic avian influenza (HPAI) H5N1 in birds and humans highlights the urgent need to develop a cross-protective H5N1 vaccine. We here report our development of a multiple-clade H5N1 influenza vaccine tested for immunogenicity and efficacy to confer cross-protection in an animal model.
Mice received two doses of influenza split vaccine with oil-in-water emulsion adjuvant SP01 by intranasal administration separated by two weeks. Single vaccines (3 µg HA per dose) included rg-A/Vietnam/1203/2004(Clade 1), rg-A/Indonesia/05/2005(Clade 2.1), and rg-A/Anhui/1/2005(Clade 2.3.4). The trivalent vaccine contained 1 µg HA per dose of each single vaccine. Importantly, complete cross-protection was observed in mice immunized using trivalent vaccine with oil-in-water emulsion adjuvant SP01 that was subsequently challenged with the lethal A/OT/SZ/097/03 influenza strain (Clade 0), whereas only the survival rate was up to 60% in single A/Anhui/1/2005 vaccine group.
Our findings demonstrated that the multiple-clade H5N1 influenza vaccine was able to elicit a cross-protective immune response to heterologous HPAI H5N1 virus, thus giving rise to a broadly cross-reactive vaccine to potential prevention use ahead of the strain-specific pandemic influenza vaccine in the event of an HPAI H5N1 influenza outbreak. Also, the multiple-clade adjuvanted vaccine could be useful in allowing timely initiation of vaccination against unknown pandemic virus.
... Not only does this create tremendous economic burdens to produce vaccines from these seed strains, but it is also becoming less clear which seed strains are the most relevant to a given geographic region. To overcome the burden and uncertainties, several new approaches have been evaluated (1,3,7,10,13,14,18,20,26,27,32 Thus, although these approaches have been shown to elicit more cross-reactive antibody responses than an antigen derived from a single strain, most of these studies were based on phylogenetic data or limited antigenic data and only evaluated a limited number of H5 clades and subclades. We hypothesized that by systematically analyzing the crossreactivity of neutralizing antibody responses among all H5 clades and subclades, one may determine antigenic clusters among H5 clades and subclades. ...
... For H5N1 viruses, during the past 15 years viruses have evolved into genetically distinct clades and subclades. To cover this diversity, the WHO has already created 20 seed recombinant vaccine strains (38), and many new approaches that have applied both reverse and analytic vaccinology have been evaluated (2,7,13,15,18,26,27,30,36,37). For reverse vaccinology, H5 HA sequence diversity was analyzed, and phylogenetic trees were constructed. ...
Because of their rapid evolution, genetic diversity, broad host range, ongoing circulation in birds, and potential human-to-human transmission, H5N1 influenza viruses remain a major global health concern. Their high degree of genetic diversity also poses enormous burdens and uncertainties in developing effective vaccines. To overcome this, we took a new approach, i.e., the development of immunogens based on a comprehensive serologic study. We constructed DNA plasmids encoding codon-optimized hemagglutinin (HA) from 17 representative strains covering all reported clades and subclades of highly pathogenic avian influenza H5N1 viruses. Using DNA plasmids, we generated the corresponding H5N1 pseudotypes and immune sera. We performed an across-the-board pseudotype-based neutralization assay and determined antigenic clusters by cartography. We then designed a triclade DNA vaccine and evaluated its immunogenicity and protection in mice. We report here that (sub)clades 0, 1, 3, 4, 5, 6, 7.1, and 9 were grouped into antigenic cluster 1, (sub)clades 2.1.3.2, 2.3.4, 2.4, 2.5, and 8 were grouped into another antigenic cluster, with subclade 2.2.1 loosely connected to it, and each of subclades 2.3.2.1 and 7.2 was by itself. Importantly, the triclade DNA vaccine encoding HAs of (sub)clades 0, 2.3.2.1, and 7.2 elicited broadly neutralizing antibody responses against all H5 clades and subclades and protected mice against high-lethal-dose heterologous H5N1 challenge. Thus, we conclude that broadly neutralizing antibodies against all H5 clades and subclades can indeed be elicited with immunogens on the basis of a comprehensive serologic study. Further evaluation and optimization of such an approach in ferrets and in humans is warranted.
... 36,37 Ferret infection studies of heterotypic protection are performed by first infecting or vaccinating with a representative construct of one influenza subtype and then challenging with a more lethal subtype virus. 33,[38][39][40][41] Alternately, heterotypic antibody responses generated to one subtype can be passively transferred to infection-naive animals for evaluation of protection against challenge by a different subtype. 40 Early studies in ferrets, however, indicated that although beneficial for limiting viral shedding, prior exposure to an earlier different virus, despite showing some protection, did not block infection. ...
... Notably, the secondary and quaternary structure of the important immunogenic and protective M2e determinant is unresolved, as is the majority of the PB1 structure and the stalk of NA. dominant regions (and possibly NA-globular-head determinants) traditionally selected for seasonal vaccines. Alternatively, presentation of those conserved Ags in an environment (for example, using MF-59 adjuvant) conducive to enhance the antigenicity of otherwise immunorecessive conserved sites (Supplementary Information S3) may also help overcome the immune imbalance between poorly immunogenic conserved domains 39,[86][87][88] and immunodominant epitopes of the HA and NA globular heads(s). Such approaches are necessary to provide a less-distracted immune focus for the stimulation of broadly protective antibodies to conserved determinants across the proteins of all (or many) influenza subtypes, for which a summation of many different heterotypic and heterosubtypic antibody effects could affect blanket protection. ...
Fundamentally new approaches are required for the development of vaccines to pre-empt and protect against emerging and pandemic influenzas. Current strategies involve post-emergent homotypic vaccines that are modelled upon select circulating 'seasonal' influenzas, but cannot induce cross-strain protection against newly evolved or zoonotically introduced highly pathogenic influenza (HPI). Avian H5N1 and the less-lethal 2009 H1N1 and their reassortants loom as candidates to seed a future HPI pandemic. Therefore, more universal 'seasoned' vaccine approaches are urgently needed for heterotypic protection ahead of time. Pivotal to this is the need to understand mechanisms that can deliver broad strain protection. Heterotypic and heterosubtypic humoral immunities have largely been overlooked for influenza cross-protection, with most 'seasoned' vaccine efforts for humans focussed on heterotypic cellular immunity. However, 5 years ago we began to identify direct and indirect indicators of humoral-herd immunity to protein sites preserved among H1N1, H3N2 and H5N1 influenzas. Since then the evidence for cross-protective antibodies in humans has been accumulating. Now proposed is a rationale to stimulate and enhance pre-existing heterotypic humoral responses that, together with cell-mediated initiatives, will deliver pre-emptive and universal human protection against emerging epidemic and pandemic influenzas.
... A previous study also showed that a vaccine containing H5N1 antigens (clade 1) is effective in immunizing chickens against heterologous challenges (clade 2.3.4) when vaccinated with 5.0 µg of HA antigens [16]. Another study conducted using mice and ferrets also demonstrated the cross-protective immunity elicited by H5N1 based vaccines [17,18]. The high cross-clade immune activity of our vaccine may be caused by its ability to induce the production of antibodies targeting the stalk region of the HA protein (HA2). ...
Background:
Over the last 20 years, circulating highly pathogenic (HP) Asian H5 subtype avian influenza viruses have caused global pandemics in poultry and sporadic infections in humans. Vaccines are a desirable solution to prevent viral infections in poultry and reduce transmission to humans. Herein, we investigated the efficacy of an oil-adjuvanted inactivated H5N6 vaccine against highly pathogenic H5N6 and H5N1 influenza virus infections in chickens.
Methods:
The polybasic amino acid cleavage site depleted HA gene and NA gene of A/Waterfowl/Korea/S57/2016 (clade 2.3.4.4) (H5N6) was assembled with the rest of the A/PR/8/34 (H1N1) genes to construct the vaccine virus. The vaccine virus was propagated in fertilized eggs, partially purified using a tangential flow filtration (TFF) system, and inactivated using formalin. The chickens were intramuscularly immunized with 384 HA, 192HA, and 96HA units of oil-adjuvanted inactivated H5N6 vaccine. Antibody titer, survival rate, and lung pathology were evaluated against the homologous H5N6: A/waterfowl/Korea/S57/2016 (clade 2.3.4.4) and heterologous H5N1: A/Hong Kong/213/2003 (clade 1) viruses 12 and 4 weeks post-vaccination (p.v.), respectively. Data were statistically analyzed using the Mann-Whitney U test.
Results:
The 384HA (n = 10) and 192HA (n = 5) antigen-immunized chickens showed 100% survival after lethal infections with homologous H5N6, and no virus shedding was observed from tracheal and cloacal routes. All chickens that received the 384HA vaccine survived the challenge of heterologous H5N1 after 4 weeks of immunization. The chickens that received the 384HA vaccine showed mean HI titers of 60 and 240 after 12 and 4 weeks of vaccination, respectively, against HP H5N6, whereas a mean HI titer of 80 was observed in sera collected 4 weeks after vaccination against HP H5N1.
Conclusions:
Our findings indicate that one dose of 384HA oil-adjuvanted inactivated H5N6 vaccine can induce a long-lasting immune response against both homologous H5N6 and heterologous H5N1 infections in chickens.
... This inconsistent correlation between low HAI titers and survival appears to be systemic for H5-specific vaccines. Previous studies have also discovered increased survival rates and decreased pathogenicity without detectable serum antibody titers [32,33]. This non-HAI protection has been attributed to stem-based antibodies [34] and/or anti-NA antibodies [35]. ...
H5N1 COBRA hemagglutinin (HA) sequences, termed human COBRA-2 HA, were constructed through layering of HA sequences from viruses isolated from humans collected between 2004–2007 using only clade 2 strains. These COBRA HA proteins, when expressed on the surface of virus-like particles (VLP), elicited protective immune responses in mice, ferrets, and non-human primates. However, these vaccines were not as effective at inducing neutralizing antibodies against newly circulating viruses. Therefore, COBRA HA-based vaccines were updated in order to elicit protective antibodies against the current circulating clades of H5Nx viruses. Next-generation COBRA HA vaccines were designed to encompass the newly emerging viruses circulating in wild avian populations. HA amino acid sequences from avian and human H5 influenza viruses isolated between 2011-2017 were downloaded from the GISAID (Global Initiative on Sharing All Influenza Data). Mice were vaccinated with H5 COBRA rHA that elicited antibodies with hemagglutinin inhibition (HAI) activity against H5Nx viruses from five clades. The H5 COBRA rHA vaccine, termed IAN8, elicited protective immune responses against mice challenged with A/Sichuan/26621/2014 and A/Vietnam/1203/2004. This vaccine elicited antibodies with HAI activity against viruses from clades 2.2, 2.3.2.1, 2.3.4.2, 2.2.1 and 2.2.2. Lungs from vaccinated mice had decreased viral titers and the levels of cellular infiltration in mice vaccinated with IAN-8 rHA were similar to mice vaccinated with wild-type HA comparator vaccines or mock vaccinated controls. Overall, these next-generation H5 COBRA HA vaccines elicited protective antibodies against both historical H5Nx influenza viruses, as well as currently circulating clades of H5N1, H5N6, and H5N8 influenza viruses.
... Enzyme-linked lectin assays (ELLAs), used to determine the NA inhibition (NI) activity of mouse sera, were performed as described previously (47)(48)(49). Virus-neutralizing (VN) antibody titers in mouse sera were determined in MDCK cells as described previously (50). ...
Human influenza A and B viruses are highly contagious and cause similar illnesses and seasonal epidemics. Currently available antiviral drugs have limited efficacy in humans with compromised immune systems; therefore, alternative strategies for protection are needed. Here, we investigated whether monoclonal antibodies (mAbs) targeting hemagglutinin (HA) and/or neuraminidase (NA) proteins would protect immunosuppressed mice from severe infections with influenza B virus. Pharmacologically immunosuppressed BALB/c mice were inoculated with B/Brisbane/60/2008 (BR/08) influenza virus and were treated with a single dose of 1, 5, or 25 mg/kg/day of either an anti-HA mAb (1D2) or an anti-NA mAb (1F2) starting at 24 hours post inoculation (hpi). Monotherapy with 1D2 or 1F2 mAbs provided dose-dependent protection of mice, with decreased BR/08 virus replication and spread in the mouse lungs, as compared to those of controls. Combination treatment with 1D2 and 1F2 provided greater protection than did monotherapy, even when started at 48 hpi. Virus spread was also efficiently restrained within the lungs, being limited to 6%, 10%, and 10% of that seen in active infection when treatment was initiated at 24, 48, and 72 hpi, respectively. In most cases, the expression of cytokines and chemokines was altered according to when treatment was initiated. Higher expression of pro-inflammatory IP-10 and MCP-1 in combination-treatment groups, but not in monotherapy groups, to some extent, promoted better control of virus spread within the lungs. This study demonstrates the potential value of mAb immunotherapy in treating influenza in immunocompromised hosts who are at increased risk of severe disease.
... One approach to improving vaccine effectiveness may therefore to elicit a broader antibody response to compensate for potential strain mismatch [100]. Adding adjuvants such as MF59 and AS03 has been shown to induce higher antibody titres that have greater cross-reactive properties [55,56,101,102]. Quantitative comparisons of cross reactivity profiles, as we have provided here, could be a useful tool in comparing the effectiveness of different adjuvants, which would provide a measurable benefit to trade-off against safety and immunogenicity concerns [103,104]. ...
The strength and breadth of an individual's antibody repertoire is an important predictor of their response to influenza infection or vaccination. Although progress has been made in understanding qualitatively how repeated exposures shape the antibody mediated immune response, quantitative understanding remains limited. We developed a set of mathematical models describing short-term antibody kinetics following influenza infection or vaccination and fit them to haemagglutination inhibition (HI) titres from 5 groups of ferrets which were exposed to different combinations of trivalent inactivated influenza vaccine (TIV with or without adjuvant), A/H3N2 priming inoculation and post-vaccination A/H1N1 inoculation. We fit models with various immunological mechanisms that have been empirically observed but have not previously been included in mathematical models of antibody landscapes, including; titre ceiling effects, antigenic seniority and exposure-type specific cross reactivity. Based on the parameter estimates of the best supported models, we describe a number of key immunological features. We found quantifiable differences in the degree of homologous and cross-reactive antibody boosting elicited by different exposure types. Infection and adjuvanted vaccination generally resulted in strong, broadly reactive responses whereas unadjuvanted vaccination resulted in a weak, narrow response. We found that the order of exposure mattered: priming with A/H3N2 improved subsequent vaccine response, and the second dose of adjuvanted vaccination resulted in substantially greater antibody boosting than the first. Either antigenic seniority or a titre ceiling effect were included in the two best fitting models, suggesting a role for a mechanism describing diminishing antibody boosting with repeated exposures. Although there was considerable uncertainty in our estimates of antibody waning parameters, our results suggest that both short and long term waning were present and would be identifiable with a larger set of experiments. These results highlight the potential use of repeat exposure animal models in revealing short-term, strain-specific immune dynamics of influenza.
... Multiple-clade influenza H5N1 vaccines using strains from different H5N1 virus clades as inactivated whole virus vaccine with adjuvants were tested in different animals and revealed broad cross protection against viruses from distant clades [25,26]. However, the inclusion of more than one strain from each influenza subtype has been limited by the need to include large doses of antigen [27]. ...
... However, it is difficult to predict whether an A/H5 antigen will protect against A/H5 viruses from distinct clades. Vaccination with a mixture of antigens from different H5 GsGd clades has been explored in animal models as a multivalent vaccination approach while utilizing different vaccine platforms: whole inactivated vaccines [37], DNA vaccines [38] or baculovirus vectors [39]. In general, multivalent vaccines induced broader antibody responses than monovalent vaccines. ...
Influenza A viruses can infect a wide range of hosts, creating opportunities for zoonotic transmission, i.e., transmission from animals to humans, and placing the human population at constant risk of potential pandemics. In the last hundred years, four influenza A virus pandemics have had a devastating effect, especially the 1918 influenza pandemic that took the lives of at least 40 million people. There is a constant risk that currently circulating avian influenza A viruses (e.g., H5N1, H7N9) will cause a new pandemic. Vaccines are the cornerstone in preparing for and combating potential pandemics. Despite exceptional advances in the design and development of (pre-)pandemic vaccines, there are still serious challenges to overcome, mainly caused by intrinsic characteristics of influenza A viruses: Rapid evolution and a broad host range combined with maintenance in animal reservoirs, making it near impossible to predict the nature and source of the next pandemic virus. Here, recent advances in the development of vaccination strategies to prepare against a pandemic virus coming from the avian reservoir will be discussed. Furthermore, remaining challenges will be addressed, setting the agenda for future research in the development of new vaccination strategies against potentially pandemic influenza A viruses.
... Some of these conserved epitopes have been described by other authors in the type 1, 2, 4, and 6 HA proteins [7,56]. Cross-reactions are frequent and have been observed against different influenza viruses with the same HA protein (A/H5N1 and A/H5N3) [57,58], and also between viruses from the same clade [59,60]. However, as far as we know, this is the first work that shows heterotypic seroconversion against AIV after seasonal vaccination in a European elderly population by using HA1 globular head-specific Abs. ...
Avian influenza viruses are currently one of the main threats to human health in the world.
Although there are some screening reports of antibodies against these viruses in humans fromWestern
countries, most of these types of studies are conducted in poultry and market workers of Asian
populations. The presence of antibodies against avian influenza viruses was evaluated in an elderly
European population. An experimental study was conducted, including pre- and post-vaccine serum
samples obtained from 174 elderly people vaccinated with seasonal influenza vaccines of 2006–2007,
2008–2009, 2009–2010, and 2010–2011 Northern Hemisphere vaccine campaigns. The presence of
antibodies against A/H5N1, A/H7N3, and A/H9N2 avian influenza viruses were tested by using
haemaglutination inhibition assays. Globally, heterotypic antibodies were found before vaccination
in 2.9% of individuals against A/H5N1, 1.2% against A/H7N3, and 25.9% against A/H9N2. These
pre-vaccination antibodies were present at titers �1/40 in 1.1% of individuals against A/H5N1,
in 1.1% against H7N3, and in 0.6% against the A/H9N2 subtype. One 76 year-old male showed
pre-vaccine antibodies (Abs) against those three avian influenza viruses, and another three individuals
presented Abs against two different viruses. Seasonal influenza vaccination induced a significant
number of heterotypic seroconversions against A/H5N1 (14.4%) and A/H9N2 (10.9%) viruses,
but only one seroconversion was observed against the A/H7N3 subtype. After vaccination, four
individuals showed Abs titers �1/40 against those three avian viruses, and 55 individuals against
both A/H5N1 and A/H9N2. Seasonal vaccination is able to induce some weak heterotypic responses
to viruses of avian origin in elderly individuals with no previous exposure to them. However,
this response did not accomplish the European Medicament Agency criteria for influenza vaccine
efficacy. The results of this study show that seasonal vaccines induce a broad response of heterotypic
antibodies against avian influenza viruses, albeit at a low level.
... Any detectable HAI antibodies (by the limits of current assays) appeared to prevent neurological involvementsomething that even high IgG-titers were not sufficient for (as summarized in Table 1). However, as seen in ours' and others' studies, the absence of an HAI titer does not necessarily preclude protection: vaccinated ferrets appear to be partially protected despite the absence of HAI antibodies when challenged with H5N1 or H7N9 virus 7,18,19 . We show here that this protection is mediated by the induction of cellular immunity and facilitated by high titers of pre-existing influenza-specific IgG-antibodies. ...
Because of the pathogenicity and low incidence of avian influenza virus infections in humans, the immune correlates of protection for avian influenza vaccines cannot be determined from clinical studies. Here, we used the ferret model to address this for an avian influenza H5N1 vaccine. Using oil-in-water adjuvants, we generated groups of ferrets with undetectable (geometric mean titer [GMT] < 10), low (GMT = 28.3), or high (GMT > 761.1) hemagglutination-inhibition (HAI) titers to the A/Viet Nam/1203/2004 (H5N1) virus. Ferrets were then challenged with the wild-type virus and disease severity and immunologic parameters were studied. The severity of infection and symptom profile were inversely associated with pre-challenge HAI titers in a dose-dependent manner. A vaccinated ferret with no detectable HAI-antibodies but high flu-specific IgG-antibody titers mounted rapid functional antibodies after infection and experienced milder disease compared to other ferrets in the group. Compared to naïve ferrets, all vaccinated ferrets showed improved cellular immunity in the lungs and peripheral blood. High number of IFNγ+ CD8- T cells in the airways was associated with early viral clearance. Thus, while neutralizing antibodies are the best correlate of protection, non-neutralizing antibodies can also be protective. This should be taken into consideration in future avian influenza vaccine trials.
... 332 In pre-clinical and clinical studies it thus has been demonstrated that the adjuvant MF59 has an antigen-sparing effect and broadens the intra-subtypic antibody response against influenza viruses upon vaccination. 330,[333][334][335] Therefore, we investigated the potential of an MF59-adjuvanted trivalent seasonal influenza vaccine to elicit protection against pH1N1 infection in ferrets, since in this vac-cine a virus strain is represented that shares an ancestor with pH1N1. 336 Recently, we have shown that immunization with an MF59-adjuvanted seasonal influenza vaccine did prime ferrets for the protective antibody response induced upon a second immunization with the MF59 adjuvanted pH1N1 vaccine. ...
In humans, viral infections causing respiratory disease have been known for many years. Every now and then such viruses may cause epidemics involving large groups of people or even pandemics with spread across the world. At the end of last century and at the beginning of this century zoonotic viruses emerged that were of serious risk for the human population: severe acute respiratory syndrome (SARS) caused by SARS coronavirus (CoV), highly pathogenic avian influenza (HPAI) virus H5N1, and pandemic influenza virus A(H1N1)pdm09 (pH1N1). Both SARS-CoV and influenza A viruses cause respiratory disease that may lead to severe and even fatal cases of pneumonia. The course and outcome of the infections is related to their pathogenesis, which can be explored by describing and comparing pathology, virology, and genomics. Understanding the pathogenesis of SARS and influenza is valuable for development of therapeutic and preventive strategies. Since the pathology of acute human fatal cases in SARS and influenza is rarely described, there is a need for animal models to provide information about the early stage of the disease. Also, pathological description of human cases with uncomplicated viral pneumonia is sparse because patients have multiple therapeutic interventions and secondary co-infections that may alter the pathology. Interestingly, the pathology of SARS-CoV and influenza virus infections has similar features; however, there are also differences in disease outcome. This thesis focusses on the pathology of SARS-CoV and influenza A virus infections in experimental animals. The pathology of these virus infections in animals is compared to that in humans and is related to the pathogenesis. The animal species that are used in this thesis to study the pathology of SARS-CoV infection are; cynomolgus macaques, African Green monkeys, ferrets, and cats. The pathology of influenza virus infection was studied in cynomolgus macaques, ferrets, and cats. Additionally, temporal and spatial dynamics for the pathology of different influenza virus infections in ferrets is described in a time course experiment. Finally, the effect of vaccination on the pathology is studied in ferrets and cynomolgus macaques for SARS-CoV infection and in ferrets for pH1N1 infection.
... Vaccination reduced febrile responses, severe weight loss and prevented the local and systemic spread of the virus. Similarly, recent studies have shown that two doses of AS03 or MF59-adjuvanted H5N1 vaccines induce protective immune responses in ferrets and reduce virus shedding in the upper respiratory tract [35,36]. The reduced virus shedding as observed in our and other studies could be an important factor in limiting the horizontal transmission of the virus to susceptible hosts. ...
Highly pathogenic avian influenza (HPAI) viruses constitute a pandemic threat and the development of effective vaccines is a global priority. Sixty adults were recruited into a randomized clinical trial and were intramuscularly immunized with two virosomal vaccine H5N1 (NIBRG-14) doses (21 days apart) of 30μg HA alone or 1.5, 7.5 or 30μg HA adjuvanted with Matrix M. The kinetics and longevity of the serological responses against NIBRG-14 were determined by haemagglutination inhibition (HI), single radial haemolysis (SRH), microneutralization (MN) and ELISA assays. The cross-H5 clade responses in sera were determined by HI and the antibody-secreting (ASC) cell ELISPOT assays. The protective efficacy of the vaccine against homologous HPAI challenge was evaluated in ferrets.
The serological responses against the homologous and cross-reactive strains generally peaked one week after the second dose, and formulation with Matrix M augmented the responses. The NIBRG-14-specific seroprotection rates fell significantly by six months and were low against cross-reactive strains although the adjuvant appeared to prolong the longevity of the protective responses in some subjects. By 12 months post-vaccination, nearly all vaccinees had NIBRG-14-specific antibody titres below the protective thresholds. The Matrix M adjuvant was shown to greatly improve ASC and serum IgG responses following vaccination. In a HPAI ferret challenge model, the vaccine protected the animals from febrile responses, severe weight loss and local and systemic spread of the virus.
Our findings show that the Matrix M-adjuvanted virosomal H5N1 vaccine is a promising pre-pandemic vaccine candidate.
ClinicalTrials.gov NCT00868218.
... Multiple-clade influenza H5N1 vaccines using strains from different H5N1 virus clades as inactivated whole virus vaccine with adjuvants were tested in different animals and revealed broad cross protection against viruses from distant clades [25,26]. However, the inclusion of more than one strain from each influenza subtype has been limited by the need to include large doses of antigen [27]. ...
The H5N1 highly pathogenic avian influenza (HPAI) virus was isolated for the first time in Egypt in 2006, since then, the virus has become endemic causing a significant threat to the poultry industry and humans. H5N1 HPAI outbreaks continue to occur despite extensive vaccination programs that have been implemented nationwide in different poultry species. Several studies showed that the co-circulating H5N1 viruses in Egypt are genetically and antigenically distant raising a question on the cross protective efficacy of commercial vaccines. In this study, we introduced mutations at the antigenic sites of the hemagglutinin (HA) to broaden reactivity of the Egyptian H5N1 virus. A reverse genetically created variant H5N1 virus (A/chicken/Egypt/1063/2010) with five amino acid mutations (G140R, Y144F, I190L, K192Q, D43N) in the HA gene showed enhanced cross reactivity. This virus showed up to 16 fold increase in reactivity to the classic-lineageH5N1viruses measured by hemagglutination inhibition (HI) assay while maintaining similar level of reactivity with the variant-lineage viruses compared to wild-type virus. In addition, a single amino acid substitution (N165H), which removes potential glycosylation site at the HA globular head of two classic strains (A/chicken/Egypt/527/2012 and A/chicken/Egypt/102d/2010) broadened the reactivity to antisera generated against H5N1 viruses from different clusters. The broadened reactivity of the mutant viruses were also confirmed by testing reactivity of antisera prepared from the mutant viruses against reference viruses from both classic and variant clades. The virus neutralization test using selected antisera and viruses further confirmed the cross HI results. This study highlights that targeted mutation in the HA may be effectively used as a tool to develop broadly reactive influenza vaccines to cope with the continuous antigenic evolution of viruses.
Copyright © 2015. Published by Elsevier Ltd.
... Although preexisting HI titers were well correlated to reduced viral load, the absence of an HI titer does not preclude the absence of protection. Observations from other studies [33,34], as well as our own finding of early viral clearance in the unadjuvanted 45-µg group, suggest that other immune mechanism, such as cellular immunity, may be important in limiting virus infection after vaccination. ...
Background:
An effective vaccine is urgently needed against the H7N9 avian influenza virus. We evaluated the immunogenicity and protective efficacy of a split-virion H7N9 vaccine with or without the oil-in-water adjuvants in ferrets.
Methods:
Ferrets were vaccinated with 2 doses of unadjuvanted, MF59 or AS03-adjuvanted A/Shanghai/2/2013 (H7N9) vaccine, and the induction of antibodies to hemagglutinin (HA) or neuraminidase proteins was evaluated. Ferrets were then challenged with wild-type H7N9 virus to assess the vaccine's protective efficacy. The vaccine composition and integrity was also evaluated in vitro.
Results:
Adjuvanted vaccines stimulated robust serum antibody titers against HA and neuraminidase compared with the unadjuvanted vaccines. Although there was a difference in adjuvanticity between AS03 and MF59 at a lower dose (3.75 µg of HA), both adjuvants induced comparable antibody responses after 2 doses of 15 µg. On challenge, ferrets that received adjuvanted vaccines showed lower viral burden than the control or unadjuvanted vaccine group. In vitro examinations revealed that the vaccine contained visible split-virus particles and retained the native conformation of HA recognizable by polyclonal and monoclonal antibodies.
Conclusions:
The adjuvanted H7N9 vaccines demonstrated superior immunogenicity and protective efficacy against H7N9 infection in ferrets and hold potential as a vaccination regimen.
... GlaxoSmithKline has also developed a line of adjuvants: AS03 (squalenebased), AS04 (aluminum hydroxide-based) that have been combined with their seasonal influenza vaccines. Alternatively, MF59 (Fluad, Chiron Vaccine) is an oil-in-water emulsion that has also demonstrated immunogenicity when combined with seasonal inactivated vaccines (Forrest et al., 2009). Immune stimulating complexes (ISCOMs) are derived from the bark of the Quillaia saponaria Molina tree and have also shown to stimulate strong humoral and cellular immune responses (Sjolander et al., 1998). ...
... [Haidari et al., 2009] Reassortant H5N1 viruses generated by reverse genetics (identified by the prefix rg) from A/Vietnam/1203/04 (VN/04), an isolate from a fatal human case, and from the avian isolates A/Duck/Hunan/795/02 (Dk/HN/02), A/Duck/Laos/3295/06 (Dk/LS/06), and A/Japanese White Eye/Hong Kong/1038/06 (JWE/HK/06). The reassortant H5N1 viruses were constructed with PR8 and expressed the H5 (from which polybasic amino acids that are associated with high virulence were removed) and N1 of the original isolates [Forrest et al., 2009]. ...
... Influenza H5N1 vaccines in ferrets have primarily been delivered by the intramuscular route [9], [10], [37], [38], [39] with fewer by the intranasal route [40], [41]. Many of these studies have demonstrated protection from a lethal challenge that was delivered either intranasally or intratracheally. ...
We investigated the protective efficacy of two intranasal chitosan (CSN and TM-CSN) adjuvanted H5N1 Influenza vaccines against highly pathogenic avian Influenza (HPAI) intratracheal and intranasal challenge in a ferret model.
Six groups of 6 ferrets were intranasally vaccinated twice, 21 days apart, with either placebo, antigen alone, CSN adjuvanted antigen, or TM-CSN adjuvanted antigen. Homologous and intra-subtypic antibody cross-reacting responses were assessed. Ferrets were inoculated intratracheally (all treatments) or intranasally (CSN adjuvanted and placebo treatments only) with clade 1 HPAI A/Vietnam/1194/2004 (H5N1) virus 28 days after the second vaccination and subsequently monitored for morbidity and mortality outcomes. Clinical signs were assessed and nasal as well as throat swabs were taken daily for virology. Samples of lung tissue, nasal turbinates, brain, and olfactory bulb were analysed for the presence of virus and examined for histolopathological findings.
In contrast to animals vaccinated with antigen alone, the CSN and TM-CSN adjuvanted vaccines induced high levels of antibodies, protected ferrets from death, reduced viral replication and abrogated disease after intratracheal challenge, and in the case of CSN after intranasal challenge. In particular, the TM-CSN adjuvanted vaccine was highly effective at eliciting protective immunity from intratracheal challenge; serologically, protective titres were demonstrable after one vaccination. The 2-dose schedule with TM-CSN vaccine also induced cross-reactive antibodies to clade 2.1 and 2.2 H5N1 viruses. Furthermore ferrets immunised with TM-CSN had no detectable virus in the respiratory tract or brain, whereas there were signs of virus in the throat and lungs, albeit at significantly reduced levels, in CSN vaccinated animals.
This study demonstrated for the first time that CSN and in particular TM-CSN adjuvanted intranasal vaccines have the potential to protect against significant mortality and morbidity arising from infection with HPAI H5N1 virus.
... Arguably the most noteworthy attempts involve the use of AS03 [8], MF59 [9], and the immune stimulating complex adjuvant Matrix M [10]. Other cross-protection strategies include the use of inactivated virus vaccines containing multi-clade [11,12] or ancestral H5N1 virus strains [13]. DNA vaccines for inducing cross-clade neutralizing antibodies associated with multi-clade HA or consensus HA gene(s) are also in various stages of development [14][15][16][17][18]. ...
The highly pathogenic avian influenza (HPAI) H5N1 virus, a known trigger of diseases in poultry and humans, is perceived as a serious threat to public health. There is a clear need for a broadly protective H5N1 vaccine or vaccines for inducing neutralizing antibodies against multiple clades/subclades. We constructed single, double, and triple mutants of glycan-masked hemagglutiinin (HA) antigens at residues 83, 127 and 138 (i.e., g83, g127, g138, g83+g127, g127+g138, g83+g138 and g83+g127+g138), and then obtained their corresponding HA-expressing adenovirus vectors and recombinant HA proteins using a prime-boost immunization strategy. Our results indicate that the glycan-masked g127+g138 double mutant induced more potent HA-inhibition, virus neutralization antibodies, cross-clade protection against heterologous H5N1 clades, correlated with the enhanced bindings to the receptor binding sites and the highly conserved stem region of HA. The immune refocusing stem-specific antibodies elicited by the glycan-masked H5HA g127+g138 and g83+g127+g138 mutants overlapped with broadly neutralizing epitopes of the CR6261 monoclonal antibody that neutralizes most group 1 subtypes. These findings may provide useful information in the development of a broadly protective H5N1 influenza vaccine.
... Subclade 2.1 is further divided into 2. To deal with this high level of genetic diversity, 2 general vaccine approaches are currently explored. The first approach is based on using particular strains or combinations of strains (multivalent) selected from a geographic region where the vaccine is intended for use [8][9][10][11][12][13]. For example, the World Health Organization (WHO) has already created 22 seed vaccine strains for use in different geographic regions. ...
Since 1996, highly pathogenic avian influenza (HPAI) H5N1 virus has presented a persistent threat to public health. Its high
degree of genetic diversity also poses enormous challenges in developing effective vaccines. To search for vaccine regimens
that could elicit broadly neutralizing antibody responses against diverse HPAI H5N1 strains, in the present study we tested
H5 hemagglutinin (HA) from an A/Thailand/1(KAN)-1/2004 strain in a heterologous prime-boost vaccination. We demonstrated that
priming mice with DNA and boosting with virus-like particle induced antibody responses that cross-neutralize all reported
clades and subclades of HPAI H5N1 viruses and protect mice from high lethal dose HPAI H5N1 challenge in both active and passive
immunizations. Unexpectedly, cross-divergent H5 neutralizing antibodies are directed to the HA head and block both attachment
and postattachment of virus entry. Thus, we conclude that as a promising pan-H5 vaccine candidate this prime-boost regimen
could be further developed in ferrets and in humans.
... Arguably the most noteworthy attempts involve the use of AS03 [8], MF59 [9], and the immune stimulating complex adjuvant Matrix M [10]. Other cross-protection strategies include the use of inactivated virus vaccines containing multi-clade [11,12] or ancestral H5N1 virus strains [13]. DNA vaccines for inducing cross-clade neutralizing antibodies associated with multi-clade HA or consensus HA gene(s) are also in various stages of development [14][15][16][17][18]. ...
Highly pathogenic avian influenza (HPAI) H5N1 viruses continue to trigger severe diseases in poultry and humans, prompting many efforts to develop an effective vaccine. Toward that goal, we employed two modern sophisticated techniques namely construction of recombinant adenovirus vector encoding HA (rAd-HA) and flagellin-containing virus-like particle (FliC-VLP). Using a murine model, we investigated a heterologous prime-boost regimen of rAd-HA vector and FliC-VLP in contrast to two-dose immunizations using rAd-HA or FliC-VLP alone. Our results indicate that priming with rAd-HA vector followed by a FliC-VLP booster induced the highest levels of HA-specific total IgG, IgG1, IgG2a, hemagglutination inhibition and neutralizing antibody titers against homologous and heterologous clades of H5N1 virus strains. These results provide useful information to support the development of more effective H5N1 vaccines.
... Of note, ferret antiserum did not distinguish the two viruses (Rowe, et al., 1999). The validity of this twodimensional antigenic dendrogram is further illustrated by its ability to distinguish A/HK/ 213/2003 (clade 1) and A/VMN/1203/2004 (clade 1), as immunization with either of these viruses failed to cross-protect reciprocally in a ferret model (Forrest, et al., 2009) Clustering is an assignment of multiple objects with similar properties into groups or clusters. Hierarchical algorithms find successive clusters using previously established clusters. ...
The hemagglutination-inhibition (HI) titers of a panel of 25 mouse monoclonal antibodies (MAbs) against 44 isolates of highly pathogenic avian influenza virus H5N1 were determined. A two-dimensional antigenic dendrogram was constructed by hierarchical clustering of HI titers. Viruses with similar reactivity patterns were clustered horizontally, whereas MAbs were clustered vertically. In this 2-D dendrogram, with 40% similarity as a cut-off, four virus clusters and four MAbs clusters were identified. A phylogenetic tree based on the deduced amino acid sequence of the hemagglutinin gene of tested viruses was constructed and its topology was compared to the antigenic dendrogram. Interestingly, viruses with high genetic homology in the phylogenetic tree also had high similarity in their reactivity patterns, as indicated by their relatedness in the tree and close clustering in the dendrogram, respectively. However, the reverse and the converse were also true. Of the five pairs of viruses in the dendrogram with bootstrap values higher than 75, four pairs were in concordance with their genetic relatedness. However, one pair contained viruses belong to two distinct genetic clades. These results were discussed in the context of antigenic variation, genetic polymorphism, and the potential application of MAbs in antigenic analysis.
... Ferrets are particularly useful as a bridging model for measuring protection offered by H5N1 vaccines in which experimental challenge and vaccine efficacy studies are not feasible in humans. However, the use of H5N1 IIV without adjuvants in ferrets has had mixed results, and although in most cases ferrets were protected from severe disease after homologous and heterologous virus challenge, only low levels of serum antibodies were detected [29][30][31]. Similarly, in serologically naive ferrets, seasonal IIV vaccination without adjuvant, even after 2 doses, results in only modest serum antibody responses [32]. Both AlOH and proprietary oil-in-water emulsion adjuvants increase H5N1 vaccine immunogenicity and cross-reactivity [8,33]. ...
Continued H5N1 virus infection in humans highlights the need for vaccine strategies that provide cross-clade protection against
this rapidly evolving virus. We report a comparative evaluation in ferrets of the immunogenicity and cross-protective efficacy
of isogenic mammalian cell-grown, live attenuated influenza vaccine (LAIV) and adjuvanted, whole-virus, inactivated influenza
vaccine (IIV), produced from a clade 1 H5N1 6:2 reassortant vaccine candidate (caVN1203-Len17rg) based on the cold-adapted
A/Leningrad/134/17/57 (H2N2) master donor virus. Two doses of LAIV or IIV provided complete protection against lethal homologous
H5N1 virus challenge and a reduction in virus shedding and disease severity after heterologous clade 2.2.1 H5N1 virus challenge
and increased virus-specific serum and nasal wash antibody levels. Although both vaccines demonstrated cross-protective efficacy,
LAIV induced higher levels of nasal wash IgA and reduction of heterologous virus shedding, compared with IIV. Thus, enhanced
respiratory tract antibody responses elicited by LAIV were associated with improved cross-clade protection.
... Aluminum adjuvants enhanced the immunogenicity of H5N1 vaccine in ferrets [11,29] but failed to enhance vaccine immunogenicity in humans [38,39,40,41]. MF59, a squalene oil emulsion adjuvant, was shown in ferrets to enhance multi-clade protection against H5N1 strains [42], and in humans improve immunogenicity of H5N1 antigens and H1N1 2009 pandemic vaccines [43,44]. Nevertheless, MF59 has not been approved for use by the FDA, leaving a major need to identify suitable safe and effective adjuvants for use in pandemic influenza vaccines. ...
The reduced immunogenicity of the H5 hemagglutinin (HA), compared to seasonal HA serotypes, has stimulated searches for effective adjuvants to improve H5 vaccine efficacy. This study examined the immunogenicity and protective efficacy in ferrets immunized with a split-virion H5N1 vaccine combined with Advax™, a novel delta inulin-based polysaccharide adjuvant technology that has previously demonstrated ability to augment humoral and cellular immunity to co-administered antigens.
Ferrets were vaccinated twice 21 days apart with 7.5 μg or 22.5 μg of a split-virion preparation of A/Vietnam/1203/2004 with or without adjuvant. An additional group received just one immunization with 22.5 μg HA plus adjuvant. Serum antibodies were measured by hemagglutination inhibition and microneutralization assays. Vaccinated animals were challenged intranasally 21 days after the last immunization with 10(6) EID(50) of the homologous strain. Morbidity was assessed by observed behavior, weight loss, temperature, cytopenias, histopathology, and viral load.
No serum neutralization antibody was detected after two immunizations with unadjuvanted vaccine. Two immunizations with high or low dose adjuvanted vaccine stimulated high neutralizing antibody titers. Survival was 100% in all groups receiving adjuvanted-vaccine including the single dose group, compared to 67% survival with unadjuvanted vaccine, and 0% survival in saline or adjuvant-alone controls. Minimal morbidity was seen in all animals receiving adjuvanted vaccine, and was limited to rhinorrhea and mild thrombocytopenia, without fever, weight loss, or reduced activity. H5N1 virus was cleared from the nasal wash by day 4 post-challenge only in animals receiving adjuvanted vaccine which also prevented viral invasion of the brain in most animals.
In this initial study, Advax™ adjuvant formulations improved the protective efficacy of a split-virion H5N1 vaccine as measured by significantly enhanced immunogenicity, survival, and reduced morbidity.
... (i) A/Puerto Rico/8/34 (H1N1) (PR8) and the reassortant virus A/HK/X31 (H3N2) (X31), which expressed the H3 and N2 of A/Aichi/2/68 on the PR8 background (Kilbourne et al., 1971) with PR8 and expressed H5 (from which polybasic amino acids that are associated with high virulence were removed) and N1 of the original isolates (Forrest et al., 2009). (iii) A reassortant (PR8 background) expressing the H1 and N1 of A/California/7/09 (CA/09), a human isolate of the novel swine-origin A(H1N1) virus that spread globally in 2009 and generated a pandemic alert (Maines et al., 2009). ...
Pomegranates have high levels of polyphenols (PPs) and may be a rich source of compounds with antiviral activity. We evaluated the direct anti-influenza activity of three commercially available pomegranate extracts: pomegranate juice (PJ), a concentrated liquid extract (POMxl), and a 93% PP powder extract (POMxp). The acidity of PJ and POMxl solutions contributed to rapid anti-influenza activity, but this was not a factor with POMxp. Studies using POMxp showed that 5min treatment at room temperature with 800μg/ml PPs resulted in at least a 3log reduction in the titers of influenza viruses PR8 (H1N1), X31 (H3N2), and a reassortant H5N1 virus derived from a human isolate. However, the antiviral activity was less against a coronavirus and reassortant H5N1 influenza viruses derived from avian isolates. The loss of influenza infectivity was frequently accompanied by loss of hemagglutinating activity. PP treatment decreased Ab binding to viral surface molecules, suggesting some coating of particles, but this did not always correlate with loss of infectivity. Electron microscopic analysis indicated that viral inactivation by PPs was primarily a consequence of virion structural damage. Our findings demonstrate that the direct anti-influenza activity of pomegranate PPs is substantially modulated by small changes in envelope glycoproteins.
... This perception has arisen from classic experiments that showed priming by infection with one type of influenza skewed a subsequent response to a second infection, favoring the production of antibodies to the first virus, a phenomenon termed "Original Antigenic Sin" (OAS) [7,8]. More recent experiments suggest this does not apply to human subjects after seasonal influenza subunit vaccination [9], and immunization with experimental H5 vaccines in animal models show broad cross protection to multiple influenza subtypes [10][11][12][13]. ...
B cell responses after immunization with a drifted H5 influenza/A/Vietnam/1203/04 vaccine were characterized in the peripheral blood of human subjects primed with experimental recombinant H5 influenza A/Hong Kong/156/97 vaccine. Antibody secreting cells were assayed by ELISPOT against a panel of recombinant hemagglutinin and control proteins. Increased frequencies of H5 HA-specific antibody secreting and memory B cells could be observed within 7 days of re-vaccination. Furthermore, these responses were cross-reactive to both H5 HA variants, but not H3 or avian H6 HA strains. These observations suggest prior vaccination against H5 influenza HA induces cellular immune responses that cross-react among drifted variants, without precluding a response to new, or existing HA strains.
Highly pathogenic H5N1 influenza viruses continue to spread around the globe and reassort with low pathogenic avian influenza viruses often resulting in morbidity and mortality to not only waterfowl, but also poultry. Our group previously developed two hemagglutinin (HA) based vaccines using a methodology termed computationally optimized broadly reactive antigen (COBRA). Each of these HA antigens, Human COBRA 2 (Hu-CO) and Human-Avian COBRA 2 (Hu-Av CO) elicit antibodies with hemagglutination-inhibition (HAI) activity against viruses from various clades, but not always the same viruses. Here, we have sought to identify residues in these two HA molecules that are critical fordifferential HAI activity against various H5Nx influenza viruses. The two HA antigens are remarkedly similar in the globular head region, except for 4 residues at amino acids 140, 141, 155, and 156. By mutating these amino acids in each HA antigen, chimeric HA proteins were used to elicit immune responses in mice. When the Asn-Thr pair at position 155-156 in the Hu-CO HA was converted to the Ser-Ala residues found in the Hu-Av CO HA, the elicited antibodies lost HAI activity against clade 2.3.2.1 H5Nx viruses, such as A/Hubei/01/2010. When this Asn-Thr motif was added at these positions in the Hu-Av CO HA molecule, HAI activity in the elicited sera against A/Hubei/01/2010 was significantly increased. We speculate that a putative N-linked glycosylation at this location in the Hu-CO HA antigen is a key driver in the elicitation of antibodies with HAI activity to different locations on wild-type H5 HA molecules resulting in differential neutralization of viral infection and protection in vivo against H5 influenza virus induced disease.
The strength and breadth of an individual’s antibody repertoire are important predictors of their response to influenza infection or vaccination. Although progress has been made in understanding qualitatively how repeated exposures shape the antibody mediated immune response, quantitative understanding remains limited. We developed a set of mathematical models describing short-term antibody kinetics following influenza infection or vaccination and fit them to haemagglutination inhibition (HI) titres from 5 groups of ferrets which were exposed to different combinations of trivalent inactivated influenza vaccine (TIV with or without adjuvant), A/H3N2 priming inoculation and post-vaccination A/H1N1 inoculation. We fit models with various immunological mechanisms that have been empirically observed but are yet to be included in mathematical models of antibody landscapes, including titre ceiling effects, antigenic seniority and exposure-type specific cross reactivity. Based on the parameter estimates of the best supported models, we describe a number of key immunological features. We found quantifiable differences in the degree of homologous and cross-reactive antibody boosting elicited by different exposure types. Infection and adjuvanted vaccination generally resulted in strong, broadly reactive responses whereas unadjuvanted vaccination resulted in a weak, narrow response. We found that the order of exposure mattered: priming with A/H3N2 improved subsequent vaccine response, and the second dose of adjuvanted vaccination resulted in substantially greater antibody boosting than the first. Either antigenic seniority or a titre ceiling effect were included in the two best fitting models, suggesting that a mechanism describing diminishing antibody boosting with repeated exposures improved the predictive power of the model. Although there was considerable uncertainty in our estimates of antibody waning parameters, our results suggest that both short and long term waning were present and would be identifiable with a larger set of experiments. These results highlight the potential use of repeat exposure animal models in revealing short-term, strain-specific immune dynamics of influenza.
Author summary
Despite most individuals having some preexisting immunity from past influenza infections and vaccinations, a significant proportion of the human population is infected with influenza each year. Predicting how an individual’s antibody profile will change following exposure is therefore useful for evaluating which populations are at greatest risk and how effective vaccination strategies might be. However, interpretation of antibody data from humans is complicated by immunological interactions between all previous, unobserved exposures in an individual’s life. We developed a mathematical model to describe short-term antibody kinetics that are important in building an individual’s immune profile but are difficult to observe in human populations. We validated this model using antibody data from ferrets with known, varied infection and vaccination histories. We were able to quantify the independent contributions of various exposures and immunological mechanisms in generating observed antibody titres. These results suggest that data from experimental systems may be included in models of human antibody dynamics, which may improve predictions of vaccination strategy effectiveness and how population susceptibility changes over time.
The evolution of highly pathogenic H5N1 avian influenza viruses (HPAI-H5N1) has resulted in the appearance of a number of diverse groups of HPAI-H5N1 based on the presence of genetically similar clusters of their haemagglutinin sequences (clades). An H5 antigen encoded by a recombinant baculovirus and expressed in insect cells, was used for oil-emulsion-based vaccine prototypes. In several experiments, vaccination was performed at 10 days of age, followed by challenge infection on day 21 post vaccination (PV) with HPAI-H5N1 clades 2.2, 2.2.1, and 2.3.2. A further challenge infection with HPAI-H5N1 clade 2.2.1 was performed at day 42 PV. High haemagglutination inhibition (HI) titres were observed for the recH5 vaccine antigen, and lower HI titres for the challenge virus antigens. Nevertheless, the rate of protection from mortality and clinical signs was 100% when challenged at 21 d PV and 42 d PV, indicating protection over the entire broiler chicken rearing period without a second vaccination. The unvaccinated control chickens mostly died between two and five days after challenge infection. A low level of viral RNA was detected by RT-qPCR in a limited number of birds for a short period after challenge infection, indicating a limited spread of HPAI-H5N1 at flock level. Furthermore, it was observed that the vaccine can be used in a DIVA approach, based on the detection of NP antibodies in vaccinated/challenged chickens. The vaccine fulfilled all expectations of an inactivated vaccine after one vaccination against challenge with different clades of H5N1-HPAI and is suitable for a DIVA approach.
Highly pathogenic avian influenza H5N1 virus may cause serious fatal diseases and constitute a grave threat to human health. Although the current H5N1 influenza strains appear not to be transmissible from human to human, it is of major concern that mixing with human influenza strains could convert H5N1 to a strain that would spread that would spread from human to human and cause a serious pandemic. In addition, avian influenza H5N1 virus after continuous variation may break through the species barrier and spread to mammals and humans, thereby cause disease even death under the influence of various virus and host factors. This paper reviews the progress in research on pandemic influenza vaccine.
H5N1 HPAI virus continues to be a severe threat for public health, as well as for the poultry industry, due to its high mortality and antigenic drift rate. There is no monovalent vaccine available which provides broad protection against those major circulating strains. In the present study, a monovalent H5 vaccine strain was developed with antigenic sequence analysis and epitope mutations. H5 from Indonesia strain (A/Indonesia/CDC669/2006) was used as backbone sequence. Three amino acids were mutated to express immunogenic epitopes from other circulating H5N1s in the backbone. RG influenza virus expressing the epitope-chimeric H5 can react in HI with multiple H5 monoclonal antibodies which fail to neutralize wild type CDC669. High titers in HI and virus neutralization against different clades H5N1s (clade 1, 2, 4 and 7) were detected using sera from mice immunized with the epitope-chimeric H5N1. The monovalent vaccine with RG-epitope-chimeric H5N1 protected mice from lethal challenge with H5N1s of different clades, including clade 1.0, 2.1, 2.2 and 2.3. This study indicates that the broad immune response elicited by this single H5N1 virus allows it to be a promising candidate for a monovalent H5 universal vaccine.
MF59 is an oil-in-water (o/w) emulsion, pre-pared with a low content of the biodegradable oil, squalene (4.3% w/w), which is a naturally occur-ring substance normally found in plants and ani-mals including humans. In humans, squalene is an intermediate in the steroid hormone biosyn-thetic pathway and is a direct synthetic precursor to cholesterol. A normal human is estimated to produce in excess of 1 g of squalene per day, while the amount of squalene contained in the licensed MF59-adjuvanted seasonal influ-enza vaccine is approximately 10 mg per dose. In comparison, the normal dietary intake of squalene in humans is 50–200 mg/day, depend-ing on diet. MF59 is a well-established, safe and potent vaccine adjuvant that has been licensed in more than 20 countries, for more than 13 years for use in an influenza vaccine focused on elderly subjects (Fluad ® , Novartis, Cambridge, MA, USA). During the 2009 H1N1 influenza pandemic, two MF59-adjuvanted vaccines were licensed and used safely in all age groups (down to 6 months of age) including pregnant women. Furthermore, MF59 has been shown to be safe in a seasonal influenza vaccine in infants and children and increased vaccine efficacy from 43 to 89% [1–3]. The overall safety profile of MF59 has been clinically established with a large safety database (>40,000 subjects) and through an extensive pharmacovigilance evalu-ation in excess of 60 million doses distributed commercially as Fluad ® . In addition, approxi-mately 100 million doses of H1N1 pandemic vaccines (Focetria ® and Celtura ® , Novartis) were distributed. The MF59 adjuvant significantly enhances the immunogenicity of influenza vaccines in the elderly, who typically respond poorly to traditional influenza vaccines, owing to age-related impairment of their immune sys-tems (immuno senescence) [4]. Moving beyond seasonal influenza vaccines, MF59 has also been shown to significantly improve the immunoge-nicity of pandemic influenza vaccines and has enabled vaccines with a relatively low antigen content to achieve titers expected to offer pro-tection, and with fewer doses [5–7]. Moreover, the addition of MF59 to the vaccine allows for greater cross-reactivity against viral strains not actually included in the vaccine (Figures 1–4) [6,8]. This is a key attribute, since it is difficult to pre-dict exactly which strain might emerge and cause a pandemic. Following the emergence of the H1N1 pandemic in 2009, an MF59-adjuvanted influenza vaccine received approval for licensure
The response to the first influenza pandemic of the twenty-first century was facilitated by years of preparation for a possible pandemic caused by the avian influenza H5N1. The threat of an H5N1 pandemic had led to an increase in manufacturing capacity, to the development of influenza vaccines made in cell culture instead of eggs, to the development of innovative adjuvants and to the establishment of clear rules to license pandemic vaccines. Most of these tools have been used and validated by the H1N1 pandemic. The main lesson learned is that oil-in-water adjuvants can be safely used in large scale and in all ages and conditions, including pregnant women. Adjuvants increase the titer of the antibody responses and broaden the epitopes recognized by antibodies so that they can neutralize also drifted viruses. In addition, they induce long lasting B- and T-memory cells. A further advantage of the use of adjuvants is the ability to use lower doses of vaccine, thus multiplying the manufacturing capacity up to fourfold. Cell-based vaccines have been established as a new technology to produce influenza vaccines.
Both adjuvants and cell cultures are expected to change not only the way we will address future pandemics but also the way we approach seasonal influenza, changing a field that has been stagnant for too many decades.
Viral diseases are important threats to public health worldwide. With the number of emerging viral diseases increasing the last decades, there is a growing need for appropriate animal models for virus studies. The relevance of animal models can be limited in terms of mimicking human pathophysiology. In this review, we discuss the utility of animal models for studies of influenza A viruses, HIV and SARS-CoV in light of viral emergence, assessment of infection and transmission risks, and regulatory decision making. We address their relevance and limitations. The susceptibility, immune responses, pathogenesis, and pharmacokinetics may differ between the various animal models. These complexities may thwart translating results from animal experiments to the humans. Within these constraints, animal models are very informative for studying virus immunopathology and transmission modes and for translation of virus research into clinical benefit. Insight in the limitations of the various models may facilitate further improvements of the models.
Emulsions have a long history of use as potent and effective adjuvants in humans for a range of vaccines, particularly for
influenza. Although older mineral oil- and water-in-oil-based emulsion adjuvants did not have an overall safety and tolerability
profile to allow them to be acceptable for widespread use, a newer generation of oil-in-water adjuvants has been recently
developed, based on the use of the biodegradable oil squalene. These adjuvants have shown particular value in the development
of new generation vaccines to offer enhanced protection against both seasonal and pandemic strains of influenza virus. The
first oil-in-water emulsion adjuvant included in an approved flu vaccine was MF59, which was originally licensed in Europe
in 1997 as an improved influenza vaccine for the elderly. In the very recent past, MF59 and related adjuvants have shown their
value by offering the possibility of significant antigen dose reductions and higher potency products in the face of the H1N1
pandemic emergency and other pandemic threats. The recent H1N1 global problem allowed the opportunity for widespread use of
emulsion-based adjuvants in a range of population groups in a number of countries, in which strict monitoring of safety was
the norm. Importantly, this widespread use allowed the safety profile of squalene-based emulsion adjuvants to be further substantiated
in large and diverse populations of humans, including young children and pregnant women. It is our confident prediction that
the coming years will see wider use and further licensures for oil-in-water emulsion adjuvants, particularly for improved
flu vaccines.
Influenza vaccines have been in use for more than 60 years and have proven to be efficacious in protecting from influenza infections during epidemics and the recent H1N1 pandemic. The development of influenza vaccines has so far been largely based on empirical grounds, which leaves room for vaccine improvement by implementation of recent insights in innate and adaptive immunity. Also, evaluation and approval of new vaccines rely on rather broad correlates of protection such as the hemagglutination inhibition titre, thereby neglecting qualitative aspects of the immune response. Here we discuss how current inactivated influenza vaccine formulations differ in the type of immune response they elicit, their protective capacity, and what causes these differences. Finally, we will discuss how this knowledge can guide the development of new adjuvants that optimize the protective efficacy of influenza vaccines.
Highly pathogenic H5N1 avian influenza viruses emerged in 1996 and have since evolved so extensively that a single strain
can no longer be used as a prepandemic vaccine or diagnostic reagent. We therefore sought to identify the H5N1 strains that
may best serve as cross-reactive diagnostic reagents. We compared the cross-reactivity of 27 viruses of clades 0, 1, 2.1,
2.2, 2.3, and 4 and of four computationally designed ancestral H5N1 strains by hemagglutination inhibition (HI) and microneutralization
(MN) assays. Antigenic cartography was used to analyze the large quantity of resulting data. Cartographs of HI titers with
chicken red blood cells were similar to those of MN titers, but HI with horse red blood cells decreased antigenic distances
among the H5N1 strains studied. Thus, HI with horse red blood cells seems to be the assay of choice for H5N1 diagnostics.
Whereas clade 2.2 antigens were able to detect antibodies raised to most of the tested H5N1 viruses (and clade 2.2-specific
antisera detected most of the H5N1 antigens), ancestral strain A exhibited the widest reactivity pattern and hence was the
best candidate diagnostic reagent for broad detection of H5N1 strains.
MF59 is a well-established, safe and potent vaccine adjuvant that has been licensed for more than 13 years for use in an influenza vaccine focused on elderly subjects (Fluad®), Novartis, Cambridge, MA, USA). Recently, MF59 was shown to be safe in a seasonal influenza vaccine for young children and was able to increase vaccine efficacy from 43 to 89%. A key and consistent feature of MF59 is the ability of the emulsion to induce fast priming of influenza antigen-specific CD4(+) T-cell responses, to induce strong and long-lasting memory T- and B-cell responses and to broaden the immune response beyond the influenza strains actually included in the vaccine. The enhanced breadth of response is valuable in the seasonal setting, but is particularly valuable in a (pre-) pandemic setting, when it is difficult to predict which strain will emerge to cause the pandemic. We have shown that the ability of MF59 to increase the breadth of immune response against influenza vaccines is mainly due to the spreading of the repertoire of the B-cell epitopes recognized on the hemagglutinin and neuraminidase of the influenza virus.
Serum antibodies induced by seasonal influenza or seasonal influenza vaccination exhibit limited or no cross-reactivity against
the 2009 pandemic swine-origin influenza virus of the H1N1 subtype (pH1N1). Ferrets immunized once or twice with MF59-adjuvanted
seasonal influenza vaccine exhibited significantly reduced lung virus titers but no substantial clinical protection against
pH1N1-associated disease. However, priming with MF59-adjuvanted seasonal influenza vaccine significantly increased the efficacy
of a pandemic MF59-adjuvanted influenza vaccine against pH1N1 challenge. Elucidating the mechanism involved in this priming
principle will contribute to our understanding of vaccine- and infection-induced correlates of protection. Furthermore, a
practical consequence of these findings is that during an emerging pandemic, the implementation of a priming strategy with
an available adjuvanted seasonal vaccine to precede the eventual pandemic vaccination campaign may be useful and life-saving.
Since the reemergence of highly pathogenic H5N1 influenza viruses in humans in 2003, these viruses have spread throughout avian species in Asia, Europe, and Africa. Their sustained circulation has resulted in the evolution of phylogenetically diverse lineages. Viruses from these lineages show considerable antigenic variation, which has confounded vaccine planning efforts. We reconstructed ancestral protein sequences at several nodes of the hemagglutinin (HA) and neuraminidase (NA) gene phylogenies that represent ancestors to diverse H5N1 virus clades. By using the same methods that have been used to generate currently licensed inactivated H5N1 vaccines, we were able to produce a panel of replication competent influenza viruses containing synthesized HA and NA genes representing the reconstructed ancestral proteins. We identified two of these viruses that showed promising in vitro cross-reactivity with clade 1, 2.1, 2.2, 2.3.4, and 4 viruses. To confirm that vaccine antigens derived from these viruses were able to elicit functional antibodies following immunization, we created whole-virus vaccines and compared their protective efficacy versus that of antigens from positive control, naturally occurring, and broadly reactive H5N1 viruses. The ancestral viruses' vaccines provided robust protection against morbidity and mortality in ferrets challenged with H5N1 strains from clades 1, 2.1, and 2.2 in a manner similar to those based on the control strains. These findings provide proof of principle that viable, computationally derived vaccine seed viruses can be constructed within the context of currently licensed vaccine platforms. Such technologies should be explored to enhance the cross reactivity and availability of H5N1 influenza vaccines.
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.
The avian H5N1 influenza virus has the potential to cause a new pandemic. The increasing number of recent outbreaks of highly pathogenic avian influenza H5N1 in birds and humans emphasizes the urgent need to develop a potent H5N1 vaccine. Here, we studied the immunogenicity and protective effect of a vaccine prepared from H5N1 inactivated whole virus. This vaccine was intranasally co-administered in mice with phosphate buffered saline, recombinant cholera toxin B subunit (rCTB), cholera toxin (CT), rCTB containing a trace amount of holotoxin (rCTB/CT), polyinosinic:polycytidylic acid double-stranded RNA (polyI:C), or MF59 as an adjuvant. Intranasal administration of H5N1 inactivated whole virus vaccine with rCTB, CT, rCTB/CT, polyI:C, and MF59 elicited an immunological response with both secretory IgA (sIgA) in nasal, lung, and vaginal lavage, and IgG antibody in serum, showing protective immunity against lethal H5N1 infection. Cross-clade protection was also observed in animals immunized with a vaccine derived from Anhui/01/2005(H5N1) with rCTB, CT, rCTB/CT, polyI:C, or MF59 as adjuvants that were subsequently challenged with the A/OT/SZ/097/03 influenza strain.
Outbreaks of highly pathogenic H5N1 influenza viruses have been reported in many countries worldwide. The possibility of pandemics caused by H5N1 influenza viruses is high since human infections by H5N1 viruses continually occur. In this study we determined the immune response and efficacy of inactivated H5N1 vaccine developed by reverse genetics in ferrets. Ferrets intramuscularly inoculated with two doses of H5N1 vaccine survived the lethal challenge with homologous or heterologous H5N1 influenza viruses, while 75% and 25% of ferrets immunized with one dose of H5N1 vaccine survived the lethal challenge with homologous and heterologous H5N1 influenza viruses, respectively. When we determined antibody subtypes specific for H5N1 influenza viruses in tissues and sera of vaccinated ferrets, IgG antibodies were detected mainly in the trachea, nostril, lung, heart, liver, kidney, intestine, spleen, and serum. Our results suggest that IgG antibodies may play a major role in protecting ferrets immunized with the inactivated H5N1 vaccine from lethal challenge with H5N1 influenza viruses.
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.
In Hong Kong in 1997, a highly lethal H5N1 avian influenza virus was apparently transmitted directly from chickens to humans with no intermediate mammalian host and caused 18 confirmed infections and six deaths. Strategies must be developed to deal with this virus if it should reappear, and prospective vaccines must be developed to anticipate a future pandemic. We have determined that unadapted H5N1 viruses are pathogenic in mice, which provides a well-defined mammalian system for immunological studies of lethal avian influenza virus infection. We report that a DNA vaccine encoding hemagglutinin from the index human influenza isolate A/HK/156/97 provides immunity against H5N1 infection of mice. This immunity was induced against both the homologous A/HK/156/97 (H5N1) virus, which has no glycosylation site at residue 154, and chicken isolate A/Ck/HK/258/97 (H5N1), which does have a glycosylation site at residue 154. The mouse model system should allow rapid evaluation of the vaccine's protective efficacy in a mammalian host. In our previous study using an avian model, DNA encoding hemagglutinin conferred protection against challenge with antigenic variants that differed from the primary antigen by 11 to 13% in the HA1 region. However, in our current study we found that a DNA vaccine encoding the hemagglutinin from A/Ty/Ir/1/83 (H5N8), which differs from A/HK/156/97 (H5N1) by 12% in HA1, prevented death but not H5N1 infection in mice. Therefore, a DNA vaccine made with a heterologous H5 strain did not prevent infection by H5N1 avian influenza viruses in mice but was useful in preventing death.
The antigenic variation of influenza A virus hemagglutinin (HA) and neuraminidase (NA) glycoproteins requires frequent changes in vaccine formulation. The classical method of creating influenza virus seed strains for vaccine production is to generate 6 + 2 reassortants that contain six genes from a high-yield virus, such as A/PR/8/34 (H1N1) and the HA and NA genes of the circulating strains. The techniques currently used are time-consuming because of the selection process required to isolate the reassortant virus. We generated the high-yield virus A/PR/8/34 (H1N1) entirely from eight plasmids. Its growth phenotype in embryonated chicken eggs was equivalent to that of the wild-type virus. By using this DNA-based cotransfection technique, we generated 6 + 2 reassortants that had the antigenic determinants of the influenza virus strains A/New Caledonia/20/99 (H1N1), A/Panama/2007/99 (H3N2), A/teal/HK/W312 (H6N1), and A/quail/HK/G1/97 (H9N2). Our findings demonstrate that the eight-plasmid system allows the rapid and reproducible generation of reassortant influenza A viruses for use in the manufacture of vaccines.
If H5N1 influenza viruses become transmissible among humans, vaccination will offer the most effective option to limit their spread. Two human vaccine candidates recently generated by reverse genetics are based on antigenically different hemagglutinin (HA) glycoproteins derived from the A/HK/213/03 (H5N1) and A/Vietnam/1203/04 (H5N1) viruses. Their HA1 amino acid sequences differ at 10 positions, one of which (N154) introduces a potential glycosylation site in A/Vietnam/1203/04 (H5N1). To assess the impact of five amino acids in the putative antigenic sites on immunogenicity and immune protection, we generated a series of whole-virus vaccines that differed only in one or two HA amino acids. Sera from ferrets vaccinated with these inactivated preparations had high virus neutralization titers, but their hemagglutination inhibition (HI) titers were usually low. Interestingly, a recombinant virus in which the HA amino acid S223 (characteristic of 2004 viruses) was converted to N223 (as in A/HK/213/03) resulted in higher HI titers. This observation indicates that specific HA residues, such as N223, increase the sensitivity of the HI assay by altering receptor specificity and/or antibody-antigen binding. Ferrets vaccinated with mutant vaccine viruses were protected against lethal challenge with wild-type A/Vietnam/1203/04 virus. Our results suggest that inclusion of the N223 residue in the HA glycoproteins of diagnostic reference viruses may facilitate the evaluation of vaccine efficacy in humans.
• reverse genetics
• receptor specificity
The recent emergence of highly pathogenic avian influenza virus (HPAI) strains in poultry and their subsequent transmission to humans in Southeast Asia have raised concerns about the potential pandemic spread of lethal disease. In this paper we describe the development and testing of an adenovirus-based influenza A virus vaccine directed against the hemagglutinin (HA) protein of the A/Vietnam/1203/2004 (H5N1) (VN/1203/04) strain isolated during the lethal human outbreak in Vietnam from 2003 to 2005. We expressed different portions of HA from a recombinant replication-incompetent adenoviral vector, achieving vaccine production within 36 days of acquiring the virus sequence. BALB/c mice were immunized with a prime-boost vaccine and exposed to a lethal intranasal dose of VN/1203/04 H5N1 virus 70 days later. Vaccination induced both HA-specific antibodies and cellular immunity likely to provide heterotypic immunity. Mice vaccinated with full-length HA were fully protected from challenge with VN/1203/04. We next evaluated the efficacy of adenovirus-based vaccination in domestic chickens, given the critical role of fowl species in the spread of HPAI worldwide. A single subcutaneous immunization completely protected chickens from an intranasal challenge 21 days later with VN/1203/04, which proved lethal to all control-vaccinated chickens within 2 days. These data indicate that the rapid production and subsequent administration of recombinant adenovirus-based vaccines to both birds and high-risk individuals in the face of an outbreak may serve to control the pandemic spread of lethal avian influenza.
Influenza A (H5N1) viruses could cause a severe worldwide epidemic, with high attack rates, large numbers of deaths and hospitalizations, and wide disruption. Effective vaccines against these viruses in humans are urgently needed.
We conducted a multicenter, double-blind two-stage study involving 451 healthy adults 18 to 64 years of age who were randomly assigned in a 2:2:2:2:1 ratio to receive two intramuscular doses of a subvirion influenza A (H5N1) vaccine of 90, 45, 15, or 7.5 microg of hemagglutinin antigen or placebo. The subjects were followed for the safety analysis for 56 days. Serum samples obtained before each vaccination and again 28 days after the second vaccination were tested for H5 antibody by microneutralization and hemagglutination inhibition.
Mild pain at the injection site was the most common adverse event for all doses of vaccine. The frequency of a serum antibody response was highest among subjects receiving doses of 45 microg or 90 microg. Among those who received two doses of 90 microg, neutralization antibody titers reached 1:40 or greater in 54 percent, and hemagglutination-inhibition titers reached 1:40 or greater in 58 percent. Neutralization titers of 1:40 or greater were seen in 43 percent, 22 percent, and 9 percent of the subjects receiving two doses of 45, 15, and 7.5 microg, respectively. No responses were seen in placebo recipients.
A two-dose regimen of 90 mug of subvirion influenza A (H5N1) vaccine does not cause severe side effects and, in the majority of recipients, generates neutralizing antibody responses typically associated with protection against influenza. A conventional subvirion H5 influenza vaccine may be effective in preventing influenza A (H5N1) disease in humans. (ClinicalTrials.gov number, NCT00115986.).
Squalene is a naturally occurring oil which has been used in the development of vaccine adjuvants, such as the oil-in-water emulsion MF59. In past years, by use of noncontrolled and nonvalidated assays, a claim was made that antisqualene antibodies were detectable in the sera of individuals with the so-called Gulf War syndrome. Using a validated enzyme-linked immunosorbent assay for the quantitation of immunoglobulin G (IgG) and IgM antibodies against squalene, we demonstrated that antisqualene antibodies are frequently detectable at very low titers in the sera of subjects who were never immunized with vaccines containing squalene. More importantly, vaccination with a subunit influenza vaccine with the MF59 adjuvant neither induced antisqualene antibodies nor enhanced preexisting antisqualene antibody titers. In conclusion, antisqualene antibodies are not increased by immunization with vaccines with the MF59 adjuvant. These data extend the safety profile of the MF59 emulsion adjuvant.
The goals of antiviral treatment for influenza are to decrease symptoms and functional disability and, more important, to decrease associated complications, hospitalizations, and mortality. Four drugs have been approved for treatment of and prophylaxis against influenza in the United States, but they are underutilized. The M2 ion channel inhibitors amantadine and rimantadine are effective for prophylaxis, and they decrease the duration of symptoms if they are used for early treatment of influenza A. The rapid emergence of resistance during therapy and, recently, the circulation of resistant H3N2 viruses in the community have decreased the usefulness of these M2 ion channel inhibitors. Early therapy with neuraminidase (NA) inhibitors, either oseltamivir or zanamivir, reduces the duration of symptoms, the duration of disability, and the risk of lower respiratory tract complications. Oseltamivir has been shown to decrease antibiotic use, the number of hospitalizations, and, probably, the risk of death after influenza. NA inhibitors might provide substantial benefits in the treatment of pandemic influenza, with reductions in the numbers of hospitalizations and deaths occurring if such treatment (1) is made available in sufficient time, through rapid distribution, and (2) is available in sufficient quantities as a result of stockpiling. Both of the aforementioned NA inhibitors are highly effective for prophylaxis. Geographically targeted mass chemoprophylaxis might contain the spread of a pandemic virus, but multiple hurdles to successful implementation exist. Resistance to oseltamivir occurs with the H274Y variant in viruses that contain N1; however, to date, such variants have been less fit, have not been transmitted from person to person, and have retained susceptibility to zanamivir. Alternative agents and approaches, including parenteral and combination therapy, for the treatment of influenza are needed in the near and long term.
The response to the next influenza pandemic will likely include extensive use of antiviral drugs (mainly oseltamivir), combined with other transmission-reducing measures. Animal and in vitro studies suggest that some strains of influenza may become resistant to oseltamivir while maintaining infectiousness (fitness). Use of antiviral agents on the scale anticipated for the control of pandemic influenza will create an unprecedented selective pressure for the emergence and spread of these strains. Nonetheless, antiviral resistance has received little attention when evaluating these plans.
We designed and analyzed a deterministic compartmental model of the transmission of oseltamivir-sensitive and -resistant influenza infections during a pandemic. The model predicts that even if antiviral treatment or prophylaxis leads to the emergence of a transmissible resistant strain in as few as 1 in 50,000 treated persons and 1 in 500,000 prophylaxed persons, widespread use of antivirals may strongly promote the spread of resistant strains at the population level, leading to a prevalence of tens of percent by the end of a pandemic. On the other hand, even in circumstances in which a resistant strain spreads widely, the use of antivirals may significantly delay and/or reduce the total size of the pandemic. If resistant strains carry some fitness cost, then, despite widespread emergence of resistance, antivirals could slow pandemic spread by months or more, and buy time for vaccine development; this delay would be prolonged by nondrug control measures (e.g., social distancing) that reduce transmission, or use of a stockpiled suboptimal vaccine. Surprisingly, the model suggests that such nondrug control measures would increase the proportion of the epidemic caused by resistant strains.
The benefits of antiviral drug use to control an influenza pandemic may be reduced, although not completely offset, by drug resistance in the virus. Therefore, the risk of resistance should be considered in pandemic planning and monitored closely during a pandemic.
Antigen sparing is regarded as crucial for pandemic vaccine development because worldwide influenza vaccine production capacity is limited. Adjuvantation is an important antigen-sparing strategy. We assessed the safety and immunogenicity of a recombinant H5N1 split-virion vaccine formulated with a proprietary adjuvant system and investigated whether it can induce cross-reactive immunity.
Two doses of an inactivated split A/Vietnam/1194/2004 NIBRG-14 (recombinant H5N1 engineered by reverse genetics) vaccine were administered 21 days apart to eight groups of 50 volunteers aged 18-60 years. We studied four antigen doses (3.8 microg, 7.5 microg, 15 microg, and 30 microg haemagglutinin) given with or without adjuvant. Blood samples were collected to analyse humoral immune response. Adverse events were recorded up through study day 51. Safety analyses were of the whole vaccinated cohort and immunogenicity analyses per protocol. This trial is registered with the ClinicalTrials.gov, number NCT00309634.
All eight vaccine formulations had a good safety profile. No serious adverse events were reported. The adjuvanted vaccines induced more injection-site symptoms and general symptoms than did the non-adjuvanted vaccines, but most were mild to moderate in intensity and transient in nature. The adjuvanted formulations were significantly more immunogenic than the non-adjuvanted formulations at all antigen doses. At the lowest antigenic dose (3.8 microg), immune responses for the adjuvanted vaccine against the recombinant homologous vaccine strain (A/Vietnam/1194/2004 NIBRG-14, clade 1) met or exceeded all US Food and Drug Administration and European Union licensure criteria. Furthermore, 37 of 48 (77%) participants receiving 3.8 microg of the adjuvanted vaccine seroconverted for neutralising antibodies against a strain derived by reverse genetics from a drifted H5N1 isolate (A/Indonesia/5/2005, clade 2).
Adjuvantation conferred significant antigen sparing that could increase the production capacity of pandemic influenza vaccine. Moreover, the cross-clade neutralising antibody responses recorded imply that such a vaccine could be deployed for immunisation before a pandemic.
In preclinical studies, MF59 adjuvant offered improved protection against influenza virus challenge and significantly reduced the viral load in the lungs of challenged mice. In humans, MF59 is a safe and potent vaccine adjuvant that has been licensed in more than 20 countries (Fluad [Novartis Vaccines and Diagnostics Inc., MA, USA]). The safety profile of an MF59-adjuvanted vaccine is well established through a large safety database. MF59 adjuvant has had a significant impact on the immunogenicity of influenza vaccines in the elderly and in adults who are chronically ill. MF59 has also been shown to have a significant impact on the immunogenicity of pandemic influenza vaccines. MF59 allows for broader cross-reactivity against viral strains not included in the vaccine. MF59 has been shown to be more potent for both antibody and T-cell responses than aluminum-based adjuvants. MF59 has broad potential to be used as a safe and effective vaccine adjuvant for a wide range of vaccine types.
Unprecedented spread between birds and mammals of highly pathogenic avian influenza viruses (HPAI) of the H5N1 subtype has resulted in hundreds of human infections with a high fatality rate. This has highlighted the urgent need for the development of H5N1 vaccines that can be produced rapidly and in sufficient quantities. Potential pandemic inactivated vaccines will ideally induce substantial intra-subtypic cross-protection in humans to warrant the option of use, either prior to or just after the start of a pandemic outbreak. In the present study, we evaluated a split H5N1 A/H5N1/Vietnam/1194/04, clade 1 candidate vaccine, adjuvanted with a proprietary oil-in- water emulsion based Adjuvant System proven to be well-tolerated and highly immunogenic in the human (Leroux-Roels et al. (2007) The Lancet 370:580-589), for its ability to induce intra-subtypic cross-protection against clade 2 H5N1/A/Indonesia/5/05 challenge in ferrets.
All ferrets in control groups receiving non-adjuvanted vaccine or adjuvant alone failed to develop specific or cross-reactive neutralizing antibodies and all died or had to be euthanized within four days of virus challenge. Two doses of adjuvanted split H5N1 vaccine containing >or=1.7 microg HA induced neutralizing antibodies in the majority of ferrets to both clade 1 (17/23 (74%) responders) and clade 2 viruses (14/23 (61%) responders), and 96% (22/23) of vaccinees survived the lethal challenge. Furthermore lung virus loads and viral shedding in the upper respiratory tract were reduced in vaccinated animals relative to controls suggesting that vaccination might also confer a reduced risk of viral transmission.
These protection data in a stringent challenge model in association with an excellent clinical profile highlight the potential of this adjuvanted H5N1 candidate vaccine as an effective tool in pandemic preparedness.
Avian influenza A (H5N1) viruses are entrenched among poultry in parts of Asia and Africa and continue to cause disease with high mortality in humans. This update summarizes recent information including research on the transmission and pathogenesis of the infection and on the current strategies for treatment and prevention.
Recent outbreaks of avian influenza in humans have demonstrated the need for vaccines for influenza viruses with pandemic potential. Recombinant hemagglutinins are an attractive option for such vaccines because they do not require handling potentially highly ...
Between December 2003 and January 2004 highly pathogenic avian influenza (HPAI) H5N1 infections of poultry were declared in China, Japan, South Korea, Laos, Thailand, Cambodia, Vietnam, and Indonesia. In 2004 an outbreak was reported in Malaysia. In 2005 H5N1 outbreaks were recorded in poultry in Russia, Kazakhstan, Mongolia, Romania, Turkey, and Ukraine, and virus was isolated from swans in Croatia. In 2004 HPAI H5N1 virus was isolated from smuggled eagles detected at the Brussels Airport and in 2005 imported caged birds held in quarantine in England. In 2006 HPAI was reported in poultry in Iraq, India, Azerbaijan, Pakistan, Myanmar, Afghanistan, and Israel in Asia; Albania, France, and Sweden in Europe; and Nigeria, Cameroon, and Niger in Africa; as well as in wild birds in some 24 countries across Asia and Europe. In 2003, over 25,000,000 birds were slaughtered because of 241 outbreaks of HPAI caused by virus of H7N7 subtype in the Netherlands. The virus spread into Belgium (eight outbreaks) and Germany (one outbreak). HPAI H5N2 virus was responsible for outbreaks in ostriches in South Africa during 2005. HPAI H7N3 virus was isolated in Pakistan in 2004. Low-pathogeniciry avian influenza (LPAI) H5 or H7 viruses were isolated from poultry in Italy (H7N3 2002-2003; H5N2 2005), the Netherlands (H7N3 2002), France (H5N2 2003), Denmark (H5N7 2003), Taiwan (H5N2 2004), and Japan (H5N2 2005). Many isolations of LPAI viruses of other subtypes were reported from domestic and wild birds. Infections with H9N2 subtype viruses have been widespread across Asia during 2002-06.
The rules for T-cell-mediated control of viruses that infect via the respiratory mucosae show both common themes and differences depending on the nature of the pathogen. Virus-specific CD8+ cytotoxic T lymphocytes (CTLs) are the key effectors of virus clearance in mice infected with both negative strand RNA viruses (influenza and Sendai) and a DNA virus, the murine γ-herpesvirus68 (MHV-68). Recently completed experiments establish that these activated CD8+ T cells indeed operate primarily via contact-dependent lysis, Perform-mediated cytotoxicity seems to be the preferred mode, though a Fas-based mechanism can apparently serve as an alternative mechanism. Immune CD4+ T cells functioning in the absence of the CD8+ subset cannot eliminate MHV-68 from lung epithelial cells, are somewhat less efficient than the CD8+ CTLs at clearing the RNA viruses, and are generally ineffectual in mice that lack B lymphocytes. Though cytokine secretion by CD4+ and CD8+ T cells in the virus-infected king may promote both T-cell extravasation and macrophage activation, such processes are not alone sufficient to deal consistently with any d these infections. However, CD4+ T help is mandatory for an effective B-cell response, and can operate lo promote the clonal expansion of virus-specific CD8+ T cells in the lymph nodes and spleen. Furthermore, a concurrent CD4+ T-cell response seems to be essential for maintaining continued CD8+ T-cell surveillance and effector capacity through the persistent, latent phase of MHV-68 infection in B cells. Thus, the evidence to date supports a very traditional view: CD8+ T cells function mainly as killers and the CD4+ T cells as helpers in these respiratory virus infections.
An improved single-radial-diffusion technique for the assay of influenza haemagglutinin antigen is described. The modified method enables the results of assays of antigen to be obtained more rapidly and with greater precision than previously. The use of immunoplates containing varied, pre-selected concentrations of anti-haemagglutinin antibody allows accurate assays to be performed over a wide range of antigen concentrations. Concentrations of haemagglutinin as low as 40 i.u./ml could be assayed with accuracy and reproducibility using immunoplates containing low antibody levels. The method is applicable to the accurate determination of haemagglutinin concentrations over the ranges likely to be present in inactivated influenza vaccines.In tests on ‘whole virus’ antigen preparations, it was found that the ratio between haemagglutination titre (i.u./ml) and haemagglutinin antigen activity (μg/ml) determined by single-radial-diffusion was relatively constant for antigens containing a given strain but showed variation between strains (range 16·5–26·8 i.u./μg HA activity). For the subunit vaccines examined this ratio showed a large degree of variation (range 1·4–16·6 i.u./μg HA activity) and in general was considerably lower than for whole virus antigens.These findings support the conclusion that techniques based on the agglutination of erythrocytes may provide data on vaccine potency which are not directly comparable from strain to strain for ‘whole virus’ vaccines and that these methods are entirely inappropriate to potency assays of split-product or subunit vaccines. In contrast, single-radial-diffusion may be of value for assays of both ‘whole virus’ vaccines and those containing disrupted virions.
The ferret model of influenza A infection was evaluated to determine whether physical signs of influenza illness in addition to fever could be adequately followed. Ferrets were evaluated for nasal and systemic signs of influenza infection in a blinded, randomized protocol. Nasal signs were scored depending on the degree of nasal discharge and congestion. Systemic signs were evaluated on the basis of the activity level of ferrets. Nasal and systemic signs in ferrets challenged with influenza began to rise at peak virus shedding. This rise was coincident with the onset of the nasal inflammatory cell response. Nasal and systemic signs were statistically higher in challenged ferrets as compared with controls from 28 to 52 h after infection [P = 0.002 except at 28 h (P = 0.01)]. Despite precautions against influenza transmission, controls shed influenza virus associated with mild increases in nasal and systemic signs late in the course of the study. Our results suggest that severity of influenza illness can be adequately assessed in the ferret model using collective measurements of nasal and systemic signs, temperatures, and nasal cellular infiltration.
In a study designed to determine whether cytotoxic T lymphocytes contribute to immunity against influenza virus infection, we inoculated 63 volunteers intranasally with live unattenuated influenza A/Munich/1/79 virus. Over the next seven days clinical observations were made, and the amount of virus shed was measured. The protective effects of preinfection serum antibody and of cytotoxic T-cell immunity against influenza A virus were assessed for each participant. All subjects with demonstrable T-cell responses cleared virus effectively. This response was observed in volunteers in all age groups, including those born after 1956, who did not have specific antibody and hence had probably not been exposed to this subtype of influenza A virus before. Cytotoxic T cells show cross-reactivity in their recognition of the different subtypes of influenza A virus, in contrast to the antibody response that is specific for each virus subtype. We conclude that cytotoxic T cells play a part in recovery from influenza virus infection.
Recent outbreaks of avian influenza in humans have demonstrated the need for vaccines for influenza viruses with pandemic potential. Recombinant hemagglutinins are an attractive option for such vaccines because they do not require handling potentially highly pathogenic influenza viruses for vaccine production. In order to evaluate the immunogenicity, optimum dosing and timing of administration of a recombinant baculovirus-expressed H5 HA (rH5) in humans, 147 healthy adults were assigned randomly to receive intramuscular rH5 as two doses of 25, 45 or 90 microg each, one dose of 90 microg followed by a dose of 10 microg, or two doses of placebo, at intervals between doses of 21, 28 or 42 days. All doses of rH5 were well tolerated. The rH5 vaccine was modestly immunogenic at high dose. Neutralizing antibody responses to a titer of 1:80 or greater were seen in 23% (14/60) of individuals after a single dose of 90 microg, and in 52% (15/29) after two doses of 90 microg. Varying intervals between doses from 21 to 42 days had no significant effect on antibody responses to vaccination. These results suggest that baculovirus-expressed H5 HA can induce functional antibody in individuals who have not had prior exposure to H5 viruses, but that further studies to improve the immunogenicity of the vaccine are needed.
In 1997, pathogenic avian influenza A/Hong Kong/97 (H5N1) viruses emerged as a pandemic threat to human beings. A non-pathogenic variant, influenza A/Duck/Singapore/97 (H5N3), was identified as a leading vaccine candidate. We did an observer-blind, phase I, randomised trial in healthy volunteers to assess safety, tolerability, and antigenicity of MF59-adjuvanted and non-adjuvanted vaccines.
32 participants were randomly assigned MF59, and 33 non-adjuvanted vaccine. Two doses were given 3 weeks apart, of 7.5, 15, or 30 microg haemagglutinin surface-antigen influenza A H5N3 vaccine. Antibody responses were measured by haemagglutination inhibition, microneutralisation, and single radial haemolysis (SRH). The primary outcome was geometric mean antibody titre 21 days after vaccination.
The A/Duck/SIngapore vaccines were safe and well tolerated. Antibody response to non-adjuvanted vaccine was poor, the best response occurring after two 30 microgram doses: one, four, four, and one person of eleven seroconverted by haemagglutination inhibition, microneutralisation, H5N3 SRH, and H5N1 SRH, respectively. The geometric mean titres of antibody, and seroconversion rates, were significantly higher after MF59 adjuvanted vaccine. Two 7.5 microg doses of MF59 adjuvanted vaccine gave the highest seroconversion rates: haemagglutination inhibition, six of ten; microneutralisation, eight of ten; H5N3 SRH, ten of ten; H5N1 SRH, nine of ten. Geometric mean titre of antibody to the pathogenic virus, A/Hong Kong/489/97 (H5N1), was about half that to A/Duck/Singapore virus.
Non-adjuvanted A/Duck/Singapore/97 (H5N3) vaccines are poorly immunogenic and doses of 7.5-30 microg haemagglutinin alone are unlikely to give protection from A/Hong Kong/97 (H5N1) virus. Addition of MF59 to A/Duck/Singapore/97 vaccines boost the antibody response to protection levels. Our findings have implications for development and assessment of vaccines for future pandemics.
In 1997, influenza A/Hong Kong/97 (H5N1) emerged as a potential human threat. In 1999, a randomised study comparing two doses of MF59-adjuvanted and non-adjuvanted influenza A/Duck/Singapore/97 (H5N3) surface-antigen vaccine found non-adjuvanted vaccine was poorly immunogenic. Addition of MF59 significantly boosted antibody to H5N1 to levels associated with protection. At 16 months, we undertook a follow-up study to assess the effect of H5N3 revaccination. Geometric mean titres (GMTs) of antibody by haemagglutination-inhibition (HI), microneutralisation (MN) and single radial haemolysis (SRH) indicated that protective antibody titres did not exist at 16 months after two-dose priming. Twenty-one days after revaccination, there was significant boosting of antibody compared to GMTs achieved 21 days after two-dose priming in the original study (P<0.001). MF59 significantly increased GMTs of antibody when compared to non-adjuvanted vaccine (P<0.001).
Antivirals are effective in the prophylaxis and therapy of influenza and are likely to be active against a new pandemic variant. They can be divided into the M2 inhibitors, amantadine and rimantadine, and the neuraminidase inhibitors (NIs), zanamivir and oseltamivir. The former are limited in activity to type A viruses, while the latter are also active against type B viruses. Both classes of drugs are approximately 70-90% efficacious when used as prophylaxis. However, the use of M2 inhibitors in therapy is frequently limited by side effects, more common with amantadine, by the emergence of antiviral resistance and by the lack of demonstrated prevention of complications. In contrast, the NIs are better tolerated, antiviral resistance has not emerged as a significant problem and limited evidence suggests they may reduce the frequency of influenza complications. Antiviral agents have not been widely used for either prophylaxis or treatment of annual influenza epidemics. During the early months of the next pandemic they will be the only specific agents that could be used for prevention and treatment. Their availability will depend entirely on the creation of stockpiles of these agents well in advance of the arrival of the pandemic.
In response to the emergence of severe infection capable of rapid global spread, WHO will issue a pandemic alert. Such alerts are rare; however, on Feb 19, 2003, a pandemic alert was issued in response to human infections caused by an avian H5N1 influenza virus, A/Hong Kong/213/03. H5N1 had been noted once before in human beings in 1997 and killed a third (6/18) of infected people. The 2003 variant seemed to have been transmitted directly from birds to human beings and caused fatal pneumonia in one of two infected individuals. Candidate vaccines were sought, but no avirulent viruses antigenically similar to the pathogen were available, and the isolate killed embryonated chicken eggs. Since traditional strategies of vaccine production were not viable, we sought to produce a candidate reference virus using reverse genetics.
We removed the polybasic aminoacids that are associated with high virulence from the haemagglutinin cleavage site of A/Hong Kong/213/03 using influenza reverse genetics techniques. A reference vaccine virus was then produced on an A/Puerto Rico/8/34 (PR8) backbone on WHO-approved Vero cells. We assessed this reference virus for pathogenicity in in-vivo and in-vitro assays.
A reference vaccine virus was produced in Good Manufacturing Practice (GMP)-grade facilities in less than 4 weeks from the time of virus isolation. This virus proved to be non-pathogenic in chickens and ferrets and was shown to be stable after multiple passages in embryonated chicken eggs.
The ability to produce a candidate reference virus in such a short period of time sets a new standard for rapid response to emerging infectious disease threats and clearly shows the usefulness of reverse genetics for influenza vaccine development. The same technologies and procedures are currently being used to create reference vaccine viruses against the 2004 H5N1 viruses circulating in Asia.
Over the past eight years, cases of human infection with highly pathogenic avian influenza viruses have raised international concern that we could be on the brink of a global influenza pandemic. Many of these human infections have proved fatal and if the viruses had been able to transmit efficiently from person to person, the effects would have been devastating. How can we arm ourselves against this pandemic threat when these viruses are too dangerous to use in conventional vaccine production? Recent technological developments (reverse genetics) have allowed us to manipulate the influenza virus genome so that we can construct safe, high-yielding vaccine strains. However, the transition of reverse-genetic technologies from the research laboratory to the manufacturing environment has presented new challenges for vaccine manufacturers as well as veterinary and public health authorities.
Antigenically well-matched vaccines against highly pathogenic avian influenza H5N1 viruses are urgently required. Human serum
samples after immunization with MF59 or nonadjuvanted A/duck/Singapore/97 (H5N3) vaccine were tested for antibody to 1997–2004
human H5N1 viruses. Antibody responses to 3 doses of nonadjuvanted vaccine were poor and were higher after MF59-adjuvanted
vaccine, with seroconversion rates to A/HongKong/156/97, A/HongKong/213/03, A/Thailand/16/04, and A/Vietnam/1203/04 of 100%
(P<.0001), 100% (P<.0001), 71% (P=.0004), and 43% (P=.0128) in 14 subjects, respectively, compared with 27%, 27%, 0%, and
0% in 11 who received nonadjuvanted vaccine. These findings have implications for the rational design of pandemic vaccines
against influenza H5
Recombinant subunit protein vaccines generally elicit good humoral immune responses, weak helper T cell responses and no cytotoxic T cell responses. Certain adjuvants are known to enhance humoral and cellular immune responses. This study evaluated the humoral, CD4+ T helper and CTL responses induced by the recombinant SL* protein adjuvanted with AS02A in comparison with non-adjuvanted SL* in PBS in two groups of 15 healthy adult volunteers. The AS02A adjuvant contains monophosphoryl lipid A (MPL), QS21 and an oil in water emulsion. The adjuvanted vaccine induced fast and vigorous humoral and helper T cell responses of the Th1 type. Using a pool of overlapping 20mer peptides a cytotoxic response was detected in 6 out of 14 HLA-A2-positive (+) and HLA-A2-negative (-) recipients of the adjuvanted vaccine. All HLA-A2-positive subjects in the adjuvanted group and up to 30% of the subjects in the SL* PBS group displayed a CTL response against selected HLA-A2-restricted CD8+ T cell epitopes. The non-adjuvanted vaccine induced a very weak antibody response and no helper T cell responses. Local and general reactions were more frequently reported by AS02A recipients than in the non-adjuvanted group but the safety profile was considered acceptable. AS02A can be considered as a useful adjuvant that strongly enhances the cellular and humoral responses of subunit protein vaccines.
The threat of pandemic human influenza looms as we survey the ongoing avian influenza pandemic and wonder if and when it will jump species. What are the risks and how can we plan? The nub of the problem lies in the inherent variability of the virus, which makes prediction difficult. However, it is not impossible; mathematical models can help determine and quantify critical parameters and thresholds in the relationships of those parameters, even if the relationships are nonlinear and obscure to simple reasoning. Mathematical models can derive estimates for the levels of drug stockpiles needed to buy time, how and when to modify vaccines, whom to target with vaccines and drugs, and when to enforce quarantine measures. Regardless, the models used for pandemic planning must be tested, and for this we must continue to gather data, not just for exceptional scenarios but also for seasonal influenza.
It is commonly held that increased risk of influenza in the elderly is due to a decline in the Ab response to influenza vaccination. This study prospectively evaluated the relationship between the development of influenza illness, and serum Ab titers and ex vivo cellular immune responses to influenza vaccination in community dwelling older adults including those with congestive heart failure (CHF). Adults age 60 years and older (90 subjects), and 10 healthy young adult controls received the 2003-04 trivalent inactivated influenza vaccine. Laboratory diagnosed influenza (LDI) was documented in 9 of 90 older adults. Pre- and postvaccination Ab titers did not distinguish between subjects who would subsequently develop influenza illness (LDI subjects) and those who would not (non-LDI subjects). In contrast, PBMC restimulated ex vivo with live influenza virus preparations showed statistically significant differences between LDI and non-LDI subjects. The mean IFN-gamma:IL-10 ratio in influenza A/H3N2-stimulated PBMC was 10-fold lower in LDI vs non-LDI subjects. Pre-and postvaccination granzyme B levels were significantly lower in CHF subjects with LDI compared with subjects without LDI. In non-CHF subjects with LDI, granzyme B levels increased to high levels at the time of influenza infection. In conclusion, measures of the ex vivo cellular immune response to influenza are correlated with protection against influenza while serum Ab responses may be limited as a sole measure of vaccine efficacy in older people. Ex vivo measures of the cell-mediated immune response should be incorporated into evaluation of new vaccines for older adults.
Pathogenic avian influenza A virus H5N1 has caused outbreaks in poultry and migratory birds in Asia, Africa, and Europe, and caused disease and death in people. Although person-to-person spread of current H5N1 strains is unlikely, the virus is a potential source of a future influenza pandemic. Our aim was to assess the safety and immunogenicity of a vaccine against the H5N1 strain.
We did a randomised, open-label, non-controlled phase I trial in 300 volunteers aged 18-40 years and assigned one of six inactivated split influenza A/Vietnam/1194/2004 (H5N1) influenza vaccine formulations, comprising 7.5 microg (with adjuvant n=50, without adjuvant n=49), 15 microg (n=50, n=50), or 30 microg (n=51, n=50) of haemagglutinin with or without aluminium hydroxide adjuvant. Individuals received two vaccinations (on days 0 and 21) and provided blood samples (on days 0, 21, and 42) for analysis by haemagglutination inhibition and microneutralisation. We recorded all adverse events. Analyses were descriptive.
All formulations were well tolerated, with no serious adverse events, few severe reactions, and no oral temperatures of more than 38 degrees C. All formulations induced an immune response, and responses were detectable in some individuals after only one dose. The adjuvanted 30 microg formulation induced the greatest response (67% haemagglutinin-inhibition seroconversion rate after two vaccinations). Adjuvant did not improve the response to the lower doses. Two vaccinations of non-adjuvanted 7.5 microg, adjuvanted 15 microg, or non-adjuvanted 15 microg seroconverted more than 40% of participants (haemagglutinin-inhibition test only). Haemagglutinin inhibition and neutralising results were comparable.
A two-dose regimen with an adjuvanted 30 microg inactivated H5N1 vaccine was safe and showed an immune response consistent with European regulatory requirements for licensure of seasonal influenza vaccine. We noted encouraging responses with lower doses of antigen that need further study to ascertain their relevance for the choice of the final pandemic vaccine.
Multiple cases of transmission of avian H5N1 influenza viruses to humans illustrate the urgent need for an efficacious, cross-protective vaccine.
Ferrets were immunized with inactivated whole-virus vaccine produced by reverse genetics with the hemagglutinin (HA) and neuraminidase genes of A/HK/213/03 virus. Ferrets received a single dose of vaccine (7 or 15 microg of HA) with aluminum hydroxide adjuvant or 2 doses (7 microg of HA each) without adjuvant and were challenged with 10(6) 50% egg infectious doses of A/HK/213/03, A/HK/156/97, or A/Vietnam/1203/04 virus.
One or 2 doses of vaccine induced a protective antibody response to the vaccine strain. All immunization regimens completely protected ferrets from challenge with homologous wild-type A/HK/213/03 virus: no clinical signs of infection were observed, virus replication was significantly reduced (P<.05) and was restricted to the upper respiratory tract, and spread of virus to the brain was prevented. Importantly, all vaccinated ferrets were protected against lethal challenge with the highly pathogenic strain A/Vietnam/1203/04. The 2-dose schedule induced higher levels of antibodies that were cross-reactive to antigenically distinct H5N1 viruses.
H5N1 vaccines may stimulate an immune response that is more cross-protective than what might be predicted by in vitro assays and, thus, hold potential for being stockpiled as "initial" pandemic vaccines.
Avian influenza A virus H5N1 has caused widespread infections that have resulted in severe disease or death in poultry and wild birds as well as human beings. This virus has the potential to emerge as a pandemic threat and H5N1 vaccines are being developed in many countries. Our aim was to assess the safety and immunogenicity of an inactivated adjuvanted whole-virion H5N1 vaccine.
A stratified randomised, placebo-controlled, double-blind phase I clinical trial was done in 120 volunteers aged 18-60 years. Volunteers were assigned to receive two doses of placebo (n=24) or an inactivated whole-virion influenza A (H5N1) vaccine with 1.25 microg (24), 2.5 microg (24), 5 microg (24), or 10 microg (24) haemagglutinin per dose with aluminium hydroxide adjuvant on day 0 and 28. Serum samples were obtained on day 0, 14, 28, 42, and 56 for haemagglutination inhibition and virus neutralisation assays. This trial is registered with the ClinicalTrials.gov registry with the number NCT00356798.
All four formulations of vaccines were well tolerated. No serious adverse event was reported and most local and systemic reactions were mild and transient. All formulations induced antibody responses after the first dose; the highest immune response of 78% seropositivity was seen in the 10 mug group after two vaccine doses. Two individuals dropped out: one in the 1.25 microg group (withdrew consent) and one in the 10 microg group (discontinued); one individual was also excluded from the final analysis.
A two-dose regimen of an adjuvanted 10 microg inactivated whole-virion H5N1 vaccine met all European regulatory requirements for annual licensing of seasonal influenza vaccine. Lower doses of this vaccine could achieve immune responses equivalent to those elicited by adjuvanted or non-adjuvanted split-virion vaccines. The use of a whole virion vaccine could be more adaptable to the antigen-sparing strategy recommended by WHO for protection against an influenza pandemic.
M. J. Mulligan, D. I. Bernstein, S. Frey, P. Winokur, N. Rouphael, M. Dickey, S. Edupuganti, P. Spearman, E. Anderson, I. Graham, D. L. Noah, B. Mangal, S. Kim, H. Hill, , J. Whitaker, W. Emery, A. Beck, K. Stephens, B. Hartwell, M. Ogilvie, N. Rimann, E. Osinski, E. Destefano, T. Gajadhar, A. Strudwick, K. Pierce, L. Lai, L. Yue, D. Wang, C. Ying, A. Cline, T. Foltz, N. Wagner, G. Dull, T. Pacatte, B. Taggart, V. Johnson, L. Haller, C. Looney, S. Li, M. May, B. Myers, R. May, L. Parker, N. Cochran, D. Bowen, M. Bell, J. Scoggins, A. Burns, C. Stablein, M. Wolff, B. Jolles, B. Leung, L. Lambert, S. Shorer, W. Buchanan, S. Murray, S. Chang, R. Gorman. (2014) Point-of-Use Mixing of Influenza H5N1 Vaccine and MF59 Adjuvant for Pandemic Vaccination Preparedness: Antibody Responses and Safety. A Phase 1 Clinical Trial. Open Forum Infectious Diseases 1, ofu102-ofu102
CrossRef
The increasing number of reports of direct transmission of avian influenza viruses to humans underscores the need for control strategies to prevent an influenza pandemic. Vaccination is the key strategy to prevent severe illness and death from pandemic influenza. Despite long-term experience with vaccines against human influenza viruses, researchers face several additional challenges in developing human vaccines against avian influenza viruses. In this Review, we discuss the features of avian influenza viruses, the gaps in our understanding of infections caused by these viruses in humans and of the immune response to them that distinguishes them from human influenza viruses, and the current status of vaccine development.
We report the first safety and immunogenicity trial of the Plasmodium falciparum vaccine candidate FMP2.1/AS02A, a recombinant E. coli-expressed protein based upon the apical membrane antigen-1 (AMA-1) of the 3D7 clone formulated with the AS02A adjuvant. We conducted an open-label, staggered-start, dose-escalating Phase I trial in 23 malaria-naïve volunteers who received 8, 20 or 40microg of FMP2.1 in a fixed volume of 0.5mL of AS02A on a 0, 1, and 2 month schedule. Nineteen of 23 volunteers received all three scheduled immunizations. The most frequent solicited local and systemic adverse events associated with immunization were injection site pain (68%) and headache (29%). There were no significant laboratory abnormalities or vaccine-related serious adverse events. All volunteers seroconverted after second immunization as determined by ELISA. Immune sera recognized sporozoites and merozoites by immunofluorescence assay (IFA), and exhibited both growth inhibition and processing inhibition activity against homologous (3D7) asexual stage parasites. Post-immunization, peripheral blood mononuculear cells exhibited FMP2.1-specific lymphoproliferation and IFN-gamma and IL-5 ELISPOT assay responses. This is the first PfAMA-1-based vaccine shown to elicit both potent humoral and cellular immunity in humans. Encouraged by the potential of FMP1/AS02A to target host immunity against PfAMA-1 that is known to be expressed by sporozoite, hepatic and erythrocytic stages, we have initiated field trials of FMP2.1/AS02A in an endemic population in the Republic of Mali.
Between December 2003 and January 2004 highly pathogenic avian influenza (HPAI) H5N1 infections of poultry were declared in China, Japan, South Korea, Laos, Thailand, Cambodia, Vietnam, and Indonesia. In 2004 an outbreak was reported in Malaysia. In 2005 H5N1 outbreaks were recorded in poultry in Russia, Kazakhstan, Mongolia, Romania, Turkey, and Ukraine, and virus was isolated from swans in Croatia. In 2004 HPAI H5N1 virus was isolated from smuggled eagles detected at the Brussels Airport and in 2005 imported caged birds held in quarantine in England. In 2006 HPAI was reported in poultry in Iraq, India, Azerbaijan, Pakistan, Myanmar, Afghanistan, and Israel in Asia; Albania, France, and Sweden in Europe; and Nigeria, Cameroon, and Niger in Africa; as well as in wild birds in some 24 countries across Asia and Europe. In 2003, over 25,000,000 birds were slaughtered because of 241 outbreaks of HPAI caused by virus of H7N7 subtype in the Netherlands. The virus spread into Belgium (eight outbreaks) and Germany (one outbreak). HPAI H5N2 virus was responsible for outbreaks in ostriches in South Africa during 2005. HPAI H7N3 virus was isolated in Pakistan in 2004. Low-pathogenicity avian influenza (LPAI) H5 or H7 viruses were isolated from poultry in Italy (H7N3 2002-2003; H5N2 2005), The Netherlands (H7N3 2002), France (H5N2 2003), Denmark (H5N7 2003), Taiwan (H5N2 2004), and Japan (H5N2 2005). Many isolations of LPAI viruses of other subtypes were reported from domestic and wild birds. Infections with H9N2 subtype viruses have been widespread across Asia during 2002-06.
Influenza is controlled by protective titres of neutralizing antibodies, induced with the help of CD4 T-cells, and by antiviral T-cell effector function. Adjuvants are essential for the efficient vaccination of a naïve population against avian influenza. We evaluated a range of adjuvants for their ability to enhance, in naïve mice, protective hemagglutination inhibition (HI) titres, which represent the generally accepted correlate of protection, virus-neutralizing titres and T-cell responses to a new generation influenza vaccine produced in cell culture. The selected adjuvants include alum, calcium phosphate (CAP), MF59, the delivery system poly-(lactide co-glycolide) (PLG) and the immune potentiator CpG. MF59 was clearly the most potent single adjuvant and induced significantly enhanced, long-lasting HI and neutralizing titres and T-cell responses in comparison to all alternatives. The combination of alum, MF59, CAP or PLG with CpG generally induced slightly more potent titres. The addition of CpG to MF59 also induced a more potent Th1 cellular immune response, represented by higher IgG2a titres and the induction of a strongly enhanced IFN-gamma response in splenocytes from immunized mice. These observations have significant implications for the development of new and improved flu vaccines against pandemic and inter-pandemic influenza virus strains.
Influenza A H5N1 viruses pose a significant threat to human health.
We conducted a multicenter, randomized, double-blind study in 394 healthy adults. Subjects were randomly assigned to receive 2 intramuscular doses of either saline placebo; influenza A/Vietnam/1203/2004(H5N1) vaccine alone at 45, 30, or 15 microg per dose; vaccine at 15 or 7.5 microg per dose with MF59; or vaccine at 30, 15, or 7.5 microg per dose with aluminum hydroxide. Subjects were followed up for safety and blood samples were obtained to determine antibody responses.
The vaccine formulations were well tolerated but local adverse effects were common; the incidence of these effects increased in a dose-dependent manner and was increased by the addition of adjuvants. The addition of MF59 increased the antibody response, whereas the addition of aluminum hydroxide did not. The highest antibody responses were seen in the group that received 15 microg of vaccine per dose with MF59, in which 63% of subjects achieved the predetermined endpoint (hemagglutination-inhibition titer > or =40) 28 days after the second dose, compared with 29% in the group that received the highest dose (45 microg per dose) of vaccine alone.
A 2-dose regimen of subvirion influenza A (H5N1) vaccine was well tolerated. The antibody responses to 15 microg of A/H5 vaccine with MF59 were higher than the responses to 45 microg of vaccine alone.
ClincalTrials.gov identifier: http://www.clinicaltrials.gov/ct2/show/NCT00280033?term= NCT00280033&rank=1 NCT00280033 .
MF59 is a safe and potent vaccine adjuvant that enhances protection against influenza virus infection
- O Hagan
- Dt
O'Hagan DT. MF59 is a safe and potent vaccine adjuvant that enhances protection against influenza virus infection. Expert Review of Vaccines 2007;6(October (5)):699–710.
MF59 adjuvant emulsion
- A Podda
- G D Giudice
- M M Levine
- J B Kaper
- R Rappuoli
- M A Liu
- M F Good
Podda A, Giudice GD. MF59 adjuvant emulsion. In: Levine MM, Kaper JB, Rap-puoli R, Liu MA, Good MF, editors. New generation vaccines, third ed. New York: Marcel Dekker, Inc.; 2004. p. 225–35.
Update on avian influenza A (H5N1) virus infection in humans
- Abdel-Ghafar An
- T Chotpitayasunondh
- Hayden Z Fg Gao
- Dh Nguyen
- De Jong
- Md
Abdel-Ghafar AN, Chotpitayasunondh T, Gao Z, Hayden FG, Nguyen DH, de Jong MD, et al. Update on avian influenza A (H5N1) virus infection in humans. The New England Journal of Medicine 2008;358(January (3)):261–73.
Summary of avian influenza activity in Europe
- Dj Alexander
- Asia
- Australasia Africa
Alexander DJ. Summary of avian influenza activity in Europe, Asia, Africa, and Australasia, 2002–
2006. Avian diseases 2007 Mar;51(1 Suppl):161–6. [PubMed: 17494548]