Introduction
Influenza virus infections are a major cause of acute seasonal respiratory illness, which causes significant morbidity and mortality. Vaccination is an effective way of reducing the numbers of infected individuals provided there is a good match between the vaccine and the circulating strains. Every February in the Northern Hemisphere the circulating strains for the next season are chosen by the World Health Organization, which allows approximately 6 months for sufficient vaccine to be generated. Annual influenza vaccines need to provide protection against the predominant influenza strains that are present in each season’s outbreaks. Since 1977, A/H1N1, A/H3N2, and B viruses are the major virus types that are in circulation. Choosing only one B strain was not always accurate as indicated during the period of 2001–2010 in the USA when the trivalent influenza strain only partially matched the circulating B strain 50% of the time [
1]. Licensed vaccines today use three (two A and one B) or four (two A and two B) strains that are predicted to be circulating in the population. The additional B strain in the quadrivalent vaccine is due to the fact that there are two B lineages (B/Yamagata and B/Victoria) currently in co-circulation and there is limited cross protection between strains from each lineage [
2]. In a study using UK data, it was estimated that the inclusion of an additional B strain to generate a quadrivalent vaccine could be expected to reduce the number of influenza cases by 17,088 and deaths by 168 in the first year of use [
3]. Although adding four strains may reduce the possibility of B strain mismatch, strain mismatch can still occur as was the case for the 2014/15 Northern Hemisphere influenza season, where the predominant H3N2 strain drifted [
34], with the result that the vaccine was only partially protective (vaccine effectiveness of only 23%) [
35]. While four strains may be better than one, strain selection is by no means perfect.
An additional issue that arises with multivalent vaccines is the possibility of immunological interference effects between the individual vaccine components. Interference can result in a diminished or an enhanced response to one component in the presence of another. This has been documented for live attenuated influenza vaccines [
4], and where it occurs, dosing may have to be modified to counteract this interference [
5]. As new quadrivalent vaccines are developed, interference effects need to be studied for each new vaccine.
We have been developing an oral-based adenovirus platform for vaccine delivery. The vaccine platform consists of a replication-incompetent adenovirus vector bearing two expression cassettes that express a vaccine antigen and a double-stranded RNA (dsRNA) adjuvant [
6]. The vaccine antigen that we have chosen for the influenza vaccine is influenza hemagglutinin (HA), an envelope glycoprotein involved in receptor binding and viral fusion. Antibodies that block the ability of HA to bind to target cells, as measured by the HA inhibition (HAI) titer, have been found to correlate with protection [
7]. This oral vaccine approach has a number of advantages over traditional influenza vaccines. These include ease of manufacturing, using standard recombinant techniques and established viral purification methods; ease of delivery without needles or other devices that need qualified medical support; long-term stability at room temperature negating the need for cold storage. This oral platform also generates minimal anti-vector immunity thus permitting revaccination and reuse [
6]. We have generated HA-based recombinant adenovirus (rAd) vaccine vectors to individual influenza strains and demonstrated either protection in small animals [
6] or immunogenicity in humans [
8,
9]. Results from these studies demonstrate the ability of the approach to generate neutralizing antibody responses to influenza, with additional advantages such as antibody durability and T cell responses shown in the studies as well. Given the commercial need for a seasonal vaccine that covers more than one strain, we have explored the utility of our platform to vaccinate against multiple influenza strains simultaneously and have evaluated whether significant interference exists between the vaccine antigens. Here, we model oral tablet delivery by measuring the ability of ferrets to mount immune responses to multiple strains following intestinal delivery to a single site and establish a potential strategy for eventual vaccine commercialization.
Discussion
Seasonal influenza vaccines have advanced to provide greater protection against multiple flu strains. The original influenza vaccines were monovalent vaccines against single influenza subtypes [
16]; however, due to co-circulation of multiple strains and the unpredictable nature of which strain might become the dominant strain in an influenza season, influenza vaccines have become more complex to account for these risks. This evolution led to the development and approval of quadrivalent influenza vaccines in the last four years (Fluarix
® Quadrivalent, GlaxoSmithKline Biologicals; FluLaval
® Quadrivalent, ID Biomedical Corp; Fluzone
® Quadrivalent, Sanofi Pasteur Inc.); and FluMist
® Quadrivalent, Medimmune, LLC) [
36]. This development has also been fueled by improvements in the capacity of production, which can accommodate these additional components. Additional improvements in technology have led to further advances in influenza vaccines. In 2012, the first cell culture-derived influenza vaccine, Flucelvax
® (Novartis Vaccines & Diagnostics, Inc.), rather than egg-derived vaccine, was approved, which can bypass potential limitations on egg availability. A number of novel subunit approaches based on injectable HAs have or are in the process of being commercialized and include the use of baculovirus-derived HA, Flublock
® (Protein Sciences Corporation), baculovirus-derived influenza vaccine-like particles [
17], or the use of bacterial expressed flagellin-HA chimeras as injectable vaccines [
18]. Similarly, our approach is a departure from traditional egg-derived influenza vaccines, using cell culture-derived adenovirus as a vector. Instead of injecting a subunit of HA, our approach delivers a vector that co-expresses influenza HA and a dsRNA adjuvant to improve immune recognition of the HA [
6]. We have delivered vaccine to humans in tablet or in liquid form, with the tablet showing slightly better HAI responses (Liebowitz et al. [
8] and Kim et al., unpublished data). The current study demonstrates that this platform can be used to generate multivalent adenovirus vaccines, and is achieved by blending multiple monovalent vaccine vectors. This study is unique as it is the first published study using adenoviruses as vaccine vectors for a quadrivalent influenza vaccine and using an oral model of delivery.
This study primarily demonstrates that a blended quadrivalent vaccine is effective at inducing antibody responses with minimal interference to four influenza HA antigens (H1, H3, B/Victoria, and B/Wisconsin) after endoscopic delivery in ferrets. To model a quadrivalent tablet vaccine, all four monovalent vaccines were mixed and delivered to the same location in the ferret intestine by endoscope. In this study, the quadrivalent vaccine induced 90–100% responders by ELISA, which was comparable to the percent responders induced for the monovalent vectors (70–100%) and 30–70% responders with a 4× rise in HAI for the quad vaccines (70–100%) responders with a 2× rise. This immunogenicity compares well to the immunogenicity of commercial vaccines in humans [
37]. We expect the vaccine would provide protection even in the absence of HAI responses as we previously demonstrated that protection against H5N1 using this platform correlated with total antibody and not HAI [
6]. The rAd doses tested in ferrets in this study were relatively low (1–10%) compared to human doses where we have seen substantial immune responses in clinical trials [
8,
9] and compare well with the immune response generated to a full human dose of TIV that we evaluated concurrently. The HA protein made from a gene-based vaccine, such as ours, cannot be measured because it is made inside each subject or animal, so the best way to compare is in equivalent human doses.
Of interest was the fact that the TIV vaccine at a full human dose, by the recommended route of delivery, only induced HAI responses to B/Brisbane, and no HAI responses to A/H1N1 or A/H3N2. A published study by Skowronski et al. [
19] has also noted that TIV vaccines are poor HAI inducers in influenza-naive ferrets. Pearce et al. [
20] found in ferrets that the best HAI response in the TIV was to B/Brisbane/60/08, (Titers 20–80) after a single immunization and generated a low HAI response to the other seasonal components after two immunizations. In contrast, our vaccine induced HAI responses to all antigens and a similar response to the B/Brisbane/60/08 antigen as TIV. A major question is what accounts for the difference in HAI observed in ferrets between our vaccine and the tested TIV. As the ferrets used in these studies are screened and selected for animals that have negative HAI titers to major influenza strains, influenza vaccine studies in ferrets are essentially priming studies. Given that the results obtained with 1:100 (1 × 10
9 IU) of a rAd human dose (1 × 10
11 IU) were equal or better than a full TIV dose in inducing HAI responses, it is possible that rAd makes for a superior priming vaccine in naïve individuals. One possible explanation for the priming difference is that a vector-based approach elicits a superior T cell response (reviewed in [
21,
22]), and that this can improve HAI titers in the absence of pre-exposure to the matched influenza strain. Several investigators have demonstrated the ability of adenovirus to induce strong T cell responses in animals and humans [
9,
23]. Observations for the role of T cell help in improved vaccine responses have been made for H5 and H1 pandemic strains in humans, where enhanced CD4 T cell responses to HA promoted higher HAI titers [
24,
25]. One possibility is that the effector T cell help induced at each immunization provides for higher antibody responses at each time point so the boost is much more effective. The other possibility is that memory CD4 T cell responses induced after the first immunization allows for better HAI responses upon the second immunization. T cell responses in ferrets are not easy to measure, so careful measurements of T cell responses and HAI post-immunization will need to be performed in HAI-naïve humans to demonstrate priming potential.
The approach we have taken of co-delivering a blend of four vaccine vectors to a specific location in the ferret intestine proved effective at eliciting immune responses, but the use of rAd in a tablet format allows for alternative approaches. For delivery to humans we could take a similar approach to the ferret study by blending all four strains together in a single tablet. Alternatively, a quadrivalent vaccine could be administered as four tablets with each tablet delivering a vaccine for one of the targeted quadrivalent strains. The advantage of this single strain per tablet approach is that if late season changes were needed on one strain, the other three strain tablets would not be impacted. Each strain would be individually released and kept, and all four vaccines could be handed out at once or given as they were made. It would be difficult to take the same approach with an injected vaccine because taking four separate shots for influenza would be difficult to sell, and if all strains were blended together, all components would need to be discarded after a single strain change. An additional advantage of the rAd-based approach is that multiple antigens could fit on one vector. This was demonstrated for a tetravalent adenovirus dengue vaccine generated by a combination of blending and expressing multiple antigens on the same adenovirus backbone, and provided significant protection to four dengue serotypes in rhesus monkeys [
26]. We have explored a similar approach and have expressed multiple genes including two HA genes on single Ad vectors using the foot and mouth 2A linker sequence, which permits the expression of multiple transcripts from a single ORF ([
27] and data not shown). This approach has the advantage of reducing the production requirements of the individual vector components.
Pre-existing immunity to adenovirus, or to any vector, can be problematic when using that vector to deliver and express a vaccine antigen. In studies by Ledgerwood et al. [
28], an adenovirus used to express an Ebola virus antigen was not as potent in humans with pre-existing neutralizing antibody responses to the adenovirus as in humans without neutralizing antibody responses. One method to potentially circumvent this problem is to choose a vector with low seroprevalence in humans such as simian adenoviruses or engineered chimeric adenoviruses [
29,
30]. There are problems with this novel vector approach in that not all vectors are equally immunogenic as adenovirus type 5, and once the vaccine vector is used in a human, that vector will not be useful for a new indication or a boost. Injected adenoviruses elicit a very strong anti-vector response that can block subsequent uses or administrations. The situation would get extremely complicated if adenoviral vectors were in wide-scale human use, and vaccine developers were forced to screen people for specific antibody titers, before deciding whether the vaccine could be given. A different alternative is to deliver adenoviruses orally, which appears to circumvent neutralizing antibody responses and selectively elicit anti-transgene immune responses (not anti-vector antibody responses) [
6,
31]. In our human tablet study, pre-existing immunity to adenovirus 5 had no effect on the ability to elicit HAI responses; the subject with the highest HAI response to the vaccine also had the highest pre-existing anti-adenovirus neutralizing antibody response [
8].
Future human trials will likely follow a similar path as our ferret studies, first testing A and B monovalent vaccines and then testing combinations to evaluate efficacy and possible interference effects. Multivalent approaches using either DNA or viral vectors such as Ad are not new. A DNA vaccination approach in mice using blends of HA expressing plasmids against multiple H3 strains increased the breath of protection to multiple strains compared to monovalent vaccines alone [
32]. Blends of adenovirus vaccine vectors expressing HIV antigens have previously been tested in animals without signs of immunological interference between vaccine antigens [
33]. Given our success of this oral platform in humans, plus the encouraging response described here in ferrets, it is our intention to evaluate such blends in future human trials. Only through clinical trials can it be determined whether such multivalent vaccines will prove efficacious as vaccines.