Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2018 Mar 7.
Published in final edited form as: Vaccine. 2017 Feb 8;35(10):1455–1463. doi: 10.1016/j.vaccine.2017.01.026

Evaluation of Multivalent H2 Influenza Pandemic Vaccines in Mice

Brian J Lenny a,b,#, Stephanie Sonnberg a,*,#, Angela F Danner a, Kimberly Friedman a, Richard J Webby a, Robert G Webster a, Jeremy C Jones a,#
PMCID: PMC5336516  NIHMSID: NIHMS847399  PMID: 28189402

Abstract

Subtype H2 Influenza A viruses were the cause of a severe pandemic in the winter of 1957. However, this subtype no longer circulates in humans and is no longer included in seasonal vaccines. As a result, individuals under 50 years of age are immunologically naïve. H2 viruses persist in aquatic birds, which were a contributing source for the 1957 pandemic, and have also been isolated from swine. Reintroduction of the H2 via zoonotic transmission has been identified as a pandemic risk, so pre-pandemic planning should include preparation and testing of vaccine candidates against this subtype. We evaluated the immunogenicity of two inactivated, whole virus influenza vaccines (IVV) in mice: a monovalent IVV containing human pandemic virus A/Singapore/1/1957 (H2N2), and a multivalent IVV containing human A/Singapore/1/1957, avian A/Duck/HongKong/319/1978 (H2N2), and swine A/Swine/Missouri/2124514/2006 (H2N3) viruses. While both vaccines induced protective immunity compared to naïve animals, the multivalent formulation was advantageous over the monovalent in terms of level and breadth of serological responses, neutralization of infectious virus, and reduction of clinical disease and respiratory tissue replication in mice. Therefore, multivalent pandemic H2 vaccines containing diverse viruses from animal reservoirs, are a potential option to improve the immune responses in a pre-pandemic scenario where antigenic identity cannot be predicted.

Keywords: Influenza, Vaccine, Pandemic, H2N2, Monovalent, Multivalent

1. INTRODUCTION

Influenza A viruses are a significant disease burden to humans and animals [1]. Vaccination is the most effective tool for prevention [2-4], but reassortment events and mutations in the influenza antigenic surface proteins necessitate frequent reevaluation of vaccine components [2, 5]. Occasionally, a novel influenza virus arises from animal reservoirs for which current vaccines are also ineffective [6, 7]. This may result in pandemic spread, with significant morbidity and mortality worldwide [6].

Previous pandemics were caused by H1N1 and H3N2 subtype viruses that later established stable lineages in humans [6] and have been included in seasonal vaccines [2, 4]. A third subtype, H2N2, caused a pandemic in 1957 that resulted in an estimated 1-4 million deaths [8]. H2N2 no longer circulates in humans, and current vaccines do not contain H2N2 antigen [9]. Thus, individuals under ≈50 years of age are immunologically naïve and would likely be unprotected should it return [10]. Though it has disappeared from humans, H2 viruses are found in aquatic and domestic birds [11-14], and pigs [15]. These zoonotic isolates are capable of replicating in mammals and causing severe disease [11, 16]. The 1957 H2N2 pandemic virus was likely introduced to humans from birds [17, 18]; therefore, reemergence of H2 from animals should be considered a significant threat to human health.

An important component of H2 pre-pandemic planning is identification and testing of vaccine virus candidates [19-21]. This is complicated by the inability to accurately predict the antigenic identity of a pandemic virus and carries the inherent risk of a mismatch between stockpiled vaccines and emergent strains.

Broadening the immune coverage and response of a putative H2 vaccine is an important goal to avoid mismatch; this can be accomplished by increasing the dosage, adding adjuvants, or using alternate formulations like live-attenuated influenza vaccines (LAIV). In animals, LAIV vaccines induce broad H2 cross-reactive immune responses [22, 23], but clinical implementation is less effective due to poor replication of the vaccine virus in humans [24] and nonhuman primates [25]. Further LAIV drawbacks include reassortment potential between the vaccine and pandemic virus [20], increased risk of clinical side effects in first time users [26], and a lower safety profile [27] that prohibits use in the immunocompromised or individuals outside the ages of 2-49 [28, 29].

The alternative to LAIV are inactivated virus vaccines (IVV) which are more widely produced and distributed [5, 30-32]. Previous H2N2 IVV vaccine studies in mice have focused on survival from lethal, mouse-adapted virus challenge and less on immune correlates of protection [33, 34]. Human clinical trials using whole-virion IVV H2 vaccines induced protective antibody titers, but were dependent on multiple doses and adjuvants [35, 36]. In these published studies, the IVVs have been monovalent (single H2 antigen) formulations. One method to improve upon IVV immune coverage is to include multiple, divergent viruses including those from animal reservoirs and represents an additional approach to avoid antigenic mismatch in a pandemic scenario.

To test the hypothesis that a multivalent H2 vaccine provides enhanced protection and cross-reactive immune responses compared to monovalent vaccine, we formulated two whole-virion IVV H2N2/3 preparations: 1) a monovalent vaccine containing putative human influenza virus seed strain A/Singapore/1/1957 [Singapore/1957, H2N2, [35-38]], and 2) a multivalent vaccine containing Singapore/1957 with an avian (A/Duck/HongKong/319/1978, H2N2) and swine virus (A/Swine/Missouri/2124514/2006, H2N3). The vaccines were tested in mice for the induction of homologous and heterologous humoral and cell mediated immunity, protection against virus challenge, and the influence of single or multiple dose regimens.

2. MATERIALS & METHODS

2.1. Ethics statement

Animal experiments were approved by the St. Jude Children's Research Hospital Animal Care and Use Committee in compliance with the National Institutes of Health and the Animal Welfare Act.

2.2. Laboratory facilities

Experiments involving human H2N2 viruses or animal challenges were performed in ABSL3+ containment (USDA 9 CFR 121; 7 CFR 331.)

2.3.

Viruses and cells Viruses were obtained from the St. Jude children's Research Hospital Repository (H2NX, H1N1, H3N2 subtypes) or from the World Health Organization Global Influenza Program (reverse genetics H5N1 subtype), propagated in embryonated chicken eggs, and titered by method of Reed and Muench [39] in Madin-Darby canine kidney (MDCKs, ATCC, Manassas, VA).

2.4. Vaccine formulation

Egg propagated virus was 0.1% formalin treated (4°C, 5 days) [40, 41], concentrated over a Pellicon Ultrafiltration Unit (Millipore, Billerica, MA) and sucrose purified. Dosages (1μg HA content) were standardized by densitometry (Image J, NIH) quantitation of HA to total protein ratios after SDS-PAGE and as described previously [41].

2.5. Animals, vaccination, challenge

6 wk old female BALB/c mice (Charles Rivers, Wilmington, MA) (n=30/vaccine group; n=10/challenge group) were bled for sera before vaccination (prime) and 1 day before challenge. Mice were administered 50μl of vaccine, intramuscularly into the hind limb with or without a boost 15 days later. 31 days post-prime, animals were anesthetized with isoflurane, inoculated with 105.5 EID50 units of challenge virus, and monitored for morbidity (weight loss). 3 days post-virus inoculation (dpi), mice (n=3) were sacrificed for tissues that were homogenized and titered in MDCKs.

2.6. Virus specific (vs) IgG Serum IgG titers were determined by enzyme-linked immunosorbent assay (ELISA) plates coated with each individual vaccine-virus antigen (10μg)

[42-47] plates (Nunc Maxisorp) blocked with 4% bovine serum albumin (BSA)/ 0.1% tween-20 in PBS (PBST). Serum doubling dilutions (beginning 1:200) were prepared in 1% BSA/PBST, incubated in the coated plates, followed by extensive washing and incubation with anti-mouse HRP IgG (1:5000, Jackson Labs, West Grove, PA). Antigen specific IgG titers were quantified by addition of tetramethylbenzidine (TMB, R&D systems, Minneapolis, MN). Reactions were stopped with 2N H2SO4 and absorbance values (A450) were measured by Synergy 2 Reader (Biotek, Winooski, VT). The reciprocal of the last dilution with A450 >2-fold above background (virus coated wells, no sera) was recorded as the (vs)IgG titer.

2.7. Hemagglutination inhibition titers (HAI) HAI assays [48] were performed with RDE (Denka-Seiken, Tokyo) treated sera (beginning 1:20) and 1% horse or 0.5% chicken erythrocytes

The reciprocal of the last dilution that inhibited hemagglutination was recorded as the HAI titer.

2.8. Neuraminidase inhibition assay (NAI)

NA antibodies in sera (doubling dilutions beginning 1:10) were measured by enzyme-linked lectin assay (ELLA) as described previously [49]. The reciprocal of the last dilution with absorbance values >2-fold below maximal NA activity (virus coated wells, no sera) was recorded as the NAI titer.

2.9.

Micronuetralization Assay (MN) MN titers were determined as described previously [50] using serum dilutions beginning 1:10, 100 TCID50 units of each virus, and detection with erythrocytes as in the HAI assay.

2.10. IFN-γ ELISPOT

10 days post-prime, spleens were taken from mice (n=5/group) and single cell populations were prepared in R10 medium (RPMI 1640, 10% FBS, 1 mM sodium pyruvate, penicillin/streptomycin). Erythrocytes were lysed with AKC buffer (0.15 M NH4Cl, 1.0 mM KHCO3, 0.1 mM EDTA). Splenocytes (1×10 per well) were added to ELISpot plates (Mabtech, Cincinnati, OH) and stimulated for 24 hr with R10 medium alone, or vaccine antigen (0.5μg). IFN-γ secreting splenocytes were counted on a dissecting scope. Concanavlin A (10μg/ml) stimulation as a positive control yielded >200 spots (data not shown).

2.11. Pathology

Lung from Swine/MO/2006 inoculated animals (n=3/group, 3dpi) was sectioned and influenza nucleoprotein (NP) was detected by immunohistochemistry (Veterinary Pathology, St. Jude Children's Hospital). Strong, diffuse NP staining was quantified by Aperio Pathology Software (Leica Biosystems, Buffalo Grove, IL).

2.12. Statistics

Experiments are representative of n=30 animals per vaccine group with matched naïve controls (n=30) for each regimen. The results represent the means and where indicated, ± standard deviations of at least triplicate determinations. Statistical significance was determined by 1-way ANOVA with Tukey's post-test using Prism 6 software (GraphPad Software Inc., La Jolla, CA) or one-tailed student's t-test (Microsoft Excel 2013, Redmond, WA).

3. RESULTS

3.1. Virus selection, formulation, and vaccine regimen

Monovalent and multivalent H2 vaccines were prepared from formalin-inactivated viruses of human, avian and swine origin [11]. Singapore/1957 (H2N2) is a prototype vaccine seed candidate [35-38] and 1957 pandemic isolate. Swine/MO/2006 (H2N3) is a contemporary mammalian H2 [15] virus that replicates and transmits in animal models [11, 15], has a heterologous N3 protein. Duck/HK/1978 (H2N2) was also identified as high-risk for replication and transmission in mammals [11]. The monovalent vaccine contained Singapore/1957 and the while the multivalent contained equal parts all three viruses. BALB/c mice were administered vaccine or PBS (naïve, negative control) intramuscularly in single dose (prime) or two doses (prime and boost) two weeks later. Unless specified, measurement of immune correlates was performed pre-challenge (31 days post-prime).

3.2. Hemagglutination inhibition (HAI) titers

The HAI assay is a standard test of vaccine efficacy used by several national health agencies [51, 52]. An titer of 1:40 is considered protective in humans [53], though this relationship is less well understood in animal models. Nevertheless, mice serve an important role and are widely used for pre-clinical influenza vaccine evaluation [54].

All formulations and regimens induced higher HAI against each antigen compared to naïve animals (p≤0.0001, Fig. 1A, B). No difference between monovalent and multivalent HAI titers were observed with swine or human antigens. The multivalent vaccine was superior to the monovalent against the avian antigen, inducing ≥2 fold higher HAI titers (p≤0.001, Fig. 1A, B). A boost failed to improve low Swine/MO/2006 HAI titers, but did increase HAI geometric mean titer (GMT) for the human and avian antigens in most cases (p≤0.05, Supplemental Table 2).

Figure 1. Cross-reactive, Humoral Responses to Monovalent and Multivalent H2 Vaccines.

Figure 1

Open in a new tab

Mice were vaccinated with one dose (A, C, E) or two doses (B, D, F) of monovalent or multivalent H2 vaccine as indicated. Sera (31 days post-prime) was evaluated in the A, B) HAI assay (n=15 mice/group), C, D) vsIgG ELISA (n=30 mice/group) and E, F) NAI assay (n=14 mice/group) against the indicated test antigens. Dotted lines indicate the assay limit of detection. Statistical significance was determined by the Log2 conversion and comparison of the GMTs. Samples below the assay cutoffs (HAI 1:20, NAI 1:10, vsIgG 1:200 serum dilutions) were assigned a value of 1 for calculations. *p≤0.05, **p≤0.01, p≤0.001, ****p≤0.0001, ns = not significant

3.3. Virus specific (vs) serum IgG

Both vaccine formulations induced serum vsIgG that cross-reacted with all test antigens better than naïve animals (p≤0.001, Fig. 1C, D). A single dose of multivalent vaccine induced 1-4 fold higher GMT (Log2) vsIgG for all antigens compared to the monovalent (p≤0.05, Fig. 1C); this included higher titers to human antigen that is present in both formulations. All animals receiving multivalent vaccine had swine vsIgG above background, compared to <50% with monovalent vaccine. In boosted groups, multivalent vaccine induced higher GMT vsIgG for swine and avian antigens (p≤0.0001) compared to monovalent (Fig. 1D) and significantly improved overall vsIgG titers when comparing individual antigens: swine (p≤0.0001), human (p≤0.05), and avian (p≤0.05, Supplemental Table 2).

3.4. Neuraminidase inhibition (NAI) titers

Significant anti-N2 neuraminidase (NA) titers were induced by all vaccines compared to controls. The multivalent formulation induced higher titers than the monovalent in a prime only scenario (p≤0.05, Fig. 1E), though this was not observed in the prime/boost regimen (Fig. 1F). The single dose, monovalent vaccine induced N2 antibodies in half of the animals, compared to 86% with multivalent (Fig. 1E), though no difference was observed when animals were boosted. The monovalent vaccine failed to produce detectable N3 NAI titers, but the multivalent formulation produced NAI titers in 64%-93% of mice depending on regimen (Fig. 1E, F).

3.5.

Neutralizing Titers With the exception of single dose, monovalent, all vaccines induced higher MN titers than naïve animals (p≤0.05, Fig. 2). In most cases, the multivalent formulation provided higher neutralizing titers against all virus antigens, and only the multivalent vaccine induced avian MN in the prime only regimen.

Figure 2.

Figure 2

Open in a new tab

Cross-reactive, Virus Neutralizing Responses to Monovalent and Multivalent H2 Vaccines. Mice were vaccinated with one dose (A) or two doses (B) of monovalent or multivalent H2 vaccine as indicated. Sera (31 days post-prime) was evaluated in microneutralization assay (n=15 mice/group) against the indicated test antigens. Dotted lines indicate the assay limit of detection. Statistical significance was determined by the Log2 conversion and comparison of the GMTs. Samples below the assay cutoff (1:10 serum dilution) were assigned a value of 1 for calculations. *p≤0.05, **p≤0.01, p≤0.001, ****p≤0.0001, ns = not significant

3.6. Heterologous humoral immunity

The breadth of vaccine HAI and MN titers were determined against heterologous antigens (phylogenetic relatedness discussed in detail in [17]) using pooled mouse (n=10) sera and H1, H2, H3, and H5 virus subtypes. Naïve mice showed low or no reactions, while titers were detected against most H2 antigens in vaccinated animals. Against avian H2 viruses, a single dose of multivalent vaccine induced a 2.8 fold (Log2) higher GMT HAI titer (p≤0.05) and an 18.0 fold (Log2) higher GMT MN titer (p≤0.0001) compared to monovalent. Qualitative, but not statistically significant differences were noted with human viruses, with a trend towards higher reactivity with multivalent vaccinate. Multivalent vaccine also produced HAI and MN antibodies reactive against all avian viruses, whereas monovalent failed to produce H2N3 HAI reactive antibodies, and generally required a boost to produce MN antibodies. While boosted animal sera of both vaccine regimens reacted to all avian viruses tested, the multivalent formulation produced modestly higher cumulative titers [HAI, 1.2 fold (Log2), p≤0.001; MN, 1.7 fold (Log2), p≤0.001]. No vaccine formulation or regimen induced HAI or MN titers against clinically relevant, non-H2 HA group 1 (H1N1, H5N1) or group 2 (H3N2) viruses (Table 1, 2).

Table 1.

Cross-reactive Serum HAI Titers Against Heterologous Influenza A Viruses

Virus Subtype/Lineage Host Species HI Titer and Vaccine Formulationa
Naïve Monovalent Prime Only Monovalent Prime & Boost Trivalent Prime Only Trivalent Prime and Boost
A/Japan/305/1957 H2N2 (pandemic) Human < < 20 40 80
A/AnnArbor/23/1957 H2N2 (pandemic) Human < 20 40 40 80
A/Netherlands/84/1968 H2N2 (seasonal) Human < < < < <
A/NorthCarolina/1/68 H2N2 (seasonal) Human < 20 40 20 40
Human H2 GMT (Log2)b 0.0 1.1 2.3 2.3 2.8
A/Mallard/MT/Y61 H2N2 Avian < 20 40 40 160
A/Mallard/Potsdam/179/1983 H2N2 Avian < 20 40 20 80
A/Chicken/Jena/4836/1983 H2N2 Avian 20 40 80 40 160
A/Mallard/NewYork/6750/1978 H2N2 Avian < 20 40 40 80
A/Chicken/Connecticut/13657/1990 H2N2 Avian 20 20 80 40 80
A/GuineaFowl/NewYork/13824/1995 H2N2 Avian < 20 40 20 80
A/Chicken/Ohio/494832/2007 H2N3 Avian < < 40 20 80
A/Widgeon/Denmark/66174/G18/2004 H2N3 Avian < < 40 20 80
A/Mallard/Alberta/35/2009 H2N3 Avian < < 20 20 80
Avian H2 GMT (Log2)b 0.4 1.7 5.4 4.7 6.5
A/Brisbane/10/2007c H1N1 (pre-pandemic) Human < < < < <
A/California/04/2009c H1N1 (pandemic) Human < < < < <
A/Perth/16/2009c H3N2 (seasonal) Human < < < < <
rgA/Egypt/N03072/2010c, d H5N1 (clade 2.2.1) Human < < < < <
rgA/Guizhou/1/2013c, d H5N1 (clade 2.3.4.2) Human < < < < <
Open in a new tab

< Below the assay limit of detection (1:20 serum dilution), GMT - Geometric Mean Titer

a

Individual HI values are expressed as the reciprocal of the highest dilution that inhibited 4 agglutinating units of the indicated test virus with horse erythrocytes unless otherwise specified.

b

GMT was determined by assigning 1 (Log2) values for samples below the assay limit of detection and subtracting 1 from the final GMT

c

HAI assays were performed with chicken erythrocytes

d

Represents reverse genetics (rg) vaccine viruses containing HA and N A surface proteins on A/Puerto/Rico/8/1934 virus backbone

Table 2.

Cross-reactive Serum Microneutralization Titers Against Heterologous Influenza A Viruses

Virus Subtype/Lineage Host Species MN Titer and Vaccine Formulationa
Naïve Monovalent Prime Only Monovalent Prime & Boost Trivalent Prime Only Trivalent Prime and Boost
A/Japan/305/1957 H2N2 (pandemic) Human < < 10 20 20
A/AnnArbor/23/1957 H2N2 (pandemic) Human ND ND ND ND ND
A/Netherlands/84/1968 H2N2 (seasonal) Human < < 20 40 40
A/NorthCarolina/1/68 H2N2 (seasonal) Human < < 40 40 40
Human H2 GMT (Log2)b 0.0 0.0 3.2 4.0 4.0
A/Mallard/MT/Y61 H2N2 Avian < < 10 20 40
A/Mallard/Potsdam/179/1983 H2N2 Avian < 20 40 320 320
A/Chicken/Jena/4836/1983 H2N2 Avian < < 10 10 40
A/Mallard/NewYork/6750/1978 H2N2 Avian < < 20 40 40
A/Chicken/Connecticut/13657/1990 H2N2 Avian < < 40 40 80
A/GuineaFowl/NewYork/13824/1995 H2N2 Avian < < 10 10 160
A/Chicken/Ohio/494832/2007 H2N3 Avian < < 10 20 40
A/Widgeon/Denmark/66174/G18/2004 H2N3 Avian < < 10 20 40
A/Mallard/Alberta/35/2009 H2N3 Avian < < 10 20 20
Avian H2 GMT (Log2)b 0.0 0.2 2.8 3.6 4.8
A/Brisbane/10/2007c H1N1 (pre-pandemic) Human < < < < <
A/California/04/2009c H1N1 (pandemic) Human < < < < <
A/Perth/16/2009c H3N2 (seasonal) Human < < < < <
rgA/Egypt/N03072/2010c, d H5N1 (clade 2.2.1) Human < < < < <
rgA/Guizhou/1/2013(H5N1)c, d H5N1 (clade 2.3.4.2) Human < < < < <
Open in a new tab

< Below the assay limit of detection (1:10 serum dilution), GMT - Geometric Mean Titer, ND - not determined

a

Individual MN values are expressed as the reciprocal of the highest dilution that inhibited 100 TCID50 units of the indicated test virus with horse erythrocytes unless otherwise specified.

b

GMT was determined by assigning 1 (Log2) values for samples below the assay limit of detection and subtracting 1 from the final GMT

c

MN assays were performed with chicken erythrocytes

d

Represents reverse genetics (rg) vaccine viruses containing HA and NA surface proteins on A/Puerto/Rico/8/1934 virus backbone

3.7. Cell-mediated immunity

Humoral immunity is an important correlate of immune protection from influenza infection, but cell-mediated responses contribute towards cross-reactive immunity and viral clearance [55]. To examine cell-mediated responses of monovalent and multivalent vaccines, IFN-γ ELISpot assays were performed with splenocytes from vaccinated mice stimulated with each vaccine component. The monovalent vaccine induced higher IFN-γ spot-forming units (SPU) compared to naïve controls with the exception of Swine/MO/2006 antigen stimulation (Fig. 3). The multivalent vaccine induced SPU in response to all antigens. However, statistical tests showed no difference between the monovalent or multivalent groups.

Figure 3. Cross-reactive, Cell Mediated Response to Monovalent and multivalent H2 Vaccines.

Figure 3

Open in a new tab

Mice (n=5) were vaccinated with monovalent or multivalent H2 vaccines. Splenocytes (1×106, 10 days post-prime) were stimulated with the indicated vaccine antigens. IFN-γ secreting splenocytes were determined by ELISpot. Statistical significance was determined by comparison of the mean values of triplicate measures from each animal. **p≤0.01, ***p≤0.001, ****p≤0.0001, ns = not significant.

3.8. Protection from challenge

BALB/c mice are routinely used to assess protection from influenza virus challenge [54]. While non-mouse adapted viruses may induce no clinical signs of disease, efficient replication may occur in respiratory tissues [15, 23]. To determine the protective efficacy of monovalent or multivalent vaccination, mice (n=10/virus group) were virus challenged 4 weeks post-prime [22, 23]. Monovalent and multivalent vaccines were assessed independently with appropriate naïve controls, and starting weights were statistically similar (data not shown). No morbidity (weight loss) was observed in challenge with Singapore/1957 or Duck/HK/1978 (Fig. 4B, C, E, F). Swine/MO/2006 caused clinical illness (ruffled fur, lethargy) and ≈15-20% weight loss in naïve animals (Fig. 4A, D) [15]. Mice administered one or two doses of monovalent vaccine showed reduced or no clinical signs of disease, though statistical significance between weight loss curves of the naïve animals was only observed in boosted groups (p≤0.05, Fig. 4A). When boosted with monovalent vaccine, mice still lost 6% of body weight before recovering. Mice administered a single dose of multivalent vaccine showed no clinical signs and both regimens yielded statistically significant differences in weight loss compared to naïve animals (p≤0.001, Fig. 4D).

Figure 4. Post-virus Challenge Weight Loss in Vaccinated Mice.

Figure 4

Open in a new tab

Mice (n=10/virus group) were vaccinated with monovalent (A-C) or multivalent (D-F) with one (primed) or two vaccine doses (boosted) and challenged 31 days post-prime with Swine/MO/2006 (A,D), Singapore/1957 (B, E) or Duck/HK/1978 (C,F). Singapore/1957 and Duck/HK/1978 virus challenge did not induce weight loss. Animals were weighed daily as an indicator of morbidity and data are presented as the mean % starting weight ± SD. *p≤0.05, ***p≤0.001, ****p≤0.0001.

We assessed vaccine influence on virus replication in the airway (nasal turbinates, trachea, lung) 3 dpi (Fig. 5). Though samples sizes were modest, (n=3 animals/titer), qualitative differences between groups were notable and all challenge viruses replicated in all tissues of naïve mice (Fig. 5). In nasal turbinates, a single dose of multivalent vaccine reduced mean Swine/MO/2006 virus titers (p≤0.001) compared to a single dose of monovalent vaccine. When boosted, replication in the nasal turbinates persisted in animals that had received the monovalent vaccine (2/3) but not in the groups receiving the multivalent vaccine. The multivalent vaccine also restricted Singapore/1957 replication with a single dose, and comparable effects were not seen with the monovalent vaccine until mice were boosted. Both vaccines and regimens were equally effective at reducing nasal turbinate replication of Duck/HK/1978, though it replicated to comparatively lower titers (Fig. 5A, C). In tracheas, no differences in mean titers between vaccine formulation or regimen were observed, but a single dose or double dose of multivalent vaccine reduced the number of animals with productive tracheal replication of Swine/MO/2006 by 1 and 2 respectively (data not shown). In the lung, both vaccines in either regimen were most effective against Duck/HK/1978, though it replicated to lower titers than the other two challenge viruses. In vaccinated animals, only a single mouse in the monovalent boosted group showed replication of Duck/HK/1978 (Fig. 5B). A single dose of multivalent vaccine inhibited lung replication in 2 of 3 mice. When boosted, both regimens inhibited lung replication with the exception of one outlier animal in the multivalent vaccine group (Fig. 5B, D). Swine/MO/2006 replicated in the lungs of all animals in monovalent vaccine groups regardless of a boost. The multivalent vaccine better restricted replication with 2 of 3 and 1 of 3 mice with lung titers in the prime or prime and boost groups, respectively (Figure 5B, D).

Fig. 5. Post-challenge Virus Replication in Tissues from Vaccinated Mice.

Fig. 5

Open in a new tab

Mice (n=3/virus group) were vaccinated with monovalent (A, B) or multivalent (C, D) with one (primed) or two (boosted) doses of vaccine. Animals were challenged (31 days post-prime) with 5.5×106 EID50 units of the indicated virus. At 3 dpi, virus replication in the nasal turbinates (A, C) or lungs (B, D) was determined in the TCID50. Data are presented as individual TCID50 titers for each animal. Dotted lines indicate the limit of assay detection (1Log10TCID50); values below the cutoff are indicative of 0 and for graphical presentation only.

To determine if these differences were attributed to reduced virus spread, the right half of a whole lung (n=3/virus group) from Swine/MO/2006 inoculated mice were stained for influenza nucleoprotein (NP). Both monovalent and multivalent vaccines reduced NP staining compared to naïve animals, but no differences were observed between the regimens (Supplemental Fig. 1).

4. DISCUSSION

Influenza A H2 viruses should be elevated to a priority concern for a future reemergence in humans because of this subtype's proven ability to cause a pandemic, a lack of H2 population immunity [10, 17, 56], and the presence of zoonotic H2 viruses capable of replicating and transmitting in mammalian models [11, 15, 16, 56-58]. Here we addressed the need for reliable vaccine candidates and novel formulations to address this threat. With multiple groups developing LAIV H2N2 vaccines [22-25, 59], we focused on IVV formulations composed of a single or multiple H2 viruses from diverse host species, and characterized of immune correlates of protection in greater detail than previously reported [33, 34].

Both formulations, even as a single dose, induced humoral and cell-mediated immunity and reduced disease burden upon challenge. The differences between the formulations in terms of serology, clinical disease, and virus replication favored the multivalent vaccine where higher levels of systemic vsIgG, the presence of heterologous NA antibodies, and enhanced HAI and MN titers to the avian virus component were present. vsIgG titers are a hallmark of IVV vaccination and may lessen disease severity [55, 60, 61]. The multivalent's broader anti-NA antibody may have provide better protection against reassortant H2NX viruses as observed in the H2N3 Swine/MO/2006 challenge, while the enhanced HAI and MN response to the avian virus antigen is particularly important given that H2N2 is hypothesized to have entered humans from birds [14, 17, 18]. Additionally, the multivalent vaccine induced higher HAI and MN GMT titers against a broader range of avian viruses irrespective of regimen. With respect to the vaccine components, the multivalent formulation provided superior MN GMT with the exception of the prime/boost regimen and the human and avian antigen, which could be a result of the shared human virus antigen and poor reactivity/detection MN antibodies against Duck/HK/1978 in this assay.

The advantages of the multivalent vaccine did not extend beyond antibody mediated immunity in our study. There were no differences observed between the two formulations in terms of ELISpot measured cell-mediated immunity; this may be due to the high level of conservation of T-cell epitopes shared amongst viruses in the vaccines.

Both the monovalent and multivalent H2 vaccine induced heterologous H2 immunity in multiple assays. However, a comparison of HAI and MN values and cross-reactivity to a recent study by Chen et al. [23] demonstrate that even our multivalent IVV produced lower titers than those induced by a monovalent LAIV. These results are not surprising given the mechanisms employed by LAIV vaccines to induce immunity [5, 61]. However, live virus vaccines have a narrower approved usage, and they are dependent on robust replication to induce an immune response. Further, disparities may exist between pre-clinical [22, 23, 25] and clinical efficacy [24] that may delay availability in a pandemic scenario. While this should not be considered disqualifying for LAIVs, they alone cannot be relied upon in preparation for a potential H2 pandemic. Stockpiling traditional IVV H2 vaccines in conjunction with LAIV should be considered [10, 20, 21]. Multivalent IVV could serve as a “priming option” administered immediately from such stockpiles during early phases of a pandemic. If significant antigenic drift occurs, pandemic-matched vaccine could later be administered when available; a heterologous prime/boost may actually be more immunogenic than homologous prime/boost scenario [62]. Unfortunately, H2N2/3 stockpiling is not currently under consideration in the US [63] despite calls by some strongly urging vaccination against H2 viruses [10].

We acknowledge the concern of increased costs associated with a multivalent vaccine despite the modest benefits we observed over monovalent [4]. A recent study comparing seasonal multivalent vs quadravalent influenza vaccines noted an ≈1.56 fold difference in the dosage cost per each additional antigen [64]. However, mathematical modeling [65] and clinical data suggest a high dose vaccine that cost 2.6 fold more per dose [66] was actually more cost-effective than a standard dose due to a reduction of influenza complications and subsequent hospital admissions. Thus, the overall cost increase in producing a multivalent vaccine may be offset by lower medical and societal economic burdens in a pandemic [67]. Should multivalent vaccine costs and production be deemed unfeasible, an alternative approach would be the use monovalent vaccine with an intended boost. Our study suggests a boost with monovalent vaccine can produce similar or equal results to multivalent, and a similar benefit with monovalent LAIV were demonstrated by Chen et al. in mice and ferrets [23]. However, patient non-compliance can complicate administration of vaccines requiring a boost. Ultimately, human clinical studies will best assess the differences between monovalent and multivalent H2 IVV and to understand how additional parameters (dose ratios of vaccine components, additional vaccine viruses, etc) warrants production and stockpiling of multivalent formulations. However, at this time, even pre-clinical H2 IVV studies such as ours are rare. Ferrets animal studies evaluating the vaccines’ effect on virus transmission, alternate preparations (virus inactivation post- purification, [68]) and formulations (split, subunit, VPL), and comparisons of monovalent regimens with the swine and duck viruses are logical continuations of this pre-clinical IVV trial in mice.

In conclusion, we present here the relative ability of a monovalent and a multivalent H2 IVV to induce an appropriate immune response in mice, finding partial protection that increases with valence of the vaccine and additional dosing. Our study adds to the growing body of H2N2 vaccine evaluation that is critical in preparing for a potential pandemic.

Supplementary Material

1

Highlights.

  • Subtype H2 influenza viruses are a significant public health threat.

  • H2 vaccine development is a critical component of pre-pandemic planning.

  • Monovalent and multivalent H2 vaccines were tested for immunogenicity in mice.

  • Both formulations induce protection against heterologous, zoonotic H2 viruses.

  • Multivalent vaccine exhibits greater breadth and magnitude of immune responses.

ACKNOWLEDGMENTS

The authors wish to thank Sook-Son Wong, Chelsi Stultz, and Sharon Lokey for experimental support, James Knowles for administrative assistance, and Faith Gallian for helpful discussion of the manuscript. This work was supported by Contract No. HHSN272201400006C (R.J. Webby) from the U.S. National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, and by the American Lebanese Syrian Associated Charities (R.J. Webby). The funders had no role in study design, data collection, interpretation, or the decision to submit the work for publication.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

AUTHOR CONTRIBUTIONS

All authors have contributed significantly to this work and approve of the submission.

CONFLICTS OF INTEREST STATEMENT

Dr. Sonnberg is currently an employee at Takeda Vaccines. Takeda Vaccines had no financial or intellectual role and did not contribute to this work.

REFERENCES

  • 1.WHO . Influenza. In: WHO, editor. Immunization, Vaccines and Biologicals. WHO; 2008. [Google Scholar]
  • 2.CDC. Key Facts About Seasonal Flu Vaccine. In: CDC, editor. Influenza (Flu) CDC; Atlanta, GA, USA: 2015. [Google Scholar]
  • 3.Soema PC, Kompier R, Amorij JP, Kersten GF. Current and next generation influenza vaccines: Formulation and production strategies. Eur J Pharm Biopharm. 2015;94:251–63. doi: 10.1016/j.ejpb.2015.05.023. [DOI] [PubMed] [Google Scholar]
  • 4.WHO . Influenza Vaccines: WHO Position Paper. In: WHO, editor. Weekly Epidemiological Record. WHO; 2005. pp. 279–87. [Google Scholar]
  • 5.Krammer F, Palese P. Advances in the development of influenza virus vaccines. Nat Rev Drug Discov. 2015;14:167–82. doi: 10.1038/nrd4529. [DOI] [PubMed] [Google Scholar]
  • 6.Herfst SI,M, Kawaoka Y, Fouchier RAM. Avian Influenza Virus Transmission to Mammals. In: Compans RO,M, editor. Current Topics in Microbiology and Immunology. Springer International Publishing; Switzerland: 2014. pp. 137–55. [DOI] [PubMed] [Google Scholar]
  • 7.Neumann G, Noda T, Kawaoka Y. Emergence and pandemic potential of swine-origin H1N1 influenza virus. Nature. 2009;459:931–9. doi: 10.1038/nature08157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis. 1998;178:53–60. doi: 10.1086/515616. [DOI] [PubMed] [Google Scholar]
  • 9.Palese P, Wang TT. Why do influenza virus subtypes die out? A hypothesis. MBio. 2011:2. doi: 10.1128/mBio.00150-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Nabel GJ, Wei CJ, Ledgerwood JE. Vaccinate for the next H2N2 pandemic now. Nature. 2011;471:157–8. doi: 10.1038/471157a. [DOI] [PubMed] [Google Scholar]
  • 11.Jones JC, Baranovich T, Marathe BM, Danner AF, Seiler JP, Franks J, et al. Risk assessment of H2N2 influenza viruses from the avian reservoir. J Virol. 2014;88:1175–88. doi: 10.1128/JVI.02526-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Schafer J, Khristova ML, Busse TL, Sinnecker R, Kharitonenkov IG, Schrader C, et al. Analysis of internal proteins of influenza A (H2N2) viruses isolated from birds in East Germany in 1983. Acta Virol. 1992;36:113–20. [PubMed] [Google Scholar]
  • 13.Makarova NV, Kaverin NV, Krauss S, Senne D, Webster RG. Transmission of Eurasian avian H2 influenza virus to shorebirds in North America. J Gen Virol. 1999;80(Pt 12):3167–71. doi: 10.1099/0022-1317-80-12-3167. [DOI] [PubMed] [Google Scholar]
  • 14.Schafer JR, Kawaoka Y, Bean WJ, Suss J, Senne D, Webster RG. Origin of the pandemic 1957 H2 influenza A virus and the persistence of its possible progenitors in the avian reservoir. Virology. 1993;194:781–8. doi: 10.1006/viro.1993.1319. [DOI] [PubMed] [Google Scholar]
  • 15.Ma W, Vincent AL, Gramer MR, Brockwell CB, Lager KM, Janke BH, et al. Identification of H2N3 influenza A viruses from swine in the United States. Proc Natl Acad Sci U S A. 2007;104:20949–54. doi: 10.1073/pnas.0710286104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Richt JA, Rockx B, Ma W, Feldmann F, Safronetz D, Marzi A, et al. Recently emerged swine influenza A virus (H2N3) causes severe pneumonia in Cynomolgus macaques. PLoS One. 2012;7:e39990. doi: 10.1371/journal.pone.0039990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Joseph U, Linster M, Suzuki Y, Krauss S, Halpin RA, Vijaykrishna D, et al. Adaptation of pandemic H2N2 influenza A viruses in humans. J Virol. 2015;89:2442–7. doi: 10.1128/JVI.02590-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Kawaoka Y, Krauss S, Webster RG. Avian-to-human transmission of the PB1 gene of influenza A viruses in the 1957 and 1968 pandemics. J Virol. 1989;63:4603–8. doi: 10.1128/jvi.63.11.4603-4608.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Rockman S, Brown L. Pre-pandemic and pandemic influenza vaccines. Hum Vaccin. 2010;6:792–801. doi: 10.4161/hv.6.10.12915. [DOI] [PubMed] [Google Scholar]
  • 20.Luke CJ, Subbarao K. Vaccines for pandemic influenza. Emerg Infect Dis. 2006;12:66–72. doi: 10.3201/eid1201.051147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Jennings LC, Monto AS, Chan PK, Szucs TD, Nicholson KG. Stockpiling prepandemic influenza vaccines: a new cornerstone of pandemic preparedness plans. Lancet Infect Dis. 2008;8:650–8. doi: 10.1016/S1473-3099(08)70232-9. [DOI] [PubMed] [Google Scholar]
  • 22.Chen GL, Lamirande EW, Yang CF, Jin H, Kemble G, Subbarao K. Evaluation of replication and cross-reactive antibody responses of H2 subtype influenza viruses in mice and ferrets. J Virol. 2010;84:7695–702. doi: 10.1128/JVI.00511-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Chen GL, Lamirande EW, Cheng X, Torres-Velez F, Orandle M, Jin H, et al. Evaluation of three live attenuated H2 pandemic influenza vaccine candidates in mice and ferrets. J Virol. 2014;88:2867–76. doi: 10.1128/JVI.01829-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Talaat KR, Karron RA, Liang PH, McMahon BA, Luke CJ, Thumar B, et al. An open-label phase I trial of a live attenuated H2N2 influenza virus vaccine in healthy adults. Influenza Other Respir Viruses. 2013;7:66–73. doi: 10.1111/j.1750-2659.2012.00350.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Broadbent AJ, Santos CP, Paskel M, Matsuoka Y, Lu J, Chen Z, et al. Replication of live attenuated cold-adapted H2N2 influenza virus vaccine candidates in non human primates. Vaccine. 2015;33:193–200. doi: 10.1016/j.vaccine.2014.10.065. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Ohmit SE, Gross J, Victor JC, Monto AS. Reduced reaction frequencies with repeated inactivated or live-attenuated influenza vaccination. Vaccine. 2009;27:1050–4. doi: 10.1016/j.vaccine.2008.11.100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.WHO . Live attenuated vaccines (LAV). In: WHO, editor. Vaccine Safety Basics: e-learning Course, Module 2. Module 2 ed. WHO; [Google Scholar]
  • 28.MedImmune. FluMist: Package Insert and Label Information. 2012 [Google Scholar]
  • 29.CDC . Live Attenuated Influenza Vaccine [LAIV] (The Nasal Spray Flu Vaccine) In: CDC, editor. Influenza (Flu) CDC; Atlanta, GA: 2015. [Google Scholar]
  • 30.Hannoun C. The evolving history of influenza viruses and influenza vaccines. Expert Rev Vaccines. 2013;12:1085–94. doi: 10.1586/14760584.2013.824709. [DOI] [PubMed] [Google Scholar]
  • 31.Francis T, Salk JE, Pearson HE, Brown PN. Protective Effect of Vaccination against Induced Influenza A. J Clin Invest. 1945;24:536–46. doi: 10.1172/JCI101633. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Nichol KL, Treanor JJ. Vaccines for seasonal and pandemic influenza. J Infect Dis. 2006;194(Suppl 2):S111–8. doi: 10.1086/507544. [DOI] [PubMed] [Google Scholar]
  • 33.Govorkova EA, Smirnov Yu A. Cross-protection of mice immunized with different influenza A (H2) strains and challenged with viruses of the same HA subtype. Acta Virol. 1997;41:251–7. [PubMed] [Google Scholar]
  • 34.Kaverin NV, Smirnov YA, Govorkova EA, Rudneva IA, Gitelman AK, Lipatov AS, et al. Cross-protection and reassortment studies with avian H2 influenza viruses. Arch Virol. 2000;145:1059–66. doi: 10.1007/s007050070109. [DOI] [PubMed] [Google Scholar]
  • 35.Hehme N, Engelmann H, Kunzel W, Neumeier E, Sanger R. Pandemic preparedness: lessons learnt from H2N2 and H9N2 candidate vaccines. Med Microbiol Immunol. 2002;191:203–8. doi: 10.1007/s00430-002-0147-9. [DOI] [PubMed] [Google Scholar]
  • 36.Hehme N, Engelmann H, Kuenzel W, Neumeier E, Saenger R. Immunogenicity of a monovalent, aluminum-adjuvanted influenza whole virus vaccine for pandemic use. Virus Res. 2004;103:163–71. doi: 10.1016/j.virusres.2004.02.029. [DOI] [PubMed] [Google Scholar]
  • 37.Control NIfBSa Control NIfBSa, editor. Influenza Reagent Influenza Antigen A/Singapore/1/57 (H2N2) 2008 [Google Scholar]
  • 38.Pyhala R. Antibody status to influenza A/Singapore/1/57(H2N2) in Finland during a period of outbreaks caused by H3N2 and H1N1 subtype viruses. J Hyg (Lond) 1985;95:437–45. doi: 10.1017/s0022172400062860. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Reed LM,H. A simple method for estimating fifty percent endpoints. American Journal of Hygiene. 1938;27:493–7. [Google Scholar]
  • 40.Jones JC, Settles EW, Brandt CR, Schultz-Cherry S. Virus aggregating peptide enhances the cell-mediated response to influenza virus vaccine. Vaccine. 2011;29:7696–703. doi: 10.1016/j.vaccine.2011.07.133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Ninomiya A, Imai M, Tashiro M, Odagiri T. Inactivated influenza H5N1 whole-virus vaccine with aluminum adjuvant induces homologous and heterologous protective immunities against lethal challenge with highly pathogenic H5N1 avian influenza viruses in a mouse model. Vaccine. 2007;25:3554–60. doi: 10.1016/j.vaccine.2007.01.083. [DOI] [PubMed] [Google Scholar]
  • 42.Katz JM, Lu X, Young SA, Galphin JC. Adjuvant activity of the heat-labile enterotoxin from enterotoxigenic Escherichia coli for oral administration of inactivated influenza virus vaccine. J Infect Dis. 1997;175:352–63. doi: 10.1093/infdis/175.2.352. [DOI] [PubMed] [Google Scholar]
  • 43.Huber VC, McKeon RM, Brackin MN, Miller LA, Keating R, Brown SA, et al. Distinct contributions of vaccine-induced immunoglobulin G1 (IgG1) and IgG2a antibodies to protective immunity against influenza. Clin Vaccine Immunol. 2006;13:981–90. doi: 10.1128/CVI.00156-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Zhang L, Lu J, Chen Y, Shi F, Yu H, Huang C, et al. Characterization of Humoral Responses Induced by an H7N9 Influenza Virus-Like Particle Vaccine in BALB/C Mice. Viruses. 2015;7:4369–84. doi: 10.3390/v7082821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Proudfoot O, Esparon S, Tang CK, Laurie K, Barr I, Pietersz G. Mannan adjuvants intranasally administered inactivated influenza virus in mice rendering low doses inductive of strong serum IgG and IgA in the lung. BMC Infect Dis. 2015;15:101. doi: 10.1186/s12879-015-0838-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Trondsen M, Aqrawi LA, Zhou F, Pedersen G, Trieu MC, Zhou P, et al. Induction of Local Secretory IgA and Multifunctional CD4(+) T-helper Cells Following Intranasal Immunization with a H5N1 Whole Inactivated Influenza Virus Vaccine in BALB/c Mice. Scand J Immunol. 2015;81:305–17. doi: 10.1111/sji.12288. [DOI] [PubMed] [Google Scholar]
  • 47.Quan FS, Huang C, Compans RW, Kang SM. Virus-like particle vaccine induces protective immunity against homologous and heterologous strains of influenza virus. J Virol. 2007;81:3514–24. doi: 10.1128/JVI.02052-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Kim JK, Kayali G, Walker D, Forrest HL, Ellebedy AH, Griffin YS, et al. Puzzling inefficiency of H5N1 influenza vaccines in Egyptian poultry. Proc Natl Acad Sci U S A. 2010;107:11044–9. doi: 10.1073/pnas.1006419107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Lambre CR, Terzidis H, Greffard A, Webster RG. An enzyme-linked lectin assay for sialidase. Clin Chim Acta. 1991;198:183–93. doi: 10.1016/0009-8981(91)90352-d. [DOI] [PubMed] [Google Scholar]
  • 50.Zanin M, Keck ZY, Rainey GJ, Lam CY, Boon AC, Rubrum A, et al. An anti-H5N1 influenza virus FcDART antibody is a highly efficacious therapeutic agent and prophylactic against H5N1 influenza virus infection. J Virol. 2015;89:4549–61. doi: 10.1128/JVI.00078-15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.USDA . In: Clinical Data Needed to Support the Licensure of Seasonal Inactivated Influenza Vaccines. Research CfBEa, editor. U.S. Department of Health and Human Services; 2009. [Google Scholar]
  • 52.Noah DL, Hill H, Hines D, White EL, Wolff MC. Qualification of the hemagglutination inhibition assay in support of pandemic influenza vaccine licensure. Clin Vaccine Immunol. 2009;16:558–66. doi: 10.1128/CVI.00368-08. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53.Coudeville L, Bailleux F, Riche B, Megas F, Andre P, Ecochard R. Relationship between haemagglutination-inhibiting antibody titres and clinical protection against influenza: development and application of a bayesian random-effects model. BMC Med Res Methodol. 2010;10:18. doi: 10.1186/1471-2288-10-18. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54.van der Laan JW, Herberts C, Lambkin-Williams R, Boyers A, Mann AJ, Oxford J. Animal models in influenza vaccine testing. Expert Rev Vaccines. 2008;7:783–93. doi: 10.1586/14760584.7.6.783. [DOI] [PubMed] [Google Scholar]
  • 55.Reber A, Katz J. Immunological assessment of influenza vaccines and immune correlates of protection. Expert Rev Vaccines. 2013;12:519–36. doi: 10.1586/erv.13.35. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 56.Xu R, McBride R, Paulson JC, Basler CF, Wilson IA. Structure, receptor binding, and antigenicity of influenza virus hemagglutinins from the 1957 H2N2 pandemic. J Virol. 2010;84:1715–21. doi: 10.1128/JVI.02162-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 57.Pappas C, Viswanathan K, Chandrasekaran A, Raman R, Katz JM, Sasisekharan R, et al. Receptor specificity and transmission of H2N2 subtype viruses isolated from the pandemic of 1957. PLoS One. 2010;5:e11158. doi: 10.1371/journal.pone.0011158. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 58.Pappas C, Yang H, Carney PJ, Pearce MB, Katz JM, Stevens J, et al. Assessment of transmission, pathogenesis and adaptation of H2 subtype influenza viruses in ferrets. Virology. 2015;477:61–71. doi: 10.1016/j.virol.2015.01.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 59.Isakova-Sivak I, Stukova M, Erofeeva M, Naykhin A, Donina S, Petukhova G, et al. H2N2 live attenuated influenza vaccine is safe and immunogenic for healthy adult volunteers. Hum Vacc Immunother. 2015;11:970–82. doi: 10.1080/21645515.2015.1010859. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 60.Schultz-Cherry S, Jones JC. Influenza vaccines: the good, the bad, and the eggs. Adv Virus Res. 2010;77:63–84. doi: 10.1016/B978-0-12-385034-8.00003-X. [DOI] [PubMed] [Google Scholar]
  • 61.Sridhar S, Brokstad KA, Cox RJ. Influenza Vaccination Strategies: Comparing Inactivated and Live Attenuated Influenza Vaccines. Vaccines (Basel) 2015;3:373–89. doi: 10.3390/vaccines3020373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 62.Lu S. Heterologous prime-boost vaccination. Curr Opin Immunol. 2009;21:346–51. doi: 10.1016/j.coi.2009.05.016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 63.WHO . Antigenic and genetic characteristics of zoonotic influenza viruses and development of candidate vaccine viruses for pandemic preparedness. WHO; Geneva, Switzerland: 2015. [PubMed] [Google Scholar]
  • 64.Thommes EW, Ismaila A, Chit A, Meier G, Bauch CT. Cost-effectiveness evaluation of quadrivalent influenza vaccines for seasonal influenza prevention: a dynamic modeling study of Canada and the United Kingdom. BMC Infect Dis. 2015;15:465. doi: 10.1186/s12879-015-1193-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 65.Chit A, Roiz J, Briquet B, Greenberg DP. Expected cost effectiveness of high-dose trivalent influenza vaccine in US seniors. Vaccine. 2015;33:734–41. doi: 10.1016/j.vaccine.2014.10.079. [DOI] [PubMed] [Google Scholar]
  • 66.Chit A, Becker DL, DiazGranados CA, Maschio M, Yau E, Drummond M. Cost-effectiveness of high-dose versus standard-dose inactivated influenza vaccine in adults aged 65 years and older: an economic evaluation of data from a randomised controlled trial. Lancet Infect Dis. 2015 doi: 10.1016/S1473-3099(15)00249-2. [DOI] [PubMed] [Google Scholar]
  • 67.Meltzer MI, Cox NJ, Fukuda K. The economic impact of pandemic influenza in the United States: priorities for intervention. Emerg Infect Dis. 1999;5:659–71. doi: 10.3201/eid0505.990507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 68.WHO. Recommendations for the production and control of influenza vaccine (inactivated) 2005:99–134. Report 927. http://www.who.int/biologicals/publications/trs/areas/vaccines/influenza/ANNEX%203%20InfluenzaP99-134.pdf?ua=1.

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS847399-supplement-1.docx (2.4MB, docx)

RESOURCES