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The Spanish influenza virus pandemic of 1918 was responsible for 40 million to 50 million deaths and is antigenically similar to the swine lineage 2009 pandemic influenza virus. Emergence of the 2009 pandemic from swine into humans has raised the possibility that low levels of cross-protective immunity to past shared epitopes could confer protection. In this study, influenza viruslike particles (VLPs) were engineered to express the hemagglutinin (HA) and genes from the 1918 influenza virus to evaluate the duration of cross-protection to the H1N1 pandemic strain by vaccinating young mice (8 to 12 weeks) and then allowing the animals to age to 20 months. This immunity was long lasting, with homologous receptor-blocking antibodies detected throughout the lifespan of vaccinated mice. Furthermore, the 1918 VLPs fully protected aged mice from 2009 pandemic H1N1 virus challenge 16 months after vaccination. Histopathological assessment showed that aged vaccinated mice had significant protection from alveolar infection but less protection of the bronchial tissue than adult vaccinated mice. Additionally, passive transfer of immune serum from aged vaccinated mice resulted in protection from death but not morbidity. This is the first report describing the lifelong duration of cross-reactive immune responses elicited by a 1918 VLP vaccine in a murine model. Importantly, these lifelong immune responses did not result in decreased total viral replication but did prevent infection of the lower respiratory tract. These findings show that immunity acquired early in life can restrict the anatomical location of influenza viral replication, rather than preventing infection, in the aged.
Infections with influenza virus resulted in ~200,000 hospitalizations and estimated annual averages of approximately 36,000 deaths during the period of 1990 to 1999 in the United States (65). In 2009, a novel strain of H1N1 influenza virus emerged from swine and quickly spread among humans, resulting in the World Health Organization declaring the first pandemic of the 21st century (13). The 1918 Spanish influenza virus pandemic was the worst pandemic in recorded history and caused severe disease and mortality (675,000 total deaths) in the United States (64) and killed up to 50 million people worldwide (33). In comparison to the 1918 pandemic, the 2009 pandemic was much more moderate, with the majority of cases being uncomplicated (4). The most common feature of fatal disease was various degrees of alveolar infection and damage (25, 42, 58). This differed from seasonal influenza virus, as fatal cases rarely involve alveolar cells, with virus located primarily in the major airways, such as the trachea and bronchioles (27, 37). Interestingly, the majority of severe cases from the 2009 H1N1 pandemic were reported in children and young adults, while the elderly population was relatively protected from infection and severe disease (4). This pattern of susceptibility to severe disease is in direct contrast to what is normally observed during seasonal influenza virus epidemics but is similar to what was reported for the 1918 pandemic (1). Although the 1918 pandemic is believed to have emerged from avian species into both swine and humans nearly simultaneously, the human and swine lineages quickly diverged. Sequence analysis indicated that the 2009 H1N1 pandemic virus is related to the 1918 H1N1 virus, and it has been proposed that the swine population has maintained an “antigenically frozen” H1N1 lineage (39). Structural analysis demonstrated conservation within antigenic regions of 1918 and 2009 pandemic hemagglutinin (HA) proteins that is not present in contemporary seasonal H1N1 viruses (73, 77).
Antigenic similarities and serologic evidence of cross-reactive antibody in older adults have led to the hypothesis that exposure to 1918-like viruses confer cross-protective immune responses (29, 32). Several studies have identified cross-reactive antibodies to the 2009 pandemic H1N1 viruses in elderly human populations (15, 78), with monoclonal antibodies derived from survivors of the 1918 pandemic cross-neutralizing the 2009 pandemic viruses (36). Humans repeatedly experience different influenza virus antigens either by infection or vaccination. Although the exposure to multiple drifted antigens likely broadens the circulating antibody repertoire, the unique history for any one individual or age group is difficult to ascertain and simulate in an animal model. Additionally, direct evidence of the cross-protective efficacy elicited by exposure to a single 1918-like virus has been demonstrated in small-animal models (39, 59), but the duration of this cross-protective immune response(s) has not been evaluated. We therefore hypothesized that cross-protection elicited by a single antigen early in life would be long lasting and sufficient to protect animals years after exposure.
Aging is associated with the decreasing ability of the immune system to respond to new antigens (7). Although elderly individuals are impaired in their immune responses, immunological memory responses to antigens experienced prior to the onset of decreased immune function can be retained and offer protection against reexposure to similar pathogens. Indeed, successful vaccines are usually antibody based, and memory B cell responses, at both the cellular and antibody levels, can persist for a lifetime (53). After exposure to antigen, B cells can develop into two main types of long-lived cells: memory B cells (MBC) and long-lived plasma cells (LLPC) (74). Both subsets generally develop in the germinal center and go through differential degrees of affinity maturation: MBC differentiate earlier and have fewer somatic mutations, while LLPC emerge later with higher affinity (19, 60, 74). Both subsets play important roles in preventing reinfection: LLPC produce high-affinity antibody in the absence of antigen stimulation, and MBC respond to a second infection that escapes circulating antibody (20). Protection of the elderly population from the 2009 pandemic virus has been attributed largely to circulating antibody derived from the LLPC arm of the B cell memory response. Interestingly, infection with the 2009 pandemic virus in humans led to development of broadly neutralizing antibody-producing cells, and it has been suggested that these cells were derived from preexisting MBC (71). Animal models have confirmed the hypothesis that prior exposure to various H1N1 virus strains can indeed protect from 2009 pandemic challenge, but mechanistic inquiries into the reason for the cross-protection have been largely antibody focused and have thus far failed to evaluate the involvement of the cellular component (39, 59).
Our research group has developed viruslike particle (VLP) vaccines against a variety of viral pathogens (8, 9, 24, 43, 44, 75). These vaccines are composed of noninfectious, nonreplicating VLPs that present functional HA and NA on the surface of a viral particle. VLPs efficiently elicit high-titer immune responses that protect small animals against lethal viral challenge (8, 24). In this study, HA and NA from the 1918 influenza virus, A/South Carolina/1/1918, were expressed on the surface of a VLP that was expressed and purified from mammalian cells. Mice were vaccinated at a young age (8 to 12 weeks) and allowed to age to elderly status (20 months). Antibody titer to the 1918 antigens persisted for the lifetime of the animals, indicating that the nonreplicating VLP vaccine elicits enduring antibody titers. The 1918 VLPs efficiently protected the aged mice from 2009 pandemic challenge and prevented alveolar infection. Additionally, the protection involved not only the previously described cross-reactive antibodies but also a robust cellular recall response.
Human embryonic kidney (HEK) 293T cells (1 × 106) were transiently transfected with plasmids expressing Gagp24 alone or together with HA and NA and incubated for 72 h at 37°C. Plasmids expressing the HIV-1NL4-3 Gag gene products only, pGagp24, were derived from codon-optimized sequences (phGag), as previously described (31). pGagp24 encodes for an immature, unprocessed HIV-1 Gag particle. Plasmids expressing HA (A/South Carolina/1/1918) and NA (A/Brevig Mission/1/1918) genes were kindly provided by A. Garcia-Sastre. Supernatants were collected and cell debris removed by low-speed centrifugation followed by vacuum filtration through a 0.22-μm sterile filter. VLPs were purified via ultracentrifugation (100,000 × g through 20% [wt/vol] glycerol) for 4 h at 4°C. The pellets were subsequently resuspended in phosphate-buffered saline (PBS) and stored at −80°C until use. Protein concentration was determined by the Micro BCA protein assay reagent kit (Pierce Biotechnology, Rockford, IL).
BALB/c mice (Mus musculis, females, 6 to 8 weeks) were purchased from Harlan Sprague Dawley (Indianapolis, IN), housed in microisolator units, allowed free access to food and water, and cared for under USDA guidelines for laboratory animals. Mice were vaccinated with 3 μg of purified VLPs based upon total protein via intramuscular injection at weeks 0 and 3. Vaccination experiments were initially prepared with and without 10 μg CpG oligonucleotides (Sigma-Aldrich, St. Louis, MO). Due to no observed effect of the adjuvant, subsequent vaccinations investigating longevity and cross-reactivity were performed without inclusion of adjuvant. Fourteen to 21 days after each vaccination, blood was collected from anesthetized mice via the retro-orbital plexus and transferred to a microcentrifuge tube. Tubes were centrifuged and sera were removed and frozen at −20°C. A subset of vaccinated mice was allowed to age to a final age of 20 months (~17 months after final vaccination), with blood collected at 10 months and 20 months of age. All procedures were in accordance with the NRC Guide for the Care and Use of Laboratory Animals, the Animal Welfare Act, and CDC/NIH Biosafety in Microbiological and Biomedical Laboratories.
The enzyme-linked immunosorbent assay (ELISA) was used to assess total antibody titer and IgG isotype titer to the 1918 HA. High-binding, 96-well polystyrene plates (Costar, Lowell, MA) were coated overnight with 50 ng/well of recombinant 1918 HA. Plates were blocked with 5% milk diluted in PBS with 0.05% Tween 20. Serum samples were diluted in blocking buffer and added to plates. Serum was 2-fold serially diluted and allowed to incubate for 1 h at room temperature. Plates were washed, and species-specific antibody against IgG, IgG1, IgG2a, IgG2b, or IgG3 and linked to horseradish peroxidase (HRP) (Southern Biotech, Birmingham, AL) were diluted in blocking buffer and added to plates. Plates were incubated for 1 h at room temperature. Plates were washed, and HRP was developed with substrate (Sigma-Aldrich, St. Louis, MO). Plates were incubated in the dark for 30 min, and then the reaction was stopped with 2 N H2SO4. Optical densities at a wavelength of 450 nm (OD450) were read by a spectrophotometer (BioTek, Winooski, VT), and endpoint dilution titers were determined. Endpoint titers were determined as the reciprocal dilution of the last well which had an OD450 above the mean OD450 plus two standard deviations of naïve animal serum.
The hemagglutination inhibition (HAI) assay was used to assess functional antibodies to the HA able to inhibit agglutination of turkey erythrocytes. The protocol was adapted from the CDC laboratory-based influenza virus surveillance manual (35). To inactivate nonspecific inhibitors, sera were treated with receptor-destroying enzyme (RDE) prior to being tested (10–12, 46, 55). Briefly, three parts RDE was added to one part serum and incubated overnight at 37°C. RDE was inactivated by incubation at 56°C for ~30 min. RDE-treated serum was 2-fold serially diluted in V-bottom microtiter plates. Equal volumes of either 1918 VLP or wild-type pandemic H1N1 virus, adjusted to approximately 4 hemagglutinating units (HAU)/25 μl, were added to each well. 1918 VLPs were produced as described above. Wild-type viruses were propagated in eggs and included the strains A/California/07/2009 and A/Mexico/4108/2009. The plates were covered and incubated at room temperature for 20 min, followed by the addition of turkey erythrocytes (RBC) (Lampire Biologicals, Pipersville, PA) in PBS for a final concentration of 0.5% RBC. Red blood cells were stored at 4°C and used within 72 h of preparation. The plates were mixed by agitation and covered, and the RBCs were allowed to settle for 30 min at room temperature (2). The HAI titer was determined by the reciprocal dilution of the last well which contained nonagglutinated RBC. Positive and negative serum controls were included for each plate. All mice were negative (HAI ≤ 10) for preexisting antibodies to currently circulating human influenza viruses prior to vaccination.
To assess the ability of mouse immune antiserum to inhibit replication of live 1918 virus, the virus microneutralization assay was used as previously described (47). Briefly, sera were 2-fold serially diluted and then incubated with 100 50% tissue culture infective doses (TCID50) of 1918 virus for 60 min at room temperature. The serum-virus mixture was then added to Madin-Darby canine kidney (MDCK) cells and allowed to incubate for 2 days at 37°C. Specific neutralizing activity was calculated as the lowest concentration of serum that displayed neutralizing activity.
To assess the binding properties of serum antibodies, surface plasmon resonance (SPR) technology was performed using a Biacore 3000 (GE/Biacore AB, Uppsala, Sweden). Protein A (Pierce, Rockford, IL) was immobilized to the surface of a CM5 sensor chip (GE/Biacore, Inc., Piscataway, NJ) using standard amine coupling chemistry. The surface of the chip was activated using a 1:1 mixture of N-hydroxysuccinimide and 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC) (Biacore, Inc.). Protein A (75 μg/ml) was immobilized on experimental and reference (adjacent) flow cells at a high level of density (approximately 5,000 response units (RU). Remaining active carboxyl groups were inactivated with an injection of ethanolamine. Pooled polyclonal IgG from vaccinated mice was diluted in HBS-EP buffer (GE Healthcare/Biacore, Inc., Piscataway, NJ) and captured at approximately 300 RU. After capture of IgG, various concentrations (0.8 to 66 nM, series of 3-fold dilutions) of recombinant HA (rHA) representing the California/04/2009 strain (Protein Sciences, Meriden, CT) were passed sequentially over both flow cells. A blank (0 nM) injection was also included for double referencing. Binding isotherms were then analyzed using BIAevaluation 4.1.1 software (Biacore AB). Polyclonal serum appeared to yield monospecific binding, and hence a 1:1 Langmuir fit was utilized for kinetic determinations. However, since kinetic rates returned using these binding models for polyclonal serum represent only apparent rates of binding due to the multiple specificities inherent to a polyclonal response, we referred to the results as “relative association” and “relative dissociation.” The percentage ([RL/RU captured serum] × 100) of HA-specific antibody captured was calculated by determining the RL of the binding reaction utilizing the Rmax from the BIAevaluation parameters of the run.
To determine the homologous efficacy of the 1918 vaccine, mice were challenged with the reconstructed 1918 virus (51, 69). Briefly, 2 weeks after the final vaccination, adult animals were challenged intranasally with 50 LD50 of 1918 virus in a volume of 50 μl. Mice were monitored daily for disease signs and death for 16 days postinfection (dpi). Body weights were recorded for individual mice at various days postinoculation. All virus challenge experiments were performed under the guidance of the U.S. National Select Agent Program in negative-pressure HEPA-filtered biosafety level 3+ (BSL3+) enhanced laboratories with the use of a battery-powered Racal HEPA filter respirator and according to Biomedical Microbiological and Biomedical Laboratory procedures (70).
To determine the cross-protective efficacy of the 1918 vaccine, mice were infected with a 2009 pandemic H1N1 isolate: A/Mexico/4108/2009. Although this virus is not highly lethal in adult BALB/c mice, it does cause 10 to 20% weight loss and development of clinical illness (data not shown). Briefly, animals were challenged intranasally with 1 × 106 PFU of A/Mexico/4108/2009 virus in a volume of 50 μl. Mice were monitored daily for disease signs and death for 14 days postinfection. Body weights and sickness scores were recorded for individual mice at various days postinfection. Sickness scores were determined by evaluating activity (0 = normal, 1 = reduced, 2 = severely reduced), hunched back (0 = absent, 1 = present), and ruffled fur (0 = absent, 1 = present) (66). Any animal reaching >20% weight loss was humanely euthanized. All experiments using 2009 pandemic H1N1 virus were performed under biosafety level 2 (BSL2) conditions.
On day 4 after 1918 virus challenge, four mice per group were exsanguinated and euthanized, and their lungs were removed for virus titration. Lungs were homogenized in 1 ml of sterile PBS, and clarified homogenate virus titers were determined using a 50% egg infectious dose (EID50) method. Homogenates were titrated for virus infectivity in eggs from initial dilutions of 1:10, and EID50 was calculated using the method of Reed and Muench (54). The limit of virus detection was 101.5 EID50/ml.
For 2009 pandemic H1N1 virus infections, lung virus titers were determined using a plaque assay (67, 68). Briefly, lungs from infected mice were harvested 4 dpi, snap-frozen, and stored at −80°C until use. Samples were thawed and weighed, and single-cell suspensions were prepared via passage through a 70-μm-pore-size mesh (BD Falcon, Bedford, MA) in an appropriate volume of PBS as to achieve a 100-mg/ml final concentration. Cell suspensions were centrifuged at 2,000 rpm for 5 min, and the supernatants were collected. MDCK cells were plated (5 × 105) in each well of a 6-well plate. Lung supernatants were diluted (dilution factors of 1 × 101 to 1 × 106) and overlaid onto the cells in 100 μl of Dulbecco's modified Eagle's medium (DMEM) supplemented with penicillin-streptomycin and incubated for 1 h. Virus-containing medium was removed and replaced with 2 ml of L12 medium plus 0.8% agarose (Cambrex, East Rutherford, NJ) and incubated for 96 h at 37°C with 5% CO2. Agarose was removed and discarded. Cells were fixed with 10% buffered formalin and then stained with 1% crystal violet for 15 min. After being thoroughly washed in distilled water (dH2O) to remove excess crystal violet, plates were allowed to dry, plaques were counted, and PFU/g was calculated.
Left lobes of lungs from infected mice were collected 4 days postinfection and placed into 10% buffered formalin. After fixation, lungs were paraffin embedded, and 6-μm sections were prepared for histopathological analysis. Tissue sections were stained with hematoxylin and eosin and examined for bronchial inflammation and denudation and alveolar infiltration.
Immunohistochemistry was performed as described before (6). Sections containing lung were stained using antibodies against influenza A virus (1:500; Maine Biotechnology Services, Portland, ME), Iba1 (1:500; Wako Pure Chemical Industries, Osaka, Japan), CD3 (1:500; Dako, Carpinteria, CA), myeloperoxidase (1:250; Abcam, Cambridge, MA), IgA (1:500; Sigma), IgG (1:500; Sigma), and IgM (1:1,000; Sigma) followed by species-appropriate secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, and Rockland Immunochemicals, Gilbertsville, PA) conjugated to a fluorophore for fluorescent stains or biotin for peroxidase-based stains. Stains were assessed and scored for frequency of influenza virus-infected cells and abundance of CD3+ T cells, Iba1-positive macrophages, myeloperoxidase-labeled neutrophils, and IgA expressing cells.
For in situ hybridization (ISH), vectors containing 760 bp of Influenza/California/04/2009 matrix protein and full-length murine interferon-β (Open Biosystems, Huntsville, AL) were linearized to create antisense and sense templates. 35S-labeled riboprobes were generated using a MAXIscript in vitro transcription kit (Ambion, Austin, TX). ISH was performed as described before (5). Control riboprobes did not hybridize to lung tissue at any time point postinfection, and noninfected tissue did not show hybridization with viral probes. Hybridized slides were assessed and scored for abundance of foci.
The number of anti-influenza virus-specific cells secreting gamma interferon (IFN-γ) was determined by enzyme-linked immunospot (ELISpot) assay (R&D systems, Minneapolis, MN) by following the manufacturer's protocol. Mice were sacrificed at 6 dpi, and spleens and lungs were harvested and prepared in single-cell suspensions. Briefly, precoated anti-IFN-γ plates were blocked with RPMI plus 10% fetal calf serum (FCS) and antibiotics (cRPMI) for 30 min at room temperature. Medium was removed from wells, and 105 cells were added to each well. Cells were stimulated with 1918 recombinant HA (truncated at residue 530; 1 μg/well), inactivated A/Mexico/4108/2009 virus (1:100 dilution of inactivated stock; 100 μl/well), or the immunodominant H2-Kd CD8+ T cell epitope in H1 HA: HA533 (IYSTVASSL; 1 μg/well) (Pepscan Presto, Leystad, Netherlands). Additional wells were stimulated with phorbol myristate acetate (PMA) (50 ng/well) and ionomycin (500 ng/well) as positive controls or Ova257 (SIINFEKL; 1 μg/well) (Pepscan Presto, Leystad, Netherlands) as a negative control. Additionally, interleukin 2 (IL-2) (10 U/ml) was added to each well. Plates were incubated at 37°C for 48 h. After incubation, plates were washed four times with R&D wash buffer and incubated at 4°C overnight with biotinylated anti-mouse IFN-γ. Plates were washed as described before and incubated at room temperature for 2 h with streptavidin conjugated to alkaline phosphatase. Plates were washed as described before, and spots were developed by incubating at room temperature for 1 h in the dark with BCIP (5-bromo-4-chloro-3-indolylphosphate)/NBT chromogen substrate. The plates were washed extensively with DI H2O and allowed to dry overnight prior to spots being counted using an ImmunoSpot ELISpot reader (Cellular Technology Ltd., Cleveland, OH).
The number of anti-1918 HA-specific antibody-secreting cells was determined by B cell ELISpot assay as previously described (34, 56, 57). Mice were sacrificed at 6 dpi, and spleens and lungs were harvested and prepared in single-cell suspensions. Briefly, membrane plates with 0.45 μm polyvinylidene difluoride (PVDF) (Millipore, Billerica, MA) were coated with 1918 recombinant HA (250 ng/well) and incubated at 4°C overnight. Plates were washed three times with PBS and blocked with cRPMI at 37°C for 3 to 4 h. Medium was removed from wells, and 105 cells were added to each well. Plates were incubated at 37°C for 48 h. After incubation, plates were washed as described before and incubated at room temperature for 2 h with horseradish peroxidase-conjugated anti-mouse IgG (Southern Biotech, Birmingham, AL). Plates were washed as described before, and spots were developed at room temperature for 1 h in the dark with detection substrate (NovaRED; Vector Labs, Burlingame, CA). The plates were washed extensively with DI H2O and allowed to dry overnight prior to spots being counted using an ImmunoSpot ELISpot reader (Cellular Technology Ltd., Cleveland, OH).
Sera from adult and aged vaccinated mice were pooled within the respective age group and passively transferred into 9-week-old recipient BALB/c mice (n = 5/group). Equal amounts of serum from each mouse in a particular vaccine/age group were pooled and heat inactivated for 30 min at 56°C. A total of 200 μl of pooled and inactivated serum was transferred to recipient mice via intraperitoneal (i.p.) injection. Twenty-four hours posttransfer, mice were infected with 2009 pandemic H1N1 virus as described above.
Statistical analyses of immune responses were performed using a one-way analysis of variance (ANOVA) with Dunn's posttest to compare each group. A P value of <0.05 was considered significant. For challenge experiments, statistical analysis was performed using a two-way ANOVA with Bonferroni's posttest to compare each group at multiple time points. A P value of <0.05 was considered significant. All statistical analyses were performed using GraphPad Prism software.
BALB/c mice (6 to 8 weeks) were vaccinated twice at weeks 0 and 3 via intramuscular injection with purified influenza VLP vaccines plus CpG adjuvant. Two weeks after the final vaccination, serum was analyzed for antibody responses. All mice vaccinated with 1918 VLPs had HAI antibodies to the homologous test antigen with a geometric mean titer (GMT) of 1:113 (Fig. 1A). In contrast, mice receiving Gag VLPs or CpG adjuvant alone failed to generate any HAI antibodies to the 1918 VLPs (Fig. 1A). To evaluate the ability of the elicited antibody response to block live virus infection, serum was tested in a live virus neutralization assay. Similar to the 1918 VLP HAI results, all mice vaccinated with 1918 VLPs had neutralizing antibodies with a GMT of 1:208, while mice receiving Gag VLPs or CpG alone failed to generate any detectable neutralizing antibodies (Fig. 1B). Additional vaccines administered without the CpG adjuvant elicited similar HAI titers, and therefore CpG adjuvant was removed from subsequent vaccine preparations (data not shown).
Two weeks after the final vaccination, mice were challenged intranasally with a lethal dose of live 1918 virus to evaluate the protective efficacy of the vaccines. 1918 VLP-vaccinated mice were protected from weight loss (maximum, 94.8% at 2 dpi), while Gag VLP- and CpG-vaccinated animals rapidly lost weight (maximum, 85.8% at 5 dpi and 86.3% at 5 dpi, respectively) (data not shown). Furthermore, mice receiving the 1918 VLP vaccine were completely protected from death, while Gag VLP- and CpG-vaccinated animals completely succumbed to infection by 7 dpi (Fig. 1C). To determine the ability of the vaccines to control viral replication in the respiratory tract, lungs of infected mice (n = 3) were collected at 4 dpi and analyzed for viral titers (Fig. 1D). Mice vaccinated with 1918 VLPs did not have detectable virus, while mice receiving the Gag VLPs or CpG alone had significantly higher viral loads (P < 0.001).
To determine the duration of the antibody response elicited by the 1918 VLP, mice (n = 15) were vaccinated twice with 3 μg of purified 1918 VLPs without adjuvant and allowed to age. Serum was collected at 3 months (two weeks postfinal vaccination), 10 months, and 20 months of age and analyzed for antibody responses to the 1918 VLPs (Fig. 2A). Homologous antibody responses were detected in vaccinated mice over the lifetime of the animals. Although the HAI GMT decreased from 1:127 to 1:88 to 1:54 at 3, 10, and 20 months, respectively, the observed differences were not significant (P > 0.05), indicating that the 1918 VLPs elicited lifelong homologous antibody responses.
To evaluate if antibodies elicited by the 1918 VLPs cross-reacted with 2009 pandemic viruses, sera from 1918 VLP-vaccinated adult (3 to 4 months; n = 13) and aged (20 months; n = 14) animals were analyzed for heterologous antibody responses. Sera from adult mice were collected 2 weeks after the final vaccination, while sera from aged mice were collected 16 months (~64 weeks) after the final vaccination. Both adult and aged animals had 2009 pandemic cross-reactive antibodies, and no significant differences between the age groups were detected (P > 0.05; Fig. 2B). Although the 2009 pandemic HAI titers in the 1918 VLP-vaccinated animals failed to achieve significance compared to the titer of the mock-vaccinated animals, 100% of adult animals and 50% of the aged animals receiving the 1918 VLP vaccine had detectable HAI titers (range of 1:20 to 1:160 and 1:10 to 1:320 for adult and aged mice, respectively), while the mock-vaccinated animals were all negative (1:10). Multiple 2009 pandemic viral isolates were tested to ensure that the observed cross-reactivity was not unique to a single virus, and similar titers were found between strains. Additionally, adult and aged 1918 VLP-vaccinated animals had equivalent titers to the homologous 1918 test antigen (P > 0.05), and these titers were significantly increased compared to age-matched mock-vaccinated controls (P < 0.05).
To evaluate the relative binding kinetic profile of the antibody elicited by the 1918 VLPs, sera from adult and aged mice were evaluated via SPR using recombinant HA protein representing the novel H1N1. Serum samples were diluted, polyclonal IgG was captured, and binding experiments were carried out as previously described with minor modifications, detailed in Materials and Methods (8, 61, 62). Sensograms demonstrating the specifics of the binding response for the adult and aged mouse sera are shown in Fig. 2C and D. Sera from adult mice collected at 3 months postvaccination had an apparent single population of antibody that bound specifically to the HA at a relative association rate (Ka) of 1.84 × 105 (Fig. 2E). Similarly, the apparently monospecific relative association rate for sera collected at 20 months postvaccination from aged mice was 2.98 × 105. However, approximately a log10 difference in relative dissociation rates (Kd) was observed between sera collected from adult and aged mice. Sera collected from adult mice had a relative dissociation rate of 3.03 × 10−4, and sera from aged mice had a dissociation rate of 6.33 × 10−5. These differences in binding resulted in approximately a log10 lower affinity for HA of the adult sera than that of the aged sera, 1.65 × 10−9 and 2.12 × 10−10, respectively. Although similar RU levels were captured from the adult and aged mouse serum samples, the percentage of HA-specific antibody collected from aged mice vaccinated with the 1918 VLPs (8.5%) was greater than 2-fold higher than sera collected from 1918 VLP-vaccinated adult mice (3.8%) (Fig. 2F). Therefore, the aged mouse sera compared to the adult mouse sera had higher concentrations of HA-specific antibody that demonstrated a log10 lower dissociation rate, which resulted in a log10 higher affinity for the HA protein.
Adult (2 weeks after final vaccination) and aged (16 months after final vaccination) vaccinated mice were then challenged intranasally with 1 × 106 PFU of the 2009 pandemic virus A/Mexico/4108/2009. This isolate is not mouse adapted and does not cause a lethal infection in adult or aged BALB/c mice, but the dose used resulted in significant weight loss and development of clinical disease. Both adult and aged animals receiving the 1918 VLPs were protected from development of clinical sickness and weight loss, while mock-vaccinated animals became ill and rapidly lost weight (Fig. 3A and B). Furthermore, 1918 VLP-vaccinated animals, regardless of age, were significantly protected from sickness (P < 0.05; 4 to 9 dpi and 3 to 13 dpi for adult and aged mice, respectively) and weight loss (P < 0.05; 3 to 12 dpi and 3 to 14 dpi for adult and aged mice, respectively) compared to the age-matched mock-vaccinated control animals. No significant differences were found between adult and aged animals within the 1918 VLP or mock vaccine groups. To evaluate viral burden, lungs were harvested from infected mice at 4 dpi (n = 3/group), and viral replication was determined (Fig. 3C). Adult 1918 VLP-vaccinated mice had significantly decreased lung viral titers compared to those of the adult mock-vaccinated animals (P = 0.0071). In contrast, aged 1918 VLP- and mock-vaccinated mice had equivalent viral titers despite the differences observed for disease signs and weight loss (P = 0.1579).
To determine if histopathological features after the A/Mexico/4108/2009 challenge could explain the dichotomy observed in aged 1918 VLP-vaccinated mouse protection from disease but increased viral titers, lung sections were assessed for histopathological changes, presence of influenza virus, and differences in immune response. At 4 dpi, the lungs of adult 1918 VLP-vaccinated mice had minimal bronchial and alveolar inflammation compared to those of adult mock-vaccinated mice, which showed bronchial epithelial intracellular edema and necrosis with moderate inflammatory infiltration and areas of intraalveolar exudate and cellular consolidation (Fig. 4A and B). Similar to adult 1918 VLP-vaccinated mice, aged 1918 VLP-vaccinated mice showed minor alveolar involvement; however, bronchial epithelium had moderate intracellular edema, with necrotic epithelium sloughing into airway spaces (Fig. 4C). Aged mock-vaccinated mice showed severe bronchial inflammation and epithelial necrosis, but alveolar spaces were less involved than adult mock-vaccinated mice (Fig. 4D).
To evaluate the location and severity of influenza viral antigen and viral replication, immunohistochemical staining using an antibody against influenza virus A and ISH for influenza virus A was scored on 4-dpi lung sections. Adult 1918 VLP-vaccinated animals had occasional bronchial epithelium infection and viral replication and even less infection in alveolar spaces (Fig. 4E, I, and M to P). This was in contrast to significant bronchial epithelium infection and replication observed in adult mock-vaccinated and aged animals regardless of vaccination (Fig. 4F to H, J to M, and O). Alveolar spaces in adult and aged mock-vaccinated animals had pronounced influenza virus antigen and RNA, but aged VLP-vaccinated mice showed less alveolar infection than mock-vaccinated animals (Fig. 4F to H, J to L, N, and P).
To assess differences in immune responses in situ, lung sections from challenged mice were scored for the presence of macrophages, T cells, neutrophils, IgA-secreting cells, and IFN-β transcription. Macrophage and T cell infiltrates were less abundant in adult 1918 VLP-vaccinated mice than in adult mock-vaccinated and aged animals, regardless of vaccination (Fig. 5A to J). Similar results were observed with neutrophils (data not shown). Total numbers of IgA-positive cells (not influenza virus specific) were higher in aged animals than in adult mice, regardless of vaccination status (Fig. 5K to O). Immunohistochemistry for IgG and IgM was attempted, but the level of background staining precluded analysis. Adult 1918 VLP-vaccinated mice showed few IFN-β RNA foci compared to adult and aged mock-vaccinated and aged 1918 VLP-vaccinated mice (Fig. 5P to T).
To determine the magnitude of influenza virus-specific cellular responses postinfection, spleens and lungs from vaccinated animals (n = 3/group) were harvested 6 dpi, and both antibody-secreting cells (ASC) and IFN-γ-producing cells were analyzed by ELISpot assay. Although the vaccines were administered via intramuscular injection, anti-1918 HA IgG-secreting cells were not detected in significant quantity in the spleens of any animals, but 1918 VLP-specific ASC were detected in the lungs of 1918 VLP-vaccinated mice regardless of age (Fig. 6A). Importantly, both adult and aged 1918 VLP-vaccinated animals had equivalent numbers of lung ASC, which were significantly increased compared to the age-matched controls (P < 0.05).
Additionally, 1918 VLP vaccine-primed influenza virus-specific IFN-γ-producing cells were analyzed (Fig. 6B). IFN-γ production in the spleen was low to undetectable, and no significant differences were found between any of the groups regardless of stimulating antigen. Adult mice receiving the 1918 VLPs had significantly more lung IFN-γ-producing cells responding to the immunodominant peptide HA533 than adult mock-vaccinated animals (P < 0.05). Aged mice vaccinated with 1918 VLPs and stimulated with HA533 peptide had increased numbers of IFN-γ-producing cells compared to those of aged mock-vaccinated animals (P < 0.05). Similar to the ASC numbers, both adult and aged 1918 VLP-vaccinated animals had equivalent numbers of IFN-γ-producing cells after infection. Although IFN-γ-producing cells were detected in the lung using truncated 1918 HA protein or intact virus as stimulating antigens, no significant differences between groups were observed.
To evaluate the contribution of serum factors to protection, 9-week-old recipient animals (n = 5/group) were administered pooled sera via i.p. injection from adult (2 weeks postvaccination) and aged (16 months postvaccination) 1918 VLP-vaccinated animals and aged mock-vaccinated animals. At 24 h after serum transfer, mice were challenged intranasally with the 2009 pandemic virus A/Mexico/4108/2009 (1 × 106 PFU). Although this isolate does not cause a lethal infection in adult or aged BALB/c mice, the disease course in 9-week-old mice is more severe and, as such, the young mice are a more sensitive model for evaluating protective efficacy (unpublished observations). Pooled serum from adult and aged 1918 VLP-vaccinated animals was confirmed to have equivalent levels of anti-1918 antibody titers prior to transfer (total IgG endpoint dilution of 1:3,200 and HAI of 1:80 for each VLP group). Mice receiving either adult or aged 1918 VLP-vaccinated serum developed mild clinical illness, while mice receiving mock-vaccinated serum developed more severe disease (P < 0.05; 4 to 8 dpi) (Fig. 7A). Interestingly, adult 1918 VLP serum recipients resolved the clinical symptoms more rapidly than the aged 1918 VLP serum recipients (P < 0.05; 7 to 8 dpi). Mice receiving adult 1918 VLP serum had significantly less weight loss compared to both mock serum recipients (P < 0.05; 4 to 8 dpi) and aged 1918 VLP serum recipients (P < 0.05; 6 to 8 dpi) (Fig. 7B). Aged 1918 VLP serum recipients had equivalent weight loss compared to the mock serum recipients at every time point except for 8 dpi. Although the aged 1918 VLP serum recipient mice developed longer-lasting disease and lost more weight than the adult 1918 VLP serum recipients, both adult and aged 1918 VLP recipients were completely protected from death, while 80% of the mock recipients had reached experimental endpoint by 8 dpi (P < 0.01; Fig. 7C). Interestingly, both adult and aged 1918 VLP transferred serum had similar levels of IgG1, IgG2a, and IgG2b, but only the adult 1918 VLP serum had detectable IgG3 (Fig. 7D).
In this study, we evaluated the efficacy of preimmunity to 1918-derived antigens in protecting against 2009 pandemic H1N1 virus in aged mice. Sequence analysis indicated that the HA protein from the 2009 H1N1 pandemic virus is more closely related to the 1918 virus than the H1N1 strains that reemerged in humans in 1977 (39). Indeed, structural similarities between 1918 HA and 2009 pandemic HA have been elegantly described and indicate conservation within antigenic sites that is not present in contemporary seasonal HA molecules (73). Additionally, neutralizing antibodies derived from human survivors of the 1918 pandemic were able to cross-neutralize the 2009 pandemic virus, confirming the antigenic predictions (36, 76). Although severe seasonal influenza virus infections usually occur in the very young and older age groups, epidemiological evidence has indicated that elderly populations were unusually protected from severe infections during the 2009 H1N1 pandemic. Our group and others have found high levels of cross-reactive antibodies in the older populations, and it is hypothesized that this is due to prior exposure(s) to antigenically similar influenza virus (29, 32, 78). Furthermore, there is direct evidence of the protective efficacy in mice with prior exposure to 1918-like or classical swine H1N1 influenza virus to 2009 pandemic infection (39, 59). Cross-protective efficacy of older viruses to 2009 pandemic infection in animal models has provided mechanistic evidence for the observed phenomenon of decreased disease severity in the older human population but has not directly evaluated the duration of the cross-reactive immune responses. We sought to confirm and expand these findings to aged animals in order to more closely mimic the findings in elderly humans. Although our model is unable to account for multiple exposures to drifted influenza virus antigens, the results indicate that anti-1918 influenza virus immunity acquired early in life can indeed retain its cross-protective efficacy against a 2009 pandemic challenge in later stages of life.
Age-associated defects in immune responses generally lead to increased susceptibility to infectious disease and decreased responsiveness to vaccines (7). Furthermore, intrinsic defects within the B cell response directly cause decreased responses to influenza virus vaccine in elderly humans (22). Although several studies have established a defect in immune responses to vaccination in the elderly (18, 22, 23, 49), we sought to evaluate the durability and efficacy of immune responses initially elicited in young animals. We found that vaccine responses were produced efficiently in adult mice (Fig. 1A and B) and were robust enough to protect against the highly lethal reconstructed 1918 virus challenge (Fig. 1C and D). The duration of homologous receptor-blocking antibody was evaluated, and titers elicited as adults were maintained throughout the lifespan of the mouse (Fig. 2A). Although the antibody titers tended to decrease with age, these differences were not statistically significant (P > 0.05). Furthermore, we found not only that aged animals maintained equivalent levels of cross-reactive antibody titers compared to adult mice but also that sera from aged vaccinated animals had increased relative antibody avidity to the novel H1N1 HA protein compared to that of sera from adult vaccinated mice (Fig. 2). Shortly after vaccination, the antibody response is dominated by low-affinity responses produced by short-lived plasma cells that may have the benefit of being more cross-reactive due to reduced somatic mutation in response to a specific antigen (60). Prior studies have evaluated only antibody responses at 2 to 4 weeks after antigen exposure, and as such the observed cross-reactivity may be due to the kinetics of the antibody response and not completely indicative of the long-lasting antibody repertoire. Our results indicate that the cross-reactive antibodies observed in adult animals are long lasting and therefore probably not only produced by short-lived plasma cells generated immediately in response to vaccination but also maintained by LLPC that continue to produce high-affinity antibody for an entire lifetime.
An important caveat of this study is that we evaluated a single antigen and its role in eliciting cross-protective immunity. A more realistic scenario is one that includes multiple exposures, via infection or vaccination, of antigenically distinct viruses over a lifetime, as happens in the human population. Although LLPC would be unable to respond to the new antigens, an accumulation of diverse LLPC and resulting high-affinity serum antibodies could lead to even more robust cross-reactivity. In support of this idea, serologic data from humans suggest that those individuals who have anti-2009 pandemic cross-reactive antibodies are more likely to be positive for other historic viruses (78). A second, but not mutually exclusive, possibility is that memory B cells (MBC) may respond to the new antigens, and a cross-reactive epitope(s) could be specifically boosted by sequential exposure. Indeed, the increased numbers of broadly neutralizing antibody-secreting cells in response to 2009 pandemic infection in humans supports the notion that the MBC specific for a cross-reactive epitope can be activated during heterologous infection (71). The increased avidity in the aged mice supports the hypothesis of LLPC-derived antibody mediating cross-protection. Additional antigen exposures would be predicted to increase the numbers and diversity of LLPC and therefore drive an even more cross-reactive antibody profile. Therefore, the use of a single antigen in our model provides a stringent evaluation of the duration of anti-1918 immunity and its protective efficacy against 2009 pandemic challenge.
Most human influenza viruses require adaptation to cause disease in mice, except for highly pathogenic viruses, including 1918 and H5N1 isolates (3). The 2009 pandemic virus also readily infects mice, although lethality differences have been reported when comparing multiple virus isolates (39). The strain used for challenge infections in these studies (A/Mexico/4108/2009) is not lethal to adult mice but does cause significant morbidity, with infected cells being detected by in situ hybridization at 3 dpi or earlier in both bronchial and alveolar spaces. The predominant bronchiolar infection peaks at 3 dpi, while infection in alveolar spaces peaks at 3 to 5 dpi. By 10 dpi, rare infected cells are observed, and virus is cleared by 14 dpi (unpublished observations). We found that naïve aged and adult animals had morbidity profiles similar to those of aged animals displaying prolonged signs of disease (Fig. 3A). This could be due to a delayed immune response in the aged animals at both the initiation and contraction stages, leading to prolonged inflammation in the lungs (66). Vaccinated animals from both age groups did not develop any signs of morbidity in response to the infection even though high levels of virus were recovered from lungs of aged vaccinated animals 4 dpi (Fig. 3C). Although the differences were not significant between the vaccinated groups, the observation was confirmed by immunohistochemistry for influenza virus antigen and in situ hybridization for influenza virus RNA. In situ analysis of pathological responses and viral replication revealed that adult vaccinated animals were protected from infection of both bronchial and alveolar spaces, while aged vaccinated animals were protected only from alveolar infection (Fig. 4). One possible explanation for why virus was detected in the bronchial epithelium in aged vaccinated animals, but not in adult vaccinated animals, is that the initial immune response is delayed in the aged cohort (66). Similar numbers of innate immune cells were found in aged and adult animals at 4 dpi, but it is possible that the differential occurs earlier in the infection than was evaluated in these studies. Alternatively, the time from vaccination could also contribute to the differences in viral replication between adult and aged vaccinated mice. Adult vaccinated mice were challenged only 2 weeks after the final vaccination, while aged mice were challenged 16 months after final vaccination. Because of the shorter time frame, larger numbers of effector cells in adult mice could be available at the time of infection and therefore more efficiently control virus replication.
Overall in the histopathological analysis of the immune response, we observed greater infiltrates of macrophages, T cells, neutrophils, and IFN-β RNA-expressing cells in the aged and adult mock-vaccinated mice than in the adult vaccinated mice. This suggests that the intensity of the observed immune response is a reflection of the level of viral replication in the lungs. Indeed, aged vaccinated mice had viral burdens and immune infiltrate similar to those of unvaccinated mice, but surprisingly they did not lose weight or display any signs of disease. Therefore, restriction of viral replication to the bronchial spaces in aged vaccinated mice likely contributed to the less severe disease than that observed in the naïve adult or aged mice. We propose that the lack of prechallenge effector cells in the aged vaccinated animals permits bronchial infection and inflammation, but the high-affinity antibody efficiently restricts the virus from reaching the alveolar spaces. Furthermore, the high-affinity nature of the antibody from aged mice is likely critical in this scenario to overcome the initial bronchial infection and prevent alveolar spread due to the high ratio of virus to antibody. These findings suggest that despite the predominance of bronchiolar infection in this model, alveolar inflammation and/or infection play a greater contribution to the development of morbidity than bronchial infection, which is consistent with postmortem analysis of fatal human cases (25, 42, 58).
Influenza virus-specific CD8+ T cells are primarily responsible for the clearance of virus-infected cells after influenza virus infection and are detectable after primary infection by day 5 (45). One defect that is associated with the aging immune response is the reduction in CD8+ T cell function (28). Our results indicate that IFN-γ-producing T cells specific to a class I immunodominant peptide are recruited to the site of infection as efficiently in vaccinated aged animals as in adult animals (Fig. 6). Consistent with aging-associated T cell defects, a reduced, albeit not significant, number of IFN-γ-producing cells was found in naïve aged animals compared to that found in the adult controls. In addition to T cell-related age defects, B cells are also impaired (14, 21). Vaccinated animals in both age groups had equivalent levels of serum antibody prechallenge and similar numbers of antigen-specific antibody-secreting cells detectable in the lungs 6 days postinfection, while unvaccinated animals did not have any detectable antigen-specific antibody-secreting cells (Fig. 6). Interestingly, antigen-specific antibody-secreting cells were not detectable in any group prior to challenge (data not shown). The rapid recall of both T and B cells in the lungs of vaccinated animals indicates that the adaptive immune response that was primed in young animals is maintained late in life, even though the ability to respond to new antigens might be impaired.
Serum surveillance of humans has indicated that the elderly population has an increased frequency of 2009 pandemic cross-reactive antibodies (29, 32, 78). To determine if cross-reactive systemic antibody is sufficient to protect from 2009 pandemic challenge, we passively transferred immune serum from both adult and aged mice into naïve recipient mice prior to challenge. Consistent with published results, young mice (<10 weeks of age) are highly susceptible to 2009 pandemic challenge and therefore provide a sensitive model for evaluating protective efficacy (63). Serum from vaccinated mice, regardless of age group, protected mice from death, while naïve animal serum did not (Fig. 7C). Interestingly, adult serum recipients lost less weight and had reduced morbidity compared to aged serum recipients. This was not due to differences in administered serum antibody titer, as adult and aged serum had equivalent levels of both total anti-HA antibody and receptor-blocking antibody. Furthermore, the avidity of serum from aged mice for HA had a slower dissociation rate than the serum from adult mice, demonstrating that the antibody remained bound longer to HA protein. The IgG subclass profile indicated that although the adult and aged animals had equivalent levels of the dominant isotypes (IgG1, IgG2a, and IgG2b), the adult mice had low but increased amounts of IgG3 compared to those of the aged mice (Fig. 7D). IgG3 is a minor fraction of antibody to T-cell-dependent antigens and is the major isotype to T cell-independent antigens (50). Additionally, IgG3 is a potent activator of the classical complement pathway, likely due to the properties of cooperative binding (17, 26). Enhanced complement fixation mediated by the HA-specific IgG3 in adult sera could be a mechanism responsible for the decreased morbidity observed in the recipient mice. It is interesting to speculate that the loss of IgG3 in aged mice is due to LLPC being more efficiently generated in the presence of T cell help, whereas it is retained in the adult mice because of the shorter kinetics of the vaccine regimen. In addition to IgG3 variability, non-HA antibodies could also explain the observed morbidity differences. Antibodies to the NA protein of the 2009 pandemic virus are increased in elderly humans (40), and the protective role of anti-NA immunity cannot be excluded in this study. The finding that directly vaccinated animals did not display any signs of morbidity implies a critical protective role for the cellular component of the immune response in addition to the cross-reactive receptor-blocking antibodies. Cellular immunity in the presence of protective antibodies is also more predictive of protection in the context of highly pathogenic 1918 influenza virus challenge (52). Therefore, although serum antibody is sufficient to protect from severe disease and death, cellular immune responses in both the T and B cell compartments likely contribute to protection from the morbidity associated with 2009 pandemic infection in both aged and adult mice.
Viruslike particles are an intriguing platform for developing new influenza virus vaccines (9, 30, 38, 41). The VLPs are self-assembling and completely nonpathogenic particles similar in morphology to intact virions (72). For the vaccines used in this study, the influenza virus proteins HA and NA were pseudotyped onto the surface of HIV Gag particles. This strategy has been used for multiple applications and takes advantage of the robust budding properties of the Gag protein (30, 48). Lifelong antibody responses are more efficiently produced by virus infection than by nonreplicating antigens (16). We found that vaccination with 1918 influenza VLPs elicited robust lifelong immunity that was effective at protecting against heterologous 2009 pandemic virus challenge. Importantly, the cohort of vaccinated animals allowed to age was not vaccinated in the presence of adjuvant, indicating that the duration of the elicited immune response was not a function of an adjuvant. These studies were performed in mice and evaluated only a single HA antigen; however, the findings reported here indicate that VLP-based vaccinations are capable of eliciting lifelong immunity, as measured by both serum antibody (Fig. 2) and cellular recall to infection (Fig. 6).
This is the first evaluation of cross-reactive immunity to 2009 pandemic influenza virus in an aged-animal model. Antigenic similarities between the pandemic influenza virus strains of 1918 and 2009 have been demonstrated at the structural level, indicated by human data and confirmed in adult-animal models (29, 39, 73). Here, we show that animals that experience 1918 influenza virus antigens during adulthood maintain the cross-protective immunity to 2009 pandemic H1N1 influenza virus late into life. The aged animals were not protected from viral replication but restricted the virus to larger airways and did not show signs of alveolar infection, which is the most common feature of fatal human disease. The lifelong immunity evaluated in these studies was established by vaccination with a nonreplicating VLP rather than by infection and included B and T cell cross-reactive responses in addition to serum antibody. The studies reported here confirm prior work by others that 1918 influenza virus can elicit cross-reactive antibody responses to 2009 pandemic influenza virus and expands those findings to aged animals, further validating the hypothesis that decreased disease severity in the elderly human population observed during the 2009 H1N1 pandemic may be due to prior exposure to antigenically similar viruses.
This work was supported by an NIH training grant award, T32AI060525, to B.M.G., grants U01AI077771 and GM083602 to T.M.R., and the Center for Vaccine Research. In addition, T.M.R. was partially supported by a grant from the Pennsylvania Department of Health. The department specifically disclaims responsibility for any analyses, interpretations, or conclusions.
We thank Brooke Pierce for technical assistance and Hermancia Eugene and Nitin Bhardwaj for helpful discussions.
Published ahead of print 30 November 2011