Immunological Investigations, 2014; 43(3): 236–254 ! Informa Healthcare USA, Inc. ISSN: 0882-0139 print / 1532-4311 online DOI: 10.3109/08820139.2013.864665
A pandemic H1N1 influenza virus-like particle vaccine induces cross-protection in mice Kyung-Soo Inn,1 Gi-Ja Lee,2,3 and Fu-Shi Quan4 Department of Pharmaceutical Science, College of Pharmacy, Kyung Hee University, Seoul, Korea 130-701, 2 Department of Biomedical Engineering & Healthcare Industry Research Institute, College of Medicine, Kyung Hee University, Seoul, Korea 130-701, 3 Department of Medical Engineering, Kyung Hee University, Seoul, Korea 130-701, 4 Department of Medical Zoology, Kyung Hee University School of Medicine, Seoul, Korea 130-701 Influenza virus-like particles (VLPs) represent promising alternative vaccines. However, it is necessary to demonstrate that influenza VLPs confer cross-protection against antigenically distinct viruses. In this study, a VLP vaccine comprising hemagglutinin (HA) and M1 from the A/California/04/2009 (H1N1) were used and its ability to induce cross-protective efficacy against heterologous viruses A/PR/8/34 (H1N1) and A/New Caledonia/20/99 (H1N1) in mice was assessed. Vaccination with 2009 H1 VLPs induced significantly higher levels of IgG cross-reactive with these heterologous viruses after the second boost compared to after the prime or first boost. Lung virus titers also decreased significantly and the lung cross-reactive IgG response after lethal virus challenge was significantly greater in immunized mice compared to naı¨ve mice. Vaccinated mice showed 100% protection against A/PR/8/34 and A/Caledonia/20/99 viruses with only moderate body weight loss and induction of cross-reactive recall, IgG antibody-secreting cell responses. The variations in HA amino acid sequences and antigenic sites were determined and correlated with induction of cross-protective immunity. These results indicate that VLPs can be used as an effective vaccine that confers cross-protection against antigenically distinct viruses. Keywords H1N1 influenza, protection, virus-like particles
INTRODUCTION
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1
Influenza is a serious global respiratory disease, which causes seasonal epidemics and recurrent outbreaks, resulting in more than 220 000 hospitalizations annually. Approximately 36 000 people die in the United States each year, with up to millions succumbing in pandemic years (Pence et al., 2012; Perrone et al., 2009). Because current vaccines provide protection only against antigenically matched viruses, seasonal epidemics caused by antigenic variants of circulating influenza viruses or a new pandemic strain may emerge at any time.
Correspondence: Fu-Shi Quan, Department of Medical Zoology, Kyung Hee University School of Medicine, Seoul, Korea 130-701. E-mail: [email protected], [email protected]
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Influenza VLP Vaccines Induce Cross-Protection
The novel 2009 H1N1 virus spread rapidly to over 74 countries, resulting in the first influenza pandemic of the 21st century (Itoh et al., 2009; Naffakh & van der Werf, 2009). The experience with the 2009 H1N1 virus demonstrated that conventional vaccination significantly delays control of the spread of a new pandemic. Critical shortages and delays in supply of the 2009 pandemic vaccine occurred, due in part to inferior growth in egg substrates compared to seasonal vaccines. New approaches, therefore, are urgently needed to develop an effective influenza vaccine that can be rapidly produced on a large scale with low production cost. Most importantly, vaccines that induce broader cross-reactive immunity against antigenically distinct viruses should be developed. Virus-like particles are noninfectious, and so require no exceptional biosafety containment and can be manufactured rapidly. They present structurally native, immunologically relevant viral antigens. Influenza viruslike particles (VLPs), as promising vaccine candidates, induce high neutralizing antibody titers and strong protective immunity (Bright et al., 2007; Quan et al., 2007; Quan et al., 2008; Quan et al., 2009; Quan et al., 2010a; Quan et al., 2010b; Song et al., 2010). The 2009 H1N1 pandemic influenza VLPs induced robust protective immunity in mice and ferrets compared to the commercial split vaccine (Hossain et al., 2011), suggesting that influenza VLPs may represent a new vaccine platform (Hossain et al., 2011; Quan et al., 2010a). Identification of a new pandemic strain, and development and production of effective vaccines, are costly and time consuming. Thus, development of VLP vaccines that induce broad cross-reactivity against antigenically distinct viruses and provide protection against various influenza strains is highly desirable. In this study, we focused on 2009 H1N1 pandemic influenza VLPs and investigated their cross-protective efficacy against antigenically distant viruses (A/PR/8/34, A/New Caledonia/20/99) in mice. We compared amino acid sequence homologies, and antigenic sites among viruses. The 2009 H1N1 pandemic influenza VLPs provided complete protection against antigenically distinct viruses; the levels of cross-protective immune response was correlated with the amino acid sequence homology and antigenic site variation. Furthermore, we discuss potential mechanisms of the crossprotective immunity induced by influenza VLPs and strategies to improve VLP vaccines.
MATERIALS AND METHODS Viruses and cells A/California/04/2009 (H1N1) virus was kindly provided by Dr. Richard Webby and A/New Caledonia/20/99 (H1N1) virus was provided by Dr. Donald F. Smee. A/PR/8/34 (H1N1) and A/New Caledonia/20/99 (H1N1) were grown in 11-day-old embryonated hen eggs. Egg allantoic fluid was harvested and purified using a discontinuous sucrose gradient of 15%, 30% and 60%. The purified virus was inactivated by mixing with formalin at a 1:4000 (v/v) ratio, as described elsewhere (Quan et al., 2008). Inactivation of the virus was confirmed by plaque assay using confluent monolayer Madin-Darby canine kidney (MDCK) cells and inoculation into 10-day-old embryonated hen eggs. For use in challenge
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experiments, mouse-adapted A/PR/8/34, A/New Caledonia/20/99 and A/ California/04/2009 viruses were prepared as lung homogenates of infected mice. Spodoptera frugiperda Sf9 cells were maintained in suspension in serumfree SF900II medium (GIBCO-BRL, Carlsbad, CA, USA) at 27 C in spinner flasks. MDCK cells were cultured and maintained in Dulbecco’s modified Eagle’s medium (DMEM). VLP generation The PCI plasmid containing cDNA encoding hemagglutinin (HA) derived from H1N1 (A/California/04/2009) was used for polymerase chain reaction (PCR) amplification as described elsewhere (Quan et al., 2010a). Primers contained flanking restriction enzyme sites for cloning into the pFastBac plasmid expression vector. For M1 gene cloning, A/California/04/2009 virus was inoculated into MDCK cells and total viral RNA was extracted using an RNeasy Mini kit (Qiagen, Hilden Germany). Reverse transcription (RT) and PCR were performed on extracted viral RNA using the One-Step RT-PCR system (Invitrogen, Carlsbad, CA, USA) with gene-specific oligonucleotide primers, as described elsewhere (Quan et al., 2010a). Following RT-PCR, a cDNA fragment containing the M1 gene was cloned into the pFastBac vector (Invitrogen, Carlsbad, CA, USA). The nucleotide sequences of the HA and M1 genes were identical to those published previously. Recombinant baculoviruses (rBVs) expressing HA and M1 of A/California/04/2009 virus were generated as described previously (Quan et al., 2007; Quan et al., 2010a). VLPs were produced in Sf9 insect cells co-infected with recombinant BVs expressing HA and M1. VLPs released into culture supernatants were harvested, concentrated using a hollow-fiber filtration system, and subsequently purified by sucrose gradient ultracentrifugation. HA incorporated into VLPs was quantified by Western blotting and hemagglutination assay, as described previously (Quan et al., 2007; Quan et al., 2010a). VLP vaccine was characterized by Western Blotting and EM, as described previously (Quan et al., 2010a). For negative staining, sucrose gradient-purified VLPs were applied to a carbon-coated formvar grid, and stained with 1% phosphotungstic acid. Immunization and challenge Female inbred BALB/c mice (Charles River) aged 6–8 weeks were used. Groups of mice (six mice per group) were immunized intramuscularly with 10 mg (total protein) of VLPs three times at 4-week intervals. For homologous reactive control, single immunization was used. For challenge studies, naı¨ve or vaccinated mice were isoflurane-anesthetized and infected intranasally with a lethal dose (10 LD50) of A/PR/8/1934, A/New Caledonia/20/99 or A/California/ 04/2009 viruses in 50 ml of phosphate-buffered saline (PBS). Mice were observed daily to monitor changes in body weight and to record mortality, defined as a 25% loss in body weight by the Institutional Animal Care and Use Committee (IACUC) endpoint. All animal experiments and husbandry were conducted under the guidelines of the Emory University IACUC. Emory IACUC operates under the federal Animal Welfare Law, administered by the United States Department of Agriculture, and regulations of the Department of Health and Human Services.
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Influenza VLP Vaccines Induce Cross-Protection
Antibody responses, virus neutralizing activities and hemagglutination inhibition (HAI) titers Blood samples were collected by retro-orbital plexus puncture at week 3 after prime, first boost and second boost. Lung homogenates were obtained at day 4 after challenge. Influenza virus-specific serum IgG, IgG1 and IgG2a crossreactive with A/PR/8/1934 or A/New Caledonia/20/99 were determined by enzyme-linked immunosorbent assay (ELISA), as described previously (Quan et al., 2009). Serum IgG, IgG1 and IgG2a responses reactive to A/California/04/ 2009 were also determined as controls. Cross-reactive lung IgG level was also determined. For assays of cross reactive antibodies, inactivated egg-grown A/PR/8/34 or A/Caledonia/20/99 viruses were coated onto 96-well microtiter plates (Nunc Life Technologies, Rochester, NY). Cross-reactive serum neutralizing activities against A/PR/8/34 and A/New Caledonia/99 were determined by plaque assay using MDCK cells following a procedure described previously (Quan et al., 2012). For homologous response controls, inactivated egg-grown A/California/04/2009 virus was used. Lung viral titers A lung virus titer assay was performed using MDCK cells following a procedure described previously (Quan et al. 2007, Quan et al. 2009). The whole lung extracts prepared as homogenates using frosted glass slides were centrifuged at 1000 rpm for 10 min to collect supernatants. Serially diluted lung homogenates were added to the cell monolayers. After incubation for 2–3 days, the cells were fixed with 0.25% glutaraldehyde and stained with 1% crystal violet to visualize plaques. Detection of cross-reactive recall, IgG antibody-secreting cell responses in the spleen and bone marrow To detect influenza virus-specific antibody-secreting cells, inactivated viral antigens (A/PR8, A/Caledonia/20/99) were used to coat 96-well culture plates (Costar, Corning, NY, USA). Inactivated A/California/04/2009 was coated for the homologous reactive control. Freshly isolated cells from the spleen (1 106 cells) were added to each well and incubated for 2 days at 37 C in 5% CO2. After removing cells from the culture plates, horseradish peroxidase (HRP)conjugated secondary goat-anti-mouse antibodies were added. As a measure of HRP activity, the substrate O-phenylendiamine (Zymed, San Francisco, CA) was used and the optical density at 450 nm was read. Cytokine assays All antibodies against mouse cytokines used in cytokine ELISPOT assays were purchased from BD/PharMingen (San Diego, Calif.). Anti-mouse gamma interferon (IFN-g), interleukin-2 (IL-2), IL-4, and IL-5 antibodies (3mg/ml in coating buffer) were used to coat Multiscreen 96-well filtration plates (Millipore). Freshly isolated splenocytes (1.5 106 cells) were added to each well and stimulated with a pool of peptide arrays of hemagglutinin protein from A/PR/8/34 (NR-18973), A/New Caledonia/20/1999 (H1N1) (NR-2703) or A/California/04/2009 (NR-19244) at a concentration of 10 mg/ml (Quan et al., 2007). These reagents were obtained through BEI Resources, NIAID, NIH. The plates were incubated for 36 h at 37 C with 5% CO2. Development and
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counting of cytokine ELISPOTs were performed as described previously (Quan et al., 2007). Sequence data analyses The sequence homology and divergence between A/California/04/2009 (ACP41105) and A/PR/8/34 (ABD77675) and A/Caledonia/20/99 (AJ344014) were determined by aligning HA amino acid sequences using ClustalW (http:// www.ebi.ac.uk/Tools/clustalw2/index.html). To determine variations in sites of viral attachment to host cells, the amino acid sequences of HA glycosylation sites were compared using the NetNGlyc 1.0 software (http://www.cbs.dtu.dk/ services/NetNGlyc/). The antigenic divergence was calculated by the CTL epitope prediction method (http://www.imtech.res.in/raghava/ctlpred/) as described (Bhasin & Raghava, 2004). Statistics All parameters were recorded for all individuals within groups. Statistical comparisons of data were carried out using PC-SAS system. p values 50.05 were considered to indicate statistical significance.
RESULTS Production and characterization of VLPs Pandemic H1N1 influenza VLPs were produced in insect cells co-infected with rBVs expressing the M1 matrix and HA glycoprotein derived from A/California/04/09 (H1N1) virus as described previously (Quan et al., 2010b). Incorporation of HA and M1 into VLPs was confirmed by silver-stained SDSPAGE and Western blotting using immune sera from mice infected with the A/California/2009 virus (data not shown). Hemagglutination activity and electron microscopy indicated that influenza VLPs produced in insect cells were structurally intact, resembling influenza virions in both morphology and size (data not shown). VLP vaccination elicits cross-reactive serum antibody responses Induction of cross-reactive antibodies is an important goal of vaccination. To determine antibody cross-reactivity with antigenically different H1N1 viruses (A/PR/8/34 and A/New Caledonia/20/99), groups of mice (n ¼ 6) were immunized intramuscularly with 10 mg of VLPs. Vaccine was administered as three immunizations at 4-week intervals, and the levels of total IgG crossreactive to A/PR/8/34 and A/New Caledonia/20/99 were determined (Figure 1). Homologous reactive serum antibody response was also determined as control (Figure 1B). Higher levels of cross-reactive IgG antibodies against heterologous viruses (A/PR/8/34, A/New Caledonia/20/99) were identified in the second boost immune sera compared to those after the prime or first boost (Figure 1A,C,D), indicating cross-reactivity between 2009 H1N1 and A/PR/8/ 34 and A/New Caledonia/20/99. Higher levels of virus-specific IgG crossreactive to A/PR/8/34 were elicited than cross-reactive to A/New Caledonia/20/ 99 (Figure 1A,C,D) after prime, first boost, and second boost, indicating that A/PR/8/34 is antigenically more closely related to A/California/04/09
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Figure 1. Cross-reactive serum IgG antibody responses. Mice (n ¼ 6) were immunized intramuscularly with 2009 pandemic H1N1 VLPs. Blood samples were collected by retroorbital plexus puncture at week 3 after prime, first boost and second boost. Crossreactive serum IgG responses against A/PR/8/34 or A/New Caledonia/20/99 viruses were determined by ELISA after prime (A), first boost (C) and second boost (D). Homologous reactive serum IgG responses against A/California/04/2009 were determined by ELISA after prime (B).
than A/Caledonia/20/99. In terms of antibody isotype, IgG2a cross-reactive with A/PR/8/34 and A/New Caledonia/20/99 was induced at higher levels (Figure 2A–F), indicating T helper type 1 (Th1) antibody responses. Higher levels of IgG2a cross-reactive with A/PR8/1934 were observed in immune sera than those cross-reactive with A/New Caledonia/20/99 after prime, first boost, and second boost. Taken together, these results indicate that 2009 H1N1 VLPs induce cross-reactive, virus-specific total IgG and IgG2a dominant antibodies against antigenically distinct influenza viruses. Induction of cross-reactive serum viral neutralizing antibodies We determined cross-reactive serum viral neutralizing activity (Figure 3A,B) against A/PR/8/34 and A/New Caledonia/20/99 viruses. Immune sera from immunized mice against A/PR/8/34 showed lower reduction of plaque formation than that against A/New Caledonia/20/99 at 5 and 25 serum dilution. However, immune sera showed higher plaque reduction against A/new Caledonia/20/99 than A/PR8/8/34 at 125 serum dilution. Significantly lower cross-reactive viral neutralizing antibodies were detected against A/PR/8/34 and A/New Caledonia/20/99 (Figure 3A,B) than homologous viral neutralizing antibodies against A/California/04/2009 viruses (Figure 3C). Cross-reactive HAI activities against A/PR/8/34 or A/Caledonia/20/99 viruses were also low (data not shown). Serum neutralizing activity and HAI may not be effective against antigenically distinct viruses.
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Figure 2. Cross-reactive serum IgG2a and IgG1 responses. Mice (n ¼ 6) were immunized intramuscularly with 2009 pandemic H1N1 VLPs. Blood samples were collected by retro-orbital plexus puncture at week 3 after prime, first boost and second boost. IgG2a and IgG1 serum antibodies cross-reactive to A/PR/8/34 (A–C) or A/Caledonia/20/99 (D–F) viruses were determined. Homologous reactive serum IgG2a and IgG1 antibody responses against A/California/04/2009 were determined by ELISA after prime (G).
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Influenza VLP Vaccines Induce Cross-Protection A/PR8/34
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Figure 3. Cross-reactive viral neutralizing antibodies. Sera from 6 vaccinated mice at week 3 after the final immunization were used for cross-reactive neutralizing activity. Cross-reactive viral neutralizing activities against A/PR/8/34 and A/New Caledonia/20/99 were expressed as the percentage of plaque reduction compared to that of naı¨ve serum control. Homologous reactive viral neutralizing antibodies against A/California/04/ 2009 were determined (C).
Protection against lethal challenge with A/PR/8/34 or A/New Caledonia/ 20/99 viruses To determine the cross-protective efficacy of VLP vaccination, vaccinated and naı¨ve mice were challenged with a lethal dose of the antigenically distinct strains, A/PR/8/34 (10 LD50) or A/New Caledonia/20/99 (10 LD50). A/California/04/2009 (10 LD50) was used to determine homologous protection. All naı¨ve mice showed progressive reductions in body weight and all died by post-infection day 8 after challenge with A/PR/8/34 or A/New Caledonia/20/99 (Figure 4A,B,D,E). In contrast, 100% of vaccinated mice survived and exhibited only 5% and 15% transient reductions in body weight after challenge with A/PR/8/34 and A/New Caledonia/20/99, respectively (Figure 4A,B,D,E). Complete protections were induced after homologous A/California/04/2009 virus challenge infection (Figure 4C,F). These results suggest that the influenza HA VLP vaccine induces cross-protective immunity, which was more effective against A/PR/8/34 than against A/New Caledonia/20/ 99 (Figure 4A–D).
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Figure 4. Cross-protective efficacies against A/PR/8/34 and A/New Caledonia/20/99 viruses. Vaccinated mice (n ¼ 6) were challenged with A/PR/8/34 (A, D) or A/New Caledonia/20/99 (B, E) viruses after the second boost and body weight (A, B) and survival rate (D, E) were measured daily. Body weight loss greater than 25% was recorded as dead. Naı¨ve mice were used as controls. Protective efficacies against homologous A/California/04/2009 were measured (CF).
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Effective control of lung viral replication The efficiency of virus clearance from the lung provides a sensitive indicator of protective efficacy. Mice were sacrificed and viral titers in lung extracts determined at day 4 post-challenge with A/PR/8/34 or A/New Caledonia/20/99 (Figure 5). All naı¨ve-infected controls showed high viral loads in the lungs. In contrast, VLP-vaccinated mice showed significantly lower lung viral titers. Vaccinated mice challenged with A/PR/8/34 and A/New Caledonia/20/99 viruses showed 10- and 3.5-fold reductions in lung viral titers, respectively, compared to naı¨ve-infected controls, indicating induction of greater protective efficacy against A/PR/8/34 than against A/New Caledonia/20/99 (Figure 5). No virus was detected after homologous A/California/04/2009 virus challenge infection (Figure 5). Therefore, VLP vaccination can induce protective immune responses, which can control the replication of antigenically distant viruses. VLP vaccination induces cross-reactive lung IgG antibody responses We determined the levels of antibodies in lungs at day 4 post-challenge (Figure 6A). Higher levels of IgG antibodies cross-reactive to A/PR/8/1934 or A/New Caledonia/20/99 viruses were determined. As controls, much higher lung IgG antibodies were detected against homologous A/California viruses (Figure 6A). Therefore, VLP vaccination induces cross-reactive IgG in the lungs at an early timepoint post-challenge, indicating that IgG may lead to a reduction in the lung viral load. 90 Lung virus titer (PFU/ml) (X104)
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Influenza VLP Vaccines Induce Cross-Protection
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A pandemic H1N1 influenza virus-like particle vaccine induces cross-protection in mice Kyung-Soo Inn,1 Gi-Ja Lee,2,3 and Fu-Shi Quan4 Department of Pharmaceutical Science, College of Pharmacy, Kyung Hee University, Seoul, Korea 130-701, 2 Department of Biomedical Engineering & Healthcare Industry Research Institute, College of Medicine, Kyung Hee University, Seoul, Korea 130-701, 3 Department of Medical Engineering, Kyung Hee University, Seoul, Korea 130-701, 4 Department of Medical Zoology, Kyung Hee University School of Medicine, Seoul, Korea 130-701 Influenza virus-like particles (VLPs) represent promising alternative vaccines. However, it is necessary to demonstrate that influenza VLPs confer cross-protection against antigenically distinct viruses. In this study, a VLP vaccine comprising hemagglutinin (HA) and M1 from the A/California/04/2009 (H1N1) were used and its ability to induce cross-protective efficacy against heterologous viruses A/PR/8/34 (H1N1) and A/New Caledonia/20/99 (H1N1) in mice was assessed. Vaccination with 2009 H1 VLPs induced significantly higher levels of IgG cross-reactive with these heterologous viruses after the second boost compared to after the prime or first boost. Lung virus titers also decreased significantly and the lung cross-reactive IgG response after lethal virus challenge was significantly greater in immunized mice compared to naı¨ve mice. Vaccinated mice showed 100% protection against A/PR/8/34 and A/Caledonia/20/99 viruses with only moderate body weight loss and induction of cross-reactive recall, IgG antibody-secreting cell responses. The variations in HA amino acid sequences and antigenic sites were determined and correlated with induction of cross-protective immunity. These results indicate that VLPs can be used as an effective vaccine that confers cross-protection against antigenically distinct viruses. Keywords H1N1 influenza, protection, virus-like particles
INTRODUCTION
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1
Influenza is a serious global respiratory disease, which causes seasonal epidemics and recurrent outbreaks, resulting in more than 220 000 hospitalizations annually. Approximately 36 000 people die in the United States each year, with up to millions succumbing in pandemic years (Pence et al., 2012; Perrone et al., 2009). Because current vaccines provide protection only against antigenically matched viruses, seasonal epidemics caused by antigenic variants of circulating influenza viruses or a new pandemic strain may emerge at any time.
Correspondence: Fu-Shi Quan, Department of Medical Zoology, Kyung Hee University School of Medicine, Seoul, Korea 130-701. E-mail: [email protected], [email protected]
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Influenza VLP Vaccines Induce Cross-Protection
The novel 2009 H1N1 virus spread rapidly to over 74 countries, resulting in the first influenza pandemic of the 21st century (Itoh et al., 2009; Naffakh & van der Werf, 2009). The experience with the 2009 H1N1 virus demonstrated that conventional vaccination significantly delays control of the spread of a new pandemic. Critical shortages and delays in supply of the 2009 pandemic vaccine occurred, due in part to inferior growth in egg substrates compared to seasonal vaccines. New approaches, therefore, are urgently needed to develop an effective influenza vaccine that can be rapidly produced on a large scale with low production cost. Most importantly, vaccines that induce broader cross-reactive immunity against antigenically distinct viruses should be developed. Virus-like particles are noninfectious, and so require no exceptional biosafety containment and can be manufactured rapidly. They present structurally native, immunologically relevant viral antigens. Influenza viruslike particles (VLPs), as promising vaccine candidates, induce high neutralizing antibody titers and strong protective immunity (Bright et al., 2007; Quan et al., 2007; Quan et al., 2008; Quan et al., 2009; Quan et al., 2010a; Quan et al., 2010b; Song et al., 2010). The 2009 H1N1 pandemic influenza VLPs induced robust protective immunity in mice and ferrets compared to the commercial split vaccine (Hossain et al., 2011), suggesting that influenza VLPs may represent a new vaccine platform (Hossain et al., 2011; Quan et al., 2010a). Identification of a new pandemic strain, and development and production of effective vaccines, are costly and time consuming. Thus, development of VLP vaccines that induce broad cross-reactivity against antigenically distinct viruses and provide protection against various influenza strains is highly desirable. In this study, we focused on 2009 H1N1 pandemic influenza VLPs and investigated their cross-protective efficacy against antigenically distant viruses (A/PR/8/34, A/New Caledonia/20/99) in mice. We compared amino acid sequence homologies, and antigenic sites among viruses. The 2009 H1N1 pandemic influenza VLPs provided complete protection against antigenically distinct viruses; the levels of cross-protective immune response was correlated with the amino acid sequence homology and antigenic site variation. Furthermore, we discuss potential mechanisms of the crossprotective immunity induced by influenza VLPs and strategies to improve VLP vaccines.
MATERIALS AND METHODS Viruses and cells A/California/04/2009 (H1N1) virus was kindly provided by Dr. Richard Webby and A/New Caledonia/20/99 (H1N1) virus was provided by Dr. Donald F. Smee. A/PR/8/34 (H1N1) and A/New Caledonia/20/99 (H1N1) were grown in 11-day-old embryonated hen eggs. Egg allantoic fluid was harvested and purified using a discontinuous sucrose gradient of 15%, 30% and 60%. The purified virus was inactivated by mixing with formalin at a 1:4000 (v/v) ratio, as described elsewhere (Quan et al., 2008). Inactivation of the virus was confirmed by plaque assay using confluent monolayer Madin-Darby canine kidney (MDCK) cells and inoculation into 10-day-old embryonated hen eggs. For use in challenge
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experiments, mouse-adapted A/PR/8/34, A/New Caledonia/20/99 and A/ California/04/2009 viruses were prepared as lung homogenates of infected mice. Spodoptera frugiperda Sf9 cells were maintained in suspension in serumfree SF900II medium (GIBCO-BRL, Carlsbad, CA, USA) at 27 C in spinner flasks. MDCK cells were cultured and maintained in Dulbecco’s modified Eagle’s medium (DMEM). VLP generation The PCI plasmid containing cDNA encoding hemagglutinin (HA) derived from H1N1 (A/California/04/2009) was used for polymerase chain reaction (PCR) amplification as described elsewhere (Quan et al., 2010a). Primers contained flanking restriction enzyme sites for cloning into the pFastBac plasmid expression vector. For M1 gene cloning, A/California/04/2009 virus was inoculated into MDCK cells and total viral RNA was extracted using an RNeasy Mini kit (Qiagen, Hilden Germany). Reverse transcription (RT) and PCR were performed on extracted viral RNA using the One-Step RT-PCR system (Invitrogen, Carlsbad, CA, USA) with gene-specific oligonucleotide primers, as described elsewhere (Quan et al., 2010a). Following RT-PCR, a cDNA fragment containing the M1 gene was cloned into the pFastBac vector (Invitrogen, Carlsbad, CA, USA). The nucleotide sequences of the HA and M1 genes were identical to those published previously. Recombinant baculoviruses (rBVs) expressing HA and M1 of A/California/04/2009 virus were generated as described previously (Quan et al., 2007; Quan et al., 2010a). VLPs were produced in Sf9 insect cells co-infected with recombinant BVs expressing HA and M1. VLPs released into culture supernatants were harvested, concentrated using a hollow-fiber filtration system, and subsequently purified by sucrose gradient ultracentrifugation. HA incorporated into VLPs was quantified by Western blotting and hemagglutination assay, as described previously (Quan et al., 2007; Quan et al., 2010a). VLP vaccine was characterized by Western Blotting and EM, as described previously (Quan et al., 2010a). For negative staining, sucrose gradient-purified VLPs were applied to a carbon-coated formvar grid, and stained with 1% phosphotungstic acid. Immunization and challenge Female inbred BALB/c mice (Charles River) aged 6–8 weeks were used. Groups of mice (six mice per group) were immunized intramuscularly with 10 mg (total protein) of VLPs three times at 4-week intervals. For homologous reactive control, single immunization was used. For challenge studies, naı¨ve or vaccinated mice were isoflurane-anesthetized and infected intranasally with a lethal dose (10 LD50) of A/PR/8/1934, A/New Caledonia/20/99 or A/California/ 04/2009 viruses in 50 ml of phosphate-buffered saline (PBS). Mice were observed daily to monitor changes in body weight and to record mortality, defined as a 25% loss in body weight by the Institutional Animal Care and Use Committee (IACUC) endpoint. All animal experiments and husbandry were conducted under the guidelines of the Emory University IACUC. Emory IACUC operates under the federal Animal Welfare Law, administered by the United States Department of Agriculture, and regulations of the Department of Health and Human Services.
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Influenza VLP Vaccines Induce Cross-Protection
Antibody responses, virus neutralizing activities and hemagglutination inhibition (HAI) titers Blood samples were collected by retro-orbital plexus puncture at week 3 after prime, first boost and second boost. Lung homogenates were obtained at day 4 after challenge. Influenza virus-specific serum IgG, IgG1 and IgG2a crossreactive with A/PR/8/1934 or A/New Caledonia/20/99 were determined by enzyme-linked immunosorbent assay (ELISA), as described previously (Quan et al., 2009). Serum IgG, IgG1 and IgG2a responses reactive to A/California/04/ 2009 were also determined as controls. Cross-reactive lung IgG level was also determined. For assays of cross reactive antibodies, inactivated egg-grown A/PR/8/34 or A/Caledonia/20/99 viruses were coated onto 96-well microtiter plates (Nunc Life Technologies, Rochester, NY). Cross-reactive serum neutralizing activities against A/PR/8/34 and A/New Caledonia/99 were determined by plaque assay using MDCK cells following a procedure described previously (Quan et al., 2012). For homologous response controls, inactivated egg-grown A/California/04/2009 virus was used. Lung viral titers A lung virus titer assay was performed using MDCK cells following a procedure described previously (Quan et al. 2007, Quan et al. 2009). The whole lung extracts prepared as homogenates using frosted glass slides were centrifuged at 1000 rpm for 10 min to collect supernatants. Serially diluted lung homogenates were added to the cell monolayers. After incubation for 2–3 days, the cells were fixed with 0.25% glutaraldehyde and stained with 1% crystal violet to visualize plaques. Detection of cross-reactive recall, IgG antibody-secreting cell responses in the spleen and bone marrow To detect influenza virus-specific antibody-secreting cells, inactivated viral antigens (A/PR8, A/Caledonia/20/99) were used to coat 96-well culture plates (Costar, Corning, NY, USA). Inactivated A/California/04/2009 was coated for the homologous reactive control. Freshly isolated cells from the spleen (1 106 cells) were added to each well and incubated for 2 days at 37 C in 5% CO2. After removing cells from the culture plates, horseradish peroxidase (HRP)conjugated secondary goat-anti-mouse antibodies were added. As a measure of HRP activity, the substrate O-phenylendiamine (Zymed, San Francisco, CA) was used and the optical density at 450 nm was read. Cytokine assays All antibodies against mouse cytokines used in cytokine ELISPOT assays were purchased from BD/PharMingen (San Diego, Calif.). Anti-mouse gamma interferon (IFN-g), interleukin-2 (IL-2), IL-4, and IL-5 antibodies (3mg/ml in coating buffer) were used to coat Multiscreen 96-well filtration plates (Millipore). Freshly isolated splenocytes (1.5 106 cells) were added to each well and stimulated with a pool of peptide arrays of hemagglutinin protein from A/PR/8/34 (NR-18973), A/New Caledonia/20/1999 (H1N1) (NR-2703) or A/California/04/2009 (NR-19244) at a concentration of 10 mg/ml (Quan et al., 2007). These reagents were obtained through BEI Resources, NIAID, NIH. The plates were incubated for 36 h at 37 C with 5% CO2. Development and
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counting of cytokine ELISPOTs were performed as described previously (Quan et al., 2007). Sequence data analyses The sequence homology and divergence between A/California/04/2009 (ACP41105) and A/PR/8/34 (ABD77675) and A/Caledonia/20/99 (AJ344014) were determined by aligning HA amino acid sequences using ClustalW (http:// www.ebi.ac.uk/Tools/clustalw2/index.html). To determine variations in sites of viral attachment to host cells, the amino acid sequences of HA glycosylation sites were compared using the NetNGlyc 1.0 software (http://www.cbs.dtu.dk/ services/NetNGlyc/). The antigenic divergence was calculated by the CTL epitope prediction method (http://www.imtech.res.in/raghava/ctlpred/) as described (Bhasin & Raghava, 2004). Statistics All parameters were recorded for all individuals within groups. Statistical comparisons of data were carried out using PC-SAS system. p values 50.05 were considered to indicate statistical significance.
RESULTS Production and characterization of VLPs Pandemic H1N1 influenza VLPs were produced in insect cells co-infected with rBVs expressing the M1 matrix and HA glycoprotein derived from A/California/04/09 (H1N1) virus as described previously (Quan et al., 2010b). Incorporation of HA and M1 into VLPs was confirmed by silver-stained SDSPAGE and Western blotting using immune sera from mice infected with the A/California/2009 virus (data not shown). Hemagglutination activity and electron microscopy indicated that influenza VLPs produced in insect cells were structurally intact, resembling influenza virions in both morphology and size (data not shown). VLP vaccination elicits cross-reactive serum antibody responses Induction of cross-reactive antibodies is an important goal of vaccination. To determine antibody cross-reactivity with antigenically different H1N1 viruses (A/PR/8/34 and A/New Caledonia/20/99), groups of mice (n ¼ 6) were immunized intramuscularly with 10 mg of VLPs. Vaccine was administered as three immunizations at 4-week intervals, and the levels of total IgG crossreactive to A/PR/8/34 and A/New Caledonia/20/99 were determined (Figure 1). Homologous reactive serum antibody response was also determined as control (Figure 1B). Higher levels of cross-reactive IgG antibodies against heterologous viruses (A/PR/8/34, A/New Caledonia/20/99) were identified in the second boost immune sera compared to those after the prime or first boost (Figure 1A,C,D), indicating cross-reactivity between 2009 H1N1 and A/PR/8/ 34 and A/New Caledonia/20/99. Higher levels of virus-specific IgG crossreactive to A/PR/8/34 were elicited than cross-reactive to A/New Caledonia/20/ 99 (Figure 1A,C,D) after prime, first boost, and second boost, indicating that A/PR/8/34 is antigenically more closely related to A/California/04/09
Influenza VLP Vaccines Induce Cross-Protection Prime
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Figure 1. Cross-reactive serum IgG antibody responses. Mice (n ¼ 6) were immunized intramuscularly with 2009 pandemic H1N1 VLPs. Blood samples were collected by retroorbital plexus puncture at week 3 after prime, first boost and second boost. Crossreactive serum IgG responses against A/PR/8/34 or A/New Caledonia/20/99 viruses were determined by ELISA after prime (A), first boost (C) and second boost (D). Homologous reactive serum IgG responses against A/California/04/2009 were determined by ELISA after prime (B).
than A/Caledonia/20/99. In terms of antibody isotype, IgG2a cross-reactive with A/PR/8/34 and A/New Caledonia/20/99 was induced at higher levels (Figure 2A–F), indicating T helper type 1 (Th1) antibody responses. Higher levels of IgG2a cross-reactive with A/PR8/1934 were observed in immune sera than those cross-reactive with A/New Caledonia/20/99 after prime, first boost, and second boost. Taken together, these results indicate that 2009 H1N1 VLPs induce cross-reactive, virus-specific total IgG and IgG2a dominant antibodies against antigenically distinct influenza viruses. Induction of cross-reactive serum viral neutralizing antibodies We determined cross-reactive serum viral neutralizing activity (Figure 3A,B) against A/PR/8/34 and A/New Caledonia/20/99 viruses. Immune sera from immunized mice against A/PR/8/34 showed lower reduction of plaque formation than that against A/New Caledonia/20/99 at 5 and 25 serum dilution. However, immune sera showed higher plaque reduction against A/new Caledonia/20/99 than A/PR8/8/34 at 125 serum dilution. Significantly lower cross-reactive viral neutralizing antibodies were detected against A/PR/8/34 and A/New Caledonia/20/99 (Figure 3A,B) than homologous viral neutralizing antibodies against A/California/04/2009 viruses (Figure 3C). Cross-reactive HAI activities against A/PR/8/34 or A/Caledonia/20/99 viruses were also low (data not shown). Serum neutralizing activity and HAI may not be effective against antigenically distinct viruses.
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Figure 2. Cross-reactive serum IgG2a and IgG1 responses. Mice (n ¼ 6) were immunized intramuscularly with 2009 pandemic H1N1 VLPs. Blood samples were collected by retro-orbital plexus puncture at week 3 after prime, first boost and second boost. IgG2a and IgG1 serum antibodies cross-reactive to A/PR/8/34 (A–C) or A/Caledonia/20/99 (D–F) viruses were determined. Homologous reactive serum IgG2a and IgG1 antibody responses against A/California/04/2009 were determined by ELISA after prime (G).
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Influenza VLP Vaccines Induce Cross-Protection A/PR8/34
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Figure 3. Cross-reactive viral neutralizing antibodies. Sera from 6 vaccinated mice at week 3 after the final immunization were used for cross-reactive neutralizing activity. Cross-reactive viral neutralizing activities against A/PR/8/34 and A/New Caledonia/20/99 were expressed as the percentage of plaque reduction compared to that of naı¨ve serum control. Homologous reactive viral neutralizing antibodies against A/California/04/ 2009 were determined (C).
Protection against lethal challenge with A/PR/8/34 or A/New Caledonia/ 20/99 viruses To determine the cross-protective efficacy of VLP vaccination, vaccinated and naı¨ve mice were challenged with a lethal dose of the antigenically distinct strains, A/PR/8/34 (10 LD50) or A/New Caledonia/20/99 (10 LD50). A/California/04/2009 (10 LD50) was used to determine homologous protection. All naı¨ve mice showed progressive reductions in body weight and all died by post-infection day 8 after challenge with A/PR/8/34 or A/New Caledonia/20/99 (Figure 4A,B,D,E). In contrast, 100% of vaccinated mice survived and exhibited only 5% and 15% transient reductions in body weight after challenge with A/PR/8/34 and A/New Caledonia/20/99, respectively (Figure 4A,B,D,E). Complete protections were induced after homologous A/California/04/2009 virus challenge infection (Figure 4C,F). These results suggest that the influenza HA VLP vaccine induces cross-protective immunity, which was more effective against A/PR/8/34 than against A/New Caledonia/20/ 99 (Figure 4A–D).
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Figure 4. Cross-protective efficacies against A/PR/8/34 and A/New Caledonia/20/99 viruses. Vaccinated mice (n ¼ 6) were challenged with A/PR/8/34 (A, D) or A/New Caledonia/20/99 (B, E) viruses after the second boost and body weight (A, B) and survival rate (D, E) were measured daily. Body weight loss greater than 25% was recorded as dead. Naı¨ve mice were used as controls. Protective efficacies against homologous A/California/04/2009 were measured (CF).
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Effective control of lung viral replication The efficiency of virus clearance from the lung provides a sensitive indicator of protective efficacy. Mice were sacrificed and viral titers in lung extracts determined at day 4 post-challenge with A/PR/8/34 or A/New Caledonia/20/99 (Figure 5). All naı¨ve-infected controls showed high viral loads in the lungs. In contrast, VLP-vaccinated mice showed significantly lower lung viral titers. Vaccinated mice challenged with A/PR/8/34 and A/New Caledonia/20/99 viruses showed 10- and 3.5-fold reductions in lung viral titers, respectively, compared to naı¨ve-infected controls, indicating induction of greater protective efficacy against A/PR/8/34 than against A/New Caledonia/20/99 (Figure 5). No virus was detected after homologous A/California/04/2009 virus challenge infection (Figure 5). Therefore, VLP vaccination can induce protective immune responses, which can control the replication of antigenically distant viruses. VLP vaccination induces cross-reactive lung IgG antibody responses We determined the levels of antibodies in lungs at day 4 post-challenge (Figure 6A). Higher levels of IgG antibodies cross-reactive to A/PR/8/1934 or A/New Caledonia/20/99 viruses were determined. As controls, much higher lung IgG antibodies were detected against homologous A/California viruses (Figure 6A). Therefore, VLP vaccination induces cross-reactive IgG in the lungs at an early timepoint post-challenge, indicating that IgG may lead to a reduction in the lung viral load. 90 Lung virus titer (PFU/ml) (X104)
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Influenza VLP Vaccines Induce Cross-Protection
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