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Intranasal immunization with influenza antigens conjugated with cholera toxin subunit B stimulates broad spectrum immunity against influenza viruses a
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Junwei Li , Maria T Arévalo , Yanping Chen , Olivia Posadas , Jacob A Smith & Mingtao Zeng
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Center of Excellence for Infectious Diseases; Paul L. Foster School of Medicine; Texas Tech University Health Sciences Center; El Paso, TX USA Published online: 14 Mar 2014.
Click for updates To cite this article: Junwei Li, Maria T Arévalo, Yanping Chen, Olivia Posadas, Jacob A Smith & Mingtao Zeng (2014) Intranasal immunization with influenza antigens conjugated with cholera toxin subunit B stimulates broad spectrum immunity against influenza viruses, Human Vaccines & Immunotherapeutics, 10:5, 1211-1220, DOI: 10.4161/hv.28407 To link to this article: http://dx.doi.org/10.4161/hv.28407
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Research Paper
Research Paper
Human Vaccines & Immunotherapeutics 10:5, 1211–1220; May 2014; © 2014 Landes Bioscience
Intranasal immunization with influenza antigens conjugated with cholera toxin subunit B stimulates broad spectrum immunity against influenza viruses Center of Excellence for Infectious Diseases; Paul L. Foster School of Medicine; Texas Tech University Health Sciences Center; El Paso, TX USA
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Keywords: broad spectrum vaccine; cholera toxin subunit B; influenza; intranasal vaccination; protective immunity
Frequent mutation of influenza viruses keep vaccinated and non-vaccinated populations vulnerable to new infections, causing serious burdens to public health and the economy. Vaccination with universal influenza vaccines would be the best way to effectively protect people from infection caused by mismatched or unforeseen influenza viruses. Presently, there is no FDA approved universal influenza vaccine. In this study, we expressed and purified a fusion protein comprising of influenza matrix 2 protein ectodomain peptides, a centralized influenza hemagglutinin stem region, and cholera toxin subunit B. Vaccination of BALB/c mice with this novel artificial antigen resulted in potent humoral immune responses, including induction of specific IgA and IgG, and broad protection against infection by multiple influenza viruses. Furthermore, our results demonstrated that when used as a mucosal antigen, cholera toxin subunit B improved antigen-stimulated T cell and memory B cell responses.
Introduction Infection by influenza viruses is a very important public health concern. The effective way to prevent influenza virus infection is through vaccination. Traditional influenza vaccine formulations are based on inactivated or attenuated influenza viruses that induce B cell responses to produce neutralizing antibodies matching the predicted seasonal influenza viruses. Sometimes, traditional or seasonal influenza vaccines fail to protect against influenza viruses due to the frequent mutations of influenza viruses, even though the vaccines are reformulated annually according to surveillance data on epidemic of influenza viruses. As an example, the 2009–2010 season influenza vaccines did not protect against the unforeseen 2009 H1N1 pandemic influenza which broke out in 215 countries and caused at least 18 000 deaths worldwide.1,2 Pandemic strains are potentially devastating because the majority of the affected population lacks potent immune responses to clear the infecting virus. It is alarming that vaccine mismatches occurred at around 50% in United States from 1997 to 2005.3 Clearly, a universal influenza vaccine that would protect against a majority of circulating or potential epidemic influenza virus strains is pressingly needed. This universal vaccine would obviate the annual reformulation and repeated annual immunization. The most important aspect of universal influenza vaccine design is selection of conserved antigens and an appropriate
adjuvant.4-9 HA2 of influenza virus is located at the C-terminal of hemagglutinin (HA), and forms a stem-like structure that mediates the anchoring and fusion of the influenza viral membrane to the endosomal membrane. HA2-specific antibodies are able to reduce the replication of influenza by inhibiting this process.10 Compared with the HA1 portion of HA, HA2 is more conserved. Therefore, HA2 may be used in the formulation of a universal vaccine as it may provide cross-protection against many different viral strains. Matrix 2 (M2) protein of influenza A virus is a conserved structural protein incorporated into the viral lipid envelope. The 23-amino acid extracellular N-terminal domain peptide of M2 (M2e) is highly conserved among all influenza A viruses.11-15 Immunization with M2e confers cross-protection against various subtypes of influenza virus.16 However, we speculate that a centralized HA2 conjugated with M2e could be the basis for an improved universal influenza vaccine to elicit a much potent immune response against a broad subtype of influenza viruses over using M2e or HA2 alone. Furthermore, it would also be advantageous to design a subunit universal vaccine to be delivered intranasally over intramuscular delivery, because (1) it would eliminate the discomfort and pain from injections and (2) the mucosal immune response is critical for clearance of respiratory tract pathogens. However, to improve the immune responses elicited by a mucosal vaccine, one would need an adjuvant. Cholera toxin B subunit (CTB) can enhance the immune response to some mucosal antigens.17 Intranasal immunization
*Correspondence to: Mingtao Zeng; Email: [email protected] Submitted: 12/23/2013; Revised: 02/13/2014; Accepted: 03/03/2014; Published Online: 03/14/2014 http://dx.doi.org/10.4161/hv.28400 www.landesbioscience.com Human Vaccines & Immunotherapeutics 1211
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Junwei Li, Maria T Arévalo, Yanping Chen, Olivia Posadas, Jacob A Smith, and Mingtao Zeng
with CTB and bovine serum albumin (BSA) or HA of influenza viruses can stimulate high levels of BSA or HA specific serum IgG antibody and IgA antibody in the nasal and pulmonary lavages.18,19 IgA responses are sufficient to inhibit the transmission of influenza virus.20 Mucosal adjuvant cholera toxin is superior to other adjuvants in its ability to prime the memory CD8 T cell response.21 CTB facilitates entry into cells via highaffinity binding to cell surface receptor GM1 ganglioside, allowing for internalization and intracellular trafficking of conjugated antigens into the cytosol of dendritic cells to enhance antigen presentation.22-25 To stimulate high levels of secretory IgA in the respiratory tract and broad anti-influenza virus immune response, we designed an artificial antigen to be used as a universal influenza vaccine. We combined a centralized HA2, 3 sequential M2e units from different influenza subtypes, and nonconventional adjuvant CTB to enhance the antigen presentation and stimulation of the T cell response. Herein, we show that this novel antigen, as tested in mice, induced potent cross immune responses and protection against multiple strains of influenza viruses.
Results Development of recombinant antigen In this study, a chimeric gene was generated by combining: the CTB gene; 3 sequential M2e sequences from H1N1, H3N2, and H5N1 (Fig. 1A) influenza subtypes to increase the density and variation of M2e epitopes; and a highly centralized HA2 gene sequence (Fig. 1B). Finally, the gene sequence was
optimized for expression in E. coli and inserted into the pET200/ D-TOPO expression vector. Schematic representations of fusion protein designs containing a 6 × His tag with or without CTB are shown (Fig. 1C). These fusion proteins were successfully expressed in the E. coli BL21 (DE3 strain) and purified by affinity chromatography using Ni-NTA agarose beads with a purity of 95% as confirmed by western-blotting using an anti-6 × His mouse monoclonal antibody (Fig. 1D). These results showed the molecular weights of CTB-3 × M2e-HA2 and 3 × M2e-HA2 were approximately 45 kD and 25 kD, respectively. Humoral responses and protection elicited by intranasal and intramuscular vaccination with CTB-3 × M2e-HA2 Next, we assessed the immunogenicity of CTB-3 × M2e-HA2 and 3 × M2e-HA2 in mice after intranasal or intramuscular immunizations. These mice were immunized on days 0, 14, and 28. The humoral immune response was assessed by measuring antigen specific IgG, IgG1, and IgG2a levels in the sera collected on days 0, 14, 28, and 42 after initial immunization. Overall, mice immunized with CTB-3 × M2e-HA2 intranasally produced higher levels of IgG, IgG1 and IgG2a antibodies than those immunized intramuscularly with CTB-3 × M2e-HA2, or with 3 × M2e-HA2 via intramuscular or intranasal delivery (Fig. 2). By days 28 and 42, after 2 and 3 respective doses of our vaccine candidates, there were significant differences observed between mice immunized with CTB-3 × M2e-HA2, mice immunized with 3 × M2e-HA2, and naïve mice. Furthermore, by days 28 and 42, mice immunized intranasally with CTB-3 × M2e-HA2 displayed significantly greater serum antibody levels than all other groups. To test if immunities induced by our vaccine candidates provided protection against heterologous influenza viral challenge, mice
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Figure 1. Design and expression of CTB-3 × M2e-HA2. (A) Amino acid sequences of 3 M2e extracted from A/California/04/2009 (H1N1), A/Viet Nam/1204/2004 (H5N1) and A/Hong Kong/1/1968 (H3N2) influenza virus and the variable amino acids were underlined. (B) Highly centralized HA2 amino acid sequence. (C) Schematics of chimeric CTB-3 × M2e-HA2 and 3 × M2e-HA2 proteins. (D) Expression of CTB-3 × M2e-HA2 and 3 × M2e-HA2 proteins were verified by Western Blot using an anti-6 × His monoclonal antibody.
were intranasally inoculated with 100 × LD50 influenza PR8 (H1N1) viruses at day 42 post-immunization. Mice in the naïve group all died by day 8 post-challenge, while immunized mice were differentially protected depending on the fusion protein and immunization route used (Fig. 2D). Those immunized with 3 × M2e-HA2 fared better than naïve controls, but survival rate was less than ideal at 25% and 38% for intranasal and intramuscular immunization routes, respectively. Intramuscular immunization with CTB-3 × M2e-HA2 provided an increased rate of survival of 75%, but intranasal immunization with CTB-3 × M2e-HA2 provided optimal results with 100% survival. Because intranasal vaccination with CTB-3 × M2e-HA2 was more protective than intramuscular vaccination with CTB-3 × M2e-HA2, we reasoned that mucosal immunity may be involved in protecting mice. Hence, antibody responses in the bronchoalveolar lavage fluid of naïve mice and mice intranasally immunized with 3 × M2e-HA2 or CTB-3 × M2e-HA2 were also assessed (Fig. 3). Indeed, intranasal immunization with CTB-3 × M2e-HA2 induced robust IgA (Fig. 3A), total IgG (Fig. 3B), IgG1 (Fig. 3C), and IgG2a (Fig. 3D) responses in the lung. Of note, the specific antibody responses induced by intranasal immunization with CTB-3 × M2e-HA2 were statistically superior to
those induced by 3 × M2e-HA2. Taken together, these data suggested that the fusion protein consisting of centralized HA and M2e was immunogenic, inducing antibodies that were potentially involved in inhibiting the replication of influenza virus in vivo. Furthermore, CTB enhanced influenza-specific humoral and protective immunities. Evaluation of CTB-3 × M2e-HA2 vaccine potential for broad, cross-strain immunity Next, we tested if the recombinant CTB-3 × M2e-HA2 provided protection against multiple influenza virus strains or subtypes. We challenged immunized and naïve mice with 100 × LD50 pandemic H1N1 CA09 or 105 PFU HK68 (H3N2) influenza viruses. In mice immunized 3 times with CTB-3 × M2e-HA2, only 1 of 8 mice succumbed to CA09-related illness and died at day 15 post-challenge (Fig. 4A). The remaining 7 mice recovered completely by day 14 post-challenge. Furthermore, mice immunized 3 times with CTB-3 × M2e-HA2 and challenged with HK68 had almost no detectable virus in the lungs at 5 d postinfection (Fig. 4B). Thus, CTB-3 × M2e-HA2 induced potent immune response that inhibited the replication of CA09 and HK68 influenza viruses in the mouse lung. To continue to assess broad-immunity elicited by our CTB-3 × M2e-HA2 vaccine
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Figure 2. Evaluation of humoral and protective immunities by vaccine candidates. Mice were intranasally (i.n.) or intramuscularly (i.m.) primed with 10 µg recombinant protein, CTB-3 × M2e-HA2 or 3 × M2e-HA2. Boosters were given on days 14 and 28. Naïve, control mice received PBS (i.n.). Blood samples were collected on days 0, 14, 28, and 42. At 42 d post-immunization, all mice were challenged with 100 × LD50 PR8 influenza virus. Serum levels of 3 × M2e-HA2 specific (A) IgG, (B) IgG1, and (C) IgG2a antibodies as measured by ELISA are shown, ***P < 0.05. (D) Survival of mice challenged with 100 × LD50 PR8 influenza virus.
candidate, we infected MDCK cells with different viral strains and used cell lysates as a source of viral proteins, including HA2 and NP. Sera from mice immunized with CTB-3 × M2e-HA2 reacted with HA2 from cell lysates derived from MDCK infected with CA09, FM47 (H1N1), PR8, Aichi68 (H3N2), and HK68 influenza viruses (Fig. 4C). The sera from immunized mice did not react with cell lysates from mock-infected cells. As expected, a monoclonal antibody against influenza NP also reacted with the cell lysates infected with the various influenza viruses, while a monoclonal antibody against β-actin reacted with all cell lysates. In summary, the results showed that a three-dose, vaccination regimen with CTB-3 × M2e-HA2 was broadly immunogenic, and mice were protected from multiple strains and subtypes of influenza virus. CTB-3 × M2e-HA2 dose response To determine the optimal dose required for CTB-3 × M2e-HA2 to induce protective immunity, mice were immunized 1–3 times with CTB-3 × M2e-HA2 and challenged with 100 × LD50 PR8 influenza virus. One immunization with CTB-3 × M2e-HA2 provided 25% protection in mice, 2 immunizations provided 62.5% protection, and 3 immunizations provided complete protection against lethal PR8 influenza virus infection (Fig. 5). Thus, protection against lethal influenza virus infection was dose-dependent, with 3 immunizations providing optimal protection.
Correlating protection conferred by immunization with CTB-3 × M2e-HA2 with humoral immunity To determine whether humoral immunity was critical for the protection provided by immunization with CTB-3 × M2e-HA2, we injected naïve mice intraperitoneally (i.p.) with 200 µl sera from mice immunized with CTB-3 × M2e-HA2, PR8, CA09, or naïve. These mice were then challenged with 100 × LD50 PR8 or CA09 influenza viruses. As expected, passive transfer of PR8 and CA09 positive immune sera protected 100% of mice challenged with corresponding influenza viruses (Fig. 6A). Passive transfer of sera from mice immunized with CTB-3 × M2e-HA2 also provided significant protection against viral challenges, 87.5% and 75% of mice were protected against CA09 and PR8 influenza viruses, respectively. In contrast, naïve mice all succumbed to viral infection. To confirm these results, we tested if muMT mice, which lack mature B cells, would be protected against influenza viral infection after immunization with CTB-3 × M2e-HA2. Groups of muMT mice were immunized 3 times with CTB-3 × M2e-HA2 or 3 × M2e-HA2, once with a low-dose or PR8 virus, or left untreated. After challenge with 100 × LD50 PR8, naïve animals and those immunized with 3 × M2e-HA2 all succumbed to infection and died. Immunization with CTB-3 × M2e-HA2 or PR8 in these muMT mice only conferred 25% and 38% protection against lethal PR8 infection, respectively (Fig. 6B). These results suggested that protection
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Figure 3. Mucosal immune responses in intranasally vaccinated mice. Mice in one group served as naïve controls, while mice in the other groups were intranasally primed at day 0 and boosted at days 14 and 28 with 10 µg CTB-3 × M2e-HA2 or 3 × M2e-HA2. Bronchoalveolar lavage fluid was collected on day 42 to measure anti-3xM2e-HA2 (A) IgA, (B) IgG, (C), IgG1, and (D) IgG2a concentrations by ELISA, ***P < 0.05.
by CTB-3 × M2e-HA2 immunization was dependent on the humoral immune response. Immunization with CTB-3 × M2e-HA2 and cellular immunities To investigate the immune response further, we evaluated antigen specific T and B cell proliferation. Expression of CD4 +, CD8 +, CD19 +, and CD27+ surface markers was assessed in mouse lung cells (Fig. 7A–C) and splenocytes (Fig. 7D–F) from mice immunized with CTB-3 × M2e-HA2 or 3 × M2e-HA2. The percentage of CD4 + T cell, CD8 + T cell, and CD19 +/CD27+ memory B cells in the mouse lung and spleen significantly increased after 3 immunizations with CTB-3 × M2e-HA2 as compared with naïve or 3 × M2e-HA2 immunized mice, indicating that CTB enhanced T cell and memory B cell immune responses. Furthermore, CTB-3 × M2e-HA2 significantly stimulated IFN-γ secretion in the lung and spleen (Fig. 8). This IFN-γ response was enhanced in mice immunized with CTB-3 × M2e-HA2 over those immunized with 3 × M2e-HA2.
Discussion In this study, we designed and constructed a novel fusion antigen, CTB-3 × M2e-HA2, to confer cross-protection against a broad range of influenza viruses. Others have already studied the immunity and protection elicited by these antigens as individuals, and the knowledge gained from these studies was incorporated into the design of a single, fusion protein. Based on previous studies, we knew that adjuvanted, single or tandem M2e repeats elicit strong serum IgG and mucosal IgA antibody responses.26-28 These studies also established that 3–4 M2e repeats in tandem improved humoral immunity over the use of a single M2e, and that immunization with tandem M2e repeats induce protection against a broad range of influenza virus subtypes. Thus, in this work, we incorporated 3 tandem M2e sequences from 3 different viruses. It is also known that conserved or centralized HA2 induces cross protection.3,6,29,30 Thus, incorporation of a highly
conserved HA2 sequence could only strengthen a vaccine strategy to stimulate broad-based immunity and protection. Previous studies also show that CTB can be used in the design of influenza vaccines to enhance immune responses and that CTB alone does not elicit significant protection against lethal influenza viral challenge.31,32 Hence, after vaccine design, the focus of this study was to show that the novel CTB-3 × M2e-HA2 protein as a whole, was broadly immunogenic and protective across multiple influenza A subtypes. Furthermore, we show that protection by this fusion protein was antibody-dependent. While protection by CTB-3 × M2e-HA2 against influenza virus was antibody-dependent, this protection was not via antibody-mediated neutralization. In fact, past studies reported that anti-M2 or HA2 do not have direct virus neutralizing or hemagglutination inhibition (HI) activity. Despite not having neutralizing or HI activity, naturally induced HA2 antibody can inhibit influenza virus replication.33 Moreover, immunization with HA2 provides for faster elimination of influenza virus in mouse lung and increases survival rates.34 Although anti-M2e antibody is not able to neutralize the virus to prevent virus entry, it is able to
Figure 5. CTB-3 × M2e-HA2 antigen dose response. Mice were immunized once, twice, or thrice with CTB-3 × M2e-HA2 and challenged on day 42 after the first immunization with 100 × LD50 PR8 influenza virus.
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Figure 4. Testing broad protection against multiple influenza strains by CTB-3 × M2e-HA2. (A) Survival of mice immunized with CTB-3 × M2e-HA2 or CA09 in comparison to naïve mice after lethal challenge with 100 × LD50 CA09 influenza virus. (B) Lung viral loads in mice immunized with CTB-3 × M2e-HA2 or HK68 in comparison to naïve mice after challenge with 105 PFU HK68 influenza virus. (C) MDCK cells were infected with different viral strains at MOI = 0.1 to be used as source of viral HA2 and NP. Western Blot analysis was used to test if serum from mice immunized with CTB-3 × M2e-HA2 reacted with HA2 (about 26 kD) from infected MDCK cell lysates (top panel). A commercial, monoclonal antibody against NP was used to verify influenza viral infection of MDCK cells (middle panel). β-actin blots were used as loading controls.
disrupt the virus life cycle26-28and promotes death of infected cells by antibody-dependent cell-mediated cytotoxicity.35,36 We performed cross-neutralization assays using sera from mice immunized with CTB-3 × M2e-HA2 and PR8 influenza virus in vitro, and similar to those studies performed with HA2 and M2e antigens, CTB-3 × M2e-HA2 immune sera lacked virus neutralization activity against PR8 and CA09 influenza viruses (data not shown). This indicated that the mechanism of cross-protection induced by CTB-3 × M2e-HA2 was not via antibody-mediated neutralization of influenza viruses, but through another antibody-mediated mechanism. Therefore, it was assumed that antiM2e or HA2 antibodies could directly bind M2 or HA2, inhibit functions during the replication of influenza virus, or lead to opsonization by macrophages and dendritic cells. To extensively prepare for the unexpected outbreak of influenza, increased efforts are being focused on adjuvants that enhance vaccine immunogenicity against emerging influenza viruses and maximize vaccine supply.37 New strategies for influenza vaccine design are under development.38-41 One strategy is to design a vaccine for mucosal delivery. The mucosa serves as the first line of defense against invading respiratory pathogens, and designing a vaccine for intranasal delivery would also bypass the need for trained professionals needed to perform injections while reducing the associated biohazardous wastes. Mucosal immunization with influenza viral antigen can elicit robust immune response, but often the adjuvants used to enhance the immune response lead to safety issues, with side effects reported in clinical testing.42,43 On the other hand, clinical trials have shown that formulations containing recombinant CTB are safe and well tolerated.44,45 In our study, CTB fused to influenza antigens enhanced protective immunity in immunized mice. The mechanism by which CTB enhances mucosal immunity is still unclear, but dendritic cell maturation and antigen presentation are stimulated.46 Immunization with different adjuvants or by different routes may elicit different immune responses. As an example, immunization with M2e-based vaccine induces a predominantly IgG1 over IgG2a response.47 In contrast, immunization with NP-M2e fusion protein induces both IgG1 and IgG2a responses.41 The
humoral immune response induced by immunization with CTB-3 × M2e-HA2 was predominantly a Th2-mediated antibody response. In addition, results in Figures 6 and 7 suggest that T cell immunity was also stimulated by immunization with CTB-3 × M2e-HA2. While T cell immunity may be important in protection against influenza virus infection, the antibody response is critical for cross anti-influenza protection.48-50 Indeed, our results showed that the humoral immune response was indispensable for protection induced by immunization with CTB-3 × M2e-HA2. However, T cell immunity was also induced in our studies. In conclusion, we have shown that CTB-3 × M2e-HA2 stimulates potent broad spectrum immunity against influenza A viruses. This antigen, as a recombinant protein, can be produced inexpensively in large-scale quantities without any need for the time-constraining culture of influenza virus. A corollary advantage is no time constraint to prepare the vaccine for a potential new influenza pandemic. In the event of a pandemic flu crisis, this novel vaccine candidate may provide relief to the public health burden.
Materials and Methods Viruses, antibodies and animals Mouse adapted A/California/04/2009(H1N1) (pandemic CA09) and A/FM/1/47 (H1N1) (FM47) were gifts from Dr Richard Webby (St. Jude Children’s Hospital).51 A /HongKong/1/68 (H3N2) (HK68, NR-346) and A/ Aichi/2/1968(H3N2) (Aichi68, NR-3177) were provided by BEI Resources, and A/Puerto Rico/8/1934 (H1N1) (PR8) was generated using plasmids received from Dr Webster. Alexa Fluor700 conjugated anti-mouse CD3e (cat#557984), Pacific Blue conjugated anti-mouse CD4 (cat#558107), V500 conjugated anti-mouse CD8a (cat#560776), APC-Cy7 conjugated anti-mouse CD19 (cat#557655), PE conjugated anti-mouse CD27 (cat#558754) for flow cytometry were purchased from BD Bioscience. Goat anti-mouse IgG-Fc (cat#A90–131A), goat
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Figure 6. Evaluating the role of humoral immunity in protection against PR8 and CA09 influenza viruses. (A) Mice were i.p. injected with serum collected from naïve mice or mice immunized with CTB-3 × M2e-HA2, and then challenged with lethal doses of CA09 or PR8 influenza viruses. Clinical symptoms and survival were monitored and recorded. (B) B cell depleted mice were immunized with CTB-3 × M2e-HA2 or 3 × M2e-HA2, and then challenged with 100 × LD50 PR8 influenza virus.
anti-mouse IgA (cat#A90–103A) goat-anti-mouse IgG1 (A90– 105A), goat anti-mouse IgG2a (cat#A90–107A) were purchased from BETHYL Laboratories Inc. Female BALB/c and muMT B cell depleted mice were purchased from Jackson Laboratory (cat# 002288). Antigen design, construction, and expression To create a highly conserved HA2 sequence, we aligned HA2 sequences from H1N1, H2N2, H3N2, H5N1, H7N1, and H9N2 influenza viruses found in the NCBI-Influenza Virus Resource database. The most common amino acids in each position were used to create an artificial, centralized, HA2 sequence. Subsequently, the conserved HA2 sequence was linked with 3 M2 protein ectodomain (M2e) sequences from A/California/04/2009(H1N1) (CA09), A/Viet Nam/1204/2004(H5N1) (VN04), and A/Hong Kong/1/1968 (H3N2) (HK68) influenza viruses in tandem at the N-terminal. Then, the DNA sequence of cholera toxin subunit B (CTB) was fused at the N-terminal of 3 × M2e-HA2 DNA sequence. The whole DNA sequence was codon optimized for expression in E. coli and synthesized by GenScript. For the expression of chimeric proteins, the DNA sequences of CTB-3 × M2e-HA2 or 3 × M2e-HA2 were inserted into the E. coli expression vector: pET200/D-TOPO (Invitrogen, cat#K10001). Finally, the plasmid constructs were transformed in E. coli BL21 (DE3) strain (Life Technologies, cat#440054) Transformed cells were cultured in Luria Broth (LB) with 50 µg/mL of kanamycin to
OD600 of 0.5, then supplied with 0.1 mM Isopropyl β-D-1thiogalactopyranoside (IPTG) and cultured for another 4 h at 37 °C. These cultures were harvested by centrifugation at 6000 rpm for 15 min. E. coli pellets were lysed with lysis buffer (50 mM NaH2PO4, 500 mM NaCl, 10 mM imidazole, pH 8.0). The lysates were collected after centrifugation at 15 000 rpm for 30 min and incubated with Ni-NTA agarose (Qiagen, cat#30230) in polypropylene columns overnight at 4 °C. The protein-bound resin was washed with washing buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 8.0) 5 times and His-tagged proteins were eluted with elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0). Purified proteins were dialyzed using ultracentrifugal filter devices (Millipore, cat#UFC900324). The eluted proteins were confirmed by western-blotting with anti-His tag mouse monoclonal antibody (Life Technologies, cat#R93025) and stocked in PBS with 10% glycerol at −80 °C prior to use for vaccination. Mouse immunization, bleeding, and challenge Six-wk-old BALB/c mice purchased from the Jackson Laboratory were divided into 3 groups at random and housed in the LARC facility of Texas Tech University Health Science Center at El Paso. All studies with experimental animals were approved by the Institutional Animal Care and Use Committee. Mice in group 1 were used as naïve controls and intranasally received 50 μL PBS. The second group was intranasally immunized with 10 μg purified antigen in 50 μL PBS. The third group was
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Figure 7. T cell and memory B cell immune responses elicited by vaccination with CTB-3 × M2e-HA2. After mice were immunized, splenocytes and lung cells were collected and CD4, CD8 T, and memory B cell percentages were analyzed by flow cytometry, ***P < 0.05. (A) Percentage of CD3e and CD4 double positive cell in lungs. (B) Percentage of CD3e and CD8 double positive T cells in lungs. (C) Percentage of CD19 and CD27 double positive cell in lungs. (D) Percentage of CD3e and CD4 double positive cell in spleens. (E) Percentage of CD3e and CD8 double positive T cells in spleens. (F) Percentage of CD19 and CD27 double positive cell in spleens.
injected with 10 μg purified antigen in hind leg thigh muscle. Immunizations were given at day 0, 14, and 28 for a total of 3 immunizations per mouse. On the day of each immunization, mice were anesthetized with ketamine and xylazine, and mouse blood was collected from the submandibular vein. Blood serum was separated, aliquoted, and stored at −80 °C for future use. Bronchoalveolar lavage fluid was collected according established method, and stored at −80 °C for IgA quantification. For infections, each mouse was anesthetized and intranasally inoculated with influenza virus and weighed daily. Mice were observed daily for clinical symptoms and survival. Animals were anesthetized for performing procedures such as intranasal immunization and viral challenge. At the end of the experiments, animals used in the experiments were euthanatized in a CO2 chamber in a manner consistent with the AVMA Guidelines for the Euthanasia of Animals (2013 Edition) through the Panel on Euthanasia of the American Veterinary Medical Association. Enzyme-linked immunosorbent assay Serum antibody titers for each mouse were determined by enzyme-linked immunosorbent assay (ELISA). 96-well plates were coated with 1 μg/mL CTB-3 × M2e-HA2 in 100 μL 50 mM sodium bicarbonate buffer (pH 9.6) overnight at 4 °C, then blocked with PBS containing 1% bovine serum albumin for 30 min at room temperature. Sera were diluted 50-fold in 100 μL PBS and incubated overnight at 4 °C. Commercial Mouse serum was taken as reference to draw a standard curve for quantifying antibody in immunized mice serum (Bethyl, cat#rs10101). After washing, 100 μL alkaline phosphatase conjugated anti-mouse IgG-Fc antibody diluted at the ratio of 1:50 000 in PBS was added into each well and incubated for 1 h at room temperature. Plates were washed and developed with 100 μL diethanolamine substrate (KPL, cat#508000). The reaction was stopped after 20 min with 100 μL EDTA stop solution per well. The plates were read at 405 nm using the PowerWaveXS2 (Biotek). HA2 and antibody binding MDCK cells seeded in 35 mm dishes were infected with CA09, A/FM/1/47(H1N1) (FM47), PR8, A/Aichi/2/1968(H3N2), HK68 of MOI (multiplicity of infection) = 0.1. At 24 h after infection, cells were harvested, and lysed in 1 × cell lysis buffer (Cell
Signaling Technology, cat#9803). Lysed MDCK cell samples in Laemmli sample buffer (without reducing agent) were loaded and separated on 10% SDS-PAGE, then transferred onto nitrocellulose membrane using a semi-dry transblot apparatus (Biorad, cat#1703940). The membrane was blocked in PBS with 1% Tween (PBST) containing 5% non-fat milk for 1 h and incubated with serum from mice immunized with CTB-3 × M2e-HA2 or NP monoclonal antibody (Abcam, cat#ab20343) at 4 °C overnight. After washing with PBST, the membrane was incubated with alkaline-phosphatase conjugated goat anti-mouse IgG antibody at room temperature for 1 h. After washing, the membrane was developed with BCPI/NBT substrate (Sigma, cat#B1911). Passive transfer of mouse serum 48 BALB/c mice were divided into 6 groups randomly, 2 naïve groups were injected i.p. with 200 μL naïve mouse serum. The third group was injected i.p. with 200 μL mouse serum from mice previously infected with PR8. The fourth group was i.p. injected with 200 μL mouse serum from mice previously infected with CA09, and the fifth and sixth groups were injected with serum from mice immunized with CTB-3 × M2e-HA2. All groups were then challenged with either 100 × LD50 PR8 or CA09. Mice were observed for clinical symptoms and survival. Flow cytometry Splenocytes and lung cells were collected from mice immunized 3 times with CTB-3 × M2e-HA2. Single cell preparations were washed, incubated with ACK lysis buffer, and then stimulated with PHA (10 μg/mL) for 4 h. GolgiStop (BD Biosciences, cat#554724) was added and cells were incubated for another 2 h. After washing with PBS, cells were permeabilized, then stained with Alexa Fluor® 700 hamster anti-mouse CD3e, Pacific Blue™ rat anti-mouse CD4 +, V500 rat anti-mouse CD8 +a, PE hamster anti-mouse CD27+, APC-Cy™7 rat anti-and mouse CD19 + antibodies. Fixed samples were acquired using the Gallios flow cytometer (Beckman Coulter, Inc.) with a minimum acquisition of 15 000 events and analyzed by using FlowJo software (TreeStar). Enzyme-linking immunospot (ELISPOT) assay MultiScreen Filter Plates (Millipore, cat#MAIPS4510) were coated with rat anti-mouse IFN-γ monoclonal antibody (BD
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Figure 8. Analysis of IFN-γ secretion by immune cells from the lung and spleen. Cellular immune responses after vaccination were evaluated using an ELISPOT assay to enumerate IFN-γ-secreting cells, ***P < 0.05. (A) IFN-γ-secreting cells in lung cells. (B) IFN-γ-secreting cells in splenocytes.
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Windows (GrahPad Software). P values < 0.05 were considered to be significant difference. Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed. Acknowledgments
This work was supported by the US Public Service research grant AI072139 (M.Z.) from the National Institute of Allergy and Infectious Diseases and an internal fund from Texas Tech University Health Sciences Center Paul L. Foster School of Medicine. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We are grateful to David Topham for providing MDCK cells, to Richard Webby for providing PR8 influenza viral plasmid system and mouse adapted H1N1(A/California/04/2009) influenza virus and A/FM/1/47(H1N1) influenza virus, and to the NIH/NIAID funded BEI Resources for supplying seasonal influenza vaccines and influenza A/HongKong/1/68 (H3N2) and A/Aichi/2/1968 (H3N2) strains.
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Bioscience, cat#553209) in coating buffer at concentration of 5 µg/mL at 4 °C overnight. Splenocytes and lung cells were isolated from mice. After being washed, cells were seeded and stimulated with PHA at concentration of 10 µg/mL at 37 °C in 5% CO2 overnight. After stimulation, plates were washed 5 times with PBS containing 0.05% Tween 20 (PBST) and incubated with biotin conjugated rat anti-mouse IFN-γ antibody (2.0 µg/mL) at room temperature for 2 h. The plates were washed and incubated with horseradish peroxidase (HRP) conjugated streptavidin (BD Bioscience, cat#555214) at room temperature for 1.5 h. Plates were washed with PBS and added with HRP substrate. Subsequently, the plates were developed for 10 min in the dark and substrate was removed. After being washed with doubledistilled water, plates were dried overnight. Finally, spots were counted under ELISpot reader (AID, Germany). Statistical analysis Comparisons between vaccinated groups were performed by using a nonparametric one-ways ANOVA with the Tukey multiple comparison test and the Fisher exact test, and survival dates were analyzed by using the log-rank test. The analyses were performed by using GraphPad Prism version 5.0 for
32. Guo L, Zheng M, Ding Y, Li D, Yang Z, Wang H, Chen Q, Sui Z, Fang F, Chen Z. Protection against multiple influenza A virus subtypes by intranasal administration of recombinant nucleoprotein. Arch Virol 2010; 155:1765-75; PMID:20652335; http:// dx.doi.org/10.1007/s00705-010-0756-3 33. Varecková E, Mucha V, Wharton SA, Kostolanský F. Inhibition of fusion activity of influenza A haemagglutinin mediated by HA2-specific monoclonal antibodies. Arch Virol 2003; 148:469-86; PMID:12607099; http://dx.doi.org/10.1007/s00705-002-0932-1 34. Gocník M, Fislová T, Mucha V, Sládková T, Russ G, Kostolansky F, Varecková E. Antibodies induced by the HA2 glycopolypeptide of influenza virus haemagglutinin improve recovery from influenza A virus infection. J Gen Virol 2008; 89:958-67; PMID:18343837; http://dx.doi.org/10.1099/ vir.0.83524-0 35. Denkers EY, Badger CC, Ledbetter JA, Bernstein ID. Influence of antibody isotype on passive serotherapy of lymphoma. J Immunol 1985; 135:2183-6; PMID:2862210 36. Jegerlehner A, Schmitz N, Storni T, Bachmann MF. Influenza A vaccine based on the extracellular domain of M2: weak protection mediated via antibody-dependent NK cell activity. J Immunol 2004; 172:5598-605; PMID:15100303 37. Clegg CH, Roque R, Van Hoeven N, Perrone L, Baldwin SL, Rininger JA, Bowen RA, Reed SG. Adjuvant solution for pandemic influenza vaccine production. Proc Natl Acad Sci U S A 2012; 109:17585-90; PMID:23045649; http://dx.doi. org/10.1073/pnas.1207308109 38. Petsch B, Schnee M, Vogel AB, Lange E, Hoffmann B, Voss D, Schlake T, Thess A, Kallen KJ, Stitz L, et al. Protective efficacy of in vitro synthesized, specific mRNA vaccines against influenza A virus infection. Nat Biotechnol 2012; 30:1210-6; PMID:23159882; http://dx.doi.org/10.1038/nbt.2436 39. Jazi MH, Dabaghian M, Tebianian M, Gharagozlou MJ, Ebrahimi SM. In vivo electroporation enhances immunogenicity and protection against influenza A virus challenge of an M2e-HSP70c DNA vaccine. Virus Res 2012; 167:219-25; PMID:22609252; http://dx.doi.org/10.1016/j.virusres.2012.05.002 40. Alexander J, Bilsel P, del Guercio MF, Stewart S, Marinkovic-Petrovic A, Southwood S, Crimi C, Vang L, Walker L, Ishioka G, et al. Universal influenza DNA vaccine encoding conserved CD4+ T cell epitopes protects against lethal viral challenge in HLA-DR transgenic mice. Vaccine 2010; 28:66472; PMID:19895924; http://dx.doi.org/10.1016/j. vaccine.2009.10.103
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41. Wang W, Huang B, Jiang T, Wang X, Qi X, Gao Y, Tan W, Ruan L. Robust immunity and heterologous protection against influenza in mice elicited by a novel recombinant NP-M2e fusion protein expressed in E. coli. PLoS One 2012; 7:e52488; PMID:23285063; http://dx.doi.org/10.1371/journal.pone.0052488 42. Sendi P, Locher R, Bucheli B, Battegay M. Intranasal influenza vaccine in a working population. Clin Infect Dis 2004; 38:974-80; PMID:15034829; http://dx.doi.org/10.1086/386330 43. Sendi P, Locher R, Bucheli B, Battegay M. The decision to get vaccinated against influenza. Am J Med 2004; 116:856-8; PMID:15178507; http://dx.doi. org/10.1016/j.amjmed.2004.02.023 44. Bharati K, Ganguly NK. Cholera toxin: a paradigm of a multifunctional protein. Indian J Med Res 2011; 133:179-87; PMID:21415492 45. Stål P, Befrits R, Rönnblom A, Danielsson A, Suhr O, Ståhlberg D, Brinkberg Lapidus A, Löfberg R. Clinical trial: the safety and short-term efficacy of recombinant cholera toxin B subunit in the treatment of active Crohn’s disease. Aliment Pharmacol Ther 2010; 31:387-95; PMID:19878149; http://dx.doi. org/10.1111/j.1365-2036.2009.04185.x 46. Reed SG, Bertholet S, Coler RN, Friede M. New horizons in adjuvants for vaccine development. Trends Immunol 2009; 30:23-32; PMID:19059004; http://dx.doi.org/10.1016/j.it.2008.09.006 47. Shim BS, Choi YK, Yun CH, Lee EG, Jeon YS, Park SM, Cheon IS, Joo DH, Cho CH, Song MS, et al. Sublingual immunization with M2-based vaccine induces broad protective immunity against influenza. PLoS One 2011; 6:e27953; PMID:22140491; http:// dx.doi.org/10.1371/journal.pone.0027953 48. Gerhard W, Mozdzanowska K, Furchner M, Washko G, Maiese K. Role of the B-cell response in recovery of mice from primary influenza virus infection. Immunol Rev 1997; 159:95-103; PMID:9416505; ht t p : //d x .doi.org /10.1111/j.16 0 0 - 0 65X .1997. tb01009.x 49. Waffarn EE, Baumgarth N. Protective B cell responses to flu--no fluke! J Immunol 2011; 186:3823-9; PMID:21422252; http://dx.doi. org/10.4049/jimmunol.1002090 50. Swain SL, McKinstry KK, Strutt TM. Expanding roles for CD4 + T cells in immunity to viruses. Nat Rev Immunol 2012; 12:136-48; PMID:22266691 51. Ilyushina NA, Khalenkov AM, Seiler JP, Forrest HL, Bovin NV, Marjuki H, Barman S, Webster RG, Webby RJ. Adaptation of pandemic H1N1 influenza viruses in mice. J Virol 2010; 84:860716; PMID:20592084; http://dx.doi.org/10.1128/ JVI.00159-10
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24. Andrew DW, Hafner LM, Beagley KW, Timms P. Partial protection against chlamydial reproductive tract infection by a recombinant major outer membrane protein/CpG/cholera toxin intranasal vaccine in the guinea pig Chlamydia caviae model. J Reprod Immunol 2011; 91:9-16; PMID:21856018; http:// dx.doi.org/10.1016/j.jri.2011.06.100 25. Datta SK, Sabet M, Nguyen KP, Valdez PA, GonzalezNavajas JM, Islam S, Mihajlov I, Fierer J, Insel PA, Webster NJ, et al. Mucosal adjuvant activity of cholera toxin requires Th17 cells and protects against inhalation anthrax. Proc Natl Acad Sci U S A 2010; 107:10638-43; PMID:20479237; http://dx.doi. org/10.1073/pnas.1002348107 26. Eliasson DG, El Bakkouri K, Schön K, Ramne A, Festjens E, Löwenadler B, Fiers W, Saelens X, Lycke N. CTA1-M2e-DD: a novel mucosal adjuvant targeted influenza vaccine. Vaccine 2008; 26:124352; PMID:18243429; http://dx.doi.org/10.1016/j. vaccine.2007.12.027 27. Turley CB, Rupp RE, Johnson C, Taylor DN, Wolfson J, Tussey L, Kavita U, Stanberry L, Shaw A. Safety and immunogenicity of a recombinant M2eflagellin influenza vaccine (STF2.4xM2e) in healthy adults. Vaccine 2011; 29:5145-52; PMID:21624416; http://dx.doi.org/10.1016/j.vaccine.2011.05.041 28. Alvarez P, Zylberman V, Ghersi G, Boado L, Palacios C, Goldbaum F, Mattion N. Tandem repeats of the extracellular domain of Matrix 2 influenza protein exposed in Brucella lumazine synthase decameric carrier molecule induce protection in mice. Vaccine 2013; 31:806-12; PMID:23246552; http://dx.doi. org/10.1016/j.vaccine.2012.11.072 29. Steel J, Lowen AC, Wang TT, Yondola M, Gao Q, Haye K, García-Sastre A, Palese P. Influenza virus vaccine based on the conserved hemagglutinin stalk domain. MBio 2010; 1:e00018-10; PMID:20689752; http://dx.doi.org/10.1128/mBio.00018-10 30. Bommakanti G, Citron MP, Hepler RW, Callahan C, Heidecker GJ, Najar TA, Lu X, Joyce JG, Shiver JW, Casimiro DR, et al. Design of an HA2-based Escherichia coli expressed influenza immunogen that protects mice from pathogenic challenge. Proc Natl Acad Sci U S A 2010; 107:13701-6; PMID:20615991; http://dx.doi.org/10.1073/pnas.1007465107 31. Prabakaran M, Velumani S, He F, Karuppannan AK, Geng GY, Yin LK, Kwang J. Protective immunity against influenza H5N1 virus challenge in mice by intranasal co-administration of baculovirus surfacedisplayed HA and recombinant CTB as an adjuvant. Virology 2008; 380:412-20; PMID:18786689; http://dx.doi.org/10.1016/j.virol.2008.08.002
Human Vaccines & Immunotherapeutics Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/khvi20
Intranasal immunization with influenza antigens conjugated with cholera toxin subunit B stimulates broad spectrum immunity against influenza viruses a
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Junwei Li , Maria T Arévalo , Yanping Chen , Olivia Posadas , Jacob A Smith & Mingtao Zeng
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Center of Excellence for Infectious Diseases; Paul L. Foster School of Medicine; Texas Tech University Health Sciences Center; El Paso, TX USA Published online: 14 Mar 2014.
Click for updates To cite this article: Junwei Li, Maria T Arévalo, Yanping Chen, Olivia Posadas, Jacob A Smith & Mingtao Zeng (2014) Intranasal immunization with influenza antigens conjugated with cholera toxin subunit B stimulates broad spectrum immunity against influenza viruses, Human Vaccines & Immunotherapeutics, 10:5, 1211-1220, DOI: 10.4161/hv.28407 To link to this article: http://dx.doi.org/10.4161/hv.28407
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Research Paper
Research Paper
Human Vaccines & Immunotherapeutics 10:5, 1211–1220; May 2014; © 2014 Landes Bioscience
Intranasal immunization with influenza antigens conjugated with cholera toxin subunit B stimulates broad spectrum immunity against influenza viruses Center of Excellence for Infectious Diseases; Paul L. Foster School of Medicine; Texas Tech University Health Sciences Center; El Paso, TX USA
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Keywords: broad spectrum vaccine; cholera toxin subunit B; influenza; intranasal vaccination; protective immunity
Frequent mutation of influenza viruses keep vaccinated and non-vaccinated populations vulnerable to new infections, causing serious burdens to public health and the economy. Vaccination with universal influenza vaccines would be the best way to effectively protect people from infection caused by mismatched or unforeseen influenza viruses. Presently, there is no FDA approved universal influenza vaccine. In this study, we expressed and purified a fusion protein comprising of influenza matrix 2 protein ectodomain peptides, a centralized influenza hemagglutinin stem region, and cholera toxin subunit B. Vaccination of BALB/c mice with this novel artificial antigen resulted in potent humoral immune responses, including induction of specific IgA and IgG, and broad protection against infection by multiple influenza viruses. Furthermore, our results demonstrated that when used as a mucosal antigen, cholera toxin subunit B improved antigen-stimulated T cell and memory B cell responses.
Introduction Infection by influenza viruses is a very important public health concern. The effective way to prevent influenza virus infection is through vaccination. Traditional influenza vaccine formulations are based on inactivated or attenuated influenza viruses that induce B cell responses to produce neutralizing antibodies matching the predicted seasonal influenza viruses. Sometimes, traditional or seasonal influenza vaccines fail to protect against influenza viruses due to the frequent mutations of influenza viruses, even though the vaccines are reformulated annually according to surveillance data on epidemic of influenza viruses. As an example, the 2009–2010 season influenza vaccines did not protect against the unforeseen 2009 H1N1 pandemic influenza which broke out in 215 countries and caused at least 18 000 deaths worldwide.1,2 Pandemic strains are potentially devastating because the majority of the affected population lacks potent immune responses to clear the infecting virus. It is alarming that vaccine mismatches occurred at around 50% in United States from 1997 to 2005.3 Clearly, a universal influenza vaccine that would protect against a majority of circulating or potential epidemic influenza virus strains is pressingly needed. This universal vaccine would obviate the annual reformulation and repeated annual immunization. The most important aspect of universal influenza vaccine design is selection of conserved antigens and an appropriate
adjuvant.4-9 HA2 of influenza virus is located at the C-terminal of hemagglutinin (HA), and forms a stem-like structure that mediates the anchoring and fusion of the influenza viral membrane to the endosomal membrane. HA2-specific antibodies are able to reduce the replication of influenza by inhibiting this process.10 Compared with the HA1 portion of HA, HA2 is more conserved. Therefore, HA2 may be used in the formulation of a universal vaccine as it may provide cross-protection against many different viral strains. Matrix 2 (M2) protein of influenza A virus is a conserved structural protein incorporated into the viral lipid envelope. The 23-amino acid extracellular N-terminal domain peptide of M2 (M2e) is highly conserved among all influenza A viruses.11-15 Immunization with M2e confers cross-protection against various subtypes of influenza virus.16 However, we speculate that a centralized HA2 conjugated with M2e could be the basis for an improved universal influenza vaccine to elicit a much potent immune response against a broad subtype of influenza viruses over using M2e or HA2 alone. Furthermore, it would also be advantageous to design a subunit universal vaccine to be delivered intranasally over intramuscular delivery, because (1) it would eliminate the discomfort and pain from injections and (2) the mucosal immune response is critical for clearance of respiratory tract pathogens. However, to improve the immune responses elicited by a mucosal vaccine, one would need an adjuvant. Cholera toxin B subunit (CTB) can enhance the immune response to some mucosal antigens.17 Intranasal immunization
*Correspondence to: Mingtao Zeng; Email: [email protected] Submitted: 12/23/2013; Revised: 02/13/2014; Accepted: 03/03/2014; Published Online: 03/14/2014 http://dx.doi.org/10.4161/hv.28400 www.landesbioscience.com Human Vaccines & Immunotherapeutics 1211
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Junwei Li, Maria T Arévalo, Yanping Chen, Olivia Posadas, Jacob A Smith, and Mingtao Zeng
with CTB and bovine serum albumin (BSA) or HA of influenza viruses can stimulate high levels of BSA or HA specific serum IgG antibody and IgA antibody in the nasal and pulmonary lavages.18,19 IgA responses are sufficient to inhibit the transmission of influenza virus.20 Mucosal adjuvant cholera toxin is superior to other adjuvants in its ability to prime the memory CD8 T cell response.21 CTB facilitates entry into cells via highaffinity binding to cell surface receptor GM1 ganglioside, allowing for internalization and intracellular trafficking of conjugated antigens into the cytosol of dendritic cells to enhance antigen presentation.22-25 To stimulate high levels of secretory IgA in the respiratory tract and broad anti-influenza virus immune response, we designed an artificial antigen to be used as a universal influenza vaccine. We combined a centralized HA2, 3 sequential M2e units from different influenza subtypes, and nonconventional adjuvant CTB to enhance the antigen presentation and stimulation of the T cell response. Herein, we show that this novel antigen, as tested in mice, induced potent cross immune responses and protection against multiple strains of influenza viruses.
Results Development of recombinant antigen In this study, a chimeric gene was generated by combining: the CTB gene; 3 sequential M2e sequences from H1N1, H3N2, and H5N1 (Fig. 1A) influenza subtypes to increase the density and variation of M2e epitopes; and a highly centralized HA2 gene sequence (Fig. 1B). Finally, the gene sequence was
optimized for expression in E. coli and inserted into the pET200/ D-TOPO expression vector. Schematic representations of fusion protein designs containing a 6 × His tag with or without CTB are shown (Fig. 1C). These fusion proteins were successfully expressed in the E. coli BL21 (DE3 strain) and purified by affinity chromatography using Ni-NTA agarose beads with a purity of 95% as confirmed by western-blotting using an anti-6 × His mouse monoclonal antibody (Fig. 1D). These results showed the molecular weights of CTB-3 × M2e-HA2 and 3 × M2e-HA2 were approximately 45 kD and 25 kD, respectively. Humoral responses and protection elicited by intranasal and intramuscular vaccination with CTB-3 × M2e-HA2 Next, we assessed the immunogenicity of CTB-3 × M2e-HA2 and 3 × M2e-HA2 in mice after intranasal or intramuscular immunizations. These mice were immunized on days 0, 14, and 28. The humoral immune response was assessed by measuring antigen specific IgG, IgG1, and IgG2a levels in the sera collected on days 0, 14, 28, and 42 after initial immunization. Overall, mice immunized with CTB-3 × M2e-HA2 intranasally produced higher levels of IgG, IgG1 and IgG2a antibodies than those immunized intramuscularly with CTB-3 × M2e-HA2, or with 3 × M2e-HA2 via intramuscular or intranasal delivery (Fig. 2). By days 28 and 42, after 2 and 3 respective doses of our vaccine candidates, there were significant differences observed between mice immunized with CTB-3 × M2e-HA2, mice immunized with 3 × M2e-HA2, and naïve mice. Furthermore, by days 28 and 42, mice immunized intranasally with CTB-3 × M2e-HA2 displayed significantly greater serum antibody levels than all other groups. To test if immunities induced by our vaccine candidates provided protection against heterologous influenza viral challenge, mice
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Figure 1. Design and expression of CTB-3 × M2e-HA2. (A) Amino acid sequences of 3 M2e extracted from A/California/04/2009 (H1N1), A/Viet Nam/1204/2004 (H5N1) and A/Hong Kong/1/1968 (H3N2) influenza virus and the variable amino acids were underlined. (B) Highly centralized HA2 amino acid sequence. (C) Schematics of chimeric CTB-3 × M2e-HA2 and 3 × M2e-HA2 proteins. (D) Expression of CTB-3 × M2e-HA2 and 3 × M2e-HA2 proteins were verified by Western Blot using an anti-6 × His monoclonal antibody.
were intranasally inoculated with 100 × LD50 influenza PR8 (H1N1) viruses at day 42 post-immunization. Mice in the naïve group all died by day 8 post-challenge, while immunized mice were differentially protected depending on the fusion protein and immunization route used (Fig. 2D). Those immunized with 3 × M2e-HA2 fared better than naïve controls, but survival rate was less than ideal at 25% and 38% for intranasal and intramuscular immunization routes, respectively. Intramuscular immunization with CTB-3 × M2e-HA2 provided an increased rate of survival of 75%, but intranasal immunization with CTB-3 × M2e-HA2 provided optimal results with 100% survival. Because intranasal vaccination with CTB-3 × M2e-HA2 was more protective than intramuscular vaccination with CTB-3 × M2e-HA2, we reasoned that mucosal immunity may be involved in protecting mice. Hence, antibody responses in the bronchoalveolar lavage fluid of naïve mice and mice intranasally immunized with 3 × M2e-HA2 or CTB-3 × M2e-HA2 were also assessed (Fig. 3). Indeed, intranasal immunization with CTB-3 × M2e-HA2 induced robust IgA (Fig. 3A), total IgG (Fig. 3B), IgG1 (Fig. 3C), and IgG2a (Fig. 3D) responses in the lung. Of note, the specific antibody responses induced by intranasal immunization with CTB-3 × M2e-HA2 were statistically superior to
those induced by 3 × M2e-HA2. Taken together, these data suggested that the fusion protein consisting of centralized HA and M2e was immunogenic, inducing antibodies that were potentially involved in inhibiting the replication of influenza virus in vivo. Furthermore, CTB enhanced influenza-specific humoral and protective immunities. Evaluation of CTB-3 × M2e-HA2 vaccine potential for broad, cross-strain immunity Next, we tested if the recombinant CTB-3 × M2e-HA2 provided protection against multiple influenza virus strains or subtypes. We challenged immunized and naïve mice with 100 × LD50 pandemic H1N1 CA09 or 105 PFU HK68 (H3N2) influenza viruses. In mice immunized 3 times with CTB-3 × M2e-HA2, only 1 of 8 mice succumbed to CA09-related illness and died at day 15 post-challenge (Fig. 4A). The remaining 7 mice recovered completely by day 14 post-challenge. Furthermore, mice immunized 3 times with CTB-3 × M2e-HA2 and challenged with HK68 had almost no detectable virus in the lungs at 5 d postinfection (Fig. 4B). Thus, CTB-3 × M2e-HA2 induced potent immune response that inhibited the replication of CA09 and HK68 influenza viruses in the mouse lung. To continue to assess broad-immunity elicited by our CTB-3 × M2e-HA2 vaccine
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Figure 2. Evaluation of humoral and protective immunities by vaccine candidates. Mice were intranasally (i.n.) or intramuscularly (i.m.) primed with 10 µg recombinant protein, CTB-3 × M2e-HA2 or 3 × M2e-HA2. Boosters were given on days 14 and 28. Naïve, control mice received PBS (i.n.). Blood samples were collected on days 0, 14, 28, and 42. At 42 d post-immunization, all mice were challenged with 100 × LD50 PR8 influenza virus. Serum levels of 3 × M2e-HA2 specific (A) IgG, (B) IgG1, and (C) IgG2a antibodies as measured by ELISA are shown, ***P < 0.05. (D) Survival of mice challenged with 100 × LD50 PR8 influenza virus.
candidate, we infected MDCK cells with different viral strains and used cell lysates as a source of viral proteins, including HA2 and NP. Sera from mice immunized with CTB-3 × M2e-HA2 reacted with HA2 from cell lysates derived from MDCK infected with CA09, FM47 (H1N1), PR8, Aichi68 (H3N2), and HK68 influenza viruses (Fig. 4C). The sera from immunized mice did not react with cell lysates from mock-infected cells. As expected, a monoclonal antibody against influenza NP also reacted with the cell lysates infected with the various influenza viruses, while a monoclonal antibody against β-actin reacted with all cell lysates. In summary, the results showed that a three-dose, vaccination regimen with CTB-3 × M2e-HA2 was broadly immunogenic, and mice were protected from multiple strains and subtypes of influenza virus. CTB-3 × M2e-HA2 dose response To determine the optimal dose required for CTB-3 × M2e-HA2 to induce protective immunity, mice were immunized 1–3 times with CTB-3 × M2e-HA2 and challenged with 100 × LD50 PR8 influenza virus. One immunization with CTB-3 × M2e-HA2 provided 25% protection in mice, 2 immunizations provided 62.5% protection, and 3 immunizations provided complete protection against lethal PR8 influenza virus infection (Fig. 5). Thus, protection against lethal influenza virus infection was dose-dependent, with 3 immunizations providing optimal protection.
Correlating protection conferred by immunization with CTB-3 × M2e-HA2 with humoral immunity To determine whether humoral immunity was critical for the protection provided by immunization with CTB-3 × M2e-HA2, we injected naïve mice intraperitoneally (i.p.) with 200 µl sera from mice immunized with CTB-3 × M2e-HA2, PR8, CA09, or naïve. These mice were then challenged with 100 × LD50 PR8 or CA09 influenza viruses. As expected, passive transfer of PR8 and CA09 positive immune sera protected 100% of mice challenged with corresponding influenza viruses (Fig. 6A). Passive transfer of sera from mice immunized with CTB-3 × M2e-HA2 also provided significant protection against viral challenges, 87.5% and 75% of mice were protected against CA09 and PR8 influenza viruses, respectively. In contrast, naïve mice all succumbed to viral infection. To confirm these results, we tested if muMT mice, which lack mature B cells, would be protected against influenza viral infection after immunization with CTB-3 × M2e-HA2. Groups of muMT mice were immunized 3 times with CTB-3 × M2e-HA2 or 3 × M2e-HA2, once with a low-dose or PR8 virus, or left untreated. After challenge with 100 × LD50 PR8, naïve animals and those immunized with 3 × M2e-HA2 all succumbed to infection and died. Immunization with CTB-3 × M2e-HA2 or PR8 in these muMT mice only conferred 25% and 38% protection against lethal PR8 infection, respectively (Fig. 6B). These results suggested that protection
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Figure 3. Mucosal immune responses in intranasally vaccinated mice. Mice in one group served as naïve controls, while mice in the other groups were intranasally primed at day 0 and boosted at days 14 and 28 with 10 µg CTB-3 × M2e-HA2 or 3 × M2e-HA2. Bronchoalveolar lavage fluid was collected on day 42 to measure anti-3xM2e-HA2 (A) IgA, (B) IgG, (C), IgG1, and (D) IgG2a concentrations by ELISA, ***P < 0.05.
by CTB-3 × M2e-HA2 immunization was dependent on the humoral immune response. Immunization with CTB-3 × M2e-HA2 and cellular immunities To investigate the immune response further, we evaluated antigen specific T and B cell proliferation. Expression of CD4 +, CD8 +, CD19 +, and CD27+ surface markers was assessed in mouse lung cells (Fig. 7A–C) and splenocytes (Fig. 7D–F) from mice immunized with CTB-3 × M2e-HA2 or 3 × M2e-HA2. The percentage of CD4 + T cell, CD8 + T cell, and CD19 +/CD27+ memory B cells in the mouse lung and spleen significantly increased after 3 immunizations with CTB-3 × M2e-HA2 as compared with naïve or 3 × M2e-HA2 immunized mice, indicating that CTB enhanced T cell and memory B cell immune responses. Furthermore, CTB-3 × M2e-HA2 significantly stimulated IFN-γ secretion in the lung and spleen (Fig. 8). This IFN-γ response was enhanced in mice immunized with CTB-3 × M2e-HA2 over those immunized with 3 × M2e-HA2.
Discussion In this study, we designed and constructed a novel fusion antigen, CTB-3 × M2e-HA2, to confer cross-protection against a broad range of influenza viruses. Others have already studied the immunity and protection elicited by these antigens as individuals, and the knowledge gained from these studies was incorporated into the design of a single, fusion protein. Based on previous studies, we knew that adjuvanted, single or tandem M2e repeats elicit strong serum IgG and mucosal IgA antibody responses.26-28 These studies also established that 3–4 M2e repeats in tandem improved humoral immunity over the use of a single M2e, and that immunization with tandem M2e repeats induce protection against a broad range of influenza virus subtypes. Thus, in this work, we incorporated 3 tandem M2e sequences from 3 different viruses. It is also known that conserved or centralized HA2 induces cross protection.3,6,29,30 Thus, incorporation of a highly
conserved HA2 sequence could only strengthen a vaccine strategy to stimulate broad-based immunity and protection. Previous studies also show that CTB can be used in the design of influenza vaccines to enhance immune responses and that CTB alone does not elicit significant protection against lethal influenza viral challenge.31,32 Hence, after vaccine design, the focus of this study was to show that the novel CTB-3 × M2e-HA2 protein as a whole, was broadly immunogenic and protective across multiple influenza A subtypes. Furthermore, we show that protection by this fusion protein was antibody-dependent. While protection by CTB-3 × M2e-HA2 against influenza virus was antibody-dependent, this protection was not via antibody-mediated neutralization. In fact, past studies reported that anti-M2 or HA2 do not have direct virus neutralizing or hemagglutination inhibition (HI) activity. Despite not having neutralizing or HI activity, naturally induced HA2 antibody can inhibit influenza virus replication.33 Moreover, immunization with HA2 provides for faster elimination of influenza virus in mouse lung and increases survival rates.34 Although anti-M2e antibody is not able to neutralize the virus to prevent virus entry, it is able to
Figure 5. CTB-3 × M2e-HA2 antigen dose response. Mice were immunized once, twice, or thrice with CTB-3 × M2e-HA2 and challenged on day 42 after the first immunization with 100 × LD50 PR8 influenza virus.
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Figure 4. Testing broad protection against multiple influenza strains by CTB-3 × M2e-HA2. (A) Survival of mice immunized with CTB-3 × M2e-HA2 or CA09 in comparison to naïve mice after lethal challenge with 100 × LD50 CA09 influenza virus. (B) Lung viral loads in mice immunized with CTB-3 × M2e-HA2 or HK68 in comparison to naïve mice after challenge with 105 PFU HK68 influenza virus. (C) MDCK cells were infected with different viral strains at MOI = 0.1 to be used as source of viral HA2 and NP. Western Blot analysis was used to test if serum from mice immunized with CTB-3 × M2e-HA2 reacted with HA2 (about 26 kD) from infected MDCK cell lysates (top panel). A commercial, monoclonal antibody against NP was used to verify influenza viral infection of MDCK cells (middle panel). β-actin blots were used as loading controls.
disrupt the virus life cycle26-28and promotes death of infected cells by antibody-dependent cell-mediated cytotoxicity.35,36 We performed cross-neutralization assays using sera from mice immunized with CTB-3 × M2e-HA2 and PR8 influenza virus in vitro, and similar to those studies performed with HA2 and M2e antigens, CTB-3 × M2e-HA2 immune sera lacked virus neutralization activity against PR8 and CA09 influenza viruses (data not shown). This indicated that the mechanism of cross-protection induced by CTB-3 × M2e-HA2 was not via antibody-mediated neutralization of influenza viruses, but through another antibody-mediated mechanism. Therefore, it was assumed that antiM2e or HA2 antibodies could directly bind M2 or HA2, inhibit functions during the replication of influenza virus, or lead to opsonization by macrophages and dendritic cells. To extensively prepare for the unexpected outbreak of influenza, increased efforts are being focused on adjuvants that enhance vaccine immunogenicity against emerging influenza viruses and maximize vaccine supply.37 New strategies for influenza vaccine design are under development.38-41 One strategy is to design a vaccine for mucosal delivery. The mucosa serves as the first line of defense against invading respiratory pathogens, and designing a vaccine for intranasal delivery would also bypass the need for trained professionals needed to perform injections while reducing the associated biohazardous wastes. Mucosal immunization with influenza viral antigen can elicit robust immune response, but often the adjuvants used to enhance the immune response lead to safety issues, with side effects reported in clinical testing.42,43 On the other hand, clinical trials have shown that formulations containing recombinant CTB are safe and well tolerated.44,45 In our study, CTB fused to influenza antigens enhanced protective immunity in immunized mice. The mechanism by which CTB enhances mucosal immunity is still unclear, but dendritic cell maturation and antigen presentation are stimulated.46 Immunization with different adjuvants or by different routes may elicit different immune responses. As an example, immunization with M2e-based vaccine induces a predominantly IgG1 over IgG2a response.47 In contrast, immunization with NP-M2e fusion protein induces both IgG1 and IgG2a responses.41 The
humoral immune response induced by immunization with CTB-3 × M2e-HA2 was predominantly a Th2-mediated antibody response. In addition, results in Figures 6 and 7 suggest that T cell immunity was also stimulated by immunization with CTB-3 × M2e-HA2. While T cell immunity may be important in protection against influenza virus infection, the antibody response is critical for cross anti-influenza protection.48-50 Indeed, our results showed that the humoral immune response was indispensable for protection induced by immunization with CTB-3 × M2e-HA2. However, T cell immunity was also induced in our studies. In conclusion, we have shown that CTB-3 × M2e-HA2 stimulates potent broad spectrum immunity against influenza A viruses. This antigen, as a recombinant protein, can be produced inexpensively in large-scale quantities without any need for the time-constraining culture of influenza virus. A corollary advantage is no time constraint to prepare the vaccine for a potential new influenza pandemic. In the event of a pandemic flu crisis, this novel vaccine candidate may provide relief to the public health burden.
Materials and Methods Viruses, antibodies and animals Mouse adapted A/California/04/2009(H1N1) (pandemic CA09) and A/FM/1/47 (H1N1) (FM47) were gifts from Dr Richard Webby (St. Jude Children’s Hospital).51 A /HongKong/1/68 (H3N2) (HK68, NR-346) and A/ Aichi/2/1968(H3N2) (Aichi68, NR-3177) were provided by BEI Resources, and A/Puerto Rico/8/1934 (H1N1) (PR8) was generated using plasmids received from Dr Webster. Alexa Fluor700 conjugated anti-mouse CD3e (cat#557984), Pacific Blue conjugated anti-mouse CD4 (cat#558107), V500 conjugated anti-mouse CD8a (cat#560776), APC-Cy7 conjugated anti-mouse CD19 (cat#557655), PE conjugated anti-mouse CD27 (cat#558754) for flow cytometry were purchased from BD Bioscience. Goat anti-mouse IgG-Fc (cat#A90–131A), goat
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Figure 6. Evaluating the role of humoral immunity in protection against PR8 and CA09 influenza viruses. (A) Mice were i.p. injected with serum collected from naïve mice or mice immunized with CTB-3 × M2e-HA2, and then challenged with lethal doses of CA09 or PR8 influenza viruses. Clinical symptoms and survival were monitored and recorded. (B) B cell depleted mice were immunized with CTB-3 × M2e-HA2 or 3 × M2e-HA2, and then challenged with 100 × LD50 PR8 influenza virus.
anti-mouse IgA (cat#A90–103A) goat-anti-mouse IgG1 (A90– 105A), goat anti-mouse IgG2a (cat#A90–107A) were purchased from BETHYL Laboratories Inc. Female BALB/c and muMT B cell depleted mice were purchased from Jackson Laboratory (cat# 002288). Antigen design, construction, and expression To create a highly conserved HA2 sequence, we aligned HA2 sequences from H1N1, H2N2, H3N2, H5N1, H7N1, and H9N2 influenza viruses found in the NCBI-Influenza Virus Resource database. The most common amino acids in each position were used to create an artificial, centralized, HA2 sequence. Subsequently, the conserved HA2 sequence was linked with 3 M2 protein ectodomain (M2e) sequences from A/California/04/2009(H1N1) (CA09), A/Viet Nam/1204/2004(H5N1) (VN04), and A/Hong Kong/1/1968 (H3N2) (HK68) influenza viruses in tandem at the N-terminal. Then, the DNA sequence of cholera toxin subunit B (CTB) was fused at the N-terminal of 3 × M2e-HA2 DNA sequence. The whole DNA sequence was codon optimized for expression in E. coli and synthesized by GenScript. For the expression of chimeric proteins, the DNA sequences of CTB-3 × M2e-HA2 or 3 × M2e-HA2 were inserted into the E. coli expression vector: pET200/D-TOPO (Invitrogen, cat#K10001). Finally, the plasmid constructs were transformed in E. coli BL21 (DE3) strain (Life Technologies, cat#440054) Transformed cells were cultured in Luria Broth (LB) with 50 µg/mL of kanamycin to
OD600 of 0.5, then supplied with 0.1 mM Isopropyl β-D-1thiogalactopyranoside (IPTG) and cultured for another 4 h at 37 °C. These cultures were harvested by centrifugation at 6000 rpm for 15 min. E. coli pellets were lysed with lysis buffer (50 mM NaH2PO4, 500 mM NaCl, 10 mM imidazole, pH 8.0). The lysates were collected after centrifugation at 15 000 rpm for 30 min and incubated with Ni-NTA agarose (Qiagen, cat#30230) in polypropylene columns overnight at 4 °C. The protein-bound resin was washed with washing buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 8.0) 5 times and His-tagged proteins were eluted with elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0). Purified proteins were dialyzed using ultracentrifugal filter devices (Millipore, cat#UFC900324). The eluted proteins were confirmed by western-blotting with anti-His tag mouse monoclonal antibody (Life Technologies, cat#R93025) and stocked in PBS with 10% glycerol at −80 °C prior to use for vaccination. Mouse immunization, bleeding, and challenge Six-wk-old BALB/c mice purchased from the Jackson Laboratory were divided into 3 groups at random and housed in the LARC facility of Texas Tech University Health Science Center at El Paso. All studies with experimental animals were approved by the Institutional Animal Care and Use Committee. Mice in group 1 were used as naïve controls and intranasally received 50 μL PBS. The second group was intranasally immunized with 10 μg purified antigen in 50 μL PBS. The third group was
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Figure 7. T cell and memory B cell immune responses elicited by vaccination with CTB-3 × M2e-HA2. After mice were immunized, splenocytes and lung cells were collected and CD4, CD8 T, and memory B cell percentages were analyzed by flow cytometry, ***P < 0.05. (A) Percentage of CD3e and CD4 double positive cell in lungs. (B) Percentage of CD3e and CD8 double positive T cells in lungs. (C) Percentage of CD19 and CD27 double positive cell in lungs. (D) Percentage of CD3e and CD4 double positive cell in spleens. (E) Percentage of CD3e and CD8 double positive T cells in spleens. (F) Percentage of CD19 and CD27 double positive cell in spleens.
injected with 10 μg purified antigen in hind leg thigh muscle. Immunizations were given at day 0, 14, and 28 for a total of 3 immunizations per mouse. On the day of each immunization, mice were anesthetized with ketamine and xylazine, and mouse blood was collected from the submandibular vein. Blood serum was separated, aliquoted, and stored at −80 °C for future use. Bronchoalveolar lavage fluid was collected according established method, and stored at −80 °C for IgA quantification. For infections, each mouse was anesthetized and intranasally inoculated with influenza virus and weighed daily. Mice were observed daily for clinical symptoms and survival. Animals were anesthetized for performing procedures such as intranasal immunization and viral challenge. At the end of the experiments, animals used in the experiments were euthanatized in a CO2 chamber in a manner consistent with the AVMA Guidelines for the Euthanasia of Animals (2013 Edition) through the Panel on Euthanasia of the American Veterinary Medical Association. Enzyme-linked immunosorbent assay Serum antibody titers for each mouse were determined by enzyme-linked immunosorbent assay (ELISA). 96-well plates were coated with 1 μg/mL CTB-3 × M2e-HA2 in 100 μL 50 mM sodium bicarbonate buffer (pH 9.6) overnight at 4 °C, then blocked with PBS containing 1% bovine serum albumin for 30 min at room temperature. Sera were diluted 50-fold in 100 μL PBS and incubated overnight at 4 °C. Commercial Mouse serum was taken as reference to draw a standard curve for quantifying antibody in immunized mice serum (Bethyl, cat#rs10101). After washing, 100 μL alkaline phosphatase conjugated anti-mouse IgG-Fc antibody diluted at the ratio of 1:50 000 in PBS was added into each well and incubated for 1 h at room temperature. Plates were washed and developed with 100 μL diethanolamine substrate (KPL, cat#508000). The reaction was stopped after 20 min with 100 μL EDTA stop solution per well. The plates were read at 405 nm using the PowerWaveXS2 (Biotek). HA2 and antibody binding MDCK cells seeded in 35 mm dishes were infected with CA09, A/FM/1/47(H1N1) (FM47), PR8, A/Aichi/2/1968(H3N2), HK68 of MOI (multiplicity of infection) = 0.1. At 24 h after infection, cells were harvested, and lysed in 1 × cell lysis buffer (Cell
Signaling Technology, cat#9803). Lysed MDCK cell samples in Laemmli sample buffer (without reducing agent) were loaded and separated on 10% SDS-PAGE, then transferred onto nitrocellulose membrane using a semi-dry transblot apparatus (Biorad, cat#1703940). The membrane was blocked in PBS with 1% Tween (PBST) containing 5% non-fat milk for 1 h and incubated with serum from mice immunized with CTB-3 × M2e-HA2 or NP monoclonal antibody (Abcam, cat#ab20343) at 4 °C overnight. After washing with PBST, the membrane was incubated with alkaline-phosphatase conjugated goat anti-mouse IgG antibody at room temperature for 1 h. After washing, the membrane was developed with BCPI/NBT substrate (Sigma, cat#B1911). Passive transfer of mouse serum 48 BALB/c mice were divided into 6 groups randomly, 2 naïve groups were injected i.p. with 200 μL naïve mouse serum. The third group was injected i.p. with 200 μL mouse serum from mice previously infected with PR8. The fourth group was i.p. injected with 200 μL mouse serum from mice previously infected with CA09, and the fifth and sixth groups were injected with serum from mice immunized with CTB-3 × M2e-HA2. All groups were then challenged with either 100 × LD50 PR8 or CA09. Mice were observed for clinical symptoms and survival. Flow cytometry Splenocytes and lung cells were collected from mice immunized 3 times with CTB-3 × M2e-HA2. Single cell preparations were washed, incubated with ACK lysis buffer, and then stimulated with PHA (10 μg/mL) for 4 h. GolgiStop (BD Biosciences, cat#554724) was added and cells were incubated for another 2 h. After washing with PBS, cells were permeabilized, then stained with Alexa Fluor® 700 hamster anti-mouse CD3e, Pacific Blue™ rat anti-mouse CD4 +, V500 rat anti-mouse CD8 +a, PE hamster anti-mouse CD27+, APC-Cy™7 rat anti-and mouse CD19 + antibodies. Fixed samples were acquired using the Gallios flow cytometer (Beckman Coulter, Inc.) with a minimum acquisition of 15 000 events and analyzed by using FlowJo software (TreeStar). Enzyme-linking immunospot (ELISPOT) assay MultiScreen Filter Plates (Millipore, cat#MAIPS4510) were coated with rat anti-mouse IFN-γ monoclonal antibody (BD
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Figure 8. Analysis of IFN-γ secretion by immune cells from the lung and spleen. Cellular immune responses after vaccination were evaluated using an ELISPOT assay to enumerate IFN-γ-secreting cells, ***P < 0.05. (A) IFN-γ-secreting cells in lung cells. (B) IFN-γ-secreting cells in splenocytes.
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Windows (GrahPad Software). P values < 0.05 were considered to be significant difference. Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed. Acknowledgments
This work was supported by the US Public Service research grant AI072139 (M.Z.) from the National Institute of Allergy and Infectious Diseases and an internal fund from Texas Tech University Health Sciences Center Paul L. Foster School of Medicine. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We are grateful to David Topham for providing MDCK cells, to Richard Webby for providing PR8 influenza viral plasmid system and mouse adapted H1N1(A/California/04/2009) influenza virus and A/FM/1/47(H1N1) influenza virus, and to the NIH/NIAID funded BEI Resources for supplying seasonal influenza vaccines and influenza A/HongKong/1/68 (H3N2) and A/Aichi/2/1968 (H3N2) strains.
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Bioscience, cat#553209) in coating buffer at concentration of 5 µg/mL at 4 °C overnight. Splenocytes and lung cells were isolated from mice. After being washed, cells were seeded and stimulated with PHA at concentration of 10 µg/mL at 37 °C in 5% CO2 overnight. After stimulation, plates were washed 5 times with PBS containing 0.05% Tween 20 (PBST) and incubated with biotin conjugated rat anti-mouse IFN-γ antibody (2.0 µg/mL) at room temperature for 2 h. The plates were washed and incubated with horseradish peroxidase (HRP) conjugated streptavidin (BD Bioscience, cat#555214) at room temperature for 1.5 h. Plates were washed with PBS and added with HRP substrate. Subsequently, the plates were developed for 10 min in the dark and substrate was removed. After being washed with doubledistilled water, plates were dried overnight. Finally, spots were counted under ELISpot reader (AID, Germany). Statistical analysis Comparisons between vaccinated groups were performed by using a nonparametric one-ways ANOVA with the Tukey multiple comparison test and the Fisher exact test, and survival dates were analyzed by using the log-rank test. The analyses were performed by using GraphPad Prism version 5.0 for
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