Antibody Response to Influenza Hemagglutinin Conserved Stalk Domain after Sequential Immunization with Old Vaccine Strains

Hemagglutinin (HA) is the major envelope glycoprotein and antigen on the surface of influenza virions. The glycoprotein comprises a globular head and a stalk region. While immunodominant epitopes on influenza HA head are highly variable, the stalk domain is conserved. The variability of the HA head causes the antigenic drift that made the requirement of annual update of vaccine strains. Induction of antibody against the stalk domain has been proposed as an approach for a broadly protective influenza vaccine strategy. Sequential exposure to influenza strains with highly diverse HA heads but conserved stalks have been shown to induce antibody to the low immunogenic stalk domain. Here, we tested this approach by using old influenza vaccine strains that are decades apart in evolution. Inactivated whole virion vaccine of influenza A/Puerto Rico/8/1934, A/USSR/92/1977, and A/Thailand/102/2009 (H1N1) was sequentially immunized into BALB/c mice in comparison to immunization using single strain (A/Thailand/102/2009 (H1N1)). The sequentially immunized mice developed higher levels of binding antibody to the stalk domain. These suggested that using old vaccine strains in sequential vaccination may be a possible approach to induce antibody to the conserved stalk domain.


Introduction
Seasonal infuenza viruses evolve under a strong positive selection by the host immune response.Tis leads to frequent changes in the viral antigenic epitopes commonly referred to as the "antigenic drift" [1].Because of this antigenic drift, the seasonal infuenza vaccine is annually updated for its viral strain composition [2].Te antigenic drift is usually caused by changes in the immune dominant and variable epitopes on the globular head of the viral hemagglutinin (HA), which is the major envelope glycoprotein responsible for receptor binding [3,4].Te HA is a trimeric protein comprising a variable globular head and a more conserved stalk domain [5].Te stalk is less immunogenic but can be a target of neutralizing antibody.Te conserved epitopes on the HA stalk are an interesting target for the development of a broadly protective infuenza vaccine [6].Various strategies have been developed to induce protective antibody targeting this conserved stalk epitope, including chimeric HA and headless HA.Te chimeric HA approach uses HA heads from avian infuenza viruses and a stalk of seasonal infuenza virus.Chimeric HA constructs with diferent heads are sequentially immunized so that only the stalk domain can induce anamnestic response, while the diferent heads can only induce a primary response in each immunization dose.Te sequential vaccination with different immunogens that share only a common target epitope has been shown to be successful in animal studies.Te concept was further supported by the observation that people infected or immunized with the 2009 H1N1 infuenza virus developed antibody to the stalk domain.Tis was explained by an anamnestic response to the stalk, which was similar to the previously circulating seasonal H1N1 infuenza, whereas the globular heads of the seasonal H1N1 and the 2009 pandemic H1N1 were too diferent for an anamnestic response [7][8][9].Following the same line of thought, it was conceivable that sequential immunization with old vaccine strains, which are highly diverse in the globular head but have a similar stalk, may be able to induce antibody against the stalk domain.Using old vaccine strains and conventional vaccine technology has an advantage of having less safety concern and requiring no change in production technology.Terefore, we tested this approach in a mice model and show here that sequential immunization with old and distantly related seasonal H1N1 vaccine strains resulted in antibody response against the stalk domain.Tis approach may provide a viable immunization strategy to raise antibody responses against conserved epitopes of infuenza HA for a broad protection.

Cells and Viruses. Madin-Darby canine kidney (MDCK)
and human embryonic kidney (HEK) 293T cells were maintained in minimal essential medium and Dulbecco's modifed Eagle's medium (DMEM) (both from Gibco).Each was supplemented with 10% fetal bovine serum (Gibco) and 100 units/ml of penicillin 100 µg/ml of gentamycin.Te MDCK cell line was kindly provided by M. Peiris, the University of Hong Kong, and the 293T cell line was obtained from ATCC and used at passage 20th and 12th, respectively.
Chimeric infuenza A virus (cH9/1) was kindly obtained from Prof. Peter Palese.Te chimeric HAs contain the globular head domain of A/guinea fowl/Hong Kong/WF10/ 99 (H9N2) HA and the common stalk domain from the A/Puerto Rico/8/1934 (H1N1) virus.
Te recombinant infuenza A virus used in this study was produced by the reverse genetics method as previously described [10].Briefy, HEK293T and MDCK cell lines were mixed and cocultured in 2 ml of opti-MEM media (Gibco) in a 6-well plate for 18 hours.Te cells were cotransfecting with pHw2000 plasmids carrying the cloned HA gene (A/Puerto Rico/8/1934, or A/Nonthaburi/102/2009 or A/USSR/92/ 1977) and the other 7 genes from the A/Puerto Rico/8/1934 (H1N1) (PR8) strain by using TranslT-LT1 transfection reagent (MirusBio, USA, Cat.No. MIR2300).At 30 hours posttransfection, 1 ml of MEM containing 2 μg/ml of L-(tosylamido-2-phenyl) ethyl chloromethyl ketone (TPCK) treated trypsin (Sigma, USA) was added.Te supernatant was sampled for virus on day 3 by hemagglutination assay.Te culture was passaged in MDCK cells for two passages until high titer viral growth.After that, viruses were propagated in the 9-day-old embryonated eggs to obtain higher viral titer.

Inactivation and Purifcation of Viruses.
Allantoic fuids (50 eggs per virus strain) were passed through a 0.22 μm flter and inactivated with 0.4% formalin for 3 days at 4 °C.Inactivated viruses were dialyzed to eliminate formalin, and virus inactivation was confrmed by inoculation into MDCK cells.Next, inactivated viruses were concentrated with 50% polyethylene glycol 8000 (PEG8000: Sigma), and the concentrated samples were purifed using sucrose step gradient ultracentrifugation (bottom 55%; top 35%) in a Beckman MLS-50 rotor at 50,000 g for 2 hours.Fractions of 500 μl were collected.HA proteins were analyzed by SDS-PAGE and western blotting, and fractions containing infuenza viruses were pooled.Purifed viruses were dialyzed to eliminate sucrose and concentrated with 50% PEG8000.Te concentrated viruses were resuspended to homogeneity in PBS and stored at −80 °C until tested.

Western Blotting.
Te purifed viruses were loaded onto a 10% polyacrylamide gel.Te gel was transferred onto a nitrocellulose membrane.Te blot was probed with 1 : 100 of infuenza anti-A/California/7/2009 (H1N1)v HA serum (NIBSC 09/152) as a primary antibody.HA proteins were detected with conjugate rabbit antisheep horseradish peroxidase (HRP; Invitrogen) and visualized with diaminobenzidine (DAB, Sigma).Te HA protein band was compared for the intensity with a standard HA protein of the virus A/California/04/20009 (H1 HA protein with cterminal histidine tag from infuenza virus A/California/ 04/20009 recombinant from baculovirus; BEI resources NR-15749) with a gel documentation system (Syngene).

Animal.
Female BALB/c mice between 6 and 8 weeks old were obtained from the National Laboratory Animal Center, Mahidol University.Experiments were performed in a biological safety cabinet in a BSL-2 facility, and the protocol was approved by the Faculty of Veterinary Science Animal Care and Use Committee (FVS-ACUC).

Animal Experimental Design.
Mice were divided into 3 groups: homologous, heterologous, and control groups.Homologous-boosted mice (n � 10) were primed with the inactivated pandemic 2009 infuenza vaccine and boosted at week 4 and 8 with the same vaccine strain.Heterologousboosted mice (n � 10) were primed with the inactivated pandemic 2009 infuenza vaccine and boosted with the inactivated A/Puerto Rico/8/1934 infuenza vaccine at week 4 and the inactivated A/USSR/92/1977 infuenza vaccine at week 8. Te control group (n � 5) received PBS at the initial prime and at week 4 and 8. Mice were anesthetized by isofurane inhalation.Ten μg antigen in PBS mixed with AddaVax (Invitrogen) adjuvant at a 1 : 1 ratio in a volume of 50 μl was administered intramuscularly for each immunization.Sera were collected 2 weeks after the third

Selection of Old Vaccine Strains.
To obtain H1N1 infuenza strains with only distantly related globular heads and highly conserved stalk domain, we performed phylogenetic analysis of pandemic and seasonal H1N1 infuenza strains by maximum-likelihood method implemented in PAUP version 4.0 and selected 3 viruses that belong to diferent branches in the phylogenetic tree as shown in Figure 1.Tis selection aimed at having HAs with the most distances from one another in the history of H1 circulation in the human population.Te three selected strains are A/Puerto Rico/8/ 1934, Russian fu 1977, and pandemic fu 2009.Te amino acid similarities in the globular head and stalk domain among the tree strain are 83.6% and 97.0%, respectively.Tis indicates that the three strains are highly diverse in their globular head but very similar in their stalk domain.To obtain infuenza HA antigen for the immunization, the reverse genetic viruses carrying the HA of A/Puerto Rico/8/ 1934, A/Nonthaburi/102/2009, or A/USSR/92/1977 viruses were grown in embryonated eggs.Allantoic fuid was harvested and checked for the titer of infuenza virus by hemagglutination assay.Te allantoic fuid yielded virus at hemagglutination titers of 32-4096/50 μl.Whole virions were inactivated, concentrated, and purifed from the harvested allantoic fuid and analyzed for the HA content by western blot after sucrose step gradient ultracentrifugation (Figure 2).

Antibody
Response against the HA Globular Head.To confrm that the immunization was efective, the antibody response against the homologous H1 strain was evaluated.Te geometric mean hemagglutination inhibition and neutralization titers against A/Tailand/102/2009 were found to be 557 and 1114 in group 1 and 640 and 1114 in Advances in Virology group 2 mice, respectively (Figure 3).Tis indicated that the immunized antigen was adequate, and the immunization was efective in inducing the normal antibody responses against the dominant globular head epitopes.

Antibody to the Stalk Domain in Mice Sequentially
Immunized with Diferent HA Strains.After completing that vaccination, mice in both heterologous-and homologousboosted groups showed binding antibody in an ELISA assay against cH6/1 and cH9/1 chimeric HA.Te chimeric HAs were used to avoid binding to the HA globular head.Immunized mice should not have antibody response to H6 or H9 as the immunized antigens were H1.Terefore, the binding antibody that was detected in this ELISA belonged to the common HA stalk in the immunized and ELISA antigens.Since the globular head of the chimeric HAs was derived from unrelated H6 and H9 viruses, which have no cross-reactivity with the globular head of H1, the binding activity indicated reactivity of the mouse sera to the H1 stalk domain in the chimeric HA molecules.All the sera were shown to be negative for hemagglutination inhibition against H9.Tis confrmed the specifcity of the ELISA signal to the stalk domain.Tese chimeric HAs have been previously used to detect the antibody to the stalk domain of the H1 in a number of studies [9].Mice in the heterologousboosted group showed a signifcantly higher binding signal in the ELISA assay (t-test, p < 0.05) as compared to the homologous-boosted group (Figure 4).Tis indicated that the heterologous boosts could induce higher antibody response against the common stalk domain.
To fnd out whether the stalk-specifc binding antibody was capable of neutralizing the virus, the sera were tested for plaque-reduction neutralization assay against reverse   Advances in Virology genetic viruses carrying the chimeric H9/1 HA.As a control for the stalk-specifc neutralization, a monoclonal antibody known to target a neutralizing epitope on the stalk domain was used.Te monoclonal antibody (MAb 6F12) was able to signifcantly reduce the plaque number of the cH9/1 virus at a concentration equal to or higher than 5 μg/ml.contrast, all the immunized mouse sera showed only a background level of neutralization activity similar to the nonimmunized mice (Figure 5).Tis suggested that the observed binding antibody against the stalk domain was either too low to neutralize the virus or targeted nonneutralizing epitopes in the stalk domain.

. Discussion
Our data clearly showed a possibility of using old seasonal infuenza vaccine strains in sequential immunization to induce an anamnestic response against the common HA stalk domain.Te sequentially immunized mice developed binding antibody against the chimeric HAs carrying the H1 stalk.Tis ELISA technique has been previously used to demonstrate binding antibody to the stalk domain [9].Te globular heads of the chimeric HAs carried unrelated avian HA heads and were, therefore, not recognized by mice immunized with H1 HA, and the detected binding, therefore, belonged to stalk-binding antibody.Our data also demonstrated that the distances between the old vaccine HAs antigens representing 4-8 decades of evolution were sufcient to suppress the anamnestic response to the globular heads and to focus the response to the common stalk domain.
Previous studies showed that sequential heterologous immunization with HA antigens with similar stalk but different globular heads could induce stalk-specifc antibody response [6].An approach of using chimeric HAs with globular heads from avian infuenza viruses combined with similar H1 stalk was tested in animal models and shown to be efective in inducing stalk-specifc neutralizing antibody.Te chimeric vaccine approach has entered clinical development with promising results [11].Tese chimeric HAs are, however, new antigens that have never been used in humans, and a number of safety profles are required to ensure safety in human use.In contrast, old H1N1 vaccine strains have the beneft of being antigens that have been used safely in humans.Reintroducing these old vaccine strains may have a lower regulatory requirement and make the development process less time-and resource-consuming.Tese H1N1 vaccine strains are highly conserved in their stalk region and thus should efectively provide the boost for stalk-specifc antibody.Te globular heads of these old vaccine strains are highly diverse across decades of antigenic drift and are unlikely to be efective in boosting antibody against highly variable epitopes.In contrast, current seasonal infuenza vaccines use strains with small changes in the globular head, which induce efective anamnestic responses against variable epitopes and little responses against conserved stalk epitopes.Our study was conducted in naïve hosts, and it is conceivable that responses in humans previously exposed to seasonal infuenza would be diferent.It is possible that older individuals may have immune memory to older viral strains and respond less efectively to the conserved stalk domain.On the other hand, young individuals who only have immune memory to the current H1N1 virus may readily respond to conserved epitopes upon immunization with an old strain.In comparison to the current approach with chimeric avian HA heads, our approach may also induce antibody responses against conserved epitopes on HA head.Our results suggested that the diference in the variable epitopes among the old vaccine strains was sufcient to allow a more efective response to conserved epitopes.Although the conserved stalk domain drew much interest in universal vaccine design, conserved epitopes in the HA1 head have been identifed [12,13].It is, however, more difcult to isolate the conserved epitopes on HA head for vaccine design.Our approach may theoretically induce responses to such epitopes, and further studies are required to test this hypothesis.
Our results did not show a signifcant neutralizing antibody against the stalk domain.While neutralizing antibodies against the stalk epitopes have gained a lot of interest, it is still unclear whether they are required for protection, and other mechanisms such as antibody-dependent cellular cytotoxicity (ADCC) have been proposed to play an important role in the protection by stalk antibody [14].It is also possible that better immunogen preparation and formulation will result in a better neutralizing response against the conserved stalk epitopes.

Control group
Homologous boost Heterologous boost

Figure 1 :Figure 2 :Figure 3 :
Figure1: Maximum-likelihood tree of the H1 amino acid sequence shows the selected old vaccine strains on diferent branches.

Figure 4 :
Figure 4: ELISA binding to the stalk domain of HA using purifed recombinant chimeric HA; chimeric H6 head with H1 stalk (a) and chimeric H9 head with H1 stalk (b).Homologous-boosted mice � 10) were primed with inactivated pandemic 2009 infuenza vaccine and boosted at week 4 and 8 with the same vaccine strain.Heterologous-boosted mice (n � 10) were primed with inactivated pandemic 2009 infuenza vaccine and boosted with inactivated A/Puerto Rico/8/1934 infuenza vaccine at week 4 and inactivated A/USSR/92/1977 infuenza vaccine at week 8. Te control group (n � 5) received PBS at the initial prime and at week 4 and 8 boost.Te chimeric HAs were used to avoid binding to the HA globular head.Immunized mice did not have antibody response to H6 or H9 as the immunized antigens were H1.Terefore, the binding antibody that was detected in this ELISA belonged to the common HA stalk in the immunized and ELISA antigens.Te asterisks indicate signifcant diferences at p < 0.05 (t test).

Figure 5 :
Figure 5: Plaque plates showing plaque-reduction neutralizing activity of sera from unimmunized control mice and immunized mice in homologous and heterologous groups.Plaque reduction was comparable in all groups, indicating the absence of neutralizing antibody against the HA stalk.

2
Advances in Virology immunization.Mice were euthanized by overdose isofurane inhalation.Blood was collected by direct cardiac puncture and allowed to clot for 1 hour at room temperature before centrifugation at 10,000 g for 15 minutes.Sera were stored at −20 °C unit tested.≥50% of the amount of viral nucleoprotein in the reaction wells as compared to the virus control wells.2.9.Plaque-Reduction Neutralization Assay.Te treated sera were two-fold serially diluted in duplicate and incubated with 30 to 50 PFU of virus for 2 hours at 37 °C.Te serumvirus mixture was inoculated on a monolayer of MDCK cells in a 12-well plate and incubated at 37 °C for 2 hours with intermittent rocking every 15 minutes.After 2 hours of incubation, the cells were covered with an agar medium consisting of a mixture of 2% agarose (Promega, USA) and MEM supplemented with trypsin TPCK.At 2 days postinfection, the monolayer was fxed with 10% formalin for 1 hour and then stained with 1% crystal violet.Plaques were counted for each serum, and the percent inhibition was calculated versus the virus control.
Ten, plates were washed twice with 0.025% tween/1X PBS (PBS-T) and blocked with 5% nonfat milk in 1X PBS for 30 minutes at RT. Te serum was diluted serially in 5% nonfat milk, incubated for 1 hour at 37 °C, and then washed 4 times with PBS-T.Te serial dilution process started by placing 146 μl of 1 : 100 diluted serum into column 1. Subsequently, 46 μl of the diluted sample was serially transferred into 100 μl of diluent in the next well.Tis resulted in a serial ½ log 10 dilution.Goat antihuman IgGhorseradish peroxidase (HRP) (Meridian Life Science Inc.) as the secondary antibody was diluted 1 : 2000 in 5% nonfat milk; 50 μl was added to each well, incubated for 1 hour at 37 °C, and then washed 4 times with PBS-T.50 μl of TMB peroxidase substrate system (KPL, USA) was added into each well and incubated in the dark for 10 min at room temperature.1M H 2 SO 4 was added to stop the reaction, and the plate was read at OD 450/630 by ELISA plate reader.nucleoprotein (Millipore, Temecula, USA) as the primary antibody and HRP-conjugated goat antimouse immunoglobulin (SouthernBiotech, Alabama, USA) as the secondary antibody.Finally, the signal of ELISA was developed with tetramethylbenzidine-H2O2 substrate (KPL, USA), and optical densities were measured at 450 nm.Antibody titer is defned as the reciprocal of the highest serum dilution that reduces 2.10.Statistical Analysis.Te comparison between groups was performed using an independent samples t-test and SPSS Statistics software (SPSS, Inc., Chicago, IL, USA).A p value ≤0.05 was taken as statistically signifcant.