Abstract
We previously demonstrated that recombinant raccoon poxvirus (RCN) could serve as a vector for an influenza vaccine. RCN constructs expressing the hemagglutinin (HA) from H5N1 viruses were immunogenic in chickens. In the current study, we generated several recombinant RCN constructs expressing influenza (H5N1) antigens and a molecular adjuvant (Heat-Labile enterotoxin B from E. coli: RCN-LTB), demonstrated their expression in vitro, and evaluated their ability to protect mice against H5N1 virus challenge. RCN-HA provided strong protection when administered intradermally (ID), but not intranasally (IN). Conversely, the RCN-neuraminidase (NA) construct was highly efficacious by the IN route and elicited high titers of neutralizing antibodies in mice. Vaccination by combined ID (RCN-HA) and IN (RCN-NA) routes offered mice the best protection against an IN challenge with heterologous H5N1 virus. However, protection was reduced when the different RCN constructs were pre-mixed, perhaps due to reduced expression of antigen.
Keywords: Raccoon poxvirus, HPAI, vaccine, mice, mucosal
1. Introduction
Influenza viruses (family: Orthomyxoviridae) are single stranded, negative sense RNA viruses of major public health and veterinary concern. In recent years, a highly pathogenic avian influenza (HPAI) H5N1 virus has emerged that is causing high mortality in avian populations [1] and is a significant public health threat. H5N1 epidemics have resulted in mass culling of hundreds of millions of birds and economic losses for southeast Asian countries totaling to 0.5 –1.5% of their gross domestic product [2]. These H5N1 viruses occasionally infect humans with a case fatality rate nearly 60% [3].
The H5N1 virus has spread throughout Asia, Europe and Africa, establishing evolutionary and geographically distinct viral strains (clades) [3]. HPAI evolves rapidly by reassorting gene segments (antigenic shift) and mutating antigens under selective pressure from the host immune system (antigenic drift) [1]. The development of highly effective HPAI vaccines is vital to control the spread of H5N1 epidemics in poultry and to prevent a potential HPAI pandemic.
Several approaches are being used to develop vaccines against HPAI, including: inactivated whole, subunit and live attenuated viral vaccines derived from influenza virus; DNA vaccines; and recombinant viral vectors engineered to express influenza antigens [4]. Inactivated vaccines are widely used and considered to be highly safe; however, they are poorly immunogenic, have a narrow protection range, and a short duration of immunity [4]. The manufacturing process for inactivated vaccines is also laborious, and the potency of stockpiled lots has been observed to decay over time [5]. Live attenuated HPAI vaccines elicit better protection, because they mimic natural influenza infection, but they are considered unsafe because of their potential to revert back to wild-type viruses [4]. Recombinant viral-vectored vaccines have the potential to overcome the risks associated with live attenuated vaccines and can also stimulate broad humoral and cellular immunity that will provide long lasting protection [4]. Furthermore, vectored vaccines can potentially differentiate infected from vaccinated animals (DIVA), unlike vaccines that contain whole virus [6].
Poxviruses have several unique features that make them highly suitable vaccine vectors for influenza [5, 7, 8]. Poxviruses infect a wide range of hosts, can be administered by a variety of routes, tolerate large size inserts (up to 20 kb), and single constructs can be designed to express multiple antigen genes [4, 9, 10]. The manufacture, lyophilization and stability of poxviruses has also been well established [10].
We recently reported on the immune response induced in chickens by a raccoon poxvirus (RCN) construct expressing the hemagglutinin (HA) gene from the HPAI isolate A/Vietnam/1203/2004 (H5N1) [11]. The present study characterizes in mice the immune responses and protection afforded by RCN constructs expressing the H5 hemagglutinin (HA), N1 neuraminidase (NA), and nucleoprotein (NP) genes. The findings highlight the importance of the route of immunization and viral vector co-administration on the immune response and protection against HPAI challenge.
2. Materials and methods
2.1. Mice
Female 6–10-week-old A/J mice were purchased from Jackson Laboratory (Bar Harbor, ME). All experiments were approved by the University of Wisconsin-Madison’s Animal Care and Use Committee.
2.2. Cells and viruses
Vero, Rat-2 and Madin-Darby canine kidney (MDCK) cells were propagated in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% fetal bovine serum (FBS). BS-C-1 and BS-C-40 cells were propagated in RPMI 1640 medium with 10% FBS. Cells were maintained in humidified air with 5% CO2 atmosphere at 37°C. Raccoon poxvirus (RCN) was obtained from the Centers for Disease Control, Atlanta and stocks were prepared as described [12]. Inactivated H5N1 influenza (A/Vietnam/1203/2004) vaccine (whole virion, Vero cell derived, non-adjuvanted) was obtained from Biodefense and Emerging Infections Research Resources Repository (NR-12148: BEI Resources, VA). H5N1 HPAI viruses A/Vietnam/1203/2004 (NCBI taxonomy ID: 284218), and A/Vietnam/UT30850/2005 (NCBI taxonomy ID: 755296) were propagated on MDCK cells with serum free DMEM supplemented with 1% bovine serum albumin and 20 mM HEPES (influenza media). HPAI work was conducted in a BSL3+ facility in compliance with the UW Madison Office of Biological Safety.
2.3. Recombinant RCN viruses
The construction of recombinant RCN expressing the HA (RCN-HA) gene from H5N1 HPAI (A/Vietnam/1203/2004) has been described [11]. Similar methods were used for the construction of RCN viruses expressing the NA gene (RCN-NA), the NP gene (RCN-NP), and the E. coli LTB gene (RCN-LTB). The NA and NP genes are expressed in frame with the tissue plasminogen activator (tPA) secretory signal, and under the control of the poxvirus late p11 promoter and internal ribosome entry site (IRES) enhancer element. The LTB gene is expressed in frame with a tPA secretory signal coding region and under the control of a poxvirus synthetic early-late (SEL) promoter (Fig. 1).
Fig. 1:
Insertion of (A) P11-IRES-tPA HA, NA, or NP (B) SEL-IRES-tPA LTB expression cassettes into RCN TK gene by homologous recombination.
2.4. Vaccinations
Groups of A/J mice were given RCN constructs or inactivated influenza diluted in pH 7.4 Phosphate Buffered Saline (PBS) at the indicated dose by the ID, IM, or IN routes. For ID vaccinations, 25 μl was injected into the footpad. For IM vaccinations, 25 μl was injected into the caudal thigh muscle. IN vaccinations were performed by anesthetizing mice with isoflurane and dropwise pipetting 10 μl of RCN into each nostril. As indicated below, certain groups were boosted (same dose and route) at 4 weeks post primary immunization.
2.5. HPAI challenge
Mice were challenged with 1 × 104 TCID50 units of H5N1 in 20 μl PBS by intranasal instillation under isoflurane anesthesia. All animals were observed over 14 days after challenge to evaluate weight loss and mortality. At day 5 post-challenge, two mice from each group were euthanized and lung tissues were collected. Lung samples were homogenized in 0.5 mL PBS by repeated passage through a syringe. Viral lung titers were determined by infecting MDCK cells and observing cytopathic effects (CPE) 3 days after infection under a light microscope and calculating the TCID50 [13].
2.6. Western blot
The expression of recombinant RCN constructs was evaluated by Western blot assays. Vero cell monolayers were infected with RCN constructs at the indicated MOI. Cells and supernatants were harvested 48 h post infection. Samples were resolved by electrophoresis using a Mini-PROTEAN 3 system (BIO-RAD, CA). Gels were electroblotted onto nitrocellulose membranes in a GENIE® Electrophoretic Transfer apparatus (Idea Scientific, MN). Membranes were blocked in 5% (W/V) skim milk and probed with goat anti H5 HA sera (BEI Resources, VA), mouse anti-NP monoclonal (Chemicon MAB8257, MA), or rabbit anti N1 NA polyclonal (Abcam ab21304, MA). For detection, membranes were probed with AP conjugated animal specific anti IgG secondary antibody (Promega, WI) and developed in BCIP/NBT chromogenic substrate (Promega, WI).
2.7. Serology
Blood samples were collected from mice by saphenous vein bleed prior to boost or challenge and serum harvested for analysis. Equal volumes of serum aliquots from each mouse were pooled and treated with receptor destroying enzyme (RDE) (Denka Seiken, TKY) prior to analysis.
Neutralizing antibody (nAb) titers against A/Vietnam/1203/04 HPAI virus were measured by microneutralization as described previously [ref 1]. A modified assay to determine antibodies that block viral entry and not spread as previously described [14] was also conducted. All microneutralization experiments were conducted two times, and the average titer is reported.
Total IgG antibody titers against HPAI or LTB were measured by ELISA as described previously [ref 1]. Plates (Costar E.I.A) were coated by adding 4 ng per well of either HA from inactivated influenza or LTB in PBS. The ELISA endpoint values are the highest reciprocal dilution with an OD450 greater than the mean plus 3 standard deviations of similarly diluted negative control sera.
2.9. Statistical Analysis
Statistical analyses were performed using GraphPad software (La Jolla, CA). Differences in survival between treatment groups was assessed using one-tailed Fisher’s exact test [15].
3. Results
3.1. Construction and characterization of RCN recombinants.
The expression of HPAI antigens by recombinant RCN was tested in vitro by western blot analysis. All RCN-Flu viruses produced an antigen specific band that was not present in cells infected with RCN-TK- (Supplementary data 1). To ensure that mice vaccinated with RCN-LTB generated an immune response against LTB protein, antibodies were measured in serum samples taken from mice by ELISA (Supplementary data 2).
3.2. Mouse studies
3.2.1. Mouse study 1: RCN-HA ID vs. IM
The first study evaluated the ability of RCN-HA to protect mice from lethal HPAI challenge and also compared the IM and ID routes of administration. Groups of 6–8 week-old female A/J mice (n=8) were vaccinated with RCN-HA (107 PFU) by the IM and ID routes (Table 1). Formalin inactivated H5N1 A/Vietnam/1203/2004 (5 ug) and RCN-TK- (107 PFU) were used as positive and negative controls respectively. Select groups were boosted four weeks post prime (Table 1). At two weeks post boost, all mice were challenged with a lethal dose of A/Vietnam/1203/2004, and survival was recorded over a period of 14 days post challenge. All mice that received a prime and boost of RCN-HA survived (8/8 survival) compared with mice that received a prime and boost of RCN-TK- (0/8 survival). Interestingly, a single dose of RCN-HA was more protective when given by the ID route (8/8 survival), than when given by the IM route (4/8 survival). Surprisingly, several of the groups that were protected from challenge by RCN-HA did not develop detectable titers of nAbs) (Table 1). However, a prime and boost of RCN-HA by the ID route elicited nAbs (1:32, Table 1). Based on these results, all subsequent parenteral vaccinations with RCN-HA were given by the ID route.
Table 1:
Studies evaluating protection of mice by RCN constructs from lethal challenge with homologous influenza virus A/Vietnam/1204/04. In study 1, protection by RCN-HA given ID or IM was compared. In studies 2 and 3, RCN-HA was mixed with RCN-NA, RCN-NP, RCN-LTB, or RCN-TK- and given by different routes.
| Study | Vaccine/Route | Dose (PFU) | Boost | Survival | nABTiter a (entry/egress) |
|---|---|---|---|---|---|
| 1 | RCN-HA /ID | 107 | No | 8/8 | <16 |
| RCN-HA /ID | 107 | Yes | 8/8 | 32 | |
| RCN-HA /IM | 107 | No | 4/8 | <16 | |
| RCN-HA /IM | 107 | Yes | 8/8 | <16 | |
| Form Inc. Flu/IM | 5 μg | Yes | 8/8 | 512 | |
| RCN-TK /ID | 107 | Yes | 0/8 | <16 | |
|
| |||||
| 2 | RCN-HA /ID | 5×106 | No | 3/6 | N.D |
| RCN-HA /ID; RCN-LTB /ID | 5×106; 5×106 | No | 4/6 | ||
| RCN-HA /ID; RCN-NA /ID | 5×106; 5×106 | No | 0/6 | ||
| RCN-HA /ID; RCN-NP /ID | 5×106; 5×106 | No | 2/6 | ||
| RCN-HA /IN | 107 | No | 0/6 | ||
| RCN-HA /IN; RCN-LTB /IN | 107 | No | 0/6 | ||
| RCN-HA /IN; RCN-NP /IN | 107 | No | 0/6 | ||
| RCN-HA /IN; RCN-NA /IN | 107 | No | 2/6 | ||
| Form Inc. Flu/IM | 5 μg | No | 6/6 | ||
| RCN-LUC /ID | 107 | No | 0/6 | ||
|
| |||||
| 3 | RCN-HA /IN | 107 | No | 0/7 | N.D |
| RCN-TK /IN | 107 | No | 0/7 | ||
| RCN-NA /IN | 107 | No | 2/7 | ||
| RCN-HA /ID | 5×106 | No | 4/7 | ||
| RCN-HA /ID; RCN-HA /IN | 5×106; 107 | No | 5/7 | ||
| RCN-HA /ID; RCN-TK /IN | 5×106; 107 | No | 4/7 | ||
| RCN-HA /ID; RCN-NA /IN | 5×106; 107 | No | 7/7 | ||
Neutralizing antibodies detected in serum from mice pre-challenge. The titer is defined as the serum dilution resulting in complete neutralization of infection, as not detectable at the lowest dilution tested (<1:16), or not done (N.D).
3.2.2. Mouse study 2: Coadministration of additional RCN constructs with RCN-HA by the ID or IN routes
The second study tested the protection conferred by a lower dose (5×106 PFU) of RCN-HA administered by the ID and IN routes, alone or in combination with additional constructs (RCN-NA RCN-NP, or RCN-LTB). As a negative control, RCN expressing the firefly luciferase gene (RCN-LUC) was used in place of RCN-TK-. Groups of female A/J mice (n=6) were vaccinated (Table 1) and challenged 5 weeks later with a lethal dose of A/Vietnam/1203/2004 as above.
Low dose RCN-HA by the ID route resulted in decreased protection (3/6 survival) that was not improved when RCN-HA was mixed with RCN-LTB and given by the ID route (4/6 survival). Surprisingly, protection appeared to be reduced when RCN-HA was mixed with either RCN-NA (0/6 survival) or RCN-NP (2/6 survival) and given by the ID route, indicating that mixing vectors might reduce protection.
Finally, groups that received RCN-HA alone or mixed with RCN-NP or RCN-LTB by the IN route were provided no protection. However, mice given RCN-HA mixed with RCN-NA by the IN route were offered minor protection from challenge (2/6 survival).
3.2.3. Mouse Study 3: Coadministration of intradermal, low dose RCN-HA with intranasal RCN
In study 2, coadministering constructs by the same route at the same site appeared to decrease protection. Our third study tested if the partial protection from RCN-HA (5×106 PFU) given by the ID route is increased when RCN-HA or RCN-NA (107 PFU) is coadministered by a different route (IN). Groups A/J mice (n=7) were vaccinated (Table 1) and challenged 5 weeks later as in previous studies.
Mice vaccinated with a single, low, ID dose of RCN-HA were again partially protected from challenge (4/7 survival), and protection did not appear to change when coadministered an IN dose of RCN-TK- (4/7 survival) or RCN-HA (5/7 survival). Thus, RCN-HA is not protective by the IN route. Surprisingly, coadministration of RCN-HA by the ID route with RCN-NA by the IN route offered full protection against challenge. The only construct that provided some protection by the IN route was again RCN-NA (2/7 survival). This suggests that RCN-NA is a potential mucosal vaccine against HPAI.
3.2.4. Study 4: Coadministration of RCN constructs
To test the hypothesis of potential viral interference resulting in reduced protection, mice were intradermally coadministered RCN-HA either pre-mixed prior to vaccination or injected separately at a different site with RCN-LTB, RCN-NA, and RCN-NP. To determine the effect of empty vector on interference, two groups of mice were also coadministered RCN-HA with RCN-TK-. To confirm the advantages of intranasal administration of RCN-NA, a group of mice was coadministered RCN-HA by the ID route and RCN-NA by the IN route. Finally, this study determined if a prime and boost of RCN-NA by the IN route would increase the partial protection observed from a single IN dose of RCN-NA.
To test the hypothesis that RCN-Flu constructs can provide broad immunity, some groups of mice were challenged with Clade 2 HPAI (A/Vietnam/UT30850/2005). For this entire study, survival rates and weight loss were monitored over 14 days after challenge. Two mice from each group were euthanized on day 5 after challenge to determine viral titers in lung tissues.
3.2.4.1. Mixing antigen expressing RCN constructs reduces protection
Weight loss measurements and survival rates show that separate administration of RCN-flu constructs provided increased protection compared to mixing recombinant viruses prior to inoculation (Table 2, Fig. 2). Interestingly, the interference from mixing constructs appears to be due to expression of antigen specifically, because no interference was observed upon coadministering RCN-TK- (empty vector) with RCN-HA. Mixing RCN-HA with RCN-TK- resulted in increased protection (5/6 survival), and significantly decreased weight loss (Fig. 2) following challenge when compared to separate coadministration at two different inoculation sites (4/7 survival).
Table 2:
Protection of mice by coadministration of RCN constructs from lethal challenge with homologous influenza virus A/Vietnam/1204/04 (groups 1–13), or heterologous A/Vietnam/UT30850/2005 (groups 14–17).
| Group | Vaccine /Route | Treatment - prime (P) boost (P/B) | Survival | Lung titer a | ELISA titer b | nAB titer c (entry/egress) | nAB titer d(entry) |
|---|---|---|---|---|---|---|---|
| 1 | RCN-HA /ID; RCN-LTB /ID | Mixed (P) | 3/7 | 4.2 ± 0.4 | 6.7 | <16 | <16 |
| 2 | RCN-HA /ID; RCN-LTB /ID | Separate (P) | 7/7 | 0 | 7.7 | <16 | <16 |
| 3 | RCN-HA /ID; RCN-TK- /ID | Mixed (P) | 5/6 | 4.3 ± 0.0 | 6.7 | <16 | <16 |
| 4 | RCN-HA /ID; RCN-TK- /ID | Separate (P) | 4/7 | 4.3 ± 0.2 | 6.7 | <16 | <16 |
| 5 | RCN-HA /ID; RCN-NA /ID | Mixed (P) | 6/7 | 3.3 ± 0.2 | 6.7 | <16 | <16 |
| 6 | RCN-HA /ID; RCN-NA /ID | Separate (P) | 5/6 | 0 | 6.7 | <16 | <16 |
| 7 | RCN-HA /ID; RCN-NP /ID | Mixed (P) | 4/7 | 4.3 ± 0.0 | 6.7 | <16 | <16 |
| 8 | RCN-HA /ID; RCN-NP /ID | Separate (P) | 5/7 | 4.9 ± 0.1 | 7.7 | <16 | <16 |
| 9 | RCN-HA /ID | (P) | 5/7 | 5.7 ± 0.1 | 6.7 | <16 | <16 |
| 10 | RCN-NP /ID | (P) | 1/7 | 4.8 ± 0.5 | 4.7 | <16 | <16 |
| 11 | RCN-NA /IN | (P/B) | 7/7 | 0 | <3.7 | 128 | 16 |
| 12 | RCN-HA /ID; RCN-NA /IN | (P) | 7/7 | 3.7 ± 0.2 | 7.7 | <16 | <16 |
| 13 | RCN-TK- /ID | (P/B) | 0/3 | 4.4 ± 0.1 | <3.7 | <16 | <16 |
| 14 | RCN-HA /ID; RCN-NA /IN | (P/B) | 7/7 | 0 | 8.7 | 256 | 16 |
| 15 | RCN-HA /ID; RCN-LTB /ID; RCN-NP /ID; RCN-NA /IN |
Mixed (P/B) |
6/7 | 2.7 ± 0.2 | 7.7 | 24 | 16 |
| 16 | RCN-HA /ID; RCN-LTB /ID; RCN-NP /ID RCN-NA /IN |
Separate (P/B) |
6/6 | 0 | 8.7 | 48 | 16 |
| 17 | RCN-TK- /ID | (P/B) | 0/3 | 4.2 ± 0.4 | <3.7 | <16 | <16 |
Infectious A/Vietnam/1203/04 virus detected in the of lungs of mice sacrificed 5 days after challenge. Log10 values represent the number of 10 fold dilutions of sample where infectivity was detected and defined as TCID50 units/ml. The assay was repeated, and the values shown are the average of two means plus or minus the standard error of the both standard deviations.
Anti A/Vietnam/1204/04 IgG detected in serum from mice pre-challenge. The end point of 4 fold dilutions detected by ELISA is shown in Log4.
Neutralizing antibodies detected in serum from mice pre-challenge. The titer is defined as the serum dilution resulting in complete neutralization of infection, or as not detectable at the lowest dilution tested (<1:16).
Fig. 2:
Comparison of separate and mixed administration of RCN constructs on average weight loss following HPAI challenge. In study 4, groups of mice (Table 2, groups 1–8) were administered a prime only of RCN-HA with antigen expressing constructs (RCN-LTB, NA, or NP), or empty vector (RCN-TK-) mixed or separately. Body weights were assessed daily for a period of 14 days post-challenge with homologous clade 1 A/Vietnam/1204/04, and the average weight loss over this 14 day period is shown for each group. The significance of the difference in average weight loss between mixed and separate administration was compared using a one-tailed T-Test, and the p value is displayed.
As in the previous studies, vaccinating with RCN-HA alone by the ID route moderately protected mice from HPAI (5/7 survival) but did not prevent replication of virus in the lungs of mice when examined at day 5 post-challenge (Table 2). Similar results were obtained following separate vaccination by the ID route with RCN-HA and RCN-NP constructs (5/7 survival). While separate coadministration of RCN-HA with RCN-NA did not increase protection (5/6 survival), it prevented viral replication in the lungs. Separate coadministration of RCN-HA and RCN-LTB by the ID route fully protected mice (7/7 survival) and prevented viral replication in the lungs, suggesting that recombinant LTB can enhance protection provided by RCN-HA when both constructs are administered intradermally.
A prime and boost vaccination with RCN-NA by the IN route offered full protection in terms of survival and lung titers (Table 2). RCN-HA by the ID route coadministered with RCN-NA by the IN route fully protected mice (7/7 survival), although it did not prevent viral replication in the lungs (Table 2).
3.2.4.2. Separate administration of RCN vectors provides full protection against a cross-clade challenge
A prime and boost of RCN-HA (ID route) administered with RCN-NA (IN route) offered mice the best protection against clade 2 challenge in terms of survival (7/7) and weight loss (<10% peak) (Fig. 3), and prevented viral replication in the lungs (Table 2) by day 5 post-challenge. Equal levels of survival (6/6) were observed by addition of RCN-LTB and RCN-NP at different ID sites, and viral replication in the lungs was also prevented. Mice that received a mixture of RCN constructs (RCN-HA, RCN-LTB, and RCN-NP) by the ID route with RCN-NA by the IN route were not fully protected against challenge (6/7 survival), and lung samples from these mice contained replicating virus on day 5 (Table 2). The null hypothesis that mixing constructs results in equal survival was tested using Fisher’s exact test and rejected at the p =.057 level (Table 3).
Fig. 3:
Weight loss following cross clade HPAI challenge. In study 4, groups of mice (Table 2, groups 14-17) were administered a prime and boost of RCN constructs and challenged with heterologous clade 2 A/Vietnam/UT30850/2005. Body weight was assessed daily for a period of 14 days after post-challenge. Error bars were calculated as the standard deviation of the mean weight.
Table 3:
The total deaths and survivals were pooled for groups where RCN constructs expressing antigen were administered at the same, or at a different site (ID only), including the clade 2 challenge. The resulting Chi-Squared value of 3.56 is statistically significant at the P = 0.056 level.
| RCN Mixed | RCN Separate | |
|---|---|---|
| Alive | 3 (grp 1), 6 (grp 5), 4 (grp 7), 6 (grp 15) = 19 total alive | 7 (grp 2), 5 (grp 6), 5 (grp 8), 7 (grp 16) = 24 total alive |
| Dead | 4 (grp 1), 1 (grp 5), 3 (grp 7), 1 (grp 15) = 9 total dead | 0 (grp 2), 1 (grp 6), 2 (grp 8), 0 (grp 16) = 3 total dead |
3.2.4.3. RCN-NA and RCN-HA elicit neutralizing antibodies against influenza
A single dose of any RCN construct did not elicit production of nAbs (Table 1, 2) at the lowest dilution tested (<1:16). A prime and boost of RCN-NA by the IN route elicited high titers of nAbs (1:128). A prime and boost of RCN-HA by the ID route with RCN-NA by the IN route elicited the highest titers (1:256) of nAbs in this study; however, mice that additionally received RCN-LTB and RCN-NP at a separate ID sites had lower nAb titers (1:48). Mixing constructs (RCN-HA, RCN-LTB, RCN-NP) and administering by ID injection resulted in lower nAb titers (1:24).
A separate microneutralization assay was conducted as described in section 2.7 to test if the nAbs detected above readily block viral entry (Table 2). A prime and boost of RCN-HA by the ID route with RCN-NA by the IN route elicited nAbs that blocked viral entry (1:16), and this was not affected with additional constructs (RCN-LTB, RCN-NP). Surprisingly, a prime and boost of RCN-NA alone by the IN route was sufficient to elicit an equal titer of nAbs against viral entry.
3.3. Coadministration of RCN constructs in vitro
Mice administered a mixture of RCN constructs had lower antigen specific IgG titers and reduced protection. Therefore we tested in vitro the effects of RCN coinfection on antigen expression levels by western blot. Coinfection of RCN-HA with RCN-NA, RCN-NP or RCN-LUC resulted in reduced expression of antigen, while coinfection of RCN-HA with empty vector (RCN-TK-) did not significantly affect HA expression levels (Supplementary data 3).
4. Discussion
In previous studies we demonstrated that RCN can express the HA antigen of HPAI viruses and induce antibody responses in chickens [11]. We now show that RCN can also efficiently express other flu antigens (NA and NP) and a molecular adjuvant (LTB). Using the mouse model, we tested protection against HPAI conferred by administration of these constructs by several routes. Our results illustrate the importance of route of administration and antigen choice on influenza vaccine efficacy. While the RCN-HA construct was highly efficacious by the ID route, it did not induce protective immune responses when administered intranasally. Interestingly, RCN-NA was highly protective by the IN route. It is unclear what mechanisms could account for the different levels of protection seen upon using the same antigen administered by different routes. However, our studies show that a combination of the ID (RCN-HA) and IN (RCN-NA) routes can provide the best protection against HPAI. Furthermore, RCN based vaccines can confer cross clade protection. Finally, our studies also confirm previous observations regarding the potential deleterious effect of mixing several vaccine constructs prior to immunization [16].
Efficacious HPAI vaccines that provide broad protection when delivered by mucosal routes are highly desired [4]. Notably, we found that RCN-NA alone conferred protection by the IN route while similar protection was not provided by the RCN-HA construct. Whether a similar result will be obtained in other animals that are susceptible to HPAI remains to be tested. We and have previously demonstrated that RCN vectored antigens are immunogenic by mucosal routes in dogs, cats, and prairie dogs. Administration of RCN-plague vaccine provided protection against lethal plague challenge in prairie dogs [17]. In addition, we have recently found that recombinant RCN-based vaccines are highly safe in severe combined immunodeficient (SCID) mice. (Osorio, et al: manuscript in preparation).
The RCN-HA construct might elicit stronger cellular immune responses when administered intradermally versus intranasally. In a recent study, administration of RCN-LUC to mice by the ID route induced the highest levels of IFN-γ secreted by splenocytes, whereas IN administration resulted in poor IFN-γ responses (Osorio, et al: unpublished results). Thus, it is possible that mice given RCN-HA by the ID route had more HA-specific IFN-γ secreting T cells than mice given RCN-HA by the IN route. The importance of IFN-γ in protecting against influenza has been previously demonstrated [18]. Future studies will determine the nature and protection of the cellular immune response elicited by RCN-Flu constructs administered by different routes.
In this study, there was a correlation between nAbs levels and protection from challenge. Similar studies have shown that pox vectors strongly stimulate localized nAbs by mucosal routes. IN vaccination was superior over the ID route for a vaccinia virus expressing the respiratory syncytial virus (RSV) F glycoprotein, and elicited the highest levels of nAbs in bronchoalveolar lavage and nasal washes in mice [19]. In that study, IN vaccination prevented replication of RSV in the upper respiratory tract [19]. Newcastle Disease Virus based H5N1 vaccines expressing either HA, or NA were evaluated in monkeys, and animals developed high levels of neutralizing antibody titers from IN delivery of NA alone [14]. Studies have shown that natural influenza infection produces mucosal immunity due to secretory IgA, which is more cross-protective than serum IgG induced by parenterally injected vaccines in mice, ferrets and humans [20, 21]. Future studies should determine the effects of route of administration on Ig isotypes generated by RCN-Flu constructs in humoral (serum, BAL, saliva) samples.
This study demonstrated that while coadministration of viral vectors can significantly increase protection when constructs were given at different sites, protection was decreased when constructs were mixed. Viral interference was previously observed in mice vaccinated with mixtures of RCN constructs expressing plague antigens [16]. A possible mechanism for this reduced immunogenicity is that coinfection might reduce antigen expression as observed in our in vitro experiments (Supplementary data 3). Different expression cassettes (Fig. 1) are likely responsible for interference, because coinfection with RCN-TK- had no effect on HA expression (Supplementary data 3). Future studies will dissect the causes of reduced expression in vitro, and whether the same phenomenon occurs in vivo.
Our studies also demonstrate that RCN can express molecular adjuvants such as LTB, and an adjuvant effect was observed when this construct was co-administered with RCN-HPAI constructs at a different site. However, since abs were elicited against LTB (Supplementary data 1) studies should be conducted to determine if preexisting abs against LTB could affect subsequent vaccination. The potential of RCN to express other adjuvants such as cytokines will be explored in future studies, but caution will be used since some of these studies demonstrated increased virulence [22]. Finally, it is possible that RCN might have intrinsic adjuvant properties. Several studies have demonstrated an adjuvanting effect inherent to other poxviruses such as MVA [23] and canarypox virus [23].
Many vaccination strategies require delivery of multiple antigens from a certain pathogen (HIV, influenza, plague) to achieve sufficient levels of protection in terms of diversity, duration and efficacy. One of the strengths of pox-vectored vaccines is their ability to tolerate large foreign inserts, and this has encouraged development of influenza vaccine constructs encoding many different antigens and adjuvants [5].. Coadministration of a pox vector encoding several HA genes from different influenza strains with a pox vector encoding several NA genes by the ID and IN routes respectively could provide strong protection against multiple clades of HPAI.
Supplementary Material
Acknowledgements
We would like to thank Dr. Ronald Schultz and Dr. Marulasiddappa Suresh for critical review of this manuscript. Dr. Schultz-Cherry kindly provided samples of inactivated H5N1 influenza and RNA. We are also grateful to Joshua Teslaa for technical and veterinary assistance. These studies were partially funded by an IEDR grant from the UW-Wisconsin Alumni Research Foundation. This research was also funded by a Robert Draper Technology Innovation Fund (TIF) Grant from the University of Wisconsin-Madison and by the National Institute of Allergy and Infectious Diseases (NIAID) Public Health Service research grants (USA) and ERATO (Japan Science and Technology Agency).
References
- [1].Loeffelholz MJ. Avian influenza A H5N1 virus. Clin Lab Med;30(1):1–20. [DOI] [PubMed] [Google Scholar]
- [2].Meltzer MI, Cox NJ, Fukuda K. The economic impact of pandemic influenza in the United States: priorities for intervention. Emerg Infect Dis 1999. Sep-Oct;5(5):659–71. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [3].Guan Y, Smith GJD, Webby R, Webster RG. Molecular epidemiology of H5N1 avian influenza. Rev Sci Tech 2009;28(1):39–47. [DOI] [PubMed] [Google Scholar]
- [4].Kopecky-Bromberg SA, Palese P. Recombinant vectors as influenza vaccines. Curr Top Microbiol Immunol 2009;333:243–67. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [5].Poon LLM, Leung YHC, Nicholls JM, Perera P-Y, Lichy JH, Yamamoto M, et al. Vaccinia virus-based multivalent H5N1 avian influenza vaccines adjuvanted with IL-15 confer sterile cross-clade protection in mice. J Immunol 2009;182(5):3063–71. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [6].Tumpey TM, Alvarez R, Swayne DE, Suarez DL. Diagnostic approach for differentiating infected from vaccinated poultry on the basis of antibodies to NS1, the nonstructural protein of influenza A virus. J Clin Microbiol 2005;43(2):676–83. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [7].Rimmelzwaan GF, Sutter G. Candidate influenza vaccines based on recombinant modified vaccinia virus Ankara. Expert Rev Vaccines 2009. Apr;8(4):447–54. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [8].Veits J, Romer-Oberdorfer A, Helferich D, Durban M, Suezer Y, Sutter G, et al. Protective efficacy of several vaccines against highly pathogenic H5N1 avian influenza virus under experimental conditions. Vaccine 2008. Mar 20;26(13):1688–96. [DOI] [PubMed] [Google Scholar]
- [9].Gherardi MM, Esteban M. Recombinant poxviruses as mucosal vaccine vectors. J Gen Virol 2005. Nov;86(Pt 11):2925–36. [DOI] [PubMed] [Google Scholar]
- [10].Carroll MW, Moss B. Poxviruses as expression vectors. Curr Opin Biotechnol 1997. Oct;8(5):573–7. [DOI] [PubMed] [Google Scholar]
- [11].Hwa SH, Iams KP, Hall JS, Kingstad BA, Osorio JE. Characterization of recombinant raccoonpox vaccine vectors in chickens. Avian Dis 2010. Dec;54(4):1157–65. [DOI] [PubMed] [Google Scholar]
- [12].Moss B. Poxvirus vectors: cytoplasmic expression of transferred genes. Curr Opin Genet Dev 1993. Feb;3(1):86–90. [DOI] [PubMed] [Google Scholar]
- [13].Gao W, Soloff AC, Lu X, Montecalvo A, Nguyen DC, Matsuoka Y, et al. Protection of Mice and Poultry from Lethal H5N1 Avian Influenza Virus through Adenovirus-Based Immunization. J Virol 2006. Feb;80(4):1959–64. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [14].DiNapoli JM, Nayak B, Yang L, Finneyfrock BW, Cook A, Andersen H, et al. Newcastle disease virus-vectored vaccines expressing the hemagglutinin or neuraminidase protein of H5N1 highly pathogenic avian influenza virus protect against virus challenge in monkeys. J Virol 2010;84(3):1489–503. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [15].Rosner B. Fundamentals of Biostatistics. Belmnont, CA: Duxbury Press, 1995. [Google Scholar]
- [16].Rocke TE, Iams KP, Dawe S, Smith SR, Williamson JL, Heisey DM, et al. Further development of raccoon poxvirus-vectored vaccines against plague (Yersinia pestis). Vaccine 2009. Dec 11;28(2):338–44. [DOI] [PubMed] [Google Scholar]
- [17].Rocke TE, Pussini N, Smith SR, Williamson J, Powell B, Osorio JE. Consumption of baits containing raccoon pox-based plague vaccines protects black-tailed prairie dogs (Cynomys ludovicianus). Vector borne and zoonotic diseases (Larchmont, NY 2010 Jan-Feb;10(1):53–8. [DOI] [PubMed] [Google Scholar]
- [18].La Gruta NL, Kedzierska K, Stambas J, Doherty PC. A question of self-preservation: immunopathology in influenza virus infection. Immunol Cell Biol 2007;85(2):85–92. [DOI] [PubMed] [Google Scholar]
- [19].Matsuoka T, Okamoto Y, Matsuzaki Z, Endo S, Ito E, Tsutsumi H, et al. Characteristics of immunity induced by viral antigen or conferred by antibody via different administration routes. Clinical and experimental immunology 2002;130(3):386–92. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [20].Couch RB, Kasel JA. Immunity to influenza in man. Annual review of microbiology 1983;37:529–49. [DOI] [PubMed] [Google Scholar]
- [21].Liew FY, Russell SM, Appleyard G, Brand CM, Beale J. Cross-protection in mice infected with influenza A virus by the respiratory route is correlated with local IgA antibody rather than serum antibody or cytotoxic T cell reactivity. Eur J Immunol 1984;14(4):350–6. [DOI] [PubMed] [Google Scholar]
- [22].Perera LP, Goldman CK, Waldmann TA. Comparative assessment of virulence of recombinant vaccinia viruses expressing IL-2 and IL-15 in immunodeficient mice. Proc Natl Acad Sci U S A 2001;98(9):5146–51. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [23].Hutchings CL, Birkett AJ, Moore AC, Hill AVS. Combination of protein and viral vaccines induces potent cellular and humoral immune responses and enhanced protection from murine malaria challenge. Infect Immun 2007;75(12):5819–26. [DOI] [PMC free article] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.