Abstract
Eastern, Venezuelan and western equine encephalitis viruses (EEEV, VEEV, and WEEV) are mosquito-borne viruses that cause substantial disease in humans and other vertebrates. Vaccines are limited and current treatment options have not proven successful. In this report, we vaccinated outbred mice with lipid-antigen-nucleic acid-complexes (LANACs) containing VEEV E1 + WEEV E1 antigen and characterized protective efficacy against lethal EEEV, VEEV, and WEEV challenge. Vaccination resulted in complete protection against EEEV, VEEV, and WEEV in CD-1 mice. Measurements of bioluminescence and plaque reduction neutralization tests (PRNTs) indicate that LANAC VEEV E1 + WEEV E1 vaccination is sterilizing against VEEV and WEEV challenge; whereas immunity to EEEV is not sterilizing. Passive transfer of rabbit VEEV E1 + WEEV E1 immune serum to naive mice extended the mean time to death (MTD) of EEEV challenged mice and provided significant protection from lethal VEEV and WEEV challenge.
Keywords: Alphavirus, Bioluminescence, Eastern equine encephalitis virus, In vivo imaging, Subunit vaccine, Togaviridae, Venezuelan equine encephalitis virus, Western equine encephalitis virus
Introduction
Alphaviruses are mosquito-borne pathogens that cause morbidity and mortality in humans and other vertebrates. Geographically, alphaviruses are divided into New World alphaviruses (NWAs) and Old World alphaviruses (OWAs). NWAs cause an encephalitic disease and are important human health, biodefense, and biosafety threats. As viral agents of encephalitis, NWAs pose the most significant threat to human health among alphaviruses, with case fatality rates as high as 70% (Zacks and Paessler, 2010). Medically important NWAs include eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis virus (VEEV), and western equine encephalitis virus (WEEV). Sequelae persist in approximately 60% of alphavirus infection survivors (Steele and Twenhafel, 2010).
Prophylactics to prevent human alphavirus infection are currently unavailable for use in the United States. Investigational-new-drug (IND) status vaccines are available for some alphaviruses, but safety concerns and poor immunogenicity limit their usage (Barrett and Stanberry, 2008). Of the unlicensed vaccines currently used under IND status, none protect against more than one alphavirus species. Furthermore, antibody interference typically precludes vaccination against other alphavirus species (Barrett and Stanberry, 2008; Reisler et al., 2012). With the spread of alphaviruses to new areas and the co-circulation of alphaviruses, the need for a pan-alphavirus vaccine is critical.
Additionally, there are currently no licensed antivirals for the treatment of alphavirus infection, and treatment options are limited to supportive care. New antivirals for the treatment of alphavirus infection are required to oppose natural, biodefense, and biosafety related alphavirus infections.
Alphavirus genomes are approximately 11,700 nucleotides with 5’ methylated caps and 3’ polyadenylated termini. The positive sense alphavirus genome is translated to produce four non-structural proteins (nsP1-nsP4) that comprise the viral replication complex. A subgenomic RNA is also produced which encodes the five structural proteins (C, E3, E2, 6K, and E1) functioning in the assembly of progeny virions (Strauss and Strauss, 1994).
Alphaviruses have two envelope proteins, E2 and E1, which form trimeric, heterodimer spikes on the surface of the virion. Both E2 and E1 are responsible for virus entry and participate in viral binding and fusion, respectively. E2 binds to a cellular receptor resulting in virus internalization through receptor mediated endocytosis. Once internalized, E1 mediates a membrane fusion event under low pH conditions which enables the viral nucleocapsid to enter the host cell cytoplasm. Vaccines directed towards either of these alphavirus envelope glycoproteins are highly protective in laboratory models (Khan et al., 2012; Phillips et al., 2014; Roy et al., 2013; Swayze et al., 2011). Antibodies against E2 are predominantly neutralizing, whereas antibodies towards E1 generally do not interact with intact virions (Schmaljohn et al., 1982). Although E2 is the major neutralizing antigen, E1 is the better candidate for a pan-alphavirus vaccine or therapeutic because it is more highly conserved among alphaviruses than E2 (Table 1).
Table 1.
Percent homology in nucleotide and amino acid sequences between E1 and E2 of challenge viruses. A. WEEV McM. B. VEEV Trinidad donkey. Challenge virus strains are WEEV McM, EEEV FL-93, and VEEV 3908.
| A |
| egion | EEV | EEV | EEV |
| 2(aa) | 00 | 4.75 | 2.18 |
| 2(nt) | 00 | 9.21 | 8.01 |
| 1(aa) | 00 | 0.00 | 9.70 |
| 1(nt) | 00 | 6.32 | 5.23 |
| B |
| egion | EEV | EEV | EEV |
| 2(aa) | 1.69 | 8.63 | 7.28 |
| 2(nt) | 1.97 | 7.33 | 5.13 |
| 1(aa) | 9.70 | 0.32 | 9.40 |
| 1(nt) | 5.17 | 2.83 | 7.34 |
Cationic liposomes coupled to nucleic acids (CLNCs) are effective activators of innate immunity (Logue et al., 2010; Troyer et al., 2009). CLNCs combined with antigen (lipid-antigen-nucleic acid-complexes: LANACs) induce both adaptive and innate immunity. LANACs, when used as vaccines, have conferred protection against a variety of infectious agents (Firouzmand et al., 2013; Jones et al., 2010; Phillips et al., 2014).
In this study, the protective potential of LANACs containing recombinant VEEV E1 and WEEV E1 was examined. Vaccination studies, described herein, establish the protective efficacy of LANAC alphavirus E1 vaccination against the NWAs. This work demonstrates that E1 antibody is non-neutralizing and is sufficient for protection in vivo. Importantly, we show that the alphavirus E1 vaccine platform is capable of accommodating multiple alphavirus E1 antigens without compromising the immune response to either. Together these studies, support the use of E1 directed prophylactics and therapeutics to combat alphavirus infections.
Methods
Virus strains
Recombinant luciferase-expressing VEEV plasmid (VEEV-3908-FLuc) and recombinant wild-type VEEV plasmid (VEEV-3908-WT) were obtained from Dr. Scott Weaver (University of Texas Medical Branch), and viruses were generated as follows. MluI linearized plasmids were purified by QIAprep Spin Miniprep Kit (Qiagen) and genomic RNA was transcribed in vitro using a T7 RNA Polymerase MAXIscript kit (Life Technologies). Vero E6 cells (4 × 106 cells in 400 μL) (ATCC) were electroporated with genomic RNA using an ECM 630 electroporator (BTX Harvard Apparatus). Two pulses (450 V, 1200 Ω, and 150 μF) were administered. Forty-eight hours post electroporation virus was recovered from supernatant. EEEV-FL93-NLuc plasmid was obtained from Dr. William Klimstra (The University of Pittsburgh) and rescued as described (Sun et al., 2014). WEEV-McM-FLuc was constructed and generated as previously described (Phillips et al., 2016). All recombinant viruses were used without further passage after rescue. All viral stocks were titered by plaque assay on Vero cells as described previously (Mirchamsy and Rapp, 1968) and stored at −80° C.
In vitro virus growth studies
Viral growth curve experiments were performed by infecting confluent Vero E6 cell cultures in 25 cm2 flasks. Cells were counted, infected with VEEV-3908-FLuc or VEEV-3908-WT at a multiplicity of infection (MOI) of 0.1, and incubated at 37° C. Aliquots of supernatant were removed at 24 hour intervals, until the monolayers became detached, and stored at −80° C. Growth curves were produced using data from triplicate experiments with viral production measured by plaque assays performed as described (Mirchamsy and Rapp, 1968).
Mouse studies
Animal use in this study was approved by the Institutional Animal Care and Use Committee at Colorado State University (CSU). Mice were handled in compliance with the Public Health Service (PHS) Policy and Guide for the Care and Use of Laboratory Animals. All experiments were conducted in a biosafety level 3 laboratory approved as a Select Agent-facility by the Centers for Disease Control and Prevention (CDC). Outbred, 4- to 6-week old female CD-1 mice (Charles River Laboratories) were allowed to acclimate to the facility for three to five days. Intranasal (IN) inoculations were performed by instilling inocula into each nostril of lightly anesthetized animals. Mice were anesthetized by administration of isoflurane (Minrad Incorporated) through an XGI-8 anesthesia system (Caliper Life Sciences). Inocula were back-titered to confirm dosages. Animals were observed twice daily for signs of morbidity. Moribund animals were euthanized by CO2 inhalation, and the day of euthanasia was taken as the day of death to calculate mean times to death (MTD). Survivorship was followed for up to 20 days.
Mouse virulence studies
CD-1 mice were challenged by the IN route with VEEV-3908-FLuc or VEEV-3908-WT. Intranasal infections were performed with a dose of 104 PFU of virus in a volume of 20 μL.
Preparation of LANAC E1 vaccines
Vaccine was prepared fresh on the day of vaccination as described. Cationic liposomes (100 mM DOTIM lipid + cholesterol) in 10% sucrose were provided by Dr. Steven Dow (CSU). Cationic- lipid- nucleic acid- complexes (CLNCs) were prepared by diluting cationic liposomes 1:5 in sterile Tris-buffered 5% dextrose water and adding dsRNA and CpG DNA (InVivoGen), to a final concentration of 1.1 mg/mL each, causing spontaneous formation of CLNCs. Recombinant, HIS-tagged versions of the WEEV McM E1 (1.2 mg/mL) and/or VEEV Trinidad donkey E1 (3.3 mg/mL) ectodomains were expressed from the baculovirus immediate early 1 promoter, purified, and added to CLNCs at a final concentration of 50 μg/mL each (10 μg/200 μL dose). The production and purification of WEEV E1 has been previously described (Phillips et al., 2014; Toth et al., 2011). Venezuelan equine encephalitis virus E1 was produced similarly. Briefly, large preparations of secreted, HIS-tagged E1 antigens (aa 1-408) were produced in 10 L WAVE bioreactors of baculovirus-infected Sf9 cells by Expression Systems LLC (Davis, CA). Cell-free media were prepared and concentrated 10-fold with buffer exchange. E1 antigen preparations then were subjected to nickel affinity purification before removal of purification tags. Purified E1 antigens were then assayed for purity and concentration. Each 10 L production run yielded approximately 5 mg of E1 free of any contamination. E1 antigens were mixed with CLNCs for 15 minutes to form LANAC complexes prior to vaccination. Each dose of vaccine consisted of 200 μL of LANAC delivered via subcutaneous (SC) injection dorsal to the cervical spine. This priming dose was followed by an identical boost vaccination two weeks later. Control animals received no vaccination treatment.
Generation of alphavirus E1 serum
For generation of sera for passive transfer experiments, rabbits were immunized four times with VEEV E1 + WEEV E1 emulsified in TiterMax Gold (Sigma) following the manufacturer’s instructions. Briefly, VEEV E1 and WEEV E1 (50 μg/mL each) and dsRNA (0.1 mg/mL) were added to the aqueous medium before emulsification. Rabbits were terminally bled 12 weeks post primary vaccination. Serum was separated, heat inactivated at 56° C for 30 minutes, and stored at −80° C until use. Non-immune serum was collected from control animals. Neutralizing, survivor serum came from animals that naturally survived infection.
Enzyme-linked immunosorbent assays
Polyvinylchloride plates were coated overnight at 4°C with 100 μl of E1ecto WEEV or VEEV antigen diluted to 2 μg/ml in PBS (pH 7). Plates were washed twice with PBS containing 0.25% Tween 20 (PBS-TW) and twice more with PBS. Plates were blocked with 200 μl of SuperBlock T20 (Thermo Scientific) for 1 hour at room temperature and then washed as described above. Serum samples were diluted 1:100 in PBS, and log2 serial dilutions were added to the plates, followed by 1 hour of incubation at room temperature. Total IgG detection was performed by 1 hour of incubation with HRP conjugated antibody to total IgG. ABTS [2,2′-azino bis(3-ethylbenzthiazolinesulfonic acid)] substrate (KPL) was added and incubated for 15 minutes, and absorbance at 405 nm was recorded. Endpoint titers were calculated as the reciprocal of the greatest dilution that was 0.200 optical density (OD) unit greater than that for the negative control.
Passive transfer of immune serum
Due to the cost associated with passive transfer of mouse serum, naïve mice received 0.1 mL of pooled VEEV E1 + WEEV E1 immune or non-immune rabbit serum (intraperitoneally) four hours prior to IN challenge. In previous studies, WEEV E1 mouse serum protected naïve mice from lethal WEEV (Phillips et al., 2014).
Mouse infections
Nine weeks post booster vaccination or four hours post passive transfer, CD-1 mice were challenged by the IN route with EEEV-FL93-NLuc, VEEV-3908-FLuc, or WEEV-McM-FLuc. Intranasal infections were performed with a dose of 104 PFU of virus in a volume of 20 μL. IVIS images were acquired every 24 hours.
Plaque reduction neutralization titer
Vero E6 cells were seeded in 6-well plates. Plaque reduction neutralization titers (PRNTs) were measured by incubating virus with dilutions of immunized mice serum, inoculating samples onto seeded cells, and incubating the plates at 37° C for 1 hour. After aspirating the inocula, cells were washed, overlaid with Tragacanth gum overlay (Mirchamsy and Rapp, 1968), and incubated at 37° C for 2 days. Controls consisting of non-pretreated virus or virus treated with neutralizing serum were included with each assay. An 80% PRNT (PRNT80) was used as the neutralization endpoint and is expressed as the reciprocal of the lowest dilution of test serum able to neutralize 80% of input virus.
Bioluminescent imaging
Starting at 24 HPI, mice were imaged using an IVIS 200 system (Perkin Elmer). Mouse imaging was performed as described previously (Phillips et al., 2014; Phillips et al., 2013; Sun et al., 2014). Living Image 3.0 software (Caliper Life Science) was used to analyze and process images. Total light emission was determined using software provide grids and collecting light emission data.
Statistics
Kaplan-Meier (log rank test) analyses were applied to survival curves using Prism version 6.00 for Windows (GraphPad). Quantitative analyses of bioluminescence and virus titer were performed by Mann-Whitney test.
Results
VEEV-3908-FLuc and VEEV-3908-WT growth kinetics and virulence
Confluent Vero E6 monolayers were infected at an MOI of 0.1 PFU/cell with VEEV-3908-FLuc or VEEV-3908-WT. These infections produced maximum titers of 1.73× 109 PFU/mL and 2.66 × 109 PFU/mL for VEEV-3908-FLuc and VEEV-3908-WT, respectively, with no significant differences in the propagation of these two viruses (Figure 1a).
Figure 1. VEEV-3908-FLuc resembles VEEV-3908-WT.
A. VEEV-3908-FLuc and VEEV-3908-WT kinetics in Vero cells (0.1 MOI). B. Survival of mice (n=7/group) infected with VEEV-3908-FLuc or VEEV-3908-WT (104 PFU IN). No significant difference between VEEV-3908-FLuc and VEEV-3908-WT were observed.
Mortality and MTD were measured in CD-1 mice following IN inoculation with 104 VEEV-3908-FLuc or VEEV-3908-WT. Mice infected with either of these viruses had comparable rates of survival and MTD (Figure 1b).
Humoral immune response to LANAC E1 immunization
Sera from immunized mice and rabbits were analyzed by ELISA to determine the overall antibody response and then for neutralization antibody titer.
Mice were prime boost immunized with LANAC VEEV E1 + WEEV E1 and sera collected nine weeks post boost. Vaccinated mice developed a robust antibody response as measured by ELISA with E1 antibody titers of 1600 for VEEV E1 and 3200 for WEEV E1 (Table 2). E1 antibody from mouse sera was non-neutralizing (Table 2).
Table 2.
ELISA and PRNT titers for mice and rabbits immunized with VEEV E1 + WEEV E1. Mice were vaccinated with LANAC VEEV E1 + WEEV E1 twice as described herein. Rabbits were vaccinated four times using TiterMax Gold according to the manufacturer’s instructions.
| WEEV ELISA |
VEEV ELISA |
WEEV PRNT80 |
VEEV PRNT80 |
|
|---|---|---|---|---|
| VEEV E1 + WEEV E1 Immunized mouse |
320 0 |
16 00 |
<40 | <40 |
| Unimmunized control mouse |
0 | 0 | <40 | <40 |
| VEEV E1 + WEEV E1 Immunized rabbit |
>25 600 |
>2 5600 |
<40 | <40 |
| Unimmunized control Rabbit |
0 | 0 | <40 | <40 |
Rabbits were immunized four times with VEEV E1 + WEEV E1 emulsified in TiterMax Gold. Sera was collected 12 weeks post primary vaccination. Vaccinated rabbits developed a robust antibody response with VEEV E1 and WEEV E1 ELISA titers of >25600 (Table 2). Similar to vaccinated mice, rabbits vaccinated with VEEV E1 + WEEV E1 did not develop a neutralizing E1 antibody response (Table 2).
These results demonstrate that CD-1 mice and rabbits immunized with VEEV E1 + WEEV E1 produce E1 specific IgG towards both VEEV E1 and WEEV E1, whereas unimmunized animals did not. Importantly, although mice and rabbits developed a strong antibody response to E1the antibody response did not produce neutralizing E1 antibodies.
Protective efficacy of LANACs containing VEEV E1 + WEEV E1 in mice
We assessed the ability of alphavirus E1 LANACs to protect mice against the following viruses: EEEV-FL93-NLuc, VEEV-3908-FLuc, and WEEV-McM-FLuc. Nine weeks post booster vaccination, protective efficacy was assessed by challenging vaccinated mice via the IN route.
LANAC WEEV E1 immunization failed to protect against IN VEEV-3908-FLuc (Supplemental Figure 1).
LANAC VEEV E1 immunization failed to protect mice against IN EEEV FL93-939 challenge (p=0.15) (Supplemental Figure 2a), but showed 85.7% protection against IN VEEV-3908-FLuc infection (p<0.0001) (Supplemental Figure 2b) and 14.3% protection from lethal IN WEEV-5’McM-FLuc challenge (p=0.0004) (Supplemental Figure 2c). Mean time to death was extended from four to five days following IN EEEV-FL930NLuc challenge and from four to six days following IN WEEV-5’McM-FLuc challenge in immunized mice.
LANAC VEEV E1 + WEEV E1 immunization fully protected mice from lethal IN EEEV-FL93-NLuc (p=0.0004) (Figure 2), VEEV-3908-FLuc (p=0.0004) (Figure 3), and WEEV-McM-FLuc challenge (p=0.0003) (Figure 4). Lethality was uniformly observed in all unvaccinated mice (Figure 3-5).
Figure 2. LANAC VEEV E1 + WEEV E1 protects against IN challenge with EEEV.
Mice (n=7/group) were prime-boost immunized with LANAC VEEV E1 + WEEV E1 and challenged with 104 PFU EEEV-FL93-NLuc. A. Bioluminescence imaging at 2, 3, 4, and 14 DPI. Images are normalized by day. B. Survival of mice infected with EEEV-FL93-NLuc. The differences in survival among mice immunized with LANAC VEEV+ WEEV E1 and control mice were statistically significant (p=0.0004).
Figure 3. LANAC VEEV E1 + WEEV E1 protects against IN challenge with VEEV.
Mice (n=7/group) were prime-boost immunized with LANAC VEEV E1 + WEEV E1 and challenged with 104 PFU VEEV-3908-FLuc. A. Bioluminescence imaging at 1, 2, 3, and 14 DPI. Images are normalized by day. B. Survival of mice infected with VEEV-3908-FLuc. The differences in survival among mice immunized with LANAC VEEV+ WEEV E1 and control mice were statistically significant (p=0.0004).
Figure 4. LANAC VEEV E1 + WEEV E1 protects against IN challenge with WEEV.
Mice (n=7/group) were prime-boost immunized with LANAC VEEV E1 + WEEV E1 and challenged with 104 PFU WEEV-McM-FLuc. A. Bioluminescence imaging at 1, 2, 3, and 14 DPI. Images are normalized by day. B. Survival of mice infected with WEEV-McM-FLuc. The differences in survival among mice immunized with LANAC VEEV+ WEEV E1 and control mice were statistically significant (p=0.0003).
Figure 5. Passive transfer of VEEV E1 + WEEV E1 serum to EEEV-FL93-NLuc IN infected mice.
Mice (n=5/group) were treated with sera (VEEV E1 + WEEV E1 or non-immune) 4 hours before infections. Treated mice were then challenged by IN inoculation with 104 PFU of EEEV-FL93-NLuc virus then imaged at 2, 3, and 4 DPI. A. Bioluminescence. Images are normalized by day. B. Survival of infected mice. No significant differences where seen in survival between mice receiving VEEV E1 + WEEV E1 serum (immune serum) or non-immune serum however, mice receiving VEEV E1 + WEEV E1 serum (immune serum) survived 2 DPI longer than mice receiving non-immune serum.
Bioluminescence imaging to gauge vaccine efficacy in mice
To assess the ability of LANAC VEEV E1 + WEEV E1 immunization to inhibit EEEV-FL93-NLuc, VEEV-3908-FLuc, and WEEV-McM-FLuc viral replication, in vivo imaging was used to examine immunized and unimmunized mice for luciferase expression upon IN infection. Imaging revealed a reduction in bioluminescence, indicative of decreased viral replication, in immunized as compared to unimmunized mice at all time points following EEEV, VEEV, and WEEV challenge (Figures 2-4).
Importantly, LANAC VEEV E1 + WEEV E1 vaccinated mice challenged with EEEV exhibited detectable virus replication and developed EEEV neutralizing antibody post-infection; whereas LANAC VEEV E1 + WEEV E1 vaccinated mice challenged with VEEV and WEEV did not develop neutralizing antibody post-infection (Table 3). LANAC alphavirus E1 vaccines provide sterilizing immunity to homologous challenge; whereas non-sterilizing but protective immunity is provided for heterologous challenge.
Table 3.
Sterilizing immunity towards EEEV, VEEV, and WEEV in mice vaccinated with VEEV E1 + WEEV E1 LANACs. PRNTs of sera from LANAC VEEV E1 + WEEV E1 vaccinated mice which survived EEEV, VEEV or WEEV challenge. Sera was tested against virus homologous to challenge virus.
| Challenge Virus |
PRNT80 Post- Vaccination Pre- Challenge |
PRNT80 Post- Vaccination Post- Challenge |
Development of Neutralizing Antibodies |
Sterilizing Immunity following Vaccination |
|---|---|---|---|---|
| WEEV | <40 | <40 | No | Yes |
| VEEV | <40 | <40 | No | Yes |
| EEEV | <40 | >200 | Yes (post challenge) |
No |
Passive transfer of E1 antibodies confers protection
VEEV E1 + WEEV E1 or non-immune rabbit sera were passively transferred to mice four hours prior to IN infection with EEEV-FL93-NLuc, VEEV-3908-FLuc, or WEEV-McM-FLuc to determine if antibody was sufficient for protection.
Mice receiving non-immune serum exhibited progressive intensification of bioluminescence and neurological deficits until euthanasia regardless of virus species (Figure 5-7). Mice receiving VEEV E1 + WEEV E1 serum and challenged with EEEV-FL93-NLuc exhibited progressive intensification of bioluminescence signal and neurological deficits until euthanasia (Figure 5a). Mice receiving VEEV E1 + WEEV E1 serum and challenged with VEEV-3908-FLuc (Figure 6a) or WEEV-McM-FLuc (Figure 7a) showed limited bioluminescence compared to mice receiving non-immune serum.
Figure 7. Passive transfer of VEEV E1 + WEEV E1 serum to WEEV-McM-FLuc IN infected mice.
Mice (n=5/group) were treated with sera (VEEV E1 + WEEV E1 or non-immune) 4 hours before infection. Treated mice were then challenged by IN inoculation with 104 PFU of WEEV-McM-FLuc virus then imaged at 2, 3, 4, and 7 DPI. A. Bioluminescence. Images are normalized by day. B. Survival of infected mice. The differences in survival between mice receiving VEEV E1 + WEEV E1 serum (immune serum) and non-immune serum were significant (p=0.0027).
Figure 6. Passive transfer of VEEV E1 + WEEV E1 serum to VEEV-3908-FLuc IN infected mice.
Mice (n=5/group) were treated with sera (VEEV E1 + WEEV E1 or non-immune) 4 hours before infection. Treated mice were then challenged by IN inoculation with 104 PFU of VEEV-3908-FLuc virus then imaged at 2, 3, 4, and 7 DPI. A. Bioluminescence. Images are normalized by day. B. Survival of infected mice. The differences in survival between mice receiving VEEV E1 + WEEV E1 serum (immune serum) and non-immune serum were significant (p=0.0027).
In the group that received VEEV E1 + WEEV E1 serum, none of the EEEV infected mice (Figure 5b), 100% of VEEV infected mice (p=0.0027) (Figure 6b), and 80% of WEEV infected mice were alive at 14 DPI (p=0.0027) (Figure 7b). EEEV-FL93-NLuc infected mice treated with VEEV E1 + WEEV E1 serum survived two days longer than mice receiving non-immune serum (p=0.0047). All mice receiving non-immune serum were euthanized prior to 7 DPI due to neurological deficits.
Discussion
This work describes a new LANAC alphavirus E1 formulation for the prevention of EEEV, VEEV, and WEEV infections. LANACs containing recombinant alphavirus E1 antigen induce a strong humoral immune response. Antibodies are non-neutralizing yet capable of inhibiting virus replication in both therapeutic and prophylactic contexts resulting in protective efficacy. These findings are not unexpected, as similar neutralizing and protection properties have been previously reported for alphavirus E1 vaccines and antibodies (Hodgson et al., 1999; Hunt and Roehrig, 1985; Phillips et al., 2014; Wust et al., 1989).
Of particular significance, is the ability of the LANAC alphavirus E1 vaccination platform to accommodate E1 antigen from more than one alphavirus species, and initiate an immune response towards each. Alphavirus E1 baculovirus-expressed protein antigens were chosen for these LANAC formulations for several reasons. First, E1 antigens were produced in the baculovirus expression vector system because this system enables authentic post-translational protein modification of E1. Post-translational modification of E1 appears to be important for eliciting an immune response, as alphavirus E1 produced in E. coli was unable to protect against WEEV challenge (Das et al., 2004); whereas, E1 produced using baculovirus expression systems, which carry out more complex post-translation modifications, have demonstrated a strong protective immune response (Hodgson et al., 1999; Phillips et al., 2014; Phillips et al., 2013). Regarding the use of E1, studies using inactivated IND status EEEV, VEEV, and WEEV vaccines have shown that prior alphavirus immunization can interfere with subsequent alphavirus immunization (Reisler et al., 2012). In these cases, the immune response to E2 may be immune-dominant and overwhelm the immune response driving the response to neutralization of specific alphavirus species rather than eliciting cross protection. E1 rather than E2 antigen was chosen because it rarely elicits neutralizing antibody responses and may drive the immune response towards cross protection rather than neutralization of specific alphavirus species and original antigenic sin. Additionally, E1 is more conserved among alphavirus species than E2 and antibodies towards E1 can be alphavirus group reactive (Hunt and Roehrig, 1985; Schmaljohn et al., 1982; Strauss and Strauss, 1994) (Table 1).
LANAC alphavirus E1 platforms, described herein and in Phillips et al. (2014) have the potential to prevent infection with the three major NWAs: EEEV, VEEV, and WEEV. LANAC WEEV E1 protects against EEEV and WEEV (Phillips et al., 2014), but not VEEV (Supplemental Figure 1); whereas LANAC VEEV E1 protects against EEEV and VEEV, but not WEEV (Supplemental Figure 2). When combined, LANAC VEEV E1 + WEEV E1 protects against EEEV, VEEV, and WEEV (Figures 2-4). Importantly, this LANAC platform is capable of accommodating multiple E1 antigens and eliciting cross-protection out to 11 weeks post vaccination. Here, we have extended our previous studies by adding multiple E1 antigens to our LANAC E1 formulation. We show that a protective immune response towards each antigen is developed without compromising the immunity achieved by single immunization. Future studies could include adding EEEV E1 antigen derived from other alphavirus species to the LANAC VEEV E1 + WEEV E1 formulation to maximize the NWA cross-protection and determine the number of E1 antigens the LANAC platform can accommodate and remain protective and efficacious.
E1 polyclonal serum is protective by passive transfer in vivo. Significantly, E1 polyclonal serum protects against VEEV and WEEV and cross-protects (extension of MTD) against EEEV. These results extend E1 cross-reactivity data (Hunt and Roehrig, 1985; Wust et al., 1989) to the context of in vivo cross-protection. Importantly, differences were observed in in vivo protection resulting from passive transfer of mouse versus rabbit antibody with mouse serum providing better protection to mice (data not shown). These differences in survival likely result from the inability of the Fc fragment of rabbit antibody to interact efficiently with mouse Fc receptors. Further studies will be required to identify the role of Fc in in vivo alphavirus protection. Previous studies have demonstrated that cytotoxicity is an unlikely mechanism for clearance of infection from neurons (Levine et al., 1991); however, Fc interactions could be utilized in transfer of antibody across the blood brain barrier (Hirsch et al., 1979) or to stimulate effector cells in a non-cytotoxic manner (DiLillo and Ravetch, 2015). Fc mediated transit of antibody across the blood brain barrier is a probable cause for the differences observed between antibody efficacy in vivo and in vitro. In vivo passively transferred antibody has to be transited to the brain via Fc interacting effector cells before it is able to control CNS infection. When Fc interactions are less efficient, rabbit Fc interacting with mouse effector cells, less antibody is transited to the brain and as a result protection is less complete particularly for heterologous viruses.
The utility of LANAC alphavirus E1 vaccination against EEEV, VEEV, and WEEV has been clearly demonstrated by showing the protective efficacy of alphavirus E1 antibodies in vivo, and progress has been made towards the characterization of their mode of action. Studies to expand and optimize LANAC alphavirus E1 vaccination to achieve the most effective pan-alphavirus vaccine are required. To meet the FDA’s animal efficacy rule, LANAC alphavirus E1 vaccination needs to be assessed in other animal models. Lastly, the humanization of the E1 antibodies described here and elsewhere (Hunt and Roehrig, 1985; Wust et al., 1989) could greatly expand current alphavirus therapeutic options. The LANAC alphavirus E1 vaccine and polyclonal E1 serum described here together constitute a promising tool for the prevention and treatment of alphavirus disease.
Supplementary Material
HIGHLIGHTS.
We developed a LANAC-based vaccine containing multiple alphavirus E1 antigens
The LANAC-based vaccine did not diminish antibody responses to each E1 antigen.
LANAC VEEV E1 + WEEV E1 vaccination protected mice against NWAs.
Passive transfer of VEEV E1 + WEEV E1 immune sera conferred protection to NWAs.
Acknowledgements
We thank Dr. Ann Toth, Melissa Stuart, and Theodore R. John at University of Wyoming, Laramie for producing the baculovirus vectors, fine-tuning the purification method, and characterizing the recombinant antigens used in the study; Dr. Steve Dow (Colorado State University) for providing liposomes; Lab Animal Resources (Colorado State University) for outstanding care of the animals used in these studies; Dr. Amanda McGuire for help with antibody ELISA titrations, and Tach Costello and Susan Rogers (Colorado State University) for administrative support.
Funding information
Our research has been funded by the National Institutes of Health (NIH) (RO1 AI46435 and NIH-RCE (A1065357)) and Colorado State University (DMIP Bridge Grant Fund). The funding sources had no role in study design; data collection and analysis; decision to publish; or preparation of the manuscript.
Footnotes
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