Research ArticleEMERGING INFECTIONS

A virus-like particle vaccine prevents equine encephalitis virus infection in nonhuman primates

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Science Translational Medicine  15 May 2019:
Vol. 11, Issue 492, eaav3113
DOI: 10.1126/scitranslmed.aav3113

A triple threat to equine encephalitis viruses

There are no approved treatments or vaccines for certain alphaviruses that can cause fatal encephalitis. Ko et al. designed a trivalent vaccine to simultaneously fend off Eastern, Western, and Venezuelan equine encephalitis viruses. The vaccine is composed of noninfectious virus-like particles and completely protected mice and nonhuman primates from all three viruses. There was no evidence of central nervous system infection or pathology in trivalent virus-like particle–immunized nonhuman primates. Passive transfer experiments revealed that humoral immunity delivered protection from viral challenge. Phase 1 studies are currently being started to test this trivalent vaccine in people.

Abstract

Western, Eastern, and Venezuelan equine encephalitis viruses (WEEV, EEEV, and VEEV, respectively) are important mosquito-borne agents that pose public health and bioterrorism threats. Despite considerable advances in understanding alphavirus replication, there are currently no available effective vaccines or antiviral treatments against these highly lethal pathogens. To develop a potential countermeasure for viral encephalitis, we generated a trivalent, or three-component, EEV vaccine composed of virus-like particles (VLPs). Monovalent VLPs elicited neutralizing antibody responses and protected mice and nonhuman primates (NHPs) against homologous challenges, but they were not cross-protective. In contrast, NHPs immunized with trivalent VLPs were completely protected against aerosol challenge by each of these three EEVs. Passive transfer of IgG from immunized NHPs protected mice against aerosolized EEV challenge, demonstrating that the mechanism of protection was humoral. Because they are replication incompetent, these trivalent VLPs represent a potentially safe and effective vaccine that can protect against diverse encephalitis viruses.

INTRODUCTION

Alphaviruses are composed of two different subclasses, Old World and New World viruses. Old World species include arthritogenic viruses such as chikungunya (CHIKV) and Ross River virus that cause inflammatory joint disease in humans. In contrast, Western, Eastern, and Venezuelan equine encephalitis viruses (WEEV, EEEV, and VEEV, respectively) are among the New World viruses that cause fatal encephalitis (1). These enveloped, positive-sense, single-stranded RNA viruses are members of the family Togaviridae and encode four nonstructural and five structural proteins (2, 3). EEEV inflicts the highest mortality rate in humans, ranging from 30 to 50%, whereas VEEV and WEEV infections cause death in 1 to 15% of cases (4, 5). These viruses are relatively stable and highly infectious by aerosol delivery, presenting a plausible bioterrorism concern (6). The need for a protective vaccine or antiviral therapy against these pathogens therefore remains substantial. Development of a multivalent vaccine offers the potential to control the spread of alphavirus encephalitic disease in animals and humans. Several vaccine platforms have been studied for previous vaccines (714); however, they either have not demonstrated complete protection in nonhuman primate (NHP) aerosol challenge models or alternatively have elicited adverse events when administered to humans (15), complicating further development.

We previously developed a virus-like particle (VLP)–based vaccine against CHIKV, another reemerging pathogenic alphavirus (16). This vaccine elicited highly protective immunity against CHIKV challenge in NHPs through its ability to elicit potent neutralizing antibodies (NAbs) (17, 18). To develop effective VLPs from this alphavirus, we expressed a single plasmid encoding the structural genes but not the regulatory nonstructural genes from this virus. We applied a similar approach by expressing equine encephalitis virus (EEV) structural genes and optimizing expression through the introduction of mutations to improve assembly and release of the VLPs.

RESULTS

Generation of WEEV, EEEV, and VEEV VLPs

To determine whether VLPs could be generated from the encephalitic alphaviruses, we prepared eukaryotic expression vectors encoding the C-E3-E2-6K-E1 genes from wild-type WEEV CBA87, EEEV PE-6, and VEEV TC-83 strains. Amino acid identity of E1 and E2 glycoproteins among the three EEVs ranges from 51 to 60% and 42 to 49%, respectively (fig. S1). The plasmids were transfected into human embryonic kidney–derived suspension cells (293F), and expression in the supernatant was measured by Western blot (Fig. 1A). Resulting expression of WEEV, EEEV, and VEEV VLPs was either low or undetectable. Specifically, expression of capsid was not detectable, suggesting that the secretion of capsid proteins differed for these viruses compared to CHIKV. Analysis of WEEV, EEEV, VEEV, and CHIKV sequences revealed a nuclear localization signal (NLS) sequence (19, 20) not present in CHIKV (Fig. 1A). To determine whether the NLS sequence affected expression, we prepared expression vectors encoding the structural protein including NLS knockout mutations (K64N for WEEV, and K67N for EEEV and VEEV). These mutations increased the VLP yield by more than 100-fold. Analysis of VLPs after buoyant density gradient sedimentation revealed maximal E1 and E2 expression at an expected density of 1.13 to 1.16 g/ml (Fig. 1B). Negative-stain transmission electron microscopy, cryogenic electron microscopy, and single particle analysis confirmed the icosahedral symmetry of the viral spikes, with an external diameter of 65 nm and a core diameter of 40 nm (Fig. 1C and fig. S3), similar to the previously described WEEV, EEEV, VEEV, or CHIKV VLPs (17, 2123).

Fig. 1 Characterization of WEEV, EEEV, and VEEV VLP vaccines.

(A) Schematic representation of the alphavirus genome and WEEV, EEEV, and VEEV VLP expression vectors. The alphavirus genome consists of the following genes: nonstructural proteins nsP1, nsP2, nsP3, and nsP4 and structural proteins capsid (C); envelope glycoproteins E3, E2, and E1; and the 6K protein. The NLS sequences in WEEV, EEEV, VEEV, and CHIKV are shown under the schematic representation. Expression of wild-type (WT) and NLS mutant VLPs in normal (−), basic pH buffers (+) (bottom). The expression was analyzed by Western blot using mouse sera against WEEV, EEEV, or VEEV. (B) Buoyant density gradient analysis of NLS mutant VLPs using OptiPrep. Each vector was transfected into 293F cells, and supernatants were collected after 4 days. VLPs were purified by density gradient centrifugation. (C) Surface rendering of three-dimensional (3D) cryogenic electron microscopy maps. The triangle in the central panel corresponds to the icosahedral symmetry unit, and the numbers mark the positions of the two-, three-, and fivefold symmetry axes. Scale bar, 10 nm.

Immunogenicity and protection of VLP vaccines in mice

To evaluate the immune response to single and trivalent VLPs, BALB/c mice were injected intramuscularly twice with a single-component (monovalent) or a three-component (trivalent) VLPs at a 3-week interval (Fig. 2A). Analysis of antibody (Ab) responses 4 weeks later using an Env-pseudotyped lentiviral reporter(s) neutralization showed that monovalent VLPs elicited high titer NAb responses against the homologous but not heterologous virus, whereas the trivalent VLPs elicited similarly high titer NAb to all viruses (50% inhibitory dilution, 1:1062, 1:5240, and 1:18,997 titers to WEEV, EEEV, and VEEV, respectively; Fig. 2B). Similar neutralization was observed using the plaque reduction neutralization test (PRNT), with VEEV and WEEV VLPs inducing the highest and lowest Ab titers, respectively, and trivalent VLPs generated NAbs against all three viruses (Fig. 2C). These data suggested that no immune competition was evident compared to single-matched VLPs.

Fig. 2 Monovalent and trivalent VLPs protected against matched WEEV, VEEV, or VEEV challenges in mice.

BALB/c mice (n = 5 per group) were immunized intramuscularly twice with 5 μg of monovalent WEEV, EEEV, or VEEV VLPs or with 15 μg of trivalent VLPs (5 μg each of WEEV, EEEV, and VEEV VLPs) at a 3-week interval. (A) Vaccination, neutralization assay, and challenge time course. (B and C) Neutralization titers of sera from the immunized mice were evaluated by WEEV, EEEV, and VEEV Env-pseudotyped lentiviral reporter neutralization assays (B) and PRNT (C). Data are representative of at least two independent experiments. Each data point represents the means ± SEM of the values from five animals per group. Overall significance of multigroups was evaluated by Kruskal-Wallis analysis of variance (ANOVA). LOD, limit of detection. (D) Mice immunized with the indicated monovalent WEEV, EEEV, or VEEV VLPs or trivalent VLPs were challenged at week 8 with aerosolized WEEV CBA87, and Kaplan-Meier survival curves are shown over time after the challenge. (E) Mice immunized with trivalent VLPs (n = 5 per group) were challenged with aerosolized virus. Kaplan-Meier survival curves are shown after lethal WEEV CBA87, EEEV FL93-939, or VEEV Trinidad challenge.

To determine whether immunization with the VLPs protected against infection, immune mice were challenged 5 weeks after the boost with a lethal dose of aerosolized WEEV CBA87 strain. Animals immunized with VEEV or EEEV VLPs rapidly became moribund and were euthanized, but those immunized with WEEV or trivalent VLPs did not show clinical signs of illness and had no detectable viremia at the end of the study (Fig. 2D and table S1), demonstrating that the WEEV VLPs, alone or in combination with other VLPs, conferred complete protection against WEEV infection.

To evaluate the breadth of protection of the trivalent VLP vaccine, immunized animals were challenged with lethal doses of aerosolized WEEV CBA87, EEEV FL93-939, or VEEV Trinidad donkey strains. Mice immunized with trivalent VLPs were completely protected against a lethal aerosol challenge by each of the three EEVs, in contrast to negative control phosphate-buffered saline (PBS)–injected animals (Fig. 2E). In summary, the trivalent VLPs elicited NAb responses to each EEV and conferred protection against lethal EEV challenges without immune competition in mice.

Immunogenicity and protection by monovalent VLPs in NHPs

To characterize immune responses in an animal model more relevant to human disease, cynomolgus macaques were immunized intramuscularly twice with WEEV, EEEV, or trivalent VLPs (Fig. 3A) at a 4-week interval. The VEEV VLP–only immunization was not performed because VEEV challenge in NHPs was not available when this study was performed. WEEV or EEEV VLPs elicited strong NAb responses against homologous strains, respectively, but did not elicit notable heterologous cross-reactivity (Fig. 3, B and C), similar to the response in mice. The boost immunization increased the NAb responses more than 20-fold compared to the prime (P < 0.05, Mann-Whitney test; Fig. 3C).

Fig. 3 Monovalent VLP vaccine induced robust NAb responses and conferred complete protection against homologous virus challenge in NHPs.

Cynomolgus macaques (WEEV VLPs, n = 3; EEEV VLPs, n = 4) were immunized intramuscularly twice with 20 μg of monovalent WEEV or EEEV VLPs at a 4-week interval. (A) Vaccination, neutralization assay, and challenge time course. (B and C) Neutralization titers of sera from the immunized macaques were evaluated by WEEV and EEEV Env-pseudotyped lentiviral reporter neutralization assay (B) and PRNT (C). Data are representative of at least two independent experiments. Each data point represents the means ± SEM of the values from three to four animals per group. Overall significance at multiple time points was evaluated by Friedman ANOVA. *P < 0.05, Mann-Whitney test. (D) The immunized macaques were aerosol challenged with homologous WEEV Fleming (left) or EEEV FL93-939 (right) with which they were immunized, and Kaplan-Meier survival curves are shown over time after the challenges.

To determine whether WEEV and EEEV VLP immunization could protect against infection, immune macaques were challenged by aerosol exposure with homologous virus, WEEV Fleming or EEEV FL93-939 strains, respectively, 4 weeks after final immunization. All macaques in the vaccinated groups were fully protected against the homologous EEV aerosol challenge (WEEV, n = 3; EEEV, n = 4; Fig. 3D).

Immunogenicity and protection by trivalent VLPs in NHPs

We next assessed the efficacy of the trivalent VLP vaccine in protecting against aerosol challenge by WEEV, EEEV, or VEEV. Macaques were immunized intramuscularly twice at a 4-week interval, and serum NAb responses were measured (Fig. 4A). NAb responses were detected (Fig. 4B) and were similar to the monovalent VLP immunization. A second immunization boosted neutralization titers by more than 20-fold (Fig. 4C). Macaques immunized with trivalent VLPs were challenged with aerosolized WEEV Fleming, EEEV FL93-939, or VEEV Trinidad donkey 4 weeks after the boost. WEEV and EEEV are highly lethal for macaques; however, because VEEV is nonlethal in macaques, hallmarks of infection including viremia, early lymphopenia, and fever were assessed after aerosolized VEEV challenge to determine protective efficacy. All negative control VEEV-exposed macaques displayed viremia, elevated fever, and early lymphopenia, whereas macaques immunized with the trivalent VLP vaccine showed no detectable viremia or fever and exhibited only mild lymphopenia (Figs. 4D and 5A). Five of nine control animals and eight of eight animals vaccinated with trivalent VLPs survived aerosolized WEEV challenge, which was significantly different based on time-to-event analysis (P = 0.0373, log-rank test; days to death of control group, 9.45 ± 1.79 days; Fig. 4E, left). Although fever responses were not distinguishing, the differences in lymphopenia were significant (P = 0.0104, two-tailed Student’s t test; Fig. 5B). Eight of eight macaques immunized with trivalent VLPs, in contrast to one of nine controls, survived against aerosolized EEEV challenge (P = 0.0003, log-rank test; Fig. 4E, right). No viremia was detected in vaccinated animals, and fever persisted for about 60 hours in controls compared to no febrile response in vaccinated recipients (P < 0.0001, two-tailed Student’s t test; Fig. 5C). These results demonstrated that the trivalent VLPs induced robust NAb responses that fully protected against aerosolized challenge with WEEV, EEEV, and VEEV in NHPs.

Fig. 4 The trivalent VLP vaccine protected against virus challenge in NHPs and IgG from immunized NHPs protected against EEV challenge in mice.

Cynomolgus macaques (n = 8 per challenge and n = 24 in total) were immunized intramuscularly twice with 60 μg of trivalent VLPs (20 μg each of WEEV, EEEV, and VEEV VLPs) vaccine at a 4-week interval. (A) Vaccination, neutralization data, and challenge time course. (B and C) NAb responses were evaluated against WEEV, EEEV, and VEEV Env-pseudotyped lentiviral reporter viruses (B) and PRNT (C). Data are representative of at least two independent experiments. Each data point represents the means ± SEM of the values from eight animals per group. Overall significance at multiple time points was evaluated by Friedman ANOVA. *P < 0.05, Mann-Whitney test. (D and E) Immunized monkeys were challenged separately with aerosolized WEEV, EEEV, or VEEV (n = 8 per challenge virus group) at 4 weeks after boost. Blood viremia was evaluated after VEEV Trinidad donkey challenge. P values were calculated by Fisher’s exact test (D). Kaplan-Mayer survival curves are shown over time after WEEV Fleming (left) and EEEV FL93-939 (right) challenges. P values were calculated by log-rank test (E).

Fig. 5 The trivalent VLP vaccine protected against clinical sequalae of viral challenge in NHPs.

Cynomolgus macaques (n = 24) were immunized intramuscularly twice with 60 μg of trivalent VLPs (20 μg each of WEEV, EEEV, and VEEV VLPs) at 4-week intervals. Immunized monkeys were challenged separately with aerosolized WEEV, EEEV, or VEEV at 4 weeks after boost (n = 8 or 9 per each challenge in test or control group, respectively). PBS was administered to control monkeys. (A) Hallmarks of human disease including number of days of lymphopenia (left) and fever duration (right) were evaluated after VEEV Trinidad donkey challenge. (B) The days of lymphopenia (left) and fever duration (right) are shown after WEEV Fleming challenge. (C) The blood viremia (left) and fever duration (right) analyses are shown after EEEV FL93-939 challenge. P values were calculated by unpaired two-tailed Student’s t test. For lymphopenia and fever analyses, survivors in the control group are designated as circles, whereas nonsurvivors are designated as squares.

Pathology

To investigate the pathology in the central nervous system (CNS) in monovalent VLP–immunized macaques, we carried out detailed histopathologic and immunohistochemical analysis of brain tissues from WEEV- and EEEV-challenged NHPs (Fig. 3A). Monovalent WEEV VLP–vaccinated animals showed no remarkable pathological lesions after WEEV exposure. The monovalent WEEV VLP vaccine protected NHPs from viral infection–related pathology. The pathologic findings in the sham-vaccinated WEEV-exposed animals were typical of WEEV-induced encephalitis with predominant pathological changes in the brain and CNS. Encephalitis was prominent throughout brain lobes, which included occipital meningitis and focal hemorrhage in the frontal lobe and within the brain stem (fig. S4, top). Similarly, the monovalent EEEV VLP–vaccinated group was protected from infectious challenge and showed no remarkable pathological lesions after exposure. The monovalent EEEV VLP vaccine protected NHPs from viral infection–related pathology. The pathologic findings in the sham-vaccinated EEEV-exposed animals were also typical of EEEV-induced infection and predominated in the brain/CNS with prominent changes including multilobe gliosis with perivascular cellularity, hemorrhage, and meningitis, consistent with viral-induced encephalitis. Changes in the cervical and thoracic spinal cord included myelitis and perineuritis (fig. S4, bottom).

Virus-induced pathology in the CNS was also studied in trivalent VLP–immunized macaques. We performed detailed histopathologic and immunohistochemical analysis of brain tissues from EEEV-, WEEV-, and VEEV-challenged NHPs (Fig. 4A). No clinically significant histopathologic lesions were observed, and no viral antigen was detected in the brain tissue sections from trivalent VLP–immunized macaques after the challenge with each EEV, whereas neuropathology (encephalitis) and viral antigen were observed in sham-vaccinated control groups when animals met euthanasia criteria. Macaques in the sham-vaccinated control group aerosol exposed to EEEV exhibited histologic lesions typical of EEEV infection, including varying degrees of neutrophilic and lymphocytic meningoencephalitis in all regions of the brain. Immunohistochemically, EEEV antigen was present in these lesions, primarily within neurons. Two of the trivalent VLP–immunized macaques exhibited mild, focal perivascular inflammation or gliosis with no detectable viral antigen. Macaques in the sham-vaccinated control group aerosol exposed to WEEV that were euthanized because of clinical signs had mild to moderate multifocal lymphocytic and histiocytic meningoencephalitis in more than one region of the brain, and viral antigen was detected immunohistochemically in neurons in and around the CNS lesions. Animals that survived until the end of study, whether sham-vaccinated controls or trivalent VLP–immunized macaques, had no detectable viral antigen and either no histologic lesions in the brain or clinically insignificant focal perivascular inflammation or gliosis. All macaques in the sham-vaccinated control group exposed to VEEV by aerosol exhibited minimal to moderate lymphoplasmacytic meningoencephalitis in one or more regions of the brain, with some animals also having gliosis. Although no antigen was detected by immunohistochemistry, the nature and distribution of the inflammation suggest that this lesion was most likely due to a resolving viral encephalitis. None of the trivalent VLP–vaccinated macaques had evidence of resolving meningoencephalitis or detectable viral antigen, although two exhibited minimal focal lymphocytic perivascular inflammation (fig. S5). Overall, trivalent VLP–vaccinated macaques survived aerosol challenge with no substantial viral-induced pathology or persistent viral infection; however, it is not possible to differentiate mild viral-induced lesions from background lesions unrelated to viral challenge. Together, these results suggest that the trivalent VLP vaccine was neuroprotective against each EEV challenge in macaques.

Mechanism of immune protection

To define the mechanism of protection by vaccination in NHPs, passive transfer studies were performed in the murine infectious challenge model. BALB/c mice were injected intraperitoneally with purified total immunoglobulin G (IgG) from trivalent VLP vaccine–immunized or control macaques. Each mouse was injected 1 day before viral challenge and again 3 days later. Separate groups received aerosolized WEEV Fleming, EEEV FL93-939, or VEEV Trinidad donkey strains. Animals inoculated with purified IgG from monkeys immunized with the trivalent VLP vaccine were completely protected against WEEV Fleming, EEEV FL93-939, or VEEV Trinidad donkey challenges compared to mice that received control IgG from naïve monkeys (P < 0.0001, WEEV; P = 0.0256, EEEV; P = 0.0004, log-rank test; Fig. 6). These results demonstrated that the Ab response induced by trivalent VLPs was sufficient to protect against EEV infections, suggesting an Ab-dependent mechanism of immune protection.

Fig. 6 IgG from immunized NHPs is sufficient to protect against EEV challenge in mice.

IgG was purified from the pooled serum of trivalent VLP–immunized or naïve cynomolgus macaques using Protein A. BALB/c mice (n = 10 mice per challenge and n = 30 in total) were administered 2 mg of IgG by intraperitoneal injection twice on days −1 and +3. The mice were exposed separately by aerosol to WEEV Fleming, EEEV FL93-939, or VEEV Trinidad donkey on day 0. Kaplan-Meier survival curves are shown. P values were calculated by log-rank test.

DISCUSSION

Global climate change, international trade, and frequent travel have expanded the distribution of mosquitoes, common viral vectors, to new geographic areas, posing the threat of outbreaks transmitted by mosquito-borne viruses including flaviviruses such as Zika and West Nile viruses and alphaviruses such as CHIKV. Several vaccine modalities for WEEV, EEEV, and VEEV have been tested, mostly in rodents, including DNA vaccines (24, 25), recombinant Ad5-based vaccines (26, 27), inactivated whole virus vaccines (9, 28), virus-like replicon particle vaccines (13, 29), chimeric Sindbis–based vaccines (30, 31), and live attenuated vaccines (32). Furthermore, no licensed vaccine or antiviral therapies are available to prevent or treat human infection. In this study, we developed a trivalent VLP vaccine that includes three viruses of public health concern—WEEV, EEEV, and VEEV. Monovalent and trivalent VLP vaccines generated potent NAb responses that completely protect against EEV aerosol challenge in mice and NHPs. These vaccines represent a marked improvement compared to previous EEV vaccines with respect to immunogenicity, adverse effects, and lack of immune interference.

Robust immune responses in in vivo models are critical for successful translation to protective immunity into humans (18, 33). Formalin-inactivated WEEV, EEEV, and VEEV vaccines are weakly immunogenic, possibly because of cross-linking of proteins and masking of important immune epitopes required for protection. These are being used for at-risk laboratory workers under Investigational New Drug status (7, 28, 3437). Another modality, a trivalent EEV replicon vaccine, failed to seroconvert all animals and protect completely against each aerosol EEV challenge in NHPs (13). Several live attenuated virus-based vaccines have been studied, but not developed further, in part, because of the potential for reversion to the wild-type virulent phenotype (38, 39). In phase 1 safety studies, a live attenuated VEEV vaccine, TC-83, caused fever, headache, and malaise in 25% of subjects and minimal immune response in 20% of subjects vaccinated (32). The mutations required to attenuate the TC-83 vaccine may result in alteration of immune epitopes (40), and the virus became highly sensitive to the inhibitory effects of interferon during passage of TC-83 (41). It is possible that a combination of antigenic site changes and increased interferon sensitivity resulted in a poorly immunogenic vaccine compared with the VLP vaccine that preserves native virus structures. Subsequently, a new live attenuated virus vaccine was developed to reduce the virulence reversion, which has shown promising immunogenicity results, but safety remained a concern in phase 1 clinical trials (42). More effective prevention modalities are therefore needed (7).

Monovalent VLP vaccine generated high titer NAb responses and afforded complete protection against the homologous virus but not heterologous virus. These results are consistent with a previously published report demonstrating that inactivated trivalent EEV prime immunization followed by inactivated monovalent EEV boost immunization failed to induce cross-reactive Abs (43). The trivalent VLP vaccine stimulated NAb responses against each EEV after prime immunization that were significantly increased after a boost immunization with the same trivalent VLP vaccine in both mouse and NHP models and were fully protective against all three aerosol EEV challenges. This suggests that the trivalent VLP vaccine has advantages over other EEV vaccine modalities discussed above. Analysis of the mechanism of protection demonstrated that the Ab response was sufficient for vaccine immunity. Protection against aerosol challenge in NHPs correlated with the induction of a NAb response and passive transfer of immune IgG into mice similarly conferred protective immunity. This finding is consistent with our previous study of a VLP vaccine against an arthritogenic alphavirus, CHIKV, that showed a similar mode of action (17).

We have demonstrated that the CHIKV VLP vaccine was well tolerated and induced robust NAb responses in humans (18). With the efficacy demonstrated in animals here, and because minimal adverse events were observed with a similar CHIKV VLP vaccine in clinical trials, a phase 1 clinical trial with the trivalent EEV VLPs was recently approved for further safety and immunogenicity studies in humans.

One limitation of this study was the use of laboratory isolates of viral strains tested in the challenge model. Although these viruses are highly conserved, it is not certain that their infectivity will be comparable to viruses transmitted in vivo in nature. Similarly, should different variants arise, the efficacy of the vaccine against divergent strains also remains unknown. Another limitation relates to the aerosol challenge model. Although it represents a challenging mode of transmission, it does not fully mimic the mode of spread through natural infection, which could involve cell-to-cell and/or mucosal modes of transmission. Last, the duration of protective immunity in humans and frequency of boosting related to this vaccine are unknown. Further characterization of human immunity and field efficacy will require clinical trials that are essential for future development and deployment of this vaccine.

In summary, we developed an effective multivalent alphavirus VLP vaccine that protects against three major alphaviruses with the potential for expanded coverage as additional alphavirus VLPs are generated. The trivalent VLP vaccine protected similarly to monovalent VLPs against matched alphaviruses, and no immune interference was detected with the combined immunogen. The inherent safety of alphavirus VLPs, together with their efficacy in a rigorous NHP aerosol model, suggests that they represent promising vaccine candidates against these zoonotic and mosquito-borne encephalitis viruses. Given the potential for natural or deliberate spread of mosquito-borne equine encephalitis pathogens and limited therapies for the long-term sequelae of encephalitic alphaviruses, a broadly neutralizing trivalent EEV vaccine would address an important public health and biosecurity need.

MATERIALS AND METHODS

Study design

The overall objective of this study was to develop VLP vaccines against WEEV, EEEV, and VEEV. Cynomolgus macaques were randomized according to gender and weight. The serum NAb responses were evaluated using an Env-pseudotyped lentiviral reporter neutralization and PRNT, and the protection efficacy was tested by aerosol challenge of the three EEVs in animal models. Three to four macaques per group were enrolled in the monovalent VLP study with a power of >99% to detect 98% survival difference between vaccine group and untreated controls at an α of 0.05. Eight macaques per vaccinated group and nine macaques per control group were allocated for the trivalent VLP study with a power of >80% to detect at least a 65% survival difference between the vaccine group and untreated controls at an α of 0.05. The challenged macaques were clinically assessed on a daily basis, and euthanasia was performed on any macaque that was determined to have clinical signs severe enough to meet the predetermined criteria for euthanasia. Animals that lived until up to 28 days after viral challenge were considered survivors and were subsequently euthanized and submitted for post-mortem examination, including histopathology. The statistical comparisons between control and test groups for survival were determined by log-rank test (GraphPad Prism 7.0). For mouse studies, the animal number of each independent experiments is indicated in each of the figure legends. Mouse passive immunization studies were conducted as described in Results. Primary data are reported in data file S1.

Vector construction

The construction of vectors that express each VLP (C-E3-E2-6K-E1) with capsid (C) and envelope glycoproteins of WEEV, EEEV, and VEEV (CBA87, PE-6, and TC-83 strain, respectively) were made as described previously (17). GenBank accession numbers of the three EEVs are ABD98014.1, AAU95735.1, and AAB02517.1 for WEEV, EEEV, and VEEV, respectively. The position of the targeted NLS in the capsids differed among the three EEVs (WEEV, 67KKKS; EEEV, 67KRKKP; and VEEV, 64KKPKK). The polymerase chain reaction (PCR) products were cloned into a mammalian expression vector after confirming the sequence. Mutants were made using the PCR-based QuikChange method (Stratagene) according to the manufacturer’s instructions. Each mutant was confirmed by sequencing.

Confirmation of VLP expression

To confirm VLP expression, the vector encoding capsid and envelope genes of WEEV, EEEV, or VEEV was transfected to the human embryonic kidney cell line 293–derived suspension cell line 293F (Thermo Fisher Scientific) by 293fectin transfection reagent (Thermo Fisher Scientific) according to the manufacturer’s instruction. Expression of VLPs was examined in normal (pH 7.0) and basic pH (pH 7.9) (44). At 24 hours after transfection, tris-HCl buffer (pH 8.8) was added to the culture medium (final concentration, 40 mM) to change the pH to 7.9. Then, the cells were incubated further for 24 hours, and the supernatant was collected. Supernatant was separated on a 4 to 15% SDS–polyacrylamide gel electrophoresis and transferred into an Immobilon-P membrane. Western blotting was performed with the sera from mice injected with a WEEV, EEEV, VEEV or purified EEV VLPs and horseradish peroxidase–conjugated goat anti-mouse IgG (Santa Cruz Biotechnology). E1 and E2 glycoproteins of WEEV and VEEV VLPs did not separate, probably because of similar molecular weights of the two glycoproteins (difference by 2 to 3 kDa) and heterogeneous posttranslational modifications, such as glycosylation, consistent with the previously published reports (45, 46).

Buoyant density gradient sedimentation analysis and purification of VLPs

Supernatants were harvested 96 hours after transfection of WEEV, EEEV, or WEEV plasmid into 293F cells, filtered through a 0.45-μm membrane filter, layered onto a 60% OptiPrep (Iodixanol) medium (Sigma-Aldrich), and then centrifuged at 50,000g for 1.5 hours with a SureSpin 630 rotor (Thermo Fisher Scientific). Supernatants were removed to leave 4 ml above the virus band and mixed to a 20% final concentration of OptiPrep. Next, they were centrifuged at 360,000g for 3.5 hours with an NVT100 rotor (Beckman Coulter) to form a density gradient. After centrifugation, 500 μl of each fraction was collected, weighed, and plotted against the densities. Total VLP concentration was measured with the bicinchoninic acid assay method (Thermo Fisher Scientific) according to the manufacturer’s instructions. Each fraction was analyzed by Western blotting as described above. For immunization in mice and NHPs, the VLPs were purified further by size exclusion chromatography using a Sephacryl S-500 HR column (GE Healthcare) in which the running buffer is PBS (pH 7.4).

Virus preparation

For the monovalent VLP vaccine study in NHPs, WEEV (strain Fleming) and EEEV (strain FL93-939) were prepared by transfecting viral RNA transcribed from plasmid into baby hamster kidney (BHK) cells, BHK-21 cells, by electroporation. Supernatants were aliquoted from the transfected cells and titrated to determine median tissue culture infectious dose (TCID50) endpoint titers using Vero cells. To produce virus for vertebrate challenge, we infected C6/36 (Aedes albopictus) cells grown to confluence in T-150 flasks with stock virus at a multiplicity of infection of 0.03. Supernatants were collected 48 hours after infection, aliquoted, and titrated to determine TCID50 endpoint titers on Vero cells.

For mouse and NHP trivalent studies, virus was prepared by sucrose purification on 20 to 60% continuous sucrose gradients from supernatants of infected BHK cells. Gradient sections containing virus were pooled and filtered through a 0.2-μm filter. Virus was aliquoted, frozen, and subsequently titrated by plaque assay. Sucrose-purified VEEV Trinidad donkey was prepared from working virus stock (L019-06-005, GP-?, CE-14, SM-1, and V-1) and had a final passage history of GP-?, CE-14, SM-1, V-1, and BHK-1. EEEV FL93-939 (previously obtained from S. Weaver, University of Texas Medical Branch) was prepared from master stock (C636-1, SMB-1, and Vero-1). The passage history for the sucrose-purified material was C636-1, SMB-1, Vero-1, and BHK-1.

Production of pseudotyped lentiviral reporter(s)

Lentiviral vectors expressing the structural proteins from WEEV, EEEV, and VEEV (CBA87, PE-6, and TC-83 strain, respectively) were created [EEV Env-pseudotyped lentiviral reporter(s)]. The method for producing recombinant lentiviral vectors expressing a luciferase reporter gene has been previously described (17). Briefly, 293 T cells were transfected with 500 ng of WEEV, EEEV, or VEEV envelope plasmid, 7 μg of a transducing vector encoding a luciferase reporter gene under the control of a cytomegalovirus (CMV) promoter (pHR′ CMV-luciferase plasmid), and 7 μg of a packaging plasmid that expresses all HIV-1 structural proteins except envelope (pCMV ΔR8.2). Two micrograms of vesicular stomatitis virus glycoprotein, 2 μg of pNGVL-4070A amphotropic murine leukemia virus gp70 expression vector, or 500 ng of empty vector served as controls for these pseudotyped lentiviral reporters. After calcium phosphate transfection (Thermo Fisher Scientific) overnight, the culture medium was replenished. Forty-eight hours later, supernatants were harvested, filtered through a 0.45-μm syringe filter, stored in aliquots, and frozen at −80°C. The viruses were standardized by the amount of HIV-1 Gag p24 (47). Three EEV Env-pseudotyped lentiviral reporters were prepared for the neutralization assay, and they showed similar luciferase activity in infected 293A cells (fig. S2).

Neutralization of WEEV, EEEV, and VEEV Env-pseudotyped lentiviral vectors/reporters by mouse and monkey antisera

Neutralization assays were performed as described previously (17). One day before infection, a total of 104 293A cells were plated into each well of a 96-well plate. Pseudotyped lentiviral vectors encoding luciferase were titrated by fourfold serial dilution starting at 1:50 serum dilution. Then, similar amounts of pseudotyped lentiviral vectors (with p24 levels of about 50 ng/ml) were incubated with the indicated dilutions of mouse antisera for 60 min at room temperature before adding the virus:sera solution to 293A cells (104 cells per well in a 96-well dish, 50 μl per well, in triplicate). Sera from nonimmune mice or monkeys served as a negative control. After 24-hour incubation, cells were lysed using cell lysis buffer (Cell Signaling Technology), and luciferase activity was measured using the MicroBeta JET (PerkinElmer) after incubation with a “Luciferase assay reagent” (Promega), according to the manufacturer’s protocol. All the experiments were performed in triplicate. We independently repeated the assays for each figure at least three times. Inhibition values were calculated as follows: inhibition (%) = {1 – [luciferase activity (cps) in pseudotyped lentiviral vector–infected cells incubated with the indicated dilutions of mouse antisera]/[luciferase activity (cps) in pseudotyped lentiviral vector–infected cells incubated with the same dilutions of nonimmune mouse serum)} × 100. We calculated the 50% inhibitory dilution with Prism 5 software.

Electron microscopy

The Electron Microscopy Laboratory at the Frederick National Laboratory examined the morphology of the VLPs.

Negative-stain transmission electron microcopy. VLPs were purified by OptiPrep density centrifugation, and negative-stain transmission electron microscopy was performed after fixing VLPs in 4% formaldehyde in PBS. Samples were adsorbed to freshly glow-discharged carbon film grids, rinsed twice with buffer, and stained with freshly made 0.75% uranyl formate. Images were recorded on a Hitachi H7650 microscope at 80 kV with a 2k × 2k Advanced Microscopy Techniques’ charge-coupled device (CCD) camera.

Cryogenic electron microscopy. Holey carbon electron microscopy grids (Quantifoil R1.2/1.3) were subjected to glow discharging, and a 2.7-μl drop of WEEV, EEEV, or VEEV VLPs at a concentration of about 2 mg/ml was applied for 15 s and removed by blotting with filter paper. Another drop was applied, and the sample was vitrified in liquid ethane using the Vitrobot Mark IV (FEI). Micrographs were recorded at a nominal magnification of 50,000× and a pixel size of 0.44 nm using an FEI T20 electron microscope with a LaB6 filament equipped with an FEI Eagle 2k × 2k CCD camera. Particles were selected manually and automatically using EMAN2 (48) and subjected to reference-free 2D classification in Relion 2.0 (49). Initial models were generated in EMAN2 using selected 2D classes and with icosahedral symmetry imposed. 3D classification and refinement were then performed in Relion 2.1. A total of 1898, 1643, and 1189 particles contributed to the final 3D reconstructions of WEEV, EEEV, and VEEV VLPs, respectively. The resolutions determined according to the gold standard approach using a Fourier shell correlation threshold of 0.143 were 24, 30, and 22 Å for WEEV, EEEV, and VEEV VLPs, respectively. For comparison, the maps were filtered to 30 Å using a low-pass Gaussian filter. The 3D maps were sharpened by applying a negative B factor (−10,000 Å2 for WEEV, −15,000 Å2 for EEEV, and −7500 Å2 for VEEV). UCSF (University of California at San Francisco) Chimera was used for visualization (50). 3D cryogenic electron microscopy maps and the central cross sections of the maps of each VLP were compared with those of wild-type or chimeric EEVs in the Electron Microscope Data Bank (EMDB) (entry numbers: WEEV, EMD-5210; EEEV, EMD-7004; and VEEV, EMD-8071).

Plaque reduction neutralization test

Serum samples were analyzed for EEV NAb using a standard PRNT (17). Briefly, mice or monkey sera was heat-inactivated at 56°C for 30 min and serially diluted in virus diluent [PBS/5% bovine serum albumin or Hank’s balanced salt solution (HBSS) complete: 2% heat-inactivated fetal bovine serum (HI-FBS), 2% penicillin-streptomycin (Pen/Strep), and 1% Hepes]. Virus stocks were diluted to a concentration of 2.0 × 103 plaque-forming units (PFU)/ml (2× virus stock) and added 1:1 to the serially diluted samples. The 2× virus stock was diluted 1:1 in PBS or HBSS complete and used as the virus-only control. All samples were incubated for 1 hour at 37°C or overnight at 4°C. Vero 76 cells seeded on six-well plates were grown to about 90 to 100% confluence. Cells were infected with 0.1 ml of each serial dilution per well in duplicate. Plates were incubated at 37°C with 5% CO2 for 1 hour, with gentle rocking about every 15 min. After 1 hour, cells were overlaid with 0.6% agarose in basal medium eagle (BME) complete (10% HI-FBS and 2% Pen/Strep) and incubated for 24 hours at 37°C with 5% CO2. A second overlay containing 0.6% agarose in BME complete and 5% of total volume neutral red vital stain was added to wells and further incubated 18 to 24 hours for visualization of plaques. Plaques were counted after incubation with stain overlay overnight. The virus-only control was counted, and the value equivalent to neutralization of 80% of the virus was determined (PRNT80). This value was used to determine the PRNT80 titer for the study samples.

Animals

Female BALB/c mice (Mus musculus), 6 to 8 weeks old, were obtained from the Jackson Laboratory. For some studies, mice were housed and bred in the animal facility of the Vaccine Research Center (VRC), National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, MD. For other studies, mice were housed at the United States Army Medical Research Institute of Infectious Diseases (USAMRIID). Cynomolgus macaques (Macaca fascicularis), animals of Chinese origin, were used in the NHP studies and housed in either Tulane National Primate Research Center (TNPRC)/Tulane or USAMRIID. All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committees (IACUCs) of the VRC/NIAID/NIH, TNPRC/Tulane (for experiments performed in TNPRC), and the USAMRIID (for experiments in USAMRIID). All animals were housed and cared for in accordance with local, state, federal, and institutional policies in facilities accredited by the American Association for Accreditation of Laboratory Animal Care International and adhering to principles stated in the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011). At USAMRIID, research was conducted under an IACUC-approved protocol in compliance with the Animal Welfare Act, U.S. Public Health Service Policy, and other federal statutes and regulations relating to animals and experiments involving animals. Animals were chosen and randomized on the basis of age and weight. Hematology parameters were evaluated from whole blood using a hematology analyzer.

Vaccine studies in mice

Mice were intramuscularly administered monovalent (5 μg) or trivalent (5 μg each of WEEV, EEEV, and VEEV VLPs; 15 μg total) VLPs without adjuvant on days 0 and 21. Control mice were administered PBS. Blood was collected from all mice on days 0, 21, and 49 for assessment of the humoral response. Mice were aerosol exposed to WEEV CBA87, EEEV FL93-939, and VEEV Trinidad donkey at target doses of 2.5 × 103 PFU, 8.9 × 103 PFU, and 1.3 × 103 PFU, respectively, on day 56. Mice were monitored daily for clinical signs of disease. Moribund animals were euthanized immediately according to the approved IACUC protocol.

Passive transfer studies in mice

Mice (n = 10 per group) were administered 2 mg of purified total IgG derived from either trivalent VLP–immunized or naïve cynomolgus macaques on days −1 and +3. Mice were exposed by aerosol to WEEV Fleming, EEEV FL93-939, or VEEV Trinidad donkey at target doses of 200 PFU, 300 PFU, and 100 PFU, respectively, on day 0. Mice were monitored daily for clinical signs of disease. Moribund animals were euthanized immediately.

Vaccine studies in NHPs

Alphavirus-naïve, mixed gender Chinese cynomolgus macaques >3 kg (3.2 to 8.4 kg) were randomized by body weight and intramuscularly injected with 20 μg of monovalent or 60 μg of trivalent VLPs (20 μg each of the monovalent VLPs) without adjuvant at days 0 and 28. PBS was administered as a negative control. Three to four macaques per group were enrolled in the monovalent VLP study; we expected all vaccinated animals to survive challenge and all controls to succumb to challenge based on the results from the challenge study in mice, which yielded a power of >99% to detect 98% survival difference between vaccine group and untreated controls by one-tailed Fisher’s exact test at an α of 0.05. Eight macaques per vaccinated group and nine macaques per control group were allocated for the trivalent VLP study: We expected all vaccinated animals to survive the challenge. Therefore, assuming no more than three controls survived (or showed no signs of infection), it yielded a power of >80% to detect at least a 65% survival difference between the vaccine group and untreated controls by one-tailed Fisher’s exact test at an α of 0.05. All macaques were challenged with aerosolized WEEV Fleming, EEEV FL93-939, or VEEV Trinidad donkey at target doses of 1 × 106 PFU, 1 × 108 PFU, and 1 × 108 PFU, respectively, at day 56. The NHPs were maintained in Animal Biosafety Level 3 facilities at either TNPRC or the USAMRIID for the duration of the in-life phase of this study.

Detection of viremia by plaque assay

Infectious virus was detected in whole blood by standard plaque assay as previously described (51). Briefly, samples were serially log-diluted starting at a dilution of 1:10 in HBSS complete. Vero 76 cells seeded on six-well plates were grown to about 90 to 100% confluence. Cells were infected with 0.1 ml of each serial dilution per well in duplicate. Plates were incubated at 37°C for 1 hour, with gentle rocking every 15 min. After 1 hour, cells were overlaid with 0.6% agarose in BME complete and incubated for 24 hours at 37°C with 5% CO2. A second overlay containing 1.2% agarose mixed 1:1 with BME complete and 5% of total volume neutral red vital stain was added to wells and further incubated for 18 to 24 hours for visualization of plaques. Unlike VEEV and EEEV, WEEV viremia in blood is not detected in cynomolgus macaques even in those NHPs where disease may lead to unresponsiveness (13, 52).

Statistical analysis

Most results are expressed as means ± SEM. Comparisons of two groups were done by unpaired two-tailed Student’s t test or Mann-Whitney test, and overall significance of multigroup comparisons was evaluated with Kruskal-Wallis ANOVA (unpaired) or Friedman ANOVA (paired). The statistical comparisons between control group and test group for infection and survival were determined by Fisher’s exact test or log-rank test, respectively. All the statistical analyses were conducted by the software GraphPad Prism 7.0.

SUPPLEMENTARY MATERIALS

stm.sciencemag.org/cgi/content/full/11/492/eaav3113/DC1

Materials and Methods

Fig. S1. Alignment and similarity of Env amino acid sequences of WEEV, EEEV, and VEEV.

Fig. S2. WEEV, EEEV, and VEEV Env-pseudotyped lentiviral reporters have similar luciferase activity.

Fig. S3. Transmission electron microscopy and cryogenic electron microscopy images of monovalent VLPs.

Fig. S4. Monovalent VLP vaccine conferred complete protection against homologous virus challenge in cynomolgus macaques.

Fig. S5. The trivalent VLP vaccine protected against virus challenge and eliminated clinically significant neuropathology and persistent viral infection in NHPs.

Table S1. Observation of clinical signs and viremia after WEEV challenge in mice.

Data file S1. Primary data.

REFERENCES AND NOTES

Acknowledgments: We thank H. Bao, A. Taylor, and J.-P. Todd for the animal work and A. Porter, K. Beck, A. Piper, R. Erwin-Cohen, A. Burkey, R. Bakken, the Division of Veterinary Medicine, and the Clinical Research Laboratory for outstanding commitment and support provided in conducting the animal studies at USAMRIID. In addition, we appreciate the work of the technical staff of the Pathology Division at USAMRIID for providing excellent necropsy, histology, and immunohistochemistry support. Funding: This work was funded in part by an Interagency Agreement with the U.S. Department of Defense (DOD) Joint Program Executive Office Chemical Biological Defense–Medical Countermeasure Systems–Joint Vaccine Acquisition Program (JPEO-CBD MCS-JVAP) and the Defense Threat Reduction Agency Program under USAMRIID project number 1882093 (H.H.0003_07_RD_B and CCR no. CB3691) (to K.C., C.W.B., S.P.H., D.K.N., L.D., and P.J.G.), federal funds from the Frederick National Laboratory for Cancer Research, NIH, under contract HHSN261200800001E, and Leidos Biomedical Research Inc. (to T.S., Y.T., and U.B.). This work was also supported by funding from the intramural program of the VRC, NIAID, and NIH (to S.-Y.K., W.A., E.S.Y., W.-P.K., G.L.S., K.C., J.R.M., G.J.N., and S.S.R.). The opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the U.S. Army. Author contributions: S.-Y.K., W.A., W.-P.K., J.R.M., G.J.N., and S.S.R. designed the study, analyzed the data, and/or prepared the manuscript. E.S.Y. performed the immunogenicity evaluation and analyzed the data. L.D., C.W.B., and P.J.G. prepared the virus stocks and conducted the vaccine and passive immunization studies in mouse model and trivalent vaccine studies in NHP model, assisting in the design, analyses, and manuscript preparation for these studies. V.T.-D. and C.J.R. prepared the challenge virus stocks and performed the monovalent vaccine study in NHP model. Y.-J.S.H., D.L.V., and S.H. prepared the virus challenge stock. S.P.H. and D.K.N. performed the pathology evaluation. G.L.S. and K.C. coordinated this project and helped to prepare the manuscript. Y.T., T.S., and U.B. performed the electron microscopy analysis. Competing interests: G.J.N., W.A., and S.S.R. are inventors on a patent describing the VLPs. All other authors declare that they have no competing interests. Data and materials availability: All data associated with this study are present in the paper or the Supplementary Materials. Expression vecors for VLPs are available from NIAID/NIH under an appropriate material transfer agreement.
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