Immunization with Low Doses of Recombinant Postfusion or Prefusion Respiratory Syncytial Virus F Primes for Vaccine-Enhanced Disease in the Cotton Rat Model Independently of the Presence of a Th1-Biasing (GLA-SE) or Th2-Biasing (Alum) Adjuvant
ABSTRACT
Respiratory syncytial virus (RSV) infection of children previously immunized with a nonlive, formalin-inactivated (FI)-RSV vaccine has been associated with serious enhanced respiratory disease (ERD). Consequently, detailed studies of potential ERD are a critical step in the development of nonlive RSV vaccines targeting RSV-naive children and infants. The fusion glycoprotein (F) of RSV in either its postfusion (post-F) or prefusion (pre-F) conformation is a target for neutralizing antibodies and therefore an attractive antigen candidate for a pediatric RSV subunit vaccine. Here, we report the evaluation of RSV post-F and pre-F in combination with glucopyranosyl lipid A (GLA) integrated into stable emulsion (SE) (GLA-SE) and alum adjuvants in the cotton rat model. Immunization with optimal doses of RSV F antigens in the presence of GLA-SE induced high titers of virus-neutralizing antibodies and conferred complete lung protection from virus challenge, with no ERD signs in the form of alveolitis. To mimic a waning immune response, and to assess priming for ERD under suboptimal conditions, an antigen dose de-escalation study was performed in the presence of either GLA-SE or alum. At low RSV F doses, alveolitis-associated histopathology was unexpectedly observed with either adjuvant at levels comparable to FI-RSV-immunized controls. This occurred despite neutralizing-antibody titers above the minimum levels required for protection and with no/low virus replication in the lungs. These results emphasize the need to investigate a pediatric RSV vaccine candidate carefully for priming of ERD over a wide dose range, even in the presence of strong neutralizing activity, Th1 bias-inducing adjuvant, and protection from virus replication in the lower respiratory tract.
IMPORTANCE RSV disease is of great importance worldwide, with the highest burden of serious disease occurring upon primary infection in infants and children. FI-RSV-induced enhanced disease, observed in the 1960s, presented a major and ongoing obstacle for the development of nonlive RSV vaccine candidates. The findings presented here underscore the need to evaluate a nonlive RSV vaccine candidate during preclinical development over a wide dose range in the cotton rat RSV enhanced-disease model, as suboptimal dosing of several RSV F subunit vaccine candidates led to the priming for ERD. These observations are relevant to the validity of the cotton rat model itself and to safe development of nonlive RSV vaccines for seronegative infants and children.
INTRODUCTION
Respiratory syncytial virus (RSV) is a viral human pathogen of the family Paramyxoviridae that causes significant respiratory pathology in young children, immunocompromised individuals, and older adults (1–3). Despite being an important disease and economic burden, prevention and treatment of RSV infection remains a major unmet medical need, and no licensed vaccine is available. To date, the clinically most advanced RSV vaccines are focused on RSV-seropositive individuals, especially pregnant women and older adults (4). For these RSV-seropositive populations, clinical development is more straightforward due to a lack of safety concerns related to enhanced respiratory disease (ERD), encouraging active investigation of vaccine platforms, such as subunit vaccines (5, 6). In contrast, vaccine development in seronegative pediatric populations has been primarily geared toward virally vectored or live-attenuated RSV vaccine platforms (4–7), due to the concern that immunization with nonlive vaccines, such as subunit vaccines, may prime for ERD. ERD was first observed in children who received a formalin-inactivated, whole-virus RSV vaccine (FI-RSV) in the 1960s (8–11). The children later naturally infected with RSV were not protected but rather were predisposed to develop severe RSV disease; 80% were hospitalized in one study versus 5% of controls, and 2 children died (11).
In-depth analyses of the immune causes of enhanced RSV disease have identified potential biomarkers associated with ERD, which when assessed in animal models of ERD provide a means to evaluate whether novel RSV vaccine candidates may be ready for human pediatric use (summarized in reference 12). FI-RSV-mediated ERD has been attributed to a number of causes, including the failure to induce a robust neutralizing-antibody response plus priming for an exaggerated Th2-biased immune response in the absence of cytotoxic T lymphocytes (reviewed in references 12 and 13). Due to a suboptimal, nonprotective immune response, upon subsequent RSV exposure, the potentially high antigen burden in the lungs could lead to the recruitment of immune cells (i.e., RSV-specific T cells, neutrophils, or eosinophils) into the lower respiratory tract, ultimately resulting in airway obstruction. Studies in animal models suggest that a safe immune profile in response to RSV immunization would combine a high neutralizing-antibody response with a cellular response that is Th1 biased (reviewed in references 14 and 15).
The fusion (F) protein of RSV is the primary target for neutralizing antibodies. RSV F exists in a metastable state (prefusion [RSV pre-F]) on the surface of the virus and functions to drive membrane fusion between the viral envelope and the host cell. To initiate fusion, RSV F undergoes a dramatic conformational change, resulting in a very stable postfusion form (RSV post-F). A recent breakthrough by McLellan et al. has been the generation of a stabilized prefusion form of RSV F by structure-based design (16). RSV neutralization activity in human sera is primarily directed against antigenic sites (such as site Ø), which are found only on RSV pre-F (17).
However, recombinant soluble RSV post-F (produced in Chinese hamster ovary [CHO] cells), when adjuvanted with the synthetic Toll-like receptor 4 (TLR4) agonist glucopyranosyl lipid A (GLA) integrated into stable emulsion (SE) (GLA-SE), has been shown to elicit a strong anti-RSV F immune response, a Th1-biased cellular immune response, and cytotoxic T lymphocytes, all of which led to protection from RSV challenge in BALB/c mice and cotton rats (18, 19). This vaccine candidate is currently in a phase 2 clinical trial in seropositive older adults (5). Because RSV post-F plus GLA-SE represents a nonlive vaccine platform, a comprehensive nonclinical assessment, including ERD studies, was conducted in the cotton rat prior to proceeding to clinical studies within pediatric populations. The cotton rat, Sigmodon hispidus, is an accepted ERD rodent model (20, 21) that has been used to support the safety evaluation of RSV vaccines developed for RSV-naive populations (reviewed in reference 22). The alveolitis and interstitial pneumonitis observed in the cotton rat model of ERD is thought to mimic the pathology observed with FI-RSV given to RSV-naive children (11); alveolitis is the primary marker in this model.
In the vaccine studies presented here, we conducted a detailed evaluation of soluble RSV F in the prefusion and postfusion conformations as the antigen, with GLA-SE or alum as the adjuvant, in the cotton rat model in order to evaluate immunogenicity, efficacy, and, more importantly, safety. In short-term studies (RSV challenge 49 days after prime immunization) and at prophylactic doses of RSV F antigen, we did not observe any increase in lung inflammation whether the animals were immunized with GLA-SE or alum as the adjuvant. At lower antigen doses, we observed ERD in long-term studies (RSV challenge 91 days after prime immunization) only when F antigen was formulated with alum. To model low titers of neutralizing antibodies due to a waning immune response or suboptimal immunization outcome, we immunized cotton rats with very low doses of antigen. Surprisingly, we observed ERD in the form of alveolar histopathology at a severity similar to that induced by FI-RSV immunization. This was true for both adjuvants regardless of Th1 or Th2 biasing properties and even when virus titers were below the lower limit of detection of our plaque assay. Furthermore, the same disease enhancement was observed after immunization of cotton rats with RSV pre-F, despite the induction of a more potent neutralizing-antibody response. Further studies in mice and cotton rats are under way to elucidate the mechanism of action responsible for the low-antigen-dose ERD. Overall, our observations are very significant for the RSV field, as demonstration of vaccine safety is paramount for RSV vaccine development.
RESULTS
Virus neutralization activity and protection from challenge after immunization with RSV pre-F and post-F in combination with GLA-SE adjuvant.
RSV F in the postfusion conformation (RSV post-F) in combination with GLA-SE is currently being developed as a vaccine candidate for older adults (5). Recently, McLellan et al. reported that a stabilized, soluble form of RSV F in the prefusion conformation (RSV pre-F) was able to induce higher levels of neutralizing antibodies than soluble RSV post-F in mice and nonhuman primates (NHP) and might serve as a preferred antigen for an RSV subunit vaccine (16, 23). We therefore included RSV pre-F, purified from the same cell substrate as RSV post-F, in our analyses. The pre-F stabilized conformation was confirmed by enzyme-linked immunosorbent assay (ELISA) using D25 monoclonal antibody (MAb), which specifically binds to site Ø (Fig. 1). As expected, no D25 binding against the post-F construct was observed, and similar binding profiles were obtained using motavizumab, which recognizes a common epitope between pre- and post-F conformations (Fig. 1).
FIG 1
To compare the immunogenicity and efficacy of soluble RSV pre-F and RSV post-F in combination with GLA-SE adjuvant, groups of cotton rats were immunized twice (days 0 and 21) intramuscularly (i.m.) with RSV F protein of either conformation at various doses (0.06, 0.3, and 1.5 μg) in the presence of GLA-SE (2.5 μg GLA, 2% SE) or without adjuvant (1.5 μg). As controls, one group of animals received the adjuvant alone and another group was immunized intranasally (i.n.) with live RSV A2 at day 0. The studies were duplicated to challenge one cohort of animals with RSV A (strain A2; homologous challenge, as our F antigen sequence was based on the A2 strain) and the other with RSV B (strain 9320; heterologous challenge) 2 weeks after the second immunization. Prior to challenge, the animals were bled to allow the neutralizing-antibody levels to be determined in a virus microneutralization assay. Four days post-RSV infection, lungs and nasal turbinates (NT) were harvested to assess tissue-specific virus replication by plaque assay.
As shown in Fig. 2A, neutralizing activity against RSV A2 was strongly induced in the adjuvanted groups, and the titers were significantly higher (5.3- to 8.0-fold) in the RSV pre-F-immunized animals than in the RSV post-F-immunized animals at all RSV F dose levels tested. In contrast, unadjuvanted RSV post-F induced detectable titers (mean titer, 8.7 log2), whereas surprisingly, responses in the animals receiving unadjuvanted RSV pre-F were not different from those in the GLA-SE alone control group, where no neutralizing activity was detectable (4 log2, which is the lower limit of detection [LLOD] of the assay). Live-RSV infection induced mean titers of 14.5 log2. To determine protective efficacy, one cohort of animals was challenged with RSV A2 (1 × 106 PFU) (Fig. 2C and E) and the second cohort with RSV B9320 (1 × 105 PFU) (Fig. 2D and F). RSV titers were determined in lung and NT homogenates by virus plaque assay. Immunization with unadjuvanted RSV post-F provided partial protection in the lungs of RSV A2-challenged animals, whereas unadjuvanted RSV pre-F did not provide any protection, as expected based on the nondetectable neutralizing activity. Vaccination in the presence of GLA-SE conferred complete protection against RSV A2 challenge in the lungs of RSV post-F- and pre-F-immunized cotton rats. RSV A2 was detectable in the NT of challenged animals in all RSV post-F-immunized dose groups (adjuvanted and unadjuvanted), albeit at much lower levels in the adjuvanted groups than in the controls treated with GLA-SE alone. RSV pre-F immunization conferred complete protection at the 1.5-μg antigen dose in the presence of GLA-SE and almost complete protection at the 0.06-μg and 0.3-μg antigen doses (5/7 and 6/7 cotton rats had no detectable virus in their NT, respectively).
FIG 2
In comparison, after challenge with RSV B9320, animals immunized with RSV pre-F and post-F in the presence of GLA-SE were protected from virus replication in their lungs (Fig. 2D). Very little or no protection was observed in the unadjuvanted groups. Virus was detectable in NT of all RSV F-immunized animals in the presence or absence of adjuvant in all protein dose groups tested, albeit greatly reduced when the protein was formulated with GLA-SE, reflecting the levels of neutralizing activity measured in the serum (Fig. 2F). Previous infection of cotton rats with live RSV A2 fully protected the animals from infection with RSV A2 or RSV B in both lungs and noses.
No signs of ERD after RSV F immunization with prophylactic antigen doses.
The cotton rat is an accepted model for the evaluation of ERD (20, 21) and has been used to support the safety evaluation of RSV vaccines prior to clinical evaluation in RSV-naive populations. In the cotton rat model, enhanced RSV disease is characterized by marked alveolitis in the lungs (21). The goal of the first ERD study was to compare the immunogenicity and protection from challenge of immunizations with RSV post-F and pre-F plus GLA-SE to that with FI-RSV and to assess whether the RSV F plus GLA-SE vaccine candidates induced alveolitis-associated histopathology in the lungs of cotton rats after virus challenge (Fig. 3). When administered with GLA-SE, the response to the 0.3-μg RSV F antigen dose chosen for this study has been demonstrated to completely block virus replication in the lower respiratory tract in cotton rats subsequently challenged with RSV (Fig. 2) (18). FI-RSV was used as a reference control for ERD. FI-mock (formalin-inactivated HEp-2 cell supernatants processed in parallel with FI-RSV and used at the same dilution) served here as a control for FI-RSV to ensure that the disease enhancement was specific to formalin-inactivated RSV and not solely due to host cell protein sensitivity generated in the cotton rats, as the RSV generated for formalin inactivation and the live challenge virus were both grown in HEp-2 cells. FI-RSV, FI-mock, and RSV post-F and pre-F plus GLA-SE were administered i.m. on days 0 and 21. Wild-type RSV A Long (1 × 106 PFU), a virus strain classically used in the cotton rat ERD model (21), was administered i.n. on day 0. As we had already determined in the previous study that immunization with GLA-SE alone had no effect, one group of animals received protein formulation buffer as a control. On day 49 post-prime immunization, animals were bled (to provide sera to determine neutralization titers) and infected with RSV A Long. Naive animals were not exposed to any treatment.
FIG 3
The day 49 virus neutralization titers were determined using RSV A2 as the target virus in the assay (Fig. 3A). FI-RSV elicited very low neutralization titers, with a group mean average of 4.8 log2. As expected from the previous study, RSV post-F and pre-F immunization in the presence of GLA-SE elicited a robust and significantly higher neutralizing-antibody response than FI-RSV, with mean averages of 12.9 and 16.7 log2, respectively. RSV A Long immunization resulted in comparatively low neutralization titers of 9.7 log2. In contrast to FI-RSV immunization, which conferred only partial protection from RSV A Long challenge (average titer, 3.8 log10 PFU/g of lung tissue) in cotton rats immunized with RSV post-F and pre-F plus GLA-SE and live RSV, the lung viral titers were reduced to below the limit of detection (Fig. 3B). The buffer- and FI-mock-immunized animals had lung viral titers that averaged 4.9 log10 and 5.0 log10 PFU/g of lung tissue, respectively. To determine if RSV post-F and pre-F plus GLA-SE immunization elicited ERD, histopathologic analysis was performed on the lungs and compared to live-RSV-, FI-RSV-, and FI-mock-immunized cotton rats (Fig. 3C). Animals immunized with protein buffer only and infected with RSV served as controls for RSV disease, and untreated, naive animals served as baseline controls. Analysis was performed by a pathologist blinded to the groups on samples collected 4 days post-RSV challenge. Inflammation in the lung was measured using a severity score defined as 0 (within normal limits), 0.5 (minimal inflammation; <5% of the lung affected), 1 (mild inflammation; <10% of the lung affected), 2 (moderate inflammation; 10 to 25% of the lung affected), 3 (marked inflammation; 25 to 50% of the lung affected), and 4 (severe inflammation; >50% of the lung affected, with tissue necrosis or damage). As alveolitis was established as the primary hallmark of ERD in the cotton rat model (21), we focused our analysis on this parameter. Multifocal alveolitis (macrophages, eosinophils, and fewer neutrophils and lymphocytes) with mild to moderate severity was consistently present in all animals in the FI-RSV control group (Fig. 3C). The lungs of animals in the group immunized with RSV F plus GLA-SE showed no or minimal/mild alveolitis regardless of the RSV F protein conformation. Alveolitis was observed in only one animal in the FI-mock-immunized group. One animal in the live-RSV-immunized group had a locally extensive infiltration of macrophages, eosinophils, and neutrophils in the alveolar space. Importantly, in this experiment, no treatment group exhibited the characteristic moderate/marked alveolitis seen in cotton rats vaccinated with FI-RSV.
Evaluation of RSV F plus GLA-SE in the cotton rat ERD model with RSV challenge 3 months after prime immunization.
Murphy and colleagues reported that a subunit RSV F- and RSV G-based vaccine did not induce enhanced alveolar and bronchiolar pulmonary histopathology when cotton rats were challenged with RSV 1 week after the last immunization (24). In contrast, RSV challenge 3 months post-prime immunization with a similar RSV F glycoprotein vaccine on alum (an adjuvant that promotes a Th2-biased immune response) induced alveolar histopathology comparable to that of animals immunized with FI-RSV upon RSV challenge (25).
To address these findings, a study was performed to determine if RSV post-F immunization in the presence of GLA-SE could prime for enhanced disease when RSV challenge occurred 3 months postprime. The immunogenicity and efficacy of RSV post-F plus GLA-SE were compared to those of FI-RSV and RSV post-F plus alum in the cotton rat model at high (5 μg RSV F) and low (0.05 μg RSV F) antigen doses. The antigen doses were chosen based on the Murphy et al. study (in which RSV F was purified from RSV-infected Vero cells [25]). FI-RSV, FI-mock, RSV post-F plus GLA-SE, RSV post-F plus alum, and live RSV A Long were administered as described above. Naive animals were not exposed to any treatment.
Animals were bled for serological analysis and infected with RSV A Long on day 91 post-prime immunization. Vaccination with FI-RSV elicited neutralization titers with a group mean average of 6.5 log2. In contrast, immunization with RSV post-F plus GLA-SE and RSV post-F plus alum elicited robust and comparable neutralizing-antibody responses with mean average titers of 14.0 and 13.5 log2 at the 5.0-μg dose and 11.6 and 11.0 log2 at the 0.05-μg dose, respectively (Fig. 4A). To compare the abilities of different immunization regimens to confer protection from challenge, individual cotton rat lung tissues were harvested 4 days postchallenge (day 95). In contrast to FI-RSV immunization, which conferred only partial protection from RSV challenge (mean titer, 3.8 log10 PFU/g of lung tissue), the lung viral titers were reduced to below the LLOD in cotton rats immunized with RSV post-F plus GLA-SE or RSV post-F plus alum at both RSV F doses tested, identical to live-virus immunization. The control animals immunized with buffer alone had mean average lung viral titers of 4.9 log10 PFU/g of lung tissue (Fig. 4B).
FIG 4
Histopathology analysis was performed as described above, and alveolitis of mild to marked severity was present in animals of the FI-RSV control group (Fig. 4C). The lungs of cotton rats immunized with RSV post-F plus GLA-SE and RSV post-F plus alum showed no or minimal/mild alveolitis at the 5.0-μg RSV F dose. At the 0.05-μg RSV F dose in the RSV post-F plus GLA-SE group, no or minimal/mild alveolitis was also observed, similar to naive animals challenged with RSV. In contrast, in animals immunized with 0.05 μg of RSV post-F plus alum, moderate to marked alveolitis was present in 4 out of 7 animals, despite the presence of a robust neutralizing-antibody response and the lack of detectable virus in the lungs. Zero or minimal/mild alveolitis was observed in the FI-mock-immunized cotton rats.
Neutralizing-antibody response elicited by RSV post-F and pre-F plus GLA-SE immunization is above the protective threshold for the lifetime of a cotton rat.
The above analysis showed that RSV F in combination with GLA-SE did not appear to prime for ERD under the tested conditions. To ensure that this is also true in the context of a waning immune response over time, we set out to determine the time point at which the neutralizing antibody falls under the “protective” threshold of 8.5 log2, which confers sterilizing immunity in the lungs of cotton rats, based on literature reports (26). To this end, cotton rats were immunized twice, with RSV post-F and pre-F (1.5 and 0.3 μg), in the presence of GLA-SE. Virus-neutralizing titers were monitored on sequential bleeds by microneutralization assay over a period of 380 days. Mean average peak neutralization titers (2 weeks after the boost immunization) were 11.8 log2 (0.3 μg RSV post-F) and 13.0 log2 (1.5 μg RSV post-F) in the animals immunized with RSV post-F plus GLA-SE and 16.1 log2 (0.3 μg RSV pre-F) and 16.4 log2 (1.5 μg RSV pre-F) in the animals immunized with RSV pre-F plus GLA-SE (Fig. 5). At the end of the study, 34% of the animals were dead due to age-related causes. The virus neutralization titers at day 380 post-prime immunization in the remaining cotton rats were long-lasting and still very robust, at 8.8 log2 (0.3 μg RSV post-F), 9.7 log2 (1.5 μg RSV post-F), and 13.3 log2 (0.3 μg and 1.5 μg RSV pre-F) and therefore above the target protective threshold of 8.5 log2 (Fig. 5). Immunizing cotton rats with the vaccination regimen described above and waiting for the neutralizing-antibody response to drop below the protective threshold was therefore not practical.
FIG 5
Low-dose RSV F antigen immunization in the presence of GLA-SE and alum results in ERD.
Hence, the waning immune response or a suboptimal dosing outcome was modeled in a follow-up study by decreasing RSV F antigen doses while keeping the adjuvant dose constant. The safety profile of low-antigen-dose RSV post-F and pre-F immunization was assessed by determining the histological changes occurring in the lungs of immunized and subsequently live-RSV-challenged cotton rats 50 days post-prime immunization.
Animals were immunized in the first dose-de-escalating study with a starting RSV post-F dose of 0.3 μg and 10-fold serially diluted RSV post-F down to 0.0003 μg of protein in the presence of GLA-SE and alum (Fig. 6). Virus neutralization titers were determined at the time of challenge. RSV post-F plus GLA-SE and RSV post-F plus alum elicited antigen dose-dependent neutralizing-antibody titers, with group mean average titers at the highest antigen dose (0.3 μg) of 13.4 log2 and 13.1 log2, respectively. At the lowest antigen dose (0.0003 μg), the group mean average titers were 7.4 log2 and 5.7 log2, respectively (Fig. 6A), and therefore below the protective threshold of 8.5 log2. Total RSV post-F IgG titers measured by ELISA were detectable in all immunized animals and mirrored the levels of neutralization antibodies detected (data not shown).
FIG 6
Virus plaque assay analysis 4 days postchallenge revealed that, in the animals immunized with live RSV, the lung viral titers were reduced to below the limit of detection, whereas the challenged unimmunized animals had average lung viral titers of 4.9 log10 PFU/g of lung tissue (Fig. 6B). In contrast to FI-RSV vaccination, which conferred only partial protection from RSV challenge, cotton rats immunized with RSV post-F plus GLA-SE at the 0.3-μg and 0.03-μg RSV F doses showed no detectable virus in their lungs by plaque assay. At the 0.003-μg dose, 1 out of 7 animals had a very low titer of detectable virus in the lung (2.0 log10 PFU/g), and at the 0.0003-μg RSV post-F dose, all animals had detectable virus present in their lungs (average titer, 4.4 log10 PFU/g). In the RSV post-F plus alum groups, 1 out of 7 animals had very low levels of detectable virus in the lung at the 0.3- and 0.03-μg RSV F doses (1.7 log10 PFU/g) and 2 out of 7 at the 0.003-μg dose (average titer, 1.8 log10 PFU/g). At the lowest antigen dose of 0.0003 μg, all animals had detectable virus in their lungs (average titer, 4.8 log10 PFU/g). In the NT, we detected RSV at very low levels in 4 out of 7 (average titer, 1.3 log10 PFU/g) animals in the 0.3-μg and 4 out of 7 (average titer, 1.4 log 10 PFU/g) in the 0.03-μg RSV post-F plus GLA-SE-immunized animals. Virus was detectable in 6 out of 7 noses in the cotton rats immunized with 0.003 μg of RSV post-F plus GLA-SE (average titer, 2.4 log10 PFU/g), and all animals in the 0.0003-μg RSV post-F plus GLA-SE group had virus titers similar to those of unimmunized, RSV-challenged animals (4.9 log10 PFU/g and 5.1 log10 PFU/g, respectively) (Fig. 6C). RSV post-F plus alum immunization conferred less protection in the noses than RSV post-F plus GLA-SE (average titers, 1.9 log10 [0.3 μg], 2.6 log10 [0.03 μg], 4.3 log10 [0.003 μg], and 4.9 log10 [0.0003 μg] PFU/g [RSV post-F dose]) (Fig. 6C).
Histopathology analysis was performed to determine if RSV post-F plus GLA-SE and alum elicited enhanced RSV disease when immunization was performed with low RSV F antigen doses in the presence of a constant dose of adjuvant (Fig. 6D and 7). Multifocal alveolitis of mild to moderate severity was present in animals of the FI-RSV control group. No or minimal/mild alveolitis was observed in the FI-mock-immunized cotton rats, with the exception of one animal, which presented with moderate alveolitis. The lungs of animals in the RSV post-F plus GLA-SE group immunized with 0.3 μg of RSV post-F showed no or minimal alveolitis, similar to previous observations (Fig. 3C). In contrast, when the animals were immunized with lower doses of RSV post-F (0.03 and 0.003 μg), multifocal alveolitis (severity score ≥ 1) was present in 12 out of 14 animals with severity similar to that observed in the FI-RSV control group (7 out of 7 animals with severity scores of ≥1), despite complete (0.03 μg RSV post-F) or almost complete (0.003 μg RSV post-F) protection from virus replication in the lung and neutralizing-antibody levels comparable to those of previously RSV-infected cotton rats. At the lowest antigen dose (0.0003 μg) in the RSV post-F plus GLA-SE group, the alveolitis observed was similar to that of the FI-mock control and significantly lower than that of the FI-RSV control. At the lowest antigen dose, the immune response, measured by neutralizing-antibody induction, was close to that of the negative-control groups (naive and FI-mock). It appears that at the lowest antigen dose the immune response induced was too low to contribute to alveolitis-associated histopathology after RSV challenge. When immunizing with decreasing doses of RSV post-F in the presence of alum, alveolar-associated histopathology was similar to that with FI-RSV at the 0.3-μg and 0.003-μg doses of RSV post-F and even more pronounced than that with FI-RSV at the 0.03-μg dose of antigen (ranging from minimal to marked severity). At the lowest antigen dose (0.0003 μg) plus alum, the cotton rats showed no or mild alveolitis, similar to what was observed in the presence of GLA-SE. Animals in the live-RSV-immunized group and the unimmunized group presented with no, minimal, or mild alveolar-associated histopathology. Overall, a similar trend was consistently observed when scoring for peribronchiolar cuffing (Fig. 6 and data not shown).
FIG 7
An identical study was performed with RSV pre-F as the antigen, with the only difference that the dose de-escalation was started with a 10-fold-lower antigen dose (0.03 μg) (Fig. 8). Neutralizing-antibody activity (Fig. 8A), virus replication in the lung postchallenge (Fig. 8B), and alveolitis-associated histopathology (Fig. 8C) were determined. Similar to the findings shown in Fig. 5, alveolar-associated histopathology of mild to marked severity was observed at the 0.03-, 0.003-, and 0.0003-μg pre-F doses in the presence of GLA-SE. Interestingly, replicating virus was not detectable in the lungs by plaque assay in the animals immunized with 0.03 and 0.003 μg of RSV pre-F, and reduced levels of replicating virus compared to nonimmunized RSV-infected animals were detectable in the 0.0003-μg RSV pre-F plus GLA-SE group (mean titer, 3.0 log10 PFU/g of lung tissue). In the presence of alum adjuvant, mild to marked alveolitis was present at the 0.03-μg RSV pre-F dose. Inflammation in the alveoli was similar to that in previously RSV-infected and rechallenged animals in the 0.003-, 0.0003-, and 0.00003-μg RSV pre-F plus alum groups. At these antigen doses, RSV titers in the lungs (mean titers, 5.0, 5.0, and 5.1 log10 PFU/g, respectively) were close to the titer observed in the nonimmunized RSV-challenged animals (mean titer, 4.6 log10 PFU/g), suggesting that little or no protective immune response was elicited, which is reflected in virus neutralization titers at or below the protective threshold of 8.5 log2 (mean titers, 7.7 log2 for the 0.003-μg dose, 4.2 log2 for the 0.0003-μg dose, and 4.0 log2 for the 0.00003-μg RSV pre-F dose).
FIG 8
DISCUSSION
Children, especially infants, entering their first RSV season are at high risk of serious RSV disease and would greatly benefit from a prophylactic vaccination approach. RSV post-F in combination with the synthetic TLR4 agonist GLA in a stable emulsion (GLA-SE) is currently under development as a prophylactic vaccine, preventing RSV disease in older adults (5). While the mechanism of action of RSV post-F plus GLA-SE to induce both a strong neutralizing-antibody response and a Th1-biased T cell response may be applicable for protection against RSV disease in infants, this type of adjuvanted recombinant subunit vaccine has never been tested in an RSV-seronegative pediatric population.
Based on the historical experience with FI-RSV vaccine that resulted in ERD in vaccinated infants following their first natural RSV infection, all new RSV vaccines must be evaluated for their potential to induce ERD prior to clinical studies on RSV-seronegative infants. In particular, nonlive vaccines, such as the subunit vaccines investigated here, have been associated with ERD in the past (25, 27).
Recently, McLellan et al. reported that a stabilized, soluble form of RSV F in the prefusion conformation was able to induce higher levels of neutralizing antibodies than soluble post-F in mice and nonhuman primates and might therefore stand out as a promising antigen for a subunit vaccine (16). In the studies presented here, we therefore included RSV pre-F, purified from the same cell substrate, in our analysis.
The first study was performed with the goal of evaluating the immunogenicity and efficacy of both forms of RSV F in combination with GLA-SE in the cotton rat. Next, we performed detailed studies to evaluate the safety of the subunit vaccine candidates.
Antibodies neutralizing RSV A2 were abundantly induced after post-F and pre-F plus GLA-SE immunization and were well above the protective threshold in cotton rats of 8.5 log2, with RSV pre-F inducing significantly higher titers than RSV post-F. These findings comparing pre-F and post-F confirm previous studies in mice and NHP, in which RSV pre-F and post-F were combined with poly(I·C) adjuvant (16). Interestingly, cross-neutralizing activities against RSV B9320 (the RSV F used in the studies presented here is based on the RSV A2 sequence) were comparable between RSV pre-F- and post-F-immunized animals in the adjuvanted and nonadjuvanted groups (Fig. 2B). This differs from the earlier findings reported in mice and NHP, where RSV pre-F-immunized animals showed higher neutralization of RSV subtypes A and B than RSV post-F-vaccinated animals (16). Our observation might be specific to cotton rats, to the type of adjuvant, or to the RSV B strain used in these studies.
Protection after challenge with either RSV A2 or RSV B9320 was reflective of the neutralizing-antibody titers induced, with complete lung protection provided by either antigen for both challenge viruses. Better protection was observed in the NT in the RSV pre-F plus GLA-SE-immunized, RSV A2-challenged cotton rats, with the two antigens providing equivalent protection in the RSV B-challenged animals (Fig. 2C to F).
The next step was to evaluate RSV post-F and pre-F in the ERD model. Immunization with 0.3 μg of RSV post-F and pre-F plus GLA-SE was associated with high levels of neutralizing antibodies, complete protection from RSV replication in the lungs, and no evidence of ERD after challenge on day 49 post-prime immunization (Fig. 3). Generally, the nonclinical safety evaluation of RSV vaccine candidates is considered sufficient at this point, but previous reports by Murphy and colleagues (25) showing the occurrence of ERD when RSV challenge was performed at a later time point prompted further investigation. A study was performed comparing RSV post-F adjuvanted with GLA-SE and alum with RSV challenge 3 months post-prime immunization in which animals immunized with RSV post-F plus alum developed alveolar histopathology, similar to data reported by Murphy et al. (25) and Connors et al. (27). This observation is potentially associated with the induction of a Th2-biased immune response by alum adjuvant, which has been identified to play a role in ERD (12). Further investigation is required to confirm this hypothesis. The absence of ERD in the presence of GLA-SE adjuvant in this study appeared to support the need for a Th1 bias-inducing adjuvant. Of note, in the experiment presented here, the highest level of alveolar histopathology in the RSV post-F plus alum groups was observed at the low dose of RSV post-F, whereas in the studies performed by Murphy and colleagues, the maximum observed histopathology was associated with the highest dose of antigen (25). This might be due to the difference in RSV F protein sources and levels of purity, which makes a direct dose comparison difficult. The levels of observed neutralization antibody after immunization in relation to natural RSV infection in the Murphy et al. study (25) were much lower than the levels induced here (Fig. 4A). During the evaluation of a pediatric RSV vaccine candidate, virus challenge is ideally performed after RSV-neutralizing titers in the cotton rat fall below the protective threshold of 8.5 log2 (26). As neutralizing-antibody titers after immunization with 1.5 or 0.3 μg RSV post-F and pre-F were stable over the lifetime of a cotton rat (Fig. 5), waiting to challenge after the antibody response had fallen below the protective threshold was not practical. Hence, dose de-escalation studies, in which groups of animals are given suboptimal doses of vaccine, were performed to address the question of ERD in the presence of low serum neutralization antibody levels. To our surprise, at very low antigen doses and in the presence of GLA-SE and alum, alveolitis-associated histopathology with severity similar to that observed in the FI-RSV-immunized control animals was observed. This observation was made in the presence of robust levels of neutralizing antibodies and complete or almost complete absence of detectable virus replication in the lung. Similar results were obtained when antigen dose-down was performed with RSV pre-F plus GLA-SE and with alum as the adjuvant. These findings indicate that, at suboptimal antigen doses, alveolitis-associated histopathology, the hallmark of enhanced RSV disease in the cotton rat model, can be induced by RSV post-F and pre-F, despite the inclusion of a strong, Th1-skewing adjuvant. Humoral responses, such as antibody avidity and affinity and cellular responses, in particular CD4+ and CD8+ T cells and regulatory T cell numbers and phenotypes, need to be investigated. Further studies, ideally in the mouse ERD model, where more immunological tools are readily available, are planned to determine the biomarkers and mechanism associated with the pathology observed after low-dose antigen vaccination. Studies in the mouse ERD model might either confirm or refute the ERD findings after low-dose antigen immunization, which is important for the validity of the cotton rat model and for pediatric RSV vaccine development.
Conclusions.
The safety signals for ERD observed in the antigen de-escalation studies raise concerns about the safety of an RSV post-F/pre-F plus adjuvant subunit vaccine in the RSV-naive pediatric population. It is critical that vaccines developed for RSV-naive populations do not represent a risk for ERD at the proposed pediatric dose, and vaccine developers need to also consider the risk for ERD with suboptimal dosing, which might occur in clinical practice and with waning of the immune response. As several nonlive vaccine candidates are in preclinical and clinical development (4), the findings presented here are of utmost importance to guide preclinical evaluation of vaccine candidates and to ensure the safety of RSV-naive infants and children.
MATERIALS AND METHODS
Vaccine components.
The soluble postfusion (RSV post-F) antigen was expressed in the CHO cell line using a fed-batch manufacturing process and was purified using a 3-step chromatography process, a nanofiltration virus removal step, and a low-pH virus inactivation step. Soluble prefusion (RSV pre-F) DS-Cav1 antigen was constructed and generated according to the method of McLellan et al. (16). Briefly, pre-F protein was expressed by transient transfections scaled up to 15 liters according to the method described by Daramola et al. using polyethylenimine (Polysciences) and purified by affinity and gel filtration chromatography steps with protease treatment for His tag removal (28).
Alum (aluminum hydroxide) adjuvant was obtained as Alhydrogel (Brenntag, Denmark). Alum was used at 100 μg or 150 μg per vaccine dose and adsorbed to protein by mixing for 60 min at 4°C. GLA-SE (29) was provided by and licensed from Immune Design Corporation (Seattle, WA). GLA-SE was used at 2.5 μg GLA in 2% SE per dose.
FI-RSV and formalin-inactivated cell supernatants (FI-mock) were obtained from Sigmovir Biosystems (Rockville, MD). HEp-2 cells (80% to 90% confluent) in T-150 tissue culture flasks were infected at a multiplicity of infection of 0.01 with RSV A Long. The cells were incubated for 1 h at 37°C. After incubation, the cells were washed twice with phosphate-buffered saline (PBS), and 25 ml of HEp-2 maintenance medium (Eagle's minimal essential medium [EMEM], antibiotics, 10% fetal bovine serum [FBS], 1% l-glutamine) was added to each flask. After the cells achieved 30% to 50% infection (days 2 to 3 postinfection), the cells were washed twice with PBS, and 20 ml of infection medium (EMEM, antibiotics, 1% l-glutamine, 10% streptomycin-penicillin-glutamate) was added to each flask, followed by overnight incubation at 37°C. The virus-containing supernatants were harvested, and 0.01 ml of a 1:40 formalin solution was added per ml of virus stock. The 1:4,000 formalin-virus solution was stored upright and incubated at 37°C for 72 h with vortexing every 24 h. The solution was subsequently stored at 4°C for at least 30 min prior to proceeding with (4-mg/ml) aluminum hydroxide adsorption overnight at room temperature. The alum-adsorbed FI-RSV was collected by centrifugation at 1,000 rpm for 10 min, resuspended to one-fourth its original volume in EMEM with penicillin-streptomycin, aliquoted, and stored at 4°C. The same procedure was followed when preparing the FI-mock control, omitting the RSV A Long infection of the HEp-2 cells. Concentrated FI-RSV and 1:5, 1:25, 1:125, and 1:625 dilutions were tested in cotton rats to determine the dilution that induced maximal alveolitis after RSV challenge (data not shown). Based on our results, FI-RSV and FI-mock were administered at 1:25 dilution.
RSV A2, RSV B9320, and RSV A Long strains (ATCC) were used for immunization and challenge. Virus was propagated in HEp-2 cells grown with EMEM. Viral supernatants were centrifuged to remove cellular debris, stabilized with 1× sucrose phosphate (0.2 M sucrose, 0.0038 M KH2PO4, and 0.0072 M KH2PO4), and snap-frozen in aliquots at −80°C until use.
ELISA assessment of pre-F antigen conformation.
Ninety-six-well plates were coated with purified monoclonal antibody (motavizumab or D25) at 1 μg/ml in PBS. The plates were blocked with 5% milk in PBS with 0.05% Tween 20. Purified F proteins were serially diluted 5-fold (10 μg/ml to 0.0001 μg/ml) in blocking buffer and added to the plates, which were then washed before incubation with horseradish peroxidase (HRP)-coupled 1331H antibody (30). After addition of 3,3′,5,5′-tetramethylbenzidine (TMB), the reactions were stopped with 2 N sulfuric acid, and absorbance was read at 450 nm. An unrelated protein was used as a control for specificity.
Animals, immunizations, and RSV challenge.
RSV-seronegative 6- to 8-week-old female cotton rats (Envigo, Dublin, VA) were housed under pathogen-free conditions at Medimmune, Gaithersburg, MD. All procedures were performed in accordance with federal, state, and institutional guidelines in an AAALAC-accredited facility and were approved by the Medimmune Institutional Animal Care and Use Committee. The animals were lightly anesthetized with isoflurane for immunizations and blood draws and euthanized with carbon dioxide for terminal organ harvests. For immunogenicity and efficacy studies comparing postfusion and prefusion RSV F, RSV F alone, or RSV F plus GLA-SE or alum, the mixtures were administered by i.m. injection on days 0 and 21. Wild-type RSV A2 (1 × 106 PFU) (positive control) was administered i.n. on day 0 under isoflurane anesthesia. As a negative control, one group of animals received GLA-SE adjuvant alone. The animals were challenged with RSV A2 (1 × 106 PFU) or RSV B9320 (1 × 105 PFU) by i.n. administration on day 35.
For studies addressing ERD, animals were administered FI-RSV (1:25), FI-mock (1:25), RSV F plus GLA-SE, and RSV F plus alum by i.m. injection on days 0 and 21. Wild-type RSV A Long (1 × 106 PFU) (positive control) was administered i.n. on day 0 under isoflurane anesthesia as a control for “natural” RSV infection. One group of animals (buffer) received protein storage buffer as a control (20 mM histidine/histidine-HCl, 23 mM potassium chloride, 7% [weight/volume] sucrose, 0.01% polysorbate 80, pH 6.5). The animals were challenged with RSV A Long (1 × 105 PFU) on day 49, day 50, or day 91 post-prime immunization. Naive animals were not exposed to any treatment prior to challenge. Organs were harvested on day 4 post-RSV challenge.
Microneutralization assay.
Serum samples at day 35, 49, or 91 were heat inactivated at 56°C for 45 min. In 96-well plates, the positive-control antibody (palivizumab) was serially diluted in 3-fold increments (starting at 8 μg/ml) in cell culture medium (minimal essential medium [MEM]) supplemented with 5% heat-inactivated FBS, 2 mM l-glutamine, 100 U of penicillin/ml, and 100 μg of streptomycin/ml [all from Invitrogen]) for a final volume of 50 μl. In duplicate, the test sera (starting dilution, 1:2) were serially diluted in 3-fold increments in cell culture medium for a final volume of 50 μl. Each serum dilution was mixed with 50 μl RSV A2 or RSV B9320 at 500 PFU per well. Following 2 h of incubation at 37°C with 5% CO2, 2.5 × 104 HEp-2 cells in a 100-μl volume were added to each well. Cells plus virus and cell only wells served as controls. After 3 days of incubation at 37°C with 5% CO2, the cell culture medium was removed and the monolayer was fixed with chilled 80% acetone. RSV replication was visualized by immunostaining with an HRP-labeled 1331H monoclonal antibody (30). The reciprocal log2 of the 50% inhibitory concentration (IC50) was determined for each serum sample using GraphPad Prism software. The LLOD of this assay is 4 log2. If an IC50 could not be calculated, the log2 of the initial dilution (1:16) was used for analysis.
Serum IgG ELISA.
Serological responses were evaluated with sera collected at day 49 by a standard ELISA on RSV post-F-coated (200 ng/well) plates. Samples were diluted in sample diluent (1× PBS with 1% bovine serum albumin [BSA] and 0.05% Tween 20) in 8 10-fold dilutions. All 8 dilutions were plated. Bound IgG was detected with chicken anti-cotton rat HRP. The serum total RSV F-specific antibody titers were calculated using the log2 of the lowest dilution recovered on samples that were <4 times the background value.
Viral plaque assays.
Lungs or NT were collected 4 days post-RSV challenge and placed in cold balanced Hanks salt solution supplemented with 1× sucrose phosphate in tissue homogenization tubes (MP Biomedicals) and homogenized using an MP FastPrep24 instrument (MP Biomedicals). The clarified supernatants were serially diluted and placed onto subconfluent HEp-2 cells in 24-well plates. After a 90-min incubation, the supernatants were removed and the cells were overlaid with MEM plus FBS and penicillin-streptomycin supplemented with 0.75% methylcellulose. After 5 days, the cells were fixed with 100% methanol for 15 min. Following fixation, the cells were blocked for 1 h in 5% nonfat milk and stained for 1 h with an RSV goat polyclonal antibody (Chemicon), followed by staining for 1 h with an HRP-conjugated rabbit anti-goat antibody (Dako). To visualize plaques, cells were incubated with AEC substrate ready-to-use (Dako), followed by a water rinsing step. The numbers of PFU per milliliter were calculated based on the number of plaques and the dilution factors.
Lung histopathology.
The histopathology of whole cotton rat lungs fixed in 10% neutral buffered formalin and routinely processed in paraffin was assessed. Tissues were sectioned at 4 μm, mounted on polarized slides, and stained with hematoxylin and eosin for histologic evaluation by a board-certified pathologist on a Nikon 80i Eclipse light microscope.
Statistical analysis.
Statistical assumptions were assessed prior to analysis. If statistical assumptions were not violated (in the case of three or more groups), an analysis of variance (ANOVA) followed by appropriate multiplicity-adjusted tests (e.g., Sidak's or Dunnett's test) was performed. If the assumptions were violated, a Kruskal-Wallis test followed by an appropriate multiplicity-adjusted test was performed. In the case of a two-group comparison with heteroscedastic variance, Welch's correction was applied. Groups at the LLOD were considered to have no activity in regard to hypothesis testing and were excluded from the analysis. Multiplicity-adjusted 2-sided P values are reported. A P value of <0.05 was considered significant. Analyses were performed with GraphPad Prism version 6.03.
ACKNOWLEDGMENTS
We thank Jorge Blanco and Marina Boukhvalova for helpful discussions and technical advice. We thank Bob Stadelman for assistance with reagent generation. We thank Amy Grenham and Tonya Villafana for critical review of the manuscript.
We declare no conflicts of interest.
REFERENCES
1.
Ebbert JO and Limper AH. 2005. Respiratory syncytial virus pneumonitis in immunocompromised adults: clinical features and outcome. Respiration72:263–269.
2.
Hall CB, Weinberg GA, Iwane MK, Blumkin AK, Edwards KM, Staat MA, Auinger P, Griffin MR, Poehling KA, Erdman D, Grijalva CG, Zhu Y, and Szilagyi P. 2009. The burden of respiratory syncytial virus infection in young children. N Engl J Med360:588–598.
3.
Widmer K, Zhu Y, Williams JV, Griffin MR, Edwards KM, and Talbot HK. 2012. Rates of hospitalizations for respiratory syncytial virus, human metapneumovirus, and influenza virus in older adults. J Infect Dis206:56–62.
4.
Higgins D, Trujillo C, and Keech C. 2016. Advances in RSV vaccine research and development; a global agenda. Vaccine34:2870–2875.
5.
Falloon J, Ji F, Curtis C, Bart S, Sheldon E, Krieger D, Dubovsky F, Lambert S, Takas T, Villafana T, and Esser MT. 2016. A phase 1a, first-in-human, randomized study of a respiratory syncytial virus F protein vaccine with and without a Toll-like receptor-4 agonist and stable emulsion adjuvant. Vaccine34:2847–2854.
6.
Glenn GM, Fries LF, Thomas DN, Smith G, Kpamegan E, Lu H, Flyer D, Jani D, Hickman SP, and Piedra PA. 2016. A randomized, blinded, controlled, dose-ranging study of a respiratory syncytial virus recombinant fusion (F) nanoparticle vaccine in healthy women of childbearing age. J Infect Dis213:411–422.
7.
Broadbent L, Groves H, Shields MD, and Power UF. 2015. Respiratory syncytial virus, an ongoing medical dilemma: an expert commentary on respiratory syncytial virus prophylactic and therapeutic pharmaceuticals currently in clinical trials. Influenza Other Respir Viruses9:169–178.
8.
Chin J, Magoffin RL, Shearer LA, Schieble JH, and Lennette EH. 1969. Field evaluation of a respiratory syncytial virus vaccine and a trivalent parainfluenza virus vaccine in a pediatric population. Am J Epidemiol89:449–463.
9.
Fulginiti VA, Eller JJ, Sieber OF, Joyner JW, Minamitani M, and Meiklejohn G. 1969. Respiratory virus immunization. I. A field trial of two inactivated respiratory virus vaccines; an aqueous trivalent parainfluenza virus vaccine and an alum-precipitated respiratory syncytial virus vaccine. Am J Epidemiol89:435–448.
10.
Kapikian AZ, Mitchell RH, Chanock RM, Shvedoff RA, and Stewart CE. 1969. An epidemiologic study of altered clinical reactivity to respiratory syncytial (RS) virus infection in children previously vaccinated with an inactivated RS virus vaccine. Am J Epidemiol89:405–421.
11.
Kim HW, Canchola JG, Brandt CD, Pyles G, Chanock RM, Jensen K, and Parrott RH. 1969. Respiratory syncytial virus disease in infants despite prior administration of antigenic inactivated vaccine. Am J Epidemiol89:422–434.
12.
Acosta PL, Caballero MT, and Polack FP. 2015. Brief history and characterization of enhanced respiratory syncytial virus disease. Clin Vaccine Immunol23:189–195.
13.
Hurwitz JL. 2011. Respiratory syncytial virus vaccine development. Expert Rev Vaccines10:1415–1433.
14.
Christiaansen AF, Knudson CJ, Weiss KA, and Varga SM. 2014. The CD4 T cell response to respiratory syncytial virus infection. Immunol Res59:109–117.
15.
Shaw CA, Ciarlet M, Cooper BW, Dionigi L, Keith P, O'Brien KB, Rafie-Kolpin M, and Dormitzer PR. 2013. The path to an RSV vaccine. Curr Opin Virol3:332–342.
16.
McLellan JS, Chen M, Joyce MG, Sastry M, Stewart-Jones GB, Yang Y, Zhang B, Chen L, Srivatsan S, Zheng A, Zhou T, Graepel KW, Kumar A, Moin S, Boyington JC, Chuang GY, Soto C, Baxa U, Bakker AQ, Spits H, Beaumont T, Zheng Z, Xia N, Ko SY, Todd JP, Rao S, Graham BS, and Kwong PD. 2013. Structure-based design of a fusion glycoprotein vaccine for respiratory syncytial virus. Science342:592–598.
17.
Ngwuta JO, Chen M, Modjarrad K, Joyce MG, Kanekiyo M, Kumar A, Yassine HM, Moin SM, Killikelly AM, Chuang GY, Druz A, Georgiev IS, Rundlet EJ, Sastry M, Stewart-Jones GB, Yang Y, Zhang B, Nason MC, Capella C, Peeples ME, Ledgerwood JE, McLellan JS, Kwong PD, and Graham BS. 2015. Prefusion F-specific antibodies determine the magnitude of RSV neutralizing activity in human sera. Sci Transl Med7:309ra162.
18.
Lambert SL, Aslam S, Stillman E, MacPhail M, Nelson C, Ro B, Sweetwood R, Lei YM, Woo JC, and Tang RS. 2015. A novel respiratory syncytial virus (RSV) F subunit vaccine adjuvanted with GLA-SE elicits robust protective TH1-type humoral and cellular immunity in rodent models. PLoS One10:e0119509.
19.
Patton K, Aslam S, Shambaugh C, Lin R, Heeke D, Frantz C, Zuo F, Esser MT, Paliard X, and Lambert SL. 2015. Enhanced immunogenicity of a respiratory syncytial virus (RSV) F subunit vaccine formulated with the adjuvant GLA-SE in cynomolgus macaques. Vaccine33:4472–4478.
20.
Byrd LG and Prince GA. 1997. Animal models of respiratory syncytial virus infection. Clin Infect Dis25:1363–1368.
21.
Prince GA, Curtis SJ, Yim KC, and Porter DD. 2001. Vaccine-enhanced respiratory syncytial virus disease in cotton rats following immunization with lot 100 or a newly prepared reference vaccine. J Gen Virol82:2881–2888.
22.
Boukhvalova MS and Blanco JC. 2013. The cotton rat Sigmodon hispidus model of respiratory syncytial virus infection. Curr Top Microbiol Immunol372:347–358.
23.
Boyington JC, Joyce MG, Sastry M, Stewart-Jones GB, Chen M, Kong WP, Ngwuta JO, Thomas PV, Tsybovsky Y, Yang Y, Zhang B, Chen L, Druz A, Georgiev IS, Ko K, Zhou T, Mascola JR, Graham BS, and Kwong PD. 2016. Structure-based design of head-only fusion glycoprotein immunogens for respiratory syncytial virus. PLoS One11:e0159709.
24.
Murphy BR, Sotnikov A, Paradiso PR, Hildreth SW, Jenson AB, Baggs RB, Lawrence L, Zubak JJ, Chanock RM, and Beeler JA. 1989. Immunization of cotton rats with the fusion (F) and large (G) glycoproteins of respiratory syncytial virus (RSV) protects against RSV challenge without potentiating RSV disease. Vaccine7:533–540.
25.
Murphy BR, Sotnikov AV, Lawrence LA, Banks SM, and Prince GA. 1990. Enhanced pulmonary histopathology is observed in cotton rats immunized with formalin-inactivated respiratory syncytial virus (RSV) or purified F glycoprotein and challenged with RSV 3-6 months after immunization. Vaccine8:497–502.
26.
Prince GA, Horswood RL, Camargo E, Koenig D, and Chanock RM. 1983. Mechanisms of immunity to respiratory syncytial virus in cotton rats. Infect Immun42:81–87.
27.
Connors M, Collins PL, Firestone CY, Sotnikov AV, Waitze A, Davis AR, Hung PP, Chanock RM, and Murphy BR. 1992. Cotton rats previously immunized with a chimeric RSV FG glycoprotein develop enhanced pulmonary pathology when infected with RSV, a phenomenon not encountered following immunization with vaccinia–RSV recombinants or RSV. Vaccine10:475–484.
28.
Daramola O, Stevenson J, Dean G, Hatton D, Pettman G, Holmes W, and Field R. 2014. A high-yielding CHO transient system: coexpression of genes encoding EBNA-1 and GS enhances transient protein expression. Biotechnol Prog30:132–141.
29.
Anderson RC, Fox CB, Dutill TS, Shaverdian N, Evers TL, Poshusta GR, Chesko J, Coler RN, Friede M, Reed SG, and Vedvick TS. 2010. Physicochemical characterization and biological activity of synthetic TLR4 agonist formulations. Colloids Surf B Biointerfaces75:123–132.
30.
Beeler JA and van Wyke Coelingh K. 1989. Neutralization epitopes of the F glycoprotein of respiratory syncytial virus: effect of mutation upon fusion function. J Virol63:2941–2950.
Information & Contributors
Information
Published In
Journal of Virology
Volume 91 • Number 8 • 15 April 2017
eLocator: 10.1128/jvi.02180-16
Editor: Terence S. Dermody, University of Pittsburgh School of Medicine
PubMed: 28148790
Copyright
© 2017 American Society for Microbiology. All Rights Reserved .
History
Received: 9 November 2016
Accepted: 23 January 2017
Published online: 29 March 2017
Keywords
Contributors
Editor
Terence S. Dermody
Editor
University of Pittsburgh School of Medicine
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Citation
2017.
Immunization with Low Doses of Recombinant Postfusion or Prefusion Respiratory Syncytial Virus F Primes for Vaccine-Enhanced Disease in the Cotton Rat Model Independently of the Presence of a Th1-Biasing (GLA-SE) or Th2-Biasing (Alum) Adjuvant. J Virol
91:10.1128/jvi.02180-16.
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