Long-Term and Memory Immune Responses in Mice against
Newcastle Disease Virus-Like Particles Containing Respiratory
Syncytial Virus Glycoprotein Ectodomains
Madelyn R. Schmidt, Lori W. McGinnes, Sarah A. Kenward, Kristen N. Willems, Robert T. Woodland, and Trudy G. Morrison
Department of Microbiology and Physiological Systems/Program in Immunology and Virology, University of Massachusetts Medical School, Worcester,
RSV cases and 160,000 to 199,000 deaths per year worldwide (28)
(www.who.int/vaccine_research/diseases/ari/en). In the United
States, 85,000 to 144,000 infants are hospitalized per year due to
the virus (42). In addition, RSV infection in children has been
erence 32). Elderly and immunocompromised populations are
the virus accounts for 10,000 deaths per year among individuals
greater than 64 years of age and 14,000 to 60,000 hospitalizations
per year (5, 6, 10). In immunocompromised populations, partic-
ularly stem cell transplant recipients (38) and individuals with
tality rates. Furthermore, RSV infections exacerbate chronic con-
ditions such as chronic obstructive airway disease, asthma, and
cystic fibrosis (reviewed in reference 8). Despite this significant
Complicating the management of RSV disease and RSV vac-
the same virus serotype multiple times over several years or even
within the same season (reviewed in references 8 and 35). The
subsequent infection in humans are not clear but the inadequate
than those from natural infection (36). The fact that many RSV
vaccine candidates have failed to stimulate long-term protective
uman respiratory syncytial virus (RSV) is a major cause of
acute respiratory disease in infants and young children
our lack of understanding of the immune mechanisms necessary
to generate long-term, protective anti-RSV immune responses.
Virus-like particles (VLPs) are increasingly recognized as safe,
effective vaccines for viral diseases (14). VLPs are virus-sized par-
ticles composed of arrays of structures on their surfaces and in
their cores, structures that mimic those of infectious viruses and
that may preserve, in a noninfectious particle, the very potent
immunogenicity of live viruses (14, 30). VLPs are formed by the
assembly of the structural proteins and sometimes lipids without
the incorporation of the viral genome, making VLPs incapable of
multiple rounds of infection. Two VLP vaccines are licensed for
use in humans, the papillomavirus vaccine and the hepatitis B
virus vaccine, and a number of other VLP vaccine candidates are
in preclinical testing and clinical trials (14).
We have recently described a novel VLP based on the New-
castle disease virus (NDV) core proteins and containing the RSV
fusion (F) protein and glycoprotein (G) ectodomains (23). This
(M) protein and the ectodomains of the RSV F and G proteins
fused to the transmembrane (TM) and cytoplasmic tail (CT) do-
mains of the NDV fusion (F) protein or hemagglutinin-neur-
aminidase (HN) protein, respectively. These particles stimulated,
Received 18 June 2012 Accepted 8 August 2012
Published ahead of print 15 August 2012
Address correspondence to Trudy G. Morrison, email@example.com.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
jvi.asm.orgJournal of Virology p. 11654–11662 November 2012 Volume 86 Number 21
in mice, robust protective immune responses (23). To determine
the durability of these protective responses, we characterized the
maintenance of neutralizing antibodies, as well as the presence of
anti-RSV F protein antibody secreting bone marrow-associated
cells, and RSV-specific memory B cells in mice 14 months after a
single VLP immunization or a single RSV infection. We report
that VLPs containing the RSV F and G glycoprotein ectodomains
stimulated germinal center B cells, long-lived bone marrow asso-
B cell responses, whereas RSV infection did not. Furthermore, we
report that the IgG response to VLPs is T cell dependent.
MATERIALS AND METHODS
Cells, virus, and plasmids. ELL-0 (avian fibroblasts), Vero cells, Hep2
cells, and COS-7 cells were obtained form the American Type Culture
Ennis. ELL-0 cells were maintained in Eagle minimal essential medium
in Dulbecco modified Eagle medium (DMEM) supplemented with peni-
cillin, streptomycin, and 10% fetal calf serum. Vero cells and Hep2 cells
were grown in DMEM supplemented with penicillin, streptomycin, and
5% fetal calf serum. RSV, A2 strain, was obtained from R. Finberg.
The cDNAs encoding the NDV NP and M protein have been previ-
ously described (33). The genes encoding the chimera proteins F/F and
proteins, respectively, and the sequences encoding the transmembrane
and cytoplasmic domains of the NDV F and HN proteins, respectively.
The construction and characterization of these chimera genes has been
previously described (23, 27).
VLP preparation, purification, and characterization. For prepara-
ELL-0 cells growing in T-150 flasks were transfected with cDNAs encod-
ing the NDV M protein and NP and the chimeric protein H/G or the
cDNAs encoding the chimera proteins H/G and F/F, as previously de-
a final concentration of 10 ?g/ml as previously described (23, 27) to in-
hibit rebinding of released VLPs to cells. At 48, 72, and 96 h posttransfec-
tion, cell supernatants were collected and VLPs were purified, by sequen-
tial pelleting and sucrose gradient fractionation as previously described
by silver-stained polyacrylamide gels and by Western analysis as previ-
ously described (23).
Antibodies. Polyclonal rabbit anti-NDV antibody was raised against
UV inactivated, purified NDV as previously described (24). Polyclonal
goat anti-RSV antibody (Biodesign) was used in Western blots to detect
the RSV G protein. RSV F monoclonal antibody (clone 131-2A; Chemi-
con) was used in plaque assays and immunofluorescence of fixed cells.
Anti-RSV HR2 antibody is a polyclonal antibody specific to the HR2 do-
main of the RSV F protein and has been previously described (23). Anti-
NDV F tail is a polyclonal antibody raised against the cytoplasmic tail
domain of the NDV F protein as previously described (24). Secondary
antibodies against goat, mouse, and rabbit IgGs were purchased from
analysis of murine spleen and bone marrow cells were anti-CD19 APC
(Biolegend), anti-GL7 fluorescein isothiocyanate, and anti-B220 PerCp
Preparation of RSV, RSV plaque assays, and antibody neutraliza-
27). RSV plaque assays were accomplished on Vero cells as previously
described (23, 27).
For antibody neutralization assays, mouse sera were complement in-
activated (56°C for 30 min) and then diluted in DMEM without serum.
RSV stocks were diluted to approximately 75 to 150 PFU in 100 ?l. Dilu-
tions of mouse serum in 100 ?l were added to the virus, followed by
incubation for 1 h at 37°C. The mixture was then added to prewashed,
confluent monolayers of Vero cells growing in 24-well tissue culture
dishes, and the cells were incubated at 37°C for 1 h. The antibody-virus
mixture was removed, and 1 ml of methylcellulose overlay was added to
days, and plaques were stained as previously described (23, 27). Neutral-
ization titer was defined as the log2of the reciprocal of the dilution of
serum that reduced virus titer by 60%.
Animals, animal immunization, and RSV challenge. Four-week-old
BALB/c mice from Jackson Laboratories or Taconic laboratories were
cages at the University of Massachusetts Medical Center animal quarters.
School) and TCR???/?mice (generously provided by Eva Szomolanyi-
Tsuda, UMass Medical School) were generated from breeding pairs
housed under pathogen free conditions and received acidified (HCl; pH
2.8–3.2) water containing trimethoprim-sulfamethoxazole (Goldline
All protocols requiring open cages were accomplished in biosafety
cabinets. BALB/c mice were immunized by intramuscular (i.m.) inocula-
tion of 10 to 30 ?g of total VLP protein in 0.05 ml of phosphate-buffered
saline (PBS) containing 10% sucrose. For infection of wild-type BALB/c
or rag?/?mice with RSV, the animals were lightly anesthetized with iso-
flurane and then infected by intranasal (i.n.) inoculation of RSV (7.5 ?
105to 22.5 ? 105PFU/mouse in 50 ?l). Mice challenged with live RSV
were lightly anesthetized as described above and infected i.n. with 7.5 ?
105PFU of virus.
Detection of virus in lung tissue. At 4 days after RSV challenge (26),
mice were sacrificed by CO2asphyxiation. The lungs were removed asep-
tically, placed in 0.5 ml of 30% sucrose in PBS, and frozen on dry ice. The
lungs were stored at ?80°C. Upon thawing, lungs were weighed and then
homogenized in the storage buffer using a Dounce homogenizer (Kon-
tes). The homogenate was centrifuged at 12,000 rpm for 15 min, and the
Determination of antibody titers by ELISA. For serum collection,
blood was obtained by tail vein nicks and centrifuged in BD Microtainer
serum separator tubes to remove red blood cells. For enzyme-linked im-
munosorbent assay (ELISA), extracts containing G protein target antigen
were prepared from 293T cells transfected with pCAGGS-G as previously
described (23). Dilutions of transfected cell extract used as target were
adjusted so that the amounts of G protein were comparable from experi-
ment to experiment as determined by Western blotting. The F protein
target used for ELISAs was purified recombinant F protein (generous gift
of Novavax, Inc.). Each well contained 25 ng of F protein. All antigens
were diluted in carbonate buffer (50 mM; pH 9.6). A total of 50 ?l was
added to each well of a microtiter plate (Costar) and incubated overnight
at 4°C. Wells were washed with PBS and blocked with 50 ?l of PBS con-
for 1 h at room temperature. The plates were washed three times and a
biotinylated anti-mouse IgG antibody (1:4,000 dilution; Sigma) in 50 ?l
radish peroxidase (HRP)-conjugated neutravidin (1:4,000 dilution;
Pierce) was added to 50 ?l of PBS-BSA (1:4,000 dilution) for 1 h at room
temperature prior to further washing. The HRP activity in each well was
detected using TMB (3,3=,5,5=-tetramethylbenzidene) substrate (Sigma)
at 50 ?l/well and incubated for 15 to 20 min, at times determined in
preliminary experiments to be within the linear range of the assay. The
reaction was stopped with 50 ?l of 1 N H2SO4, and the optical density at
450 nm (OD450) was read in a plate reader (Molecular Devices). Alterna-
tively, alkaline phosphatase coupled to streptavidin was utilized to detect
bound antibody using as a substrate 4-nitrophenyl phosphate disodium
salt hexahydrate at 1 mg/ml in 1 M diethanolamine and 0.5 ?M MgCl2.
Long-Term Immune Responses to RSV Vaccine Candidate
November 2012 Volume 86 Number 21 jvi.asm.org 11655
The OD450was determined. As previously described, the titers of anti-G
protein antibodies were defined as the reciprocal of the serum dilution
that gave an OD450of 3-fold over background (23). Titers for anti-F pro-
tein antibodies were defined as the reciprocal of the serum dilution that
gave an OD450of 0.2 since background values for this target antigen were
row cells. Splenocytes prepared from disrupted spleens were filtered
through Nytex mesh (100- to 120-?m pore size) washed in balanced salt
cells were lysed in Gey’s solution (Sigma). Washed cells, resuspended in
BSS, were counted and resuspended at a concentration of 1 ? 107to 2 ?
107live cells/ml in complete medium (RPMI) containing 10% serum,
penicillin-streptomycin, glutamine, and ?-mercaptoethanol (5 ? 10?5
For ELISpot assays, ELISpot plates (Millipore) were coated overnight
in water, drained, and incubated for 1 h in compete medium (RPMI
containing 10% serum, pen-strep, glutamine and BME at 5 ? 10?5M).
Fourfold serial dilutions of spleen or bone marrow cells were added in
triplicate to precoated wells, followed by incubation at 37°C for 6 h. The
plates were washed eight times and blocked overnight in PBS containing
followed by eight water washes. The wells were then incubated for 1 h at
room temperature with streptavidin-AP (Southern; 1:4,000 dilution) di-
luted in PBS-BSA, washed, and then developed with BCIP/NBT (5-bromo-
4-chloro-3-indolylphosphate/nitroblue tetrazolium; 1 tablet/10 ml of
autoclaved Millipore water) until purple spots appeared. The spots were
counted using a CTL immunospot S5 analyzer.
Purification of splenic B cells and adoptive transfer. Splenocytes
from disrupted spleens were washed, and red blood cells were lysed with
and incubated on ice for 45 min. The cells were then washed and treated
with rabbit complement (Pel-Freeze H2) for 45 min at 37°C (39). FACS
analysis using anti-CD19 demonstrated that B cells were ca. 90% pure.
Adoptive transfer of purified B cells to rag?/?mice (1.2 ? 107cells/
mouse) was accomplished by periorbital inoculation.
Detection of germinal center B cells. Lymph nodes (LN; deep cervi-
ment. The cell suspensions were made, the RBCs were lysed, and FACS
staining was performed to assess the presence of GL7?germinal center B
FlowJo (Treestar) software.
accomplished using GraphPad Prism 5 software.
Long-term antibody responses to VLP-H/G?F/F and RSV. To
ies to the RSV proteins, the anti-F and anti-G protein antibody
titers were measured, by ELISA, periodically over 430 days after a
single immunization and compared to mice infected intranasally
protein ectodomains (VLP-H/G?F/F) were purified and charac-
terized as previously described (23). Groups of five mice were
injected, intramuscularly (i.m.) to mimic vaccination, with either
10 or 30 ?g of total VLP/mouse, whereas another group of mice
was infected i.n. with RSV to mimic natural infection. Negative
control groups received buffer–10% sucrose i.m. Sera were col-
1A and B, respectively. VLP immunization resulted in robust
anti-F and anti-G protein IgG antibody titers that remained rela-
tively constant for 430 days. Similarly, serum anti-F and anti-G
protein anti-IgG antibody titers after i.n. infection with RSV re-
mained relatively constant over the course of the experiment, al-
nization. These results are consistent with the VLP stimulation of
long-lived antibody responses in the absence of adjuvants at both
doses of VLP tested.
Long-term neutralizing antibody responses to VLP-H/
F?F/F and infectious RSV. In humans, whereas RSV infection
may result in detectable antibody responses that persist for some
time, protective responses diminish rapidly, resulting in suscepti-
bility to subsequent infection in many individuals (reviewed in
references 8 and 35). To assess the longevity of neutralizing anti-
body responses in the murine system, the neutralization titers of
FIG 1 Titers of serum anti-F and anti-G protein antibodies with time after immunization or infection. Groups of five BALB/c mice were immunized i.m. with
VLP-H/G?F/F. One group received 10 ?g of total VLP/mouse (0.7 ?g of F protein and 0.8 ?g of G protein) or 30 ?g of total VLP protein/mouse (2.1 ?g of F
protein and 2.4 ?g of G protein). Antibody titers in sera, with time after immunization, were measured as described in Materials and Methods. Another group
titers; (B) anti-G protein antibody titers.
Schmidt et al.
jvi.asm.orgJournal of Virology
were determined. Figure 2 shows that the neutralizing antibody
titers after VLP immunization were maximal by 60 to 100 days
tralizing antibodies levels failed to reach those seen with VLP im-
munization and declined markedly by 100 days, despite the fact
that total anti-F and G protein IgG antibody titers were relatively
stable after RSV infection. These results suggest that VLP immu-
nization produced an antibody response that is different in char-
acter than that induced by RSV infection.
we challenged mice infected with RSV or immunized with VLPs
14 months previously. Mice immunized with a low dose of VLPs
controls were infected with RSV i.n. After 4 days, the mice were
sacrificed, and virus titers in the lungs are determined (Fig. 3A).
Clearly, even at the low concentration of VLPs used in this exper-
iment, there was still significant protection from RSV replication
after 14 months. In contrast, mice infected with RSV 14 months
previously showed no evidence of protection from RSV replica-
tion relative to the PBS controls upon virus challenge. These re-
from mice immunized with VLPs had significant neutralization
not. We have previously reported that mice challenged with RSV
38 days (27) or 52 days (23) after a single i.n. RSV infection were
completely protected. Thus, protective responses after RSV infec-
tion are transient.
mice. The presence of significant RSV-specific antibody levels 14
months after immunization with VLPs was consistent with the
generation of long-lived, antibody-secreting plasma cells, cells
that reside primarily in the bone marrow. To determine the fre-
row-associated cells in these mice, the bone marrows of the RSV-
challenged mice characterized in Fig. 3A were harvested, and the
numbers of anti-F protein antibody-secreting cells (ASC) were
nized mice had significant numbers of these cells relative to mice
infected with RSV or unimmunized controls. These results sug-
gest that VLPs containing the RSV F and G protein ectodomains
stimulated long-lived anti-RSV F protein ASC, whereas a single
RSV infection did not.
Assessment of memory responses to VLPs. To evaluate the
generation of memory B cells specific for RSV glycoproteins after
VLP immunization, we performed an adoptive transfer experi-
FIG 2 Serum neutralization titers with time after immunization or infection.
Sera from mice immunized with 30 ?g of VLP, described in the legend to Fig.
1, were pooled and neutralization titers were determined as described in Ma-
terials and Methods. Each point is the average of three separate plaque assays,
each performed in duplicate. The standard deviations are shown.
with 10 ?g of total VLP-H/G?F/F protein/mouse were challenged with RSV (7.5 ? 105PFU/mouse). Five mice previously infected with RSV (2.25 ? 106
PFU/mouse) were similarly challenged with RSV. (A) Four days after challenge, the lungs were removed, and the virus titers were determined as described in
Materials and Methods. Virus titers (per g of lung tissue) in each lobe of the lungs of VLP-immunized mice were determined separately, while titers in one
randomly selected lobe of the PBS- and RSV-immunized mice were determined. Means and standard deviations are shown. Bars indicate groups compared for
statistical analysis using GraphPad Prism software (t tests), with P values shown above the bar. The differences between control (PBS) and RSV-infected mice
immunized with PBS were harvested and prepared for ELISpot analysis as described in Materials and Methods. The panel shows the number of cells secreting
anti-F protein antibodies per 106bone marrow cells. The means and standard deviations are shown. The bar at the top indicates the groups compared for
statistical analysis using GraphPad Prism software (t tests), with the P value shown above the bar. The differences between PBS and RSV groups were not
Long-Term Immune Responses to RSV Vaccine Candidate
November 2012 Volume 86 Number 21jvi.asm.org 11657
ment. Groups of mice immunized with either 30 ?g of VLP/
were injected into syngeneic immunodeficient, BALB/c rag?/?
mice. After 6 days, the mice were challenged with RSV by i.n.
inoculation. At 4 days after challenge, the mice were sacrificed,
and sera, spleens, and lungs were harvested for ELISA, ELISpot,
and virus titer analysis, respectively.
Figure 4A and B show the titers of anti-F protein and anti-G
protein IgG antibody, respectively, in the sera of recipient mice.
Sera from mice that received B cells from VLP-immunized mice
whereas mice that received B cells from RSV-infected mice or
F or G proteins.
RSV-specific anti-F protein immunoglobulin-secreting B cells
in the spleens of recipient mice were readily detected by ELISpot
(Fig. 4C). However, mice that received B cells from RSV-infected
mice were devoid of immunoglobulin-secreting cells, as were the
To determine whether the transferred B cells could provide
ient mice with RSV. Figure 4D shows that RSV replication was
significantly reduced in mice that received B cells from VLP-im-
munized donors, while mice that received B cells from RSV-in-
fected mice were not protected. The titers of RSV in the lungs of
received B cells from control vaccinated mice. These combined
results are consistent with the generation in donor mice of mem-
ory B cells specific for RSV F protein by VLP immunization. In
430 days after RSV infection, a result that is consistent with the
lack of long-term protection from challenge in these mice.
It should be noted that the titers of RSV in wild-type PBS con-
similar. This unexpected result may indicate that the well-known
partial restriction of RSV replication in BALB/c mice may not be
due, entirely, to the murine adaptive immune responses to RSV.
T cell dependence of anti-G and anti-F protein responses to
VLPs. The development of long-lived, antibody-secreting plasma
cells and memory B cells is classically characteristic of T-cell-de-
pendent (TD) immune stimulation (discussed in reference 19),
responses can produce B cell memory (31; reviewed in references
2 and 43). To determine whether VLPs require T cells to produce
anti-RSV F protein IgG immune responses, a group of T-cell-
deficient C57BL/6-TCR?? knockout mice and a control group of
C57BL/6 wild-type mice were immunized with a single dose of
FIG 4 Properties of B cell recipient mice. B cells from groups of five mice immunized with 30 ?g of total VLP-H/G?F/F protein/mouse, infected with RSV
(2.25 ? 106PFU/mouse), or sham immunized with PBS were harvested and purified 430 days after immunization as described in Materials and Methods.
B cells in each mouse in each group with the means and standard deviations indicated. The bar at the top of the graph indicates groups compared for statistical
analysis using GraphPad Prism software (Student t test), with P values shown above the bar. Differences between the PBS and RSV groups were not significant.
(D) Titers of RSV in the lungs of RSV challenged B cell recipient mice were determined as described in Materials and Methods, with means and standard
deviations indicated. Bars indicate groups compared for statistical analysis using GraphPad Prism software (Student t test), with P values shown above the bar.
Schmidt et al.
jvi.asm.orgJournal of Virology
VLP-H/G?F/F. Another group of mice received buffer immuni-
zation. Anti-F protein IgG antibody responses at 21 and 29 days
after immunization were measured by ELISA. Figure 5A and B
show that while wild-type mice developed robust anti-F protein
antibody responses, no anti-F protein IgG antibody was detected
in the T-cell-deficient mice, even at low serum dilutions. Simi-
larly, to determine whether VLPs require T cells to produce anti-
RSV G protein IgG antibody responses, a group of C57BL/6-
mice were immunized with a single dose of VLP-H/G, a VLP that
contains only the ectodomain of the RSV G protein (27). Figure
5C and D show that no anti-G protein IgG antibody was detected
in mutant mouse sera, whereas sera from wild-type mice con-
tained anti-G protein IgG antibody. These results are consistent
with VLP-H/G?F/F or VLP-H/G acting as TD antigens to elicit
anti-F protein and anti-G protein IgG responses.
uration and the production of long-lived plasma cells and mem-
ory B cells during TD responses is the induction of the germinal
center reaction (GC) in spleens and lymph nodes. To assess
whether VLP immunization stimulated GC formation, cells were
immunized mice at 7 days postimmunization and analyzed by
flow cytometry for the formation of germinal center B cells
the average numbers of CD19?cells/106lymph node (LN) cells.
Although VLP immunization resulted in slightly higher levels of
CD19?LN cells, the levels were not statistically significantly dif-
ferent from those observed after PBS or RSV immunization. Fig-
ure 6C shows the average numbers of GL7?cells/106CD19?cells
in LN of these mice. The induction of GC B cells after RSV infec-
tion has been previously reported (see, for example, reference 3).
Clearly, VLP-H/G?F/F stimulated the induction of GC B cells
more effectively than did RSV infection. This result supports the
data, shown in Fig. 4 and 5, that VLPs stimulate a T-cell-depen-
dent germinal center reaction necessary for the production of
RSV-specific long-lived ASC and memory B cells.
A major problem in formulating a successful vaccine for RSV is
that natural infection in humans does not result in complete pro-
tection from subsequent infection. Immune responses are weak
and short-lived and ineffective against repeated infections with
the same strain of virus, even in the same season (8, 9, 35). Fur-
thermore, high titers of serum antibody can fail to protect a con-
siderable proportion of both adults and infants. These observa-
tions led to the comment by Pulendran and Ahmed (36) that an
better immune responses than natural infection. Development of
FIG 5 T cell dependence of anti-G and anti-F protein responses to VLPs. (A and B) C57BL/6 or C57BL/6-TCR???/?mice (five and eight mice/group,
respectively) were immunized (i.m.) with 30 ?g of total VLP-H/G?F/F protein. Five C57BL/6 mice immunized with PBS served as negative controls. At 21 (A)
and 29 (B) days after immunization, serum anti-F antibodies were measured by ELISA using neutravidin-coupled HRP. The OD450at different serum dilutions
immunized (i.m.) with 30 ?g of total VLP-H/G protein. Five C57BL/6 mice immunized with PBS served as negative controls. At 20 (C) and 30 (D) days after
immunization, sera from mice in each group were pooled, and anti-G protein antibodies in pooled sera were measured in triplicate by ELISA using alkaline
the standard deviations indicated.
Long-Term Immune Responses to RSV Vaccine Candidate
November 2012 Volume 86 Number 21 jvi.asm.org 11659
such a vaccine has thus far been unsuccessful since many clinical
trials of vaccine candidates show modest immunogenicity and
Vaccine development is further complicated by the finding
that RSV infection is reported to produce long-lasting protective
responses in rodents, the preferred preclinical model, suggesting
ever, in mice, induction of protective responses requires multiple
infections and a single infection has been shown inadequate for
long-lasting protective responses (see, for example, reference 44),
a finding more in line with observations of the course of infection
We have chosen to analyze, in mice, the longevity of responses
to a single immunization with our RSV VLP vaccine candidate
and to compare its relative effectiveness to a single infection with
prolonged serum anti-F and anti-G protein antibody responses,
ciated anti-F protein antibody-secreting cells nor B cell memory
responses, all contributing factors in making protection short-
lived. In striking contrast, a single VLP immunization resulted in
VLPs stimulate more effective protective immunity than does
RSV infection, a critical criterion for an effective RSV vaccine.
Our results showed that these long-term responses correlated
with the presence of long-lived anti-F protein antibody secreting
cells in bone marrow and memory B cells. B cell memory and
long-lived high-affinity antibody responses to conventional pro-
tein antigens arise when antigen-specific B cells proliferate and
differentiate during the germinal center (GC) reaction (reviewed
in reference 25). Within the GC, B cells with somatically mutated
helper cells (TFH). To assess the T cell requirement for VLP im-
munogenicity, we sought to determine whether antibody re-
sponses could be induced in mice that were deficient in T cells
not stimulate any anti-F or G protein IgG antibodies in T-cell-
deficient mice, indicating that the F and G chimera proteins pre-
sented by these VLPs behave as classic TD antigens. Further sup-
port for the T cell dependence of B cell responses to our VLPs is
nodes 7 days after VLP immunization at levels significantly in-
creased over those detected after buffer immunization or RSV
Our results also show that protection against RSV challenge
could be effected with memory B cells, induced as a result of VLP
immunization and adoptively transferred into BALB/c rag?/?
mice before RSV infection. Comparable numbers of B cells pre-
FIG 6 Stimulation of germinal center GL7?B cells. Groups of five mice were immunized with VLP-H/G?F/F (i.m.), RSV (i.n.), or PBS. At 7 days after
immunization, caudal, axillary, inguinal, deep cervical, mediastinal, and brachial lymph nodes were harvested and combined. B-cell-enriched cell suspensions,
prepared as described in Materials and Methods, were incubated with anti-CD19 and anti-GL7 antibodies and analyzed by flow cytometry. (A) CD19?, GL7?
106CD19?cells in each group. Means with standard deviations are shown. The bars at the top indicate groups compared for statistical analysis using GraphPad
Prism software (Student t test), with P values shown above the bar.
Schmidt et al.
jvi.asm.org Journal of Virology
pared from RSV-infected or PBS injected donors did not confer
protection. The memory B cells from VLP-primed mice were ac-
tivated in the presence of limited numbers of T cells. Studies with
model hapten-protein conjugates have demonstrated that T cells
are required for the differentiation of memory B cells into IgG
secreting plasma cells (see, for example, references 4 and 45).
However, it has been reported that virus-specific memory B cells
virus infection can be activated in the absence of cognate or by-
stander T cells upon a second exposure to viral antigen (12, 17,
47). Similarly, a complex of ovalbumin and alum, which forms a
memory B cells (21). These results led MacLeod et al. to suggest
that requirements for T cells in activation of memory B cells vary
responses to particulate antigens may be T cell independent (22).
Our results suggest that VLPs containing RSV F and G proteins
in the presence of minimal levels of T cells. In contrast, infectious
RSV, also a particulate antigen, did not generate memory B cells
with similar properties. The reasons for differences in immune
future investigation. The differences may be related to differences
in the routes of immunization. Alternatively, virus infection may
suppress innate immune responses important for long-term im-
mune memory development.
Stimulation of innate immunity is necessary for the induction
of durable B cell and T cell responses (reviewed in references 1, 7,
20, 34, and 41). Furthermore, signal pathways induced by the
stimulation of multiple Toll-like receptors (TLRs) synergize to
enhance adaptive immunity (16, 29, 37), as well as determine the
of innate immunity are often included in vaccine formulations to
enhance adaptive immune responses. Our data suggest that VLP-
H/G?F/F must be self-adjuvanting since this VLP stimulated
long-term immunity in the absence of added adjuvant. It is not
known how VLP-H/G?F/F stimulates the innate immune re-
sponses important for the long-term adaptive responses we have
observed, although it may be speculated that TLR4 and TLR7 or
TLR3 are involved. RSV F protein has been reported to activate
TLR4 (11, 18) and VLP-H/G?F/F may stimulate through TLR3,
TLR7, or TLR9, which recognize various forms of nucleic acid. It
has been reported that parainfluenza virus 5 (PIV5) VLPs assem-
via TLR pathways that respond to single-stranded RNA. It has
have found that the best stimulation of long-term antibody re-
sponses to antigen-associated nanoparticles requires both TLR4
and TLR7 ligands (16).
proteins stimulate, in murine systems, robust, long-lived neutral-
izing antibodies, anti-RSV antibody-secreting bone marrow cells,
and RSV-specific memory B cells. These responses are in contrast
to those observed after a single RSV infection, which stimulated
transient neutralizing antibodies, no detectable antibody-secret-
ing bone marrow cells, and no memory B cells.
This study was supported by grants from the National Institute of Allergy
and Infectious diseases of the National Institutes of Health (AI093791
[T.G.M.] and AI041054 and AI084800 [R.T.W. and M.R.S.]). Core re-
sources supported by the Diabetes Research Center grant DK32520 were
We thank Rachel Gerstein with help with flow cytometry analysis and
Ann Rothstein and Eva Szomolanyi-Tsuda for mutant mice.
1. Bessa J, Kopf M, Bachmann MF. 2010. Cutting edge: IL-21 and TLR
2. Defrance T, Taillardet M, Genestier L. 2011. T cell-independent B cell
memory. Curr. Opin. Immunol. 23:330–336.
Toll-like receptor stimulation leads to enhanced respiratory syncytial vi-
rus disease. Nat. Med. 15:34–41.
4. Duffy D, Yang C-P, Heath A, Garside P, Bell EB. 2006. Naive T-cell
5. Falsey AR, Hennessey PA, Formica MA, Cox C, Walsh EE. 2005.
Respiratory syncytial virus infection in elderly and high-risk adults. N.
Engl. J. Med. 352:1749–1759.
6. Falsey AR, Walsh EE. 2000. Respiratory syncytial virus infection in
adults. Clin. Microbiol. Rev. 13:371–384.
7. Guay HM, Andreyeva TA, Garcea RL, Welsh RM, Szomolanyi-Tsuda E.
2007. MyD88 is required for the formation of long-term humoral immu-
nity to virus infection. J. Immunol. 178:5124–5131.
8. Hall CB. 2001. Respiratory syncytial virus and parainfluenza virus. N.
Engl. J. Med. 344:1917–1928.
9. Hall CB, Long CE, Schnabel KD. 2001. Respiratory syncytial virus infec-
tions in previously healthy working adults. Clin. Infect. Dis. 33:792–796.
10. Han LL, Alexander JP, Anderson LJ. 1999. Respiratory syncytial virus
pneumonia among the elderly: an assessment of disease burden. J. Infect.
11. Haynes LM, et al. 2001. Involvement of Toll-like receptor 4 in innate
immunity to respiratory syncytial virus. J. Virol. 75:10730–10737.
12. Hebeis BJ, et al. 2004. Activation of virus-specific memory B cells in the
absence of T cell help. J. Exp. Med. 199:593–602.
13. Iwasaki A, Medzhitov R. 2004. Toll-like receptor control of adaptive
immune responses. Nat. Immunol. 5:987–995.
14. Jennings GT, Bachmann MF. 2008. The coming of age of virus-like
particle vaccines. Biol. Chem. 389:521–536.
15. Karron RA. 2008. Respiratory syncytial virus and parainfluenza virus
vaccines, 5th ed. Saunders-Elsevier, Philadelphia, PA.
16. Kasturi SP, et al. 2011. Programming the magnitude and persistence of
antibody responses with innate immunity. Nature 470:543–547.
17. Klenovsek K, et al. 2007. Protection from CMV infection in immunode-
mediate response to respiratory syncytial virus. Nat. Immunol. 1:398–
19. Lanzavecchia A, Sallusto F. 2009. Human B cell memory. Curr. Opin.
20. Lanzavecchia A, Sallusto F. 2007. Toll-like receptors and innate immu-
nity in B-cell activation and antibody responses. Curr. Opin. Immunol.
21. Leclerc C, et al. 1995. Stimulation of a memory B cell response does not
require primed helper T cells. Eur. J. Immunol. 25:2533–2538.
22. MacLeod MKL, Clambey ET, Kappler JW, Marrack P. 2009. CD4
memory T cells: what are they and what can they do? Semin. Immunol.
23. McGinnes LW, et al. 2011. Assembly and immunological properties of
Newcastle disease virus-like particles containing the respiratory syncytial
virus F and G proteins. J. Virol. 85:366–377.
24. McGinnes LW, Reitter JN, Gravel K, Morrison TG. 2003. Evidence for
mixed membrane topology of the Newcastle disease virus fusion protein.
J. Virol. 77:1951–1963.
25. McHeyzer-Williams M, Okitsu S, Wang N, McHeyzer-Williams L.
Long-Term Immune Responses to RSV Vaccine Candidate
November 2012 Volume 86 Number 21jvi.asm.org 11661
2011. Molecular programming of B cell memory. Nat. Rev. Immunol.
26. Murawski MR, et al. 2009. Respiratory syncytial virus activates innate
immunity through Toll-like receptor 2. J. Virol. 83:1492–1500.
27. Murawski MR, et al. 2010. Newcastle disease virus-like particles contain-
ing respiratory syncytial virus G protein induced protection in BALB/c
mice with no evidence of immunopathology. J. Virol. 84:1110–1123.
28. Nair H, et al. 2010. Global burden of acute lower respiratory infections
due to respiratory syncytial virus in young children: a systematic review
and meta-analysis. Lancet 375:1545–1555.
29. Napolitani G, Rinaldi A, Bertoni F, Sallusto F, Lanzavecchia A. 2005. Selected
Toll-like receptor agonist combinations synergistically trigger a T helper type
30. Noad R, Roy P. 2003. Virus-like particles as immunogens. Trends Mi-
31. Obukhanych TV, Nussenzweig MC. 2006. T-independent type II im-
mune responses generate memory B cells. J. Exp. Med. 203:305–310.
32. Openshaw PJ, Tregoning JS. 2005. Immune responses and disease en-
hancement during respiratory syncytial virus infection. Clin. Microbiol.
33. Pantua HD, McGinnes LW, Peeples ME, Morrison TG. 2006. Require-
ments for the assembly and release of Newcastle disease virus-like parti-
cles. J. Virol. 80:11062–11073.
34. Pasare C, Medzhitov R. 2005. Control of B-cell responses by Toll-like
receptors. Nature 438:364–368.
35. Power UF. 2008. Respiratory syncytial virus (RSV) vaccines: two steps
back for one leap forward. J. Clin. Virol. 41:38–44.
36. Pulendran B, Ahmed R. 2011. Immunological mechanisms of vaccina-
tion. Nat. Immunol. 12:509–517.
37. Querec T, et al. 2006. Yellow fever vaccine YF-17D activates multiple
dendritic cell subsets via TLR2, 7, 8, and 9 to stimulate polyvalent immu-
nity. J. Exp. Med. 203:413–424.
38. Raboni SM, et al. 2003. Respiratory tract viral infections in bone marrow
transplant patients. Transplant 76:142–146.
39. Schmidt MR, et al. 2008. Human BLyS facilitates engraftment of human
PBL derived B cells in immunodeficient mice. PLoS One 3:e3192. doi:
40. Schmitt AP, Leser GP, Waning DL, Lamb RA. 2002. Requirements for
budding of paramyxovirus simian virus 5 virus-like particles. J. Virol.
T cell responses. Eur. J. Immunol. 33:1465–1470.
42. Shay DK, et al. 1999. Bronchiolitis-associated hospitalizations among US
children, 1980–1996. JAMA 282:1440–1446.
43. Shlomchik MJ, Weisel F. 2012. Germinal centers. Immunol. Rev. 247:
44. Singleton R, Etchart N, Hou S, Hyland L. 2003. Inability to evoke a
long-lasting protective immune response to respiratory syncytial virus
infection in mice correlates with ineffective nasal antibody responses. J.
45. Vieira P, Rajewsky K. 1990. Persistence of memory B cells in mice de-
prived of T cell help. Int. Immunol. 2:487–494.
46. Walsh EE, Falsey AR, Hennessey PA. 1999. Respiratory syncytial and
other virus infections in persons with chronic cardiopulmonary disease.
Am. J. Respir. Crit. Care Med. 160:791–795.
47. Weisel FJ, et al. 2010. Unique requirements for reactivation of virus-
specific memory B lymphocytes. J. Immunol. 185:4011–4021.
Schmidt et al.
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