Following are our point-by-point responses to Reviewer’s comments:
Author’s response to reviews
Title: An Adjuvanted Inactivated Murine Cytomegalovirus (MCMV) Vaccine Induces
Potent and Long-term Protective Immunities against a Lethal Challenge with a Virulent
MCMV
Authors:
Huadong Wang (wanghd116@hotmail.com)
Yanfeng Yao (yaoyanfeng1981@yahoo.com.cn)
Chaoyang Huang (cyhuang1984@hotmail.com)
Xingxing Fu (fuxingxing1@yahoo.com.cn)
Quanjiao Chen (zuochqj@126.com)
Hongbo Zhang (hongboflood@163.com)
Jianjun Chen (chenjj126@126.com)
Fang Fang (fangfangc@263.net)
zhenyuan Xie (zhen1212@sina.com)
Ze Chen (chenze2005@263.net, chenze2005@hotmail.com)
Reviewer’s report (Second time)
Title: An Adjuvanted Inactivated Murine Cytomegalovirus (MCMV) Vaccine Induces
Potent and Long-term Protective Immunities against a Lethal Challenge with a Virulent
MCMV
Reviewer #1: Professor Luka Cicin-Sain
Version: 3 Date: 20 November 2012
Response to referee’s comments 1
We thank the reviewers for their careful review and constructive comments. We
have made extensive modifications of the manuscript according to the suggestions.
Reviewer’s report:
I have read the revised version of the manuscript. The authors of this manuscript
have addressed adequately my major compulsory revisions 1, 3, and 5.
1. There appeared to be a misunderstanding about my question 4. The authors pasted
their reply to their answer 2 of the reviewer #2, but did not address my concern about
the non-specific induction of the immune system by their vaccine formulation. While
the authors provide a detailed description of the purification conditions used to
generate their antigen, they failed to recognize that their control groups have still not
received any adjuvant in isolation. Therefore, it is not clear if the adjuvant alone
would have resulted in protection against MCMV challenge, or if this effect was due
to the increase in the induction of B-cell responses, as measured by ELISA. While I
have not requested that the authors provide a formal proof of serum-mediated
protection by adoptive transfer experiments, it is necessary that the authors show at
least that immune protection is not enhanced in control groups with adjuvant-alone
treatment.
Thanks for the reviewer’s comment and suggestion. We understand reviewer’s
concerning about the non-specific induction of the immune system by vaccine
formulation and adjuvant alone would have resulted in protection against MCMV
challenge. We agreed with reviewer that it is necessary to show that immune
protection is not enhanced in control groups with adjuvant-alone treatment. We have
conducted the suggested control experiments about adjuvant-alone treatment and mock
vaccine. The results from adjuvant-alone treatment groups showed that administration
with MF59, alum, chitosan alone intraperitoneally or intramuscularly couldn’t provide
mice specific protection against lethal viral infection and anti-MCMV IgG was also not
detected in three adjuvants alone treated mice (table 1, 4). The mice immunized with
mock vaccine were not protected from lethal MCMV challenge and no anti-MCMV
IgG was detected in these mice (table 1, 4). So the adjuvant-alone treatment or mock
vaccine immunization neither induce specific anti-MCMV humoral immune response
nor provide mice any protection against MCMV challenge.
2. The major omission in the new manuscript version is the lack of control
experiments as required in my comment 2. Arguing that intraperitoneal immunization
of mice is a common practice by referring to 10+ years old literature (which they
failed to cite in the primary submission), is not a valid rationale to decline to perform
vaccinations by a route that would mimic real-life settings, approximating clinical or
veterinary medicine standards. Since the major advance of this article is the
comparison of various adjuvants, and adjuvants may have effects which could vary by
their site of application, it is imperative that the effect of the adjuvants on immune
response as well as its toxicity are evaluated by an immunization route that may be
used in clinical trials. Therefore, I am, unfortunately, still forced to advise against the
publication of this article, unless the suggested control experiments have been
performed.
Thanks for the reviewer’s constructive suggestions. We agree with reviewer that
the major advance of this article is the comparison of various adjuvants, and adjuvants
may have effects which could vary by their site of application, it is imperative that the
effect of the adjuvants on immune response as well as its toxicity are evaluated by an
immunization route that may be used in clinical trials. We took the suggestions of
administering the vaccine in different immunization route very seriously and performed
a series of experiments to learn whether FI-MCMV vaccine administrated by
intramuscular injection could provide similar protection as i.p. immunization route.
The mice were immunized twice with 4 µg, 1µg and 0.25µg of the vaccine with or
without an adjuvant by intramuscular route. The results from i.m. immunization
experiments agreed with those from analysis by intraperitoneal immunization, whereas
the overall protection effect of i.m. immunization was slightly inferior relative to i.p.
route. Co-administration the FI-MCMV vaccine with adjuvant could significantly
enhance the immune response in mice than the adjuvant-free vaccine and MF59 was
more potent in immune enhancement relative to alum and chitosan. The corresponding
results were presented in table 4 (page 30) of the revised manuscript.
Level of interest: An article of limited interest
Quality of written English: Acceptable
Statistical review: No, the manuscript does not need to be seen by a statistician.
Declaration of competing interests: I declare that I have no competing interests.
Reviewer’s report (First time)
Title: An Adjuvanted Inactivated Murine Cytomegalovirus (MCMV) Vaccine Induces
Potent and Long-term Protective Immunities against a Lethal Challenge with a Virulent
MCMV
Reviewer # 1: Professor Luka Cicin-Sain
Version: 2 Date: 6 August 2012
Response to referee’s comments 1
We thank the reviewer for a very careful review and very thoughtful
comments. Following are our point by point response to the reviewer’s comments:
The major compulsory revisions are:
1. The authors do a poor job in explaining the state of the art in experimental
inactivated MCMV vaccines, and to place their advances in context. In fact, a
protective effect of an immunization with a formalin-inactivated MCMV was shown
in mice several times before. The authors do not refer to the seminal work by Tolpin
et al. (Tolpin et al., J Infect Dis 1980, 142(4): 569-574) and the subsequent work by
Morello et al. (Morello et al., J Virol 2002, 76 (10): 4822-4835) and Geoffroy et al.
(Geoffroy et al., Vaccine 1996, 14(17-18): 1686-1694) neither in the introduction, nor
in the discussion.
In fact, Tolpin et al showed in 1980 that administration of two doses of
formalin-inactivated MCMV reduced the mortality after challenge with a lethal
SG-MCMV, and Morello showed that FI-MCMV and alum leads to higher antibody
titers and decreased viral titer in spleen and salivary glands after lethal challenge in
comparison to a vaccination without adjuvant. While these articles are referred in the
material and method section, the citations are out of context, and do not allow the
reader to appreciate which part of the current work is a repetition of previously
published data.
Thanks for the reviewer’s comment. The research on HCMV vaccine has been
carried out for 30 years, but up to now no vaccine was approved for clinical use. As
the reviewer mentioned, there were already several studies about the performance of
inactivated MCMV vaccine in the animal experiments. As early as in 1980, Tolpin et
al. reported that twice immunizations of mice with inactivated MCMV vaccine
(immunizing dose around 107.7 pfu) could provide a protection rate of 89% against a
low-level virus challenge (2 × LD50). However, most mice still developed mild or
subclinical infections after challenge. In 1996, Geoffroy et al. inactivated MCMV
with sodium periodate or β-propiolactone. They evaluated the immunogenicity of
vaccine inactivated by these two inactivating agents by immunizing mice three times
(immunizing dose around 107 pfu) and a following 5 × LD50 challenge. Although the
protective rate of PI- or βI-MCMV reached 100%, the researchers discovered that the
long-lasting protection offered was incomplete. In 2002, Morello et al. adopted an
immunization strategy by combining pDNA pool and FI-MCMV (mixed with alum),
in which DNA immunization was performed 3 times and FI-MCMV immunization
twice. Their results demonstrated that this immunization strategy could provide
effective protection. However, they only performed a sub-lethal challenge (< 1 ×
LD50). We have added these relevant information in the manuscript.
From the above studies, we can see that the protection efficiency and efficacy
of inactivated MCMV vaccine need further improvement. Adjuvant is non-specific
immune-enhancing substance, which is a main focus of vaccine development. We
designed our study to examine the protective effect of vaccine with adjuvant. In this
study, our immunization strategy in combination with adjuvant effectively increased
the efficacy of inactivated MCMV vaccine. Furthermore, we compared the protective
immunity of three adjuvants (MF59, alum, and chitosan) and discussed the long-term
protection of this vaccine.
2. The major novelty of the manuscript is the comparison of the protective capacity of
various adjuvants. Therefore, the relevant information would be obtained by
administering the vaccine in a format that is common in vaccine asministration, (i.e.
subcutaneously, intramuscularly or intranasally). Unfortunately, the vaccine
formulation was exclusively applied intraperitoneally, a route of immunization that is
unlikely to be performed in clinical settings, and hence it is not possible to predict if
MF59 would outperform the other adjuvants when applied locally.
Thanks for the reviewer’s suggestion. In the mouse experiment for evaluating
the protective effect of inactivated vaccine, intraperitoneal injection is a common
route for vaccination. In addition, intraperitoneal injection was adopted in all the
literatures mentioned by the reviewer in the first comment. We agree with the
reviewer that it is interesting to compare different immunization routes, and we will
carry related research in future study.
3. The authors claim that the adjuvant they used is safe. However, no information is
provided about the monitoring of side-effects during immunizations. Hence it is not
clear if the intraperitoneal application of the vaccine formulation induced fever,
weight loss, or other symptoms typical of acute inflammation. Moreover, the method
used makes it impossible to monitor for local inflammatory side effects at the site of
immunization (see previous point).
Thanks for the reviewer’s comment. Based on the advice, we have changed
the conclusion in the beginning of the manuscript from “These results suggest that
inactivated CMV vaccine is safe and effective. In addition, the adjuvanted CMV
vaccine provided more effective and longer-term protection than the adjuvant-free
vaccine.” to “These results show that inactivated CMV vaccine is effective, and the
adjuvanted CMV vaccine provides more effective and longer-term protection than the
adjuvant-free vaccine.” Actually, the safety of the adjuvant was observed by the
symptoms in mice. In this study, normal gait, typical posture and behavior were
observed in all study groups. Significant abnormal signs, such as piloerection, body
weight loss, hypoactivity, and ascites, were not observed in immunized mice during
the study. Moreover, in the long-term immunogenicity study, there were no abnormal
signs or reactions observed in groups inoculated with the vaccine with or without
adjuvant at month 6 after the vaccination, which further demonstrated the safety of the
vaccine and adjuvant.
4. The authors claim that the adjuvanted CMV vaccine provides more effective
protection than the adjuvant-free vaccine, but they omitted controls with
formalin-treated lysates of mock-infected NIH-3T3 accompanied by adjuvants.
Therefore, it is not clear if the improved immune protection resulted as a boost of the
Ag-specific immune response, or is a result of nonspecific immunity provided by
peritoneal inflammation caused by the injection of the adjuvant. Similarly, due to the
absence of a mock-vaccinated control, it is not possible to define if the vaccine devoid
of adjuvant induced any increase of the antibody titer over the baseline values.
Thanks for the reviewer’s suggestion. In this study, we only harvested medium
from the infected cells, after removal of cellular debris by low speed centrifugation
(6500 g, 20 min, and 4°C) and subsequent inactivation, the virus was concentrated by
centrifugation at 26,000g for 3h at 4°C. Virus pellets were resuspended in PBS and
purified by ultracentrifugation at 72,000g in a swing-out rotor for 1 hr at 4 °C through
a sucrose gradient. The MCMV vaccine was further confirmed by SDS PAGE and
western blot analysis (see figure in the end of this document). In addition, in order to
avoid the present of cellular proteins, we didn’t split the infected cells to obtain virus.
According to Geoffroy’s report, through this procedure, cellular contamination of the
viral material was found to be less than 2% (Arch Virol 1994, 135(1-2):61-74). Thus，
we did not set controls with formalin-treated lysates of mock-infected NIH-3T3, we
use unimmunized mice as control (table 2). In addition, Ab-positive cutoff values
were set as mean ± 2SD of unimmunized sera in this study (table 1). The ELISA Ab
titer was expressed as the highest serum dilution giving a positive reaction.
Vaccination with inactivated MCMV vaccine alone could provide protection,
whereas combination of adjuvant could enhance the protective effect. In our study, the
adjuvants were proved to be able to improve the long-term protection. As for the
immuno-mechanism, adjuvant not only induces the more long-lived effects of the
adaptive immune response, but enhances innate immune response, affects the
transient reaction of the innate immune response. An adjuvant-enhanced innate
immune response leads to an enhanced adaptive immune response. So, the
inflammation caused by the injection of the adjuvant maybe the available initiation to
promote the effective immune response，and that is the sense of the inflammation
resulted from adjuvant. In this study, we observed that the antibody titers in the
adjuvanted group were higher than those in the corresponding vaccine alone groups,
and the survival rates were higher in the adjuvanted vaccine groups. The stronger
adaptive immue response was induced in groups administrated with adjuvanted
vaccine, which indicated that the adjuvant had played its role successfully. In addition,
MF59 and alum have been approved for marketing as adjuvant. We think that these
two adjuvants might not able to provide protection alone.
5. The authors should also carefully and comprehensively check the English grammar
and style, probably by inviting a native speaker to carefully peruse the manuscript.
Thanks for the reviewer’s suggestion. We have asked professional English
editing company recommended by the BMC infectious disease to check the English
grammar of this manuscript.
Fig SDS-PAGE and Western bloting of inactivated MCMV. (A) FI-MCMV proteins
were separated by SDS-PAGE and stained with Coomassie blue (M: protein marker,
lane 1: separated FI-MCMV protein bands). (B) The electrophoresed proteins were
transferred to a PVDF membrane. The membrane was incubated with serum pooled
from FI-MCMV vaccine immunized mice and then with HRP-conjugated secondary
antibodies and enhanced-chemiluminescence detection (M: protein marker).
Level of interest: An article of limited interest
Quality of written English: Not suitable for publication unless entensively edited
Statistical review: Yes, but I do not feel adequately qualified to assess the statistics.
Declaration of competing interests:
I declare that I have no competing interests.
Reviewer #2: Professor Mark Schleiss
Version: 2 Date: 23 August 2012
Response to referee’s comments 2
We thank the reviewer for a very careful review and very thoughtful
comments. Following are our point by point response to the reviewer’s comments:
Major Comments:
1. The authors could provide additional evidence that makes it more clear and certain
that the vaccine was inactivated. There is a short sentence saying that plaque assay
showed that the inactivation was successful, but few details are provided. Were any
additional studied performed to ensure adequate formalin inactivation? This is
particularly important for a translational goal of a human vaccine, where GMP issues
would require rigorous proof of inactivation.
Thanks for reviewer’s suggestion. As described in the manuscript, the viruses
were inactivated by adding 37% formalin at a ratio of 1: 4000, and mixed thoroughly at
4°C for one week with stirring. The inactivation of the virus and vaccine was proved to
be complete by three methods in this study. Firstly, the inactivated virus stock was used
to infect NIH-3T3 cells. The 3T3 cells were incubated for 7 days, and no cytopathic
effect (CPE) was observed. Secondly, the inactivated vaccine was confirmed to be
completely inactivated by plaque assay. Thirdly, the inactivated vaccine was i.p.
injected into mice, and no virus was isolated from the spleens, livers and salivary
glands of mice. Based on these results, we could confirm that our viruses were
completely inactivated by formalin. We added these information in manuscript.
2. Did the centrifugation steps to purify vaccine result in purification of actual virus
particles? Were additional studies performed to be certain that there was no cellular
protein present? This, too, would be a major regulatory issue for GMP purposes. If the
inactivation process resulted in a vaccine that still consisted of particles, the vaccine is
in some ways reminiscent of the work of Plachter (Gene Ther. 2003 Feb; 10:278-84; J
Clin Virol. 2002 Aug; 25 Suppl 2: S75-85). It would be of interest to compare the
inactivation techniques, the added benefit of adjuvant, and the vaccine procedures to
the dense body work.
Thanks for reviewer’s comment. We only harvested medium from the infected
cells, after removal of cellular debris by low speed centrifugation (6500 g, 20 min, 4 °C)
and subsequent inactivation, the virus was concentrated by centrifugation at 26,000g
for 3hr, at 4 °C. Virus pellets were resuspended in a small amount of
phosphate-buffered saline and purified by loading the virus onto a 15% sucrose/virus
standard buffer (VSB) and centrifuge at 72,000g in a swing-out rotor for 1 hr at 4 °C (J
Virol. 1983 Sep;47(3):421-33; Curr Protoc Immunol 2001, Chapter 19:Unit 19 17; Hum
Reprod 2001, 16(10):2041-2049). The MCMV vaccine was further confirmed by SDS
PAGE and western blot analysis (see figure in the end of this document). In addition, in
order to avoid the present of cellular proteins, we didn’t split the infected cells to obtain
virus. According to Geoffroy’s report, through this procedure, cellular contamination of
the viral material was found to be less than 2% (Arch Virol 1994, 135(1-2):61-74). The
preparation of the vaccine in this study was referred to previously reported methods as
mentioned by reviewer. There might be a small quantity of dense body present in the
prepared vaccine. Dense bodies are mainly composed of the tegument protein pp65,
and a lipid bilayer envelope, comparable or identical to the viral envelope, which can
induce potent immune responses. The presence of small quantity of Dense bodies in the
MCMV vaccine did not influence the results of experiment.
3. The authors should state what the LD50 dose of SG virus was. How did this
translate into PFU or actual titer of virus? This would be of interest for investigators
who define inocula not by LD50, but by titer of virus.
Thanks for reviewer’s comments. For lethal dose experiments, MCMV was
serially passaged in BALB/c mice to restore the virulence. The SG-MCMV stock was
titrated on 3T3 cells by plaque assay, which had a titer of 107.1 PFU/ml and a 50% lethal
dose (LD50) in BALB/c mice following intraperitoneal infection of 105 PFU. In our
study, challenge was performed with 5 × LD50 virus stock (5 ×105 PFU). Since the
virulence of virus differs in each passage, its LD50 also changes and should be
determined in each practical animal experiment. In this study, the 10th passage of
SG-MCMV was used, its LD50 was determined based on the mortality rate of mice and
its virus titer was obtained from cell experiments.
4. It would be of interest to compare the ELISA assay titers in vaccinated animals to
the titers of “naturally seropositive” animals, or animals challenge with non-lethal
doses of virus.
Thanks for reviewer’s suggestion. We agree with reviewer that it will be
interesting to compare the ELISA assay titers in vaccinated animals to the titers of
"naturally seropositive" animals, or animals challenge with non-lethal doses of virus.
Due to the difficulty in obtaining naturally seropositive mice, the complexity in genetic
backgrounds of mice and huge individual differences, we think it is difficult to perform
the comparison study. Since the ELISA titer is dependant on the vaccine immune dose
and virus infective dose, we think that the ELISA assay titer induced by high dose of
inactivated vaccine will be higher than that of non-lethal doses of virus.
5. It would be of interest to know what the neutralizing antibody titers were that were
induced by immunization, and how these compared to naturally infected mice.
Thanks for reviewer’s kind suggestion. Since the tendency of neutralizing
antibody titer was previously reported to be consistent with that of serum IgG titer
(Geoffroy et al., Vaccine 1996, 14(17-18):1686-1694; Morello et al., J Virol 2002,
76(10) :4822-4835), we only measured IgG antibody level induced by vaccination in
this study. Since the MCMV naturally infected mice were hard to obtain, we did not
design the comparison study. However, we will take this suggestion into consideration
in our future study.
6. Did the authors examine the western blot profile against MCMV polypeptides in
vaccinated animals?
Thanks for reviewer’s comments. Since the genome of MCMV Smith is
230,278 bp in length, which is predicted to encode 170 ORFs (J Virol. 1996
Dec;70(12):8833-49). Analysis on the targeting protein or polypeptide of the antibody
by Western blot will be huge work. In addition, the main purpose of this study is to
compare the efficiency of different adjuvants. However, we agree with the reviewer
and we will study the surface glycoprotein first in future.
7. The authors should clarify why chitosan was less protective than alum in terms of
mortality, even though they were both equally immunogenic. The authors note,
“adjuvant Alum could granulate antigens and also activate the innate immune pathway,
creating an immune active environment at the injection site, thus promoting the
co-immunized antigen to induce good antibody response ”, but while this statement is
scholarly, there are no experiments in this study that specifically address this point.
Thanks for reviewer’s suggestion. In our study, the IgG antibody levels induced
in mice of chitosan group and Alum group were close, with the Chitosan group slightly
lower than the Alum group. In addition, there was no significant difference in the
survival rates of mice from these two adjuvanted groups three weeks after the second
immunization. However, the antibody level of the Chitosan group was significantly
lower than that of the Alum group at 6 month after the third immunization, which was
corresponding to the low survival rate of the Chitosan group after the lethal challenge.
Based on these observations, we speculate that the chitosan may be less efficient in
inducing long-term immunity.
We have cited the reported and confirmed results for the immunostimulation
mechanism of Alum in this manuscript. We didn’t perform relevant experiments in this
study.
8. Do the authors have any information on cell mediated immune responses? It may
be that some of the differences identified from one adjuvant to another are related to
differences in the cell mediated immune response.
Thanks for reviewer’s suggestion. Generally, the inactivated vaccine will
mainly induce humoral immune response. The differences of adjuvants in improving
immune responses could be well illustrated by the antibody levels, we didn’t evaluate
the cell mediated immune responses in this study. However, we couldn’t exclude that
the cell mediated immune responses might have played a certain role.
9. The authors should comment on the work of Morello and Spector (for example, J
Virol. 2002; 76: 4822-35) that demonstrate really very robust sterilizing immunity
using various combination of formalin-inactivated vaccines. Although the authors do
reference this work, there is not much comment on the difference in the magnitude of
protection. Wang et al. reassuring saw a reduction in virus titer in organs of
vaccinated versus unvaccinated animals, but the level of protection appears lower than
that observed by Morello. Is there an explanation for this?
Thanks for reviewer’s careful review. The magnitude of protection induced by
the inactivated vaccine in our study was lower than that previously reported in Morello
et al, which was mainly resulted by the difference in the challenge dosages used. In our
study, we used high lethal dose of 5 × LD50 for challenge, whereas a sub-lethal dose
below 1 × LD50 was used in Morello et al study.
10. Figures 1 and 2 do not have statistical information. This would enhance the figure.
Were any of the differences observed considered to be statistically significant? The
text of the paper does not make this clear, or how the authors tested for statistical
significance in these comparisons.
Thanks for reviewer’s suggestion. There were indeed significant differences
between some experimental groups in Figure 1 and 2, and we didn’t clarify these in the
Figures. According to the reviewer’s advice, we have made necessary modifications in
these two figures in the manuscript.
Minor comments:
1. The manuscript should have page numbers on it.
Thanks for reviewer’s suggestion. We have added page numbers in the
manuscript.
2. Methods section, “Detection of HCMV-specific antibody” should be corrected to
“Detection of MCMV-specific antibody”.
Thanks for the reviewer’s careful review. We have corrected the “Detection of
HCMV-specific antibody” to “Detection of MCMV-specific antibody” in the
manuscript.
3. Although it is a different model, in another rodent CMV there were interesting and
significant differences in protection and immune response upon comparison of alum
and Freund’s adjuvant (J Infect Dis. 2004; 189: 1374-81), underscoring the critical
importance of adjuvant in optimization of a CMV vaccine.
Thanks for reviewer’s suggestion. In this study, we have used MF59, alum and
chitosan as adjuvants and compared their efficiencies in improving the immunization
effects. We agree with the reviewer that adjuvant plays an important role in
optimization of a CMV vaccine
Level of interest: An article whose findings are important to those with closely
related research interests
Quality of written English: Needs some language corrections before being published
Statistical review: No, the manuscript does not need to be seen by a statistician.
Declaration of competing interests:
I have no conflicts of interest related to this manuscript.