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Human Vaccines & Immunotherapeutics Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/khvi20
VLPNPV 2014: Session 8 (Vaccines I) a
David S. Thiriot Associate Principal Scientist a
Biologics and Vaccine Formulation, Vaccine Drug Product Development, Merck Research Laboratories, PO Box 4, West Point PA, 19486, USA Accepted author version posted online: 31 Oct 2014.Published online: 10 Oct 2014.
Click for updates To cite this article: David S. Thiriot Associate Principal Scientist (2014): VLPNPV 2014: Session 8 (Vaccines I), Human Vaccines & Immunotherapeutics, DOI: 10.4161/21645515.2014.972841 To link to this article: http://dx.doi.org/10.4161/21645515.2014.972841
Disclaimer: This is a version of an unedited manuscript that has been accepted for publication. As a service to authors and researchers we are providing this version of the accepted manuscript (AM). Copyediting, typesetting, and review of the resulting proof will be undertaken on this manuscript before final publication of the Version of Record (VoR). During production and pre-press, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal relate to this version also.
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Title VLPNPV 2014: session 8 (vaccines I) 05 June 2014, Salk Institute for Biological Studies, La Jolla, CA, USA Meeting Report written by
ip t
David S. Thiriot, Associate Principal Scientist – Biologics and Vaccine Formulation, Vaccine Drug Product Development, Merck Research Laboratories, PO Box 4, West
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ce
pt
ed
M
an
us
Email: [email protected]
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Downloaded by [University of Louisville] at 13:03 06 January 2015
Point PA, 19486, USA.
1
Abstract In Session 8 of the recent conference “Virus-Like Particle and Nano-Particle Vaccines” held at the Salk Institute in La Jolla, California (05 June 2014), four scientists described new virus-like particle (VLP) approaches, progress, and early-stage plans for vaccines
ip t
against significant human pathogens including HPV, malaria, HIV, Dengue, and RSV. A unifying theme was that displaying epitopes in an array on a virus-like particle can be a
cr
us
included display of epitopes on bacteriophage, display of epitopes as fusions with other protein multimerization domains, and self-assembly of recombinantly-expressed virus
an
coat proteins. Another theme in some of the presentations was the targeting of neutralizing epitopes that are masked or only transiently accessible during natural
ed
M
infection.
Keywords
pt
VLP (virus-like particle), bacteriophage display, E2 display, vaccine development, HPV (human papillomavirus), malaria, HIV (human immunodeficiency virus), dengue, RSV
ce
(respiratory syncytial virus)
Ac
Downloaded by [University of Louisville] at 13:03 06 January 2015
powerful approach for achieving a strong immune response. VLP approaches described
2
Main Content Bacterophage VLP-based HPV and malaria vaccine candidates Dr. Bryce Chackerian (University of New Mexico, Albuquerque, NM, USA) described work in his group on a broadly-protective bacteriophage-based VLP vaccine for HPV.
ip t
Dr. Chackerian pointed out that some good potential neutralizing epitopes for infectious diseases may be largely hidden and only transiently accessible during natural infection,
cr
us
For challenging diseases for which effective vaccines may require achieving a better (or different) immune response than natural infection, display of such “cryptic” epitopes in
an
an array on the highly accessible surface of a virus-like particle may be a useful vaccine approach. His group demonstrated this approach by displaying an epitope from the HPV
M
“L2” minor capsid protein, present across many HPV serotypes, on the surface of
ed
engineered coat proteins of RNA bacteriophages (MS2/PP7). While 6-10 amino acid length insertions into coat protein monomers disrupted VLP formation, insertions into
pt
dimers of coat proteins were much better tolerated and allowed a high yield of VLP formation. VLPs displaying the HPV L2 eptiope were highly immunogenic in mice. To
ce
demonstrate and assess the breadth of protection in mice, a HPV “pseudovirus” challenge model was employed. Virus-like particles assembled from the coat proteins of at least 10
Ac
Downloaded by [University of Louisville] at 13:03 06 January 2015
leading to lack of significant immune response to those epitopes during natural infection.
different HPV serotypes were constructed, all of which encapsulated the same reporter plasmid that enabled fluorescent detection and imaging in whole mice. VLP vaccines displaying the L2 epitope sequence from HPV type 16 showed strong protection in mice against homologous challenge with HPV type 16 pseudovirus. They also showed broad protection, significantly reducing pseudovirus radiance at the vaginal infection site for 10
3
other HPV serotypes. The antibody response was long-lived, and mice continued to show protection from challenge 13-15 months after vaccination. Dr. Chackerian then described collaborative studies with Dr. David Peabody’s laboratory at the University of New Mexico toward development of a blood-stage malaria vaccine
ip t
targeting an epitope of the Plasmodium falciparum RH5 protein (a protein involved in parasite invasion of erythrocytes). Starting with a complex MS2 bacteriophage library
cr
us
neutralizing, parasite inhibiting antibody were performed and a single predominant 8 amino acid sequence was recovered. The recovered peptide was a partial match to an N-
an
terminal sequence of mature RH5. When used as a vaccine, the selected VLPs induce sera that recognizes native Rh5 and also inhibits growth invasion by 3D7 parasites,
M
demonstrating the potential of this approach. Dr. Chackerian concluded his talk by
ed
discussing his efforts to use bacteriophage display to select peptides that mimic more complex conformational epitopes present on the P. falciparum AMA1 protein. Initial
pt
data are that some selected VLP vaccines do elicit AMA1 cross-reactive antibodies. Optimizing HIV-1 Env VLPs to elicit broad, consistent, and potent neutralizing antibody
ce
responses
Dr. James Binley (San Diego Biomedical Research Institute, San Diego, CA, USA)
Ac
Downloaded by [University of Louisville] at 13:03 06 January 2015
encoding many random peptides, multiple cycles of affinity selection against an RH5
discussed recent progress in his ongoing efforts to improve the breadth, potency, and consistency of HIV-1 envelope protein-based VLP vaccines. HIV VLPs were produced by co-transfecting 293T cells with two plasmids, one expressing Env and the second (pNL4-3.Luc.R-E-) expressing Gag, Rev and other HIV sequences for VLP formation. As vaccine antigens, such VLPs previously elicited only weak and inconsistent
4
neutralizing responses in guinea pigs. By identifying and addressing several factors that contributed to this weak response, Dr. Binley and colleagues have significantly improved the potency and consistency of HIV Env-trimer VLP vaccines. One part of this approach is to eliminate the many different non-functional forms and conformations of the Env
ip t
protein that can serve as “decoys” for the immune system. By treating VLPs with a
protease cocktail that included proteinase K, subtilisin, chymotrypsin and trypsin, these
cr
us
the more compact, functional Env trimer epitopes to which the neutralizing antibody response is desired. The protease approach proved simpler and more robust than a
an
previous approach of using antibodies to block the non-functional forms of Env. Dr. Binley and colleagues also came to recognize that species-specific issues with guinea
M
pigs, and insufficient doses of VLP vaccine had contributed to the weak responses in
ed
prior studies. Better responses to the VLP vaccine were obtained by using higher doses and testing in rabbits, which lack the serum background problem associated with guinea
pt
pigs. It was also observed that VLP preps still contain a lot of “bald”-VLPs, lacking Env, and recognized that purifying Env-VLPs from “bald”-VLPs could improve vaccine
ce
potency in the future. After vaccination with high dose “Env trimer” VLPs, Dr. Binley and colleagues induced very potent neutralizing antibodies against the JR-FL index virus
Ac
Downloaded by [University of Louisville] at 13:03 06 January 2015
more accessible but non-functional forms of Env could be removed, without damaging
in some rabbits. Mapping showed that the serum recognized an epitope present in many tier 2 strains but which is blocked by a glycan in most strains. When the glycan was removed from several resistant strains, they were rendered highly sensitive to this neutralizing serum. This reveals a possible avenue for inducing neutralization breadth by selecting neutralizing antibodies that can “navigate around” naturally occurring glycans
5
to reach the underlying target epitopes. Non-human primate immunization studies are planned, and it is hoped that their more complex IgG repertoire might be better able to respond to pure native Env trimer. Developing a VLP dengue vaccine using “E2-display”
ip t
Dr. Sean McBurney (Oregon Health Sciences University, Beaverton, OR, USA)
described plans and initial data for the development of VLP dengue vaccines. His
cr
us
dehydrogenase complex. E2 particles are formed when E2 monomers form trimers
through interactions between their catalytic domains, and 20 trimers further associate into
an
particles ~25 nm in diameter containing 60 copies of E2. Recombinant E2 from the thermophilic bacteria Bacillus stearothermophilus can be expressed in E. coli, and after
M
purification can self-assemble into particles without the need for accessory folding
ed
proteins. For epitope display, only the catalytic domain of the E2 protein is used, with a 12 amino acid spacer between the (C-terminal) catalytic domain and the (N-terminal)
pt
epitope for display. E2 particles can be disassembled and reassembled to “mix and match” E2 subunits displaying different epitopes, allowing multivalent immunogens
ce
within a single VLP particle. This capability may prove useful for a dengue vaccine, as vaccination must protect against four dengue serotypes.
Ac
Downloaded by [University of Louisville] at 13:03 06 January 2015
approach uses a technique called “E2 display”. The E2 protein is a part of the pyruvate
The dengue antigen selected for display was a portion of the capsid envelope (“E”) protein. The dengue E protein is comprised of three domains, and the third domain (amino acid 270-372) was selected as the epitope for display. Significantly, Dr. McBurney mentioned that recent work has shown that antibodies directed to this domain (dengue ED-III) demonstrate little antibody dependent enhancement of disease.
6
Expression constructs were prepared for dengue E protein domain III – B. stearothermophilus E2 protein catalytic domain fusion proteins, for each of the four dengue serotypes. In initial experiments, dengue serotype 2 and serotype 4 constructs expressed the desired fusion proteins at high levels, but dengue serotypes 1 and 3
ip t
constructs did not. Solubilization of inclusion bodies, purification, refolding and self-
assembly methods that were employed were briefly described, including DNAse/RNAse
cr
us
step-down dialysis. The resulting VLP particles for dengue serotypes 2 and 4 were shown to bind multiple specific linear and conformational anti-dengue antibodies. Plans
an
for further development include immunogenicity studies in rhesus macaques that would test co-immunization of dengue serotype 2 VLP and a dengue DNA vaccine, and future
M
immunogenicity testing of multivalent heterologous VLP particles in mice.
ed
Developing an RSV VLP vaccine
Dr. Pramila Walpita (University of Hawaii at Manoa, HI, USA) and colleagues have been
pt
producing VLPs of certain paramyxoviruses by co-expression of the surface glycoproteins (F and G) and the matrix protein (M) in mammalian cells. Following a
ce
pattern similar to her published work on a Nipah virus VLP vaccine, Dr. Walpita described her more recent work on an RSV VLP vaccine. Co-expression of the RSV F,
Ac
Downloaded by [University of Louisville] at 13:03 06 January 2015
treatment, removal of endotoxin, anion exchange and size exclusion chromatography, and
G, and M proteins led to spontaneous formation of VLPs, which were purified using sucrose gradient centrifugation. Analysis by Western blots and immunogold-labeling electron microscopy confirmed that all three proteins were present in the VLPs, and that the F protein was cleaved as typical for native RSV virions. In-vivo studies of the VLP vaccine were performed in cotton rats, testing the immunogen with and without adjuvant
7
(aluminum adjuvant plus monophosphoryl lipid A [MPLA]), and comparing two different routes of administration (intramuscular versus subcutaneous). Vaccines were administered in two doses on day 0 and day 21, after which an RSV challenge was performed on day 42. There was no significant difference in response observed between
ip t
intramuscular and subcutaneous injection. Groups that received the aluminum adjuvant
with MPLA had high total IgG titers and neutralizing titers, and showed protection from
cr
us
viral load in lung and nasal washes. Groups that did not receive adjuvant had low titers and were not protected from challenge.
an
Acknowledgement
ce
pt
ed
meeting to augment my own.
M
I thank Dr. James Cook (Merck, West Point, PA, USA) for sharing his notes from the
Ac
Downloaded by [University of Louisville] at 13:03 06 January 2015
homologous (RSV-A) and heterologous (RSV-B) challenge, with a 3-4 log reduction in
8
Human Vaccines & Immunotherapeutics Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/khvi20
VLPNPV 2014: Session 8 (Vaccines I) a
David S. Thiriot Associate Principal Scientist a
Biologics and Vaccine Formulation, Vaccine Drug Product Development, Merck Research Laboratories, PO Box 4, West Point PA, 19486, USA Accepted author version posted online: 31 Oct 2014.Published online: 10 Oct 2014.
Click for updates To cite this article: David S. Thiriot Associate Principal Scientist (2014): VLPNPV 2014: Session 8 (Vaccines I), Human Vaccines & Immunotherapeutics, DOI: 10.4161/21645515.2014.972841 To link to this article: http://dx.doi.org/10.4161/21645515.2014.972841
Disclaimer: This is a version of an unedited manuscript that has been accepted for publication. As a service to authors and researchers we are providing this version of the accepted manuscript (AM). Copyediting, typesetting, and review of the resulting proof will be undertaken on this manuscript before final publication of the Version of Record (VoR). During production and pre-press, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal relate to this version also.
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Title VLPNPV 2014: session 8 (vaccines I) 05 June 2014, Salk Institute for Biological Studies, La Jolla, CA, USA Meeting Report written by
ip t
David S. Thiriot, Associate Principal Scientist – Biologics and Vaccine Formulation, Vaccine Drug Product Development, Merck Research Laboratories, PO Box 4, West
cr
ce
pt
ed
M
an
us
Email: [email protected]
Ac
Downloaded by [University of Louisville] at 13:03 06 January 2015
Point PA, 19486, USA.
1
Abstract In Session 8 of the recent conference “Virus-Like Particle and Nano-Particle Vaccines” held at the Salk Institute in La Jolla, California (05 June 2014), four scientists described new virus-like particle (VLP) approaches, progress, and early-stage plans for vaccines
ip t
against significant human pathogens including HPV, malaria, HIV, Dengue, and RSV. A unifying theme was that displaying epitopes in an array on a virus-like particle can be a
cr
us
included display of epitopes on bacteriophage, display of epitopes as fusions with other protein multimerization domains, and self-assembly of recombinantly-expressed virus
an
coat proteins. Another theme in some of the presentations was the targeting of neutralizing epitopes that are masked or only transiently accessible during natural
ed
M
infection.
Keywords
pt
VLP (virus-like particle), bacteriophage display, E2 display, vaccine development, HPV (human papillomavirus), malaria, HIV (human immunodeficiency virus), dengue, RSV
ce
(respiratory syncytial virus)
Ac
Downloaded by [University of Louisville] at 13:03 06 January 2015
powerful approach for achieving a strong immune response. VLP approaches described
2
Main Content Bacterophage VLP-based HPV and malaria vaccine candidates Dr. Bryce Chackerian (University of New Mexico, Albuquerque, NM, USA) described work in his group on a broadly-protective bacteriophage-based VLP vaccine for HPV.
ip t
Dr. Chackerian pointed out that some good potential neutralizing epitopes for infectious diseases may be largely hidden and only transiently accessible during natural infection,
cr
us
For challenging diseases for which effective vaccines may require achieving a better (or different) immune response than natural infection, display of such “cryptic” epitopes in
an
an array on the highly accessible surface of a virus-like particle may be a useful vaccine approach. His group demonstrated this approach by displaying an epitope from the HPV
M
“L2” minor capsid protein, present across many HPV serotypes, on the surface of
ed
engineered coat proteins of RNA bacteriophages (MS2/PP7). While 6-10 amino acid length insertions into coat protein monomers disrupted VLP formation, insertions into
pt
dimers of coat proteins were much better tolerated and allowed a high yield of VLP formation. VLPs displaying the HPV L2 eptiope were highly immunogenic in mice. To
ce
demonstrate and assess the breadth of protection in mice, a HPV “pseudovirus” challenge model was employed. Virus-like particles assembled from the coat proteins of at least 10
Ac
Downloaded by [University of Louisville] at 13:03 06 January 2015
leading to lack of significant immune response to those epitopes during natural infection.
different HPV serotypes were constructed, all of which encapsulated the same reporter plasmid that enabled fluorescent detection and imaging in whole mice. VLP vaccines displaying the L2 epitope sequence from HPV type 16 showed strong protection in mice against homologous challenge with HPV type 16 pseudovirus. They also showed broad protection, significantly reducing pseudovirus radiance at the vaginal infection site for 10
3
other HPV serotypes. The antibody response was long-lived, and mice continued to show protection from challenge 13-15 months after vaccination. Dr. Chackerian then described collaborative studies with Dr. David Peabody’s laboratory at the University of New Mexico toward development of a blood-stage malaria vaccine
ip t
targeting an epitope of the Plasmodium falciparum RH5 protein (a protein involved in parasite invasion of erythrocytes). Starting with a complex MS2 bacteriophage library
cr
us
neutralizing, parasite inhibiting antibody were performed and a single predominant 8 amino acid sequence was recovered. The recovered peptide was a partial match to an N-
an
terminal sequence of mature RH5. When used as a vaccine, the selected VLPs induce sera that recognizes native Rh5 and also inhibits growth invasion by 3D7 parasites,
M
demonstrating the potential of this approach. Dr. Chackerian concluded his talk by
ed
discussing his efforts to use bacteriophage display to select peptides that mimic more complex conformational epitopes present on the P. falciparum AMA1 protein. Initial
pt
data are that some selected VLP vaccines do elicit AMA1 cross-reactive antibodies. Optimizing HIV-1 Env VLPs to elicit broad, consistent, and potent neutralizing antibody
ce
responses
Dr. James Binley (San Diego Biomedical Research Institute, San Diego, CA, USA)
Ac
Downloaded by [University of Louisville] at 13:03 06 January 2015
encoding many random peptides, multiple cycles of affinity selection against an RH5
discussed recent progress in his ongoing efforts to improve the breadth, potency, and consistency of HIV-1 envelope protein-based VLP vaccines. HIV VLPs were produced by co-transfecting 293T cells with two plasmids, one expressing Env and the second (pNL4-3.Luc.R-E-) expressing Gag, Rev and other HIV sequences for VLP formation. As vaccine antigens, such VLPs previously elicited only weak and inconsistent
4
neutralizing responses in guinea pigs. By identifying and addressing several factors that contributed to this weak response, Dr. Binley and colleagues have significantly improved the potency and consistency of HIV Env-trimer VLP vaccines. One part of this approach is to eliminate the many different non-functional forms and conformations of the Env
ip t
protein that can serve as “decoys” for the immune system. By treating VLPs with a
protease cocktail that included proteinase K, subtilisin, chymotrypsin and trypsin, these
cr
us
the more compact, functional Env trimer epitopes to which the neutralizing antibody response is desired. The protease approach proved simpler and more robust than a
an
previous approach of using antibodies to block the non-functional forms of Env. Dr. Binley and colleagues also came to recognize that species-specific issues with guinea
M
pigs, and insufficient doses of VLP vaccine had contributed to the weak responses in
ed
prior studies. Better responses to the VLP vaccine were obtained by using higher doses and testing in rabbits, which lack the serum background problem associated with guinea
pt
pigs. It was also observed that VLP preps still contain a lot of “bald”-VLPs, lacking Env, and recognized that purifying Env-VLPs from “bald”-VLPs could improve vaccine
ce
potency in the future. After vaccination with high dose “Env trimer” VLPs, Dr. Binley and colleagues induced very potent neutralizing antibodies against the JR-FL index virus
Ac
Downloaded by [University of Louisville] at 13:03 06 January 2015
more accessible but non-functional forms of Env could be removed, without damaging
in some rabbits. Mapping showed that the serum recognized an epitope present in many tier 2 strains but which is blocked by a glycan in most strains. When the glycan was removed from several resistant strains, they were rendered highly sensitive to this neutralizing serum. This reveals a possible avenue for inducing neutralization breadth by selecting neutralizing antibodies that can “navigate around” naturally occurring glycans
5
to reach the underlying target epitopes. Non-human primate immunization studies are planned, and it is hoped that their more complex IgG repertoire might be better able to respond to pure native Env trimer. Developing a VLP dengue vaccine using “E2-display”
ip t
Dr. Sean McBurney (Oregon Health Sciences University, Beaverton, OR, USA)
described plans and initial data for the development of VLP dengue vaccines. His
cr
us
dehydrogenase complex. E2 particles are formed when E2 monomers form trimers
through interactions between their catalytic domains, and 20 trimers further associate into
an
particles ~25 nm in diameter containing 60 copies of E2. Recombinant E2 from the thermophilic bacteria Bacillus stearothermophilus can be expressed in E. coli, and after
M
purification can self-assemble into particles without the need for accessory folding
ed
proteins. For epitope display, only the catalytic domain of the E2 protein is used, with a 12 amino acid spacer between the (C-terminal) catalytic domain and the (N-terminal)
pt
epitope for display. E2 particles can be disassembled and reassembled to “mix and match” E2 subunits displaying different epitopes, allowing multivalent immunogens
ce
within a single VLP particle. This capability may prove useful for a dengue vaccine, as vaccination must protect against four dengue serotypes.
Ac
Downloaded by [University of Louisville] at 13:03 06 January 2015
approach uses a technique called “E2 display”. The E2 protein is a part of the pyruvate
The dengue antigen selected for display was a portion of the capsid envelope (“E”) protein. The dengue E protein is comprised of three domains, and the third domain (amino acid 270-372) was selected as the epitope for display. Significantly, Dr. McBurney mentioned that recent work has shown that antibodies directed to this domain (dengue ED-III) demonstrate little antibody dependent enhancement of disease.
6
Expression constructs were prepared for dengue E protein domain III – B. stearothermophilus E2 protein catalytic domain fusion proteins, for each of the four dengue serotypes. In initial experiments, dengue serotype 2 and serotype 4 constructs expressed the desired fusion proteins at high levels, but dengue serotypes 1 and 3
ip t
constructs did not. Solubilization of inclusion bodies, purification, refolding and self-
assembly methods that were employed were briefly described, including DNAse/RNAse
cr
us
step-down dialysis. The resulting VLP particles for dengue serotypes 2 and 4 were shown to bind multiple specific linear and conformational anti-dengue antibodies. Plans
an
for further development include immunogenicity studies in rhesus macaques that would test co-immunization of dengue serotype 2 VLP and a dengue DNA vaccine, and future
M
immunogenicity testing of multivalent heterologous VLP particles in mice.
ed
Developing an RSV VLP vaccine
Dr. Pramila Walpita (University of Hawaii at Manoa, HI, USA) and colleagues have been
pt
producing VLPs of certain paramyxoviruses by co-expression of the surface glycoproteins (F and G) and the matrix protein (M) in mammalian cells. Following a
ce
pattern similar to her published work on a Nipah virus VLP vaccine, Dr. Walpita described her more recent work on an RSV VLP vaccine. Co-expression of the RSV F,
Ac
Downloaded by [University of Louisville] at 13:03 06 January 2015
treatment, removal of endotoxin, anion exchange and size exclusion chromatography, and
G, and M proteins led to spontaneous formation of VLPs, which were purified using sucrose gradient centrifugation. Analysis by Western blots and immunogold-labeling electron microscopy confirmed that all three proteins were present in the VLPs, and that the F protein was cleaved as typical for native RSV virions. In-vivo studies of the VLP vaccine were performed in cotton rats, testing the immunogen with and without adjuvant
7
(aluminum adjuvant plus monophosphoryl lipid A [MPLA]), and comparing two different routes of administration (intramuscular versus subcutaneous). Vaccines were administered in two doses on day 0 and day 21, after which an RSV challenge was performed on day 42. There was no significant difference in response observed between
ip t
intramuscular and subcutaneous injection. Groups that received the aluminum adjuvant
with MPLA had high total IgG titers and neutralizing titers, and showed protection from
cr
us
viral load in lung and nasal washes. Groups that did not receive adjuvant had low titers and were not protected from challenge.
an
Acknowledgement
ce
pt
ed
meeting to augment my own.
M
I thank Dr. James Cook (Merck, West Point, PA, USA) for sharing his notes from the
Ac
Downloaded by [University of Louisville] at 13:03 06 January 2015
homologous (RSV-A) and heterologous (RSV-B) challenge, with a 3-4 log reduction in
8
