Publications by authors named "Tracy J Ruckwardt"

40 Publications

COVID-19 vaccine mRNA-1273 elicits a protective immune profile in mice that is not associated with vaccine-enhanced disease upon SARS-CoV-2 challenge.

Immunity 2021 Jul 2. Epub 2021 Jul 2.

Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA. Electronic address:

Vaccine-associated enhanced respiratory disease (VAERD) was previously observed in some preclinical models of severe acute respiratory syndrome (SARS) and MERS coronavirus vaccines. We used the SARS coronavirus 2 (SARS-CoV-2) mouse-adapted, passage 10, lethal challenge virus (MA10) mouse model of acute lung injury to evaluate the immune response and potential for immunopathology in animals vaccinated with research-grade mRNA-1273. Whole-inactivated virus or heat-denatured spike protein subunit vaccines with alum designed to elicit low-potency antibodies and Th2-skewed CD4 T cells resulted in reduced viral titers and weight loss post challenge but more severe pathological changes in the lung compared to saline-immunized animals. In contrast, a protective dose of mRNA-1273 induced favorable humoral and cellular immune responses that protected from viral replication in the upper and lower respiratory tract upon challenge. A subprotective dose of mRNA-1273 reduced viral replication and limited histopathological manifestations compared to animals given saline. Overall, our findings demonstrate an immunological signature associated with antiviral protection without disease enhancement following vaccination with mRNA-1273.
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http://dx.doi.org/10.1016/j.immuni.2021.06.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8249710PMC
July 2021

Ultrapotent antibodies against diverse and highly transmissible SARS-CoV-2 variants.

Science 2021 Jul 1. Epub 2021 Jul 1.

Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.

The emergence of highly transmissible SARS-CoV-2 variants of concern (VOC) that are resistant to therapeutic antibodies highlights the need for continuing discovery of broadly reactive antibodies. We identify four receptor-binding domain targeting antibodies from three early-outbreak convalescent donors with potent neutralizing activity against 23 variants including the B.1.1.7, B.1.351, P.1, B.1.429, B.1.526 and B.1.617 VOCs. Two antibodies are ultrapotent, with sub-nanomolar neutralization titers (IC 0.3 to 11.1 ng/mL; IC 1.5 to 34.5 ng/mL). We define the structural and functional determinants of binding for all four VOC-targeting antibodies and show that combinations of two antibodies decrease the generation of escape mutants, suggesting their potential in mitigating resistance development.
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http://dx.doi.org/10.1126/science.abh1766DOI Listing
July 2021

Level of maternal respiratory syncytial virus (RSV) F antibodies in hospitalized children and correlates of protection.

Int J Infect Dis 2021 Jun 10;109:56-62. Epub 2021 Jun 10.

Biomedical Research Center, Qatar University, Qatar; College of Health Sciences, Qatar University, Qatar. Electronic address:

Background: Respiratory syncytial virus (RSV) is a major cause of lower respiratory infection among children and no vaccine is available. The stabilized form of the fusion (F) protein - pre-F - is a leading vaccine candidate to target different populations, including pregnant women. This study aimed to determine the magnitude and nature of RSV-directed maternal antibodies (matAbs) in hospitalized children with RSV infection.

Methods: Sixty-five paired blood samples were collected from RSV-infected children aged <6 months and their corresponding mothers. All pairs were screened for levels of pre-F and post-F antibodies using ELISA. The neutralizing antibodies (NAbs) in both groups were measured in vitro against mKate RSV-A2 using H28 cells.

Results: It was found that 14% of matAbs (log 12.8) were present in infants at hospitalization, with an average log EP titer of 10.2 directed to both F-protein conformations. Additionally, 61.4% of maternal NAbs (log EC = 9.4) were detected in infants (log EC = 8.7), which were mostly pre-F exclusive (81%). Pre-F antibodies in children showed a positive correlation with matAbs titers and negative correlations with age and bronchiolitis score.

Conclusions: The maintenance of neutralizing activity in infants relative to maternal titers was greater than the maintenance of antibody binding based on ELISA, suggesting that higher-potency antibodies may have a longer half-life than weakly neutralizing antibodies.
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http://dx.doi.org/10.1016/j.ijid.2021.06.015DOI Listing
June 2021

Safety, tolerability, and immunogenicity of the respiratory syncytial virus prefusion F subunit vaccine DS-Cav1: a phase 1, randomised, open-label, dose-escalation clinical trial.

Lancet Respir Med 2021 Apr 14. Epub 2021 Apr 14.

Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA. Electronic address:

Background: Multiple active vaccination approaches have proven ineffective in reducing the substantial morbidity and mortality caused by respiratory syncytial virus (RSV) in infants and older adults (aged ≥65 years). A vaccine conferring a substantial and sustainable boost in neutralising activity is required to protect against severe RSV disease. To that end, we evaluated the safety and immunogenicity of DS-Cav1, a prefusion F subunit vaccine.

Methods: In this randomised, open-label, phase 1 clinical trial, the stabilised prefusion F vaccine DS-Cav1 was evaluated for dose, safety, tolerability, and immunogenicity in healthy adults aged 18-50 years at a single US site. Participants were assigned to receive escalating doses of either 50 μg, 150 μg, or 500 μg DS-Cav1 at weeks 0 and 12, and were randomly allocated in a 1:1 ratio within each dose group to receive the vaccine with or without aluminium hydroxide (AlOH) adjuvant. After 71 participants had been randomised, the protocol was amended to allow some participants to receive a single vaccination at week 0. The primary objectives evaluated the safety and tolerability at every dose within 28 days following each injection. Neutralising activity and RSV F-binding antibodies were evaluated from week 0 to week 44 as secondary and exploratory objectives. Safety was assessed in all participants who received at least one vaccine dose; secondary and exploratory immunogenicity analysis included all participants with available data at a given visit. The trial is registered with ClinicalTrials.gov, NCT03049488, and is complete and no longer recruiting.

Findings: Between Feb 21, 2017, and Nov 29, 2018, 244 participants were screened for eligibility and 95 were enrolled to receive DS-Cav1 at the 50 μg (n=30, of which n=15 with AlOH), 150 μg (n=35, of which n=15 with AlOH), or 500 μg (n=30, of which n=15 with AlOH) doses. DS-Cav1 was safe and well tolerated and no serious vaccine-associated adverse events deemed related to the vaccine were identified. DS-Cav1 vaccination elicited robust neutralising activity and binding antibodies by 4 weeks after a single vaccination (p<0·0001 for F-binding and neutralising antibodies). In analyses of exploratory endpoints at week 44, pre-F-binding IgG and neutralising activity were significantly increased compared with baseline in all groups. At week 44, RSV A neutralising activity was 3·1 fold above baseline in the 50 μg group, 3·8 fold in the 150 μg group, and 4·5 fold in the 500 μg group (p<0·0001). RSV B neutralising activity was 2·8 fold above baseline in the 50 μg group, 3·4 fold in the 150 μg group, and 3·7 fold in the 500 μg group (p<0·0001). Pre-F-binding IgG remained significantly 3·2 fold above baseline in the 50 μg group, 3·4 fold in the 150 μg group, and 4·0 fold in the 500 μg group (p<0·0001). Pre-F-binding serum IgA remained 4·1 fold above baseline in the 50 μg group, 4·3 fold in the 150 μg group, and 4·8 fold in the 500 μg group (p<0·0001). Although a higher vaccine dose or second immunisation elicited a transient advantage compared with lower doses or a single immunisation, neither significantly impacted long-term neutralisation. There was no long-term effect of dose, number of vaccinations, or adjuvant on neutralising activity.

Interpretation: In this phase 1 study, DS-Cav1 vaccination was safe and well tolerated. DS-Cav1 vaccination elicited a robust boost in RSV F-specific antibodies and neutralising activity that was sustained above baseline for at least 44 weeks. A single low-dose of pre-F immunisation of antigen-experienced individuals might confer protection that extends throughout an entire RSV season.

Funding: The National Institutes of Allergy and Infectious Diseases.
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http://dx.doi.org/10.1016/S2213-2600(21)00098-9DOI Listing
April 2021

Vaccination with prefusion-stabilized respiratory syncytial virus fusion protein induces genetically and antigenically diverse antibody responses.

Immunity 2021 Apr 5;54(4):769-780.e6. Epub 2021 Apr 5.

Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.

An effective vaccine for respiratory syncytial virus (RSV) is an unrealized public health goal. A single dose of the prefusion-stabilized fusion (F) glycoprotein subunit vaccine (DS-Cav1) substantially increases serum-neutralizing activity in healthy adults. We sought to determine whether DS-Cav1 vaccination induces a repertoire mirroring the pre-existing diversity from natural infection or whether antibody lineages targeting specific epitopes predominate. We evaluated RSV F-specific B cell responses before and after vaccination in six participants using complementary B cell sequencing methodologies and identified 555 clonal lineages. DS-Cav1-induced lineages recognized the prefusion conformation of F (pre-F) and were genetically diverse. Expressed antibodies recognized all six antigenic sites on the pre-F trimer. We identified 34 public clonotypes, and structural analysis of two antibodies from a predominant clonotype revealed a common mode of recognition. Thus, vaccination with DS-Cav1 generates a diverse polyclonal response targeting the antigenic sites on pre-F, supporting the development and advanced testing of pre-F-based vaccines against RSV.
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http://dx.doi.org/10.1016/j.immuni.2021.03.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8099422PMC
April 2021

Antibodies with potent and broad neutralizing activity against antigenically diverse and highly transmissible SARS-CoV-2 variants.

bioRxiv 2021 Mar 1. Epub 2021 Mar 1.

Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.

The emergence of highly transmissible SARS-CoV-2 variants of concern (VOC) that are resistant to therapeutic antibodies highlights the need for continuing discovery of broadly reactive antibodies. We identify four receptor-binding domain targeting antibodies from three early-outbreak convalescent donors with potent neutralizing activity against 12 variants including the B.1.1.7 and B.1.351 VOCs. Two of them are ultrapotent, with sub-nanomolar neutralization titers (IC50 <0.0006 to 0.0102 μ g/mL; IC80 < 0.0006 to 0.0251 μ g/mL). We define the structural and functional determinants of binding for all four VOC-targeting antibodies, and show that combinations of two antibodies decrease the in vitro generation of escape mutants, suggesting potential means to mitigate resistance development. These results define the basis of therapeutic cocktails against VOCs and suggest that targeted boosting of existing immunity may increase vaccine breadth against VOCs.
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http://dx.doi.org/10.1101/2021.02.25.432969DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7924272PMC
March 2021

T cell immunity to SARS-CoV-2 following natural infection and vaccination.

Biochem Biophys Res Commun 2021 01 23;538:211-217. Epub 2020 Oct 23.

Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, 20892, USA. Electronic address:

SARS-CoV-2 first emerged in the human population in late 2019 in Wuhan, China, and in a matter of months, spread across the globe resulting in the Coronavirus Disease 19 (COVID-19) pandemic and substantial economic fallout. SARS-CoV-2 is transmitted between humans via respiratory particles, with infection presenting a spectrum of clinical manifestations ranging from asymptomatic to respiratory failure with multiorgan dysfunction and death in severe cases. Prior experiences with human pathogenic coronaviruses and respiratory virus diseases in general have revealed an important role for cellular immunity in limiting disease severity. Here, we review some of the key mechanisms underlying cell-mediated immunity to respiratory viruses and summarize our current understanding of the functional capacity and role of SARS-CoV-2-specific T cells following natural infection and vaccination.
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http://dx.doi.org/10.1016/j.bbrc.2020.10.060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7584424PMC
January 2021

Development of a potent Zika virus vaccine using self-amplifying messenger RNA.

Sci Adv 2020 Aug 7;6(32):eaba5068. Epub 2020 Aug 7.

Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.

Zika virus (ZIKV) is the cause of a pandemic associated with microcephaly in newborns and Guillain-Barre syndrome in adults. Currently, there are no available treatments or vaccines for ZIKV, and the development of a safe and effective vaccine is a high priority for many global health organizations. We describe the development of ZIKV vaccine candidates using the self-amplifying messenger RNA (SAM) platform technology delivered by cationic nanoemulsion (CNE) that allows bedside mixing and is particularly useful for rapid responses to pandemic outbreaks. Two immunizations of either of the two lead SAM (CNE) vaccine candidates elicited potent neutralizing antibody responses to ZIKV in mice and nonhuman primates. Both SAM (CNE) vaccines protected these animals from ZIKV challenge, with one candidate providing complete protection against ZIKV infection in nonhuman primates. The data provide a preclinical proof of concept that a SAM (CNE) vaccine candidate can rapidly elicit protective immunity against ZIKV.
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http://dx.doi.org/10.1126/sciadv.aba5068DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7413734PMC
August 2020

SARS-CoV-2 mRNA vaccine design enabled by prototype pathogen preparedness.

Nature 2020 10 5;586(7830):567-571. Epub 2020 Aug 5.

Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA.

A vaccine for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is needed to control the coronavirus disease 2019 (COVID-19) global pandemic. Structural studies have led to the development of mutations that stabilize Betacoronavirus spike proteins in the prefusion state, improving their expression and increasing immunogenicity. This principle has been applied to design mRNA-1273, an mRNA vaccine that encodes a SARS-CoV-2 spike protein that is stabilized in the prefusion conformation. Here we show that mRNA-1273 induces potent neutralizing antibody responses to both wild-type (D614) and D614G mutant SARS-CoV-2 as well as CD8 T cell responses, and protects against SARS-CoV-2 infection in the lungs and noses of mice without evidence of immunopathology. mRNA-1273 is currently in a phase III trial to evaluate its efficacy.
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http://dx.doi.org/10.1038/s41586-020-2622-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7581537PMC
October 2020

Evaluation of the mRNA-1273 Vaccine against SARS-CoV-2 in Nonhuman Primates.

N Engl J Med 2020 10 28;383(16):1544-1555. Epub 2020 Jul 28.

From the Vaccine Research Center (K.S.C., B. Flynn, K.E.F., J.R.F., S.B.-B., A.P.W., B. Flach, S. O'Connell, A.T.N., N.D., M.M.D., N.N.N., G.S.A., D.R.F., E.L., N.A.D.-R., B.C.L., M.K.L., S. O'Dell, S.D.S., E.P., L.A.C., C.Y., J.-P.M.T., W.S., Y.Z., O.M.A., L.W., A.P., E.S.Y., K.L., T.Z., I.-T.T., A.W., I.G., L.N., R.A.G., R.J.L., J.I.M., W.-P.K., K.M.M., T.J.R., J.E.L., M.R.G., P.D.K., J.R.M., A.M., N.J.S., M.R., R.A.S., B.S.G.), the Infectious Disease Pathogenesis Section (K.W.B., M.M., B.M.N., M.G.L.), and the Biostatistics Research Branch, Division of Clinical Research (M.C.N.), National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, and Bioqual (H.A., L.P., A.V.R., S.B., J.G., T.P.-T., A.S., T.-A.C., A. Cook, A.D., K.S., I.N.M.) and the Public Health Service Commissioned Corps (M.R.G.), Rockville - both in Maryland; the Department of Epidemiology, University of North Carolina at Chapel Hill, Chapel Hill (D.R.M., R.S.B.); Moderna, Cambridge, MA (D.K.E., G.S.-J., S.H., A. Carfi); and the Institute for Biomedical Sciences, George Washington University, Washington, DC (E.P.).

Background: Vaccines to prevent coronavirus disease 2019 (Covid-19) are urgently needed. The effect of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines on viral replication in both upper and lower airways is important to evaluate in nonhuman primates.

Methods: Nonhuman primates received 10 or 100 μg of mRNA-1273, a vaccine encoding the prefusion-stabilized spike protein of SARS-CoV-2, or no vaccine. Antibody and T-cell responses were assessed before upper- and lower-airway challenge with SARS-CoV-2. Active viral replication and viral genomes in bronchoalveolar-lavage (BAL) fluid and nasal swab specimens were assessed by polymerase chain reaction, and histopathological analysis and viral quantification were performed on lung-tissue specimens.

Results: The mRNA-1273 vaccine candidate induced antibody levels exceeding those in human convalescent-phase serum, with live-virus reciprocal 50% inhibitory dilution (ID) geometric mean titers of 501 in the 10-μg dose group and 3481 in the 100-μg dose group. Vaccination induced type 1 helper T-cell (Th1)-biased CD4 T-cell responses and low or undetectable Th2 or CD8 T-cell responses. Viral replication was not detectable in BAL fluid by day 2 after challenge in seven of eight animals in both vaccinated groups. No viral replication was detectable in the nose of any of the eight animals in the 100-μg dose group by day 2 after challenge, and limited inflammation or detectable viral genome or antigen was noted in lungs of animals in either vaccine group.

Conclusions: Vaccination of nonhuman primates with mRNA-1273 induced robust SARS-CoV-2 neutralizing activity, rapid protection in the upper and lower airways, and no pathologic changes in the lung. (Funded by the National Institutes of Health and others.).
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http://dx.doi.org/10.1056/NEJMoa2024671DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7449230PMC
October 2020

SARS-CoV-2 mRNA Vaccine Development Enabled by Prototype Pathogen Preparedness.

bioRxiv 2020 Jun 11. Epub 2020 Jun 11.

Institute for Biomedical Sciences, George Washington University, Washington, DC 20052, United States of America.

A SARS-CoV-2 vaccine is needed to control the global COVID-19 public health crisis. Atomic-level structures directed the application of prefusion-stabilizing mutations that improved expression and immunogenicity of betacoronavirus spike proteins. Using this established immunogen design, the release of SARS-CoV-2 sequences triggered immediate rapid manufacturing of an mRNA vaccine expressing the prefusion-stabilized SARS-CoV-2 spike trimer (mRNA-1273). Here, we show that mRNA-1273 induces both potent neutralizing antibody and CD8 T cell responses and protects against SARS-CoV-2 infection in lungs and noses of mice without evidence of immunopathology. mRNA-1273 is currently in a Phase 2 clinical trial with a trajectory towards Phase 3 efficacy evaluation.
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http://dx.doi.org/10.1101/2020.06.11.145920DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7301911PMC
June 2020

Distinct neutralizing antibody correlates of protection among related Zika virus vaccines identify a role for antibody quality.

Sci Transl Med 2020 06;12(547)

Laboratory of Viral Diseases, NIAID, NIH, Bethesda, MD 20892, USA.

The emergence of Zika virus (ZIKV) in the Americas stimulated the development of multiple ZIKV vaccine candidates. We previously developed two related DNA vaccine candidates encoding ZIKV structural proteins that were immunogenic in animal models and humans. We sought to identify neutralizing antibody (NAb) properties induced by each vaccine that correlated with protection in nonhuman primates (NHPs). Despite eliciting equivalent NAb titers in NHPs, these vaccines were not equally protective. The transfer of equivalent titers of vaccine-elicited NAb into AG129 mice also revealed nonequivalent protection, indicating qualitative differences among antibodies (Abs) elicited by these vaccines. Both vaccines elicited Abs with similar binding titers against envelope protein monomers and those incorporated into virus-like particles, as well as a comparable capacity to orchestrate phagocytosis. Functional analysis of vaccine-elicited NAbs from NHPs and humans revealed a capacity to neutralize the structurally mature form of the ZIKV virion that varied in magnitude among vaccine candidates. Conversely, sensitivity to the virion maturation state was not a characteristic of NAbs induced by natural or experimental infection. Passive transfer experiments in mice revealed that neutralization of mature ZIKV virions more accurately predicts protection from ZIKV infection. These findings demonstrate that NAb correlates of protection may differ among vaccine antigens when assayed using standard neutralization platforms and suggest that measurements of Ab quality, including the capacity to neutralize mature virions, will be critical for defining correlates of ZIKV vaccine-induced immunity.
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http://dx.doi.org/10.1126/scitranslmed.aaw9066DOI Listing
June 2020

Immunological Lessons from Respiratory Syncytial Virus Vaccine Development.

Immunity 2019 09;51(3):429-442

Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA. Electronic address:

Respiratory syncytial virus (RSV) has eluded active vaccination efforts for more than five decades and continues to cause substantial morbidity and mortality in infants, the immunocompromised, and older adults. Although newer approaches of passive antibody-mediated protection show promise, vaccines aimed at eliciting fusion protein (F)-targeting antibodies have repeatedly failed to meet pre-established, modest-efficacy goals. Newer candidates, including protein-based vaccines, live-attenuated viruses, and gene-based delivery platforms, incorporate structurally defined and stabilized versions of the prefusion form of the F glycoprotein and are advancing rapidly into critical efficacy studies in susceptible target populations. This review discusses the storied history of RSV vaccine development, immunological lessons learned along the way, and critical findings about protein structure that remodeled our understanding of protective immunity to this important pathogen.
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http://dx.doi.org/10.1016/j.immuni.2019.08.007DOI Listing
September 2019

OMIP-061: 20-Color Flow Cytometry Panel for High-Dimensional Characterization of Murine Antigen-Presenting Cells.

Cytometry A 2019 12 19;95(12):1226-1230. Epub 2019 Aug 19.

Viral Pathogenesis Laboratory, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland.

This 20-color flow cytometry panel was designed to resolve the cellular heterogeneity of antigen-presenting cells and was optimized for lymph node tissue. Reagents were carefully selected and optimized for identification of B cells (B220), neutrophils (Ly6G), monocytes and macrophages (Ly6C, CD169, F4/80), and dendritic cells (XCR1, CD172a, CD11c, I-A/I-E, CD24, CD64, pDCA-1, CD103, CD11b). Inclusion of additional functional markers involved in cell migration (CCR7), co-stimulation (CD80), and adhesion (ICAM-1) enabled further phenotypic characterization. Finally, this panel has been tested and is compatible with fluorescently labeled antigens such as Alexa Fluor 488 (Ax488) for the study of antigen-bearing cells in vivo. Published 2019. This article is a U.S. Government work and is in the public domain in the USA.
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http://dx.doi.org/10.1002/cyto.a.23880DOI Listing
December 2019

A proof of concept for structure-based vaccine design targeting RSV in humans.

Science 2019 08;365(6452):505-509

Technologies that define the atomic-level structure of neutralization-sensitive epitopes on viral surface proteins are transforming vaccinology and guiding new vaccine development approaches. Previously, iterative rounds of protein engineering were performed to preserve the prefusion conformation of the respiratory syncytial virus (RSV) fusion (F) glycoprotein, resulting in a stabilized subunit vaccine candidate (DS-Cav1), which showed promising results in mice and macaques. Here, phase I human immunogenicity data reveal a more than 10-fold boost in neutralizing activity in serum from antibodies targeting prefusion-specific surfaces of RSV F. These findings represent a clinical proof of concept for structure-based vaccine design, suggest that development of a successful RSV vaccine will be feasible, and portend an era of precision vaccinology.
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http://dx.doi.org/10.1126/science.aav9033DOI Listing
August 2019

Epitope-Specific Serological Assays for RSV: Conformation Matters.

Vaccines (Basel) 2019 Feb 23;7(1). Epub 2019 Feb 23.

Vaccine Research Center, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.

Respiratory syncytial virus (RSV) causes substantial morbidity and mortality in children and older adults. An effective vaccine must elicit neutralizing antibodies targeting the RSV fusion (F) protein, which exists in two major conformations, pre-fusion (pre-F) and post-fusion (post-F). Although 50% of the surface is shared, pre-F contains highly neutralization-sensitive antigenic sites not present on post-F. Recent advancement of several subunit F-based vaccine trials has spurred interest in quantifying and understanding the protective potential of antibodies directed to individual antigenic sites. Monoclonal antibody competition ELISAs are being used to measure these endpoints, but the impact of F conformation and competition from antibodies binding to adjacent antigenic sites has not been thoroughly investigated. Since this information is critical for interpreting clinical trial outcomes and defining serological correlates of protection, we optimized assays to evaluate D25-competing antibodies (DCA) to antigenic site Ø on pre-F, and compared readouts of palivizumab-competing antibodies (PCA) to site II on both pre-F and post-F. We show that antibodies to adjacent antigenic sites can contribute to DCA and PCA readouts, and that cross-competition from non-targeted sites is especially confounding when PCA is measured using a post-F substrate. While measuring DCA and PCA levels may be useful to delineate the role of antibodies targeting the apex and side of the F protein, respectively, the assay limitations and caveats should be considered when conducting immune monitoring during vaccine trials and defining correlates of protection.
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http://dx.doi.org/10.3390/vaccines7010023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6466065PMC
February 2019

Memory Inflation Drives Tissue-Resident Memory CD8 T Cell Maintenance in the Lung After Intranasal Vaccination With Murine Cytomegalovirus.

Front Immunol 2018 14;9:1861. Epub 2018 Aug 14.

Viral Pathogenesis Laboratory, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, United States.

Tissue-resident memory T (T) cells provide first-line defense against invading pathogens encountered at barrier sites. In the lungs, T cells protect against respiratory infections, but wane more quickly than T cells in other tissues. This lack of a sustained T population in the lung parenchyma explains, at least in part, why infections with some pathogens, such as influenza virus and respiratory syncytial virus (RSV), recur throughout life. Intranasal (IN) vaccination with a murine cytomegalovirus (MCMV) vector expressing the M protein of RSV (MCMV-M) has been shown to elicit robust populations of CD8 T cells that accumulate over time and mediate early viral clearance. To extend this finding, we compared the inflationary CD8 T cell population elicited by MCMV-M vaccination with a conventional CD8 T cell population elicited by an MCMV vector expressing the M2 protein of RSV (MCMV-M2). Vaccination with MCMV-M2 induced a population of M2-specific CD8 T cells that waned rapidly, akin to the M2-specific CD8 T cell population elicited by infection with RSV. In contrast to the natural immunodominance profile, however, coadministration of MCMV-M and MCMV-M2 did not suppress the M-specific CD8 T cell response, suggesting that progressive expansion was driven by continuous antigen presentation, irrespective of the competitive or regulatory effects of M2-specific CD8 T cells. Moreover, effective viral clearance mediated by M-specific CD8 T cells was not affected by the coinduction of M2-specific CD8 T cells. These data show that memory inflation is required for the maintenance of CD8 T cells in the lungs after IN vaccination with MCMV.
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http://dx.doi.org/10.3389/fimmu.2018.01861DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6102355PMC
September 2019

Use of Hemagglutinin Stem Probes Demonstrate Prevalence of Broadly Reactive Group 1 Influenza Antibodies in Human Sera.

Sci Rep 2018 06 5;8(1):8628. Epub 2018 Jun 5.

Vaccine Research Center, National Institute of Allergy and Infectious Diseases, Bethesda, MD, 20892, USA.

A better understanding of the seroprevalence and specificity of influenza HA stem-directed broadly neutralizing antibodies (bNAbs) in the human population could significantly inform influenza vaccine design efforts. Here, we utilized probes comprising headless, HA stabilized stem (SS) to determine the prevalence, binding and neutralization breadth of antibodies directed to HA stem-epitope in a cross-sectional analysis of the general population. Five group-1 HA SS probes, representing five subtypes, were chosen for this analyses. Eighty-four percent of samples analyzed had specific reactivity to at least one probe, with approximately 60% of the samples reactive to H1 probes, and up to 45% reactive to each of the non-circulating subtypes. Thirty percent of analyzed sera had cross-reactivity to at least four of five probes and this reactivity could be blocked by competing with F10 bNAb. Binding cross-reactivity in sera samples significantly correlated with frequency of H1H5 cross-reactive B cells. Interestingly, only 33% of the cross-reactive sera neutralized both H1N1 and H5N1 pseudoviruses. Cross-reactive and neutralizing antibodies were more prevalent in individuals >50 years of age. Our data demonstrate the need to use multiple HA-stem probes to assess for broadly reactive antibodies. Further, a universal vaccine could be designed to boost pre-existing B-cells expressing stem-directed bNAbs.
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http://dx.doi.org/10.1038/s41598-018-26538-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5988737PMC
June 2018

Chemical Control over T-Cell Activation in Vivo Using Deprotection of trans-Cyclooctene-Modified Epitopes.

ACS Chem Biol 2018 06 11;13(6):1569-1576. Epub 2018 May 11.

Leiden Institute of Chemistry and The Institute for Chemical Immunology , Leiden University , Einsteinweg 55 , 2333 CC Leiden , The Netherlands.

Activation of a cytotoxic T-cell is a complex multistep process, and tools to study the molecular events and their dynamics that result in T-cell activation in situ and in vivo are scarce. Here, we report the design and use of conditional epitopes for time-controlled T-cell activation in vivo. We show that trans-cyclooctene-protected SIINFEKL (with the lysine amine masked) is unable to elicit the T-cell response characteristic for the free SIINFEKL epitope. Epitope uncaging by means of an inverse-electron demand Diels-Alder (IEDDA) event restored T-cell activation and provided temporal control of T-cell proliferation in vivo.
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http://dx.doi.org/10.1021/acschembio.8b00155DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6006443PMC
June 2018

Pulmonary Dendritic Cell Subsets Shape the Respiratory Syncytial Virus-Specific CD8+ T Cell Immunodominance Hierarchy in Neonates.

J Immunol 2017 01 28;198(1):394-403. Epub 2016 Nov 28.

Viral Pathogenesis Laboratory, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892.

Young infants are generally more susceptible to viral infections and experience more severe disease than do adults. CD8 T cells are important for viral clearance, and although often ineffective in neonates they can be protective when adequately stimulated. Using a murine CB6F1/J hybrid model of respiratory syncytial virus (RSV) infection, we previously demonstrated that the CD8 T cell immunodominance hierarchy to two RSV-derived epitopes, KM2 and DM, was determined by the age at infection. To determine whether age-dependent RSV-specific CD8 T cell responses could be modified through enhanced innate signaling, we used TLR4 or TLR9 agonist treatment at the time of infection, which remarkably changed the neonatal codominant response to an adult-like KM2 CD8 T cell immunodominant response. This shift was associated with an increase in the number of conventional dendritic cells, CD11b and CD103 dendritic cells, in the lung-draining lymph node, as well as increased expression of the costimulatory molecule CD86. The magnitude of the KM2 CD8 T cell response in TLR agonist-treated neonates could be blocked with Abs against CD80 and CD86. These studies demonstrate the age-dependent function of conventional dendritic cells, their role in determining immunodominance hierarchy, and epitope-specific CD8 T cell requirements for costimulation, all of which influence the immune response magnitude. The unique impact of TLR agonists on neonatal T cell responses is important to consider for RSV vaccines designed for young infants.
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http://dx.doi.org/10.4049/jimmunol.1600486DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5849234PMC
January 2017

Thermoresponsive Polymer Nanoparticles Co-deliver RSV F Trimers with a TLR-7/8 Adjuvant.

Bioconjug Chem 2016 10 22;27(10):2372-2385. Epub 2016 Sep 22.

Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health , Bethesda, Maryland 20892, United States.

Structure-based vaccine design has been used to develop immunogens that display conserved neutralization sites on pathogens such as HIV-1, respiratory syncytial virus (RSV), and influenza. Improving the immunogenicity of these designed immunogens with adjuvants will require formulations that do not alter protein antigenicity. Here, we show that nanoparticle-forming thermoresponsive polymers (TRP) allow for co-delivery of RSV fusion (F) protein trimers with Toll-like receptor 7 and 8 agonists (TLR-7/8a) to enhance protective immunity. Although primary amine conjugation of TLR-7/8a to F trimers severely disrupted the recognition of critical neutralizing epitopes, F trimers site-selectively coupled to TRP nanoparticles retained appropriate antigenicity and elicited high titers of prefusion-specific, T1 isotype anti-RSV F antibodies following vaccination. Moreover, coupling F trimers to TRP delivering TLR-7/8a resulted in ∼3-fold higher binding and neutralizing antibody titers than soluble F trimers admixed with TLR-7/8a and conferred protection from intranasal RSV challenge. Overall, these data show that TRP nanoparticles may provide a broadly applicable platform for eliciting neutralizing antibodies to structure-dependent epitopes on RSV, influenza, HIV-1, or other pathogens.
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http://dx.doi.org/10.1021/acs.bioconjchem.6b00370DOI Listing
October 2016

Determinants of early life immune responses to RSV infection.

Curr Opin Virol 2016 Feb 15;16:151-157. Epub 2016 Mar 15.

Vaccine Research Center, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, MD, USA.

Respiratory syncytial virus causes significant morbidity and mortality in both developed and developing countries, and a vaccine that adequately protects from severe disease remains an important unmet need. RSV disease has an inordinate impact on the very young, and the physical and immunological immaturity of early life complicates vaccine design. Defining and targeting the functional capacities of early life immune responses and controlling responses during primary antigen exposure with selected vaccine delivery approaches will be important for protecting infants by active immunization. Alternatively, vaccination of older children and pregnant mothers may ameliorate disease burden indirectly until infants reach about six months of age, when they can generate more effective anti-RSV immune responses.
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http://dx.doi.org/10.1016/j.coviro.2016.01.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4821759PMC
February 2016

Phenotype and Hierarchy of Two Transgenic T Cell Lines Targeting the Respiratory Syncytial Virus KdM282-90 Epitope Is Transfer Dose-Dependent.

PLoS One 2016 11;11(1):e0146781. Epub 2016 Jan 11.

Vaccine Research Center, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, Maryland, United States of America.

In this study, we compared two lines of transgenic CD8+ T cells specific for the same KdM282-90 epitope of respiratory syncytial virus in the CB6F1 hybrid mouse model. Here we found that these two transgenic lines had similar in vivo abilities to control viral load after respiratory syncytial virus infection using adoptive transfer. Transfer of the TRBV13-2 line resulted in higher levels of IL-6 and MIP1-α in the lung than TRBV13-1 transfer. Interestingly, when large numbers of cells were co-transferred, the lines formed a hierarchy, with TRBV13-2 being immunodominant over TRBV13-1 in the mediastinal lymph node despite no identifiable difference in proliferation or apoptosis between the lines. This hierarchy was not established when lower cell numbers were transferred. The phenotype and frequency of proliferating cells were also cell transfer dose-dependent with higher percentages of CD127loCD62LloKLRG1lo and proliferating cells present when lower numbers of cells were transferred. These results illustrate the importance of cell number in adoptive transfer experiments and its influence on the phenotype and hierarchy of the subsequent T cell response.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0146781PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4708989PMC
July 2016

Viruslike Particles Encapsidating Respiratory Syncytial Virus M and M2 Proteins Induce Robust T Cell Responses.

ACS Biomater Sci Eng 2016 3;2(12):2324-2332. Epub 2016 Nov 3.

Department of Chemistry, Indiana University, 800 East Kirkwood Avenue, Bloomington, Indiana 47405, United States.

Subunit vaccines provide a safe, focused alternative to conventional vaccines. However, these vaccines often require significant adjuvants and are particularly hard to target toward cytotoxic T lymphocyte (CTL) immunity. Viruslike particles (VLPs) provide biomaterial scaffolds with pathogen-like polyvalent structures making them useful platforms for biomimetic antigen delivery to the immune system. Encapsidation of antigens within VLPs has been shown to enhance antigen availability for CD8 T cell responses. Here, we examine the potential to generate complex responses to multiple subunit antigens localized within the same VLP particle. Two proteins of respiratory syncytial virus (RSV) with well-characterized CD8 T cell responses, the matrix (M) and matrix 2 (M2) proteins, were successfully coencapsidated within the P22 VLP. Upon intranasal administration in mice, the particles stimulated CD8 T cell memory responses against both antigens. In addition, vaccination elicited tissue-resident T cell populations. Upon subsequent RSV challenge, P22-M/M2-treated mice displayed significantly reduced lung viral titers. This demonstrates the utility of the P22 VLP in directing immune responses to multiple encapsidated viral antigens, demonstrating the potential of this technology to facilitate immunity to multiple targets simultaneously.
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http://dx.doi.org/10.1021/acsbiomaterials.6b00532DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5777520PMC
November 2016

Bioorthogonal deprotection on the dendritic cell surface for chemical control of antigen cross-presentation.

Angew Chem Int Ed Engl 2015 May 17;54(19):5628-31. Epub 2015 Mar 17.

Leiden Institute of Chemistry and The Institute for Chemical Immunology, Leiden University, Einsteinweg 55, 2333 CC Leiden (The Netherlands).

The activation of CD8(+) T-cells requires the uptake of exogenous polypeptide antigens and proteolytic processing of these antigens to octamer or nonamer peptides, which are loaded on MHC-I complexes and presented to the T-cell. By using an azide as a bioorthogonal protecting group rather than as a ligation handle, masked antigens were generated-antigens that are not recognized by their cognate T-cell unless they are deprotected on the cell using a Staudinger reduction.
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http://dx.doi.org/10.1002/anie.201500301DOI Listing
May 2015

CD8+ TCR transgenic strains expressing public versus private TCR targeting the respiratory syncytial virus K(d)M2(82-90) epitope demonstrate similar functional profiles.

PLoS One 2014 4;9(6):e99249. Epub 2014 Jun 4.

Vaccine Research Center, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, Maryland, United States of America.

Our previous work has characterized the functional and clonotypic features of two respiratory syncytial virus (RSV) epitope-specific T cell responses in mice. Following single-cell sequencing, we selected T cell receptor sequences to represent both a public and a private clone specific for the dominant K(d)M2(82-90) epitope for the generation of T cell receptor transgenic (TCR Tg) mice. We evaluated cells from these TCR Tg strains for three major functions of CD8+ T cells: proliferation, cytokine production and cytolytic activity. In vitro comparisons of the functional characteristics of T cells from the newly-generated mice demonstrated many similarities in their responsiveness to cognate antigen stimulation. Cells from both TRBV13-1 (private) and TRBV13-2 (public) TCR Tg mice had similar affinity, and proliferated similarly in vitro in response to cognate antigen stimulation. When transferred to BALB/c mice, cells from both strains demonstrated extensive proliferation in mediastinal lymph nodes following RSV infection, with TRBV13-2 demonstrating better in vivo proliferation. Both strains similarly expressed cytokines and chemokines following stimulation, and had similar Granzyme B and perforin expression, however cells expressing TRBV13-1 demonstrated better cytolytic activity than TRBV13-2 cells. These new, well-characterized mouse strains provide new opportunities to study molecular mechanisms that control the phenotype and function of CD8+ T cell responses.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0099249PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4045939PMC
October 2015

Quantitative and qualitative deficits in neonatal lung-migratory dendritic cells impact the generation of the CD8+ T cell response.

PLoS Pathog 2014 Feb 13;10(2):e1003934. Epub 2014 Feb 13.

Vaccine Research Center, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, Maryland, United States of America.

CD103+ and CD11b+ populations of CD11c+MHCIIhi murine dendritic cells (DCs) have been shown to carry antigens from the lung through the afferent lymphatics to mediastinal lymph nodes (MLN). We compared the responses of these two DC populations in neonatal and adult mice following intranasal infection with respiratory syncytial virus. The response in neonates was dominated by functionally-limited CD103+ DCs, while CD11b+ DCs were diminished in both number and function compared to adults. Infecting mice at intervals through the first three weeks of life revealed an evolution in DC phenotype and function during early life. Using TCR transgenic T cells with two different specificities to measure the ability of CD103+ DC to induce epitope-specific CD8+ T cell responses, we found that neonatal CD103+ DCs stimulate proliferation in a pattern distinct from adult CD103+ DCs. Blocking CD28-mediated costimulatory signals during adult infection demonstrated that signals from this costimulatory pathway influence the hierarchy of the CD8+ T cell response to RSV, suggesting that limited costimulation provided by neonatal CD103+ DCs is one mechanism whereby neonates generate a distinct CD8+ T cell response from that of adults.
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http://dx.doi.org/10.1371/journal.ppat.1003934DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3923758PMC
February 2014

Consequences of immature and senescent immune responses for infection with respiratory syncytial virus.

Curr Top Microbiol Immunol 2013 ;372:211-31

Vaccine Research Center, NIAID, NIH, Bethesda, MD, USA.

Infants in the first 6 months of life and older adults (>65 years of age) are disproportionately burdened with respiratory syncytial virus (RSV)-associated morbidity and mortality. While other factors play a role in the risk these groups assume, shortcomings of the immune response make a substantial contribution to the predisposition to severe disease. Ineffectual antibody production with misdirected cytokine responses and excess inflammation in the airways are common to both groups. However, the mechanisms underlying these immune responses differ between infants and older adults and need to be better understood. Preventative approaches to decreasing the burden of disease are preferable to therapeutic intervention and effective vaccination strategies will need to target the strengths of the immune responses in these populations.
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http://dx.doi.org/10.1007/978-3-642-38919-1_11DOI Listing
April 2014

Neonatal CD8 T-cell hierarchy is distinct from adults and is influenced by intrinsic T cell properties in respiratory syncytial virus infected mice.

PLoS Pathog 2011 Dec 1;7(12):e1002377. Epub 2011 Dec 1.

Vaccine Research Center, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, Maryland, United States of America.

Following respiratory syncytial virus infection of adult CB6F1 hybrid mice, a predictable CD8+ T cell epitope hierarchy is established with a strongly dominant response to a K(d)-restricted peptide (SYIGSINNI) from the M2 protein. The response to K(d)M2(82-90) is ∼5-fold higher than the response to a subdominant epitope from the M protein (NAITNAKII, D(b)M(187-195)). After infection of neonatal mice, a distinctly different epitope hierarchy emerges with codominant responses to K(d)M2(82-90) and D(b)M(187-195). Adoptive transfer of naïve CD8+ T cells from adults into congenic neonates prior to infection indicates that intrinsic CD8+ T cell factors contribute to age-related differences in hierarchy. Epitope-specific precursor frequency differs between adults and neonates and influences, but does not predict the hierarchy following infection. Additionally, dominance of K(d)M2(82-90)-specific cells does not correlate with TdT activity. Epitope-specific Vβ repertoire usage is more restricted and functional avidity is lower in neonatal mice. The neonatal pattern of codominance changes after infection at 10 days of age, and rapidly shifts to the adult pattern of extreme K(d)M2(82-90)-dominance. Thus, the functional properties of T cells are selectively modified by developmental factors in an epitope-specific and age-dependent manner.
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http://dx.doi.org/10.1371/journal.ppat.1002377DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3228797PMC
December 2011

T Cell receptor clonotype influences epitope hierarchy in the CD8+ T cell response to respiratory syncytial virus infection.

J Biol Chem 2011 Feb 30;286(6):4829-41. Epub 2010 Nov 30.

Vaccine Research Center, NIAID, National Institutes of Health, Bethesda, Maryland 20892-3017, USA.

CD8+ T cell responses are important for recognizing and resolving viral infections. To better understand the selection and hierarchy of virus-specific T cell responses, we compared the T cell receptor (TCR) clonotype in parent and hybrid strains of respiratory syncytial virus-infected mice. K(d)M2(82-90) (SYIGSINNI) in BALB/c and D(b)M(187-195) (NAITNAKII) in C57Bl/6 are both dominant epitopes in parent strains but assume a distinct hierarchy, with K(d)M2(82-90) dominant to D(b)M(187-195) in hybrid CB6F1/J mice. The dominant K(d)M2(82-90) response is relatively public and is restricted primarily to the highly prevalent Vβ13.2 in BALB/c and hybrid mice, whereas D(b)M(187-195) responses in C57BL/6 mice are relatively private and involve multiple Vβ subtypes, some of which are lost in hybrids. A significant frequency of TCR CDR3 sequences in the D(b)M(187-195) response have a distinct "(D/E)WG" motif formed by a limited number of recombination strategies. Modeling of the dominant epitope suggested a flat, featureless structure, but D(b)M(187-195) showed a distinctive structure formed by Lys(7). The data suggest that common recombination events in prevalent Vβ genes may provide a numerical advantage in the T cell response and that distinct epitope structures may impose more limited options for successful TCR selection. Defining how epitope structure is interpreted to inform T cell function will improve the design of future gene-based vaccines.
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http://dx.doi.org/10.1074/jbc.M110.191437DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3039322PMC
February 2011
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