scholarly journals Immune responses to SARS-CoV-2 infection in hospitalized pediatric and adult patients

2020 ◽  
Vol 12 (564) ◽  
pp. eabd5487 ◽  
Author(s):  
Carl A. Pierce ◽  
Paula Preston-Hurlburt ◽  
Yile Dai ◽  
Clare Burn Aschner ◽  
Natalia Cheshenko ◽  
...  
Keyword(s):  
T Cells ◽  
Immune Responses ◽  
Pediatric Patients ◽  
Neutralizing Antibody ◽  
Children And Youth ◽  
Spike Protein ◽  
Serum Concentrations ◽  
Antiviral Immune Response ◽  
Antibody Titers ◽  
Ifn Γ

Children and youth infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have milder disease than do adults, and even among those with the recently described multisystem inflammatory syndrome, mortality is rare. The reasons for the differences in clinical manifestations are unknown but suggest that age-dependent factors may modulate the antiviral immune response. We compared cytokine, humoral, and cellular immune responses in pediatric (children and youth, age <24 years) (n = 65) and adult (n = 60) patients with coronavirus disease 2019 (COVID-19) at a metropolitan hospital system in New York City. The pediatric patients had a shorter length of stay, decreased requirement for mechanical ventilation, and lower mortality compared to adults. The serum concentrations of interleukin-17A (IL-17A) and interferon-γ (IFN-γ), but not tumor necrosis factor–α (TNF-α) or IL-6, were inversely related to age. Adults mounted a more robust T cell response to the viral spike protein compared to pediatric patients as evidenced by increased expression of CD25+ on CD4+ T cells and the frequency of IFN-γ+ CD4+ T cells. Moreover, serum neutralizing antibody titers and antibody-dependent cellular phagocytosis were higher in adults compared to pediatric patients with COVID-19. The neutralizing antibody titer correlated positively with age and negatively with IL-17A and IFN-γ serum concentrations. There were no differences in anti-spike protein antibody titers to other human coronaviruses. Together, these findings demonstrate that the poor outcome in hospitalized adults with COVID-19 compared to children may not be attributable to a failure to generate adaptive immune responses.

scholarly journals Severe Acute Respiratory Syndrome-Associated Coronavirus Vaccines Formulated with Delta Inulin Adjuvants Provide Enhanced Protection while Ameliorating Lung Eosinophilic Immunopathology

2014 ◽  
Vol 89 (6) ◽  
pp. 2995-3007 ◽  
Author(s):  
Yoshikazu Honda-Okubo ◽  
Dale Barnard ◽  
Chun Hao Ong ◽  
Bi-Hung Peng ◽  
Chien-Te Kent Tseng ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Neutralizing Antibodies ◽  
Case Fatality ◽  
Neutralizing Antibody ◽  
The Elderly ◽  
Spike Protein ◽  
Interleukin 4 ◽  
Unique Problem ◽  
Antibody Titers ◽  
Ifn Γ

ABSTRACTAlthough the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) epidemic was controlled by nonvaccine measures, coronaviruses remain a major threat to human health. The design of optimal coronavirus vaccines therefore remains a priority. Such vaccines present major challenges: coronavirus immunity often wanes rapidly, individuals needing to be protected include the elderly, and vaccines may exacerbate rather than prevent coronavirus lung immunopathology. To address these issues, we compared in a murine model a range of recombinant spike protein or inactivated whole-virus vaccine candidates alone or adjuvanted with either alum, CpG, or Advax, a new delta inulin-based polysaccharide adjuvant. While all vaccines protected against lethal infection, addition of adjuvant significantly increased serum neutralizing-antibody titers and reduced lung virus titers on day 3 postchallenge. Whereas unadjuvanted or alum-formulated vaccines were associated with significantly increased lung eosinophilic immunopathology on day 6 postchallenge, this was not seen in mice immunized with vaccines formulated with delta inulin adjuvant. Protection against eosinophilic immunopathology by vaccines containing delta inulin adjuvants correlated better with enhanced T-cell gamma interferon (IFN-γ) recall responses rather than reduced interleukin-4 (IL-4) responses, suggesting that immunopathology predominantly reflects an inadequate vaccine-induced Th1 response. This study highlights the critical importance for development of effective and safe coronavirus vaccines of selection of adjuvants based on the ability to induce durable IFN-γ responses.IMPORTANCECoronaviruses such as SARS-CoV and Middle East respiratory syndrome-associated coronavirus (MERS-CoV) cause high case fatality rates and remain major human public health threats, creating a need for effective vaccines. While coronavirus antigens that induce protective neutralizing antibodies have been identified, coronavirus vaccines present a unique problem in that immunized individuals when infected by virus can develop lung eosinophilic pathology, a problem that is further exacerbated by the formulation of SARS-CoV vaccines with alum adjuvants. This study shows that formulation of SARS-CoV spike protein or inactivated whole-virus vaccines with novel delta inulin-based polysaccharide adjuvants enhances neutralizing-antibody titers and protection against clinical disease but at the same time also protects against development of lung eosinophilic immunopathology. It also shows that immunity achieved with delta inulin adjuvants is long-lived, thereby overcoming the natural tendency for rapidly waning coronavirus immunity. Thus, delta inulin adjuvants may offer a unique ability to develop safer and more effective coronavirus vaccines.

scholarly journals Gamma Interferon Produced by Antigen-Specific CD4+ T Cells Regulates the Mucosal Immune Responses to Citrobacter rodentium Infection

2010 ◽  
Vol 78 (6) ◽  
pp. 2653-2666 ◽  
Author(s):  
Hideyuki Shiomi ◽  
Atsuhiro Masuda ◽  
Shin Nishiumi ◽  
Masayuki Nishida ◽  
Tetsuya Takagawa ◽  
...  
Keyword(s):  
T Cells ◽  
Immune Responses ◽  
Gamma Interferon ◽  
Immune Defense ◽  
Interleukin 4 ◽  
Mucosal Inflammation ◽  
Citrobacter Rodentium ◽  
Macrophage Phagocytosis ◽  
Ifn Γ ◽  
Deficient Mice

ABSTRACT Citrobacter rodentium, a murine model pathogen for enteropathogenic Escherichia coli, colonizes the surface of intestinal epithelial cells and causes mucosal inflammation. This bacterium is an ideal model for investigating pathogen-host immune interactions in the gut. It is well known that gene transcripts for Th1 cytokines are highly induced in colonic tissue from mice infected with C. rodentium. However, it remains to be seen whether the Th1 or Th2 cytokines produced by antigen-specific CD4+ T cells provide effective regulation of the host immune defense against C. rodentium infection. To investigate the antigen-specific immune responses, C. rodentium expressing ovalbumin (OVA-C. rodentium), a model antigen, was generated and used to define antigen-specific responses under gamma interferon (IFN-γ)-deficient or interleukin-4 (IL-4)-deficient conditions in vivo. The activation of antigen-specific CD4+ T cells and macrophage phagocytosis were evaluated in the presence of IFN-γ or IL-4 in vitro. IFN-γ-deficient mice exhibited a loss of body weight and a higher bacterial concentration in feces during OVA-C. rodentium infection than C57BL/6 (wild type) or IL-4-deficient mice. This occurred through the decreased efficiency of macrophage phagocytosis and the activation of antigen-specific CD4+ T cells. Furthermore, a deficiency in antigen-specific CD4+ T-cell-expressed IFN-γ led to a higher susceptibility to mucosal and gut-derived systemic OVA-C. rodentium infection. These results show that the IFN-γ produced by antigen-specific CD4+ T cells plays an important role in the defense against C. rodentium.

scholarly journals The effect of Kefir on The Immune Response of Healthy Volunteers In Vitro

2017 ◽  
Vol 3 (2) ◽  
pp. 28
Author(s):  
Desie Dwi Wisudanti
Keyword(s):  
Immune Response ◽  
T Cells ◽  
Immune Responses ◽  
Healthy Volunteers ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Fermented Milk ◽  
Bioactive Components ◽  
Ifn Γ

Kefir is a functional foodstuff of probiotics, made from fermented milk with kefir grains containing various types of beneficial bacteria and yeast. There have been many studies on the effects of oral kefir on the immune system, but few studies have shown the effect of bioactive components from kefir (peptides and exopolysaccharides/ kefiran), on immune responses. The purpose of this study was to prove the effect of kefir supernatant from milk goat on healthy immune volunteer response in vitro. The study was conducted on 15 healthy volunteers, then isolated PBMC from whole blood, then divided into 5 groups (K-, P1, P2, P3 and P4) before culture was done for 4 days. The harvested cells from culture were examined for the percentage of CD4+ T cells, CD8+ T cells, IFN-γ, IL-4 using flowsitometry and IL-2 levels, IL-10 using the ELISA method. The results obtained that kefir do not affect the percentage of CD4+ T cells and CD8+ T cells. The higher the concentration of kefir given, the higher levels of secreted IFN- γ and IL-4, but a decrease in IL-2 levels. Significant enhancement occurred at levels of IL-10 culture PBMC given kefir with various concentrations (p <0.01), especially at concentrations of 1%. These results also show the important effects of kefir bioactive components on immune responses. The conclusion of this study is that kefir can improve the immune response, through stimulation of IL-10 secretion in vitro.

scholarly journals Evaluation of Feline Monocyte-Derived Dendritic Cells Loaded with Internally Inactivated Virus as a Vaccine against Feline Immunodeficiency Virus

2008 ◽  
Vol 15 (3) ◽  
pp. 452-459 ◽  
Author(s):  
Giulia Freer ◽  
Donatella Matteucci ◽  
Paola Mazzetti ◽  
Francesca Tarabella ◽  
Valentina Catalucci ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
Immune Responses ◽  
Immune Functions ◽  
Peripheral Blood Mononuclear ◽  
Challenge Virus ◽  
Homologous Virus ◽  
Antibody Titers ◽  
Cellular Immune ◽  
Antigen Presenting

ABSTRACT Dendritic cells are the only antigen-presenting cells that can present exogenous antigens to both helper and cytolytic T cells and prime Th1-type or Th2-type cellular immune responses. Given their unique immune functions, dendritic cells are considered attractive “live adjuvants” for vaccination and immunotherapy against cancer and infectious diseases. The present study was carried out to assess whether the reinjection of autologous monocyte-derived dendritic cells loaded with an aldithriol-2-inactivated primary isolate of feline immune deficiency virus (FIV) was able to elicit protective immune responses against the homologous virus in naive cats. Vaccine efficacy was assessed by monitoring immune responses and, finally, by challenge with the homologous virus of vaccinated, mock-vaccinated, and healthy cats. The outcome of challenge was followed by measuring cellular and antibody responses and viral and proviral loads and quantitating FIV by isolation and a count of CD4+/CD8+ T cells in blood. Vaccinated animals exhibited clearly evident FIV-specific peripheral blood mononuclear cell proliferation and antibody titers in response to immunization; however, they became infected with the challenge virus at rates comparable to those of control animals.

scholarly journals NK T Cells Contribute to Expansion of CD8+ T Cells and Amplification of Antiviral Immune Responses to Respiratory Syncytial Virus

2002 ◽  
Vol 76 (9) ◽  
pp. 4294-4303 ◽  
Author(s):  
Teresa R. Johnson ◽  
Seokmann Hong ◽  
Luc Van Kaer ◽  
Yasuhiko Koezuka ◽  
Barney S. Graham
Keyword(s):  
T Cells ◽  
Respiratory Syncytial Virus ◽  
T Lymphocytes ◽  
Immune Responses ◽  
Natural Killer ◽  
Viral Clearance ◽  
Normal Numbers ◽  
Nk T Cells ◽  
Ifn Γ ◽  
Syncytial Virus

ABSTRACT CD1d-deficient mice have normal numbers of T lymphocytes and natural killer cells but lack Vα14+ natural killer T cells. Respiratory syncytial virus (RSV) immunopathogenesis was evaluated in 129×C57BL/6, C57BL/6, and BALB/c CD1d−/− mice. CD8+ T lymphocytes were reduced in CD1d−/− mice of all strains, as shown by cell surface staining and major histocompatibility complex class I tetramer analysis, and resulted in strain-specific alterations in illness, viral clearance, and gamma interferon (IFN-γ) production. Transient activation of NK T cells in CD1d+/+ mice by α-GalCer resulted in reduced illness and delayed viral clearance. These data suggest that early IFN-γ production and efficient induction of CD8+-T-cell responses during primary RSV infection require CD1d-dependent events. We also tested the ability of α-GalCer as an adjuvant to modulate the type 2 immune responses induced by RSV glycoprotein G or formalin-inactivated RSV immunization. However, immunized CD1-deficient or α-GalCer-treated wild-type mice did not exhibit diminished disease following RSV challenge. Rather, some disease parameters, including cytokine production, eosinophilia, and viral clearance, were increased. These findings indicate that CD1d-dependent NK T cells play a role in expansion of CD8+ T cells and amplification of antiviral responses to RSV.

scholarly journals Mechanisms of Dysregulated Humoral and Cellular Immunity by SARS-CoV-2

Pathogens ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 1027
Author(s):  
Nima Taefehshokr ◽  
Sina Taefehshokr ◽  
Bryan Heit
Keyword(s):  
Immune Response ◽  
T Cells ◽  
T Cell ◽  
Cell Function ◽  
Adaptive Immune Response ◽  
Neutralizing Antibody ◽  
Humoral And Cellular Immunity ◽  
Cell Responses ◽  
Adaptive Immune ◽  
Antibody Titers

The current coronavirus disease 2019 (COVID-19) pandemic, a disease caused by severe acute respiratory syndrome corona virus 2 (SARS-CoV-2), was first identified in December 2019 in China, and has led to thousands of mortalities globally each day. While the innate immune response serves as the first line of defense, viral clearance requires activation of adaptive immunity, which employs B and T cells to provide sanitizing immunity. SARS-CoV-2 has a potent arsenal of mechanisms used to counter this adaptive immune response through processes, such as T cells depletion and T cell exhaustion. These phenomena are most often observed in severe SARS-CoV-2 patients, pointing towards a link between T cell function and disease severity. Moreover, neutralizing antibody titers and memory B cell responses may be short lived in many SARS-CoV-2 patients, potentially exposing these patients to re-infection. In this review, we discuss our current understanding of B and T cells immune responses and activity in SARS-CoV-2 pathogenesis.

scholarly journals Vector Order Determines Protection against Pathogenic Simian Immunodeficiency Virus Infection in a Triple-Component Vaccine by Balancing CD4+ and CD8+ T-Cell Responses

2017 ◽  
Vol 91 (23) ◽  
Author(s):  
Ulrike Sauermann ◽  
Antonia Radaelli ◽  
Nicole Stolte-Leeb ◽  
Katharina Raue ◽  
Massimiliano Bissa ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
T Cell Activation ◽  
Cell Activation ◽  
T Cell Responses ◽  
Target Cells ◽  
Aids Vaccine ◽  
Cell Responses ◽  
Antibody Titers

ABSTRACT An effective AIDS vaccine should elicit strong humoral and cellular immune responses while maintaining low levels of CD4+ T-cell activation to avoid the generation of target cells for viral infection. The present study investigated two prime-boost regimens, both starting vaccination with single-cycle immunodeficiency virus, followed by two mucosal boosts with either recombinant adenovirus (rAd) or fowlpox virus (rFWPV) expressing SIVmac239 or SIVmac251 gag/pol and env genes, respectively. Finally, vectors were switched and systemically administered to the reciprocal group of animals. Only mucosal rFWPV immunizations followed by systemic rAd boost significantly protected animals against a repeated low-dose intrarectal challenge with pathogenic SIVmac251, resulting in a vaccine efficacy (i.e., risk reduction per exposure) of 68%. Delayed viral acquisition was associated with higher levels of activated CD8+ T cells and Gag-specific gamma interferon (IFN-γ)-secreting CD8+ cells, low virus-specific CD4+ T-cell responses, and low Env antibody titers. In contrast, the systemic rFWPV boost induced strong virus-specific CD4+ T-cell activity. rAd and rFWPV also induced differential patterns of the innate immune responses, thereby possibly shaping the specific immunity. Plasma CXCL10 levels after final immunization correlated directly with virus-specific CD4+ T-cell responses and inversely with the number of exposures to infection. Also, the percentage of activated CD69+ CD8+ T cells correlated with the number of exposures to infection. Differential stimulation of the immune response likely provided the basis for the diverging levels of protection afforded by the vaccine regimen. IMPORTANCE A failed phase II AIDS vaccine trial led to the hypothesis that CD4+ T-cell activation can abrogate any potentially protective effects delivered by vaccination or promote acquisition of the virus because CD4+ T helper cells, required for an effective immune response, also represent the target cells for viral infection. We compared two vaccination protocols that elicited similar levels of Gag-specific immune responses in rhesus macaques. Only the animal group that had a low level of virus-specific CD4+ T cells in combination with high levels of activated CD8+ T cells was significantly protected from infection. Notably, protection was achieved despite the lack of appreciable Env antibody titers. Moreover, we show that both the vector and the route of immunization affected the level of CD4+ T-cell responses. Thus, mucosal immunization with FWPV-based vaccines should be considered a potent prime in prime-boost vaccination protocols.

scholarly journals Diverse Myeloid and Lymphoid Cell Subpopulations Produce Gamma Interferon during Early Innate Immune Responses to Francisella tularensis Live Vaccine Strain

2008 ◽  
Vol 76 (9) ◽  
pp. 4311-4321 ◽  
Author(s):  
Roberto De Pascalis ◽  
Betsy C. Taylor ◽  
Karen L. Elkins
Keyword(s):  
T Cells ◽  
Immune Responses ◽  
Nk Cells ◽  
Francisella Tularensis ◽  
Vaccine Strain ◽  
Live Vaccine ◽  
Innate Immune ◽  
Live Vaccine Strain ◽  
Ifn Γ ◽  
Cell Subpopulations

ABSTRACT Francisella tularensis, a small gram-negative intracellular bacterium responsible for causing tularemia, is highly pathogenic and classified as a category A agent of bioterrorism. As for other intracellular pathogens, successful protective immune responses to Francisella tularensis require rapid and efficient induction of gamma interferon (IFN-γ) production. Studies using intracellular bacteria such as Listeria monocytogenes as well as Francisella suggest that natural killer (NK) and T cells are important sources of IFN-γ. However, comprehensive characterization of specific sources of IFN-γ produced during Francisella infection in vivo remains incomplete, and depletion of NK cells before infection of mice with the F. tularensis live vaccine strain (LVS) has little impact on the course or outcome of infection. In this study, we determined the cell subpopulations that respond quickly to intradermal F. tularensis LVS infection of mice by producing IFN-γ within hours to a few days. Splenic and liver lymphocytes were obtained from LVS-infected mice and analyzed for IFN-γ mRNA by reverse transcription-PCR, for intracellular cytokine expression by multiparameter flow cytometry, and for ex vivo production of IFN-γ protein by enzyme-linked immunosorbent assay. Cells producing IFN-γ were readily detectable by day 3 after infection, and numbers progressively increased through days 5 to 7. Importantly, the cell types responsible for IFN-γ production were much more varied than expected: these included not only NK cells and T cells, which might be predicted, but also other cells, including dendritic cells (DCs), “NK DCs,” NK T cells, and neutrophils. Most importantly, since RAG-1 knockout mice appeared to exhibit a frequency of IFN-γ-producing cells comparable to that of intact wild-type mice, early IFN-γ production by innate immune cells does not depend on the presence of T or B cells.

scholarly journals Molecular and Immunological Significance of Chimpanzee Major Histocompatibility Complex Haplotypes for Hepatitis C Virus Immune Response and Vaccination Studies

2002 ◽  
Vol 76 (12) ◽  
pp. 6093-6103 ◽  
Author(s):  
Eishiro Mizukoshi ◽  
Michelina Nascimbeni ◽  
Joshua B. Blaustein ◽  
Kathleen Mihalik ◽  
Charles M. Rice ◽  
...  
Keyword(s):  
Immune Response ◽  
T Cells ◽  
Major Histocompatibility Complex ◽  
Hepatitis C ◽  
Immune Responses ◽  
Cellular Immune Responses ◽  
Functional Homology ◽  
Histocompatibility Complex ◽  
Ifn Γ ◽  
Cellular Immune

ABSTRACT The chimpanzee is a critical animal model for studying cellular immune responses to infectious pathogens such as hepatitis B and C viruses, human immunodeficiency virus, and malaria. Several candidate vaccines and immunotherapies for these infections aim at the induction or enhancement of cellular immune responses against viral epitopes presented by common human major histocompatibility complex (MHC) alleles. To identify and characterize chimpanzee MHC class I molecules that are functionally related to human alleles, we sequenced 18 different Pan troglodytes (Patr) alleles of 14 chimpanzees, 2 of them previously unknown and 3 with only partially reported sequences. Comparative analysis of Patr binding pockets and binding assays with biotinylated peptides demonstrated a molecular homology between the binding grooves of individual Patr alleles and the common human alleles HLA-A1, -A2, -A3, and -B7. Using cytotoxic T cells isolated from the blood of hepatitis C virus (HCV)-infected chimpanzees, we then mapped the Patr restriction of these HCV peptides and demonstrated functional homology between the Patr-HLA orthologues in cytotoxicity and gamma interferon (IFN-γ) release assays. Based on these results, 21 HCV epitopes were selected to characterize the chimpanzees' cellular immune response to HCV. In each case, IFN-γ-producing T cells were detectable in the blood after but not prior to HCV infection and were specifically targeted against those HCV peptides predicted by Patr-HLA homology. This study demonstrates a close functional homology between individual Patr and HLA alleles and shows that HCV infection generates HCV peptides that are recognized by both chimpanzees and humans with Patr and HLA orthologues. These results are relevant for the design and evaluation of vaccines in chimpanzees that can now be selected according to the most frequent human MHC haplotypes.

Identification of the AAV2 Capsid CD8+ T Cell Epitope in C57BL/6 Mice.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3188-3188
Author(s):  
Denise E. Sabatino ◽  
Federico Mingozzi ◽  
Haifeng Chen ◽  
Peter Colosi ◽  
Hildegund C.J. Ertl ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Capsid Protein ◽  
Cd8 T Cells ◽  
Cell Epitope ◽  
T Cell Response ◽  
Cd8 Cells ◽  
Media Control ◽  
Ifn Γ

Abstract Recently, a clinical trial for adeno-associated virus serotype 2 (AAV2) mediated liver directed gene transfer of human Factor IX to subjects with severe hemophilia B revealed that two patients developed transient asymptomatic transaminitis following vector administration. Immunology studies in the second patient demonstrated a transient T cell response to AAV2 capsid peptides suggesting that the immune response to the AAV capsid may be related to the transient transaminitis. We hypothesized that the observations made in the human subjects were due to a CD8 T cell response to AAV2 capsid protein. Preclinical studies in mice and dogs, which are not naturally infected by wild type AAV2 viruses, did not predict these findings in the clinical study. Thus, we developed a mouse model in which we were able to mimic this phenomenon (Blood 102:493a). In an effort to further characterize the immune responses to AAV2 capsid proteins in this mouse model, we identified the T cell epitope in the AAV capsid protein recognized by murine C57Bl/6 CD8 T cells. A peptide library of AAV2 VP1 capsid peptides (n=145) that were synthesized as 15mers overlapping by 10 amino acids were divided into 6 pools each containing 24–25 peptides. C57Bl/6 mice were immunized intramuscularly with an adenovirus expressing AAV2 capsid protein. Nine days later the spleen was harvested and intracellular cytokine staining (ICS) was used to assess release of IFN-γ from CD8 T cells in response to 6 AAV2 capsid peptide pools. ICS demonstrated CD8 cells from mice immunized with Ad-AAV2 produced IFN-γ (3.5% of the CD8 cells) in response to Pool F (amino acid 119–145) while no IFN-γ release in CD8 cells was detected with Pool A to E (mean 0.28%±0.25%) compared to the media control (0.16%). This detection of IFN-γ release from CD8 T cells indicates a specific proliferation to a peptide(s) within this peptide pool (Pool F). A matrix approach was used to further define which peptide(s) contained the immunodominant epitope. Eleven small peptide pools of Pool F were created in which each peptide was represented in 2 pools. ICS of splenocytes from immunized (Ad-AAV2 capsid) C57Bl/6 mice demonstrated IFN-γ response from CD8 cells to 3 of the matrix pools corresponding to peptide 140 (PEIQYTSNYNKSVNV) and 141 (TSNYNKSVNVDFTVD) compared with media controls. To determine the exact peptide sequence that binds to the MHC Class I molecule, 9 amino acid peptides (n=7) were created that overlap peptide 140 and 141. Peptide SNYNKSVNV showed positive staining for both CD8 and IFN- γ(3.2%) compared with the six other peptides (0.14%±0.08%), media control (0.08%) and mice that were not immunized (0.11%). This epitope lies in the C terminus of the AAV2 VP1 capsid protein. Current studies using strains of mice with different MHC H2 haplotypes will allow us to determine which of the C57Bl/6 MHC alleles the epitope binds. These findings will provide us with a powerful tool for assessing immune responses to AAV capsid in the context of gene therapy. Specifically, they will allow us to determine how long immunologically detectable capsid sequences persist in an animal injected with AAV vectors. This in turn will provide a basis for a clinical study in which subjects are transiently immunosuppressed, from the time of vector injection until capsid epitopes are no longer detectable by the immune system.

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