GagCM9-Specific CD8+ T Cells Expressing Limited Public TCR Clonotypes Do Not Suppress SIV Replication In Vivo

Several lines of evidence suggest that HIV/SIV-specific CD8+ T cells play a critical role in the control of viral replication. Recently we observed high levels of viremia in Indian rhesus macaques vaccinated with a segment of SIVmac239 Gag (Gag45–269) that were subsequently infected with SIVsmE660. These seven Mamu-A*01+ animals developed CD8+ T cell responses against an immunodominant epitope in Gag, GagCM9, yet failed to control virus replication. We carried out a series of immunological and virological assays to understand why these Gag-specific CD8+ T cells could not control virus replication in vivo. GagCM9-specific CD8+ T cells from all of the animals were multifunctional and were found in the colonic mucosa. Additionally, GagCM9-specific CD8+ T cells accessed B cell follicles, the primary residence of SIV-infected cells in lymph nodes, with effector to target ratios between 20–250 GagCM9-specific CD8+ T cells per SIV-producing cell. Interestingly, vaccinated animals had few public TCR clonotypes within the GagCM9-specific CD8+ T cell population pre- and post-infection. The number of public TCR clonotypes expressed by GagCM9-specific CD8+ T cells post-infection significantly inversely correlated with chronic phase viral load. It is possible that these seven animals failed to control viral replication because of the narrow TCR repertoire expressed by the GagCM9-specific CD8+ T cell population elicited by vaccination and infection

Improved genetic stability of recombinant yellow fever 17D virus expressing a lentiviral Gag gene fragment

AbstractWe have previously designed a method to construct viable recombinant Yellow Fever (YF) 17D viruses expressing heterologous polypeptides including part of the Simian Immunodeficiency Virus (SIV) Gag protein. However, the expressed region, encompassing amino acid residues from 45 to 269, was genetically unstable. In this study, we improved the genetic stability of this recombinant YF 17D virus by introducing mutations in the IRES element localized at the 5' end of the SIV gag gene. The new stable recombinant virus elicited adaptive immune responses similar to those induced by the original recombinant virus. It is, therefore, possible to increase recombinant stability by removing functional motifs from the insert that may have deleterious effects on recombinant YF viral fitness

The majority of freshly sorted simian immunodeficiency virus (SIV)-specific CD8(+) T cells cannot suppress viral replication in SIV-infected macrophages

Submitted by Sandra Infurna ([email protected]) on 2016-08-30T13:22:40Z No. of bitstreams: 1 marlon_santana_etal_IOC_2012.pdf: 1100597 bytes, checksum: 08780ab83069cd89ca478db45b5676e7 (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2016-08-30T13:44:45Z (GMT) No. of bitstreams: 1 marlon_santana_etal_IOC_2012.pdf: 1100597 bytes, checksum: 08780ab83069cd89ca478db45b5676e7 (MD5)Made available in DSpace on 2016-08-30T13:44:45Z (GMT). No. of bitstreams: 1 marlon_santana_etal_IOC_2012.pdf: 1100597 bytes, checksum: 08780ab83069cd89ca478db45b5676e7 (MD5) Previous issue date: 2012University of Wisconsin—Madison. Department of Pathology and Laboratory Medicine. Madison, Wisconsin, USA.University of Wisconsin—Madison. Department of Pathology and Laboratory Medicine. Madison, Wisconsin, USA.University of Wisconsin—Madison. Department of Pathology and Laboratory Medicine. Madison, Wisconsin, USA.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular de Flavivírus. Rio de Janeiro, RJ, Brasil.Oregon Health Sciences University. Oregon National Primate Research Center. Vaccine and Gene Therapy Institute. Beaverton, Oregin, USA.University of Wisconsin—Madison. Department of Pathology and Laboratory Medicine. Madison, Wisconsin, USA.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular de Flavivírus. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto de Tecnologia em Imunobiológicos. Rio de Janeiro, RJ, Brasil.University of Miami Leonard M. Miller School of Medicine. Miami, Florida, USA.University of Wisconsin—Madison. Department of Pathology and Laboratory Medicine. Madison, Wisconsin, USA / Wisconsin National Primate Research Center. Madison, Wisconsin, USA.Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) primarily infect activated CD4(+) T cells but can infect macrophages. Surprisingly, ex vivo tetramer-sorted SIV-specific CD8(+) T cells that eliminated and suppressed viral replication in SIV-infected CD4(+) T cells failed to do so in SIV-infected macrophages. It is possible, therefore, that while AIDS virus-infected macrophages constitute only a small percentage of all virus-infected cells, they may be relatively resistant to CD8(+) T cell-mediated lysis and continue to produce virus over long periods of time

CD8+ gamma-delta TCR+ and CD4+ T cells produce IFN-γ at 5-7 days after yellow fever vaccination in Indian rhesus macaques, before the induction of classical antigen-specific T cell responses

Submitted by Sandra Infurna ([email protected]) on 2017-09-12T13:11:11Z No. of bitstreams: 1 ricardo_galler_etal_IOC_2010.pdf: 953535 bytes, checksum: f84d77c95a4d65cf2f33f15963068d41 (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2017-09-12T13:34:25Z (GMT) No. of bitstreams: 1 ricardo_galler_etal_IOC_2010.pdf: 953535 bytes, checksum: f84d77c95a4d65cf2f33f15963068d41 (MD5)Made available in DSpace on 2017-09-12T13:34:25Z (GMT). No. of bitstreams: 1 ricardo_galler_etal_IOC_2010.pdf: 953535 bytes, checksum: f84d77c95a4d65cf2f33f15963068d41 (MD5) Previous issue date: 2010Fundação Oswaldo Cruz. Bio-Manguinhos. Vice-Diretoria de Desenvolvimento Tecnológico. Rio de Janeiro, RJ, Brasil.University of Wisconsin-Madison. Department of Pathology. Laboratory Medicine. Madison, WI, USA.Fundação Oswaldo Cruz. Bio-Manguinhos. Vice-Diretoria de Desenvolvimento Tecnológico. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular de Flavivírus. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular de Flavivírus. Rio de Janeiro, RJ. Brasil.University of Wisconsin-Madison. Department of Pathology. Laboratory Medicine. Madison, WI, USA.University of Wisconsin-Madison. Department of Pathology. Laboratory Medicine. Madison, WI, USA.University of Wisconsin-Madison. Department of Pathology. Laboratory Medicine. Madison, WI, USA.University of Wisconsin-Madison. Department of Pathology. Laboratory Medicine. Madison, WI, USA.University of Wisconsin-Madison. Department of Pathology. Laboratory Medicine. Madison, WI, USA.The yellow fever 17D (YF-17D) vaccine is one of the most efficacious vaccines developed to date. Interestingly, vaccination with YF-17D induces IFN-γ production early after vaccination (days 5-7) before the development of classical antigen-specific CD8(+) and CD4(+) T cell responses. Here we investigated the cellular source of this early IFN-γ production. At days 5 and 7 post-vaccination activated CD8(+) gamma-delta TCR T cells produced IFN-γ and TNF-α. Activated CD4(+) T cells produced IFN-γ and TNF-α at day 7 post-vaccination. This early IFN-γ production was also induced after vaccination with recombinant YF-17D (rYF-17D), but was not observed after recombinant Adenovirus type 5 (rAd5) vaccination. Early IFN-γ production, therefore, might be an important aspect of yellow fever vaccination

The live-attenuated yellow fever vaccine 17D induces broad and potent T cell responses against several viral proteins in Indian rhesus macaques – implications for recombinant vaccine design

The yellow fever vaccine 17D (YF17D) is one of the most effective vaccines. Its wide use and favorable safety profile make it a prime candidate for recombinant vaccines. It is believed that neutralizing antibodies account for a large measure of the protection afforded to YF17D-vaccinated individuals, however cytotoxic T lymphocyte (CTL) responses have been described in the setting of YF17D vaccination. YF17D is an ssRNA flavivirus that is translated as a full-length polyprotein, several domains of which pass into the lumen of the endoplasmic reticulum (ER). The processing and presentation machinery for MHC class I-restricted CTL responses favor cytoplasmic peptides that are transported into the ER by the transporter associated with antigen presentation (TAP) proteins. In order to inform recombinant vaccine design, we sought to determine if YF17D-induced CTL responses preferentially targeted viral domains that remain within the cytoplasm. We performed whole YF17D proteome mapping of CTL responses in 6 Indian rhesus macaques vaccinated with YF17D using overlapping YF17D peptides. We found that the ER luminal E protein was the most immunogenic viral protein followed closely by the cytoplasmic NS3 and NS5 proteins. These results suggest that antigen processing and presentation in this model system is not preferentially affected by the subcellular location of the viral proteins that are the source of CTL epitopes. The data also suggest potential immunogenic regions of YF17D that could serve as the focus of recombinant T cell vaccine development

Recombinant Yellow Fever Vaccine Virus 17D Expressing Simian Immunodeficiency Virus SIVmac239 Gag Induces SIV-Specific CD8+ T-Cell Responses in Rhesus Macaques ▿

Here we describe a novel vaccine vector for expressing human immunodeficiency virus (HIV) antigens. We show that recombinant attenuated yellow fever vaccine virus 17D expressing simian immunodeficiency virus SIVmac239 Gag sequences can be used as a vector to generate SIV-specific CD8+ T-cell responses in the rhesus macaque. Priming with recombinant BCG expressing SIV antigens increased the frequency of these SIV-specific CD8+ T-cell responses after recombinant YF17D boosting. These recombinant YF17D-induced SIV-specific CD8+ T cells secreted several cytokines, were largely effector memory T cells, and suppressed viral replication in CD4+ T cells

Vaccination with gag, vif, and nef Gene Fragments Affords Partial Control of Viral Replication after Mucosal Challenge with SIVmac239

Made available in DSpace on 2015-05-04T17:07:28Z (GMT). No. of bitstreams: 2 license.txt: 1914 bytes, checksum: 7d48279ffeed55da8dfe2f8e81f3b81f (MD5) myrna_bonaldoetal_IOC_2014.pdf: 6308018 bytes, checksum: 8d6762258d0dfa74834cd8870c4070d5 (MD5) Previous issue date: 2014University of Miami. Miller School of Medicine. Department of Pathology. Miami, Florida, USA.University of Wisconsin. Department of Medicine. Madison, Wisconsin, USA.University of Wisconsin. Wisconsin National Primate Research Center. Madison, Wisconsin, USA.University of Wisconsin. Wisconsin National Primate Research Center. Madison, Wisconsin, USA.University of Wisconsin. Wisconsin National Primate Research Center. Madison, Wisconsin, USA.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular de Flavivírus. Rio de Janeiro, RJ, Brasil.University of Miami. Miller School of Medicine. Department of Pathology. Miami, Florida, USA.University of Wisconsin. Wisconsin National Primate Research Center. Madison, Wisconsin, USA.University of Wisconsin. Wisconsin National Primate Research Center. Madison, Wisconsin, USA.University of Alabama at Birmingham. Department of Biostatistics. Section on Statistical Genetics. Birmingham, Alabama, USA.International AIDS Vaccine Initiative. AIDS Vaccine Design and Development Laboratory. Brooklyn Army Terminal. Brooklyn, New York, USA.Leidos Biomedical Research. Inc. Frederick National Laboratory. AIDS and Cancer Virus Program. Frederick, Maryland, USA.University of Alabama at Birmingham. Department of Biostatistics. Section on Statistical Genetics. Birmingham, Alabama, USA.International AIDS Vaccine Initiative. AIDS Vaccine Design and Development Laboratory. Brooklyn Army Terminal. Brooklyn, New York, USA.Fundação Oswaldo Cruz. Instituto de Tecnologia em Imunobiológicos. Rio de Janeiro, RJ, Brasil.Leidos Biomedical Research. Inc. Frederick National Laboratory. AIDS and Cancer Virus Program. Frederick, Maryland, USA.University of Miami. Miller School of Medicine. Department of Pathology. Miami, Florida, USA.Broadly targeted cellular immune responses are thought to be important for controlling replication of human and simian immunodeficiency viruses (HIV and SIV). However, eliciting such responses by vaccination is complicated by immunodominance, the preferential targeting of only a few of the many possible epitopes of a given antigen. This phenomenon may be due to the coexpression of dominant and subdominant epitopes by the same antigen-presenting cell and may be overcome by distributing these sequences among several different vaccine constructs. Accordingly, we tested whether vaccinating rhesus macaques with “minigenes” encoding fragments of Gag, Vif, and Nef resulted in broadened cellular responses capable of controlling SIV replication.Wedelivered these minigenes through combinations of recombinant Mycobacterium bovis BCG (rBCG), electroporated recombinant DNA (rDNA) along with an interleukin-12 (IL-12)-expressing plasmid (EP rDNA plus pIL-12), yellow fever vaccine virus 17D (rYF17D), and recombinant adenovirus serotype 5 (rAd5). Although priming with EP rDNA plus pIL-12 increased the breadth of vaccine-induced T-cell responses, this effect was likely due to the improved antigen delivery afforded by electroporation rather than modulation of immunodominance. Indeed, Mamu-A*01 vaccinees mounted CD8 T cells directed against only one subdominant epitope, regardless of the vaccination regimen. After challenge with SIVmac239, vaccine efficacy was limited to a modest reduction in set point in some of the groups and did not correlate with standard T-cell measurements. These findings suggest that broad T-cell responses elicited by conventional vectors may not be sufficient to substantially contain AIDS virus replication

CD8+ gamma-delta TCR+ and CD4+ T cells produce IFN-γ at 5–7 days after yellow fever vaccination in Indian rhesus macaques, before the induction of classical antigen-specific T cell responses

The yellow fever 17D (YF-17D) vaccine is one of the most efficacious vaccines developed to date. Interestingly, vaccination with YF-17D induces IFN-γ production early after vaccination (d 5–7) before the development of classical antigen-specific CD8(+) and CD4(+) T cell responses. Here we investigated the cellular source of this early IFN-γ production. At days 5 and 7 post vaccination activated CD8(+) gamma-delta TCR T cells produced IFN-γ and TNF-α. Activated CD4(+) T cells produced IFN-γ and TNF-α at day 7 post vaccination. This early IFN-γ production was also induced after vaccination with recombinant YF-17D (rYF-17D), but was not observed after recombinant Adenovirus type 5 (rAd5) vaccination. Early IFN-γ production, therefore, might be an important aspect of yellow fever vaccination

Vaccine-Induced Simian Immunodeficiency Virus-Specific CD8 +

Certain major histocompatibility complex class I (MHC-I) alleles (e.g., HLA-B*27) are enriched among human immunodeficiency virus type 1 (HIV-1)-infected individuals who suppress viremia without treatment (termed “elite controllers” [ECs]). Likewise, Mamu-B*08 expression also predisposes rhesus macaques to control simian immunodeficiency virus (SIV) replication. Given the similarities between Mamu-B*08 and HLA-B*27, SIV-infected Mamu-B*08(+) animals provide a model to investigate HLA-B*27-mediated elite control. We have recently shown that vaccination with three immunodominant Mamu-B*08-restricted epitopes (Vif RL8, Vif RL9, and Nef RL10) increased the incidence of elite control in Mamu-B*08(+) macaques after challenge with the pathogenic SIVmac239 clone. Furthermore, a correlate analysis revealed that CD8(+) T cells targeting Nef RL10 was correlated with improved outcome. Interestingly, this epitope is conserved between SIV and HIV-1 and exhibits a delayed and atypical escape pattern. These features led us to postulate that a monotypic vaccine-induced Nef RL10-specific CD8(+) T-cell response would facilitate the development of elite control in Mamu-B*08(+) animals following repeated intrarectal challenges with SIVmac239. To test this, we vaccinated Mamu-B*08(+) animals with nef inserts in which Nef RL10 was either left intact (group 1) or disrupted by mutations (group 2). Although monkeys in both groups mounted Nef-specific cellular responses, only those in group 1 developed Nef RL10-specific CD8(+) T cells. These vaccine-induced effector memory CD8(+) T cells did not prevent infection. Escape variants emerged rapidly in the group 1 vaccinees, and ultimately, the numbers of ECs were similar in groups 1 and 2. High-frequency vaccine-induced CD8(+) T cells focused on a single conserved epitope and therefore did not prevent infection or increase the incidence of elite control in Mamu-B*08(+) macaques. IMPORTANCE Since elite control of chronic-phase viremia is a classic example of an effective immune response against HIV/SIV, elucidating the basis of this phenomenon may provide useful insights into how to elicit such responses by vaccination. We have previously established that vaccine-induced CD8(+) T-cell responses against three immunodominant epitopes can increase the incidence of elite control in SIV-infected Mamu-B*08(+) rhesus macaques—a model of HLA-B*27-mediated elite control. Here, we investigated whether a monotypic vaccine-induced CD8(+) T-cell response targeting the conserved “late-escaping” Nef RL10 epitope can increase the incidence of elite control in Mamu-B*08(+) monkeys. Surprisingly, vaccine-induced Nef RL10-specific CD8(+) T cells selected for variants within days after infection and, ultimately, did not facilitate the development of elite control. Elite control is, therefore, likely to involve CD8(+) T-cell responses against more than one epitope. Together, these results underscore the complexity and multidimensional nature of virologic control of lentivirus infection

Vaccine-Induced Simian Immunodeficiency Virus-Specific CD8 TCell Responses Focused on a Single Nef Epitope Select for Escape Variants Shortly after Infection

Submitted by sandra infurna ([email protected]) on 2016-05-24T12:52:58Z No. of bitstreams: 1 noemia_lima_etal_IOC_2015.pdf: 4329443 bytes, checksum: 07ae819d17aad8154e81d4a63ea57ee2 (MD5)Approved for entry into archive by sandra infurna ([email protected]) on 2016-06-02T13:02:47Z (GMT) No. of bitstreams: 1 noemia_lima_etal_IOC_2015.pdf: 4329443 bytes, checksum: 07ae819d17aad8154e81d4a63ea57ee2 (MD5)Made available in DSpace on 2016-06-02T13:02:47Z (GMT). No. of bitstreams: 1 noemia_lima_etal_IOC_2015.pdf: 4329443 bytes, checksum: 07ae819d17aad8154e81d4a63ea57ee2 (MD5) Previous issue date: 2015Made available in DSpace on 2016-06-03T12:34:19Z (GMT). No. of bitstreams: 2 noemia_lima_etal_IOC_2015.pdf: 4329443 bytes, checksum: 07ae819d17aad8154e81d4a63ea57ee2 (MD5) license.txt: 2991 bytes, checksum: 5a560609d32a3863062d77ff32785d58 (MD5) Previous issue date: 2015University of Miami. Department of Pathology. Miami, Florida, USA.Ragon Institute of MGH. MIT. Harvard. Cambridge. Massachusetts, USA.University of Miami. Department of Pathology. Miami, Florida, USA.Ragon Institute of MGH. MIT. Harvard. Cambridge. Massachusetts, USA.University of Miami. Department of Pathology. Miami, Florida, USA.Ragon Institute of MGH. MIT. Harvard. Cambridge. Massachusetts, USA.Ragon Institute of MGH. MIT. Harvard. Cambridge. Massachusetts, USA.Ragon Institute of MGH. MIT. Harvard. Cambridge. Massachusetts, USA.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular de Flavivírus. Rio de Janeiro, RJ, Brasil.University of Miami. Department of Pathology. Miami, Florida, USA.University of Alabama. Department of Biostatistics. Section on Statistical Genetics. Birmingham, Alabama, USA.University of Alabama. Department of Biostatistics. Section on Statistical Genetics. Birmingham, Alabama, USA.Frederick National Laboratory. Leidos Biomedical Research, Inc. AIDS and Cancer Virus Program. Frederick, Maryland, USA.Emory University. Department of Microbiology and Immunology. Atlanta, Georgia, USA.Brooklyn Army Terminal. AIDS Vaccine Design and Development Laboratory. International AIDS Vaccine Initiative. Brooklyn, New York, USA.University of Wisconsin—Madison. Wisconsin National Primate Research Center. Madison, Wisconsin, USA.University of Wisconsin—Madison. Wisconsin National Primate Research Center. Madison, Wisconsin, USA.Fundação Oswaldo Cruz. Instituto de Tecnologia em Imunobiológicos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular de Flavivírus. Rio de Janeiro, RJ, Brasil.Frederick National Laboratory. Leidos Biomedical Research, Inc. AIDS and Cancer Virus Program. Frederick, Maryland, USA.Ragon Institute of MGH. MIT. Harvard. Cambridge. Massachusetts, USA.University of Miami. Department of Pathology. Miami, Florida, USA.Certain major histocompatibility complex class I (MHC-I) alleles (e.g., HLA-B*27) are enriched among human immunodeficiency virus type 1 (HIV-1)-infected individuals who suppress viremia without treatment (termed "elite controllers" [ECs]). Likewise, Mamu-B*08 expression also predisposes rhesus macaques to control simian immunodeficiency virus (SIV) replication. Given the similarities between Mamu-B*08 and HLA-B*27, SIV-infected Mamu-B*08(+) animals provide a model to investigate HLA-B*27-mediated elite control. We have recently shown that vaccination with three immunodominant Mamu-B*08-restricted epitopes (Vif RL8, Vif RL9, and Nef RL10) increased the incidence of elite control in Mamu-B*08(+) macaques after challenge with the pathogenic SIVmac239 clone. Furthermore, a correlate analysis revealed that CD8(+) T cells targeting Nef RL10 was correlated with improved outcome. Interestingly, this epitope is conserved between SIV and HIV-1 and exhibits a delayed and atypical escape pattern. These features led us to postulate that a monotypic vaccine-induced Nef RL10-specific CD8(+) T-cell response would facilitate the development of elite control in Mamu-B*08(+) animals following repeated intrarectal challenges with SIVmac239. To test this, we vaccinated Mamu-B*08(+) animals with nef inserts in which Nef RL10 was either left intact (group 1) or disrupted by mutations (group 2). Although monkeys in both groups mounted Nef-specific cellular responses, only those in group 1 developed Nef RL10-specific CD8(+) T cells. These vaccine-induced effector memory CD8(+) T cells did not prevent infection. Escape variants emerged rapidly in the group 1 vaccinees, and ultimately, the numbers of ECs were similar in groups 1 and 2. High-frequency vaccine-induced CD8(+) T cells focused on a single conserved epitope and therefore did not prevent infection or increase the incidence of elite control in Mamu-B*08(+) macaques. IMPORTANCE: Since elite control of chronic-phase viremia is a classic example of an effective immune response against HIV/SIV, elucidating the basis of this phenomenon may provide useful insights into how to elicit such responses by vaccination. We have previously established that vaccine-induced CD8(+) T-cell responses against three immunodominant epitopes can increase the incidence of elite control in SIV-infected Mamu-B*08(+) rhesus macaques—a model of HLA-B*27-mediated elite control. Here, we investigated whether a monotypic vaccine-induced CD8(+) T-cell response targeting the conserved "late-escaping" Nef RL10 epitope can increase the incidence of elite control in Mamu-B*08(+) monkeys. Surprisingly, vaccine-induced Nef RL10-specific CD8(+) T cells selected for variants within days after infection and, ultimately, did not facilitate the development of elite control. Elite control is, therefore, likely to involve CD8(+) T-cell responses against more than one epitope. Together, these results underscore the complexity and multidimensional nature of virologic control of lentivirus infection