Variation at Extra-epitopic Amino Acid Residues Influences Suppression of Influenza Virus Replication by M158-66 Epitope-Specific CD8+ T Lymphocytes.

Influenza virus-specific CD8+ T lymphocytes (CTLs) contribute to clearance of influenza virus infections and reduce disease severity. Variation at amino acid residues located in or outside CTL epitopes has been shown to affect viral recognition by virus-specific CTLs. In the present study, we investigated the effect of naturally occurring variation at residues outside the conserved immunodominant and HLA*0201-restricted M158-66 epitope, located in the influenza virus M1 protein, on the extent of virus replication in the presence of CTLs specific for the epitope. To this end, we used isogenic viruses with an M1 gene segment derived from either an avian or a human influenza virus, HLA-transgenic human epithelial cells, human T cell clones specific for the M158-66 epitope or a control epitope, and a novel, purposely developed in vitro system to coculture influenza virus-infected cells with T cells. We found that the M gene segment of a human influenza A/H3N2 virus afforded the virus the capacity to replicate better in the presence of M158-66-specific CTLs than the M gene segment of avian viruses. These findings are in concordance with previously observed differential CTL activation, caused by variation at extra-epitopic residues, and may reflect an immune adaptation strategy of human influenza viruses that allows them to cope with potent CTL immunity to the M158-66 epitope in HLA-A*0201-positive individuals, resulting in increased virus replication and shedding and possibly increasing disease severity.IMPORTANCE Influenza viruses are among the leading causes of acute respiratory tract infections. CD8+ T lymphocytes display a high degree of cross-reactivity with influenza A viruses of various subtypes and are considered an important correlate of protection. Unraveling viral immune evasion strategies and identifying signs of immune adaptation are important for defining the role of CD8+ T lymphocytes in affording protection more accurately. Improving our insight into the interaction between influenza viruses and virus-specific CD8+ T lymphocyte immunity may help to advance our understanding of influenza virus epidemiology, aid in risk assessment of potentially pandemic influenza virus strains, and benefit the design of vaccines that induce more broadly protective immunity.

Annual vaccination against influenza virus hampers development of virus-specific CD8⁺ T cell immunity in children.

Infection with seasonal influenza A viruses induces immunity to potentially pandemic influenza A viruses of other subtypes (heterosubtypic immunity). We recently demonstrated that vaccination against seasonal influenza prevented the induction of heterosubtypic immunity against influenza A/H5N1 virus induced by infection with seasonal influenza in animal models, which correlated with the absence of virus-specific CD8(+) T cell responses. Annual vaccination of all healthy children against influenza has been recommended, but the impact of vaccination on the development of the virus-specific CD8(+) T cell immunity in children is currently unknown. Here we compared the virus-specific CD8(+) T cell immunity in children vaccinated annually with that in unvaccinated children. In the present study, we compared influenza A virus-specific cellular and humoral responses of unvaccinated healthy control children with those of children with cystic fibrosis (CF) who were vaccinated annually. Similar virus-specific CD4(+) T cell and antibody responses were observed, while an age-dependent increase of the virus-specific CD8(+) T cell response that was absent in vaccinated CF children was observed in unvaccinated healthy control children. Our results indicate that annual influenza vaccination is effective against seasonal influenza but hampers the development of virus-specific CD8(+) T cell responses. The consequences of these findings are discussed in the light of the development of protective immunity to seasonal and future pandemic influenza viruses.

FATT-CTL assay for detection of antigen-specific cell-mediated cytotoxicity.

Here we describe a flowcytometric assay that measures the defining function of virus-specific cytotoxic T lymphocytes (CTL), i.e., killing viral protein expressing cells. The fluorescent antigen-transfected target cell (FATT)-CTL assay requires no viruses, recombinant viral vectors, or radioactive isotopes to generate CTL target cells that present naturally processed epitopes. It facilitates developing standardized applications in clinical trial settings. Plasmid vectors encoding antigen-green fluorescent protein (GFP) fusion proteins were used directly to nucleofect immortalized B cells or peripheral blood mononuclear cells (PBMCs). Elimination of antigen-GFP expressing cells by cloned CTL, in vitro sensitized PBMC, or ex vivo PBMC was quantified following a 4-18-h coculture period by flowcytometry. This technology successfully detected cell-mediated cytotoxicity in studies involving human PBMC and various viral antigens, including structural proteins of influenza A virus, and structural and nonstructural HIV proteins. Standardized protocols are currently being developed in the framework of a clinical immunotherapy trial in HIV-infected individuals. The FATT-CTL assay principles facilitate standardized flowcytometric detection of antigenic protein-specific cell-mediated cytotoxicity in many different basic research and clinical trial settings. By measuring their defining function, the FATT-CTL assay contributes to a more complete assessment of antigen-specific CTL responses to infection and vaccination.

Impact of antigen expression kinetics on the effectiveness of HIV-specific cytotoxic T lymphocytes.

Recent studies indicate that the time required for virus-infected cells to become vulnerable for the activity of CTL is of significance for the capacity of CTL to control ongoing viral reproduction. To investigate whether this applies to the effectiveness of HIV-1-specific CTL, we measured virus production in cultures containing CD4(+) T cells inoculated with HIV at low multiplicity of infection, and CTL directed against an early protein, Rev, or a late protein, RT. The Rev-specific CTL prevented at least 2 log(10) more HIV-1 production, in 10 days, than similar numbers of RT-specific CTL. To study how CTL effectiveness depends on variations in the potency of effector functions and kinetics of HIV protein expression, we developed a mathematical model describing CTL-target cell interactions during successive infection cycles. The results show that substantially higher CTL-mediated target cell elimination rates are required to achieve control as there is less time for CTL to act before infected cells release progeny virions. Furthermore, in vitro experiments with HIV recombinant viruses showed that the RT-specific CTL were at least as effective as the Rev-specific CTL, but only if the RT epitope was expressed as part of the early protein Nef. Together these results indicate that CTL control ongoing HIV reproduction more effectively if they are able to recognize infected cells earlier during individual viral replication cycles. This provides rationale for immunization strategies that aim at inducing, boosting or skewing CTL responses to early regulatory proteins in AIDS vaccine development.

Comparison of the efficacy of early versus late viral proteins in vaccination against SIV.

The immune response against early regulatory proteins of simian- and human immunodeficiency virus (SIV, HIV) has been associated with a milder course of infection. Here, we directly compared vaccination with Tat/Rev versus Pol/Gag. Challenge infection with SIVmac32H (pJ5) suggested that vaccination with Tat/Rev induced cellular immune responses that enabled cynomolgus macaques to more efficiently control SIV replication than the vaccine-induced immune responses against Pol/Gag. Vaccination with Tat/Rev resulted in reduced plasma SIV loads compared with control (P=0.058) or Pol/Gag-vaccinated (P=0.089) animals, with undetectable plasma viral loads in two of the four Tat/Rev-vaccinated animals. Therefore, the results warrant further investigation of the early regulatory proteins and their potential for vaccination against HIV.

The advantage of early recognition of HIV-infected cells by cytotoxic T-lymphocytes.

Accumulating evidence indicates that cytotoxic T-lymphocytes (CTL) play an important role in the clearing of primary and control of chronic human immunodeficiency virus (HIV) infection. Here, we discuss recent findings that indicate that the timing of target cell recognition critically contributes to CTL effectiveness. In this light several problems that have troubled CTL research are discussed. The use of early proteins like Tat and Rev is proposed for future vaccines design.

Construction and characterisation of infectious recombinant HIV-1 clones containing CTL epitopes from structural proteins in Nef.

In this study the construction is described of HIV-1 molecular clones in which CTL epitopes from RT or Env late proteins were inserted into the Nef early protein. The ectopic epitopes were efficiently processed from the recombinant Nef proteins, were recognized by their cognate CTL in cytolytic assays, and did not perturb virus replication or viral protein expression in vitro. These recombinant viruses will therefore be an important tool in studying the effect of distinct epitope expression kinetics on the efficiency of CTL-mediated suppression of HIV-1 replication.