Brigitte A. Askonas

Memory T cells established by seasonal human influenza A infection cross-react with avian influenza A (H5N1) in healthy individuals

The threat of avian influenza A (H5N1) infection in humans remains a global health concern. Current influenza vaccines stimulate antibody responses against the surface glycoproteins but are ineffective against strains that have undergone significant antigenic variation. An alternative approach is to stimulate pre-existing memory T cells established by seasonal human influenza A infection that could cross-react with H5N1 by targeting highly conserved internal proteins. To determine how common cross-reactive T cells are, we performed a comprehensive ex vivo analysis of cross-reactive CD4+ and CD8+ memory T cell responses to overlapping peptides spanning the full proteome of influenza A/Viet Nam/CL26/2005 (H5N1) and influenza A/New York/232/2004 (H3N2) in healthy individuals from the United Kingdom and Viet Nam. Memory CD4+ and CD8+ T cells isolated from the majority of participants exhibited human influenza-specific responses and showed cross-recognition of at least one H5N1 internal protein. Participant CD4+ and CD8+ T cells recognized multiple synthesized influenza peptides, including peptides from the H5N1 strain. Matrix protein 1 (M1) and nucleoprotein (NP) were the immunodominant targets of cross-recognition. In addition, cross-reactive CD4+ and CD8+ T cells recognized target cells infected with recombinant vaccinia viruses expressing either H5N1 M1 or NP. Thus, vaccine formulas inducing heterosubtypic T cell-mediated immunity may confer broad protection against avian and human influenza A viruses.

Enhanced recognition of a modified peptide antigen by cytotoxic T cells specific for influenza nucleoprotein

A previously identified Kd restricted epitope of influenza A virus nucleoprotein (147-161) was modified, resulting in recognition by Kd restricted cytotoxic T cells at significantly lower concentrations than the natural peptide sequence. This was achieved by first refining the epitope to the minimum determinant 147-158. Deletion of arginine 156 resulted in a peptide that was shown to be greatly superior in both dose response titrations and in its rate of association with cells to form targets. Analog peptides were tested to determine the important amino acid changes. These data suggest that T cell epitopes can be modified to result in improved immunological recognition.

Purified Influenza Virus Nucleoprotein Protects Mice from Lethal Infection

Local administration of nucleoprotein purified from X31 (H3N2) influenza A virus primed for A virus cross-reactive cytotoxic T cells and resulted in substantial protection (75%) of mice from a lethal challenge with the heterologous mouse-adapted A/PR/8/34 (H1N1) virus. By following the course of a lethal virus challenge we found that nucleoprotein priming did not prevent virus infection but rather aided recovery. Nucleoprotein-primed mice suffered initial symptoms of infection, i.e. weight loss and surface temperature changes, but started to recover after approximately 7 days. We suggest that such heterotypic protection can be attributed to priming of A virus cross-reactive cytotoxic T cells.

Cytotoxic T cell specificity for respiratory syncytial virus proteins: fusion protein is an important target antigen

We examined the specificity of BALB/c cytotoxic T (Tc) cells for respiratory syncytial virus (RSV) components, using recombinant vaccinia viruses (VV) coding for several individual RSV proteins. We found that immunization with the different VVs yielded the following Tc memory cell populations: high levels of RSV-specific Tc cells were induced with the fusion protein VV, but low levels were induced with VV coding for the RSV nucleoprotein. Tc cell recognition of attachment glycoprotein, part of the matrix molecule or 1A internal protein was poor. While high levels of fusion protein-specific Tc cells were induced by the fusion protein VV, they showed poor cross-reactivity between the A2 and 8/60 RSV strains compared with Tc cells primed by RSV infection.

Biosynthesis of immunoglobulins: the separate classes of polyribosomes synthesizing heavy and light chains.

Studies on the biosynthesis of myeloma protein in ascitic murine plasmacytoma cells (5563) have shown that the heavy and light chains of immunoglobulin G † are formed separately and then have to be assembled to form the complete molecule. Polyribosomes of diverse sizes have been obtained from ascitic plasmacytoma cells utilizing conditions which minimize degradation. Pulse labelling of these cells with [3H]leucine has shown that polyribosomes are the predominant site of protein synthesis. By precipitation with antisera against various myeloma protein determinants, a substantial proportion of the radioactive peptide chains on polyribosomes were identified as nascent myeloma protein chains. Nascent heavy chain determinants were located by direct precipitation with antibody to the Fc part of heavy chain which precipitated about 30% of radioactive material on polyribosomes sedimenting in the region of 300 s. Immunological procedures for detection of light-chain determinants showed a much lower proportion of precipitable radioactive material, but this formed a peak reproducibly in the 120 to 180 s region. We conclude that heavy chains are synthesized on the large polyribosomes in the 300 s region; the identification of the smaller polyribosomes synthesizing light chains is less definite. On the basis of the known relationship between polyribosome size and mRNA, the maximum sizes of polyribosomes carrying either heavy- or light-chain determinants are of sufficient size to contain messenger RNA molecules coding for complete, individual heavy or light chains.

Class I MHC molecules rather than other mouse genes dictate influenza epitope recognition by cytotoxic T cells

Influenza nucleoprotein (NP) is an important target antigen for influenza A virus cross-reactive cytotoxic T cells (Tc). Here we examine the NP epitope recognized by cloned and polyclonal BALB/c Tc and the genetics of this recognition pattern. We can define NP residues 147–161 as the epitope seen in conjunction with Kd, the only H-2d class I responder allele for NP restriction. H-2d/H-2bF1 mice (C57BL × DBA/2) primed by influenza infection lyse only H-2d target cells treated with peptide 147–161 while H-2b targets are recognized only after treatment with NP residues 365–379 (previously found to be recognized by Db restricted Tc cells). Tc cell recognition of NP peptide 147–161 is entirely dictated by expression of Kdand not by other B10 or OH background genes of congenic mice. Restriction of a unique NP sequence by each responder class I major histocompatibility complex (MHC) allele suggests that antigen and class I MHC interact for Tc recognition.

Membrane fractions of trypanosomes mimic the immunosuppressive and mitogenic effects of living parasites on the host.

Summary African trypanosomiasis in mice leads to profound changes in lymphoid tissues. In an attempt to define the nature of the trypanosome stimulus, we have studied the effect of radio‐attenuated trypanosomes and their subcellular fractions in vivo. We find that relatively low doses of irradiated Trypanosoma brucei S42 injected into (CBA/H × C57B1/ 6) F1 mice mimicked the previously reported effects of infective parasites. 2 × 107 irradiated trypanosomes caused a greater than two‐fold increase in spleen weight accompanied by a roughly 10‐fold increase in background plaque forming cells (PFC) to sheep red blood cells (SRBC). The primary response to SRBC was significantly enhanced when priming was carried out on the day of trypanosome injection, but significantly suppressed when carried out 3 days later. Disruption of trypanosomes by freeze‐thawing did not destroy their mitogenic or immunosuppressive activities. A membrane fraction collected by high speed centrifugation (150 000 × g) after removal of larger organelles at 12 000 × g retained both mitogenic and suppressive activities. The high speed supernatant lost the ability to enhance background PFC, but still caused partial immunosuppression with a much lower potency than the membrane pellet. Whether immunosuppression and enhanced PFC levels relate to the same parasite product is not clear as yet, but both effects can be ascribed to a membrane fraction of the parasite.

Correlation of grain counts with radioactivity (125I and tritium) in autoradiography.

Summary The approximate yield per radioactive disintegration was measured for autoragiography with 125I and tritium, using various photographic emulsions. When the isotopes were attached to red blood cells, and autoradiographs made of smears of the cells, the mean grain yields for γ-disintegration from 125I were about 0.44 for L4 and NTB2 emulsions and about 0.21 for AR10 stripping film. In the case of tritium the mean grain yield per β-disintegration was about 0.12 for L4 emulsion. Since the distribution of the isotopes on the surface of red blood cells differs from that likely to be encountered when the label is present within cells in a histological section, grain yields were also examined for these isotopes uniformly distributed in protein films of known thickness and density. Under these circumstances the grain yields from very thin films were very much greater than from thicker films. Over the range of thickness 1 to 5 μ the grain yields from 125I varied very little with the thickness. Using assumptions described in the text, the mean grain yield/disintegration of the isotopes in tissue section cut at 3–5μ, and exposed to L4 emulsion, are estimated to be approximately as follows:—0.23 for 125I and about 0.03 for tritium. The grain yields from constant amounts of isotope incorporated in protein films of varying thickness were also compared with the number of electrons emerging from the films, counted directly in a windowless gas flow counter. With 125I and to a lesser extent with tritium, the number of grains per emergent electron increased with film thickness. It is suggested that this is because the electrons emerging from the thicker films were mainly those in the higher energy ranges given off by each isotope. The implications are discussed for the choice of radioactive isotope in autoradiography of histological sections aimed at detection and detailed localization of small amounts of material.

Helper T Cell Recognition of Respiratory Syncytial Virus in Mice

In this study we aimed to define the protein and viral subtype specificities of helper Th cells to respiratory syncytial virus (RSV). BALB/c mice were primed by infection with RSV, or with vaccinia viruses (VV) containing genes encoding several individual RSV proteins. Priming for Th cell memory was assayed by stimulating spleen cells in vitro with different RSV isolates and measuring RSV-specific interleukin 2 (IL-2) release by T cells into supernatants using an IL-2-dependent CTLL cell line. Splenocytes from mice primed intranasally with RSV exhibited RSV-specific Th cell memory, whereas those from unprimed mice did not. Th cell recognition was in part specific to the strain of RSV used in priming and in part cross-reactive between RSV strains. Intraperitoneal priming with RSV fusion protein-expressing VV or nucleoprotein-expressing VV induced a stronger RSV-specific Th cell response than the attachment glycoprotein-expressing VV which produced only slight Th recognition. No Th cell recognition of two non-structural proteins (1A and 1B) could be demonstrated.

Low responder MHC alleles for Tc recognition of influenza nucleoprotein

Since virus nucleoprotein is an important target antigen for antiinfluenza cytotoxic T cells (Tc), we examined the genetics of Tc responses to this single viral protein to find three nonresponder alleles (Dd, Dk, and Kb) in three haplotypes and their recombinants so far tested. B10.A(5R) mice bearing nonresponder MHC class I antigen in the D and K regions show no anti-NP Tc responses, however they do show a strong A-virus cross-reactive anti-influenza cytotoxicity. The high frequency of nonresponsiveness to a single viral component, as compared with the entire virus, has implications for the development of simple vaccines.