Abstract Effective vaccines should confer long-term protection against future outbreaks of severe acute respiratory syndrome (SARS) caused by a novel zoonotic coronavirus (SARS-CoV) with unknown animal reservoirs. We conducted a cohort study examining multiple parameters of immune responses to SARS-CoV infection, aiming to identify the immune correlates of protection. We used a matrix of overlapping peptides spanning whole SARS-CoV proteome to determine T cell responses from 128 SARS convalescent samples by ex vivo IFN-γ ELISPOT assays. Approximately 50% of convalescent SARS patients were positive for T cell responses, and 90% possessed strongly neutralizing Abs. Fifty-five novel T cell epitopes were identified, with spike protein dominating total T cell responses. CD8+ T cell responses were more frequent and of a greater magnitude than CD4+ T cell responses (p < 0.001). Polychromatic cytometry analysis indicated that the virus-specific T cells from the severe group tended to be a central memory phenotype (CD27+/CD45RO+) with a significantly higher frequency of polyfunctional CD4+ T cells producing IFN-γ, TNF-α, and IL-2, and CD8+ T cells producing IFN-γ, TNF-α, and CD107a (degranulation), as compared with the mild-moderate group. Strong T cell responses correlated significantly (p < 0.05) with higher neutralizing Ab. The serum cytokine profile during acute infection indicated a significant elevation of innate immune responses. Increased Th2 cytokines were observed in patients with fatal infection. Our study provides a roadmap for the immunogenicity of SARS-CoV and types of immune responses that may be responsible for the virus clearance, and should serve as a benchmark for SARS-CoV vaccine design and evaluation.
Inoculation of macaques with live attenuated SIV strains has been shown to protect against subsequent challenge with wild-type SIV. The protective mechanism(s) remain obscure. To study the effect in more detail, we have investigated the role of virus-specific CTL responses in macaques infected with an attenuated SIV strain (pC8), which has a four–amino acid deletion in the nef gene, as compared with the wild-type SIVmac32H clone (pJ5). Cynomolgus macaques infected with pC8 were protected against subsequent challenge with pJ5 and did not develop any AIDS-like symptoms in the 12 months after infection. The pC8-induced protection was associated with high levels of virus-specific CTL responses to a variety of viral antigens. In contrast, pJ5-infected macaques had little, if any, detectable CTL response to the viral proteins after three months. The latter group of macaques also showed increased Fas expression and apoptotic cell death in both the CD4+ and CD8+ populations. In vitro, pJ5 but not pC8 leads to an increase in FasL expression on infected cells. Thus the expression of FasL may protect infected cells from CTL attack, killing viral-specific CTLs in the process, and providing a route for escaping the immune response, leading to the increased pathogenicity of pJ5. pC8, on the other hand does not induce FasL expression, allowing the development of a protective CTL response. Furthermore, interruption of the Fas-FasL interaction allows the regeneration of viral-specific CTL responses in pJ5-infected animals. This observation suggests an additional therapeutic approach to the treatment of AIDS.
ABSTRACT The genome of the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) contains eight open reading frames (ORFs) that encode novel proteins. These accessory proteins are dispensable for in vitro and in vivo replication and thus may be important for other aspects of virus-host interactions. We investigated the functions of the largest of the accessory proteins, the ORF 3a protein, using a 3a-deficient strain of SARS-CoV. Cell death of Vero cells after infection with SARS-CoV was reduced upon deletion of ORF 3a. Electron microscopy of infected cells revealed a role for ORF 3a in SARS-CoV induced vesicle formation, a prominent feature of cells from SARS patients. In addition, we report that ORF 3a is both necessary and sufficient for SARS-CoV-induced Golgi fragmentation and that the 3a protein accumulates and localizes to vesicles containing markers for late endosomes. Finally, overexpression of ADP-ribosylation factor 1 (Arf1), a small GTPase essential for the maintenance of the Golgi apparatus, restored Golgi morphology during infection. These results establish an important role for ORF 3a in SARS-CoV-induced cell death, Golgi fragmentation, and the accumulation of intracellular vesicles.
Significance The function of the T-cell coreceptor CD4 presents a long-standing puzzle. Although it is among the most potent modulators of immune responses, CD4 interacts with its binding partner, peptide-major histocompatibility class II (pMHC II), with previously unmeasurably low affinity. Here, we set a new upper limit for the solution affinity of CD4 and pMHC II and show that the two-dimensional dissociation constant in supported lipid bilayers is as much as two to three orders of magnitude higher than that for other interacting leukocyte surface proteins. These findings extend the known physical limits of functional protein interactions at the cell surface and suggest new ways that T cells may use differential receptor affinities during antigen recognition and discrimination.
Fas and TNF-R1 are cysteine-rich cell surface receptors related to the low-affinity nerve growth factor receptor family. Engagement of these receptors by their respective ligands, FasL and tumor necrosis factor, leads to apoptosis that is signaled through a conserved intracellular portion of the receptor termed the "death domain." We have cloned a new member of this family, lymphocyte-associated receptor of death (LARD), which leads to spontaneous apoptosis when expressed in 293T cells. The expression of LARD is more tightly regulated than that of either Fas or TNF-R1 as it is found predominantly on lymphocytes (T and B cells) but not on macrophages or a number of transformed lymphocyte cell lines. Alternative pre-mRNA splicing generates at least 11 distinct isoforms of LARD. The full-length isoform, LARD-1, extends to include the transmembrane and death domains, whereas the other isoforms encode potentially secreted molecules. Naive B and T cells express very little LARD-1 but express combinations of the other isoforms. Upon T cell activation, a programmed change in alternative splicing occurs so that the full-length, membrane-bound LARD-1 predominates. This may have implications for the control of lymphocyte proliferation following activation.
Antibodies play a major role in the protection against influenza virus in human. However, the antibody level is usually short-lived and the cellular mechanisms underlying influenza virus-specific antibody response to acute infection remain unclear. We studied the kinetics and magnitude of influenza virus-specific B-cell and serum antibody responses in relation to virus replication during the course of influenza infection in healthy adult volunteers who were previously seronegative and experimentally infected with seasonal influenza H1N1 A/Brisbane/59/07 virus. Our data demonstrated a robust expansion of the virus-specific antibody-secreting cells (ASCs) and memory B cells in the peripheral blood, which correlated with both the throat viral load and the duration of viral shedding. The ASC response was obviously detected on day 7 post-infection when the virus was completely cleared from nasal samples, and serum hemagglutination-inhibition antibodies were still undetectable. On day 28 postinfection, influenza virus-specific B cells were further identified from the circulating compartment of isotype-switched B cells.Virus-specific ASCs could be the earliest marker of B-cell response to a new flu virus infection, such as H7N9 in humans.
We describe therapeutic monoclonal antibodies isolated from human volunteers vaccinated with recombinant adenovirus expressing Ebola virus glycoprotein (EBOV GP) and boosted with modified vaccinia virus Ankara. Among 82 antibodies isolated from peripheral blood B cells, almost half neutralized GP pseudotyped influenza virus. The antibody response was diverse in gene usage and epitope recognition. Although close to germline in sequence, neutralizing antibodies with binding affinities in the nano- to pico-molar range, similar to "affinity matured" antibodies from convalescent donors, were found. They recognized the mucin-like domain, glycan cap, receptor binding region, and the base of the glycoprotein. A cross-reactive cocktail of four antibodies, targeting the latter three non-overlapping epitopes, given on day 3 of EBOV infection, completely protected guinea pigs. This study highlights the value of experimental vaccine trials as a rich source of therapeutic human monoclonal antibodies.
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