Chris Ka-fai Li

Preexisting influenza-specific CD4 + T cells correlate with disease protection against influenza challenge in humans

Protective immunity against influenza virus infection is mediated by neutralizing antibodies, but the precise role of T cells in human influenza immunity is uncertain. We conducted influenza infection studies in healthy volunteers with no detectable antibodies to the challenge viruses H3N2 or H1N1. We mapped T cell responses to influenza before and during infection. We found a large increase in influenza-specific T cell responses by day 7, when virus was completely cleared from nasal samples and serum antibodies were still undetectable. Preexisting CD4+, but not CD8+, T cells responding to influenza internal proteins were associated with lower virus shedding and less severe illness. These CD4+ cells also responded to pandemic H1N1 (A/CA/07/2009) peptides and showed evidence of cytotoxic activity. These cells are an important statistical correlate of homotypic and heterotypic response and may limit severity of influenza infection by new strains in the absence of specific antibody responses. Our results provide information that may aid the design of future vaccines against emerging influenza strains.

Immunogenicity and Protective Efficacy of a Live Attenuated H5N1 Vaccine in Nonhuman Primates

The continued spread of highly pathogenic H5N1 influenza viruses among poultry and wild birds, together with the emergence of drug-resistant variants and the possibility of human-to-human transmission, has spurred attempts to develop an effective vaccine. Inactivated subvirion or whole-virion H5N1 vaccines have shown promising immunogenicity in clinical trials, but their ability to elicit protective immunity in unprimed human populations remains unknown. A cold-adapted, live attenuated vaccine with the hemagglutinin (HA) and neuraminidase (NA) genes of an H5N1 virus A/VN/1203/2004 (clade 1) was protective against the pulmonary replication of homologous and heterologous wild-type H5N1 viruses in mice and ferrets. In this study, we used reverse genetics to produce a cold-adapted, live attenuated H5N1 vaccine (AH/AAca) that contains HA and NA genes from a recent H5N1 isolate, A/Anhui/2/05 virus (AH/05) (clade 2.3), and the backbone of the cold-adapted influenza H2N2 A/AnnArbor/6/60 virus (AAca). AH/AAca was attenuated in chickens, mice, and monkeys, and it induced robust neutralizing antibody responses as well as HA-specific CD4+ T cell immune responses in rhesus macaques immunized twice intranasally. Importantly, the vaccinated macaques were fully protected from challenge with either the homologous AH/05 virus or a heterologous H5N1 virus, A/bar-headed goose/Qinghai/3/05 (BHG/05; clade 2.2). These results demonstrate for the first time that a cold-adapted H5N1 vaccine can elicit protective immunity against highly pathogenic H5N1 virus infection in a nonhuman primate model and provide a compelling argument for further testing of double immunization with live attenuated H5N1 vaccines in human trials.

Circulating chemokine (C‐X‐C Motif) receptor 5+CD4+ T cells benefit hepatitis B e antigen seroconversion through IL‐21 in patients with chronic hepatitis B virus infection

Given the clinical significance of hepatitis B e antigen (HBeAg) seroconversion in chronic hepatitis B virus (HBV) infection, it is critical to elucidate the mechanisms regulating this process. In the present study, we found that the frequency of circulating chemokine (C‐X‐C motif) receptor 5 (CXCR5)+CD4+ T cells was higher in patients who had achieved HBeAg seroconversion in both cross‐sectional (P < 0.001) and longitudinal (P = 0.009) studies. These cells were able to produce a significantly higher level of intracellular interleukin 21 (IL‐21) after stimulation with HBV peptides in patients with telbivudine‐induced HBeAg seroconversion (P = 0.007). Furthermore, sorted CXCR5+CD4+ T cells from HBeAg seroconverters boosted a higher frequency of antibody against hepatitis B e antigen (anti‐HBe)‐secreting B cells in coculture assay (P = 0.011). Of note, the increase in frequency of anti‐HBe‐secreting B cells was abrogated by soluble recombinant IL‐21 receptor‐Fc chimera (P = 0.027), whereas exogenous recombinant IL‐21 enhanced this effect (P = 0.043). Additionally, circulating CXCR5+CD4+ T cells shared similar phenotypic markers, and were positively correlated in frequency with, splenic follicular T helper cells. Conclusion: Circulating CXCR5+CD4+ T cells, by producing IL‐21, may have a significant role in facilitating HBeAg seroconversion in patients with chronic HBV infection. (Hepatology 2013;58:1277–1286)

Reduction of Natural Killer but Not Effector CD8 T Lymphoyctes in Three Consecutive Cases of Severe/Lethal H1N1/09 Influenza A Virus Infection

Background The cause of severe disease in some patients infected with pandemic influenza A virus is unclear. Methodology/Principal Findings We present the cellular immunology profile in the blood, and detailed clinical (and post-mortem) findings of three patients with rapidly progressive infection, including a pregnant patient who died. The striking finding is of reduction in natural killer (NK) cells but preservation of activated effector CD8 T lymphocytes; with viraemia in the patient who had no NK cells. Comparison with control groups suggests that the reduction of NK cells is unique to these severely ill patients. Conclusion/Significance Our report shows markedly reduced NK cells in the three patients that we sampled and raises the hypothesis that NK may have a more significant role than T lymphocytes in controlling viral burden when the host is confronted with a new influenza A virus subtype.

T Cell Responses to Whole SARS Coronavirus in Humans

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.

Correlates of protection against influenza infection in humans — on the path to a universal vaccine?

Influenza is an acute respiratory viral infection with high mutation rate and pandemic potential. Vaccination is an effective means of prevention and control of influenza, but the challenges of vaccine mismatches for the next influenza seasons and adequate global supply of influenza vaccines limit its effectiveness. Protective immunity in vaccination or natural infection is primarily mediated by antibody responses against surface proteins of influenza including haemagglutinin (HA) as the major neutralizing target, whereas strong T cell responses to internal viral proteins are associated with reduced disease severity. Recently, identification of broadly neutralizing antibodies against the conserved stem region of HA from influenza infected individuals has invigorated interest in development of a universal vaccine against different subtypes of influenza. Moreover, because of the cross-reactive nature of T cell recognition and more conserved internal antigens of influenza, strategies that boost memory T cell responses to these internal antigens may provide not only help for antibody-mediated protection but also limit the cell damage caused by viral infection directly. This is particularly important in acute infection with new pandemic viruses or antibody-escape variants where there are no pre-existing neutralizing antibodies. Here, we review the protective immune correlates against human influenza infection and discuss current status of universal influenza vaccine development.

A Systematic Molecular Pathology Study of a Laboratory Confirmed H5N1 Human Case

Autopsy studies have shown that human highly pathogenic avian influenza virus (H5N1) can infect multiple human organs other than just the lungs, and that possible causes of organ damage are either viral replication and/or dysregulation of cytokines and chemokines. Uncertainty still exists, partly because of the limited number of cases analysed. In this study, a full autopsy including 5 organ systems was conducted on a confirmed H5N1 human fatal case (male, 42 years old) within 18 hours of death. In addition to the respiratory system (lungs, bronchus and trachea), virus was isolated from cerebral cortex, cerebral medullary substance, cerebellum, brain stem, hippocampus ileum, colon, rectum, ureter, aortopulmonary vessel and lymph-node. Real time RT-PCR evidence showed that matrix and hemagglutinin genes were positive in liver and spleen in addition to positive tissues with virus isolation. Immunohistochemistry and in-situ hybridization stains showed accordant evidence of viral infection with real time RT-PCR except bronchus. Quantitative RT-PCR suggested that a high viral load was associated with increased host responses, though the viral load was significantly different in various organs. Cells of the immunologic system could also be a target for virus infection. Overall, the pathogenesis of HPAI H5N1 virus was associated both with virus replication and with immunopathologic lesions. In addition, immune cells cannot be excluded from playing a role in dissemination of the virus in vivo.

Optimal vaccination strategies for 2009 pandemic H1N1 and seasonal influenza vaccines in humans.

A randomized clinical trial was conducted to assess whether the immunogenicity of seasonal and pandemic (H1N1/09) influenza vaccines is affected by the order of vaccine administration. 151 healthy adult volunteers were randomized into three groups. All groups received one dose (15 μg haemagglutinin) each of a pandemic H1N1 vaccine and a seasonal trivalent vaccine. Group 1 received the pandemic H1N1 vaccine first, followed by the seasonal vaccine 21 days later. Group 2 received vaccinations in vice versa and Group 3 received both vaccines simultaneously. Post-vaccination blood samples were collected to determine the immunogenicity by hemagglutination-inhibition (HI), microneutralization (MN), and B cell ELISPOT assays. All three vaccination strategies were well-tolerated and generated specific immune responses. However, we found a significant difference in magnitude of antibody responses to pandemic H1N1 between the three groups. Pre- or co-vaccination with the seasonal flu vaccine led to a significant reduction by 50% in HI titre to pandemic H1N1 virus after pandemic vaccination. Pre- or co-vaccination of pandemic H1N1 vaccine had no effect on seasonal flu vaccination. MN and ELISPOT assays showed a similar effect. Vaccination with pandemic H1N1 vaccine first is recommended to avoid an associated inhibitory effect by the seasonal trivalent flu vaccine.

Avian influenza A(H5N1) viruses can directly infect and replicate in human gut tissues.

The human respiratory tract is a major site of avian influenza A(H5N1) infection. However, many humans infected with H5N1 present with gastrointestinal tract symptoms, suggesting that this may also be a target for the virus. In this study, we demonstrated that the human gut expresses abundant avian H5N1 receptors, is readily infected ex vivo by the H5N1 virus, and produces infectious viral particles in organ culture. An autopsy colonic sample from an H5N1-infected patient showed evidence of viral antigen expression in the gut epithelium. Our results provide the first evidence, to our knowledge, that H5N1 can directly target human gut tissues.

Virus-Specific Antibody Secreting Cell, Memory B-cell, and Sero-Antibody Responses in the Human Influenza Challenge Model

BACKGROUND  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. METHODS  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. RESULTS  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. CONCLUSIONS Virus-specific ASCs could be the earliest marker of B-cell response to a new flu virus infection, such as H7N9 in humans.