William J. Liu

CD8+ T Cell Immune Response in Immunocompetent Mice during Zika Virus Infection

ABSTRACT Zika virus (ZIKV) infection causees neurologic complications, including Guillain-Barré syndrome in adults and central nervous system (CNS) abnormalities in fetuses. We investigated the immune response, especially the CD8+ T cell response in C57BL/6 (B6) wild-type (WT) mice, during ZIKV infection. We found that a robust CD8+ T cell response was elicited, major histocompatibility complex class I-restricted CD8+ T cell epitopes were identified, a tetramer that recognizes ZIKV-specific CD8+ T cells was developed, and virus-specific memory CD8+ T cells were generated in these mice. The CD8+ T cells from these infected mice were functional, as evidenced by the fact that the adoptive transfer of ZIKV-specific CD8+ T cells could prevent ZIKV infection in the CNS and was cross protective against dengue virus infection. Our findings provide comprehensive insight into immune responses against ZIKV and further demonstrate that WT mice could be a natural and easy-access model for evaluating immune responses to ZIKV infection. IMPORTANCE ZIKV infection has severe clinical consequences, including Guillain-Barré syndrome in adults, microcephaly, and congenital malformations in fetuses and newborn infants. Therefore, study of the immune response, especially the adaptive immune response to ZIKV infection, is important for understanding diseases caused by ZIKV infection. Here, we characterized the CD8+ T cell immune response to ZIKV in a comprehensive manner and identified ZIKV epitopes. Using the identified immunodominant epitopes, we developed a tetramer that recognizes ZIKV-specific CD8+ T cells in vivo, which simplified the detection and evaluation of ZIKV-specific immune responses. In addition, the finding that tetramer-positive memory CD8+ T cell responses were generated and that CD8+ T cells can traffic to a ZIKV-infected brain greatly enhances our understanding of ZIKV infection and provides important insights for ZIKV vaccine design.

CASCIRE surveillance network and work on avian influenza viruses

CASCIRE surveillance network and work on avian influenza viruses Yuhai Bi, Weifeng Shi, Jianjun Chen, Quanjiao Chen, Zhenghai Ma, Gary Wong, Wenxia Tian, Renfu Yin, Guanghua Fu, Yongchun Yang, William J. Liu, Chuansong Quan, Qianli Wang, Shenghu He, Xiangdong Li, Qianfeng Xia, Lixin Wang, Zhaohui Pan, Laixing Li, Hong Li, Wen Xu, Ying Luo, Hui Zeng, Lianpan Dai, Haixia Xiao, Kirill Sharshov, Alexander Shestopalov, Yi Shi, Jinghua Yan, Xuebing Li, Yingxia Liu, Fumin Lei, Wenjun Liu & George F. Gao

Conserved peptides enhance immune efficiency of inactive vaccines against emerging avian influenza viruses in chicken

Avian influenza viruses (AIVs) such as H5N1 and H7N9 are a great threat to poultry economics and public health. Vaccination can effectively inhibit the spread of AIV in poultry, which is also a viable strategy for controlling virus transmission from poultry to human. Adjuvants that are commonly used in current inactivated vaccines to provide stronger anti-AIV immune responses are often limited in their capacity to quantitatively induce both humoral and cellular immune responses. Herein, we assessed the levels of immune responses generated by a vaccine formulation comprising inactivated H5N1 antigen and synthetic peptides covering conserved CD4+, CD8+ T cell, and B cell epitopes. We found that the synthetic peptides enhanced the antibody responses against conserved influenza virus antigen M2e. Notably, the hemagglutination inhibition test results indicated that the peptides significantly augmented the antibody responses of inactivated H5N1 antigen even in the 1/10 or 1/5 dose group, in the identical antibody level as antigen alone used at the full dose. This indicates that the peptide can significantly reduce the use of inactivated virus, lowering the cost of the vaccine. Moreover, the peptides increased the transcript levels of interleukin-4 and interferon-γ cytokines in chicken peripheral blood mononuclear cells, which may facilitate both humoral and cellular immune responses. Our data suggest that this peptide combined with inactivated H5N1 antigen enhances both the humoral and cellular immune responses, which may benefit the prediction and design of synthetic peptide-based adjuvants for vaccines in chicken.

Hemagglutinin-specific CD4+ T-cell responses following 2009-pH1N1 inactivated split-vaccine inoculation in humans

Influenza A virus remains a major threat to public health, and the inactivated split-virus vaccine is the most prevalent vaccine used worldwide. However, our knowledge about cellular immune responses to the inactivated influenza virus vaccine and its correlation with humoral responses are yet limited, which has restricted our understanding of the vaccines protective mechanisms. Herein, in two clinical trials, T-cell responses specific for both previously identified human leucocyte antigen (HLA)-I-restricted epitopes from influenza virus and hemagglutinin (HA) protein were longitudinally investigated before, during, and after a two-dose vaccination with the inactivated 2009 pandemic H1N1 (2009-pH1N1) vaccine. A robust antibody response in all of the donors after vaccination was observed. Though no CD8+ T-cell responses to known epitopes were detected, HA-specific T-cell responses were primed following vaccination, and the responses were found to be mainly CD4+ T-cell dependent. However, HA-specific T-cells circulating in peripheral blood dropped to baseline levels 6weeks after vaccination, but humoral immune responses maintained a high level for 4months post-vaccination. Significant correlations between the magnitude of the HA-specific T-cell responses and hemagglutination inhibition antibody titers were demonstrated, indicating a priming role of HA-specific T-cells for humoral immune responses. In conclusion, our study indicates that HA-specific CD4+ T-cell responses can be primed by the inactivated 2009-pH1N1 vaccine, which may coordinate with the elicitation of antibody protection. These findings would benefit a better understanding of the immune protective mechanisms of the widely used inactivated 2009-pH1N1 vaccine.

Human T-cell immunity against the emerging and re-emerging viruses

Over the past decade, we have seen an alarming number of high-profile outbreaks of newly emerging and re-emerging viruses. Recent outbreaks of avian influenza viruses, Middle East respiratory syndrome coronaviruses, Zika virus and Ebola virus present great threats to global health. Considering the pivotal role of host T-cell immunity in the alleviation of symptoms and the clearance of viruses in patients, there are three issues to be primarily concerned about T-cell immunity when a new virus emerges: first, does the population possess pre-existing T-cells against the new virus through previous infections of genetically relevant viruses; second, does a proper immune response arise in the patients to provide protection through an immunopathogenic effect; lastly, how long can the virus-specific immune memory persist. Herein, we summarize the current updates on the characteristics of human T-cell immunological responses against recently emerged or re-emerged viruses, and emphasize the necessity for timely investigation on the T-cell features of these viral diseases, which may provide beneficial recommendations for clinical diagnosis and vaccine development.

Development of risk reduction behavioral counseling for Ebola virus disease survivors enrolled in the Sierra Leone Ebola Virus Persistence Study, 2015-2016.

Background During the 2014–2016 West Africa Ebola Virus Disease (EVD) epidemic, the public health community had concerns that sexual transmission of the Ebola virus (EBOV) from EVD survivors was a risk, due to EBOV persistence in body fluids of EVD survivors, particularly semen. The Sierra Leone Ebola Virus Persistence Study was initiated to investigate this risk by assessing EBOV persistence in numerous body fluids of EVD survivors and providing risk reduction counseling based on test results for semen, vaginal fluid, menstrual blood, urine, rectal fluid, sweat, tears, saliva, and breast milk. This publication describes implementation of the counseling protocol and the key lessons learned. Methodology/Principal findings The Ebola Virus Persistence Risk Reduction Behavioral Counseling Protocol was developed from a framework used to prevent transmission of HIV and other sexually transmitted infections. The framework helped to identify barriers to risk reduction and facilitated the development of a personalized risk-reduction plan, particularly around condom use and abstinence. Pre-test and post-test counseling sessions included risk reduction guidance, and post-test counseling was based on the participants’ individual test results. The behavioral counseling protocol enabled study staff to translate the study’s body fluid test results into individualized information for study participants. Conclusions/Significance The Ebola Virus Persistence Risk Reduction Behavioral Counseling Protocol provided guidance to mitigate the risk of EBOV transmission from EVD survivors. It has since been shared with and adapted by other EVD survivor body fluid testing programs and studies in Ebola-affected countries.

On the ground in Western Africa: from the outbreak to the elapse of Ebola

I was sitting in the chair of the airplane back to Beijing when I started writing the memoirs to recollect my three journeys to Western Africa. All of the trips were related to the Ebola epidemic and covered different stages, from the outbreak peak to the post-Ebola era. Each journey was taken for a different task, but all of them represented the urgent and specific support from the Chinese Center for Disease Control and Prevention (China CDC) during the Ebola epidemic in Western African. On the August 8th, 2014, the World Health Organization (WHO) declared the Ebola outbreak in West Africa a Public Health Emergency of International Concern (PHEIC) (http:// who.int/mediacentre/news/statements/2014/ebola-20140808/ en/). Three days later, together with two colleagues, I was on the ground of Conakry (Fig. 1), the capital of Guinea, in which country the first Ebola case was traced back to the end of 2013 (Baize et al., 2014). As the first international support force during this epidemic, China rapidly reacted to help the three major affected countries, Guinea, Sierra Leone and Liberia, to fight against Ebola. Three experts were sent to each of these countries, respectively, to train the local health workers to use freely offered sanitary and biological protection supplies. The training went smoothly under the active cooperation of local health workers, and the supplies were soon delivered to the Ebola Treatment Units (ETUs) and the diagnostic laboratories in Guinea. This was the first time that China has sent public health specialists to countries outside of Asia to support disease control. In September, 2014, as the Ebola-related support to Western Africa went deeper, additional Chinese specialists were sent to Sierra Leone. Clinical treatment teams in ETUs, Ebola test teams based in a mobile biological safety level 3 laboratory (BSL-3 or P3 lab) and public health training teams working in the field were included. The Deputy DirectorGeneral of the China CDC, Dr. George F. Gao, worked in Sierra Leone as the co-team leader of the first team of the mobile laboratory for 2 months (Gao and Feng, 2014). He represented an idol to recruit more young health experts to work on the ground in Africa (Fig. 2), and I was one of them. Aside from the mobile laboratory, taking into consideration a long-term goal to support disease control in Western Africa, the Chinese government has built the first fixed P3 lab in West Africa (Jui, Freetown, Sierra Leone). In the middle of April, 2015, I came back to Africa with the second team for the fixed P3 lab, working as the main operator in the core area for two and a half months. Every time that I opened specimen packages in the hood, I reminded myself that the tube in my hand might contain the Ebola virus at a sky-high titer. Indeed, all of my colleagues recognized that biosafety is the priority of the laboratory testing for Ebola. During this period, Ebola virus genomes were quickly assembled and analyzed by the Chinese deep sequencing platform in Western Africa (Tong et al., 2015). This platform in the field makes it possible to trace the origin and transmission chain of the Ebola viruses soon after any new case is reported (Fig. 3). With the elapse of new Ebola cases, the Ebola diagnostic laboratories run on international aid were closed one by one. In contrast, China CDC strengthened the laboratory work force to avoid a potential flare-up of the Ebola epidemic. In the end of 2015, I came back to Freetown as the lead of the fourth team for the fixed P3 lab to perform Ebola testing together with nine other Chinese colleagues. The Ebola surveillance strategy in Sierra Leone was a community-based swab test of dead individuals, together with hospital-based blood testing of suspected cases. As the number of specimens to be tested increased, the human resources in the laboratory became insufficient. However, our team accomplished its Ebola testing job and established good coordination with Dr. Abdul Kamara, who is the National Laboratory Services Manager of the Ministry of Health and Sanitation (MOHS), Sierra Leone (Fig. 4). In the end of January, 2016, we have helped to diagnose the latest and hopefully the last Ebola case in Sierra Leone.

Ebola Virus Persistence in Ocular Tissues and Fluids (EVICT) Study: Reverse Transcription-Polymerase Chain Reaction and Cataract Surgery Outcomes of Ebola Survivors in Sierra Leone

Background Ebola virus disease (EVD) survivors are at risk for uveitis during convalescence. Vision loss has been observed following uveitis due to cataracts. Since Ebola virus (EBOV) may persist in the ocular fluid of EVD survivors for an unknown duration, there are questions about the safety and feasibility of vision restorative cataract surgery in EVD survivors. Methods We conducted a cross-sectional study of EVD survivors anticipating cataract surgery and patients with active uveitis to evaluate EBOV RNA persistence in ocular fluid, as well as vision outcomes post cataract surgery. Patients with aqueous humor that tested negative for EBOV RNA were eligible to proceed with manual small incision cataract surgery (MSICS). Findings We screened 137 EVD survivors from June 2016 – August 2017 for enrolment. We enrolled 50 EVD survivors; 46 with visually significant cataract, 1 with a subluxated lens, 2 with active uveitis and 1 with a blind painful eye due to uveitis. The median age was 24.0 years (IQR 17–35) and 35 patients (70%) were female. The median logMAR visual acuity (VA) was 3.0 (Snellen VA Hand motions; Interquartile Range, IQR: 1.2-3.0, Snellen VA 20/320 – Hand motions). All patients tested negative for EBOV RNA by RT-PCR in aqueous humor/vitreous fluid and conjunctiva at a median of 19 months (IQR 18-20) from EVD diagnosis in Phase 1 of ocular fluid sampling and 34 months (IQR 32-36) from EVD diagnosis in Phase 2 of ocular fluid sampling. Thirty-four patients underwent MSICS, with a preoperative median VA improvement from hand motions to 20/30 at three-month postoperative follow-up (P < 0.001). Interpretation EBOV persistence by RT-PCR was not identified in ocular fluid or conjunctivae of fifty EVD survivors with ocular disease. Cataract surgery can be performed safely with vision restorative outcomes in patients who test negative for EBOV RNA in ocular fluid specimens. These findings impact the thousands of West African EVD survivors at-risk for ocular complications who may also require eye surgery during EVD convalescence.

Clinical and immunological characteristics of human infections with H5N6 avian influenza virus

BACKGROUNDnH5N6 avian influenza virus (AIV) has caused sporadic, recurring outbreaks in China and Southeast Asia since 2013, with 19 human infections and 13 deaths. Seventeen of these infections occurred since December 2015, indicating a recent rise in the frequency of H5N6 cases.nnnMETHODSnTo assess the relative threat of H5N6 virus to humans, we summarized and compared clinical data from patients infected with H5N6 (n = 19) against data from 2 subtypes of major public health concern, H5N1 (n = 53) and H7N9 (n = 160). To assess immune responses indicative of prognosis, we compared concentrations of serum cytokines/chemokines in patients infected with H5N6, H5N1, H7N9, and 2009 pandemic H1N1 and characterized specific immune responses from 1 surviving and 2 nonsurviving H5N6 patients.nnnRESULTSnH5N6 patients were found to have higher incidences of lymphopenia and elevated alanine aminotransferase and lactate dehydrogenase levels compared with H5N1 and H7N9 patients. Hypercytokinemia was detected at substantially higher frequencies from H5N6 patients compared to those infected with other AIV subtypes. Evaluation of adaptive immunity showed that both humoral and cellular responses could be detected in the H5N6-infected survivor, but cellular responses were absent in the nonsurvivors. In addition, the surviving patient had lower concentrations of both pro- and anti-inflammatory cytokines/chemokines compared to the nonsurvivors.nnnCONCLUSIONSnOur results support that H5N6 virus could potentially be a major public health threat, and suggest it is possible that the earlier acquisition of cellular immunity and lower concentrations of cytokines/chemokines contributed to survival in our patient. Analysis of more patient samples will be needed to draw concrete conclusions.

CTL immunogenicity of Rv3615c antigen and diagnostic performances of an ESAT-6/CFP-10/Rv3615c antigen cocktail for Mycobacterium tuberculosis infection

T cell immune responses have played pivotal roles in host immune protection against Mycobacterium tuberculosis (MTB) infection. MTB specific antigen, Rv3615c (EspC), was identified to be as immunodominant as the well-known ESAT-6 and CFP-10, and has brought promising expectations to more sensitive T-cell based diagnosis and vaccine development. However, limited knowledge about the immunogenicity and diagnostic values of this antigen has restricted its application in clinical practice. Herein, the Rv3615c antigen was identified as a robust CTL immunoantigen with broadly cross-human leucocyte antigen (HLA) allele recognized peptides which may contribute to the broad recognition of Rv3615c antigen among the population. A three-antigen-cocktail (3-Ag-cocktail) comprising of ESAT-6, CFP-10 and Rv3615c was investigated in a multicenter, randomized and double-blinded study to evaluate its clinical diagnostic performances. A significantly improved sensitivity was demonstrated against the 3-Ag-cocktail compared with that against ESAT-6 and CFP-10. Both responsive magnitude and sensitivity were significantly lower in patients concurrently suffering from cancer, indicating its restriction in diagnosis of immunocomprised patients. In conclusion, inclusion of the Rv3615c antigen with multiple HLA restricted CTL epitopes would benefit the T-cell based diagnosis of MTB infection.