Severity of Infectious Mononucleosis (IM) Correlates with the Frequency of Crossreactive Influenza A Virus (IAV)-M1 and Epstein Barr Virus (EBV)-BMLF-1-specific CD8 T Cells

During EBV-associated IM IAV-specific crossreactive memory T cells are activated and play a role in disease severity. In HLA-A2+ IM patients, influenza M158 (IAV-M1)-specific CD8 memory T cell responses crossreacted with two different EBV lytic epitopes, BMLF1280 (17/29) and BRLF1190 (19/20). Furthermore, 11/22 IM patients demonstrated some intra-viral crossreactivity between EBV-BRLF1 and -BMLF1 responses. Disease severity of IM directly correlated with significantly increased frequencies of crossreactive IAV-M1/EBV-BMLF1, IAV-M1, and EBV-BMLF1 specific CD8 cells, and with mean viral load over the first 5 weeks of infection. Disease severity did not correlate with BRLF1 or M1/BRLF1 crossreactive responses. When severity of IM was scored and patients were assigned to either mild or severe groups, disease severity correlated with specific TCR Vb usage in IAV-M1 population suggesting that TcR selection is driving disease outcome. Consistent with IAV-M1 and EBV-BMLF1 responses driving increased immunopathology was the observation that patients with severe disease had significantly more IAV-M1 and EBV-BMLF1 cells producing IFNg/MIP1-b in response to antigen as compared to patients with mild disease. These results suggest that T cell crossreactivity impacts T cell selection and function and ultimately disease outcome. Insights on these issues are important for the intelligent design of vaccines and to develop therapeutic interventions for virally induced disease (NIHAI49320)

Crossreactive Epstein-Barr Virus (EBV)-Influenza A Virus (IAV) Specific CD8 Memory T Cells During Acute Symptomatic IAV Infection

We previously showed that crossreactivity is common between IAV and EBV in HLA-A2+ patients during infectious mononucleosis. IAV-M1-GIL58-66 specific CD8 T cells, along with expanded populations of IAV-M1-GIL58-66/EBV-BRLF-1109-117 -YVL and IAV-M1-GIL58-66/EBV-BMLF1280-288-GLC double-tetramer+ cells were detected directly ex-vivo in 5 HLA-A2+ patients. Altered IAV-M158-66, EBV-BRLF1119-117 and -BMLF1280-288 TCR repertoires were observed over the course of infection and in comparison to healthy donors. After culture, cells were sorted and analyzed by gene array in order to assess global changes in immune responses following different stimulations, either cognate or crossreactive, in different patient populations. M1-GIL and BRLF1-YVL specific cells had similar immune-response gene signatures, but the -GLC specific CD8 cells were more similar to the two-crossreactive populations. Crossreactive M1-GIL/BRLF1-YVL cells from the BRLF1-YVL line were different in their activation status than the BRLF1-specific cells, consistent with BRLF1-YVL ligand stimulation of different gene activation profiles in these two populations. These results suggest that during symptomatic IAV infection there is an expansion of EBV/IAV crossreactive memory CD8 T cell responses. Ongoing studies are investigating whether EBV-IAV cross-reactive CD8+ T cells may contribute to immunopathology during acute IAV infection (NIH / NIAID PO1 AI 049320)

Epstein-Barr Virus (EBV)-lytic Cross-reactive Influenza-A (IAV) Memory CD8 T-cells in EBV Sero-negative Middle-aged Adults

EBV is a common human pathogen, which infects ~90% of people and establishes a life-long chronic infection. The clinical outcomes of acute infection can range from asymptomatic to severe immunopathology such as infectious mononucleosis (IM). However, for unknown reasons 5-10% of middle-aged adults (\u3e35 years) remain EBV-seronegative (EBV-SN) when the virus infects the vast majority of people, and is actively shed at high titers during chronic infection. Here we show that EBV-SN (ASN) HLA-A2+ middle-aged adults possess a unique IAV-M1-GIL58-66 memory CD8 T-cell response that cross-reacts with EBV lytic epitopes that differs from teenage EBV-SN (TSN) (18-19 years) and EBV-seropositive (EBV-SP) adult donors. The five tested HLA-A2+ EBV-SN middle-aged adults had a significantly increased IAV-M158-66-GIL tetramer+ CD8 frequency compared to EBV-SP donors. Upon exposure to EBV antigens in vitro both IAV-M158-66GIL/EBV-BMLF1280-288-GLC and IAV-M158-66-GIL/EBV-BRLF1109-117-YVL, functionally cross-reactive CD8+ responses could be detected in the peripheral blood of middle-aged EBV-SN donors, while only IAV-M1/EBV-YVL cross-reactive responses were detected in some teenage EBV-SN or EBV-seropositive people . Surprisingly, these IAV-M1-GIL-specific CD8 T-cells in middle-aged EBV-SN adults expanded dramatically to EBV lytic antigens and produced cytokines at high functional avidity. They lysed EBV-infected targets and showed potential (by CD103 expression) to enter mucosal epithelial tissue where infection initiates. Additionally, these cross-reactive cells had an oligo-clonal T-cell receptor repertoire different than EBV-SP donors. Taken together these data suggest that an altered cross-reactive T cell repertoire could mediate protective immunity against viral infection. Our results imply that sero-negative adults might have the ability to resist viral infection via heterologous immunity. (NIH-AI49320)

Epstein-Barr Virus Epitope-Major Histocompatibility Complex Interaction Combined with Convergent Recombination Drives Selection of Diverse T Cell Receptor alpha and beta Repertoires

Recognition modes of individual T cell receptors (TCRs) are well studied, but factors driving the selection of TCR repertoires from primary through persistent human virus infections are less well understood. Using deep sequencing, we demonstrate a high degree of diversity of Epstein-Barr virus (EBV)-specific clonotypes in acute infectious mononucleosis (AIM). Only 9% of unique clonotypes detected in AIM persisted into convalescence; the majority (91%) of unique clonotypes detected in AIM were not detected in convalescence and were seeming replaced by equally diverse de novo clonotypes. The persistent clonotypes had a greater probability of being generated than nonpersistent clonotypes due to convergence recombination of multiple nucleotide sequences to encode the same amino acid sequence, as well as the use of shorter complementarity-determining regions 3 (CDR3s) with fewer nucleotide additions (i.e., sequences closer to germ line). Moreover, the two most immunodominant HLA-A2-restricted EBV epitopes, BRLF1109 and BMLF1280, show highly distinct antigen-specific public (i.e., shared between individuals) features. In fact, TCRalpha CDR3 motifs played a dominant role, while TCRbeta played a minimal role, in the selection of TCR repertoire to an immunodominant EBV epitope, BRLF1. This contrasts with the majority of previously reported repertoires, which appear to be selected either on TCRbeta CDR3 interactions with peptide/major histocompatibility complex (MHC) or in combination with TCRalpha CDR3. Understanding of how TCR-peptide-MHC complex interactions drive repertoire selection can be used to develop optimal strategies for vaccine design or generation of appropriate adoptive immunotherapies for viral infections in transplant settings or for cancer. IMPORTANCE Several lines of evidence suggest that TCRalpha and TCRbeta repertoires play a role in disease outcomes and treatment strategies during viral infections in transplant patients and in cancer and autoimmune disease therapy. Our data suggest that it is essential that we understand the basic principles of how to drive optimum repertoires for both TCR chains, alpha and beta. We address this important issue by characterizing the CD8 TCR repertoire to a common persistent human viral infection (EBV), which is controlled by appropriate CD8 T cell responses. The ultimate goal would be to determine if the individuals who are infected asymptomatically develop a different TCR repertoire than those that develop the immunopathology of AIM. Here, we begin by doing an in-depth characterization of both CD8 T cell TCRalpha and TCRbeta repertoires to two immunodominant EBV epitopes over the course of AIM, identifying potential factors that may be driving their selection

Early Epstein-Barr Virus Genomic Diversity and Convergence toward the B95.8 Genome in Primary Infection

Over 90% of the world\u27s population is persistently infected with Epstein-Barr virus. While EBV does not cause disease in most individuals, it is the common cause of acute infectious mononucleosis (AIM) and has been associated with several cancers and autoimmune diseases, highlighting a need for a preventive vaccine. At present, very few primary, circulating EBV genomes have been sequenced directly from infected individuals. While low levels of diversity and low viral evolution rates have been predicted for double-stranded DNA (dsDNA) viruses, recent studies have demonstrated appreciable diversity in common dsDNA pathogens (e.g., cytomegalovirus). Here, we report 40 full-length EBV genome sequences obtained from matched oral wash and B cell fractions from a cohort of 10 AIM patients. Both intra- and interpatient diversity were observed across the length of the entire viral genome. Diversity was most pronounced in viral genes required for establishing latent infection and persistence, with appreciable levels of diversity also detected in structural genes, including envelope glycoproteins. Interestingly, intrapatient diversity declined significantly over time (P \u3c 0.01), and this was particularly evident on comparison of viral genomes sequenced from B cell fractions in early primary infection and convalescence (P \u3c 0.001). B cell-associated viral genomes were observed to converge, becoming nearly identical to the B95.8 reference genome over time (Spearman rank-order correlation test; r = -0.5589, P = 0.0264). The reduction in diversity was most marked in the EBV latency genes. In summary, our data suggest independent convergence of diverse viral genome sequences toward a reference-like strain within a relatively short period following primary EBV infection. IMPORTANCE Identification of viral proteins with low variability and high immunogenicity is important for the development of a protective vaccine. Knowledge of genome diversity within circulating viral populations is a key step in this process, as is the expansion of intrahost genomic variation during infection. We report full-length EBV genomes sequenced from the blood and oral wash of 10 individuals early in primary infection and during convalescence. Our data demonstrate considerable diversity within the pool of circulating EBV strains, as well as within individual patients. Overall viral diversity decreased from early to persistent infection, particularly in latently infected B cells, which serve as the viral reservoir. Reduction in B cell-associated viral genome diversity coincided with a convergence toward a reference-like EBV genotype. Greater convergence positively correlated with time after infection, suggesting that the reference-like genome is the result of selection

High Epstein-Barr Virus Load and Genomic Diversity Are Associated with Generation of gp350-Specific Neutralizing Antibodies following Acute Infectious Mononucleosis

The Epstein-Barr virus (EBV) gp350 glycoprotein interacts with the cellular receptor to mediate viral entry and is thought to be the major target for neutralizing antibodies. To better understand the role of EBV-specific antibodies in the control of viral replication and the evolution of sequence diversity, we measured EBV gp350-specific antibody responses and sequenced the gp350 gene in samples obtained from individuals experiencing primary EBV infection (acute infectious mononucleosis [AIM]) and again 6 months later (during convalescence [CONV]). EBV gp350-specific IgG was detected in the sera of 17 (71%) of 24 individuals at the time of AIM and all 24 (100%) individuals during CONV; binding antibody titers increased from AIM through CONV, reaching levels equivalent to those in age-matched, chronically infected individuals. Antibody-dependent cell-mediated phagocytosis (ADCP) was rarely detected during AIM (4 of 24 individuals; 17%) but was commonly detected during CONV (19 of 24 individuals; 79%). The majority (83%) of samples taken during AIM neutralized infection of primary B cells; all samples obtained at 6 months postdiagnosis neutralized EBV infection of cultured and primary target cells. Deep sequencing revealed interpatient gp350 sequence variation but conservation of the CR2-binding site. The levels of gp350-specific neutralizing activity directly correlated with higher peripheral blood EBV DNA levels during AIM and a greater evolution of diversity in gp350 nucleotide sequences from AIM to CONV. In summary, we conclude that the viral load and EBV gp350 diversity during early infection are associated with the development of neutralizing antibody responses following AIM. IMPORTANCE: Antibodies against viral surface proteins can blunt the spread of viral infection by coating viral particles, mediating uptake by immune cells, or blocking interaction with host cell receptors, making them a desirable component of a sterilizing vaccine. The EBV surface protein gp350 is a major target for antibodies. We report the detection of EBV gp350-specific antibodies capable of neutralizing EBV infection in vitro The majority of gp350-directed vaccines focus on glycoproteins from lab-adapted strains, which may poorly reflect primary viral envelope diversity. We report some of the first primary gp350 sequences, noting that the gp350 host receptor binding site is remarkably stable across patients and time. However, changes in overall gene diversity were detectable during infection. Patients with higher peripheral blood viral loads in primary infection and greater changes in viral diversity generated more efficient antibodies. Our findings provide insight into the generation of functional antibodies, necessary for vaccine development

Strong HIV-1-Specific T Cell Responses in HIV-1-Exposed Uninfected Infants and Neonates Revealed after Regulatory T Cell Removal

BACKGROUND: In utero transmission of HIV-1 occurs on average in only 3%–15% of HIV-1-exposed neonates born to mothers not on antiretroviral drug therapy. Thus, despite potential exposure, the majority of infants remain uninfected. Weak HIV-1-specific T-cell responses have been detected in children exposed to HIV-1, and potentially contribute to protection against infection. We, and others, have recently shown that the removal of CD4(+)CD25(+) T-regulatory (Treg) cells can reveal strong HIV-1 specific T-cell responses in some HIV-1 infected adults. Here, we hypothesized that Treg cells could suppress HIV-1-specific immune responses in young children. METHODOLOGY/PRINCIPAL FINDINGS: We studied two cohorts of children. The first group included HIV-1-exposed-uninfected (EU) as well as unexposed (UNEX) neonates. The second group comprised HIV-1-infected and HIV-1-EU children. We quantified the frequency of Treg cells, T-cell activation, and cell-mediated immune responses. We detected high levels of CD4(+)CD25(+)CD127(−) Treg cells and low levels of CD4(+) and CD8(+) T cell activation in the cord blood of the EU neonates. We observed HIV-1-specific T cell immune responses in all of the children exposed to the virus. These T-cell responses were not seen in the cord blood of control HIV-1 unexposed neonates. Moreover, the depletion of CD4(+)CD25(+) Treg cells from the cord blood of EU newborns strikingly augmented both CD4(+) and CD8(+) HIV-1-specific immune responses. CONCLUSIONS/SIGNIFICANCE: This study provides new evidence that EU infants can mount strong HIV-1-specific T cell responses, and that in utero CD4(+)CD25(+) T-regulatory cells may be contributing to the lack of vertical transmission by reducing T cell activation

Epidemiological, socio-demographic and clinical features of the early phase of the COVID-19 epidemic in Ecuador

The SARS-CoV-2 virus has spread rapidly around the globe. Nevertheless, there is limited information describing the characteristics and outcomes of COVID-19 patients in Latin America. We conducted a cross-sectional analysis of 9,468 confirmed COVID-19 cases reported in Ecuador. We calculated overall incidence, mortality, case fatality rates, disability adjusted life years, attack and crude mortality rates, as well as relative risk and relative odds of death, adjusted for age, sex and presence of comorbidities. A total of 9,468 positive COVID-19 cases and 474 deaths were included in the analysis. Men accounted for 55.4% (n = 5, 247) of cases and women for 44.6% (n = 4, 221). We found the presence of comorbidities, being male and older than 65 years were important determinants of mortality. Coastal regions were most affected by COVID-19, with higher mortality rates than the highlands. Fatigue was reported in 53.2% of the patients, followed by headache (43%), dry cough (41.7%), ageusia (37.1%) and anosmia (36.1%). We present an analysis of the burden of COVID-19 in Ecuador. Our findings show that men are at higher risk of dying from COVID-19 than women, and risk increases with age and the presence of comorbidities. We also found that blue-collar workers and the unemployed are at greater risk of dying. These early observations offer clinical insights for the medical community to help improve patient care and for public health officials to strengthen Ecuador’s response to the outbreak