Eduardo J. M. Nascimento

Alternative complement pathway deregulation is correlated with dengue severity.

Background The complement system, a key component that links the innate and adaptive immune responses, has three pathways: the classical, lectin, and alternative pathways. In the present study, we have analyzed the levels of various complement components in blood samples from dengue fever (DF) and dengue hemorrhagic fever (DHF) patients and found that the level of complement activation is associated with disease severity. Methods and Results Patients with DHF had lower levels of complement factor 3 (C3; p = 0.002) and increased levels of C3a, C4a and C5a (p<0.0001) when compared to those with the less severe form, DF. There were no significant differences between DF and DHF patients in the levels of C1q, immunocomplexes (CIC-CIq) and CRP. However, small but statistically significant differences were detected in the levels of MBL. In contrast, the levels of two regulatory proteins of the alternative pathway varied widely between DF and DHF patients: DHF patients had higher levels of factor D (p = 0.01), which cleaves factor B to yield the active (C3bBb) C3 convertase, and lower levels of factor H (p = 0.03), which inactivates the (C3bBb) C3 convertase, than did DF patients. When we considered the levels of factors D and H together as an indicator of (C3bBb) C3 convertase regulation, we found that the plasma levels of these regulatory proteins in DHF patients favored the formation of the (C3bBb) C3 convertase, whereas its formation was inhibited in DF patients (p<0.0001). Conclusion The data suggest that an imbalance in the levels of regulatory factors D and H is associated with an abnormal regulation of complement activity in DHF patients.

Conservation and variability of dengue virus proteins: Implications for vaccine design

Background Genetic variation and rapid evolution are hallmarks of RNA viruses, the result of high mutation rates in RNA replication and selection of mutants that enhance viral adaptation, including the escape from host immune responses. Variability is uneven across the genome because mutations resulting in a deleterious effect on viral fitness are restricted. RNA viruses are thus marked by protein sites permissive to multiple mutations and sites critical to viral structure-function that are evolutionarily robust and highly conserved. Identification and characterization of the historical dynamics of the conserved sites have relevance to multiple applications, including potential targets for diagnosis, and prophylactic and therapeutic purposes. Methodology/Principal Findings We describe a large-scale identification and analysis of evolutionarily highly conserved amino acid sequences of the entire dengue virus (DENV) proteome, with a focus on sequences of 9 amino acids or more, and thus immune-relevant as potential T-cell determinants. DENV protein sequence data were collected from the NCBI Entrez protein database in 2005 (9,512 sequences) and again in 2007 (12,404 sequences). Forty-four (44) sequences (pan-DENV sequences), mainly those of nonstructural proteins and representing ∼15% of the DENV polyprotein length, were identical in 80% or more of all recorded DENV sequences. Of these 44 sequences, 34 (∼77%) were present in ≥95% of sequences of each DENV type, and 27 (∼61%) were conserved in other Flaviviruses. The frequencies of variants of the pan-DENV sequences were low (0 to ∼5%), as compared to variant frequencies of ∼60 to ∼85% in the non pan-DENV sequence regions. We further showed that the majority of the conserved sequences were immunologically relevant: 34 contained numerous predicted human leukocyte antigen (HLA) supertype-restricted peptide sequences, and 26 contained T-cell determinants identified by studies with HLA-transgenic mice and/or reported to be immunogenic in humans. Conclusions/Significance Forty-four (44) pan-DENV sequences of at least 9 amino acids were highly conserved and identical in 80% or more of all recorded DENV sequences, and the majority were found to be immune-relevant by their correspondence to known or putative HLA-restricted T-cell determinants. The conservation of these sequences through the entire recorded DENV genetic history supports their possible value for diagnosis, prophylactic and/or therapeutic applications. The combination of bioinformatics and experimental approaches applied herein provides a framework for large-scale and systematic analysis of conserved and variable sequences of other pathogens, in particular, for rapidly mutating viruses, such as influenza A virus and HIV.

Gene Expression Profiling during Early Acute Febrile Stage of Dengue Infection Can Predict the Disease Outcome

Background We report the detailed development of biomarkers to predict the clinical outcome under dengue infection. Transcriptional signatures from purified peripheral blood mononuclear cells were derived from whole-genome gene-expression microarray data, validated by quantitative PCR and tested in independent samples. Methodology/Principal Findings The study was performed on patients of a well-characterized dengue cohort from Recife, Brazil. The samples analyzed were collected prospectively from acute febrile dengue patients who evolved with different degrees of disease severity: classic dengue fever or dengue hemorrhagic fever (DHF) samples were compared with similar samples from other non-dengue febrile illnesses. The DHF samples were collected 2–3 days before the presentation of the plasma leakage symptoms. Differentially-expressed genes were selected by univariate statistical tests as well as multivariate classification techniques. The results showed that at early stages of dengue infection, the genes involved in effector mechanisms of innate immune response presented a weaker activation on patients who later developed hemorrhagic fever, whereas the genes involved in apoptosis were expressed in higher levels. Conclusions/Significance Some of the gene expression signatures displayed estimated accuracy rates of more than 95%, indicating that expression profiling with these signatures may provide a useful means of DHF prognosis at early stages of infection.

Association between Magnitude of the Virus-Specific Plasmablast Response and Disease Severity in Dengue Patients

Dengue is a globally expanding disease caused by infection with dengue virus (DENV) that ranges from febrile illness to acute disease with serious complications. Secondary infection predisposes individuals to more severe disease, and B lymphocytes may play a role in this phenomenon through production of Ab that enhance infection. To better define the acute B cell response during dengue, we analyzed peripheral B cells from an adult Brazilian hospital cohort with primary and secondary DENV infections of varying clinical severity. Circulating B cells in dengue patients were proliferating, activated, and apoptotic relative to individuals with other febrile illnesses. Severe secondary DENV infection was associated with extraordinary peak plasmablast frequencies between 4 and 7 d of illness, averaging 46% and reaching 87% of B cells, significantly greater than those seen in mild illness or primary infections. On average >70% of IgG-secreting cells in individuals with severe secondary DENV infection were DENV specific. Plasmablasts produced Ab that cross-reacted with heterotypic DENV serotypes, but with a 3-fold greater reactivity to DENV-3, the infecting serotype. Plasmablast frequency did not correlate with acute serum-neutralizing Ab titers to any DENV serotype regardless of severity of disease. These findings indicate that massive expansion of DENV-specific and serotype cross-reactive plasmablasts occurs in acute secondary DENV infection of adults in Brazil, which is associated with increasing disease severity.

Sequential Seasonal H1N1 Influenza Virus Infections Protect Ferrets against Novel 2009 H1N1 Influenza Virus

ABSTRACT Individuals <60 years of age had the lowest incidence of infection, with ∼25% of these people having preexisting, cross-reactive antibodies to novel 2009 H1N1 influenza. Many people >60 years old also had preexisting antibodies to novel H1N1. These observations are puzzling because the seasonal H1N1 viruses circulating during the last 60 years were not antigenically similar to novel H1N1. We therefore hypothesized that a sequence of exposures to antigenically different seasonal H1N1 viruses can elicit an antibody response that protects against novel 2009 H1N1. Ferrets were preinfected with seasonal H1N1 viruses and assessed for cross-reactive antibodies to novel H1N1. Serum from infected ferrets was assayed for cross-reactivity to both seasonal and novel 2009 H1N1 strains. These results were compared to those of ferrets that were sequentially infected with H1N1 viruses isolated prior to 1957 or more-recently isolated viruses. Following seroconversion, ferrets were challenged with novel H1N1 influenza virus and assessed for viral titers in the nasal wash, morbidity, and mortality. There was no hemagglutination inhibition (HAI) cross-reactivity in ferrets infected with any single seasonal H1N1 influenza viruses, with limited protection to challenge. However, sequential H1N1 influenza infections reduced the incidence of disease and elicited cross-reactive antibodies to novel H1N1 isolates. The amount and duration of virus shedding and the frequency of transmission following novel H1N1 challenge were reduced. Exposure to multiple seasonal H1N1 influenza viruses, and not to any single H1N1 influenza virus, elicits a breadth of antibodies that neutralize novel H1N1 even though the host was never exposed to the novel H1N1 influenza viruses.

Identification of Continuous Human B-Cell Epitopes in the Envelope Glycoprotein of Dengue Virus Type 3 (DENV-3)

Background Dengue virus infection is a growing global public health concern in tropical and subtropical regions of the world. Dengue vaccine development has been hampered by concerns that cross-reactive immunological memory elicited by a candidate vaccine could increase the risk of development of more severe clinical forms. One possible strategy to reduce risks associated with a dengue vaccine is the development of a vaccine composed of selected critical epitopes of each of the serotypes. Methodology/Principal Findings Synthetic peptides were used to identify B-cell epitopes in the envelope (E) glycoprotein of dengue virus type 3 (DENV-3). Eleven linear, immunodominant epitopes distributed in five regions at amino acid (aa) positions: 51–65, 71–90, 131–170, 196–210 and 246–260 were identified by employing an enzyme- linked immunosorbent assay (ELISA), using a pool of human sera from dengue type 3 infected individuals. Peptides 11 (aa51–65), 27 and 28 (aa131–150) also reacted with dengue 1 (DENV-1) and dengue 2 (DENV-2) patient sera as analyzed through the ROC curves generated for each peptide by ELISA and might have serotype specific diagnostic potential. Mice immunized against each one of the five immunogenic regions showed epitopes 51–65, 131–170, 196–210 and 246–260 elicited the highest antibody response and epitopes131–170, 196–210 and 246–260, elicited IFN-γ production and T CD4+ cell response, as evaluated by ELISA and ELISPOT assays respectively. Conclusions/Significance Our study identified several useful immunodominant IgG-specific epitopes on the envelope of DENV-3. They are important tools for understanding the mechanisms involved in antibody dependent enhancement and immunity. If proven protective and safe, in conjunction with others well-documented epitopes, they might be included into a candidate epitope-based vaccine.

Human Leukocyte Antigen (HLA) Class I Restricted Epitope Discovery in Yellow Fewer and Dengue Viruses: Importance of HLA Binding Strength

Epitopes from all available full-length sequences of yellow fever virus (YFV) and dengue fever virus (DENV) restricted by Human Leukocyte Antigen class I (HLA-I) alleles covering 12 HLA-I supertypes were predicted using the NetCTL algorithm. A subset of 179 predicted YFV and 158 predicted DENV epitopes were selected using the EpiSelect algorithm to allow for optimal coverage of viral strains. The selected predicted epitopes were synthesized and approximately 75% were found to bind the predicted restricting HLA molecule with an affinity, KD, stronger than 500 nM. The immunogenicity of 25 HLA-A*02:01, 28 HLA-A*24:02 and 28 HLA-B*07:02 binding peptides was tested in three HLA-transgenic mice models and led to the identification of 17 HLA-A*02:01, 4 HLA-A*2402 and 4 HLA-B*07:02 immunogenic peptides. The immunogenic peptides bound HLA significantly stronger than the non-immunogenic peptides. All except one of the immunogenic peptides had KD below 100 nM and the peptides with KD below 5 nM were more likely to be immunogenic. In addition, all the immunogenic peptides that were identified as having a high functional avidity had KD below 20 nM. A*02:01 transgenic mice were also inoculated twice with the 17DD YFV vaccine strain. Three of the YFV A*02:01 restricted peptides activated T-cells from the infected mice in vitro. All three peptides that elicited responses had an HLA binding affinity of 2 nM or less. The results indicate the importance of the strength of HLA binding in shaping the immune response.

Identification of Conserved and HLA Promiscuous DENV3 T-Cell Epitopes

Anti-dengue T-cell responses have been implicated in both protection and immunopathology. However, most of the T-cell studies for dengue include few epitopes, with limited knowledge of their inter-serotype variation and the breadth of their human leukocyte antigen (HLA) affinity. In order to expand our knowledge of HLA-restricted dengue epitopes, we screened T-cell responses against 477 overlapping peptides derived from structural and non-structural proteins of the dengue virus serotype 3 (DENV3) by use of HLA class I and II transgenic mice (TgM): A2, A24, B7, DR2, DR3 and DR4. TgM were inoculated with peptides pools and the T-cell immunogenic peptides were identified by ELISPOT. Nine HLA class I and 97 HLA class II novel DENV3 epitopes were identified based on immunogenicity in TgM and their HLA affinity was further confirmed by binding assays analysis. A subset of these epitopes activated memory T-cells from DENV3 immune volunteers and was also capable of priming naïve T-cells, ex vivo, from dengue IgG negative individuals. Analysis of inter- and intra-serotype variation of such an epitope (A02-restricted) allowed us to identify altered peptide ligands not only in DENV3 but also in other DENV serotypes. These studies also characterized the HLA promiscuity of 23 HLA class II epitopes bearing highly conserved sequences, six of which could bind to more than 10 different HLA molecules representing a large percentage of the global population. These epitope data are invaluable to investigate the role of T-cells in dengue immunity/pathogenesis and vaccine design.

T-Cell Memory Responses Elicited by Yellow Fever Vaccine are Targeted to Overlapping Epitopes Containing Multiple HLA-I and -II Binding Motifs

The yellow fever vaccines (YF-17D-204 and 17DD) are considered to be among the safest vaccines and the presence of neutralizing antibodies is correlated with protection, although other immune effector mechanisms are known to be involved. T-cell responses are known to play an important role modulating antibody production and the killing of infected cells. However, little is known about the repertoire of T-cell responses elicited by the YF-17DD vaccine in humans. In this report, a library of 653 partially overlapping 15-mer peptides covering the envelope (Env) and nonstructural (NS) proteins 1 to 5 of the vaccine was utilized to perform a comprehensive analysis of the virus-specific CD4+ and CD8+ T-cell responses. The T-cell responses were screened ex-vivo by IFN-γ ELISPOT assays using blood samples from 220 YF-17DD vaccinees collected two months to four years after immunization. Each peptide was tested in 75 to 208 separate individuals of the cohort. The screening identified sixteen immunodominant antigens that elicited activation of circulating memory T-cells in 10% to 33% of the individuals. Biochemical in-vitro binding assays and immunogenetic and immunogenicity studies indicated that each of the sixteen immunogenic 15-mer peptides contained two or more partially overlapping epitopes that could bind with high affinity to molecules of different HLAs. The prevalence of the immunogenicity of a peptide in the cohort was correlated with the diversity of HLA-II alleles that they could bind. These findings suggest that overlapping of HLA binding motifs within a peptide enhances its T-cell immunogenicity and the prevalence of the response in the population. In summary, the results suggests that in addition to factors of the innate immunity, “promiscuous” T-cell antigens might contribute to the high efficacy of the yellow fever vaccines.

West Nile Virus T-cell Ligand Sequences Shared with Other Flaviviruses: A Multitude of Variant Sequences as Potential Altered Peptide Ligands

ABSTRACT Phylogenetic relatedness and cocirculation of several major human pathogen flaviviruses are recognized as a possible cause of deleterious immune responses to mixed infection or immunization and call for a greater understanding of the inter-Flavivirus protein homologies. This study focused on the identification of human leukocyte antigen (HLA)-restricted West Nile virus (WNV) T-cell ligands and characterization of their distribution in reported sequence data of WNV and other flaviviruses. H-2-deficient mice transgenic for either A2, A24, B7, DR2, DR3, or DR4 HLA alleles were immunized with overlapping peptides of the WNV proteome, and peptide-specific T-cell activation was measured by gamma interferon (IFN-γ) enzyme-linked immunosorbent spot (ELISpot) assays. Approximately 30% (137) of the WNV proteome peptides were identified as HLA-restricted T-cell ligands. The majority of these ligands were conserved in ∼≥88% of analyzed WNV sequences. Notably, only 51 were WNV specific, and the remaining 86, chiefly of E, NS3, and NS5, shared an identity of nine or more consecutive amino acids with sequences of 64 other flaviviruses, including several major human pathogens. Many of the shared ligands had an incidence of >50% in the analyzed sequences of one or more of six major flaviviruses. The multitude of WNV sequences shared with other flaviviruses as interspecies variants highlights the possible hazard of defective T-cell activation by altered peptide ligands in the event of dual exposure to WNV and other flaviviruses, by either infection or immunization. The data suggest the possible preferred use of sequences that are pathogen specific with minimum interspecies sequence homology for the design of Flavivirus vaccines.