Ana B. Pérez

Tumor necrosis factor–alpha, transforming growth factor–β1, and interleukin-10 gene polymorphisms: implication in protection or susceptibility to dengue hemorrhagic fever

Dengue virus infection has emerged as one of the most important arthropod-borne viral diseases. Some dengue infected individuals develop the severe, life-threatening form of the disease, dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Host genetic factors may be relevant and may predispose some individuals to the severe illness. Human leukocyte antigen (HLA), FcγR, tumor necrosis factor (TNF)-α, and dendritic cell-specific intracellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN), among others genes have been associated with the pathogenesis of dengue. Little is known, however, about the predictive value of cytokine genotypes for the clinical outcome of dengue infection. In this study, the TNF-α, interleukin (IL)-6, interferon (IFN)-γ, IL-10 and transforming growth factor (TGF)-β1 gene single nucleotide polymorphisms (SNP) were studied by polymerase chain reaction-sequence-specific primer in a group of individuals with the antecedent of DHF during a secondary infection in the sequence dengue 1/dengue 2. A control group was also included. TNF-α (-308) A allele and IL-10 (-1082/-819/-592) ACC/ATA haplotype were significantly associated with DHF. TNF-α (-308) GG and TGF-β1 (c25) GG genotypes were associated with protection. Our results suggest that genetic predisposition to a high TNF-α production and a low IL-10 production seems to increase the susceptibility to DHF during a secondary dengue 2 infection, whereas TGF-β1 high producers might be protected for developing DHF.

Asymptomatic dengue infection in a Cuban population confirms the protective role of the RR variant of the FcγRIIa polymorphism.

The role of human Fcgamma receptors (FcgammaR) has been recognized considerably over the last years. These receptors vary in their affinity for IgG subclasses and the intracellular signals elicited by them. Allelic variants of FcgammaR genes may influence the biological phagocyte activity, accounting for an inherited pre-disposition to disease. The specific FcgammaRIIa (CD32) contains a polymorphic variant (H/R131) that has been associated to a reduced risk for developing dengue hemorrhagic fever (DHF). Here, we investigated the role of this polymorphism in a very well-characterized group of Cuban individuals with antecedents of DHF, dengue fever (DF), or subclinical dengue infection. The HH131 genotype was significantly associated with dengue disease, either DF (*P = 0.016; odds ratio = 4.425; 95% confidence interval = 1.10-20.52) or DHF (P = 0.00018; odds ratio = 10.56; 95% confidence interval = 2.33-54.64) with respect to the subclinical infection.

Immune response to synthetic peptides of dengue prM protein

The immunological activities of five synthetic peptides of the prM protein of dengue-2 (DEN-2) virus containing B cell epitopes were evaluated in BALB/c mice. Two peptides elicited neutralizing antibodies against all four DEN serotypes. Virus-specific proliferative responses were demonstrated in mice immunized with four of the five peptides, demonstrating the presence of T cell epitopes. Mice immunized with three of the five peptides conjugated with bovine albumin showed statistically significant levels (P<0.05) of protection when challenged with DEN-2 virus. These results could constitute the basis for the establishment of the role of DEN virus pre and M antigens in the development of anti-flaviviral vaccines.

Long-term persistence of clinical symptoms in dengue-infected persons and its association with immunological disorders

OBJECTIVES The acute manifestations of dengue are well known. The clinical symptoms that present during the convalescent phase of infection are less well characterized, but may be autoimmune-based. This study was undertaken to determine the prevalence of persistent clinical symptoms among individuals infected during the 2006 Cuban epidemic and to evaluate the immunological and genetic factors associated with their occurrence. METHODS In 2008, clinical data and blood samples were collected from a random sample of adult individuals diagnosed during the 2006 epidemic with dengue fever (DF, n=68), dengue hemorrhagic fever (DHF, n=29), or an asymptomatic infection (AI, n=42). The presence of persistent symptoms was evaluated in all individuals and a psychological assessment was performed. IgG titers and the Fc receptor (FcR) were also evaluated. The following autoimmune markers were assessed in a subset (n=26) of symptomatic individuals: complement factors C3/C4, rheumatoid factor (RF), C-reactive protein (CRP), antinuclear antibodies (ANA), and immune complex (IC). RESULTS Over half (55/97) the individuals with a prior of diagnosis of DF or DHF had persistent clinical symptoms in the 2 years following infection. The sequelae were unrelated to the initial diagnosis and were more common among women (44/55). No symptoms were reported in the AI group and all study participants had normal mental and cognitive function. Persistent clinical symptoms were associated with HH polymorphic variant (p=0.027) and high IgG titer (p=0.041). Autoimmune marker alterations were common (20/26) in the subset of symptomatic individuals evaluated. CONCLUSIONS Clinical sequelae after recovery from an acute dengue virus infection are common in the 2 years following infection. The results obtained in this study suggest that persistent symptoms are associated with alterations in some immunological parameters and FcγRIIa gene polymorphism. This could suggest an autoimmune-based disturbance.

Long-term memory cellular immune response to dengue virus after a natural primary infection

OBJECTIVES This study was conducted to examine the memory T-cell response to dengue virus 20 years after a primary infection. We took advantage of the exceptional epidemiologic situation in Cuba, where the population initially suffered two large successive epidemics due to dengue virus 1 and 2 respectively over a 4-year period. Thereafter, no dengue virus circulation was subsequently observed, except for the Santiago de Cuba municipality. DESIGN T-cell response was evaluated in peripheral blood mononuclear cells (PBMCs) from 20 individuals with history of a primary infection by dengue virus 1 or 2. Methods previously shown to induce lymphoproliferation of CD4+ memory T-cell subpopulations were used. We evaluated the proliferative responses generated in those PBMCs after stimulation with dengue virus 1, 2, 3 and 4 antigens in a serotype-specific and serotype-crossreactive way. RESULTS Serotype-specific and serotype-crossreactive lymphoproliferative responses in all PBMCs donated by dengue immune donors were observed. The serotype-crossreactive response for dengue 2 was stronger than for the rest of the serotypes. CONCLUSIONS This is the first report of cellular memory lymphocyte response specific for dengue virus detected 20 years after a primary infection by dengue.

Dengue, one of the great emerging health challenges of the 21st century

Dengue, one of the great emerging health challenges of the 21st Century 31 May-3 June 2004, 2nd International Congress on Dengue and Yellow Fever, Havana, Cuba Maria G Guzman, Gustavo Kouri, Manuel Diaz, Alina Llop, Susana Vazquez, Daniel Gonzalez, Osvaldo Castro, Angel Alvarez, Omar Fuentes, Domingo Montada, Harish Padmanabha, Beatriz Sierra, Ana B Perez, Delfina Rosario, Maritza Pupo, Cristina Diaz and Lisette Sanchez

Secondary heterologous dengue infection risk: Disequilibrium between immune regulation and inflammation?

Increased serum levels of cytokines released by cells of the immune response have been detected in patients suffering from dengue disease. Likewise, secondary infections by a different dengue virus serotype result in a highest risk of development of the severe dengue disease. Both findings suggest that the memory immune response is one of the key players in the pathogenesis of this disease. Here we take advantage of the particular Cuban epidemiological situation in dengue to analyze a broad spectrum of cell-mediated immune response mediators at mRNA and protein level. Evidences for a regulatory immune pattern in homologous (TGF-beta, IL-10) vs. pro-inflammatory pattern (IFN-gamma, TNF-alpha) in heterologous dengue virus re-challenge were found, suggesting a possible association with the higher incidence of severe dengue cases in the latter case.

Superiority of intramuscular route and full length glycoprotein D for DNA vaccination against herpes simplex 2. Enhancement of protection by the co-delivery of the GM-CSF gene

Immunization with naked DNA has been analyzed in two critical variables: the site of injection and the cellular compartment to which the coded protein is directed. The gene for the full length of the glycoprotein D (gD) of HSV-2 under the control of the citomegalovirus (CMV) promoter was injected via the intradermal (i.d.) or the intramuscular (i.m.) routes in mice. Immunization in the quadricep muscle was superior to the intradermal immunization in the footpads. A stronger activation of IFN-gamma-secreting cells in the spleen and draining lymph nodes (DLN) was induced, resulting in a more efficient protection against an intravaginal challenge. In order to analyze the effect of the cellular localizations of the coded protein, the DNA for the truncated form of the gD (DeltagD) was injected via the i.m. route. Immunization with a vector encoding for DeltagD resulted in higher antibody levels in serum and vaginal washes than immunization with the gene for the full length gD. However, immunization with the DeltagD DNA elicited a much weaker cell-mediated immune response and was inferior to gD DNA in providing protection against a lethal intravaginal challenge with HSV. Co-injection of an expression cassette for the granulocyte-macrophage colony-stimulating factor (GM-CSF) increased both the humoral and cell-mediated immune response with both gD and DeltagD. A strong activation of IL-4-secreting cells was observed in the spleen and DLN together with an increase in the number of IFN-gamma-secreting cells. In addition, a reduction in the vaginal virus titers after an intravaginal challenge was observed in mice co-injected with the GM-CSF gene as compared to those immunized with pCDNAgD only.

Association of MICA and MICB alleles with symptomatic dengue infection

Dengue viruses (DV) are one of the most important arthropod-borne viral diseases in the developing world. DV can cause syndromes that are either self-limiting or severe. Allelic variants of human leukocyte antigen (HLA) genes have been demonstrated to be associated with disease susceptibility. Here we report the association of nonclassical HLA class I MICA-MICB genes with disease outcome during DV infection. A sequencing-based typing method and genotyping of MICA and MICB in a well-characterized group of Cuban individuals with dengue hemorrhagic fever (DHF), dengue fever (DF), or asymptomatic dengue infection (ADI) was performed. Statistical analysis revealed a tendency for MICA*008 and MICB*008 to associate with susceptibility to illness when symptomatic versus asymptomatic cases (odds ratio [OR] = 2.1, p(v) = 0.03, and OR = 10.4, p = 0.0096, respectively) were compared. Surprisingly, a stronger association of both allelic forms was observed for the DF patients compared with the ADI group (MICA*008, OR = 5.2, p = 0.0001; and MICB*008, OR = 13.2, p = 0.0025) rather than the severe cases. Major histocompatibility class I-related gene-related natural killer cells and/or γδ and αβ T-cell activation might regulate the development of symptomatic DF and DHF.

CRF19_cpx is an Evolutionary fit HIV-1 Variant Strongly Associated With Rapid Progression to AIDS in Cuba

Background Clinicians reported an increasing trend of rapid progression (RP) (AIDS within 3 years of infection) in Cuba. Methods Recently infected patients were prospectively sampled, 52 RP at AIDS diagnosis (AIDS-RP) and 21 without AIDS in the same time frame (non-AIDS). 22 patients were sampled at AIDS diagnosis (chronic-AIDS) retrospectively assessed as > 3 years infected. Clinical, demographic, virological, epidemiological and immunological data were collected. Pol and env sequences were used for subtyping, transmission cluster analysis, and prediction of resistance, co-receptor use and evolutionary fitness. Host, immunological and viral predictors of RP were explored through data mining. Findings Subtyping revealed 26 subtype B strains, 6 C, 6 CRF18_cpx, 9 CRF19_cpx, 29 BG-recombinants and other subtypes/URFs. All patients infected with CRF19 belonged to the AIDS-RP group. Data mining identified CRF19, oral candidiasis and RANTES levels as the strongest predictors of AIDS-RP. CRF19 was more frequently predicted to use the CXCR4 co-receptor, had higher fitness scores in the protease region, and patients had higher viral load at diagnosis. Interpretation CRF19 is a recombinant of subtype D (C-part of Gag, PR, RT and nef), subtype A (N-part of Gag, Integrase, Env) and subtype G (Vif, Vpr, Vpu and C-part of Env). Since subtypes D and A have been associated with respectively faster and slower disease progression, our findings might indicate a fit PR driving high viral load, which in combination with co-infections may boost RANTES levels and thus CXCR4 use, potentially explaining the fast progression. We propose that CRF19 is evolutionary very fit and causing rapid progression to AIDS in many newly infected patients in Cuba.