Association of TLR7 Variants with AIDS-Like Disease and AIDS Vaccine Efficacy in Rhesus Macaques

In HIV infection, TLR7-triggered IFN-α production exerts a direct antiviral effect through the inhibition of viral replication, but may also be involved in immune pathogenesis leading to AIDS. TLR7 could also be an important mediator of vaccine efficacy. In this study, we analyzed polymorphisms in the X-linked TLR7 gene in the rhesus macaque model of AIDS. Upon resequencing of the TLR7 gene in 36 rhesus macaques of Indian origin, 12 polymorphic sites were detected. Next, we identified three tightly linked single nucleotide polymorphisms (SNP) as being associated with survival time. Genotyping of 119 untreated, simian immunodeficiency virus (SIV)-infected male rhesus macaques, including an ‘MHC adjusted’ subset, revealed that the three TLR7 SNPs are also significantly associated with set-point viral load. Surprisingly, this effect was not observed in 72 immunized SIV-infected male monkeys. We hypothesize (i) that SNP c.13G>A in the leader peptide is causative for the observed genotype-phenotype association and that (ii) the underlying mechanism is related to RNA secondary structure formation. Therefore, we investigated a fourth SNP (c.-17C>T), located 17 bp upstream of the ATG translation initiation codon, that is also potentially capable of influencing RNA structure. In c.13A carriers, neither set-point viral load nor survival time were related to the c.-17C>T genotype. In c.13G carriers, by contrast, the c.-17C allele was significantly associated with prolonged survival. Again, no such association was detected among immunized SIV-infected macaques. Our results highlight the dual role of TLR7 in immunodeficiency virus infection and vaccination and imply that it may be important to control human AIDS vaccine trials, not only for MHC genotype, but also for TLR7 genotype

Induction of neutralizing antibody response against four dengue viruses in mice by intramuscular electroporation of tetravalent DNA vaccines.

DNA vaccine against dengue is an interesting strategy for a prime/boost approach. This study evaluated neutralizing antibody (NAb) induction of a dengue tetravalent DNA (TDNA) vaccine candidate administered by intramuscular-electroporation (IM-EP) and the benefit of homologous TDNA boosting in mice. Consensus humanized pre-membrane (prM) and envelope (E) of each serotypes, based on isolates from year 1962-2003, were separately cloned into a pCMVkan expression vector. ICR mice, five-six per group were immunized for three times (2-week interval) with TDNA at 100 µg (group I; 25 µg/monovalent) or 10 µg (group II; 2.5 µg/monovalent). In group I, mice received an additional TDNA boosting 13 weeks later. Plaque reduction neutralization tests (PRNT) were performed at 4 weeks post-last immunization. Both 100 µg and 10 µg doses of TDNA induced high NAb levels against all DENV serotypes. The median PRNT50 titers were comparable among four serotypes of DENV after TDNA immunization. Median PRNT50 titers ranged 240-320 in 100 µg and 160-240 in 10 µg groups (p = ns). A time course study of the 100 µg dose of TDNA showed detectable NAb at 2 weeks after the second injection. The NAb peaked at 4 weeks after the third injection then declined over time but remained detectable up to 13 weeks. An additional homologous TDNA boosting significantly enhanced the level of NAb from the nadir for at least ten-fold (p<0.05). Of interest, we have found that the use of more recent dengue viral strain for both vaccine immunogen design and neutralization assays is critical to avoid a mismatching outcome. In summary, this TDNA vaccine candidate induced good neutralizing antibody responses in mice; and the DNA/DNA prime/boost strategy is promising and warranted further evaluation in non-human primates

NAb responses against reference viruses used in PRNT in mice immunized with TDNA.

<p>NAb activities in sera of individual mouse following an IM-EP immunization with 100 µg/dose (opened diamond) and 10 µg/dose (grey square) for three times were determined at week 4 after the third dose. Horizontal lines represent the median PRNT50 titer for each group of mice (<i>n</i> = 5–6). ns: no statistically significant difference.</p

P5339Association between high-sensitive troponin I and subclinical coronary atherosclerosis in well-controlled HIV-infected adults

Abstract Background and objectives Patients with human immunodeficiency virus (HIV) infection live longer and the prevalence of coronary heart disease is increasing among them. High-sensitive troponin I (hs-TnI) is associated with coronary artery calcification as determined by non-contrast cardiac computed tomography (CT) in general population without established cardiovascular disease (CVD). Nevertheless, the relationship in well-controlled HIV-infected patients has not been validated. Design and methods A cross-sectional study among HIV-infected adults aged &gt;50 years free from known CVDs. All subjects underwent non-contrast cardiac CT and blood test for serum hs-TnI was concomitantly performed. Relationship between Agatston score, a parameter used to quantify coronary artery calcification and serum hs-TnI level was analysed using spearman correlation and logistic regression models. Results A total of 338 HIV-infected adults (median age 54 years, 62% men) were included. All of them were in antiretroviral therapy with a median 18 years of exposure. The median CD4 cell count was 614 cell/mm3, 98% were virologically suppressed. Hs-TnI was correlated with coronary artery calcification with the correlation coefficient of 0.287 (p&lt;0.0001). Multivariated logistic regression analysis demonstrated that serum hs-TnI concentration was associated with an increased odd of coronary artery calcification (Agatston score&gt;0) (OR 1.64; 95% CI, 1.05–2.56, p=0.029). To detect coronary artery calcification, using the hs-TnI in addition to Thai CV risk score slightly increased the ROCAUC from 0.6827 to 0.692 (p=0.45). Distribution of CAC score over hs-TnI Conclusion Among well-controlled HIV-infected patients without established CVDs, hs-TnI concentration was associated with coronary artery calcification. This could be a potential biomarker for an early risk stratification of subclinical coronary atherosclerosis in this population. The association with long-term adverse cardiovascular outcome needs to be validated in the future study. </jats:sec

Kinetics of NAb responses following TDNA prime-boost immunization.

<p>NAb titers of individual mice sera (<i>n</i> = 6), against each of the four dengue serotypes were shown separately. Opened circle and grey square represent the median PRNT50 titer with inter-quartile ranges for the TDNA group and the empty vector group, respectively. Arrows represent the injection of each dose of TDNA. * indicates <i>p</i><0.05.</p

DataSheet_1_Feasibility of plant-expression system for production of recombinant anti-human IgE: An alternative production platform for therapeutic monoclonal antibodies.docx

Omalizumab, the anti-immunoglobulin IgE antibody is the only approved and available monoclonal antibody as an auxiliary medicament for the severe respiratory allergic reactions. It forms small size immune complexes by binding to free IgE, thereby inhibiting the interaction of IgE with its receptors. Additionally, the anti-IgE can also differently shape the airflow by impeding the stimulation of IgE receptors present on structural cells in the respiratory tract. The present study aimed to use plants as an expression system for anti-human IgE antibody production, using Nicotiana benthamiana as hosts. Recombinant Agrobacterium tumefaciens containing heavy chain (HC) and light chain (LC) domains of anti-human IgE were co-transformed in N. benthamiana. The assembling of the antibody and its expression was detected by SDS-PAGE and Western blot analysis. The functional ability of the anti-IgE antibody was determined via its binding capacity with target IgE by ELISA and the inhibition of basophil activation. The anti-human IgE mAb generated in plants was shown to be effective in binding to its target IgE and inhibit the IgE-crosslink in RS-ATL8 reporter cells. Although, antibody yield and purification process have to be further optimized, this study demonstrates the use of plant expression system as a promising platform for the production of Omalizumab which showed a comparable in vitro function to that of commercial Omalizumab (Xolair) in the inhibition of basophil activation.</p

Image_1_Feasibility of plant-expression system for production of recombinant anti-human IgE: An alternative production platform for therapeutic monoclonal antibodies.jpeg

Omalizumab, the anti-immunoglobulin IgE antibody is the only approved and available monoclonal antibody as an auxiliary medicament for the severe respiratory allergic reactions. It forms small size immune complexes by binding to free IgE, thereby inhibiting the interaction of IgE with its receptors. Additionally, the anti-IgE can also differently shape the airflow by impeding the stimulation of IgE receptors present on structural cells in the respiratory tract. The present study aimed to use plants as an expression system for anti-human IgE antibody production, using Nicotiana benthamiana as hosts. Recombinant Agrobacterium tumefaciens containing heavy chain (HC) and light chain (LC) domains of anti-human IgE were co-transformed in N. benthamiana. The assembling of the antibody and its expression was detected by SDS-PAGE and Western blot analysis. The functional ability of the anti-IgE antibody was determined via its binding capacity with target IgE by ELISA and the inhibition of basophil activation. The anti-human IgE mAb generated in plants was shown to be effective in binding to its target IgE and inhibit the IgE-crosslink in RS-ATL8 reporter cells. Although, antibody yield and purification process have to be further optimized, this study demonstrates the use of plant expression system as a promising platform for the production of Omalizumab which showed a comparable in vitro function to that of commercial Omalizumab (Xolair) in the inhibition of basophil activation.</p