Genetic Diversity in HIV-1 Subtype C LTR from Brazil and Mozambique Generates New Transcription Factor-Binding Sites

The HIV-1 subtype C has been substituting the subtype B population in southern Brazil. This phenomenon has been previously described in other countries, suggesting that subtype C may possess greater fitness than other subtypes. The HIV-1 long-terminal repeat (LTR) is an important regulatory region critical for the viral life cycle. Sequence insertions immediately upstream of the viral enhancer are known as the most frequent naturally occurring length polimorphisms (MFNLP). Previous reports demonstrated that the MFNLP could lead to the duplication of transcription factor binding sites (TFBS) enhancing the activity of the HIV-1 subtype C LTR. Here, we amplified and sequenced the LTR obtained from proviral DNA samples collected from patients infected with subtype C from the Southern Region of Brazil (naïve or treatment failure) and Mozambique (only naïve). We confirm the presence of different types of insertions in the LTR sequences of both the countries leading to the creation of additional TFBS. In the Brazilian clinical samples, the frequency of the sequence insertion was significantly higher in subjects experiencing treatment failure than in antiretroviral naïve patients

The Grafting of Universal T-Helper Epitopes Enhances Immunogenicity of HIV-1 Tat Concurrently Improving Its Safety Profile

<div><p>Extracellular Tat (eTat) plays an important role in HIV-1 pathogenesis. The presence of anti-Tat antibodies is negatively correlated with disease progression, hence making Tat a potential vaccine candidate. The cytotoxicity and moderate immunogenicity of Tat however remain impediments for developing Tat-based vaccines. Here, we report a novel strategy to concurrently enhance the immunogenicity and safety profile of Tat. The grafting of universal helper T-lymphocyte (HTL) epitopes, Pan DR Epitope (PADRE) and Pol₇₁₁ into the cysteine rich domain (CRD) and the basic domain (BD) abolished the transactivation potential of the Tat protein. The HTL-Tat proteins elicited a significantly higher titer of antibodies as compared to the wild-type Tat in BALB/c mice. While the N-terminal epitope remained immunodominant in HTL-Tat immunizations, an additional epitope in exon-2 was recognized with comparable magnitude suggesting a broader immune recognition. Additionally, the HTL-Tat proteins induced cross-reactive antibodies of high avidity that efficiently neutralized exogenous Tat, thus blocking the activation of a Tat-defective provirus. With advantages such as presentation of multiple B-cell epitopes, enhanced antibody response and importantly, transactivation-deficient Tat protein, this approach has potential application for the generation of Tat-based HIV/AIDS vaccines.</p></div

Mapping of the B-cell epitopes in Tat.

<p>The antisera collected from mice immunized with (A) WT-Tat or (B) PADRE-CRD proteins were diluted 500 and 1,000 times, respectively, and used in the pepscan analysis. Twenty-mer synthetic peptides with a 10-residue overlap and encompassing the full-length C-Tat consensus sequence were used in the assay. Peptides 1-9 represent full-length subtype C Tat protein. The gray bars (A and B) represent the amino acid sequences generated due to the grafting of the HTL in Tat. The data are presented as the mean absorbance + SD on the x-axis with corresponding peptides on the y-axis. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114155#pone.0114155.s002" target="_blank">Figure S2</a> for data pertaining to the other three HTL-Tat antisera.</p

Cross-clade reactivity of the anti-Tat antibodies.

<p>The antibody titers were determined by incubating serially diluted antisera in triplicate microtiter wells coated with recombinant Tat from subtypes A, B, C and D. The x-axis represents the serum dilution and the y-axis to mean absorbance + SD. Representative data of two or more independent experiments are shown.</p

The HTL-Tat proteins elicit a strong immune response.

<p>The immunization protocol is represented in the inset schema. (A) Antisera collected 14 days after the final booster were used for the titer determination. X-axis corresponds to antibody titer and y-axis to the Tat protein used in immunizations. (B) Splenocytes harvested from mice 14 days after the final booster immunization were stained with CFSE and dilution of the dye in CD4+ cells was evaluated and the mean stimulation index (SI) + SD was plotted on the x-axis. SI  =  Percent CFSE-low (Peptide-stimulated cells)/Percent CFSE-low (DMSO-treated cells). The data presented are representative of two independent experiments (n = 10, * P<0.05).</p