Current HIV Research (v.15, #1)

Meet Our Editorial Board Member by Guido Ferrari (1-1).

Preface by Charles Wood, Avi Nath (2-2).

Effect of the Modification of p24 Peptide Antigen on Dendritic Cell Uptake and T Cell Activation by Peter L. Smith, Kirstie J. Norgate, Eve Hegarty, Nathalie Gregeda, Edward Heelas, Maja Sommerfelt, Tonnes Lange, Arnt-Ove Hovden, Birger Sorenson, Angus G. Dalgleish, Mark Bodman-Smith (3-14).
Background: Vacc-4x is a candidate therapeutic vaccine consisting of 4 modified peptides based on conserved regions of HIV-1 p24Gag. Vacc4x has been shown to induce long term cellular immunity in immunized infected individuals resulting a reduction in viral load on treatment interruption.

Objective: Vacc-4x peptides are modified. In this study the effect of modification on uptake of the peptides into PBMC, their subsequent presentation and antigenicity was tested. The feasibility of using an in vitro culture system for testing immunogenicity of peptides using PBMC from uninfected donors was also assessed.

Methods: Labelled peptides were evaluated for uptake into PBMC using flow cytometry or confocal microscopy. Monocyte derived dendritic cells (DC) and autologous T cells were co-cultured with native and modified peptide antigens derived from p24. Activation was measured by flow cytometry and IFN-? ELISPOT.

Results: Peptide modifications significantly increased peptide uptake by monocyte derived dendritic cells. Both the native (unmodified) and Vacc-4x (modified) peptide loaded DC could activate CD4+ and CD8+ T cell responses in vitro. Individual modified peptides induced greater responses than their native counterparts. The grouped Vacc-4x peptides elicited greater IFN ? responses than their native grouped counterparts at a lower concentration, however this effect was not detected at higher concentrations.

Conclusion: These data indicate that the modifications increase uptake, alter the antigenicity of HIV- 1 p24 Vacc-4x peptides and increase the breadth of the response to the Vacc-4x peptides. The in vitro cell culture system is a suitable model for the antigenic assessment of peptide antigens.

Background: Sterile alpha motif and histidine aspartate domain containing deoxynucleoside triphosphate triphosphohydrolase 1 (SAMHD1) is one of the novel restriction factors that potently supresses HIV-1 infection in myeloid cells at an early stage in the viral replication cycle. SAMHD1 activity is blocked by the action of viral accessory protein x (Vpx), which targets and recruits SAMHD1 for proteasomal degradation, in the SIVsm/HIV-2 lineage.

Methods: The impact of SAMHD1 polymorphisms on viral replication in Chinese-origin rhesus macaques (CR) and cynomolgus macaques of Vietnamese origin (CM) have not been reported until now. Therefore, we aimed to explore the polymorphisms, as well as the impact of polymorphisms, on HIV- 2 and SIV infections among CR and CM.

Results: We found two variants, T168C and T320C, located in the SAM domain of CR SAMHD1, which were significantly correlated with the HIV-2ROD/SIVmac239 virus load, suggesting that T168C and T320C probably affected HIV-2ROD and anti-SIVmac239 replication in CR, respectively. Conversely, T320C possibly affected CM SAMHD1-mediated HIV-2ROD restriction. However, none of the variants were correlated with CM SAMHD1-mediated SIVmac239 restriction.

Conclusion: Based on these results, we concluded that SAMHD1 polymorphisms did not affect SIVmac239 replication in CM, but perhaps altered HIV-2ROD infection; however, different sites of the SAM domain of SAMHD1 were responsible for restricting the replication of different viruses in CR.

Comparison of Antibody Immune Responses to Different HIV-1 Envelope Glycoprotein Mutants by Jian-Dong Liu, Li Ren, Bin Ju, Wei Song, Xiang-Yang Ge, Kun-Xue Hong, Ying Liu, Wei Xu, Yan-Ling Hao, Yi-Ming Shao (23-30).
Background: The identification of immunogens is crucial for human immunodeficiency virus type 1 (HIV-1) vaccine development. In our previous study, we demonstrated that HIV-1 envelope glycoprotein mutants based on the equine infectious anemia virus (EIAV) attenuated vaccine enhance immunogenicity, both for DNA immunization alone and as a combined DNA prime-vaccinia boost immunization. An RV144 clinical trial has demonstrated that an envelope protein boost may provide some degree of protection against HIV-1 infection.

Methods: In order to explore the antibody immune responses to two HIV-1 envelope glycoprotein mutants based on the EIAV vaccine and wild-type envelope glycoprotein, mice and guinea pigs were immunized using a DNA prime-protein boost immunization strategy.

Results: The result showed, compared with wild-type gp140, gp140 2M (which contained 2 sites amino acid mutations) and gp140 5M (which contained 5 sites amino acid mutations) increased envspecific IgG and IgG3 binding antibody titers. Gp140 2M resulted in a slight improvement in the neutralizing antibody response against sensitive HIV-1 isolates compared with gp140.

Conclusion: These findings have implications for HIV-1 vaccine development based on the HIV-1 CN54 envelope glycoprotein.

The Changes of Positive Selection Within env Gene of HIV-1 B', CRF07_BC and CRF08_BC from China Over Time by Tingting Li, Binlian Sun, Yanyan Jiang, Haiyan Zeng, Yanpeng Li, Yan Wang, Rongge Yang (31-37).
Background: It is not clear about the possible evolutionary changes of the three predominant strains of HIV-1 in China over the course of the epidemic. Envelope (env) gene of HIV-1 is a good target for this evolutionary pressure for its enriched epitopes.

Methods: We collected 159 full or part of env sequences sampled between 1997 and 2010 from database of China, we calculated the genetic distance, detected the positively selected sites by PAML suite and then compared the number using Fisher's exact test between the early period 1997 to 2003 and the late period 2004 to 2010.

Results: We found that the diversity of env genes had increased significantly and the positively selected sites were significantly becoming more over time. V2, V4, and C3 regions had suffered an increase positive selection pressure, while V1, V3, V5 and other conserved regions were relatively stable. Several sites were widely selected and compactly located in C3 and V4, five sites were consecutive in V4.There were two common positively selected sites in all groups: 413T in gp120 and 619L in gp41.

Conclusion and Discussion: The common positively selected sites identified in our study implied that they are important in viral survival and adaptation. Based on the role of V3 region in coreceptor determination and disease progression, our results suggested that the virulence of HIV-1 in China might be stable in the short time span. Given the overall increased tendency of positively selection sites in env, we might predict a less virulent state in the long run.

Background: South Africa's Prevention of Mother-to-Child Transmission (PMTCT) of Human Immunodeficiency Virus (HIV) program has been implemented for over ten years. This has led to a considerable reduction in mother-to-child transmissions. In 2010, the Northern Cape Province had a 1.4% transmission rate, being the lowest in the country.

Objective: To describe the implementation of the PMTCT program in the Frances Baard district, Northern Cape Province; South Africa and identify factors that may influence the effectiveness of this program.

Method: 100 Mother-child pairs from four clinics participated in this cross-sectional study. Information was collected on socio demographics, antenatal clinic attendance, feeding counseling, knowledge and practices, CD4 cell count and HIV stage, antiretroviral treatment (ART) initiation/adherence and partner involvement.

Results: Almost 25% of mothers first attended an antenatal clinic during their third trimester, out of them, 52.2% were tested for HIV infection for the first time. Most of the mothers received counseling on feeding practices during antenatal visits (97%), after labor (64%) and during post natal visits (84%). Most mothers knew the definition (77%) and recommended duration (76%) of exclusive breastfeeding and the dangers of defaulting ART when breastfeeding (75%), but only two mothers knew the risk of mixed feeding. Fifteen mothers were pressured to stop breastfeeding by healthcare professionals before 12 months. More than half (52%) of the mothers started ART during their last pregnancy, among them, 50% only started treatment after 14 weeks of gestation. The children who received ART, 13.7% defaulted their treatment for one day or more. All three HIV infected children's mothers defaulted their own ART while breastfeeding.

Conclusion: The PMTCT program is implemented according to the national policy in this district. Earlier ART initiation and improved compliance, education of healthcare workers, more focused counseling sessions and improving community awareness of early antenatal visits may improve effectiveness of this program.

Conjugated Linoleic Acid Isomers Exert Differential Effects on an Adipocyte Model of HIV-associated Lipodystrophy by Cathríona R. Loonam, Sandra D. O';Dell, Paul A. Sharp, Anne Mullen (46-55).
Background: HIV-associated lipodystrophy is associated with decreased expression of PPAR-γ in adipose tissue. Conjugated linoleic acid (CLA) isomers (cis9, trans11 and trans10, cis12) are putative PPAR-γ agonists, but have not previously been investigated in the context of HIVassociated lipodystrophy.

Method: 3T3-L1 pre-adipocytes were differentiated in the presence of ritonavir (20 μM as per previous experimental models) and 100 μM cis9,trans11, trans10,cis12 or vehicle control, DMSO. Microarray analysis, RT-PCR, DNA binding ELISA and Oil Red O staining were used to investigate adipocyte gene expression and binding, protein secretion and triglyceride storage.

Results: trans10, cis12 + ritonavir altered the expression of 2160 gene transcripts greater than 1.5-fold compared with control, while 257 gene transcripts were altered by cis9,trans11 + ritonavir (P<0.001). trans10,cis12 + ritonavir down-regulated Pparg (-1.55) and Adipoq (-2.95), as well as differentiation (Fcor (-4.78-fold), Arl4a (-4.84), Itga6 (-2.45), Id4 (-2.01)) and triglyceride storage genes (Mrap (- 8.25), Scd1 (-4.34), Lipin1 (-2.52)). Changes in Adipoq were confirmed by RT-PCR (P=0.038) and adiponectin secretion by ELISA (P= 0.003). cis9,trans11 + ritonavir increased PPAR-γ nuclear binding to its gene response element (P=0.038). Both isomers increased triglyceride storage in the presence of ritonavir (P<0.001).

Conclusion: In the presence of ritonavir, trans10, cis12 appears to be detrimental, while cis9, trans11 was beneficial and may mediate its effects via PPAR-γ. Further research is required to determine the potential role of CLA isomers as therapeutic agents in the management of HIV-associated lipodystrophy.

Trimethyl Chitosan Improves Anti-HIV Effects of Atripla as a New Nanoformulated Drug by Sepideh Shohani, Mahdieh Mondanizadeh, Asghar Abdoli, Behzad Khansarinejad, Mohammad Salimi-Asl, Mehdi Shafiee Ardestani, Maryam Ghanbari, Mehrdad Sadeghi Haj, Rezvan Zabihollahi (56-65).
Background: Highly active antiretroviral therapy (HAART) has been commonly used for HIV treatment. Its main drawbacks like drug resistance and side effects raised researcher's interest to find new approaches for its treatment. Trimethyl chitosan is one of the drug carriers which has been introduced recently.

Materials and Methods: the conjugated atripla-trimethyl chitosan was designed and characterized by zetasizer, AFM and FTIR techniques. The drug conjugation with trimethyl chitosan and cellular uptake of nano-conjugate were determined by spectrophotometry. XTT test was used to measure the cytotoxicity. Anti-retroviral efficiency was studied by ELISA test.

Results: Zetasizer Results proved that the average size of nano-conjugate particles agglomeration was 493.4±24.6 nm but the size of the majority of the particles was 177.2±7.8 nm with the intensity of 87.9%. AFM technique revealed that the sizes of nano-conjugate and trimethyl chitosan were 129 nm and 59.78 nm, respectively. Zeta potential was -1.35±0.04 mv for nano-conjugate and -7.69±0.3 mv for drug. Conjugation efficiency of atripla with trimethyl chitosan was 5.27%. Measured cellular uptake with spectrophotometry for nano-conjugate was about twice of the free drug in examined concentrations (P=0.007). Compared to atripla, the nano-conjugate showed a higher inhibitory effect on HIV replication (P=0.0001).

Conclusion: The result showed that atripla-TMC conjugate does not have a significant cytotoxicity effect. Due to the higher inhibitory effect of nano-conjugate on viral replication, it can be used in lower concentration for antiviral treatment, which resulted in reduction of drug resistance and other side effects.

HIV-1 Drug Susceptibility to Potential Second- and Third-Line Antiretroviral Regimens among Cameroonian Patients: Evidence from a Crosssectional Design by Aubin J. Nanfack, Desire Takou, Joseph Fokam, Romina Salpini, Maria M. Santoro, Giulia Cappelli, Martin Baane, Suzie M. Tetang, Josef Eberle, Lutz Gurtler, Francesca Ceccherini-Silberstein, Judith N. Torimiro, Vittorio Colizzi, Carlo-Federico Perno, Alexis Ndjolo (66-73).
Background: Scale-up of antiretroviral therapy (ART) and the growing number of longterm treated patients may favor multi-HIV drug resistance (HIVDR) in resource-limited settings. Understanding the burden of HIVDR with ART-exposure may provide new insights for an effective long-term management of infected patients.

Methods: Sixty-six HIV-infected individuals (18 ART-naïve, 24 failing first-line, 24 failing secondline ART) living in Yaounde-Cameroon were evaluated by sequencing protease-reverse transcriptase (PR-RT, n=62), envelope-V3 loop (V3, n=58) and integrase (IN, n=30) regions. Drug resistance mutations (DRMs) were interpreted using Stanford University HIV drug resistance database and geno2pheno, while viral tropism prediction was done using geno2pheno, position-specific scoring matrices (PSSM) and Net charge rule.

Results: Participants, from naïve, first- to second-line, had respectively 5.30, 4.85 and 4.66 log HIV RNA, and 532, 203 and 146 CD4 cells/mm3), and infected with diverse HIV-1 non-B clades (58.1% CRF02_AG). Among ART-naïve patients, 6.7% harbored K103N, 28.6% had IN accessory-mutations (L74I, E157Q) and 26.7% carried CXCR4-tropic viruses. At first-line failure, 79.2% harbored DRMs to nucleoside and non-nucleoside RT inhibitors, 33.3% had IN accessory-mutations (L68I, L74I, T97A, E157Q), and 47.4% carried CXCR4-tropic viruses. At second-line failure, 91.3% harbored multi-DRMs to PR-RT inhibitors (with 52.2% and 4.3% DRMs to second-generation NNRTIs and darunavir/ r, respectively), 27.3% had IN accessory-mutations (L74I, T97A, E157EQ), and 37.5% carried CXCR4-tropic viruses.

Conclusion: Levels of PR-RT resistance increases with ART-exposure, with needs for new ART-options following second-line failure. IN inhibitors and darunavir/r are potentially suitable for a third-line regimen, while the use of maraviroc, etravirine or rilpivirine, requires individual genotypic testing.