Absence Of Antiretroviral Drugs Research Articles

Isolation of Leukocytes from Human Breast Milk for Use in an Antibody-dependent Cellular Phagocytosis Assay of HIV Targets.

Even in the absence of antiretroviral drugs, only ~15% of infants breastfed by HIV-infected mothers become infected, suggesting a strong protective effect of breast milk (BM). Unless access to clean water and appropriate infant formula is reliable, the WHO does not recommend cessation of breastfeeding for HIV-infected mothers. Numerous factors likely work in tandem to reduce BM transmission. Breastfed infants ingest ~105-108 maternal leukocytes daily, though what remains largely unclear is the contribution of these cells to the antiviral qualities of BM. Presently we aimed to isolate cells from human BM in order to measure antibody-dependent cellular phagocytosis (ADCP), one of the most essential and pervasive innate immune responses, by BM phagocytes against HIV targets. Cells were isolated from 5 human BM samples obtained at various stages of lactation. Isolation was carried out via gentle centrifugation followed by careful removal of milk fat and repeated washing of the cell pellet. Fluorescent beads coated with HIV envelope (Env) epitope were used as targets for analysis of ADCP. Cells were stained with the CD45 surface marker to identify leukocytes. It was found that ADCP activity was significant above control experiments and reproducibly measurable using an HIV-specific antibody 830A.

Chimeric antigen receptors against HIV: Bispecific CD4-based designs for enhanced breadth/potency and minimal immunogenicity

Abstract Chimeric antigen receptor (CAR) technology has potential to achieve durable (life-long?) control of HIV in the absence of antiretroviral drugs, i.e. a “functional cure”. Thus in targeting the HIV-1 Env glycoprotein expressed on the surface of virus-producing cells, CAR design must strive not only for high potency and HIV specificity, but also to approach the ideal qualities of inescapablilty and non-immunogenicity. Our strategy involves bispecific targeting domains consisting of an extracellular region of human CD4, the primary HIV receptor, attached to a second motif that recognizes a distinct highly conserved region on the otherwise highly variable Env. The second motif is intended both to enhance CAR potency, and to prevent the CD4 from acting as an HIV entry receptor on CAR-expressing CD8+ T cells. We have focused on second motifs predicted to lack the immunogenicity associated with antibody-based CARs. A particularly favorable construct contains CD4 linked to the carbohydrate recognition domain of human mannose-binding lectin (MBL), which recognizes the highly conserved oligomannose patch on gp120 (Ghanem et al., Cytotherapy). In another variant, CD4 is linked to the N-terminal portion of the CCR5 co-receptor. We found that the bispecific CARs broadly suppressed infection by genetically diverse HIV-1 isolates, with significantly greater potency than a monospecifc CD4 CAR. Similar results were obtained using rhesus macaque PBMCs and a rhesus version of the CD4-MBL CAR. These results set the stage for testing the activities of the CD4-based bispecific CARs in relevant animal models including rhesus macaques and humanized mice.

Genetic and phenotypic analyses of sequential vpu alleles from HIV-infected IFN-treated patients

Treatment of HIV-infected patients with IFN-α results in significant, but clinically insufficient, reductions of viremia. IFN induces the expression of several antiviral proteins including BST-2, which inhibits HIV by multiple mechanisms. The viral protein Vpu counteracts different effects of BST-2. We thus asked if Vpu proteins from IFN-treated patients displayed improved anti-BST-2 activities as compared to Vpu from baseline. Deep-sequencing analyses revealed that in five of seven patients treated by IFN-α for a concomitant HCV infection in the absence of antiretroviral drugs, the dominant Vpu sequences differed before and during treatment. In three patients, vpu alleles that emerged during treatment improved virus replication in the presence of IFN-α, and two of them conferred improved virus budding from cells expressing BST-2. Differences were observed for the ability to down-regulate CD4, while all Vpu variants potently down-modulated BST-2 from the cell surface. This report discloses relevant consequences of IFN-treatment on HIV properties.

Vectored antibody gene delivery for the prevention or treatment of HIV infection.

To discuss recent progress in the use of vectors to produce antibodies in vivo as an alternative form of HIV prophylaxis or therapy. Instead of passive transfer of monoclonal antibody proteins, a transgene encoding an antibody is delivered to cells by the vector, resulting in expression and secretion by the host cell. This review will emphasize adeno-associated virus (AAV)-based strategies and summarize the evidence in support of this strategy as an alternative to traditional vaccines. We will highlight the major findings in the field and discuss the impact that this approach could have on the prevention, treatment and possibly eradication of HIV in patients. In this emerging field, the emphasis has been on the use of vectors delivering antibodies as an alternative to the development of an HIV vaccine. However, recent findings suggest that AAV-delivered broadly neutralizing antibodies can suppress HIV replication. As such, a single injection of AAV could mediate long-term antibody expression to act as a long-lived therapeutic in the absence of antiretroviral drugs. Vector-mediated antibody expression can both prevent transmission and inhibit the replication of established HIV infections. As such, it offers an alternative to immunogen-based vaccine design and a novel therapeutic intervention by enabling precise manipulation of humoral immunity. Success may enable not only the development of effective prevention against HIV but may also provide an alternative to a lifetime of antiretroviral drugs taken by those who are already infected.

Towards the embodiment of biosocial resistance? How to account for the unexpected effects of antiretroviral scale-up in the Central African Republic

At the fringes of the unprecedented medication scale-up in the treatment of HIV, many African countries have experienced dramatic antiretroviral drug stock-outs. Usually considered the result of irrational decisions on behalf of local politicians, programme managers and even patients (who are stigmatised as immoral), these problems seem not to be so exceptional. However, ethnographic attention to the social consequences of the presence and absence of antiretroviral drugs in the Central African Republic (CAR) suggests that these stock-outs entail far more than logistical failures. In 2010 and 2011 in the CAR, major antiretroviral treatment (ARV) stock-outs resulted in the renewal of ‘therapeutic’ social ties and also significant social resistance and defiance. While this paper explores reasons for the shortage, its focus is on subsequent popular reactions to it, particularly among people who are HIV-positive and dependent on ARVs. The exceptional and ambiguous consequences of these drug stock-outs raise new concerns relevant to the politics of global public health.

Restriction of HIV-1 Genotypes in Breast Milk Does Not Account for the Population Transmission Genetic Bottleneck That Occurs following Transmission

BackgroundBreast milk transmission of HIV-1 remains a major route of pediatric infection. Defining the characteristics of viral variants to which breastfeeding infants are exposed is important for understanding the genetic bottleneck that occurs in the majority of mother-to-child transmissions. The blood-milk epithelial barrier markedly restricts the quantity of HIV-1 in breast milk, even in the absence of antiretroviral drugs. The basis of this restriction and the genetic relationship between breast milk and blood variants are not well established.Methodology/Principal FindingsWe compared 356 HIV-1 subtype C gp160 envelope (env) gene sequences from the plasma and breast milk of 13 breastfeeding women. A trend towards lower viral population diversity and divergence in breast milk was observed, potentially indicative of clonal expansion within the breast. No differences in potential N-linked glycosylation site numbers or in gp160 variable loop amino acid lengths were identified. Genetic compartmentalization was evident in only one out of six subjects in whom contemporaneously obtained samples were studied. However, in samples that were collected 10 or more days apart, six of seven subjects were classified as having compartmentalized viral populations, highlighting the necessity of contemporaneous sampling for genetic compartmentalization studies. We found evidence of CXCR4 co-receptor using viruses in breast milk and blood in nine out of the thirteen subjects, but no evidence of preferential localization of these variants in either tissue.Conclusions/SignificanceDespite marked restriction of HIV-1 quantities in milk, our data indicate intermixing of virus between blood and breast milk. Thus, we found no evidence that a restriction in viral genotype diversity in breast milk accounts for the genetic bottleneck observed following transmission. In addition, our results highlight the rapidity of HIV-1 env evolution and the importance of sample timing in analyses of gene flow.

Replication and drug resistant mutation of HIV-1 subtype B' (Thailand B) variants isolated from HAART treatment individuals in China

BackgroundDrug resistant HIV-1 variants were emergent more and more in AIDS individuals with highly active antiretroviral therapy (HAART) treatment. Understanding the replication and drug resistant mutation of HIV-1 variants isolated from HAART treatment individuals of China could help to design appropriate therapeutic strategies for these individuals.MethodsUse GHOST cell lines to analysis the coreceptor usage of HIV-1 variants. Coculture with PBMCs to analysis the replication capacity. Use RT-PCR to analysis the drug resistant mutation of pol gene.Results13 HIV-1 variants experienced HAART were included in this study. 5 HIV-1 variants used CCR5 coreceptor (R5), while 8 use both CCR5 and CXCR4 coreceptor (R5X4). The replication capacity of R5X4 variants was no difference with R5 variants in vitro without antiretroviral drugs. Compare the drug resistant mutation between first HIV-1 variants and fourth variants; there were 37 drug resistant mutations in first variants and 32 drug resistant mutations in fourth variants. Only 7 drug resistance mutations were lost after coculture for 4 weeks, and 2 drug resistance mutations were emerged.ConclusionThese data suggested that the drug resistant level could not reduce in vitro in absence of antiretroviral drugs in few weeks. And maybe helpful for these HAART experienced individuals when change antiretroviral drugs.

Inverse Relationship between Viral Load and Genotypic Resistance Mutations in Korean Patients with Primary HIV Type 1 Infections

The transmission of antiretroviral-resistant HIV-1 strains is associated with suboptimal virological responses to initial antiretroviral therapy. However, certain types of resistance mutations are known to be associated with decreased viral fitness, which confers a lower replication capacity than that of the wild-type virus in the absence of antiretroviral drugs. Therefore, we evaluated the relationship between antiretroviral resistance mutations and viral replication in the primary HIV-1 infection (PHI) period. From January 2002 to March 2005, 52 PHI patients were identified in the Republic of Korea. HIV-1 RNA genotyping was performed, and the resistance mutation score was obtained from the HIV Drug Resistance Database of Stanford University. We defined the sum of the average resistance mutation scores (SARMS) for each antiretroviral drug class as a measure of the degree of resistance of any specific strain. The overall mean SARMS was 2.00 +/- 2.74, and the annual mean did not change significantly during the study period. No critical resistance mutation gene was identified in the study group. The SARMS showed a weak negative correlation with the viral load log10 during PHI, but without statistical significance (r = -0.274, p = 0.051). But the mean SARMS of patients with a viral load exceeding 100,000 copies/ml was significantly lower than that of patients with a viral load of less than 100,000 copies/ml (p = 0.03). Evaluation of the potency of antiretroviral resistance revealed a weak negative correlation with viral replication in the PHI period. This could be one reason why the transmission of resistant strains in PHI patients is not increasing significantly despite the widespread use of highly active antiretroviral therapy (HAART).

Use of a novel assay based on intact recombinant viruses expressing green (EGFP) or red (DsRed2) fluorescent proteins to examine the contribution of pol and env genes to overall HIV-1 replicative fitness

Multiple studies have described a reduction in the replicative fitness of HIV-1 isolates harboring mutations that confer resistance to antiretroviral drugs. Contradictory results, however, have been obtained depending on the methodology used in each study (Quinones-Mateu, M.E., Arts, E.J., 2002. Fitness of drug resistant HIV-I: methodology and clinical implications. Drug Resist. Update 5, 224–233), affecting our understanding of the potential relationship of viral replicative fitness with HIV-1 disease. It has been demonstrated previously that both pol and env genes play a major role in HIV-1 replicative fitness of clinical isolates. Therefore, measuring clinically relevant replicative fitness using recombinant viruses where a single mutation and/or viral gene have been introduced does not seem like a reasonable approach in this era of multi-target antiretroviral therapy. A novel method was developed to measure HIV-1 replicative fitness based on recombinant viruses expressing the enhanced green fluorescent (EGFP) or the Discosoma sp. red fluorescent (DsRed2) proteins in a HIV-1 NL4-3 backbone. Contrary to previous designs to analyze HIV-1 fitness, these replication competent viruses were created in an intact viral genetic background (without deleting or affecting the expression of any viral gene). This new system was used to evaluate the contribution of drug-resistance mutations in the pol and env genes to overall viral replicative fitness (in the presence and absence of drug pressure) using direct growth competition experiments. Mutations in pol showed a stronger effect on HIV-1 replicative fitness than mutations in the env gene associated with resistance to enfuvirtide, corroborating the plasticity of the later gene to accept mutations and the sensibility of the protease and reverse transcriptase enzymes to drug-associated primary mutations. In conclusion, a new protocol was used to measure HIV-1 replicative fitness in either the presence or absence of antiretroviral drugs, which may be used as a high-throughput assay to help us understand the clinical significance of viral fitness.

An international screening tool for HIV dementia

In this issue of AIDS, Sacktor and colleagues [1] describe a relatively simple bedside screening tool for HIV dementia. Despite the advent of HAART, dementia remains both common and debilitating in the HIV-infected population. HIV dementia will ultimately affect 10–15% of all HIV infected individuals and even higher rates pertain, perhaps approaching 60% [2], for any degree of cognitive impairment occurring by the time of death in the absence of antiretroviral drugs. Along with HIV-associated peripheral neuropathy, HIV dementia represents one of the most common of the neurological disorders resulting from this viral infection. In general, studies comparing the incidence of this disorder from the period of time before to that after the introduction of HAART indicated that its incidence had declined, at least as an AIDS defining event [3–5]. However, when the DANA (pre-HAART) and NEAD (post-HAART) cohorts were compared, the cumulative incidence of HIV dementia was virtually identical at 1 and 2 years [6]. These cumulative incidences were 25% in both studies at one year and 40% and 38%, respectively, at 2 years. Despite the decline in incidence, the prevalence of HIV dementia appeared to increase; however, these findings have been inconsistently demonstrated, perhaps because of differing populations. Some investigators have proposed that HAART has changed the pattern of neuropsychological abnormality observed in HIV infection and, possibly, that in HIV dementia [7]. In its advanced form, diagnosing HIV dementia is not difficult. The illness is characterized by features chiefly reflective of a frontal subcortical dementing process typically associated with a severe degree of immunodeficiency. A decline in mental alacrity (bradyphrenia) and slowness of movement (bradykinesia) are salient components of the disorder and the patient frequently complains of difficulty with memory. When, as is often the case, the disorder occurs in association with other illnesses attending advanced immunodeficiency, it may be overlooked. Less advanced dementia may be difficult to diagnose. In general, detailed neuropsychological batteries are employed to determine the degree and nature of the cognitive impairment and to identify contributing or obfuscating co-morbidities such as depression and anxiety. Unfortunately, these batteries require professionals skilled in their performance and are both time consuming and costly. They are not widely available and certainly not applicable for the AIDS populations in developing countries. In an effort to develop a bedside screening tool, Power and colleagues [8] devised a test that could be administered in minutes consisting of four components. The test focuses on tasks that are likely to be affected by a frontal subcortical disorder and includes components that assess verbal memory recall, writing speed for the alphabet in capital letters, speed and ability to copy a three dimensional figure, and suppression of ocular saccadic refixation on an image. Sacktor and colleagues [1] have essentially revised this set of tasks to a total of three which are more universally applicable than the original battery. In essence, they include tasks of recall, non-dominant finger tapping speed, and psychomotor speed determined by the correct repetitive replication of a sequence of hand movements. Currently, it is estimated that there are 42 million people worldwide infected with HIV (http://www.who.int/hiv/pub/epidemiology/epi2002/en/). Fewer than 10% of these individuals live in developed countries. Therefore, access to advanced diagnostic tools and the most highly effective antiretroviral regimens is limited in at least 38 million people. If 10% of this population (a conservative estimate), ultimately develops HIV dementia, this is a staggering burden of this disorder. Reliable bedside methods for determining whether a patient has features of HIV dementia and for following the impact of the disorder would be very important among these populations. In that respect, the International HIV Dementia Scale proposed by Sacktor and colleagues is an important first step. One must bear in mind that this scale appears to be, at best, only a screening tool. The absolute difference in mean scores between the demented and non-demented subjects was small, 1.3 points, on a scale of 12, albeit statistically significant. Not surprisingly, with a cut off score of 10, which provided a sensitivity of 80%, the test lacked specificity. These results parallel those observed with the original HIV dementia scale, but neither sensitivity or specificity seem to be as robust as that observed with the former [8,9]. The relatively low specificity indicates a risk of misclassifying patients without dementia. Additionally, the number of subjects in this study was relatively small and only one site in an underdeveloped country was compared to a US population. Therefore, additional studies will be needed before this test is widely adopted. Furthermore, issues such as re-test validity and practice effect need to be defined. As the authors correctly assert, the International HIV Dementia Scale is but a screening tool and has not been designed to be a substitute for detailed neuropsychological testing. That said, it is a valuable contribution to the field.