The human immunodeficiency virus (HIV) and acquired immune deficiency syndrome (AIDS) is a worldwide pandemic that affects the lives of millions of people across all ages. Its devastating effects are far-reaching and affect all aspects of an individual’s daily life. HIV/AIDS is responsible for widespread clinical manifestations involving the head and neck. Disorders of the auditory and vestibular systems are often associated with HIV/AIDS, however the extent and nature of these vestibular manifestations is still largely unknown. The main aim of this research study was to investigate vestibular functioning and pathology in adults with HIV/AIDS. This was achieved through three main research steps: a systematic literature review of the body of peer-reviewed literature on HIV/AIDS related vestibular manifestations and pathology, a description and comparison of vestibular involvement in adults with and without HIV/AIDS and an investigation to determine if HIV/AIDS influence the vestibulocollic reflex (VCR) pathways. For the first study a systematic literature review related to vestibular findings in individuals with HIV infection and AIDS was conducted. A varied search strategy was used across several electronic databases to identify relevant peer-reviewed reports in English. Several databases (Medline, Scopus and PubMed) and search strategies were employed. Where abstracts were not available, the full paper was reviewed, and excluded if not directly relevant to the study’s aims. Articles were reviewed for any HIV/AIDS associated vestibular symptoms and pathologies reported. For the second and third study, a cross-sectional, quasi-experimental comparative research design was employed. A convenience sampling method was used to recruit subjects. The sample consisted of 53 adults (29 male, 24 female, aged 23-49 years, mean = 38.5, SD = 4.4) infected with HIV, compared to a control group of 38 HIV negative adults (18 male, 20 female, aged 20-49 years, mean = 36.9, SD = 8.2). A structured interview probed the subjective perception of vestibular complaints and symptoms. Medical records were reviewed for cluster of differentiation 4+ (CD4+) cell counts and the use of antiretroviral (ARV) medication. An otologic assessment and a comprehensive vestibular assessment (bedside assessments, vestibular evoked myogenic potentials, ocular motor and positional tests and bithermal caloric irrigation) were conducted on all subjects. The systematic literature review identified 442 records, reduced to 210 after excluding duplicates, reviews, editorials, notes, letters and short surveys. These were reviewed for relevance to the scope of the study. There were only 13 reports investigating vestibular functioning and pathology in individuals affected by HIV/AIDS. This condition can affect both the peripheral and central vestibular system, irrespective of age and viral disease stage. Post-mortem studies suggest direct involvement of the entire vestibular system, while opportunistic infections such as oto- and neurosyphilis and encephalitis cause secondary vestibular dysfunction resulting in vertigo, dizziness and imbalance. The second study showed an overall vestibular involvement in 79.2% of subjects with HIV in all categories of disease progression, compared to 18.4% in those without HIV. Vestibular involvement increased from 18.9% in the Centers for Disease Control and Prevention (CDC) category 1 to 30.2% in category 2. Vestibular involvement was 30.1% in category 3. There was vestibular involvement in 35.9% of symptomatic HIV positive subjects and 41.5% in asymptomatic HIV positive subjects. Individuals with HIV were 16.6 times more likely to develop vestibular involvement during their lifetime, than among individuals without this disease. Vestibular involvement may occur despite being asymptomatic. The third study showed that abnormal cervical vestibular evoked myogenic potentials and caloric results were significantly higher in the HIV positive group (p=.001), with an odds ratio of 10.2. Vestibulocollic reflex and vestibulo-ocular reflex involvement increased with progression of the disease. There were more abnormal test results in subjects using ARV therapies (66.7%) compared to those not using ARV therapies (63.6%), but this difference was not statistically significant. Vestibular involvement was significantly more common in subjects with HIV than among those without this disease. This disease and its associated risk profile include direct effects of the virus on the vestibular system as demonstrated by postmortem studies. Opportunistic infections may compromise the functioning of the sensory and neural structures of hearing and the vestibular system indirectly, causing vertigo, dizziness or disequilibrium. Ototoxicity may also be related to vestibular dysfunction, due to the ototoxic nature of certain ARV medications. HIV/AIDS influence not only the vestibulo-ocular reflex, but also the vestibulocollic reflex pathways. Primary health care providers could screen HIV positive patients to ascertain if there are symptoms of vestibular involvement. If there are any, then they may consider further vestibular assessments and subsequent vestibular rehabilitation therapy, to minimize functional limitations of quality of life. / Thesis (DPhil)--University of Pretoria, 2014. / lk2014 / Speech-Language Pathology and Audiology / DPhil / Unrestricted

Objetivo: Evaluar si existe asociación entre el abandono con la falla terapéutica en inmigrantes venezolanos con infección por el virus de inmunodeficiencia humana (VIH) que reinicien tratamiento antirretroviral de gran actividad (TARGA). Métodos: Llevamos a cabo una cohorte retrospectiva en el Hospital Nacional Arzobispo Loayza. Incluimos pacientes que reiniciaron tratamiento TARGA. La variable de resultado fue la falla terapéutica (FT), compuesta por falla inmunológica (FI), virológica (FV) y clínica (FC). La variable de exposición fue el abandono terapéutico, aquellos que no recibieron tratamiento por 30 días, de 30 días a seis meses y de seis meses a más. Las variables control fueron el sexo, la orientación sexual, el nivel de instrucción, la edad y comorbilidades. Utilizamos modelos lineales generalizados de Poisson con errores estándar robustos para calcular riesgo relativo a nivel crudo (RR) y ajustado por criterio estadístico (RRa1) y epidemiológico (RRa2). Resultados: Incluimos 294 pacientes, 47,7% de ellos abandonaron TARGA, 32,7% abandono menos de seis meses, 15% abandonó mas de seis meses y el 27,9% tenían FT. Comparado con aquellos que no abandonaron, un abandono menor a 6 meses [RRa1: 1,98 (IC95%: 1,27 a 3,09);] y de seis meses a más [RRa1: 3,17 (IC95%: 2,02 a 4,95);] incrementó el riesgo de FT. El abandono de hasta seis meses [RRa1: 2,32 (IC95%: 1,40 a 3,84)] y de seis meses a más [RRa1: 3,93 (IC95%: 2,39 a 6,45)]; aumentó el riesgo de FV. En el caso de la FC [RRa1: 1,96 (IC95%: 0,67 a 5,79)] y la FI [RRa1: 2,99 (IC95%: 0,87 a 10,30)] no encontramos evidencia estadística de asociación con el abandono terapéutico. Conclusiones: El abandono del TARGA incrementa el riesgo de FT y FV en pacientes inmigrantes venezolanos. Dentro de la valoración integral que los pacientes con VIH debe explorarse el abandono como una variable de alta a importancia. / Objective: To evaluate the association between antiretroviral treatment interruption with the treatment failure in adults with human immunodeficiency virus (HIV) infection that reinitiates (HAART). Methods: We carried out a retrospective cohort study at the Hospital Nacional Arzobispo Loayza. We included immigrant patients who reinitiate HAART. The outcome was treatment failure (TF) may be immunological (IF), virological (VF), and clinical (CF) failure. The exposure variable was antiretroviral treatment interruption. The control variables measured were sex, sexual orientation, educational level, age, and comorbidities. We performed linear Poisson models with robust standard errors to calculate relative risk at the crude level (RR) and adjusted by statistical and epidemiological criteria. Results: We included 294 in the analysis. 47.7% of them defaulted treatment, 32.7% abandoned less than six months, 15% abandoned more than six months and 27.9% did TF; 24.6% VF; 6.8% IF, and 6.4% CF. A treatment interruption less than 6 months [aRR: 1.98 (95% CI: 1.27 to 3.09);] and from six months to more [aRR: 3.17 (95% CI: 2.02 to 4.95)] increased the risk of TF. Likewise, treatment interruption of up to six months [aRR: 2.85 2.32 (95% CI: 1.40 to 3.84)] and from six months to more [aRR: 3.93 (95% CI: 2.39 to 6,45)]; increased the risk of VF concerning patients who did not abandon treatment. In the CF [aRR: 1,96 (IC95%: 0,67 a 5,79)] and IF [aRR: 2,99 (IC95%: 0,87 a 10,30)] case, we did not find statistical evidence of an association with the abandonment time. Conclusions: The HAART treatment interruption is associated with the development of therapeutic and virologic failure. Treatment interruption should be explored as a variable of high importance in the assessment of patients with HIV. / Tesis

Introduction International guidelines endorse the screening of patients for human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV) and Chlamydia trachomatis before assisted reproductive techniques (ART). At present no such guidelines exists in South Africa. At the Reproductive and Endocrine Unit (referred to as “the Unit”) of Steve Biko Academic Hospital, all patients with unknown HIV status are counselled and a blood sample is collected during the initial visit for automated laboratory based HIV screening. These HIV results are not available before semen samples are processed. Furthermore, patients are not screened for HBV, HCV and Chlamydia trachomatis. Couples attending the Unit are of a low to middle socio-economic status and experience financial constraints. Moreover, automated laboratory based assays are expensive to perform. Rapid testing is a cost effective and practical method from screening patients, with a 20–30 minute result turnover time. Until screening at the Unit is improved, the possible identification of semen characteristics that could indicate HIV infection would be a useful tool. Materials and Methods The following rapid point-of-care assays were evaluated: Determine® HIV-1/2 combo test (n=100), Determine® HBsAg test (n=100), DIAQUICK HCV kit (n=74), and the DIAQUICK Chlamydia trachomatis kit (n=30). For profiling, parameters from a basic semen analysis of HIV-positive males (n=60) were compared with HIV-negative males (n=60). Information pertaining to CD4 count, antiretroviral treatment and plasma viral load of HIV-positive males were analysed. Results From all patients included in the study, 8% tested positive for HIV. The risk of a female being HIV-positive was 3.73 times higher than for males. In the pilot study to explore rapid testing for HBV and HCV, 1% and 1.4% of patients tested positive respectively. When testing for Chlamydia trachomatis 31.3% of females, but no males tested positive. Comparing semen profiles, no significant differences were found between samples from HIV positive and negative males or between HIV positive males categorised by CD4 cell count (p>0.05). For the HIV-positive group with a detectable plasma HIV viral load (>40 copies/ml), a significant difference was observed in the semen viscosity (p=0.0460). Significant differences were noted in the sperm motility (immotile sperm p=0.0456, progressive sperm p=0.0192) of patients receiving antiretroviral (ARV) therapy. Discussion and Conclusion The use of rapid testing is an acceptable and feasible option for improving current screening protocols at the Unit. The absence of definite alterations in the semen characteristics of HIV-positive men further motivates the need for a simpler, point-of-care screening protocol. The prevalence of HBV was lower than that reported in the general population of South Africa and further investigation is needed. Although the sample size was small, HCV prevalence was similar to that of the general population. One third of females tested positive for Chlamydia trachomatis. The methodology used was possibly not appropriate for males. This study highlighted the need for guidelines that address the specialised needs of ART clinics in resource-limited and developing countries with a high HIV prevalence. / Dissertation (MSc)--University of Pretoria, 2013. / gm2014 / Obstetrics and Gynaecology / unrestricted

Laser application in the field of biological and medical sciences has significantly grown, thereby strengthening the field of Biophotonics. Research conducted in Biophotonics focuses on the concept of using light especially in the visible and near infrared regions of the electromagnetic radiation for the evaluation of living systems. In this thesis new discoveries are presented about low level laser therapy, optical trapping, transmission spectroscopy, luminescence spectroscopy and structured illumination microscopy (SIM), displaying the impact each technique has on HIV infected cells. The results showed that the irradiation of HIV-1 infected TZM-bl cells with low power red laser reduces HIV-1 infection. The outcomes of this study further proved that when irradiation is used in conjunction with efavirenz, an antiretroviral drug, HIV-1 infection could be reduced to undetectable levels in TZM-bl cells. Through the coupling of transmission spectroscopy with optical trapping, and separately, use of luminescence spectroscopy, label free diagnosis of HIV in infected cell samples was achieved. This finding affirms that HIV-1 infection can be detected in a label free manner when using laser based techniques. Furthermore, the photoluminescence spectrometer system was employed to generate a decay curve, which was necessary so as to have some understanding on lifetime of the luminescent signal in infected TZM-bl cells. Finally, in order to confirm that indeed TZM-bl cells were infected, an established super-resolution microscopy system SIM was used to detect HIV-1 infection in TZM-bl cells. Indeed in the infected cells viral molecules p24 and gp41 were detected through SIM, while they were not detected in uninfected cells. In future studies, super resolution microscopy would be coupled to an optical trapping system in order to confirm that each trapped cells is whether infected or uninfected so as to improve HIV diagnosis. / College of Science, Engineering and Technology / Ph. D. (Science, Engineering and Technology)

Human immunodeficiency virus type 1 (HIV-1) is a lentivirus that causes acquired immunodeficiency syndrome (AIDS). Since the first cases of AIDS were described in 1981, HIV-1 has become one of the most serious public health threats in the world. There are approximately 38 million people worldwide currently living with HIV-1. 28 million of these people have access to antiretroviral therapy (ART) that is highly effective in reducing viral load to undetectable levels, thereby curbing the risk of viral transmission and preventing progression to AIDS. Despite their effectiveness in suppressing HIV-1 viremia, systemic inflammation remains as a hallmark of HIV-1 infection in vivo. This persistent immune activation is often associated with non-AIDS related complications, including elevated risk of neurocognitive and cardiovascular disorders. Several different mechanisms may contribute to this chronic immune activation and inflammation in people living with HIV-1 on ART. One of the contributing factors might be HIV-1 RNA expressed from the provirus. Even though ART potently suppresses HIV-1 replication, it fails to eradicate proviruses established prior to initiation of ART. Ongoing activation of CD4+ T cells and macrophages by HIV-1 proviral transcripts might contribute to the persistent inflammation that remains even after HIV-1 suppression by ART. Previously, our laboratory has shown that induction of innate immune signaling after HIV-1 challenge of primary human dendritic cells (DCs), macrophages, or CD4+ T cells requires integration, transcription from the nascent provirus, and nuclear export of intron-containing HIV-1 RNA through the Rev-CRM1 pathway. However, these studies failed to identify the innate immune sensor of intron-containing HIV-1 RNA. Here we conducted a targeted loss-of-function screen, using shRNA-expressing lentivectors in human DCs to identify this innate immune receptor. Of the twenty-one candidate genes targeted for knockdown by shRNA, the innate immune response to HIV-1 was inhibited only by knockdown of IFIH1, MAVS, and XPO1. The effect of IFIH1 and MAVS knockdowns on HIV-1-induced immune activation was confirmed in macrophages, and rescue of the knockdown with non-targetable coding sequence showed that IFIH1 protein was required. IFIH1 mutants that are defective for interaction with MAVS blocked activation, demonstrating that MAVS acts downstream of IFIH1 in this system. Since both IFIH1 and DDX58 signal via MAVS, the specificity of HIV-1 RNA detection by IFIH1 was demonstrated by the fact that DDX58 knockdown had no effect on activation; the IFIH1-specific inhibitor Nipah virus V protein blocked the activation by HIV-1. RNA-Seq showed that IFIH1-knockdown in DCs globally disrupted the induction of IFN-1-stimulated genes. Altogether, results presented in this thesis reveal that IFIH1 is required for innate immune activation by intron-containing RNA from the HIV-1 provirus, and potentially contributes to chronic inflammation in people living with HIV-1.

Vpu has been shown to possess two distinct roles in the pathogenesis of HIV. First, Vpu has been shown to down-regulate the expression of CD4 molecules at the plasma membrane of infected cells by targeting CD4 molecules for degradation in the endoplasmic reticulum. Second, Vpu promotes viral egress in specific cell lines termed non-permissive cells by mechanism that remain relatively unclear. Therefore, experiments were conducted in order to identify cellular factors involved in the Vpu-dependent phenotype. Using full-length Vpu as bait in yeast two-hybrid experiments, several candidate cellular factors were identified. One protein, SNAPIN, was identified as a cellular factor putatively involved in the Vpu-dependent phenotype. Further experiments determined that not only do SNAPIN and Vpu interact, but that Vpu also leads to the degradation of SNAPIN by both proteasomal and lysosomal degradation pathways. Over-expression of SNAPIN in cell lines that do not normally require Vpu expression for viral production resulted in a Vpu-dependent phenotype. While over-expression of SNAPIN in otherwise permissive cell lines significantly reduced Vpu-deficient virus production, wild type levels remained relatively constant. Importantly, no defective viral structural protein production was observed; however, intracellular p24/p55 did not accumulate suggesting that in SNAPIN expressing cells, Gag is also targeted for degradation. In addition, the reduction of SNAPIN expression in non-permissive cell lines significantly increased viral titers in supernatants. Of particular interest, even in cells expressing Bst-2 (a previously identified cellular factor involved in the Vpu-phenotype), siRNA mediated knockdown of SNAPIN led to increased viral titers. In addition, the co-transfection of siRNAs targeting both SNAPIN and Bst-2 resulted in an additive effect, in which Vpu-deficient viral titers were nearly equivalent to wild-type titers. Surprisingly, siRNA-mediated knockdown of SNAPIN in Jurkat cells was sufficient to overcome any restriction in viral egress imposed by the deletion of Vpu. Conversely, siRNA targeting Bst-2 had little or no effect on viral titers in Jurkat cells regardless of whether it was transfected alone or in combination with siRNAs targeting SNAPIN. These experiments provide evidence of an alternate cellular restriction mechanism involved in viral egress that is countered by the HIV-1 accessory protein, Vpu. In addition, this research may provide further insight into the complex cellular networks involved in the trafficking of Gag through cellular endosomal pathways.

The best known correlate of protection provided by vaccines is the presence of pathogen specific antibodies after immunization. However, against the Human Immunodeficiency Virus-1 (HIV-1) the mere presence of antibodies specific for the viral Envelope (Env) protein is not sufficient to provide protection. This necessitates in depth study of the humoral responses elicited during infection and by vaccination. While a significant amount of effort has been invested in studying the evolution of antibody responses to viral infection, only limited progress in understanding antibody responses elicited through vaccination has been made. In the studies described here, I attempt to rectify this deficiency by investigating how the quality of a humoral response is altered with the use of different immunization regimens, in particular a DNA prime-protein boost regimen, or with the use of different model HIV-1 Env gp120 immunogens. In a New Zealand White (NZW) rabbit model, we demonstrate that the broader neutralizing activity elicited with the DNA prime-protein boost regimen may be the result of the elicitation of a higher avidity antibody response and a unique profile of antibody specificities. Specifically, use of a DNA prime-protein boost regimen elicits antibodies targeted to the CD4 binding domain of the HIV-1 Env, a specificity that was not frequently observed when only protein based immunizations were administered. We extended this analysis to sera from healthy human volunteers who participated in early phase HIV vaccine trials utilizing either a protein alone immunization regimen, a canarypox prime-protein boost immunization regimen, or a DNA prime-protein boost immunization regimen. Evaluation of sera from these trials demonstrated that the use of a DNA prime-protein boost regimen results in an antibody response with greater neutralization breadth characterized by an increased frequency and titer of antibodies targeted toward the CD4 binding site (CD4bs). In addition to this, the antibody response elicited by the DNA prime-protein boost regimen also exhibited the capability to mediate antibody dependent cell-mediated cytotoxicity (ADCC) activity as well as activation of the complement system. Additionally, in an attempt to better understand the capabilities of antibodies elicited by a DNA prime-protein boost regimen, we generated gp120 specific monoclonal antibodies (mAbs) from a single DNA primed-protein boosted NZW rabbit. Analysis of mAbs produced from this animal revealed that use of this immunization regimen elicits an antibody repertoire with diverse epitope specificity and cross reactivity. Furthermore, these select mAbs are capable of neutralizing heterologous HIV isolates. Further application of mAb generation in rabbits may provide a valuable tool to study immunogenicity of different vaccines and immunization regimens. Concurrently, while demonstrating that a DNA prime-protein boost regimen elicits a higher quality antibody response than that observed with other leading techniques, we also demonstrated that immunogen selection can play a vital role in the quality of the resulting antibody response. By immunizing with two closely related but phenotypically distinct model gp120 immunogens, known as B33 and LN40, we demonstrated that disparate gp120s have different intrinsic abilities to raise a heterologous neutralizing antibody response. Additionally, we showed that residues found within and flanking the b12 and CD4 binding sites play critical roles in modulating neutralizing activity of sera from animals immunized with LN40 gp120, indicating that the broader neutralizing activity seen with this immunogen may be due to differential elicitation of antibodies to this domain.

Nef is an accessory protein encoded by human and simian immunodeficiency viruses (HIV and SIV), and is critical for viral pathogenicity in vivo.The structure of Nef has been resolved and the major cellular activities of Nef are generally described as down-regulation of cell surface molecules, enhancement of virus infectivity and regulation of cell signaling and activation. Macrophages represent a key target of HIV-1 infection and may contribute significantly to viral pathogenesis by facilitating viral propagation, maintaining a viral reservoir and regulating viral replication. During HIV-1 infection, various cytokines and chemokines are induced for viral advantages more than for host defense. We have previously demonstrated that HIV-1 Nef regulates the release of chemokines, MIP-1α and MIP-1ß, from infected macrophages and have proposed that this may enhance conditions for viral replication by promoting recruitment of substrate lymphocytes to sites of infection (1). However, the molecular basis for this Nef activity remains to be defined. The main goals of this thesis are to identify the functional motif in Nef that is responsible for chemokine induction in macrophages and to elucidate the relevance of this motif to other Nef functions. Using a mutagenesis approach, we have eventually identified a novel motif (KEK) that regulates chemokine production in infected macrophages after we excluded several previously described Nef motifs. This motif is conserved in both HIV-1 and SIV Nef proteins. Mutations in this domain abrogated MIP-1ß induction as well as the Nef-dependent release of other secretory factors by macrophages. However, disruption of this motif did not affect other Nef-ascribed activities such as CD4 and MHC-I down-regulation. In addition, we have determined the involvement of viral Env proteins in Nef-induced chemokine production. Distinct signaling pathways that regulate chemokine release in macrophage will also be described. Finally, several possible roles of the KEK motif are proposed and some preliminary results of co-immunoprecipitation experiments will be presented which aim to characterize cellular proteins involved in chemokine regulation by Nef. Collectively, our studies reveal a specific determinant within Nef that is critical for chemokine release by Nef. Identification of this motif paves the way for future studies to explore the molecular machanisms of Nef-regulated cell signaling pathways. Such knowledge may point to new therapeutic strategies that interrupt Nef function and limit the course of HIV-1 infection.

The process of HIV-1 particle production is a multi-step process directed by the viral structural protein Gag. As Gag is the only viral protein required to form virus-like particles, it presents a viable target for anti-viral therapeutics of which there are currently none. Although the functions of Gag during the particle assembly process have been well characterized, one of the least known parts of the assembly process is how Gag is targeted to the site of virus assembly. Two main virus assembly sites have been identified in cells that support HIV-1 replication: the plasma membrane or multivesicular bodies (MVBs). However the mechanism by which Gag is targeted to either of these sites remains unknown. The δ subunit of Adaptor Protein Complex 3 has previously been identified as a cellular co-factor for HIV-1 Gag and was reported to mediate Gag trafficking to MVBs, providing a mechanism for Gag targeting to this assembly site. Additionally, AP-3δ was reported to be required for HIV-1 production, suggesting that Gag to MVB targeting is also required for HIV-1 production. The work presented in this thesis further investigates the role of AP-3δ in Gag trafficking to MVBs and its role in HIV-1 production in previously unexplored host environments. Through the use of RNA interference-mediated depletion of AP-3δ, we determined that AP-3δ is dispensible for virus replication in infected HeLa cells, chronically infected HeLa-LAV cells and infected primary human monocyte-derived macrophages. We concomitantly disrupted AP-3 function by disrupting its association with membranes and observed no effect on virus production. Collectively, these results demonstrate that AP-3δ is not required for HIV-1 replication. However, AP-3δ was demonstrated to be required for Gag targeting to MVBs thus presenting a new model for the function of AP-3δ in the context of HIV-1 replication.

The Human Immunodeficiency Virus (HIV) is able to infect CD4+ T cells as well as macrophages. Macrophage-tropism has been linked to determinants in the envelope of HIV. These determinants allow envelopes to exploit low levels of CD4 for infection. Macrophages are an important reservoir of virus, especially during chronic infection, and are likely responsible for the bulk of virus produced after CD4+T cells have declined. Viral factors that may impact the ability to infect macrophages are worth studying because this cell type is so important in infection. It was previously reported that the macrophage-tropic primary isolate AD8 was vpu-independent. The molecular clone YU-2, derived from brain tissue without culture, was also reported to be macrophage-tropic despite having a mutation in the vpu start codon. It was therefore possible that vpu-independent envelopes could evolve in vivo. To examine this possibility, I constructed chimeras containing wild type or defective vpu start codons, and gp160 sequences from AD8, YU-2 or SF162 (a vpu-dependent control). I also used full length AD8 and YU-2 with wild type or defective vpu start codons. I infected macrophages with equal amounts of virus, and measured viral output over two weeks. Viruses with defective vpu start codons were released to lower levels compared to their wild type vpucounterparts. In contrast to previous reports, the AD8 envelope is not vpu-independent for replication in macrophages. The YU-2 envelope is also not vpu-independent. Macrophage-tropic envelopes from late stages of infection can be sensitive to antibodies that bind the CD4 binding site on gp120, implying that macrophage-tropic envelopes have more exposed CD4 binding sites. Neutralizing antibodies may act as modulators of macrophage-tropism over the course of infection. Using chimeras containing gp120 sequences derived from the PBMC of four HIV+patients, I examined the capacity for envelopes to infect macrophages. Three patients (MM1, 4, and 8) had macrophage-tropic envelopes before and after developing autologous neutralizing antibodies. Three patients (MM1, 4, and 23) developed heterologous antibodies against IIIB, an easily neutralized T-cell line adapted strain of HIV-1. This data indicates that macrophage-tropism in these patients is not modulated by the presence of neutralizing antibodies. The macrophage-tropism of envelopes tends to segregate depending on the tissue origin of the virus. Envelopes from two separate tissues from the same patient exhibit very different infectivity characteristics. The B33 envelope, from brain tissue, is very infectious and is macrophage-tropic, while the LN40 envelope, from lymph node tissue, is weakly infectious and is not macrophage-tropic. Replacing the entire gp41 of LN40 with that of B33 restores some infectivity to LN40. The cytoplasmic domain of gp41 contains many motifs important for assembly and infectivity. To examine which motifs are responsible for the weak infectivity of LN40, I made chimeras of gp41, as well as point mutations in gp41. The LN40 chimera containing the entire gp41 of B33 restored the most infectivity. Point mutations in the palmitoylation site, Pr55gagbinding region, and dileucine motif at the C-terminus also restored infectivity when combined. Determinants in the gp41 cytoplasmic domain are responsible for the weak infectivity of LN40; however, it is possible that there are contributing determinants in gp120, such as the ability to use low levels of CD4. Here, I examined how changes in the vpu and env genes of HIV-1 can impact infectivity, especially infectivity of macrophages. Changes that adversely impact the virus’ ability to infect macrophages may also impact the overall course of disease. However, the data here show that retaining the ability to infect, and replicate in, macrophages give HIV an advantage. I speculate that retaining the ability to infect macrophages gives the virus a reservoir for later in disease, when CD4+ T cells have been depleted, as well as way of avoiding neutralizing antibodies. This work further defines the importance of macrophages in HIV-1 infectivity and disease.