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- 6.5.6 Tracking of sexual contacts and social networks
- 6.5.7 Brain MRI and MRS
- 6.5.8 Neuropsychological assessment, comprehensive neurological examination and psychological assessment and assessment of function
- 6.6 Specimen Collection and Testing
- Scientific rationale for leukopheresis
- 6.6.2 HIV RNA PCR and HIV DNA PCR
- 6.6.3 Lymphocyte polyfunctional studies and immunophenotyping
- 6.6.5 Testing for syphilis, hepatitis C and hepatitis B
6.5.5 Study Questionnaires: Standardized questionnaires on risk behavior and willingness to participate in vaccine and prevention trials will be used every 24 weeks. In addition, the first visit will have a demographic questionnaire. Trained study staff will be available to assist study volunteers with any questions or concerns. Persons who cannot read will have the questionnaires read to them by a study staff. The questionnaire will be similar to those used in previous WRAIR vaccine cohort studies. Data from questionnaires will not contain any personal identifiers and identified only with the volunteer’s unique study ID. 6.5.6 Tracking of sexual contacts and social networks: With the high viral load shortly after infection, acute HIV-infected subjects are at extremely high risk of transmitting HIV. VCT has been shown in several studies to reduce short term risk behavior [19]. In this study, with permission from the volunteers (consent form)trained peer counselors will counsel the volunteers’ sexual partners within the past 30 days about exposure to HIV, risk of acquiring HIV and advise them to receive VCT. This will be done without informing the sexual contacts from whom we received their contact information unless the study volunteer will allow it. Under no circumstances will we inform the sexual contacts of the subject’s test results and medical information. Counseling regarding risk behavior reduction will be done. If the sexual contacts are HIV positive, the investigators will be responsible for referring them for appropriate health services. Sexual contacts with acute HIV infection can enroll in this study if they fulfill the inclusion and exclusion criteria. A separate consent form will be used for enrolling sexual contacts into a sub-study in order to evaluate the prevalence of HIV infection, and assess by interview and questionnaires their risk behavior and willingness to participate in HIV vaccine and prevention trials. There are no other sub-studies planned for sexual contacts. In order to prevent HIV spread at a community level, identifying the acute HIV-infected volunteers’ social networks is crucial [39]. With permission from the acutely HIV-infected volunteers, trained peer counselors will collect information on where and how they met their sexual partners, particularly within the past 4 months. This will be done by collecting information during counseling sessions. Areas of sexual contacts, particularly venue-based, will be mapped to help guide outreach, risk behavior reduction education and condom distribution. Local community-based organizations and non-governmental organizations are actively working within the most at-risk populations (MSM, transgender, sex worker). Advice and assistance will be sought from these organizations and the information obtained on venue-based or other potential areas for high transmission may also help focus their efforts to reduce the risk of HIV-transmission among at-risk persons. We will encourage the acute HIV infected volunteers to refer persons in their social networks for VCT. 6.5.7 Brain MRI and MRS: MRI and MRS are non-invasive methods to detect brain pathology (MRI) and to determine the in-vivo concentration of brain metabolites (MRS). Several cell types are distinguishable with commonly measured metabolites, including neurons (N-acetyl-aspartate [NAA]) and glial or inflammatory cells (myoinositol [MI] and choline [Cho]), often measured as ratios to brain creatine (Cr) [40]. HIV infection is associated with increased MI/Cr and Cho/Cr ratios, indicating direct effects on microglial cell concentrations [41, 42]. These changes occur most prominently in subcortical structures including the frontal white matter, basal ganglia and thalamus [43, 44]. 6.5.8 Neuropsychological assessment, comprehensive neurological examination and psychological assessment and assessment of function: Brain injury may occur in this very early stage, a factor that is sometime clinically evident with neurological manifestations of disease and is thought to have long-standing consequences. To date, our understanding of these earliest CNS effects is limited since capturing clinical data in the weeks following infection requires extensive resources and because such individuals seldom succumb to autopsy. Information relating to the first few weeks following infection is particularly lacking as few centers are able to identify infection in this very early phase. Intensive neurological and psychological characterization of acute HIV-infected individuals would greatly advance our understanding of brain injury in HIV. In particular, the CSF and MRS components will establish the timing of HIV CNS infection and injury and can document viral strains that may be more neurovirulent. The neuropsychological assessment will be important to document how acute HIV infection impacts cognitive function while the comprehensive neurological examination will document neurological symptoms and signs during acute infection and seroconversion. The psychological assessment and assessment of function are required to adequately interpret neuropsychological data given the impact of anxiety and depression on neuropsychological performance. 6.6 Specimen Collection and Testing As part of the TRCAC routine procedures, 6 ml of blood is collected in EDTA-treated tube and processed within one hour. A total of approximately 2 ml of plasma is separated from blood by centrifugation, for HIV-1 testing. The remaining plasma is stored. For this protocol, part of the blood will be used for pooled NAT or sequential EIA. Left over samples will be stored at -20 degrees Centigrade. Procedures specific to this study are below: Venipuncture will be done using standard sterile technique; substitutions of tube types may be made as long as these are appropriate and do not interfere with the performance of the studies in question. Plasma will be separated within 24 hours. PBMCs will be processed and cryopreserved (-135oC or lower) within 8 hours. Remaining blood specimens will be coded and archived without personal identifiers. These samples may be used for future studies that may provide additional scientific information with respect to the protocol’s primary and secondary objectives with the appropriate IRB approvals. The archived specimens from volunteers will be stored in restricted-access and locked storage at AFRIMS, Dept. of Retrovirology, Bangkok or the TRCARC for up to 10 years. Leukopheresis will be done to obtain PBMCs and plasma storage at weeks 0, 2, 4, 8, 12, 24, 48, and 96 using a continuous flow apheresis device. During leukopheresis, whole blood will be withdrawn from the peripheral vein and channeled into a cell separator where the cellular fractions are separated by centrifugation. The lymphocyte fraction is directed into a collection bag and the red cells and platelets are returned to the patient. The cell separator kit is anticoagulated with sodium citrate. Some plasma from leukopheresis will be stored. The blood volume loss from red blood cell loss and plasma storage will be about 50 ml. The procedure takes about 2-3 hours, and requires one or two needle access sites. Leukopheresis will allow the collection of 5 x 109 to 10 X 1010PMBCs while minimizing the total blood volume taken from participants. The leukopheresis will be performed in the Blood Bank Unit at Chulalongkorn University Hospital by experienced apheresis technician. Each leukopac (bag of white blood cells) will be sent to TRCARC for PMBC isolation. PBMCs will be divided into aliquots and cryopreserved (-135°C or lower) within 8 hours. Subjects who wish to not have leukopheresis will instead have phlebotomy of 50ml for each of the leukopheresis visits. The samples from the individuals who sign the information sheet but are found not to have acute HIV infection will be stored at the TRCARC laboratory in accordance with routine TRCARC procedures (stored for up to 5 years from the time of study completion and subsequently destroyed by autoclave). The laboratory will not have access to the data on the information sheet. Some samples will be sent to WRAIR for additional testing not available in Thailand. This testing may include, but may not be limited to, immunohistochemistry of gut tissue to assess lymphocyte subsets and full-length, single genome analysis of viral sequences and host genetic factors from peripheral blood and genital compartments, cerebrospinal fluid and colon. Fluorescence activated cell sorting of HIV specific T cells will permit T-Cell receptor sequencing to determine T helper and suppressor cell antigen specificities. Technology transfer to Thailand is planned if feasible. Scientific rationale for leukopheresis HIV immunopathogenesis involves persistent immune activation, and a wide range of T, B, NK and dendritic cell defects. There is increased turnover of T cells, monocytes, natural killer cells and polyclonal hypergammaglobulinemia. The result is immune exhaustion, CD4 decline and loss of HIV viremic control. The specifics of how HIV drives the immune system into a cycle of activation resulting in immune deficiency remains unclear. There is growing evidence, however, that how the immune system interacts with the virus at the very early stages in the infection sets the stage for disease outcome in the chronic phase [45]. Since early time points of acute HIV infections are very difficult to study in humans due to an inability to identify acutely-infected subjects by routine HIV testing. This study allows us to study the complex immunopathogenesis in a more comprehensive manner within the same individual at a particular Fiebig stage. This removes the variability associated with the limited human data reported thus far. This study will, therefore, significantly contribute to the understanding of the interactions between HIV and its host which are crucial for the design of effective prevention and treatment modalities. However, the ability to do such studies is limited by the number of PBMCs obtained from blood. In general, 1 million PBMCs can be obtained from 1 ml of blood. With the addition of leukopheresis, we will be able to adequately study prospectively the immunology and virology of acute HIV infection and its progression to chronic infection. The protocol will focus on studying different T, B, dendritic and NK cell populations and their functions, evaluating host genetic factors associated with immune response and assessing evolution of HIV by sequencing. Below describes the scientific information that supports the proposed immunologic and virologic studies in this protocol. The rapid T cell loss following acute HIV infection is believed to be effected directly by HIV, while the continued T cell loss in the chronic phase is believed to be a result of chronic immune activation [46]. Activation of dendritic cells and natural killer cells (NK) of the innate immune system through toll-like receptors (TLR 7/8) may be the main driver to further immune activation and T cell depletion in HIV [47-49]. HLA class I subtype-dependent expansion of specific KIR(+) NK cells during an acute infection in humans was recently reported [50]. In vitro experiments show that T cell activation occurs within the first 24 hours before any HIV-specific immune response [51]. Understanding innate immune cell functions may be the key to blocking HIV infection; however, these cells exist in such low concentrations in the blood and large numbers of PBMCs are required. For example, within the NK cell population, which is generally about 14-20% of lymphocytes for Thai subjects, CD56 bright NK cells can be as low as 3.9%, making functional studies on this subset very difficult from a 50ml blood draw, which would provide only approximately 351,000 CD56 bright NK cells (assuming a median total NK cell count of 300/µl blood and a 40% lymphocyte loss following cryopreservation and thawing). In the acute phase of HIV infection and pathogenic SIV infection, it is shown that the specific loss of memory CD4 T cells occurs in every compartment (blood, lymph node, GI tract) but to a far greater extent in the GI tract [45]. Recent studies have discovered active recruitment of peripheral lymphocytes to the gut via 4β7 integrins or via other gut homing receptors such as CCR7, but how this plays a role in acute HIV infection is unclear [52]. Downregulation and depletion of Th17, memory CD4 T cells that produce IL17 and IL22 necessary for bacterial and fungal infection control in the gut and possibly in the periphery, following acute HIV infection is seen in HIV-infected patients and SIV-infected rhesus macaques but not in natural SIV hosts, sooty mangabeys and African green monkeys [52-54]. Such depletion may be the cause of bacterial translocation that triggers immune activation in chronic HIV infection. African green monkeys and sooty mangabeys do not progress to AIDS despite significant T cell loss following acute infection. The low level of immune activation, lipopolysaccharide and possible differences in expression of genes associated with cell cycle regulation and lipid metabolism may play a role [10]. With the gut biopsy in our study, we will be able to decipher the different lymphocytes subsets in the blood during acute infection and correlate them to those in the gut and other compartments. Upregulation of expression of surface immune activation markers such as CD38, HLA DR, CD71, and intracellular Ki67 expression are found to be associated with level of HIV viremia and disease progression [55]. How early these occur is unclear and whether therapeutic intervention targeted at these cell types could dampen immune activation and further T cell loss is speculative. HIV- and SIV-specific CD8+ T cells or CTLs are the very first cells involved in combating HIV and SIV. Why CTLs cannot contain HIV infection is not well understood. Exciting work by Streeck et al demonstrated that the immunodominance patterns of HIV-1-specific CTL responses detected in primary, but not in chronic HIV-1 infection, were significantly associated with the subsequent set point of viral replication and slower CD4+ T cell decline. These data suggest that the specificity of the initial CTL response to HIV is critical for the subsequent control of viremia, and have important implications for the rational selection of antigens for future HIV-1 vaccines [56]. The upregulation of the programmed death-1 (PD-1) surface receptor on CTLs necessary for apoptosis likely contributes to CTL intrinsic defects [57]. Long term nonprogressors have CTLs with lower expression of PD-1 and lower viral load. Blocking PD-1 increased T cell proliferation in vitro [58]. There is some evidence that PD-1 may be less expressed in primary HIV infection. Giving anti-PD1 antibody to SIV-infected rhesus macaques improved their CTL response and survival [59]. PD-1 blocking antibody is now been taken into clinical trials in cancer patients. Migueles et al found that lytic granule contents of memory cells are a critical determinant of cytotoxicity and that defective CTLs of progressors could be restored after treatment with phorbol ester and calcium ionophore [60]. It will be important to perform such studies in acutely infected persons to understand whether similar events occur and whether induction of cells able to undergo rapid expansion and mediate cytotoxicity upon antigen encounter are possible goals for HIV vaccines. The role of regulatory T cells (Tregs) in HIV is still elusive. Tregs are suppressive T cells that can exhibit opposite functions by either suppressing and controlling detrimental immune activation or suppressing HIV-specific T cell response and allowing for HIV viremia. Studies in monkeys have highlighted the potential important role of Tregs in acute HIV infection. Immediate emergence of Tregs in the gut is seen in acute nonpathogenic SIV infection while in pathogenic SIV infection, Tregs are rapidly depleted [46, 61, 62]. Difficulty in studying Tregs is partly because of cell number limitation. This can be overcome with leukopheresis. This complex immune and viral interplay can be studied by determining the quantity of different cell types by flow cytometry and by determining cellular functional responses by intracellular cytokine staining. Through these, researchers have identified polyfunctional T cells that are found in larger fractions in non progressors compared to progressors. These polyfunctional T cells appear to have a broader response to epitopes [63]. A few studies have found evidence of an important role of polyfunctional T cells in primary HIV infection particularly in relation to viral load set point [64, 65], however, no studies have been done in a large enough populations that are in the very early stages of acute HIV infection. Ability to respond to epitopes and epitope repertoires that induce more polyfunctional T cells is also important to study. Longterm changes of epitope-specific responses and polyfunctional T cells after acute HIV infection are not well understood. A study of 21 acutely infected subjects suggest that epitope-specific T cell responses expanded rapidly and peaked as early as 5 days but were limited to 1 to 2 epitope-containing regions. The limited epitope breadth may influence the efficiency with which viral replication is contained in early HIV infection [66]. Polyfunctional T cells are between central memory and effector memory T cell differentiation. Understanding how the various phenotypes of T cells, as defined by their functional differentiation as naïve, central memory, effector memory and terminal effector T cells, play a role from very early HIV to chronic HIV will help shed light on the viral-immune interactions. Recent studies in humans suggest that early activation has a major role in shaping B-cell (memory and plasma cell) repertoire and trafficking. A recent study showed that during the acute phase of the disease in SIVmac251-infected cynomolgus macaques, SIV-specific antibodies were detectable only from day 21 post infection. A better understanding of the inflammatory signals that triggers HIV-specific antibody production should help to design of new therapeutic strategies [67]. In another study, high-titer, broadly reactive V3-specific antibodies are among the first to be elicited during acute and early HIV-1 infection but these antibodies lack neutralizing potency against primary HIV-1 viruses, which effectively shield V3 from antibody binding to the functional Env trimer. There might also be possible synergism between neutralizing antibodies and polyfunctional T cells for control of HIV/SIV replication. In macaques challenged with SIVmac239 and immunized with neutralizing antibody one week after, polyfunctionality of Gag-specific CD4(+) T cells was markedly elevated in the acute phase and was maintained through the chronic phase with viral control [68]. By single-genome amplification (SGA) followed by direct sequencing of uncloned DNA amplicons in samples of Zambian subjects within 3 months of seroconversion and found that 8/12 were infected with a single virus while the other 4 had more than one virus. Strong immunologic selection can be seen in the Env and overlapping Rev sequences. The mechanism of how transmitted virus establishes infection is not clearly understood. Because most infected individuals are identified after the acute period, molecular diversification of the virus has already occurred. Some initial studies have suggested that virus in the early phase is fairly homogenous but most of these studies are done in seroconverters and almost none are done in the very acute phase as in our study. Kinetic analysis and mathematical modeling of virus immune escape showed that the contribution of CD8 T cell-mediated killing of productively infected cells was earlier and much greater than previously recognized and that it contributed to the initial decline of plasma virus in acute infection. After virus escape, these first T cell responses often rapidly waned, leaving or being succeeded by T cell responses to epitopes which escaped more slowly or were invariant [69]. Interpersonal variability in immune responses to HIV, and occurrence of escape mutants exist; therefore, determining host genetic factors in this study such Fc gamma, APOBEC, cyclophilin, TRIM5a, and KIR APOBEC3s, TRIM5α and TLRs will aid in the understanding of host responses to HIV. The host restriction factors Cyclophilin A/TRIM5α, APOBECs, and Tetherins interfere with crucial post-entry viral replication steps, and their naturally occurring genetic variants have been associated with varying rates of HIV acquisition [70] and disease progression [71]. On the other hand, variants of the HIV genes that are targeted by these host restriction factors (i.e., gag, vif, and vpu, respectively) have shown varying in vitro susceptibilities to these restriction barriers [72-74], suggesting that the genetic background of some hosts may make them more susceptible or resistant to infections with given HIV genetic variants. In conclusion, our failure to eradicate the virus via current available treatment options or to adequately prevent infection via vaccines is partly due to our limited knowledge of the immune – viral interplay in the very early stages of HIV infection. This study will provide an opportunity to study these complex interactions and to identify potential targets for new therapeutics and vaccine interventions, and to develop targeted individualized interventions. Such studies will be greatly limited without an adequate cell samples obtained by leukopheresis. 6.6.1 HIV serology: Serology will be performed at TRCARC using their standard algorithm for HIV testing. The testing algorithm is described 6.5.3.1 and in Appendix A. 6.6.2 HIV RNA PCR and HIV DNA PCR: Plasma and cells will be collected in EDTA or another suitable anti-coagulated tube. HIV RNA PCR will be assayed for HIV viral load using the Roche Amplicor® 1.5 kit or another nucleic acid amplification method. HIV DNA PCR will be assayed according to published methods [75]. Nucleic acid testing will be performed at either AFRIMS Bangkok or the TRC facility or at VGTI laboratory depending on the assay used. 6.6.3 Lymphocyte polyfunctional studies and immunophenotyping: The following lineage and differentiation markers will be used for the immunophenotyping studies: CD16/56, CD19 and CD3 (or Tetramer), with CD4 and CD8 to further define T cell subsets. Differentiation markers will include: CD27, CD28, CD45RA, CD45RO, CD57, CD95 and CCR7 and HLA-DR, CCR9 and alpha-4/beta-7 integrin. Additional markers for CD8+ T effector activity will include IFNg, TNFa, IL-2, MIP1b, CD107 surface expression. Other immunologic markers of HIV acute infection such as those of T cell turnover and proliferation may be assessed depending on the current scientific literature. Blood will be drawn in ACD, heparin or equivalent anti-coagulated blood tubes and flow cytometry will be performed in accordance with established guidelines. These tests will be performed at AFRIMS, Bangkok. 6.6.4 Chemistry: Total bilirubin and direct bilirubin, ALT, GGT, creatinine, triglyceride, cholesterol, HDL, LDL will be performed at the TRCARC laboratory. 6.6.5 Testing for syphilis, hepatitis C and hepatitis B: RPR and TPHA, HCV antibody, HBs Ag and anti HBs antibody will be performed at the TRCARC laboratory. 6.6.6 HIV genotyping/selected full-length sequencing: HIV-1 subtype analysis will be performed in all confirmed acute HIV-1 infection positive samples, at the collaborative AFRIMS/TRCARC Molecular Epidemiology Research Lab at the TRCARC or the Retrovirology laboratory at WRAIR, using full-length sequencing as previously described [76, 77]. Full length, single genome analysis of 'transmitted' HIV in multiple compartments and in individual infected cells will also be performed. Viruses may be obtained by co-culturing with activated PBMC, if other molecular methods for virus sequence determination fail [76]. Latent reservoirs in different compartments may be determined. CCR5 and CXCR4 tropism at baseline will also be determined on stored samples using the Trofile(TM) co-receptor tropism assay (Monogram Biosciences, Inc, California, USA) or other validated methods using stored samples. The results of tropism will not be available in real time and will not influence treatment choice. Download 0.91 Mb. Share with your friends:
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