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Walker Progress Report Abstracts continued

 
Submitted February 3, 2011 (Interim Report)

Project Goal: To study HIV infected people who have been able to maintain low viral loads without the use of antiretroviral medications and use this knowledge for the design of a global HIV vaccine.

This project has four interdependent elements: patient recruitment for the cohort, immune response characterization in situations of spontaneous control of HIV, identification of the predictable patterns of viral evolution in response to host immune pressures in situations of spontaneous control of HIV and the contribution of viral replicative fitness and antigen processing to viral control and immunodominance, and genome-wide association studies.

The foundation of the grant is cohort development. We have completed recruitment of >1500 HIV (aviremic and viremic) controllers to support activities 2 through 6. From this large cohort we have identified a select subgroup of individuals for large volume blood donations and longitudinal clinical follow-up and biological samples. We have recruited 58 individuals with acute HIV infection.

The second component, which focuses on adaptive responses, is designed to understand how T cell responses mediate viral control. We have used new technologies to demonstrate that epitope specific immune responses differ markedly in their ability to inhibit virus replication, and that these differences are not associated with cytokine secretion such as IFN╬│ (manuscript submitted). Moreover, we have recently shifted our focus on HIV-specific CD4 T cell responses as these might ultimately be pivotal for the generation of life-long anti-HIV immunity. In collaboration with Alessandro Sette, we made a large effort to comprehensively determine HIV-specific CD4 T cell responses and their HLA class II restriction. We have screened 100 HIV infected individuals for their HIV-specific CD4 cell responses, representing the largest assessment performed to date. Finally, we have identified key functions of CD4 T cells for CD8 T cell help that will allow us to better understand which CD4 T cell responses are most important to induce in terms of HIV vaccine design.

The third element, viral evolution and antigen processing, has made great strides in technology development and scientific results. We have made considerable progress in terms of development of the new 454 pyrosequencing technology for viral sequencing, through a series of meetings between Broad and Ragon Institute members, chaired by Eric Lander and Bruce Walker. This approach, which includes the development of novel assembly and error detection algorithms, has enabled an unprecedented in-depth analysis of the viral quasi-species in over 100 subjects, and will be scaleable to much larger throughput in the next few months. We have also identified a unique set of linked mutations in Gag and Nef which dramatically impair viral replication and are associated with protective HLA class I alleles, supporting the importance of vaccine targeting of only the most conserved regions of HIV.

The project on HIV antigen processing led to the identification of multiple motifs within and outside epitopes driving the efficiency of epitope processing. Accordingly the degradation patterns of HIV proteins vary within and among proteins, although the reasons for these variations and consequences forepitopes or located within epitopes evolve toward motifs that either reduce the trimming of extened epitopes, or increase the degradation of the epitopes, thus showing strong immune pressure toward antigen processing mutations.

The fourth element is a genome-wide study to identify host genetic factors associated with persistent control of the virus. Our initial focus has been on common SNPs in the human genome. We have recently published in Science Magazine on the identification of specific amino acid positions within the HLA-B protein, involved in antigen peptide presentation to CD8+ cells (or other cellular entities that interact with HLA-B). Our current focus has shifted towards testing the role of rare variants in selected genes implicated in the HIV life cycle.

Submitted August 10, 2010

Project Goal: To study HIV infected people who have been able to maintain low viral loads without the
use of antiretroviral medications and use this knowledge for the design of a global HIV vaccine.
This project has four interdependent elements: patient recruitment for the cohort, immune response
characterization in situations of spontaneous control of HIV, identification of the predictable patterns of
viral evolution in response to host immune pressures in situations of spontaneous control of HIV and the
contribution of viral replicative fitness and antigen processing to viral control and immunodominance, and
genome-wide association studies.


This and other HIV research is critically dependent upon the availability of well-pedigreed human blood
samples, and to this end we have used this funding to create an international resource to benefit science
well into the future, in that we have completed recruitment of >1500 HIV (aviremic and viremic)
controllers, and from this large cohort we have identified a select subgroup of individuals for large volume
blood donations and longitudinal clinical follow-up and biological samples. We have also recruited 150
individuals with acute HIV infection.


The path forward to an HIV vaccine depends on eliciting immune responses that have a direct antiviral
effect. A major accomplishment to date has been the identification of immune responses that are
associated with viral control, and equally importantly the identification of a large proportion of responses
that in fact appear to provide no benefit to controlling HIV, but rather serve as decoys. Those that are
antiviral appear to function at least in part by forcing the virus to mutate in ways that render it less able to
replicate rapidly and to cause disease. These studies have immediate and broad implications for
immunogen design.


The design of an effective vaccine will require that we be able to protect against all HIV strains, a
scientific challenge of unprecedented proportions given the immense sequence variability fostered by the
poor proofreading of the RT as new viruses are made. We have developed a novel technology in
collaboration with the Broad Institute, which allows us to obtain detailed sequence information on the
infecting strains, and the evolution of these strains under immune selection pressure. We have also
developed methods to understand how virus infected cells are sensitized for recognition by the immune
system, and the factors governing this process. These data are critical to define the sequence spacewithin which HIV must exist, as they directly dictate the design of effective immunogens to counter HIV
diversity.


The fourth element is a genome wide association study to determine the host genetic factors associated
with persistent viral control. We have found a strong signal in the MHC region regardless of ethnic
background of the subjects, and of the three billion nucleotides that make up the human genome, have
found six amino acids, all related to HLA class I presentation of viral peptides, that account for the
positive and negative associations with viral load. These data indicate that it is the nature of the
presentation of peptide that influences outcome, and paves the way for a mechanistic understanding of
what constitutes a good immune responses that can then be applied to vaccine design.

 
 
 
Submitted February 1, 2010 (Interim Report)

Project Goal: To study HIV infected people who have been able to maintain low viral loads without the use of antiretroviral medications and use this knowledge for the design of a global HIV vaccine.

This project has four interdependent elements: patient recruitment for the cohort, immune response characterization in situations of spontaneous control of HIV, identification of the predictable patterns of viral evolution in response to host immune pressures in situations of spontaneous control of HIV and the contribution of viral replicative fitness and antigen processing to viral control and immunodominance, and genome-wide association studies.

The foundation of the grant is cohort development. We have completed recruitment of >1500 HIV (aviremic and viremic) controllers to support activities 2 through 6. From this large cohort we have identified a select subgroup of individuals for large volume blood donations and longitudinal clinical follow-up and biological samples. We have recruited 58 individuals with acute HIV infection.

The second component, which focuses on adaptive responses, is designed to understand how T cell responses mediate viral control. We have used new technologies to demonstrate that epitope-specific immune responses differ markedly in their ability to inhibit virus replication, and that pathways to immune escape differ in elite controllers.  Moreover, we have examined eighteen rare cases of acute HIV-1 infected subjects who subsequently became elite controllers, showing that viral fitness in the earliest stages of infection is associated with outcome.  We are also currently performing a detailed analysis of the interaction of CD4+ T cell help and CD8+ T cell responses, and we are in the process of further characterizing HIV-1-specific CD4+ T cells.

The third element, viral evolution and antigen processing, has made great strides in technology development and scientific results. We have made considerable progress in terms of development of the new 454 pyrosequencing technology for viral sequencing..  This approach, which included the development of novel assembly and error detection algorithms will enable an unprecedented in-depth analysis of the viral quasi-species in each individual. We have also identified a unique set of mutations in Gag which dramatically impair viral replication and are associated with protective HLA class I alleles, supporting the importance of vaccine targeting of only the most conserved regions of HIV.

The project on HIV antigen processing has generated new data indicating that it may possible to control HIV epitope production. We have identified N-flanking motifs regulating the kinetics and the amount of antigenic peptides produced, and in altering the amount of epitopes available for loading onto MHC-I. Additionally we have identified HLA-restricted polymorphisms that limit either the trimming of peptides into epitopes or the pool of epitopes produced inside cells and reduce recognition by epitope-specific CTL, suggesting that naturally occurring HIV mutations may alter various steps of antigen processing to limit immune recognition.

The fourth element is a genome wide association study to determine the host genetic factors associated with persistent viral control.  We have found a strong signal in the MHC region regardless of ethnic background of the subjects, and have found over 60 single nucleotide polymorphisms that reach genome wide significance, indicating that host immune responses are likely critical in persistent containment of HIV.

 

Submitted August 14, 2009

Project Goal: To identify the immunological, virological, and host genetic factors that contribute to the spontaneous control of HIV, and to use this knowledge to design an effective global HIV vaccine.

This project has four interdependent elements: patient recruitment, characterization of how the immune system recognizes HIV, characterization of how the virus mutates as it reproduces itself, and characterization of human genes that are involved in allowing some people to coexist with the virus without becoming ill, and without the need for medication to treat HIV.

The first component involves the creation of the International HIV controllers consortium, which seeks to recruit persons who have no detectable virus in the blood by standard commercial assays (elite controllers) as well as persons who maintain viral loads in the blood of less than 2000 RNA copies/ml (viremic controllers), a level so low that they are unlikely to develop progressive infection and unlikely to transmit to others. The project now includes over 245 IRB-approved health care providers that enroll subjects to our study, as well as numerous additional collaborators. To date we have recruited >1400 HIV controllers (elite and viremic) into this cohort.

The second component has generated new data indicating that not all immune responses to HIV are equal, with up to 1000 fold differences in their ability to kill virus infected cells.  Importantly, these new inhibition assays capture these differences which are not detected using traditional assays of immune function like the IFN gamma Elispot assay. We have also made considerable progress on studies of innate immunity (the first line of cellular defense by the immune system), demonstrating the important role that natural killer (NK) cells can play in
modulating dendritic cell (DC) function, and the ability of HIV to interfere with this NK-DC cross-talk.

The third element has now successfully implemented the new next generation 454 pyrosequencing technology, enabling us to sequence full length HIV genomes at an unprecedented depth of coverage. Through this work we have now identified particular immune selected escape mutations that significantly impair viral replication and may contribute to immune control. We have also made considerable progress in defining particular sequence signatures and factors involved in altered processing and presentation of HIV epitopes.

The final component, through a genome-wide association study, has been able to identify a set of genetic markers and host genes that are strongly associated with control of HIV. We are currently evaluating the contribution of genetic variants in the MHC loci to HIV control where the strongest markers of control of HIV were found. The data thus far indicate that the host genes that modulate HIV control are all within the region of the human genome that governs immune function.

Taken together, our work suggests that both immunological, virological and host genetic factors are contributing to the spontaneous control of HIV, which is helping to guide HIV vaccine design.

 
Submitted February 2, 2009 (Interim Report)

Project Goal: To study HIV infected people who have been able to maintain low viral loads without the use of antiretroviral medications and use this knowledge for the design of a global HIV vaccine.

This project has four interdependent elements: patient recruitment for the cohort, immune response in situations of spontaneous control of HIV, identification of the predictable patterns of viral evolution in response to host immune pressures in situations of spontaneous control of HIV and the contribution of viral replicative fitness and antigen processing to viral control and immunodominance, and genome-wide association studies.

This foundation of the entire grant is the cohort development. We have expanded the International HIV controllers consortium to include over 260 IRB-approved clinical collaborators that refer subjects to our study as well as utilize our repository, and to date we have recruited >1300 HIV controllers. As the cohort grows, so does our potential to make novel discoveries in the areas of adaptive immunity, viral evolution, and host genomics.

The second component, focused on adaptive and innate responses, has generated new data indicating that all immune responses to HIV are not equal in their ability to kill virus infected cells.  Our data show up to 1000 fold differences when we examine these responses in vitro.  Importantly, using new assays that measure the ability of CD8 T cells to inhibit virus replication in tissue culture, we see that traditional Elispot assays do not capture these differences.   We are now performing a detailed analysis of the optimal epitopes targeted and the relationship of epitope targeting to immune control, and using these data to reassess the STEP trial data.   We have also made considerable progress on studies of innate immunity, demonstrating the important role that natural killer (NK) cells can play in modulating dendritic cell (DC) function, and the ability of HIV-1 to interfere with this NK-DC cross-talk.

The third element, viral evolution and antigen processing, has made great strides in technology development and scientific results. New 454 pyrosequencing technology has been developed for viral sequencing which will enable unprecedented in-depth sequencing of the viral quasi-species in each individual, as well as a high-throughput viral sequencing platform to enable the sequencing of over 2000 sample isolates. We have also made considerable progress in defining factors involved in efficient or impaired processing and presentation of HIV epitopes, defining intraepitopic and flanking sequence signatures contributing to efficient production or degradation of HIV epitopes.  

 The final component was to conduct a genome-wide association study to define the host genetic factors that contribute to durable control of HIV. We have been able to generate a large enough group to be able to stratify by ethnicity, and are finding  SNPs that are statistically associated with HIV control.  We are currently evaluating the contribution of genetic variants in the MHC in HIV controllers of non-European descent.

 
Submitted August 26, 2008

Project Goal: To use population based studies of immune responses, viral evolution, and host genetics to achieve effective immunogen design for a global HIV vaccine.

This overarching goal has four principle components. One, we have expanded the International HIV controllers consortium to include over 200 HIV providers and scientists that refer subjects to our study, and we have recruited >1000 HIV controllers. This recruitment effort provides the foundation for the project.

The second component was to define the functionality and critical targets of the innate and adaptive cellular immune responses in situations of spontaneous control of HIV. This entailed defining the breadth and specificity of HIV-specific CD8 T cell responses in elite controllers, viremic controllers and progressors, via Elispot screening of over 300 samples. We also concentrated on standardizing assays to characterize the role of NK cells in viral control and in modulating the adaptive immune response to HIV-1 infection through their interactions with DCs.

The third component was to identify the predictable patterns of viral evolution in response to host immune pressures in situations of spontaneous control of HIV and the contribution of viral replicative fitness and antigen processing to viral control and immunodominance. We sought to do this via viral sequencing and measuring antigen processing activities.

The final component was to conduct a WGAS to define the genetic factors that impact durable control of HIV. Our statistical analysis on these data (from >1300 subjects) demonstrates replication of previously reported associations between SNPs in the major histocompatibility complex and viral control.

 
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