HIV/AIDS Research:

Its History and Future

Genentech Center for the History of Molecular Biology and Biotechnology
at Cold Spring Harbor Laboratory Archives

Meeting: 13–16 October 2016

Organizers: Robert C. Gallo, John M. Coffin, Mila Pollock & Bruce D. Walker

Posters

ASSESSING INTRA-PATIENT HIV GENETIC DIVERSITY TO IDENTIFY GENOMIC REGIONS WITH APPROPRIATE PHYLOGENETIC SIGNAL FOR TARGETED NGS SEQUENCING

Michael J Bale1, Jon Spindler1, Ann Wiegand1, Frank Maldarelli1, John W Mellors2, John M Coffin3, Wei Shao4, Mary F Kearney1

1NCI, HIV dynamics and Replication Program, Frederick, MD, 2University of Pittsburgh, , Pittsburgh, PA, 3Tufts University, , Boston, MA, 4 Leidos Biomedical, Inc, , Frederick, MD

Background: Targeted Next Generation Sequencing (NGS) is becoming an increasingly popular method to assess intra-patient HIV genetics because of its ability to generate sequences from thousands of HIV genomes in one sample. This approach is limited, however, by the short sequence reads (i.e. ~500bp by Illumina) that may not be adequate to differentiate among variants in an HIV quasispecies. We therefore sought to identify 500 bp segments of the HIV genome that provided phylogenetic signal similar to data obtained by the gold-standard assay method of single-genome sequencing (SGS) of >1.0kb segments of the HIV genome.

Methods: SGS datasets were obtained from plasma HIV RNA and DNA for 2 segments of the HIV-1 genome, a 2.6kb region in env (single time points from 5 donors) and a 1.2kb region encoding p6-PR-RT (single time points from 16 donors and 2-3 time points from 5 donors). The data sets were scanned for intra-patient HIV diversity using a sliding window protocol (SWP). The window size (N) was varied from 300 to 600nt with a step size (k) of 25nt to 100nt. The relative phylogenetic signal for window (i) of size N(P’N,i) was calculated by the ratio of the number of unique sequences in the window (S’N,i) to the number of unique sequences in the larger SGS segment. The calculated phylogenetic signals (P’N,i) for each window were averaged across all the SGS data sets and plotted to identify short genomic regions with the highest phylogenetic signal that can be used for targeted NGS.

Results: As expected, phylogenetic signal increased with the size of the window analyzed but was not affected by the step size used. Only one 500nt segment in the P6-PR-RT region (PR: 74-99 + RT: 1-141) had phylogenetic signal comparable to the larger 1.2 kb SGS segment (92.8% retention compared to the SGS segment). For env, 4 windows of ~300nt showed good signal for targeted NGS: gp120: 60-164 (C1/V1) (88.5% retention), 139-233 (V1/V2) (88.7% retention), 275-376 (C2/V3) (88.7% retention), and gp41: 45-152 (N-Term/RRE) (88.5% retention).

Conclusion: Careful selection of the 500 bp HIV genomic region targeted by NGS can result in phylogenetic signal similar to that obtained from longer segments (1.2-2.6 kb) by SGS. Similar sequence analyses of longitudinal plasma samples are needed to determine if the phylogenetic signal obtained from shorter segments of the HIV genome is sustained over the course of HIV-1 infection and across HIV-1 subtypes.


CSHL AND THE HIV FIELD THROUGH 35 YEARS: SCIENCE, RESEARCH, & EDUCATION

Clare Clark

Cold Spring Harbor Laboratory, Library & Archives, Cold Spring Harbor, NY

CSHL is a significant contributor to the history of HIV. From early meetings on vaccines, public presentations from early pioneers of AIDS, to a children’s book on HIV in Africa; all are a significant subject of interest to scientists and non-scientific communities. CSHL scientists work on HIV related research, hosts significant meetings on modern vaccine and virus HIV-related topics, public talks from investigators on the front lines of HIV research, and published children’s books on the topics including “Staying Alive — Fighting HIV/AIDS” and “You, Me and HIV: With Knowledge We have Hope.” This poster reflects a brief overview of HIV at CSHL through science, research, and education.


THE CONTRIBUTION OF COLD SPRING HARBOR LABORATORY SCIENTISTS TO HIV/AIDS RESEARCH

Matthew Covey

Cold Spring Harbor Laboratory, Library & Archives, Cold Spring Harbor, NY

While not associated with large scale HIV/AIDS research Cold Spring Harbor Laboratory nevertheless had a number of Scientists who made important contributions towards better understanding HIV/AIDS during their time at the Laboratory.

Beginning in the late 1980’s Michael Laspia, Michael Mathews, Jacek Skowronski, Robert Franza, and Nouria Hernandez, with funding from NIH, the American Foundation for AIDS Research, and AMFAR, led research at the Laboratory that examined the effect of the HIV-1 virus on gene expression and transcription, as well the immune response to the HIV-1 virus. In this poster we will highlight the Cold Spring Harbor Laboratory researchers and their most important published work that contributed to the field of HIV/AIDS research.


DEVELOPMENT OF THE FULL LENGTH SINGLE CHAIN GP120-CD4 (FLSC), A NOVEL VACCINE FOR HIV PREVENTION

Timothy Fouts1, Ilia Prado1, Kathryn Bobb1, Wenlei Zhang1, Jennifer Schwartz1, Terry Higgins1, Anthony Cristillo2, Ranajit Pal2, Ian Collins4, Greg Bleck4, Brian Woodrow4, Ronald Salerno5, Melanie Hartsough5, Robert Gallo6, Bruce Gilliam6, Robert Redfield6, George Lewis6, Anthony DeVico6

1Profectus BioSciences, , Baltimore, MD, 2ABL, , Rockville, MD, 3Bioqual, , Rockville, MD, 4Catalent Pharma Solutions, , Madison, WI, 5BCG, , Alexandria, VA, 6IHV, , Baltimore, MD

We have developed the FLSC in order to induce broader immunity against HIV, including against new conserved epitopes, as compared to conventional gp120 or trimeric gp140s. The FLSC is a fusion protein consisting of a modified gp120 protein and the first two domains (D1D2) of human CD4, genetically fused via a 20 amino acid linker. Immunization with FLSC was able to protect rhesus macaques against rectal challenge of SHIV(162P3) and SIVmac251. Efficacy tracked with Fc-mediated effector function of the antibody provided that the concurrent anti-vaccine T cell response is minimal. To advance the FLSC into clinical trials, a master cell bank expressing human FLSC was prepared from G293H, a derivative of HEK-293 cell that grows in suspension, using the GPEx® retrovector transduction system. Bioreactor production rates were consistent from the 2L to the 200L scale, yielding approximately 1 gms/liter after downstream purification. Drug substance was predominately monomeric, and expressed the expected CD4 induced structure. Potency studies with the drug product demonstrated a significant relationship between the dose of the FLSC/Alum (IHV01) and the induction of the desired CD4i directed immune response. Immunogenicity studies performed in rhesus macaques showed that the drug product induced antibodies directed to CD4i epitopes and mediated ADCC activity while T cell responses were modest. Toxicity studies performed in rabbits and cynomolgus macaques demonstrated that the vaccine was safe and did not induce any deleterious autoimmune effects directed to CD4. A dose escalation phase 1 clinical trial with IHV01 is ongoing.


THE HIV/AIDS PANDEMIC AND ITS LARGER PLACE IN HISTORY

Monica H Green

Arizona State University, History, Tempe, AZ

The HIV/AIDS Pandemic of the past century spent half of its life in silence. Not formally described as a syndrome until 1981, and not pinned to a specific infectious entity until 1983, it likely spread to all five continents between the time HIV-1 group M initially emerged in its first human victim in Cameroon and when it became visible to modern science. HIV is also the first known pandemic of a retrovirus, and also the first real pandemic of the jet age, relying on rapidly increased human mobility to allow its quick spread throughout the world.

But was it, is it, a pandemic like no other? This poster puts HIV/AIDS into a larger context of humankind’s history with infectious diseases, which may in some instances extend deep into our hominin ancestry. At the moment, we know of no other retroviruses that have caused largescale disease. But neither do we have any real mechanism of determining such an impact if there were one. What we can do readily now – because of new findings coming out of paleogenetics, paleopathology, and renewed historical researches of globally distributed infectious diseases – is trace the deep history of several other pandemics of the past, pinpointing the circumstances of disease origin, epidemiological dissemination, and, most importantly, the ways in which human behavior has facilitated the spread both of rapid killers like smallpox and plague, but also slower and more insidious scourges like malaria, TB, and leprosy.


ESTIMATES OF ACHIEVING HIV CURE WITH ANTI-PROLIFERATIVE THERAPY

Daniel B Reeves1, Elizabeth R Duke1, Martin Prlic1, Florian Hladik1,2,3, Joshua T Schiffer1,3

1Fred Hutchinson Cancer Research Center, Vaccine and Infectious Disease Division, Seattle, WA, 2University of Washington, Obstetrics and Gynecology, Seattle, WA, 3University of Washington, Department of Medicine, Seattle, WA

In the modern era of HIV treatment, antiretroviral therapy (ART) can prevent the progression of HIV to AIDS. However, proviral latency prevents true cure. Here we demonstrate with a simple mathematical model that strategies aiming to eradicate the latent pool are advantageous if they can be given continuously. An analogy is made to compounding interest: modest improvements in the rate of HIV clearance are more rapidly curative than abrupt, one-time reductions in reservoir size. We suggest coupling anti-proliferative medications with ART to prevent latent cells from sustaining the reservoir. Contingent on subtypes of cells that may make up the reservoir, and their respective proliferation rates, our model predicts that coupling clinically available, anti-proliferative therapies with ART would result in functional cure within 2-10 years, rather than many decades on ART alone.


DOCUMENTING THE EPIDEMIC: UCSF ARCHIVES EXPERIENCE BUILDING AIDS HISTORY COLLECTION

Polina Ilieva

University of California, San Francisco, , San Francisco, CA

The UCSF Archives & Special Collections was the pioneering repository that started collecting materials documenting the HIV/AIDS epidemic. The UCSF AIDS History Project (AHP) began in 1987 as a joint effort of historians, archivists, AIDS activists, health care providers, scientists, and others to secure historically significant resources of the response to the AIDS crisis in the city of San Francisco.

In 1989, a conference “AIDS and the Historian” was organized with the goal “to produce documentary strategy and recommendations for issues relating to AIDS that could benefit from historical inquiry.” Early on, the UCSF archives decided to develop a collection policy that will allow researchers to examine diverse aspects of the AIDS epidemic, including political, social, economic, cultural, and biomedical aspects.

UCSF has been archiving papers of clinicians, health care workers, and researchers working at San Francisco General Hospital and UCSF. Their laboratory research notebooks, correspondence, and reports reflect how scientific and treatment discoveries were made. San Francisco was the birthplace of the community based organization (CBO) model of AIDS care that emerged in response to the needs of sick and dying, their family members and friends. CBOs records help understand how they were organized, their evolution and diverse array of activities, including information dissemination and fundraising for research projects. Other important components are papers of social scientists, journalists, and science writers. Their research on social implications and effect of the epidemic on minorities in the US and around the world, interviews with different constituencies help recreate the unfolding of the crisis and its subsequent development.

UCSF also organized an oral history program to record and preserve stories of doctors, nurses, and researchers-first respondents to AIDS epidemic in the San Francisco Bay Area. This multifaceted and multidisciplinary approach to collection development led UCSF Archives & Special Collections to creating a complex and comprehensive AIDS history research collection that documents not only medical or social aspects of the epidemic, but changes in cultural values, shifts in policy and social response.


EVIDENCE FOR RETROVIRAL ACTIVITY IN DOGS AND WILD CANIDS

Abigail S Jarosz1, Julia H Wildschutte1,2, Malika L Day1, Amanda L Pendelton2, Thomas Marques-Bonet3, Adam R Boyko4, Jeffrey M Kidd2,5

1Bowling Green State University, Dept. of Biological Sciences, Bowling Green, OH, 2University of Michigan Medical School, Dept. of Human Genetics, Ann Arbor, MI, 3CSIC-University Pompeu Fabra & ICREA, Institut de Biologia Evolutiva, Barcelona, Spain, 4 Cornell University, Dept. of Biomedical Sciences, College of Veterinary Medicine, Ithaca, NY, 5University of Michigan Medical School, Dept. of Computational Medicine & Bioinformatics, Ann Arbor, MI

In the canine reference genome, there is only a 0.15% presence of endogenous retroviruses (ERVs), known as CfERVs. This is a relatively low percentage considering ERVs make up 8% of the human genome. Of the ERVs found in canids, most are believed to be from considerably old infections of retroviruses. Due to the old age of these insertions, there are a multitude of accumulated mutations that prevent the provirus from coding for functional genes. With low representation in the canine genome as well as the apparent lack of present exogenous retroviruses, it has been previously assumed that the integration of ERVs in canines was extremely rare or nonexistent. Despite this, there have been reports of retroviral activity in tissues taken from canine tumors and cancer cell lines. In recent findings, young copies of ERVs, known as CfERVFc1, have been identified that have sequence similarity to the human ERV-Fc group. In fact, CfERVFc1 is present in ~151 copies, including ~20 proviral and ~130 solo LTRs. This raises the possibility of the presence of more ERVs that have been recently integrated have yet to be identified.

To begin to study the distribution of recently integrated CfERVs, we searched the whole genome of ~90 dogs as well as closely related canid. De novo assembly of CfERV reads at each site allowed the identification of 37 CfERV LTRs, which include 8 intronic copies. Divergence of these elements from subfamily consensus fluctuated from 0.0 to 9.7%. A total of 59 non-reference, unfixed polymorphic loci has been identified within these samples alone, suggesting levels of retroviral germline infection actually surpasses that of humans and challenges what was previously hypothesized in canines. Insertion presence varied from <0.5%, or from a single sample, to >85% of samples. PCR validations have confirmed at least 5 proviruses of the 59 non-reference insertions. These insertions do not seem to be caused by duplications, meaning that it appears each is from a separate germline event. This leads us to believe that these are more recent insertions that can have an impact on the genome structure of canines which can possibly help us understand what these CfERVs have had on canine phenotypes and evolution.


RESOURCES FOR RESEARCHING THE HISTORY OF HIV/AIDS AT THE NATIONAL INSTITUTES OF HEALTH

Michele T Lyons, Barbara F Harkins

National Institutes of Health, Office of NIH History and Stetten Museum, Bethesda, MD

As the epicenter for HIV/AIDS research in the United States, the National Institutes of Health recognized the importance of preserving the history of its identification of this disease and the subsequent development of treatments for it. The Office of NIH History and Stetten Museum has become the repository for much of that collection, which includes scientific instruments, photographs, oral histories, and other documents. This poster will describe some of the content of that collection and how researchers can access it.


A SMALL FRACTION OF PROVIRUSES IN EXPANDED CELL CLONES EXPRESS UNSPLICED HIV RNA IN VIVO

A. T .Musick1, J. Spindler1, M. Sobolewski2, M. J .Bale1, W. Shao3, A. Weigand1, S. Hughes1, J. Mellors2, J. M .Coffin4, F. Maldarelli1, M. F .Kearney1

1CCR, NCI-Frederick, HIV Dynamics and Replication Program, Frederick, MD, 2University of Pittsburgh, Department of Medicine, Pittsburgh, PA, 3Leidos Biomedical, Inc, Advanced Biomedical Computing Center, Frederick, MD, 4Tufts University, Department of Molecular Biology and Microbiology, Boston, MA

Introduction: The vast majority of proviruses in infected cells that persist in vivo after long-term ART are defective. Of the minority that are intact (~2%), what fraction are latent or transcriptionally active is not known. To address this question, we investigated the fraction of proviruses that express HIV RNA in vivo in cell populations carrying either intact or defective proviruses.

Methods: PBMCs were obtained from Patient #1 reported in Maldarelli, et al. This donor had multiple clones of cells that contain intact or defective proviruses. Fractional proviral expression was determined by single-genome sequencing (SGS) of cell-associated HIV DNA and RNA (CARD-SGS) in individual, infected PBMCs. CARD-SGS was performed by extracting, amplifying and sequencing a 1.3 kb p6-pro-RT fragment of HIV DNA or RNA from multiple aliquots of PBMCs diluted to an endpoint such that each aliquot contained a few HIV RNA expressing cells. Intact proviruses that could be activated to produce replication-competent virus were identified using a limiting dilution VOA.

Results: 77 million PBMC were analyzed, of which 10,450 contained HIV p6-pro-RT sequences. CARD-SGS identified 412 different proviruses in infected cells that were likely in expanded clones. Of these 412 different populations, 3 carried intact proviruses, 81 had defective proviruses, and 328 had proviruses that were likely defective (did not grow out in VOA but did not have stop codons in the region analyzed). The median fraction of cells in the 3 clonal populations that carried intact proviruses that expressed HIV RNA was 2.3% (range 1.2%-8.8%). For clones carrying obviously defective proviruses, the median fraction expressing HIV RNA was 3.5% (range 0.9%-7.0%), and for those likely to carry defective proviruses, the median was 6.6% (range 1.3%-66.7%). The small differences in fractional expression across the 3 different groups of proviruses were not statistically significant (p =0.51)

Conclusion: The large majority (>90%) of infected cells that persist on ART are either latent or incapable of HIV RNA expression. A small fraction of the proviruses in infected clones expressed HIV RNA, but this fraction was not significantly different between clones carrying replication-competent proviruses and those containing obviously defective proviruses, indicating that expression of HIV RNA from intact and defective proviruses have similar consequences (negative, neutral, or positive) for the infected cells in people on long-term ART.


DOCUMENTED IN TIME: A LOOK AT HIV/AIDS FROM THE DESKS OF JAMES D. WATSON & SYDNEY BRENNER

Stephanie Satalino

Cold Spring Harbor Laboratory, Library & Archives, Cold Spring Harbor, NY

The James D. Watson Collection of CSHL Archives and the Sydney Brenner Collection of the Genentech Center for the History of Molecular Biology and Biotechnology at CSHL Archives contain historical correspondence, memorandums, clippings, and meetings summaries on the topic of HIV/AIDS. The selected material displays the attitudes towards and knowledge of HIV/AIDS during the late 1980s and early 1990s.


CD62L FUNCTIONS AS AN HIV ADHESION RECEPTOR ON CD4 T CELLS AND THE VIRUS INDUCES ITS SHEDDING FOR RELEASE

Joseph Kononchik1, Joanna Ireland1, Zhongcheng Zou1, Genevieve Holzapfel1, Ashley Chastain1, Nicole Stutzman1, James Arthos2, Tae-Wook Chun2, Susan Moir2, Peter Sun1

1NIAID/NIH, Structural Immunology Section, Rockville, MD, 2NIAID/NIH, Lab of Immunoregulation, Bethesda, MD

The mechanisms that dictate preferential infection of central memory CD4+ T cells by HIV-1, as well as the factors contributing to the persistence of viral reservoirs, are not well understood but are central to controlling HIV-1 infections. Here, we identified L-selectin/CD62L as a viral adhesion receptor on CD4+ T cells. HIV-1 envelope glycan bound CD62L resulting in preferential infection of central memory T cells. HIV infection induced shedding of CD62L and downregulation of CCR7, explaining the preferential loss of CD62L+ central memory T cells in HIV patients. The infected effector memory CD4+ T cells were, however, competent in cytokine production, suggesting that the observed effector memory dysfunction in HIV patients is related to counting the CD62L-shedded central memory as effector memory T cells. Importantly, inhibition of CD62L shedding dramatically reduced HIV-1BAL infection and inhibited viral release in both viremic and aviremic patient CD4+ T cells. The requirement of CD62L shedding in HIV viral release opens new possibilities for antiretroviral treatments.


THE PREVALENCE AND PATTERNS OF HIV DIAGNOSIS IN A RURAL ZAMBIAN COMMUNITY

Mei Tan1, Joseph Mwaba2, Phil Thuma2, Elena Grigorenko3

1University of Houston, Texas Institute of Measurement, Evaluation & Statistics, Houston, TX, 2Johns Hopkins University, Macha Research Trust, Baltimore, MD, 3University of Houston , Baylor College of Medicine, Houston, TX

The HIV/AIDS pandemic remains a major public health concern, especially in sub-Saharan Africa (SSA). Much of this burden of illness is borne by children, as 90% of the world’s HIV-positive (HIV 1) children under the age of 15 years and 84% of AIDS-orphaned children (HIV-OVC) live in SSA. To better understand this vulnerable population and how to best support these children’s growth and learning potential, we are engaged in a large-scale, longitudinal study funded by the NIH to 1) identify and evaluate 1,000 OVC, particularly with respect to developmental and learning outcomes; and identify and evaluate all interventions delivered to these children, in a well-defined, rural area of Southern Province, Zambia. Our ultimate aim is to gauge the collective and differential effectiveness of these interventions.

Here, we intend to present the first set of screening data from this project. Surveying a set of representative regions within a 20km radius of a rural hospital, we screened 1,512 households, including one orphanage—the only one in existence within the designated area— to identify children orphaned or made vulnerable by HIV. We define these OVC as children who have been diagnosed with HIV, who have had one or both parents diagnosed with HIV, or one or both parents dead from HIV. In addition, they are considered OVC if their primary caregiver in the household has been diagnosed with HIV.

In identifying households in which HIV-diagnosed adults and/or children are present, whether siblings tend to be affected, which diagnosed individuals are ill, and where diagnosed parents may be located (within or outside the community), we gain a useful snapshot of the current dispersal of HIV in this area. This provides a preliminary glimpse of the prevalence of HIV in this rural area of Southern Zambia, but also indicates the occurrence of illness as a result of diagnoses (reflecting non-access to or non-adherence to ART), and patterns of diagnoses within families and geographical locations that might help us understand the continued persistence of HIV in this rural context.