How many human immune systems are there? How old are they? Why is there no AIDS vaccine? Is a new approach needed? Why is public opinion growing skeptical of the scientific community after three decades of public awareness about HIV/AIDS? Consider answers to these puzzling questions. Learn from the decades of experience of two senior scholars: Dr. Omar Bagasra (an eminent molecular biologist, immunologist, and retrovirologist) and Dr. Donald Gene Pace (a highly published writer who examines public health policy). Explore intriguing new possibilities about human immunity, and the development of an effective AIDS vaccine. Read Immunology and the Quest for an HIV Vaccine. Benefit from an informed synthesis backed by a wealth of peer-reviewed scientific references. Review basic concepts of immunology, and stretch your perspective by contemplating this creative synthesis that provides a provocative treatise on the origin, evolution, and etiology of several forms of immune systems. Consider compelling ideas that will have an important bearing on HIV vaccine development. Enjoy this informative volume designed for anyone interested in the development of a safe AIDS vaccine, for anyone curious about present knowledge about human immunity, and for anyone yearning for a solution to the global pandemic that is AIDS.

Immunology and the Quest for an HIV Vaccine

A New PerspectiveBy Omar Bagasra Donald Gene Pace

AuthorHouse

Copyright © 2012 Dr. Omar Bagasra, MD, PhD and Dr. Donald Gene Pace, PhD.
All right reserved.
ISBN: 978-1-4685-0830-7

Contents

PREFACE..............................................................................................ixACKNOWLEDGMENTS......................................................................................xvLIST OF FIGURES AND TABLES...........................................................................xviiCHAPTER 1 A SHIFT IN THE CURRENT CONCEPT OF HIV VACCINE..............................................1Evolution of Intracellular Molecular Defense.........................................................3Protective Role of Endogenous Retroviruses in Sheep..................................................5Evolution of Classical Immunity......................................................................8Why Move Beyond Classical Immunity?..................................................................11Broadly Neutralizing Antibodies: Development of Antibodies against HIV-1.............................12Do Neutralizing Antibodies Protect African Non-human Primates?.......................................14The Problem..........................................................................................17Examples of Long-term Nonprogressors (LTNPs), Elite Suppressors (ES).................................19Exposed-but-Resistant: Kenyan Sex Workers Appear Immune to HIV Infection.............................19Future Direction of miRNA Research: miRNA Delivery...................................................20A Strange Case of HIV Cure...........................................................................35CHAPTER 2 IMMUNITY: OLD PARADIGM VERSUS NEW..........................................................41The Foundation of Molecular Immunity.................................................................45Inadequate Current Paradigm: Nature, Origin, and Functions of Classical Immunity.....................48The HLA Genetic Determinants Are Not the Only Protective Mechanism...................................52Molecular Immunity Arrived from Transposons and Retroelements........................................54Recent Evidence Related to Origins of RNAi and miRNA Silencing Systems...............................55Endogenous Retroviruses: Protective Lessons..........................................................59Innate Immunity and Immune Defense Based on Pattern Recognition......................................61Evolution of Anti-transposon Resistance in Bacteria to Large Mammals.................................63Perspective from Fungal Molecular Defense............................................................64Immunity's Big Bang..................................................................................72Summary..............................................................................................79CHAPTER 3 THE ARCHITECTURE OF THE CLASSICAL IMMUNE SYSTEM............................................81The Innate Immune System.............................................................................82The Classic, or Adaptive, Immune System..............................................................86EPILOGUE.............................................................................................107REFERENCES...........................................................................................111BIBLIOGRAPHY.........................................................................................135

Chapter One

A SHIFT IN THE CURRENT CONCEPT OF HIV VACCINE

"The cellular immune response (via cytotoxic T lymphocytes, or CTLs) plays a significant role in modulating the level of infected cells but is incapable of clearing them altogether for a couple of reasons. For one, as the number of infected cells declines, the number of CTLs also declines. For another, HIV replication targets the CD4+ T helper cells – the very cells that are essential for the most effective CTL killing. This leaves only a relatively weak helper cell-independent response. The upshot is that even when antiviral therapy has reduced the number of productively infected cells by as much as 6 orders of magnitude, the immune system cannot clear the small residue of infected cells. If therapy is discontinued, even after many years of treatment, the infection always recovers to previous levels."

John Coffin, "Searching For Achilles' Heel." Microbe 2009; 4 (5), 224. Thomas S. Kuhn's Structure of Scientific Revolutions shows that paradigms shift when diverse aggregated evidence invites revision. Recent decades have witnessed the failure to formulate a classical immunity-based anti-HIV vaccine. We argue that resistance against an invading retrovirus such as HIV-1 is based on intracellular immunity that evolved in the form of small dsRNA (e.g., miRNA, RNAi, etc.). Lentiviruses (LVs), specifically SIVs, endemically infect over 40 different African non-human primates (reviewed in 2-6), and provide useful models for HIV-1 molecular studies. Although natural hosts to SIVs, African non-human primates typically do not develop immunodeficiency or AIDS, although they exhibit high degrees of viremia. It should be noted that the high degree of viremia does not kill the African nonhuman primates, including chimpanzees that harbor almost the same virus as HIV-1, or make them very immunodeficient. And, more pointedly, why is our criteria for treating HI-infected individuals with highly active anti-retroviral therapy (called HAART) mainly based on measuring the viral load and CD4+ T cell count? More, importantly, only certain Asian macaques generally exhibit viremia and progressive loss of CD+ Tlymphocytes. Why differences in pathogenicity exist has been subject of much speculation, and no conventional immunological approach has deciphered which viral and/or host factors account for African nonhuman primates resistance. We hypothesize that African non-human primates find protection through selective and differential expressions of SIV homologous microRNA (miRNA) that form stable complexes with the virus. We also believe that a similar protective mechanism is operational in a small group of infected humans resistant to HIV-1 and others that experience long-term latency. Although retroelements have infected life forms that predate classical immunity's evolution (4 billion versus 350 million years), these intracellular invaders are checked by intracellular small double-stranded nucleic acid-based defense mechanisms (i.e., miRNAs and/or non-coding ncRNAs). We will describe various aspects of this immunity throughout this book.

Evolution of Intracellular Molecular Defense

Many believe early life on earth was RNA in nature, as were primitive parasitic life forms (i.e., retroelements) that invaded them. RNA molecules performed enzymatic functions as ribozymes, and bioinformation storage functions as genomes. Early RNAs were invaded by primitive retroelements – retrotransposons – whose ribozymes served as polymerase for self-replication. As evolution moved forward, RNA evolved into more stable DNA genomic storage molecules, and proteins assumed complex enzymatic roles when multiple structural molecules evolved. RNA branched-off into specialized molecules serving as biodefense network and gene regulator. Current research clearly supports the notion that the majority of the human intracellular defenses derived from miRNAs have their origins in transposable elements (TEs) and retrotransposons.

Our current understanding of the evolutionary history of the incorporation of retrolements in the higher life forms is just beginning to develop. However, it appears that as early as the emergence of Archea, retroelements were being incorporated into the host genome that served as intracellular defense for survival. As evolution progressed, many of the retroelements became extinct due to their inability to find suitable hosts, and bolstered miRNA-based defenses that would not allow them to invade or infect the host. The integrated retroelements now serve as a vanguard against any retrovirus or lentivirus that already has a unique genetic footprint in the genome of a host. As we will show later, exposures to lentiviruses and retroviruses are very common in higher animals, but they do not develop any illness from them. One of the reasons for this is that specific fragments of the integrated retroelements, which include retroviruses and lentiviruses in higher life forms, are strategically expressed in the forms of miRNAs, introns and other small anti-sense RNA that look for homologous sequences in the invading viruses or microorgamisms, and disable them by various mechanisms. As the time passed these same integrated retroelements were co-opted to serve other gene regularity functions, in addition to the molecular immunity function. Therefore, today, these former retroelements serve as regulators of cellular differentiation, chromatin restructuring agents, and myriad other functions. Retroelements have profoundly affected the evolution of prokaryotic and eukaryotic forms. The evidence of such evolutionary events can be seen in the presence of ~50% gene sequences in the human genome, and a significant perecentage of the genomes of most contemporary life forms that share genetic similarities to transposable elements and retroelements, or their remnants.

Going back to the origins of life, amid this scene of small RNA-based defenses and invading retroelements, the eternal host-parasite struggle blossomed into innumerable fauna and flora, while defensive means against parasites did likewise. The prime directive of speciation is maintenance of genomeintegritybutthiscouldnotbeachievewithoutsymbiosisofprimitive retroelements and pre-transposons with the evolving host genomes. Both miRNA- and RNA-interference have been recorded in the earlieast existing life forms that are present today in Archea. Perhaps one of the firsts among the successful protein-based defense systems, which apparently evolved in prokaryotic forms, used restriction enzymes to counter and destroy foreign DNA, all the while guarding and protecting their own through help from methylation and miRNA. Brouns et al. indicate that bacteria began to defend themselves against retroelements as retroelement fragments established genomic regions, systematic clusters of regularly interspaced short palindromic repeats (CRISPRs). CRISPRs carry out the vital task of creating a veritable pathogenic danger list, a heritable collection of memories about prior infections. In Escherichia coli, the CRISR region is transcribed. Moreover, casE, the CRISPR-associated gene, promotes transcript cleavage into small ~57-nucleotide CRISPR-RNAs (crRNAs).

This molecular pattern recognition system pioneered in distinguishing between self and non-self (21). As DNA size increased, and as prokaryote evolution took a quantum leap, and evolved into eukaryotes, gene regulation mushroomed, and protection of self-DNA through restriction enzymes or CRISPRs became difficult. These defenses lacked effectiveness against retroelements that integrated into their genomes, and had the ability to jump in and out of the host DNA, thereby creating occasional havoc. Many life forms accommodated retroelements rather than fight them. This gave birth to "molecular immunity" (MI) (small dsRNA-based bio-defensive systems), where small hairpin retroelements were expressed as double-stranded (ds) microRNAs, or small interfering RNAs (siRNAs), to bind homologous sequences of invading retroelements, split them via DICER-like (DCL) enzymatic systems, or block integration through triplex-formation (TF). This immunity has its origin in archea and prokaryotes.

Protective Role of Endogenous Retroviruses in Sheep

Vertebrate genomes are heavily colonized by "endogenous" retroviruses that share ~50% of their genomic DNA. Endogenous retroviruses derive from retroviral infections of host germ cells during evolution, which facilitates permanent integration of viral genomes into host DNAs, and multigenerational transmission. Endogenous retroviruses block replication cycles of horizontally transmitted "exogenous" pathogenic retroviruses. We hypothesize that ERVs have protected hosts against pathogenic RVs that share genetic sequences like those of integrated viruses. Recently, Arnaud et al. characterized the evolutionary history and molecular virology of endogenous betaretroviruses of sheep (enJSRVs), and described the pivotal role that integrated retroviral genes play in countering "exogenous" retroviruses. They discovered that (i) two enJSRV loci that entered the host genome before speciation within sheep (genus Ovis) about 3 million years ago acquired, after integration, a mutated defective viral protein capable of blocking exogenous homologous retroviruses; (ii) both transdominant enJSRV loci became fixed in the host genome before or around the time of sheep domestication (~10,000 years ago); (iii) the invasion by ERVs of JSRV/enJSRVs continues; and (iv) new viruses have emerged less than 200 years ago, the same period in which sheep from many parts of the world were acquired to create better breeds, and most likely introduced multiple variants of retroviruses that evaded the transdominant enJSRV loci. The host-virus struggle continues; hosts fight retroviral infections by expression of previously endogenized retroviral genetic sequnces, and invading retroviruses find the right time and cell type to invade the host genome, and subsequently enter the germ lines. The endogenization of a new retrovirus secures the host's future. Moreover, this new endogenous retrovirus provides molecular defense against selected new invaders.

As life forms gained complexity, more sophisticated parasites emerged; some avoided integration into host DNA and replicated outside the nuclear system, cannibalizing the host's raw materials and synthetic machinery. Defense against these new DNA and RNA viruses became imperative! Therefore, siRNAs and triplex forming miRNAs (tfmiRNAs) responded by interfering with viral replication cycles. Hosts tried to destroy the functionality of the invaders' genes by several unusual enzyme-based defences like APOBEG3G enzymes, an editing system that modifies the viral genetic material. Viruses countered with their own miRNAs (called vmiRNAs) to disable and feast on host miRNAs. Balance emerged and host genomes accommodated more retroelements until a significant percent of the host genomes became retroelements in origin (e.g., ~50% of the human genome contained retroelements); this may have provided a critical balance of mass of small dsRNA permutations to quell invading retroelements and other viruses, and also to regulate the endogenous genes. It can be seem in evolutionary terms, where retroelements occupy 40% of mouse, 15-22% of fruit fly, 12% of nematode, and 8.6% of chicken genomes. However, in some plants, 90% of their genome is made of retroelements, suggesting that evolutionary history of a particular life form may reflect the critical mass of retroelements that were needed to be integrated to avoid extinction of a particular species. It should be kept in mind that only 2-3 % of the whole genome is required to expression and code for the proteins (24). So, theoretically, >90% of the genes can be utilized for regulatory and defense purposes. Increasing retroelement numbers demanded control of thousands of potentially active endogenous retroelements, and coding genes without harming host replication. The current mammalian gene regulation mechanism resembles a gigantic orchestra that creates the music of life with advanced synchronic balance. Early in evolution, hosts accumulated large numbers of retroelements and began co-opting endogenized retroelemnt-miRNAs for internal and external regulation. As life forms developed multiple layers and organs, cellular specialization and differentiation prompted expressions of selected genes in differentiated cell types, which began to express non-coding genes at differential levels, to create spatial and temporal cell-specific expressions of miRNA. Evolution provoked life forms to conserve resources, to acquire cell surface receptors to express for differentiation and resource gathering, and to invite specific retroelements and viruses to precisely express potentially neutralizing miRNA arrays.

Evolving host cells came to routinely block the entry of retroelements and other viruses from replication, but to function properly required surface receptors to communicate signals, and regulate nutrition. Receptors constitute the interface of cells to their external environment. These molecules bind specific ligands involved in multiple processes, such as signal transduction and nutrient transport. All viruses and retroviruses target host cells' most vulnerable parts, surface molecules expressed on specialized cells that are essential for their survival. Therefore, retroviruses can utilize a variety of cell-surface receptors, and gain entry into cells. Almost all viruses utilize single transmembrane proteins receptors, whereas all retroviruses utilize multi-transmembrane receptors that govern solute transports, and show the longer evolutionary history of retroviruses as opposed to other RNA and DNA viruses. Generally, a single receptor appears to be necessary and sufficient for entry of many retroviruses, but HIV requires two receptors, a CD4 molecule (found on surfaces of CD4+ T cells and monocyte/macrophage), and a CCR5 or CXCR co-receptor (found on each of these cells for cell entry). Also, two retroviruses can use different receptors in some cells but use the same receptor for entry into other cells. Why does HIV need two receptors while the majority of the retroviruses only need one receptor? Is it because it is a chimera virus?

Did it arise from a massive recombinant event of several SIVs during the preparation of an experimental vaccine (2)? The battle still rages, as seen in positive and negative usages of entry routes for HIV-1, SIVs, human herpesvirus-6 and -7, and GBV-C, a non-pathogenic variant of hepatitis C. All utilize CD4 molecules, which are essential for CD4+ T and other immune cells. In some cases (notably Caenorhabditis elegans), miRNA deterrence became so effective that no natural viruses could invade this life form. C. elegans remain susceptible to unnatural viruses, including human viruses, which can prove deadly. Like humans and Asian macaques, unnatural hosts for SIVs and HIVs respectively, these worms lack requisite miRNAs to quell unnatural zoonotic infections.

Evolution of Classical Immunity

As parasitic cellular invasion became more difficult, a new kind of parasites evolved that inhabited body cavities, fluids, and blood in larger life forms to intercept raw materials. These parasites were immune to miRNAs; having never entered inside the cells, they were unseen by miRNA! New defenses responded to these invisible insurgents; perhaps as long as 300 MYs ago, jawfish began developing antibodies to counter invading antigens, giving rise to classical immunity.

(Continues...)

Excerpted from Immunology and the Quest for an HIV Vaccineby Omar Bagasra Donald Gene Pace Copyright © 2012 by Dr. Omar Bagasra, MD, PhD and Dr. Donald Gene Pace, PhD.. Excerpted by permission of AuthorHouse. All rights reserved. No part of this excerpt may be reproduced or reprinted without permission in writing from the publisher.
Excerpts are provided by Dial-A-Book Inc. solely for the personal use of visitors to this web site.