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6 The Biology of HIV and Biomedical Research Needs Since the publication of Confronting AIDS in October 1986, there has been appreciable progress in elucidating certain structural and functional attributes of HIV. Early research efforts were productive in defining the HIV genetic structure and important aspects of viral replication in vitro. Less progress has been made in understanding the behavior of HIV in viva and the interaction of the virus with its human host. This chapter describes new knowledge gained from AIDS research efforts, ways in which that knowledge may be used against HIV infection, the difficulties of developing drugs and vaccines to combat the epidemic, and the variety of resources needed for the task. HIV BIOLOGY Viral Taxonomy and Disease Over the past 2 years, researchers have begun to appreciate more fully the nature and diversity of HIV, especially following the discovery in 1986 and subsequent characterization of a second human retrovirus, HIV-2. The first retrovirus found to cause AIDS, now referred to as HIV-1, is responsible for the vast majority of AIDS cases reported to date, and it accounts for the most prevalent and geographically dispersed occurrences of human retroviral infection now known (IOM/NAS, 19861. HIV-2 is most prevalent in certain regions of West Africa, where its transmission and pathogenesis mirror those of HIV-1 (Clavel et al., 1986a, 123

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124 CONFRONTING AIDS: UPDATE 1988 1987; Kanki et al., 19861; it has been linked to a growing number of cases of immunodeficiency diseases that are clinically indistinguishable from HIV-1-associated AIDS (Clavel et al., 1986a, 1987; Kanki et al., 1986, 19871. It is not known whether HIV-2 infection leads to AIDS at the same rate or with the same frequency as HIV-1 infection, but it is clearly a pathogenic retrovirus. HIV-2 is not as effectively detected as HIV-1 by the screening tests now available. Through morphologic and genetic analyses of HIV-1 and HIV-2, their taxonomic assignment has been further defined as the subfamily of retroviruses known as lentiviruses (Gonda et al., 1985; Sonigo et al., 1985; Clavel et al., 1986b; Guyader et al., 19871. Lentiviruses are the causative agents of a number of diseases in other mammalian species that are characterized by persistence of infection, the effective impotence of the host immune response to clear the infection, long incubation periods, and protracted symptomatic phases. Although HIV-1 was the first lentivirus to be described with profoundly immunosuppressive properties, a growing number of retroviruses of this type have since been discovered. Shortly after the isolation of HIV-1, a related lentivirus, simian immunodeficiency virus, or SIV, was identified in a variety of monkey species (Daniel et al., 1985; Kanki et al., 1985~. HIV-2, in fact, is more closely related in its genetic structure to this simian virus than to HIV-1 (Chakrabarti et al., 1987; Franchini et al., 1987; Guyader et al., 1987; Hirsch et al., 19871. Recently, researchers have identified other lentiviruses as potential agents of immunodeficiency disease in cows (bovine immunodeficiency virus) and cats (feline immu- nodeficiency virus) (Gonda et al., 1987; Pederson et al., 1987~. The continued study of these simian, bovine, and feline lentiviruses may provide valuable models of the pathogenesis and therefore potential treatment of HIV infection in humans. In April 1988 scientists from Gabon announced the isolation of a newly identified virus from chimpan- zees that appears to be closely related to HIV-1 (R. Widdus, World Health Organization, personal communication, 19881. Using molecular cloning and nucleotide sequence analysis of multiple isolates of HIV-1, researchers have defined an exceptionally complex retroviral genome with characteristics not previously seen in retroviruses (Muesing et al., 1985; Ratner et al., 1985; Sanchez-Pescador et al., 1985; Wain-Hobson et al., 19851. Studies have shown that the HIV-1 genome contains a number of novel genes in addition to those that encode the usual structural and enzymatic functions required for retroviral replica- tion. These studies have identified the protein products of HIV-l's unusual genes, and, as they do not appear to be present in mature virus particles, it has been postulated that these gene products have regulatory rather than structural roles in viral replication. The importance of such

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BIOLOGY OF HIV AND BIOMEDICAL RESEARCH NEEDS 125 findings lies in our improved understanding of the mechanisms of action of the HIV regulatory genes, an understanding that could speed the development of specific, effective means to inhibit HIV replication. The nucleotide sequences of independent isolates of HIV-1 are also notable for their degree of variation and the patterns of these variations (Alizon et al.3 1986; Coffin, 1986; Starcich et al., 1986~. As discussed in Confronting AIDS, a fuller understanding of the origin and immunologic significance of variations in the nucleotide sequences of HIV is central to efforts to engender protective immunity through vaccination. Work in this area has brought improved definition of conserved and variable domains in the envelope glycoproteins, but little is known as yet about the biological processes that may give rise to and possibly select for sequence divergence (Coffin, 1986; IOM/NAS, 1986~. Several independent isolates of HIV-2 have now been molecularly cloned and sequenced, and they appear to demon- strate a similar degree of variation (Clavel et al., 1986b; Guyader et al., 19871. Molecular studies of the HIV-1 genome have delineated the basic struc- ture of the genes that encode the necessary replicative functions provided by the viral core proteins (gag), polymerase ~ poll, and surface glycoproteins (ens). They have also defined the structure of the genetic regulatory sequences in the retroviral long terminal repeats (LTRs). In addition to these expected retroviral genes, the studies have shown that HIV-1 contains genes that are without known counterparts in other retroviruses: the so-called open reading frames, referred to as sor and 3'-orf. These open reading frames give rise to proteins that are produced during the course of HIV infection in vitro and in viva (Allen et al., 1985; Lee et al., 1985; Kan et al., 1986~. Recently, Alizon and coworkers (1986) and Wong-Staal and colleagues (1987) have demonstrated that another conserved open reading frame, R. also encodes a protein product. Functional studies and mutagenic analyses of biologically active molecular clones of HIV-1 have resulted in the discovery of two additional viral genes, tat-III and art/trs (Sodroski et al., 1984; Arya et al., 1985; Dayton et al., 1986; Feinberg et al., 1986; A. G. Fisher et al., 1986b; Sodroski et al., 1986a). Thus, whereas many other naturally occurring, replication-competent retroviruses have only three genes, HIV-1 contains at least eight (Figure 21. Of these, the tat-III and art/trs genes are known to be essential for HIV-1 replication, and the sor gene product appears to be important for efficient viral transmission; the R and 3'-orf genes appear to be dispensable for replication in vitro (A. G. Fisher et al., 1986a, 1987; Luciw et al., 1987; Strebel et al., 1987~. All of the structural (gag, pot, and envy and what are presumed to be the regulatory (tat-III, art/trs, sor, R. and 3'-oj) genes encode proteins that are recognized by sera from infected individuals, indicating that the proteins are produced in viva and are immunogenic (Allen et al., 1985; Lee et al.,1985; Aldovini et al.,1986; Knight et al.,1987; Wong-Staal et al.,19871.

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BIOLOGY OF HIV AND BIOMEDICAL RESEARCH NEEDS 127 The genome of HIV-2 has now been molecularly cloned and studied in detail (Guyader et al., 19871. In addition to the requisite gag, pol, ens, and regulatory genes, HIV-2 encodes an additional centrally located open reading frame (X) that is not found in the genome of HIV-1. Control of Viral Gene Expression Like all other retroviruses, HIV uses double-stranded DNA copies of the viral genome as templates for the synthesis of viral RNA molecules. The transcription of retroviral RNA uses the host cell's synthetic machin- ery, but it is regulated by genetic elements resident in the viral long terminal repeats, or LTRs. The complex behavior of HIV is thought to include mechanisms that facilitate both the amplification and attenuation of viral expression (production). Although there is still much to be learned about these regulatory processes, it appears likely that they are mediated in large part through specific nucleotide sequences in the HIV LTRs. Studies have shown that HIV production in vitro is enhanced by a variety of immunologic stimuli and specific cytokines (proteins produced by host cells) (Hoxie et al., 1985; Zagury et al., 1986; Folks et al., 19871. Whether or not increased production follows similar stimulation in viva is unknown; if so, such stimuli (other infections, perhaps) might be impor- tant factors mediating the pace of progression of immunocompromise and disease following initial HIV infection. The basic mechanisms regulating gene expression in human (and other eukaryotic) cells are only beginning to be understood. Early studies have clearly shown, however, that the cellular transcription apparatus may be preferentially activated to express specific subsets of cellular genes within the context of a given cell's differentiation program. Recently, evidence has been presented suggest- ing that the activation of HIV production by cellular stimulation is mediated by the activation of a host regulatory protein binding to the genetic regulatory elements of the viral LTR, which previously had been shown to function as transcriptional enhancer elements. Additional studies have identified a number of other host cell proteins that bind to specific sequences in the HIV LTR and that may also play important roles in controlling the transcription of HIV RNA (Jones et al., 1986; Franza et al., 1987; Garcia et al., 1987~. Regulation and Production of HIV As discussed in the previous section, the double-stranded DNA (pro- viral) form of HIV residing in the nucleus of an infected cell serves as the template for synthesizing full-length viral RNA transcripts. Some of the transcripts remain intact and enter the cytoplasm for incorporation into

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128 CONFRONTING AIDS: UPDATE aged virus particles and transmission to other cells. Researchers also believe that the full-length transcripts are used in the translation of HIV gag and pol proteins, which derive from proteolytic cleavage of a large polypro- tein precursor (IOM/NAS, 1986~. Because the reading frames of gag and pol are adjacent but discontinuous, until recently it was unclear how the precursor protein was produced. Now, studies have described the process, called ribosomal frameshifting, that HIV uses to generate the precursor of the mature gag and pol proteins (Jacks et al., 1988~. Researchers have also recently identified a virally encoded protease responsible for the proteolytic cleavage of the gag-pol precursor into its mature functional constituents. The constituents include the viral core structural proteins and the reverse transcriptase, endonuclease, and RNase H activities of the HIV polymerase complex (Debouck et al., 19881. The remaining HIV proteins, including the ens glycoproteins and the regulatory proteins, are all translated from subgenomic RNA transcripts derived by an unusually complicated pattern of RNA splicing (Muesing et al., 19851. What is yet unknown are the mechanisms by which retroviruses achieve an appropriate balance between sufficient unspliced RNA tran- scripts to provide new genomic RNA molecules for additional cycles of infection and spliced mRNAs to encode the requisite viral structural and regulatory proteins. It appears that HIV may encode a specific, novel gene to regulate the pattern of spliced RNAs produced in infected cells and, consequently, the amount of virus particles produced (Feinberg et al., 19861. HIV envelope glycoproteins play an essential role in the replication of HIV and may be responsible for many of the cytopathic consequences of viral infection (Lifson et al., 1986a; Sodroski et al., 1986a; Somasundaran and Robinson, 1987; McCune et al., 1988~. The envelope protein is translated from a spliced mRNA and synthesized as a large precursor protein; this precursor protein is then heavily glycosylated (sugar residues are added) to a form known as gpl60 and proteolytically cleaved into two associated subunits, gpl20 and gp41 (IOM/NAS, 1986~. Recent work has shown definitively that the gp120 molecule mediates viral attachment to the cellular receptor, the host cell surface protein CD4 (Dalgleish et al., 1984; Klatzmann et al., 1984; Maddon et al., 19861. The gp41 anchors the envelope glycoprotein complex in the lipid bilayer of the viral membrane. The glycosylation and proteolytic processing of the gpl60 envelope precursor are host cell enzymatic functions (IOM/NAS, 19861. Now, researchers also understand that glycosylation of the HIV envelope proteir1 is necessary for receptor binding and infectivity, and may play an additional role in masking critical envelope domains from host immuno- logic attack (Matthews et al., 19874. Proteolytic cleavage of the envelope precursor has been shown to be essential for the generation of infectious, cytopathic HIV particles.

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BIOLOGY OF HIV AND BIOMEDICAL RESEARCH NEEDS 129 The protein products of the HIV genes sor, R. tat-III, art/trs, and 3'-orf (and X in the case of HIV-2) are synthesized from multiply spliced viral mRNA transcripts. These regulatory proteins have been identified, but a good deal of uncertainty and controversy surrounds any definition of their exact functions. For example, early studies have demonstrated that the tat-III and art/trs proteins play roles in HIV replication and appear to involve genetic processes that are without known precedents in human cells. It is likely that the R. 3'-orf, and X genes also play important roles in the biology of HIV-1 and HIV-2, but a more thorough understanding of these genes has been limited by the lack of in vitro assays of their functions. The tat-III protein greatly amplifies (or transactivates) the level of replication of HIV-1 (Sodroski et al., 1984; Arya et al., 19851. Studies have shown that this enhancement requires specific nucleotide sequences (the transactivation response element, or TAR) that are contained within the viral LTR; following transcription, these sequences are included at the 5'-end of all viral RNA transcripts (Rosen et al., 1985; Muesing et al., 1987~. Whether the tat-III protein directly binds to the TAR regions is not yet known, but researchers have concluded that the TAR region assumes a complex RNA secondary structure whose specific topology is essential for proper functioning (Hauber and Cullen, 19881. Reportedly, the tat-III protein enhances both the transcription and translation of HIV-1 RNA (sullen, 1986; Feinberg et al., 1986; Peterlin et al., 1986; Rosen et al., 1986; Wright et al., 1986; Hauber et al., 1987; Kao et al., 1987; Muesing et al., 19871. Defining the relative importance of several proposed mechanisms of tat-III action awaits additional experimental evaluation. Nevertheless, although the function of the tat-III protein is still poorly defined, it has been shown to assume a predominantly nuclear localization in infected cells (a possible clue to its function), and important aspects of its molecular structure are emerging (Hauber et al., 1987; Frankel et al., 1988~. The art/trs protein of HIV-1 also has an essential although poorly understood function in viral replication (Feinberg et al., 1986; Sodroski et al., 1986b; Knight et al., 19871. The art/trs gene appears to control, directly or indirectly, the pattern of HIV-1 RNA transcripts seen in infected cells. In the absence of art/trs expression, the smaller, multiply spliced mRNA species that encode the viral regulation proteins predom- inate, at the expense of the full-length and singly spliced ens transcripts that provide viral genomes and specify translation of the HIV-1 virion structural components (Feinberg et al., 1986; Sadaie et al., 19881. The art/trs function may specifically affect the splicing of viral RNA and thus permit important differential regulation of HIV expression; additional art/trs effects on the translation of specific viral mRNAs have also been

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130 CONFRONTING AIDS: UPDATE 1988 suggested. Rosen and colleagues (1988) have postulated that the art/trs protein acts through specific, incompletely defined sequences contained in HIV-1 RNA transcripts. Like the tat-III protein, the art/trs protein is localized in the nucleus of HIV-1-infected cells, consistent with its postulated role in the splicing of viral RNA. It has been reported that the sor gene of HIV-1 is required for efficient replication. This finding is based on studies of viruses with experimentally induced mutations in the sor gene. The studies show that the viruses spread poorly and only through cell-to-cell contact in in vitro lymphocyte cultures (A. G. Fisher et al., 1987; Strebel et al., 1987~. The nature of this handicap is not well understood, however. It is thought that the sor protein is not incorporated in HIV virus particles. The 3'-orf gene of HIV-1 is not needed for replication in vitro, and recent reports have suggested that viruses that lack a functional 3'-orf gene may, in fact, replicate more efficiently than wild-type viruses (A. G. Fisher et al., 1986a; Luciw et al., 1987~. Whether the 3'-orf protein specifically inhibits HIV-1 production is not yet known. It has been suggested that the 3'-orf protein influences the expression of certain host cellular genes. The role of the R gene of HIV-1 has only recently begun to be scrutinized, but preliminary results (Wong-Staal et al., 1987) indicate that it may not be required for HIV- 1 replication in vitro. Also in need of direct evaluation are the roles of HIV-2 regulatory genes, although these are assumed to be analogous to those of HIV-1. The significance and function of the distinctive X gene of HIV-2 await elucidation. Because SIV's genetic structure is closely related to that of HIV-2, it will permit experimental analyses of the in viva function of the unusual regulatory genes of the immunodeficiency-inducing lentiviruses analyses that are not possible with the human AIDS viruses (Chakrabarti et al., 1987; Franchini et al., 1987; Hirsch et al., 1987~. Interrupting Infection by HIV The HIV replicative cycle presents a number of opportunities for interruption by antiviral interventions. Since 1986 a good deal of progress has been made; however, much of the information that emerges from ongoing studies highlights the difficulties that must be overcome before effective prophylactic or therapeutic interventions are feasible. The earliest event in the establishment of HIV infection is the binding of the virus particle through its envelope glycoprotein (gpl20) to a specific receptor on the host cell's surface. This CD4 cell receptor is found on the surface of certain members of the T lymphoid and macrophage-monocyte cell lineages (IOM/NAS, 1986; Maddon et al., 1986; Sattentau and Weiss,

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BIOLOGY OF HIV AND BIOMEDICAL RESEARCH NEEDS ~3 ~ 19881. The range of cells that are susceptible to HIV-1 infection, both in vitro and in viva, appears to parallel those that display the CD4 surface receptor. HIV-2 also uses the CD4 molecule as its receptor in the initiation of infection. Current studies are defining the interaction between the HIV-1 gpl20 envelope protein and the CD4 receptor with increasing resolution. The region of the gpl20 molecule that interacts with the CD4 molecule recently has been defined; the identification of the corresponding binding domain of CD4 is being actively pursued (Lasky et al., 19871. Potential strategies to inhibit the gpl20-CD4 interaction, and thus HIV infection, include vaccination to elicit antibodies that recognize and bind to the critical receptor-binding domain of the HIV envelope and the inhibition of cell surface binding through competition with appropriate exogenously added fragments of the CD4 protein. Recent studies have shown the feasibility of the latter approach by successfully inhibiting HIV infection in vitro with a soluble form of the CD4 molecule produced through recombinant DNA methods (Smith et al., 1987; Deen et al., 1988; R. A. Fisher et al., 1988; Traunecker et al., 1988~. It is hoped that small fragments of CD4 may soon be identified that will prevent HIV infection but that will not interfere with the critical immunologic functions of the CD4 molecule in viva. Experimental clinical trials of the soluble CD4 preparation in HIV-positive persons are expected to begin shortly; however, this approach may be limited by the inability of such prepara- tions to cross the blood-brain barrier and thus reach the important reservoir of infection within the central nervous system (Ho et al., 1985; Shaw et al., 1985; Gartner et al., 1986b; Koenig et al., 19861. After HIV binds to the CD4 receptor, it appears to enter the host target cell by direct fusion of the viral and cellular plasma membranes (Stein et al., 1987; McClure et al., 19881. It is thought that this process requires a hydrophobic domain on the HIV gp41 that assumes an active fusogenic conformation following cleavage of the gpl60 precursor molecule (Mc- Cune et al., 19881. One important manifestation of the cytopathic conse- quence of HIV infection in vitro involves a specific interaction between the HIV envelope glycoprotein complex and the CD4 molecule that results in the fusion and subsequent death of cells that have CD4 receptors (Lifson et al., 1986a,b; Sodroski et al., 1986a). This process, which is known as syncytia formation, may also involve the fusogenic domain of gp41 (McCune et al., 19881. The development of approaches to inhibit envelope-mediated membrane fusion may lead to novel ways of preventing HIV infection and its consequences. Once the virus is inside the host target cell, the HIV RNA genome is copied into a double-stranded DNA form by the viral reverse transcrip- tase. If reverse transcriptase could be inhibited, the lack of it would

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132 CONFRONTING AIDS: UPDATE 1988 disrupt an essential stage of HIV replication, and this antiviral strategy is being actively pursued (IOM/NAS, 19861. The drug zidovudine (i.e., AZT) acts to interrupt this stage of the infection. Zidovudine is a nucleoside analog that, once it has been phosphorylated by host cell kineses, is preferentially used by the HIV reverse transcriptase to synthesize the complementary strand of retroviral DNA. Once zidovu- dine is incorporated into the nascent DNA copy of HIV RNA, however, DNA polymerization stops, prematurely, and the DNA strand cannot be extended (Yarchoan and Broder, 1987~. Zidovudine effectively inhibits HIV-1 replication in vitro, and early clinical trials have shown that it improves survival in persons with severe manifestations of HIV-1 infec- tion (Fischl et al., 1987; Yarchoan et al., 19871. Although the clinical utility of zidovudine may be limited by its untoward side effects, which include anemia and neutropenia, it provides a hopeful precedent for future drug development efforts (Richman et al., 19871. A related chain- terminating nucleoside analog, 2',3'-dideoxycytidine (ddC), is an even more potent inhibitor of HIV replication in vitro and is now the subject of clinical trials (Yarchoan and Broder, 1987~. Another area of potential inhibition involves the processes that control the transcription and translation of HIV RNA. Much remains to be learned about these processes, although the requirements of the tat-III and art/trs genes in the HIV replicative cycle are clear. Still, a much better understanding of their functions is needed before studies can explore the specific inhibition of their activities. Similarly, researchers need an improved definition of the replicative role of the sor, R. X, and 3'-orf genes before they can evaluate their candidacy as targets for antiviral interventions. The processing and assembly of the HIV structural components offer a range of potential points for inhibiting HIV replication. The necessary proteolytic cleavage of the gag-pol polyprotein precursor is one possible target, and its mechanism is currently undergoing active analysis (De- bouck et al., 1988~. Likewise, the proteolytic processing of the envelope polyprotein gpl60, which is necessary for HIV infectivity, is a susceptible stage for inhibition. The glycosylation of the HIV envelope protein also appears to be necessary for viral infectivity, and drugs that interfere with this process have demonstrated antiviral activity in vitro (Gruters et al., 19871. Further studies of the pathways of viral assembly and maturation may identify additional promising antiviral targets. Natural History of HIV Infection Studies of HIV-infected persons have demonstrated the presence and expression of the virus in a number of host tissues, including peripheral

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BIOLOGY OF HIV AND BIOMEDICAL RESEARCH NEEDS 133 blood cells, lymph nodes, bone marrow, spleen, lung, retina, brain, cerebrospinal fluid, semen, cervical and vaginal secretions, saliva, and tears of infected individuals (IOM/NAS, 19864. Epidemiological analyses provide evidence that the essentially exclusive modes of HIV transmis- sion are sexual intercourse, blood and blood products, and perinatal transmission (see Chapter 2~. As in the case of other lentiviruses, studies have detected very little cell-free virus in HIV-seropositive persons. Perhaps in keeping with the lentivirus analogy, HIV may also be spread by virally infected cells carried in secretions rather than by cell-free virus. The factors that determine whether an HIV-infected person will transmit the viral infection to another individual are poorly understood (see Chapter 21. For instance, it appears that HIV-infected persons differ in their degree of infectiousness over time. Similarly, the factors that may influence an individual's susceptibility to HIV infection following a given exposure are incompletely defined. Other aspects of HIV infection are somewhat better understood, for example, which cells are targets of the virus in an infected human host. In addition to the CD4 helper-inducer cell population that is progressively depleted in the clinical development of AIDS, researchers now recognize that cells of the macrophage-monocyte lineage are important targets in the establishment, dissemination, and persistence of the infection (Gartner et al., 1986a; Ho et al., 1986; Koenig et al., 19861. Because macrophages permit HIV replication, are capable of wide-ranging migration, and are relatively resistant to the cytopathic consequences of viral infection, they probably play a major role in the pathogenesis of HIV-induced disease. Studies have found infected macrophage populations within the lymphat- ics, the central nervous system, and a variety of peripheral tissues, frequently at sites manifesting local pathology (Koenig et al., 19861. Interestingly, recent evidence suggests that different HIV isolates may replicate preferentially in either CD4-positive T lymphocytes or in macrophage populations derived from a variety of host tissues (Gartner et al., 1986a; Koyanagi et al., 19871. It is not known whether viruses with different cytotropisms give rise to specific clinical manifestations of HIV infection, nor what genetic differences account for the biological diversity of HIV isolates. Further hypotheses awaiting examination involve the possible in viva interactions among populations of HIV in an individual or the evolution of HIV in an infected individual, either of which might result in altered cytotropism or enhanced virulence during the course of disease progression. Once HIV infection is established, it persists throughout the lifetime of the infected person, avoiding clearance by the host immune response. The means by which HIV is able to avoid clearance is not known. Although HIV is found only in rare cells in the peripheral bloodstream, it

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|48 CONFRONTING AIDS: UPDATE 1988 The chimpanzee is now the animal of choice when HIV is used to challenge vaccinated animals to determine whether a vaccine provides safe, effective protection. Tests with chimpanzees are also being used to determine the number of different strains of HIV that must be included in a vaccine. Nevertheless, as discussed earlier in this chapter, experiments to date with vaccines in chimpanzees have failed to show that any vaccine candidate confers protection against HIV infection. The use of SIV to infect Old World primates such as rhesus macaques results in an animal model that quickly develops an AIDS-like disease with a subsequent high death rate. As described earlier in this chapter, the protein products and the organization of the SIV genome generally resemble those of HIV. The committee believes that SIV infection in macaques and the resulting disease are the best parallels at this time to human HIV infection and should be vigorously exploited. Studies of AIDS pathogenesis, the development of AIDS vaccines, and analyses of drug therapies will require tens of thousands of research animals (Weissman, 1988~. The lack of adequate numbers of chimpanzees and other primates for AIDS-related research is a serious problem. Currently, there are only about 1,200 chimpanzees in biomedical research colonies in the United States; another 80 are kept in pharmaceutical industry colonies in the United States (Desrosiers and Letvin, 19871. Although approximately 300 of these animals are available for breeding, current reproductive predictions indicate an annual increase of only about 35 chimpanzees for research use. Without significant increases in our understanding of primate reproduc- tion and conservation, there will be inadequate numbers of these animals available for AIDS-related research. Furthermore, the unregulated use for medical research of chimpanzees shipped from countries in which the animals exist in the wild could lead to their extinction. Sufficient lead time and funding must also be provided for the production of macaques in large numbers (Desrosiers and Letvin, 19871. The use of chimpanzees and other primates for AIDS research and, in particular, efforts to increase the available numbers of animals through breeding demand national-level coordination. Present programs for the conservation, population expansion, and optimal use of these animals appear inadequate (Gardner and Luciw, 19871. The committee recom- mends that plans for breeding, conserving, and otherwise expanding the present stock of chimpanzees be examined. This expansion may require increased funding. The committee is concerned that poorly designed studies will waste previously unexposed chimpanzees. The assistant secretary for health should ensure that this does not occur. Small animals can also be used in HIV infection research and efforts to develop animal models for AIDS. A mouse model, for example, would be

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BIOLOGY OF HIV AND BIOMEDICAL RESEARCH NEEDS 149 a crucial breakthrough in AIDS research, as laboratory mice are plentiful and well understood as research animals. In some studies, investigators have constructed transgenic mice by introducing DNA from other orga- nisms (humans or viruses) into the genomes of individual mice, which then express the foreign genetic instructions. The gene encoding the CD4 molecule, the essential regulatory elements of HIV that control viral expression, and the entire HIV genome have each been used to produce transgenic mice. If such efforts are successful, the transgenic mouse model could be valuable for determining which agents influence the expression of discrete genetic regions of HIV (M. Martin, NIAID, personal communication, 1988~. In another model of HIV infection, researchers have transplanted all elements of the human blood-forming and immune systems into geneti- cally immunodeficient mice. Investigators hope this mouse strain will prove susceptible to HIV infection and possibly to the development of AIDS (I. Weissman, Stanford University, personal communication, 1988). Whatever its final form, the development of a small animal model for AIDS is of utmost importance. A completely analogous animal model of HIV-induced human disease, especially in a small, plentiful, and well- understood animal such as the mouse, would greatly enhance vaccine and drug development. If efforts to develop a small animal model are carried out under carefully regulated, safe laboratory conditions, the committee strongly supports further work in this area. RESOURCES Facilities One of the major obstacles to further advances in research on HIV and AIDS remains the lack of adequate facilities- in particular, the shortage of laboratories that are equipped to handle HIV safely and of centers for housing and studying infected research animals. The paucity of such facilities is a problem in both the public and private sectors: it was estimated in the fall of 1987 that fewer than five pharmaceutical companies have containment facilities suitable for work with live HIV (IOM, 19874. Even some institutions that are generally in the forefront of research technology are underequipped for AIDS research (Weissman, 19881. Similar problems are apparent at the various primate centers located throughout the United States. Despite the emergence of significant data on simian retroviruses and AIDS, actual funding for primate research decreased during the several years prior to 1986 (Weissman, 1988~. Currently, five of the country's seven regional primate centers have

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150 CONFRONTING AIDS: UPDATE 1988 active AIDS research programs, and the federal government has provided additional funding to these facilities for their AIDS activities. Yet the extent to which the centers can use these funds to further increase their research output is limited by available laboratory space and resources. A case in point is the small number of biological containment facilities that are currently available in the United States for macaques inoculated with hazardous viruses (e.g., SIV) (Desrosiers and Letvin, 19874. In response to these needs, the federal government has authorized additional funds to be available beginning in 1988. About $24 million has been provided to the Division of Research Resources at the National Institutes of Health (NIH) to support infrastructure improvements at extramural research sites around the country and to purchase related equipment (letter from J. B. Wyngaarden, NIH, to T. Cooper, 19881. Of this amount, $2.8 million will be reserved for grants to the regional primate research centers for similar uses. Congress has also appropriated an additional $19 million to address NIH intramural facility needs. Plans are under way to use this money for renting office and off-campus laboratory space and for other improvements. Yet despite these encouraging funding developments, the two congres- sional allocations for renovations and infrastructure improvements are only a modest beginning to the facility upgrading necessary for productive AIDS research. More funds are needed to develop high-containment facilities at existing labs, and money for new laboratory construction is still a critical need. Such facilities are by no means inexpensive. Office space for personnel is another essential underpinning of a successful research effort. Additional funds are also needed to provide housing for experimental animals such as large primates. The committee recommends that the director of NIH, in consultation with research scientists from within and without the institutes, assess the need for and costs of new intramural and extramural facilities for AIDS research. This information should be forwarded to Congress for evaluation and subsequent action. Reagent Distribution Center To support AIDS research and provide the scientific community with the necessary biological materials for this work, NIAID has established the AIDS Research and Reference Reagent Program asking investigators to contribute appropriate materials that are needed for AIDS research. Reagents to be offered by the center include antisera, monoclonal antibodies, biological response modifiers, proteins derived from HIV and other related retroviruses, cellular proteins, bacterial and eukaryotic cell lines, HIV-related retroviruses, and other opportunistic infectious agents

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BIOLOGY OF HIV AND BIOMEDICAL RESEARCH NEEDS 151 associated with HIV infections. The committee supports this develop- ment. Currently, however, the success of the reagent center depends entirely on voluntary participation. The committee recommends that NIH stipulate that all investigators receiving NIH funds must make their AIDS-related reagents available to the distribution center, and thereby to all qualified investigators, after publication of their research. Industry is also urged to participate after the establishment of any patent rights. The committee plans to review the experience of the NIAID AIDS Research and Reference Reagent Program after it has been in operation for 1 year. Finally, the committee supports the development by NIH, perhaps through the reagent program, of an HIV/SIV research "starter kit" that would enable qualified new investigators to begin research more easily. The kit would contain sensitive cell lines, infectious virus, cloned DNAs, specific antibodies, and initial protocols for maintaining and infecting cell lines. FUNDING FOR RESEARCH Confronting AIDS recommended that federal appropriations for re- search on AIDS and HIV infection reach at least $1 billion annually by 1990. At the present rate of increase, it appears that this goal will be met. The 1988 NIH budget for AIDS research is $467.8 million, and the proposed 1989 budget is $587.6 million. The 1989 budget includes an additional $300 million (approximately) for AIDS research expenditures by CDC, HHS's Alcohol, Drug Abuse, and Mental Health Administra- tion, and FDA. The committee believes that when federal research expenditures for AIDS reach $1 billion annually, an assessment of the need for further increases should be made. It is important to ensure that other federal research programs are not penalized by a long-term disproportionate growth in the AIDS budget. REFERENCES Abrams, D., M. Gottlieb, M. Grieco, M. Speer, and S. Bernstein, eds. 1987. AmFAR Directory of Experimental Treatments for AIDS and ARC. Vol. 1. New York: Mary Ann Liebert, Inc. Aldovini, A., C. Debouck, M. B. Feinberg, M. Rosenberg, S. K. Arya, and F. Wong-Staal. 1986. Synthesis of the complete trans-activation gene product of human T-lymphotropic virus type III in Escherichia coli: Demonstration of immunogenicity in viva and expression in vitro. Proc. Natl. Acad. Sci. USA 83:6672-6676. Alizon, M., S. Wain-Hobson, L. Montagnier, and P. Sonigo. 1986. Genetic variability of the AIDS virus: Nucleotide sequence analysis of two isolates from African patients. Cell 46:63-74.

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