Below is the uncorrected machine-read text of this chapter, intended to provide our own search engines and external engines with highly rich, chapter-representative searchable text of each book. Because it is UNCORRECTED material, please consider the following text as a useful but insufficient proxy for the authoritative book pages.
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
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
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.
; 126 CD z in ~ _ CD o Z G ~ CL O: ~ ~ ~o 1~ <: Z LL ~ CD CD CO ~ O ~ > cn cn LLI z J O Cr O: 1 LL CD ~: ~: z ~: LL o G - LL CO CL cn z CC '1 1 LL CD llJ > ~: 11_O O C,' Z llJ O X~ CD ~ ~ > O C~ cn z - o rr CL llJ ~r o z O - O ~ O Z Z Y Z LL ~ cn Z LL llJ o ~o o LLI (~) Z LL · s~ e 'e c) · - ~ - o m o C) ._ 4 - CC S: 0 ~ ~ ~ Z .O oz 3 LL o~ ~D ._ Ct o o v . . C) s~ :- o a' o a, o~ a~ . cn .= h_ Ct ~ C~ r~ cc
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
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.
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
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,
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
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
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
i34 CONFRONTING AIDS: UPDATE 1988 can be more readily detected within the central nervous system (Straw et al., 1985; Harper et al., 1986; Koenig et al., 1986), where it is less accessible to immune clearance. The number and types of infected cells in HIV-positive persons remain incompletely defined. The persistence of HIV infection in viva, which is often imprecisely called a latent infection (because there is no evidence as yet that the HIV genome is integrated and not expressed), undoubtedly involves a complex interaction between host and virus. Lentiviruses, including HIV, are characterized by a low level of expression in viva (Haase, 1986), and the mechanisms by which HIV infection may be activated are not understood. Studies have shown that antibodies present in the sera of HIV-infected persons recognize and bind to the viral envelope glycoprotein. Because the glycoprotein mediates binding of the virus to host cells, it might be expected to provide a target for the neutralization of infectivity by the immune system. Yet although such neutralizing antibodies can be dem- onstrated in the sera of HIV-positive persons using in vitro assays, they do not appear to be protective in viva (Robert-Guroff et al., 1985; Weiss et al., 1985; Weber et al., 19871. A possible explanation for this lack of efficacy is the requirement, and the apparent failure, of the less avid antienvelope antibodies to overcome the extremely high-affinity interac- tion between the HIV gpl20 and the cellular CD4 receptor (Lasky et al., 1987~. The possible contribution of HIV's extensive genomic diversity to a resistance to immune clearance is also poorly defined. Such diversity has been observed in patients during the course of an infection (Hahn et al., 1986), but a process of immunologic selection for viral variation has yet to be demonstrated. Researchers have noted a number of correlations between the loss of host antibody response to specific HIV components and progression to AIDS (Laurence et al., 1987; Weber et al., 19871. It is not known, however, whether the change in serologic reactivity in these persons is the cause or consequence of their evolving immunodeficiency. The extent to which the host cellular immune system may recognize HIV infection and limit its spread is an area of great interest, although little is known about the role of cellular immunity in modulating the course of HIV infection. Some HIV-seropositive persons carry cytotoxic T lymphocytes that specifically recognize determinants on the viral envelope, gag, and polymerase proteins (Plate et al., 1987; Walker et al., 1987, 19881. These cytotoxic T lymphocytes may play a role in the host's containment of HIV infection, but they may also contribute to the inflammatory and sometimes deleterious reactions seen in HIV-infected persons (Plate et al., 19871. Researchers have also observed antibody- dependent, cell-mediated cytotoxic recognition and destruction of HIV- infected cells, but thus far they have been unable to determine their contribution to the protection of an infected person or, alternatively, to
BIOLOGY OF HIV AND BIOMEDICAL RESEARCH NEEDS 135 the progression to clinical disease (Lyerly et al., 1987~. Improved under- standing of the cell-mediated immune response to HIV infection becomes even more critical if HIV-1 is transmitted through infected cells rather than in free viral form. Research has produced little information as yet about the pathogenesis of HIV-induced disease. In addition, studies have yet to define the in vivo mechanism by which HIV destroys or inhibits cells of the immune system. Although it has been shown that HIV-induced cell fusion, or syncytia formation, exerts a dramatic cytopathic effect in vitro, its contribution to the immunologic consequences of HIV infection in viva has yet to be demonstrated (Lifson et al., 1986b; Sodroski et al., 1986a; Somasundaran and Robinson, 19871. Researchers have also suggested that autoimmune phenomena may contribute to the immune compromise that develops following HIV infection, but there are currently too few data to evaluate these suggestions (Stricker et al., 19871. Likewise, little is known about the role of host and viral factors in the development of HIV-1-induced necrologic disease, although its prevalence and severity have generated increased research attention over the past few years. Another important unresolved question is whether there are genetic or environmental cofactors that may modulate the tempo or course of disease development in HIV-infected persons. Studies have identified a variety of agents, both host immunoregulatory and other exogenous, infectious pathogens, that appear to enhance HIV-1 production in vitro, but claims of their in viva relevance find little support in the available epidemiological data (IOM/NAS, 1986~. Unfortunately, as the estimates of the percentage of HIV-infected persons who will progress to AIDS are revised upward, the likely importance of essential cofactors for disease progression--- and the possibilities they offer for ameliorative interven- tion~iminish. The Importance of Basic Research Understanding the processes and consequences of HIV infection described in the preceding pages is crucial to the development of therapies and vaccines against HIV. This understanding, in turn, is rooted in all basic research in the areas of cellular biology, virology, immunol- ogy, and genetics. As often happens in scientific investigation, serendi- pitous developments that initially are unrelated to HIV may unexpectedly contribute to progress in AIDS research. For example, many of the techniques that have been used to study HIV were developed during research in other fields. For this reason, increasing the amount of funds devoted to AIDS without a concomitant strengthening of all basic biomedical research is shortsighted. The committee recommends that
|36 CONFRONTING AIDS: UPDATE 1988 funding for basic research in all areas of biology should continue to grow rather than be curtailed in favor of AIDS-targeted research. DRUG DEVELOPMENT AND TESTING The difficulties and consequent challenges of developing drugs that alleviate the symptoms or slow the course of HIV infection lie in the complex virus-host interactions and the multiple pathologies to which immune deficiency gives rise. Yet despite these complexities, there is cause for cautious optimism. Current knowledge of the HIV proteins and their functions offers several potential targets for rational drug design. Several novel and worthwhile approaches to antiviral therapy also deserve consideration. In addition, our understanding of the immune system continues to grow, fueled in part by recent technical improve- ments in monoclonal antibody development, cell cloning, and growth factor isolation and characterization. Recent accomplishments in basic research on HIV and AIDS were described earlier in this chapter. Yet applying the results of these efforts to drug development requires coordinated action. For example, molecular virologists who describe a novel viral inhibitor active in cells in tissue culture should have ready access to pharmacologists or toxicologists who can begin to develop an agent that will function at tolerable levels in a human being. These kinds of partnerships are vital for rapid progress in drug development, but they do not occur naturally in most research or medical centers. The National Institute of Allergy and Infectious Diseases (NIAID), through its National Cooperative Drug Discovery Groups (NCDDG) program, has sought to encourage such cooperation among bench scien- tists and pharmaceutical companies with experience in drug development and evaluation. The members of each group share information, thus profiting from complementary expertise. In addition, the NCDDGs have formed an intergroup information network for the rapid exchange of news of progress in a particular strategy or agent. Individual scientists and drug companies have established similar partnerships. The search for therapeutic agents for HIV-infected persons must also encompass the screening of existing compounds. Scientists and industry leaders who are experienced in drug development predict that screening existing compounds rather than designing or attempting to discover new drugs to treat HIV infection and AIDS may prove more successful, at least in the immediate future (IOM, 1987~. A screening program is now under way at the National Cancer Institute involving the use of automated tests to determine the potential antiviral activity of compounds. When
BIOLOGY OF HIV AND BIOMEDICAL RESEARCH NEEDS 137 fully operational, this program will have the capacity to screen tens of thousands of compounds yearly. An extensive discussion of particular antiviral agents is beyond the scope of this report (interested readers are referred to the AmFAR Directory of Experimental Treatments for AIDS and ARC [Abrams et al., 198711. Yet several approaches to treatment that are currently being tested deserve mention. One is the combination of an antiviral agent such as zidovudine (i.e., AZT) with a biological response modifier such as granulocyte macrophage colony-stimulating factor. Another combination of therapies under evaluation is the alternate administration of zidovudine and a different reverse transcriptase inhibitor, ddC. The logic for this design is that zidovudine and ddC have similar intended sites of action but different side effects. Alternate dosing may keep adverse side effects to a minimum while maintaining high levels of reverse transcriptase inhibition. Other protocols of particular note involve tests of the treatment of asymptomatic HIV-infected people with zidovudine. In these trials, zidovudine is being evaluated for its ability to prolong the asymptomatic phase of HIV infection or reduce the severity of disease. Once a drug appears to be a candidate substance for the treatment of HIV infection or AIDS (or both), it begins the long journey toward licensure. The United States has the most rigorous review process for new drug approval of any country in the world. The development of any new drug is regulated by FDA and begins with preclinical testing of its activity and safety, a stage that generally requires 1 to 2 years. In the next step of the process, the drug sponsor files an investigational new drug (IND) application that includes the results of all animal testing and how the drug is made. Once the application is approved, promising drugs begin clinical (human) testing, which consists of three phases of activity. Phase I measures safety (i.e., toxicity) and establishes pharmacological profiles of (in this case) the antiviral agent. Tests during this phase involve a small number of healthy people (not patients) and are completed usually in less than 1 year. Phase II trials, which generally last 1 to 2 years, assess the drug's effectiveness using controlled studies with 200 to 300 volunteer patients. Phase III tests involve large numbers of volunteer patients in clinics and hospitals. These latter trials are necessary to confirm early efficacy studies and identify low-incidence adverse reactions; they gen- erally last about 3 years. After the completion of phase III trials, the drug sponsor files a new drug application (NDA). After FDA approves the application and autho- rizes the marketing of the drug, the drug sponsor must continue to file periodic reports of adverse reactions. For some drugs, FDA also requires postmarketing monitoring or additional studies to evaluate long-term effects (phase IV). Thus, for the average pharmaceutical, the process of
|38 CONFRONTING AIDS: UPDATE 1988 preclinical development, clinical testing, and new drug application takes approximately 7 to 10 years. The FDA drug approval process has been constantly evolving since its establishment. Although the process has been criticized for being slow and cumbersome, it has also been credited, rightly, with protecting the American public from the harmful effects of inadequately tested drugs. Yet the urgent need for antiviral agents to treat increasing numbers of AIDS patients has placed pressure on the system to change. In the committee's view, FDA has responded with commitment and energy. The agency has agreed to complete its reviews of new drug applications in 6 months or less rather than the 2 to 3 years normally required. Review of IND applications (prior to clinical trials) has also been speeded up. In addition, FDA has proposed new procedures to facilitate patients' access to promising new drugs as early as possible in the drug development process (Young, 1987~. Drug sponsors may now apply for a special treatment status for their investigational new drug when the following conditions are met: · the drug will treat an immediately life-threatening or otherwise . . serious c Disease; · no satisfactory alternative drug or therapy exists to treat the disease; · the drug is already under investigation in a controlled clinical trial under a standard IND process; and · the sponsor of the controlled trial is pursuing marketing approval of the investigational drug with due diligence. Treatment IND status allows the drug sponsor to sell the drug at cost (no profit making is allowed) for treatment purposes. Trimetrexate, a drug to treat Pneumocystis carinii pneumonia, was granted treatment IND status in February 1988. It was the first AIDS-related drug to be approved under the new regulations. Zidovudine (AZT), which was approved in September 1986 under a prototype treatment IND mechanism, received the fastest evaluation that has ever occurred within FDA. It remains the only drug proven to prolong the life of some AIDS patients. Yet the diversion of FDA personnel from other areas that was necessary to approve zidovudine resulted in a backlog of applications in FDA's Division of Anti-Infective Drug Prod- ucts. As the number of applications for treatment IND status grows, these personnel problems may become more severe. At present, FDA is not a "bottleneck" in the availability of new drugs to treat HIV infection and AIDS. The paucity of new drugs is related more to shortcomings in the science of antiviral agents than to the drug approval process. However, as more promising new drugs are discovered or designed, FDA, without additional resources, could become an imped-
BIOLOGY OF HIV AND BIOMEDICAL RESEARCH NEEDS 139 iment to speedy availability. The committee believes that FDA resources for new drug approval should be commensurate with the task. The need to borrow personnel from other parts of the agency should be relieved; the need for space, which appears to be particularly acute, should also be addressed. The committee applauds FDA's ingenuity in instituting new regulations for investigational new drugs. Nevertheless, it also believes a note of caution is warranted. The availability of treatment INDs could interfere with the ability to execute conclusive clinical trials. For example, the pharmaceutical industry has expressed concern that the use of the treatment IND mechanism may make it more difficult to enroll sufficient numbers of patients in clinical trials: people who know that the drug under investigation could be offered to them as treatment in the near future may be reluctant to participate in tests (Cooper, 1988~. There is also concern about potential issues of legal liability on the part of drug manufacturers and individual physicians: for instance, when patients who are receiving investigational new drugs under the treatment IND mechanism develop adverse reactions or when patients fail to receive an investigational drug because their physicians are unaware of its availability. In light of these concerns, the committee recommends that an outside evaluation of the treatment IND process be conducted after enough time has elapsed to determine its possible unanticipated consequences for any new drugs. HIV infection and AIDS have generated a pressing need to develop and test experimental drugs for treatment and to make effective drugs widely available as soon as possible. The committee recognizes the frustration, fear, and anger of people with HIV infection, who may feel a lack of urgency in the drug development process. However, the committee believes that once drugs are through phase I testing for toxicity, carefully controlled trials are still the fastest, most efficient way to determine what treatments work. In asymptomatic or mildly ill patients, trials should be placebo controlled (until an effective therapy is discovered for these patients); in patients with severe symptoms of HIV infection or AIDS, experimental drugs should be compared to zidovudine (AZT). Conducting well-designed trials from the beginning will benefit more patients, sooner, than any other approach. Poorly designed trials, or administering drugs without controls and "observing" the course of disease, risk being inconclusive or drawing incorrect conclusions. The wide distribution of untested drugs makes it impossible to determine whether or not they are effective, especially if the benefits are real but small. The end result of these approaches could include the continued prescribing of useless or harmful therapies. More than other diseases, AIDS has brought forth calls to include large numbers of people in clinical trials, both for humanitarian reasons (access
|40 CONFRONTING AIDS: UPDATE 1988 to potentially effective therapy as soon as possible) and to offer HIV- infected persons a chance to contribute to the solution of the problem. Yet the distinction between making proven, effective treatments widely available and enrolling as many people as possible in clinical trials is an important one. In fact, the best-designed trial enrolls the smallest number of people needed to show a significant difference between the experimen- tal and the control drugs, thereby minimizing the possibility of doing harm. The committee believes that, following scientifically sound guidelines, wider access to clinical trials can be gained by broadening their geographic base, by extending trials to previously untapped populations including women, IV drug abusers, and pediatric patients, and by testing all compounds that appear to have a possibility of ellectiveness. The budget for pediatric trials in particular must be increased. It is the responsibility of those conducting trials to communicate with the public about their availability and to encourage wide participation. In particular, NIAIO, through its AIDS clinical trial units (ACTUs), has opportunities for such communication and encouragement, in part be- cause of the role of ACTUs in the clinical evaluation of new drugs. The ACTUs, a component of the newly organized AIDS Clinical Trials Cooperative Group, are located at 35 sites around the country. Formerly called AIDS treatment evaluation units or clinical study groups, the units enlist clinical investigators and patients for large-scale, standardized collaborative clinical trials. The ACTU program is designed to evaluate promising drugs efficiently by coordinating protocol design, data collec- tion and analysis, and subject recruitment. An important goal of the program is to speed the transfer of therapies, once they are proven effective, to physicians and their patients. Research scientists or pharma- ceutical companies may submit drugs for evaluation at ACTU sites. The current test roster of the ACTUs includes agents to treat HIV infection, immune deficiency, opportunistic infection, and combinations of these conditions. In addition to the obvious advantages of drug trial coordina- tion and standardization, the ACTUs have a special utility in their capacity to test combined therapies involving compounds from different sources. The pharmaceutical industry also conducts clinical trials through its system of privately sponsored testing. The committee believes that, to the greatest extent possible, trials should take place within well-established sites for drug testing. Community-based trials need to be carefully supervised to yield useful results. Phase IV postmarketing surveillance for already approved drugs may be the most appropriate role for community-based studies. Finally, the committee abhors the exploitation of people with HIV infection and AIDS by those promoting and selling "effective" therapies that are in fact unproven.
BIOLOGY OF HIV AND BIOMEDICAL RESEARCH NEEDS 141 VACCINE DEVELOPMENT AND TESTING Although research findings afford hope for successful antiviral treat- ments, the prevention of HIV infection by vaccination continues to pose fundamental difficulties. Researchers have pursued sound experimental approaches, but so far they have been unable to prevent HIV infection in primates or humans. Nonetheless, innovative research continues and may produce more promising results in the future. The appeal of a vaccine to prevent HIV infection is straightforward. Vaccination prevents the infection of a healthy person by priming the immune system to respond rapidly to inactivate an infectious organism. A successful vaccine is a nonpathogenic derivative of the infectious orga- nism that induces a protective immune response. When a person is exposed again to the pathogen after vaccination, the protective response is triggered and amplified to prevent infection. So far, vaccination has only succeeded when the pathogen itself induces some protective immu- nity during natural infection. Today's vaccine design exploits the many technical advances in biology that have occurred in recent years. Earlier vaccines were prepared by killing or weakening the infectious organism or by extracting some immunogenic component from it. Modern techniques of genetic engineer- ing now allow researchers to identify a likely immunogenic protein and isolate the gene that encodes it. The protein can then be produced separately for use as a vaccine. This approach eliminates the possibility that the vaccine itself will be infectious, although other potential toxicities remain. Proteins that are often selected for this purpose are outer surface components of the organism, such as cell wall constituents, or viral envelope proteins. With this general strategy in mind, researchers have looked to the HIV envelope glycoprotein gpl20 and its precursor gpl60 as promising vaccine candidates. For one thing, these envelope glycoproteins induce antibody production during the course of natural HIV infection; in addition, humans generate cytotoxic T-cell responses directed toward gpl20 (Shearer, 19871. What may be even more important is that not only does gpl20 appear on the outer surface of HIV, but it is through gpl20 binding to the cell surface protein CD4 that HIV attaches to and enters T lymphocytes. The ability to bind the CD4 molecule is a characteristic that is retained across serologically distinct strains of HIV, which suggests that some HIV gpl20 epitopes will also be conserved among all HIV strains. Interference with viral entry seems a possible means of antibody- mediated prevention of infection. When tested in tissue culture, antibod- ies that bind either CD4 or gpl20 molecules prevent the infection of T cells (McDougal, 19871.
142 CONFRONTING AIDS: UPDATE 1988 Accordingly, investigators have cloned the gene that codes for gpl60 (the gpl20 precursor), inserted it into a baculovirus vector, and used the construct to produce gpl60 in insect cells. The protein is then prepared as a vaccine that, not surprisingly, induces antibody synthesis in mice and chimpanzees (Francis, 1987~. Neutralizing antibodies (i.e., those that block HIV infection in tissue culture) are among the antibodies detected. However, it appears that the production of neutralizing antibodies may be irrelevant to protection from HIV infection in viva. When immunized chimpanzees were infected with HIV, they continued to produce antibod- ies but became persistently infected. Currently, the baculovirus-derived gpl60 is being tested in humans for safety and immunogenicity. FDA has also approved a different construct of gpl60 for clinical evaluation; in this vaccine the gene for gpl60 is inserted into a vaccinia virus genome, and the vaccinia expresses gpl60 on its outer surface. So far, this vaccine does not appear to alter the course of HIV infection in chimpanzees when a challenge is presented. Another test of vaccination in chimpanzees has used immunization with purified gpl20, which induced neutralizing antibodies, followed by infec- tion with a virus of the same strain in a low dose but of sufficient strength to infect each of the three animals being tested. Viremia developed in all of the animals. Thus, neutralizing antibodies were not able to block infection by even a small quantity of infective HIV (Nara, 19881. Other experiments are consistent with these negative findings. For example, in a study using passive immunization (the transfer of antibod- ies) to test the general principle that the human response to HIV infection can be protective, researchers injected human sera containing neutraliz- ing antibodies into chimpanzees that were then inoculated with HIV. The chimpanzees became infected with HIV even in the presence of human neutralizing antibodies (Fultz, 1987b). Another result that calls into question the ability of the body's immune responses to prevent HIV infection comes from a superinfection experi- ment with chimpanzees. The procedure tested whether immune re- sponses or other interference phenomena established by infection with one strain of HIV inhibit infection by a second, serologically distinct strain. Superinfection did occur, indicating that no protective mechanism was fully effective (Fultz, 1987a). Finally, researchers have seen no correlation between the level of neutralizing antibodies and the progress of natural infection in humans. Total HIV antibody levels drop in AIDS patients in very late stages of the disease, a drop that may be the result of antigen excess. Thus, it appears that HIV infection proceeds in the presence of neutralizing antibodies and cytotoxic T cells that are capable of killing HIV-infected cells in vitro. Because naturally occurring immune responses to HIV are insufficient to
BIOLOGY OF HIV AND BIOMEDICAL RESEARCH NEEDS 143 prevent infection, it is apparent that HIV has evolved a way to coexist with the immune response of its host. Several mechanisms may explain why an HIV-infected person is unable to generate sustained protective immunity (Seligmann et al., 19871. First, HIV may escape what would ordinarily be an adequate immune response by antigenic drift (i.e., the viral structures that are recognized by the immune system may change into an unrecognizable form). Second, several serological types of HIV may infect one person, and, as the immune system attacks one type, another type may take its place. Third, although cell-mediated responses of the immune system may be respon- sible for lysing those cells that are actively producing new viral particles, if there are cells in which HIV exists in a latent form, they may escape immune detection. The frequent activation of these latently infected cells could result in persistent viral replication and infection. Fourth, as in the case of measles, HIV-infected cells may avoid immune detection by not displaying their HIV-encoded antigens. Fifth, some of the body's immune responses to HIV mask viral antigens so that protective mechanisms cannot be stimulated. Finally, the production of HIV antibodies may actually make infection more likely if a person already has circulating HIV antibodies when he or she becomes infected. The HIV particles that are bound in complexes to the HIV antibodies may be phagocytized (engulfed) by macrophages; if the particles remain infectious, they may establish infection in the macrophages when they enter these cells (Halstead, 1987~. Despite these obstacles, researchers continue to search for protective vaccines. Further analysis of earlier results has suggested new ap- proaches. Particular regions of gpl20, such as the conserved residues that contact the CD4 receptor, are being cloned and displayed in a highly immunogenic form. Normally, these residues are buried in the native gpl20 molecule and may not be immunogenic, but it is possible that antibodies against them, once produced, could block the initial gpl20 binding to CD4 and thus prevent infection from being established (Mo- reine, 1987~. Other research efforts have included the use of an antibody that binds gpl20 as an antigen to induce an anti-idiotypic response that in turn can form the basis of an idiotype-anti-idiotype network in which anti-gpl20 is endogenously induced (Kennedy, 19871. This approach has a potential side effect, however, in that a broadly specific anti-gpl20 antibody may mimic CD4 structure and thus induce anti-CD4 responses. Different kinds of antigensfor instance, gpl20 and viral core proteinscan also be combined in a vaccine that may be more effective than one composed of either alone. In addition, these antigens can be mixed with a highly immunogenic carrier, such as tetanus toxoid, for vaccine delivery.
144 CONFRONTING AIDS: UPDATE 1988 Another approach that is currently being tested involves a vaccine composed of whole killed HIV (Salk, 19871. This sort of vaccine may be useful for primary prevention, but it differs from the protective vaccines discussed earlier in that it has another intended use. Its "immunothera- peutic" effect would be to boost the responses already induced by natural HIV infection in seropositive patients rather than to protect individuals against infection. In this case, a beneficial effect may be the alleviation of symptoms or prolongation of life in HIV-infected persons. All of these approaches require long-term, large-scale testing and evaluation before candidate vaccines are available to the public. The process by which a vaccine is tested and approved for general use is similar to that for drugs. The sponsor or developer of the vaccine generally tests it first in animal models and, if the vaccine appears promising, applies to FDA for investigational new drug (IND) status to begin clinical testing. Once the application is approved, the sponsor begins three phases of human trials. Phase I tests the vaccine in a small number of persons for safety and immunogenicity at various dose levels and by various routes of administration. Phase II involves administering the vaccine to larger numbers of people to obtain further data. These two phases often overlap. Phase III consists of large-scale, controlled field trials with a sufficiently large number of subjects (at sufficiently high risk of infection) to determine whether the vaccine protects people (at a statistically significant level) against disease. It is this third phase of testing that confirms or disproves the efficacy of the vaccine in preventing infection. FDA standard practice has generally been that a vaccine must show protective efficacy in an accepted animal model before tests can progress to human volunteers. However, given the urgency surrounding the potentially disastrous effects of the AIDS epidemic, FDA has approved (to date) the precedent-setting initiation of human trials for two vaccine candidates in the absence of proof of protective efficacy in animals. It is unclear whether any future vaccine candidate will be required to demon- strate protection. There has been substantial controversy about the wisdom of proceed- ing to human trials for vaccines in the absence of evidence for efficacy in animal tests. The move's supporters offer several rationales for the departure from standard procedure. One, of course, is the pressing need for a protective vaccine against HIV. Another is the knowledge to be gained about the relationship between human and chimpanzee responses to HIV antigens, which could prove applicable to future HIV vaccine trials. Furthermore, testing vaccines composed of viral envelope antigens for safety in humans will provide information, in general, about both humoral and cell-mediated human immune responses to HIV antigens.
BIOLOGY OF HIV AND BIOMEDICAL RESEARCH NEEDS 145 Another rationale offered for human trials is that with further information about dosages, immunogenicity, and toxicity, some of the steps of clinical evaluation might be accelerated. Yet there are potential risks in a phase I trial that cannot be ignored. Some HIV proteins themselves have immunoregulatory activity, and a vaccine derived from such a protein may interfere with the normal operation of the immune system. In addition, it is unclear whether a vaccine might increase the severity of a subsequent, naturally acquired infection. As mentioned earlier in this chapter, antibody-HIV complexes may enter macrophages, but it is not known whether HIV retains its infectivity at this point. If it remains infectious, vaccination could facilitate the initial infection of macrophages or promote the later spread of the virus throughout the body. Furthermore, nonneutralizing antibod- ies induced by a vaccine that are capable of binding to key sites on HIV without inhibiting it might interfere with the subsequent recognition of the virus by other components of the immune system. Finally, with little hope (considering their negative protective results in animals) that the vaccines now being tested for safety will prove effective, public expectations may be raised unrealistically. The committee believes that human trials of HIV vaccine candidates should proceed only when (1) protection against infection has been demon- strated in chimpanzees (HIV), in macaques (SIV), or in another suitable animal model, or (2) the vaccine candidate rests on fundamental new knowledge of the relevant human response that cannot be adequately modeled in animals. The committee also believes that planning should begin now for large-scale human efficacy trials of as yet undeveloped vaccines. Such trials are complex to design, and their results are difficult to evaluate. The trials must enroll sufficiently large numbers of subjects at sufficiently high risk of infection that any decrease in the number of infected persons attributable to the experi- mental vaccine will be statistically significant. For this reason, the sites for large-scale vaccine efficacy trials will most likely include African and other developing countries. A process should be agreed on for joint decision making among the countries involved. The World Health Organization (WHO) is developing guidelines for the conduct of these trials. In general, the same criteria used in the United States for human vaccine trials should be applied to trials conducted in any other country. Ethical considerations also dictate that those who receive the vaccine must be counseled about behavior changes that diminish the chance for HIV infection. This counseling will have to be taken into consideration in calculating the required number of trial participants. The question of legal liability may be even more pressing for vaccines than for drugs. Typically, vaccine manufacturers are protected by insur-
i46 CONFRONTING AIDS: UPDATE 1988 ance to cover liability-associated damages. AIDS is so visible and so controversial, however, that vaccine manufacturers are concerned that their standard liability coverage may be inadequate for an AIDS vaccine. At the same time, clinical investigators and manufacturers are now also questioning whether liability insurance may not be needed even for experimental vaccines, which a subject has traditionally taken at his or her own risk after being fully informed of the potential problems. Fears of liability and subsequent damages may impede manufacturers from further developmental work on any vaccine. To encourage companies to continue the production of vaccines for other diseases, Congress recently established a childhood vaccine com- pensation schedule. A surcharge to vaccine costs will go toward a fund to pay medical costs and some limited damages for people injured by vaccines. Standards will be set in the near future for determining injury and compensation. This plan could serve as a model for a federal program to protect the rights of recipients of an AIDS vaccine while sheltering manufacturers from impossibly high legal damages. The pessimism expressed in Confronting AIDS about the likelihood of a licensed vaccine becoming available within the next 5 to 10 years has not been mitigated by the accomplishments of the past 2 years. The vaccine development effort has been characterized by a series of animal experi- ments that have yielded universally negative results. Future animal experi- ments should use cloned viruses and standardized neutralization tests. ROUNDTABLE ON DRUGS AND VACCINES The institute of Medicine Conference on Promoting Drug Development Against AIDS and HIV Infection (August 31-September 1, 1987) and the Conference on the Development of Vaccines Against HIV Infection and AIDS (December 14-15, 1987) brought together biomedical scientists, clinicians, drug manufacturers, and policymakers to consider ways in which to speed the availability of effective therapy for HIV infection. The participants found the conferences so valuable in fostering a productive exchange of views and information that they recommended that IOM consider convening more conferences and invitational workshops on these issues. In response, IOM plans to establish the Roundtable on the Development of Drugs and Vaccines Against AIDS. The roundtable will have approximately 15 members representing government, the pharma- ceutical industry, academia, the legal community, clinical medicine, and the public. Its goal is to facilitate progress in drug and vaccine develop- ment by providing a neutral forum in which developmental barriers can be addressed, problems can be aired, and opportunities can be identified and pursued. The committee endorses the establishment of the Roundtable on
BIOLOGY OF HIV AND BIOMEDICAL RESEARCH NEEDS 147 the Development of Drugs and Vaccines Against AIDS and encourages active participation by all sectors. ANIMAL MODELS OF AIDS Research on AIDS and HIV infection requires the development of model systems in which an animal infected with HIV develops the same symptoms and exhibits the same course of disease progression found in human patients with HIV infection. Such models not only enable inves- tigators to better understand HIV infection and AIDS but also provide them with a tool for initially testing antiviral agents and vaccines for biological activity and safety. Indeed, one of the key problems currently facing AIDS investigators is the lack of a suitable animal model. Current animal models are based on animal viruses, which can be divided into four categories of increasing relevance to human HIV infection: (1) retroviruses such as feline leukemia virus and type D retrovirus that have no obvious, close relationship to HIV but that can induce chronic disease in cats and other animals with manifestations that include immunologic abnormalities; (2) lentiviruses such as maedi-visna virus, caprine arthritis-encephalitis virus, and equine infectious anemia virus, all of which induce disease in hoofed animals; (3) HIV-related viruses of Old World primates (i.e., simian immunodeficiency virus, or SIV); and (4) HIV infection of chimpanzees (Desrosiers and Letvin, 19871. Some of the HIVs that infect humans can also infect some primates. Recent studies, for example, indicate that baboons can be infected with a human AIDS virus isolate, although the infection is transient (Letvin et al., 19871. In addition, both gibbons (Desrosiers, 1987) and rhesus macaques (Gardner and Luciw, 1987) can become persistently infected with HIV-1 and HIV-2, respectively. None of the animals in either of these studies developed a disease suggestive of AIDS, however, nor did any infected animal die. Chimpanzees can also become persistently infected with HIV, although to date they show minimal clinical or laboratory evidence of disease. Studies with chimpanzees have also demonstrated the transmission of HIV from an infected chimpanzee to an uninfected animal. To try to induce disease in HIV-infected chimpanzees, researchers use various approaches including superinfecting the animal with the same or a different virus or suppressing its immune system (Fultz, 1987a,b). An- other approach focuses on isolating a strain of HIV that may become modified when it is deliberately passed from one infected chimpanzee to another. Investigators plan to test whether using such a viral preparation to infect smaller nonhuman primates and rodents can lead to a suitable animal model.
|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
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
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
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.
152 CONFRONTING AIDS: UPDATE 1988 Allan, J. S., J. E. Coligan, T.-H. Lee, M. F. McLane, P. J. Kanki, J. E. Groopman, and M. Essex. 1985. A new HTLV-III/LAV encoded antigen detected by antibodies from AIDS patients. Science 230:810-813. Arya, S. K., C. Guo, S. F. Josephs, and F. Wong-Staal. 1985. Trans-activator gene of human T-lymphotropic virus type III (HTLV-III). Science 229:69-73. Chakrabarti, L., M. Guyader, M. Alizon, M. D. Daniel, R. C. Desrosiers, P. Tiollais, and P. Sonigo. 1987. Sequence of simian immunodeficiency virus from macaques and its relationship to other human and simian retroviruses. Nature 328:543-547. Clavel, F., D. Guetard, F. Brun-Vezinet, S. Chamaret, M.-A. Rey, M.-O. Santos-Ferreira, A. G. Laurent, C. Dauguet, C. Katlama, C. Ronzioux, D. Klatzmann, J.-L. Champali- maud, and L. Montagnier. 1986a. Isolation of a new human retrovirus from West African patients with AIDS. Science 233:343-346. Clavel, F., M. Guyader, D. Guetard, M. Salle, L. Montagnier, and M. Alizon. 1986b. Molecular cloning and polymorphism of the human immune deficiency virus type 2. Nature 324:691-695. Clavel, F., K. Mansinho, S. Chamaret, D. Guetard, V. Favier, J. Nina, M.-O. Santos- Ferreira, J.-L. Champalimaud, and L. Montagnier. 1987. Human immunodeficiency virus type 2 infection associated with AIDS in West Africa. N. Engl. J. Med. 316:1180-1185. Coffin, J. M. 1986. Genetic variation in AIDS viruses. Background paper. Committee on a National Strategy for AIDS, Washington, D.C. Cooper, T. M. 1988. Treatment IND: Making investigational new drugs available to the desperately ill. Address at the American Medical Association-Food and Drug Adminis- tration Conference, Washington, D.C., February 17. Cullen, B. R. 1986. Trans-activation of human immunodeficiency virus occurs via a bimodel mechanism. Cell 46:973-982. Dalgleish, A. G., P. C. L. Beverley, P. R. Clapham, D. H. Cra~vford, M. F. Greaves, and R. A. Weiss. 1984. The CD4 (T4) antigen is an essential component of the receptor for the AIDS retrovirus. Nature 312:763-767. Daniel, M. D., N. L. Letvin, N. W. King, M. Kannagi, P. K. Sehgal, R. D. Hunt, P. J. Kanki, M. Essex, and R. C. Desrosiers. 1985. Isolation of T-cell tropic HTLV-III-like retrovirusfrommacaques.Science228:1201-1204. Dayton, A. I., J. G. Sodroski, C. A. Rosen, W. C. Goh, and W. A. Haseltine. 1986. The trans-activator gene of the human T cell lymphotropic virus type III is required for replication. Cell 44:941-947. Debouck, C., J. G. Gorniak, J. E. Strickler, T. D. Meek, B. W. Metcalf, and M. Rosenberg. 1988. Human immunodeficiency virus protease expressed in Escherichia cold exhibits autoprocessing and specific maturation of the gag precursor. Proc. Natl. Acad. Sci. USA 84:8903-8906. Deen, K. C., J. S. McDougal, R. Inacker, G. Folena-Wasserman, J. Arthos, J. Rosenberg, P. J. Maddon, R. Axel, and R. W. Sweet. 1988. A soluble form of CD4 (T4) protein inhibits AIDS virus infection. Nature 331:82-84. Desrosiers, R. C. 1987. Simian and feline models of AIDS. Paper presented at the Institute of Medicine Conference on the Development of Vaccines Against HIV Infection and AIDS, Washington, D.C., December 14-15. Desrosiers, R. C., and N. L. Letvin. 1987. Animal models for acquired immunodeficiency syndrome. Rev. Infect. Dis. 9:438-446. Feinberg, M. B., R. F. Jarrett, A. Aldovini, R. C. Gallo, and F. Wong-Staal. 1986. HTLV-III expression and production involve complex regulation at the levels of splicing and translation of viral RNA. Cell 46:807-817. Fischl, M. A., D. D. Richman, M. H. Grieco, M. S. Gottlieb, P. A. Volberding, D. L. Laskin, J. M. Leedom, J. E. Groopman, D. Mildvan, R. T. Schooley, G. G. Jackson,
BIOLOGY OF HIV AND BIOMEDICAL RESEARCH NEEDS 153 D. T. Durack, D. King, and the AZT Collaborative Working Group. 1987. The efficacy of azidothymidine (AZT) in the treatment of patients with AIDS and AIDS-related complex: A double-blind, placebo-controlled trial. N. Engl. J. Med. 317:185-191. Fisher, A. G., M. B. Feinberg, S. F. Josephs, M. E. Harper, L. M. Marselle, G. Reyes, M. A. Gonda, A. Aldovini, C. Debouk, R. C. Gallo, and F. Wong-Staal. 1986a. The trans-activator gene of HTLV-III is essential for virus replication. Nature 320:367-371. Fisher, A. G., L. Ratner, H. Mitsuya, L. M. Marselle, M. E. Harper, S. Broder, R. C. Gallo, and F. Wong-Staal. 1986b. Infectious mutants of HTLV-III with changes in the 3' region and markedly reduced cytopathic effects. Science 233:655-659. Fisher, A. G., B. Ensoli, L. Ivanoff, M. Chamberlain, S. Petteway, L. Ratner, R. C. Gallo, and F. Wong-Staal. 1987. The sor gene of HIV-1 is required for efficient virus transmission in vitro. Science 237:888-893. Fisher, R. A., J. M. Bertonis, W. Meier, V. A. Johnson, D. S. Costopoulos, T. Liu, R. Tizard, B. D. Walker, M. S. Hirsch, R. T. Schooley, and R. A. Flavell. 1988. HIV infection is blocked in vitro by recombinant soluble CD4. Nature 331:76-78. Folks, T. M., J. Justement, A. Kinter, C. A. Dinarello, and A. S. Fauci. 1987. Cytokine- induced expression of HIV-1 in a chronically infected promonocyte cell line. Science 238:800-802. Franchini, G., C. Gurgo, H.-G. Guo, R. C. Gallo, E. Collalti, K. A. Fargnoli, L. F. Hall, F. Wong-Staal, and M. S. Reitz, Jr. 1987. Sequence of simian immunodeficiency virus and its relationship to the human immunodeficiency viruses. Nature 328:539-543. Francis, D. 1987. Current U.S. attempts at vaccine development. Paper presented at the Institute of Medicine Conference on the Development of Vaccines Against HIV Infection and AIDS, Washington, D.C., December 14-15. Prankel, A. D., D. S. Bredt, and C. O. Pabo. 1988. Tat protein from human immunodefi- ciency virus forms a metal-linked dimer. Science 240:70-73. Franza, B. R., S. F. Josephs, M. Z. Gilman, W. Ryan, and B. Clarkson. 1987. Character- ization of cellular proteins recognizing the HIV enhancer using a microscale DNA-affinity precipitation assay. Nature 300:391-395. Fultz, P. 1987a. Conspectus on primate models for AIDS. Correspondent paper. AIDS Activities Oversight Committee, Washington, D.C. Fultz, P. 1987b. Responses of chimpanzees to HIV immunization. Paper presented at the Institute of Medicine Conference on the Development of Vaccines Against HIV Infection and AIDS, Washington, D.C., December 14-15. Garcia, J. A., F. K. Wu, R. Mitsuyasu, and R. B. Gaynor. 1987. Interactions of cellular proteins involved in the transcriptional regulation of the human immunodeficiency virus. EMBO J. 6:3761-3770. Gardner, M. B., and P. Luciw. 1987. Nonhuman primate models for AIDS. Correspondent paper. AIDS Activities Oversight Committee, Washington, D.C. Gartner, S., P. Markovits, D. M. Markovitz, M. H. Kaplan, R. C. Gallo, and M. Popovic. 1986a. The role of mononuclear phagocytes in HTLV-III/LAV infection. Science 233:215-219. Gartner, S., P. Markovits, D. M. Markovitz, R. F. Betts, and M. Popovic. 1986b. Virus isolation from and identification of HTLV-III/LAV-producing cells in brain tissue from a patient with AIDS. J. Am. Med. Assoc. 256:2365-2371. Gonda, M. A., F. Wong-Staal, R. C. Gallo, J. E. Clements, O. Narayan, and R. V. Gilden. 1985. Sequence homology and morphologic similarity of HTLV-III and visna virus, a pathogenic lentivirus. Science 227:173-177. Gonda, M. A., M. J. Braun, S. G. Carter, T. A. Kost, J. W. Bess, L. O. Arthur, and M. J. Van Der Maaten. 1987. Characterization and molecular cloning of a bovine lentivirus related to human immunodeficiency virus. Nature 330:388-391.
154 CONFRONTING AIDS: UPDATE 1988 Gruters, R. A., J. J. Reeves, M. Tersmette, R. E. Y. de Goede, A. Tulp, H. G. Huisman, F. Miedema, and H. L. Ploegh. 1987. Interference with HIV-induced syncytium forma- tion and viral infectivity by inhibitors of trimming glucosidene. Nature 330:74-77. Guyader, M., M. Emerman, P. Sonigo, F. Clavel, L. Montagnier, and M. Alizon. 1987. Genome organization and transactivation of the human immunodeficiency virus type 2. Nature 326:662-669. Haase, A. T. 1986. Pathogenesis of lentivirus infections. Nature 322: 130-136. Hahn, B. H., G. M. Shaw, M. E. Taylor, R. R. Redfield, P. D. Markham, S. Z. Salahuddin, F. Wong-Staal, R. C. Gallo, E. S. Parks, and W. P. Parks. 1986. Genetic variation in HTLV-III/LAV over time in patients with AIDS or at risk for AIDS. Science 232: 1548-1553. Halstead, S. 1987. Immuno-enhancement of disease. Paper presented at the Institute of Medicine Conference on the Development of Vaccines Against HIV Infection and AIDS, Washington, D.C., December 14-15. Harper, M. E., L. M. Marselle, R. C. Gallo, and F. Wong-Staal. 1986. Detection of lymphocytes expressing human T-lymphotropic virus type III in lymph nodes and peripheral blood from infected individuals by in situ hybridization. Proc. Natl. Acad. Sci. USA 83:772-776. Hauber, J., and B. R. Cullen. 1988. Mutational analysis of the trans-activation-responsive region of the human immunodeficiency virus type I long terminal repeat. J. Virol. 62:673-679. Hauber, J., A. Perkins, E. P. Heimer, and B. R. Cullen. 1987. Trans-activation of human immunodeficiency virus gene expression is mediated by nuclear events. Proc. Natl. Acad. Sci. USA 84:6364-6368. Hirsch, V., N. Riedel, and J. I. Mullins. 1987. The genome organization of STLV-3 is similar to that of the AIDS virus except for a truncated transmembrane protein. Cell 49:307-319. Ho, D. D., T. R. Rota, R. T. Schooley, J. C. Kaplan, J. D. Allan, J. E. Groopman, L. Resnick, D. Felsenstein, C. A. Andrews, and M. S. Hirsch. 1985. Isolation of HTLV-III from cerebrospinal fluid and neural tissues of patients with neurologic syndromes related to the acquired immunodeficiency syndrome. N. Engl. J. Med. 313:1493-1497. Ho, D. D., T. R. Rota, and M. S. Hirsch. 1986. Infection of monocyte/macrophages by human T lymphotropic virus type III. J. Clin. Invest. 77:1712-1715. Hoxie, J. A., B. S. Haggarty, J. L. Rackowski, N. Pillsbury, and J. A. Levy. 1985. Persistent noncytopathic infection of normal human T lymphocytes with AIDS-associated retrovirus. Science 229:1400-1402. IOM (Institute of Medicine). 1987. An Agenda for AIDS Drug Development. Report of the Conference on Promoting Drug Development Against AIDS and HIV Infection. Wash- ington, D.C.: National Academy Press. IOM/NAS (Institute of Medicine/National Academy of Sciences). 1986. Future research needs. Pp. 177-259 in Confronting AIDS: Directions for Public Health, Health Care, and Research. Washington, D.C.: National Academy Press. Jacks, T., M. D. Power, F. R. Masiarz, P. A. Luciw, P. J. Barr, and H. E. Varmus. 1988. Characterization of ribosomal frameshifting in HIV-1 gag-pol expression. Nature 331 :280-283. Jones, K. A., J. T. Kadonaga, P. A. Luciw, and R. Tjian. 1986. Activation of the AIDS retrovirus promoter by the cellular transcription factor, Spl. Science 232:755-759. Kan, N. C., G. Franchini, F. Wong-Staal, G. C. DuBois, W. G. Robey, J. A. Lautenberger, and T. S. Papas. 1986. Identification of HTLV-III/LAV sor gene product and detection of antibodies in human sera. Science 231:1553-1555. Kanki, P. J., J. Alroy, and M. Essex. 1985. Isolation of T-lymphotropic retrovirus related to HTLV-III/LAV from wild-caught African green monkeys. Science 230:951-954.
BIOLOGY OF HIV AND BIOMEDICAL RESEARCH NEEDS 155 Kanki, P. J., F. Barin, S. M'Boup, J. S. Allen, J. L. Romet-Lemonne, R. Marlink, M. F. McLane, T.-H. Lee, B. Arbeille, F. Denis, and M. Essex. 1986. New human T- lymphotropic retrovirus related to simian T-lymphotropic virus type III (STLV-IIIAGM). Science 232:238-243. Kanki, P. J., S. M'Boup, D. Ricard, F. Barin, F. Denis, C. Boye, L. Sangare, K. Travers, M. Albaum, R. Marlink, J.-L. Romet-Lemonne, and M. Essex. 1987. Human T- lymphotropic virus type 4 and the human immunodeficiency virus in West Africa. Science 236:827-831. Kao, S.-Y., A. F. Calman, P. A. Luciw, and B. M. Peterlin. 1987. Anti-termination of transcription within the long terminal repeat of HIV-1 by tat gene product. Nature 330:489-493. Kennedy, R. 1987. Correspondent paper (untitled). AIDS Activities Oversight Committee, Washington, D.C. Klatzmann, D., E. Champagne, S. Chamaret, J. Gruest, D. Guetard, T. Hercend, J.-C. Gluckman, and L. Montagnier. 1984. T-lymphocyte T4 molecule behaves as the receptor for human retrovirus LAV. Nature 312:767-768. Knight, D. M., F. A. Flomerfelt, and J. Ghrayeb. 1987. Expression of the art/trs protein of HIV and study of its role in viral envelope synthesis. Science 236:837-840. Koenig, S., H. E. Gendelman, J. M. Orenstein, M. C. Dal Canto, G. H. Pezeshkpour, M. Yungbluth, F. Janotta, A. Aksamit, M. A. Martin, and A. S. Fauci. 1986. Detection of AIDS virus in macrophages in brain tissue from AIDS patients with encephalopathy. Science 233:1089-1093. Koyanagi, Y., S. Miles, R. T. Mitsuyasu, J. E. Merrill, H. V. Vinters, and I. S. Y. Chen. 1987. Dual infection of the central nervous system by AIDS viruses with distinct cellular tropisms. Science 236:819-822. Lasky, L. A., G. Nakamura, D. H. Smith, C. Fennie, C. Shimasaki, E. Patzer, P. Berman, T. Gregory, and D. J. Capon. 1987. Delineation of a region of the human immunodefi- ciency virus type 1 gpl20 glycoprotein critical for interaction with the CD4 receptor. Cell 50:975-985. Laurence, J., A. Saunders, and J. Kulkosky. 1987. Characterization and clinical association of antibody inhibitory to HIV reverse transcriptase activity. Science 235:1501-1504. Lee, T. H., J. E. Coligan, J. S. Allan, M. F. McLane, J. E. Groopman, and M. Essex. 1985. A new HTLV-III/LAV protein encoded by a gene found in cytopathic retroviruses. Science 236:1546-1549. Letvin, N. L., M. D. Daniel, P. K. Sehgal, J. M. Yetz, K. R. Solomon, M. Kannagh, D. K. Schmidt, D. P. Silva, L. Montagnier, and R. C. Desrosiers. 1987. Infection of baboons with human immunodeficiency virus-2 (HIV-2). J. Infect. Dis. 156:406-407. Lifson, J. D., G. R. Reyes, M. S. McGrath, B. S. Stein, and E. G. Engleman. 1986a. AIDS retrovirus induced cytopathology: Giant cell formation and involvement of CD4 antigen. Science 232:1123-1127. Lifson, J. D., M. B. Feinberg, G. R. Reyes, L. Rabin, B. Banapour, S. Chakrabarti, B. Moss, F. Wong-Staal, K. S. Steimer, and E. G. Engleman. 1986b. Induction of CD4-dependent cell fusion by the HTLV-III/LAV envelope glycoprotein. Nature 323:725-728. Luciw, P. A., C. Cheng-Mayer, and J. A. Levy. 1987. Mutational analysis of the human immunodeficiency virus: The orf-B region down-regulates virus replication. Proc. Natl. Acad. Sci. USA 84:1434-1438. Lyerly, H. K., T. J. Matthews, A. J. Langlois, D. P. Bolgonesi, and K. J. Weinhold. 1987. Human T-cell lymphotropic virus IIIB glycoprotein (gpl20) bound to CD4 determinants on normal lymphocytes and expressed by infected cells serves as target for immune attack. Proc. Natl. Acad. Sci. USA 84:4601-4605.
156 CONFRONTING AIDS: UPDATE 1988 Maddon, P. J., A. G. Dalgleish, J. S. McDougal, P. R. Clapham, R. A. Weiss, and R. Axel. 1986. The T4 gene encodes the AIDS virus receptor and is expressed in the immune system and the brain. Cell 47:333-348. Matthews, T. J., K. J. Weinhold, H. K. Lyerly, A. J. Langlois, H. Wigzell, and D. P. Bolognesi. 1987. Interaction between the human T-cell lymphotropic virus type IIIB envelope glycoprotein gpl20 and the surface antigen CD4: Role of carbohydrate in binding and cell fusion. Proc. Natl. Acad. Sci. USA 84:5424-5428. McClure, M. O., M. Marsh, and R. A. Weiss. 1988. Human immunodeficiency virus infection of CD4-bearing cells occurs by a pH-independent mechanism. EMBO J. 7:513-518. McCune, J. M., L. B. Rabin, M. B. Feinberg, M. Lieberman, J. C. Kosek, G. R. Reyes, and I. L. Weissman. 1988. Endoproteolytic cleavage of gpl60 is required for the activation of human immunodeficiency virus. Cell 53:55-67. McDougal, J. S. 1987. The immune response to HIV. Correspondent paper. AIDS Activities Oversight Committee, Washington, D.C. Moreine, B. 1987. Adjuvants and presentation of antigens. Paper presented at the Institute of Medicine Conference on the Development of Vaccines Against HIV Infection and AIDS, Washington, D.C., December 14-15. Muesing, M. A., D. H. Smith, C. D. Cabradilla, C. V. Benton, L. A. Lasky, and D. J. Capon. 1985. Nucleic acid structure and expression of the human AIDS/lymphadeno- pathy retrovirus. Nature 313 :450-458. Muesing, M. A., D. H. Smith, and D. J. Capon. 1987. Regulation of mRNA accumulation by a human immunodeficiency virus trans-activator protein. Cell 48:691-701. Nara, P. 1988. Introductory remarks at the Public Health Service Workshop on AIDS Vaccines, Bethesda, Maryland, April 25. Pederson, N. C., E. W. Ho, M. L. Brown, and J. K. Yamamoto. 1987. Isolation of a T-lymphotropic virus from domestic cats with an immunodeficiency-like syndrome. Science 235:790-793. Peterlin, B. M., P. A. Luciw, P. J. Barr, and M. D. Walker. 1986. Elevated levels of mRNA can account for the trans-activation of human immunodeficiency virus. Proc. Natl. Acad. Sci. USA 83:9734-9738. Plata, F., B. Autran, L. P. Martins, S. Wain-Hobson, M. Raphael, C. Mayaud, M. Denis, J.-M. Guillon, and P. Debre. 1987. AIDS virus-specific cytotoxic T lymphocytes in lung disorders. Nature 328:348-351. Ratner, L., W. Haseltine, R. Patarca, K. J. Livak, B. Starcich, S. F. Josephs, E. R. Doran, J. Antoni Rafalski, E. A. Whitehorn, K. Baumeister, L. Ivanoff, S. R. Petteway, M. L. Pearson, J. A. Lautenberger, T. S. Papas, J. Ghrayeb, N. T. Chang, R. C. Gallo, and F. Wong-Staal. 1985. Complete nucleotide sequence of the AIDS virus, HTLV-III. Nature 313:277-284. Richman, D. D., M. A. Fischl, M. H. Grieco, M. S. Gottlieb, P. A. Volberding, O. L. Laskin, J. M. Leedom, J. E. Groopman, D. Mildvan, M. S. Hirsch, G. G. Jackson, D. T. Durack, S. Nusinoff-Lehrman, and the AZT Collaborative Working Group. 1987. The toxicity of azidothymidine (AZT) in the treatment of patients with AIDS and AIDS-related complex: A double-blind, placebo-controlled trial. N. Engl. J. Med. 317:192-197. Robert-Guroff, M., M. Brown, and R. C. Gallo. 1985. HTLV-III-neutralizing antibodies in patients with AIDS and AIDS-related complex. Nature 316:72-74. Rosen, C. A., J. G. Sodroski, and W. A. Haseltine. 1985. The location of cis-acting regulatory sequences in the human T cell lymphotropic virus type III (HTLV-III/LAV) long terminal repeat. Cell 41:813-823. Rosen, C. A., J. G. Sodroski, W. C. Goh, A. I. Dayton, J. Lippke, and W. A. Haseltine. 1986. Post-transcriptional regulation accounts for the trans-activation of the human T-lymphotropic virus type III. Nature 319:555-559.
BIOLOGY OF HIV AND BIOMEDICAL RESEARCH NEEDS 157 Rosen, C. A., E. Terwilliger, A. Dayton, J. G. Sodroski, and W. A. Haseltine. 1988 Intragenic cis-acting art gene-responsive sequences of the human immunodeficiency virus. Proc. Natl. Acad. Sci. USA 85:2071-2075. Sadaie, M. R., T. Benter, and F. Wong-Staal. 1988. Site-directed mutagenesis of two trans-regulatory genes (tat-III, trs) of HIV-1. Science 239:910-913. Salk, J. 1987. Prospects for the control of AIDS by immunizing seropositive individuals. Nature 327:473-476. Sanchez-Pescador, R., M. D. Power, P. J. Barr, K. S. Steimer, M. M. Stempien, S. L. Brown-Shimer, W. W. Gee, A. Renard, A. Randolph, J. A. Levy, D. Dina, and P. A. Luciw. 1985. Nucleotide sequence and expression of an AIDS-associated retrovirus (ARV-2). Science 227:484-492. Sattentau, Q. J., and R. A. Weiss. 1988. The CD4 antigen: Physiological ligand and HIV receptor. Cell 52:631-633. Seligmann, M., A. J. Pinching, F. S. Rosen, J. L. Fahey, R. K. Khaitov, D. Klatzmann, S. Koenig, N. Luo, J. Ngu, G. Rietmuller, and T. J. Spiral 1987. Immunology of human immunodeficiency virus infection and the acquired immunodeficiency syndrome. Ann. Intern. Med. 107:234-242. Shaw, G. M., M. E. Harper, B. H. Hahn, L. G. Epstein, D. C. Gajdusek, R. W. Price, B. A. Navia, C. K. Petito, C. J. O'Hara, J. E. Groopman, E.-S. Cho, J. M. Oleske, F. Wong-Staal, and R. C. Gallo. 1985. HTLV-III infection in brains of children and adults with AIDS encephalopathy. Science 227:177-182. Shearer, G. 1987. Immune responses to HIV. Correspondent paper. AIDS Activities Oversight Committee, Washington, D.C. Smith, D. H., R. A. Byrn, S. A. Marsters, T. Gregory, J. E. Groopman, and D. J. Capon. 1987. Blocking of HIV-1 infectivity by a soluble, secreted form of the CD4 antigen. Science 238:1704-1707. Sodroski, J. G., C. A. Rosen, and W. A. Haseltine. 1984. Trans-acting transcriptional activation of the long terminal repeat of human T lymphotropic viruses in infected cells. Science 225:381-385. Sodroski, J., W. C. Goh, C. Rosen, K. Campbell, and W. A. Haseltine. 1986a. Role of HTLV-III/LAV envelope in syncytium formation and cytopathicity. Nature 322:470-474. Sodroski, J. G., W. C. Goh, C. Rosen, A. Dayton, E. Terwilliger, and W. Haseltine. 1986b. A second post-transcriptional trans-activator gene required for HTLV-III replication. Nature 321:412-417. Somasundaran, M., and H. L. Robinson. 1987. A major mechanism of human immunode- ficiency virus-induced cell killing does not involve cell fusion. J. Virol. 61:3114-3119. Sonigo, P., M. Alizon, K. Staskus, D. Klatzmann, S. Cole, O. Danos, E. Retzel, P. Tiollais, A. Haase, and S. Wain-Hobson. 1985. Nucleotide sequence of the visna lentivirus: Relationship to the AIDS virus. Cell 42:369-382. Starcich, B. R., B. H. Hahn, G. M. Shaw, P. D. McNeeley, S. Modrow, H. Wolf, E. S. Parks, W. P. Parks, S. F. Josephs, R. C. Gallo, and F. Wong-Staal. 1986. Identification and characterization of conserved and variable regions in the envelope gene of HTLV- III/LAV, the retrovirus of AIDS. Cell 45:637-648. Stein, B. S., S. D. Gowda, J. D. Lifson, R. C. Penhallow, K. G. Bensch, and E. G. Engleman. 1987. pH-Independent HIV entry into CD4-positive T cells via virus envelope fusion to the plasma membrane. Cell 49:659-668. Strebel, K., D. Daugherty, K. Clouse, D. Cohen, T. Folks, and M. A. Martin. 1987. The HIV "A" (sor) gene product is essential for virus infectivity. Nature 328:728-730. Stricker, R. B., T. M. McHugh, D. J. Moody, W. J. W. Morrow, D. P. Stites, M. A. Shuman, and J. A. Levy. 1987. An AIDS-related cytotoxic autoantibody reacts with a specific antigen on stimulated CD4+ T cells. Nature 327:710-713.
158 CONFRONTING AIDS: UPDATE 1988 Traunecker, A., W. Luke, and K. Karjalainen. 1988. Soluble CD4 molecules neutralize human immunodeficiency virus type 1. Nature 331:84-86. Wain-Hobson, S., P. Sonigo, O. Danos, S. Cole, and M. Alizon. 1985. Nucleotide sequence of the AIDS virus, LAV. Cell 40:9-17. Walker, B. D., S. Chakrabarti, B. Moss, T. J. Paradis, T. Flynn, A. G. Durno, R. S. Blumberg, J. C. Kaplan, M. S. Hirsch, and R. T. Schooley. 1987. HIV-specific cytotoxic T lymphocytes in seropositive individuals. Nature 328:345-348. Walker, B. D., C. Flexner, T. J. Paradis, T. C. Fuller, M. S. Hirsch, R. T. Schooley, and B. Moss. 1988. HIV-1 reverse transcriptase is a target for cytotoxic T lymphocytes in infected individuals. Science 240:64-66. Weber, J. N., P. R. Clapham, R. A. Weiss, D. Parker, C. Roberts, J. Duncan, I. Weller, C. Came, R. S. Tedder, A. J. Pinching, and R. Cheingsong-Popov. 1987. Human immuno- deficiency virus infection in two cohorts of homosexual men: Neutralizing sera and association of anti-gag antibody with prognosis. Lancet 1:119-122. Weiss, R. A., P. R. Clapham, R. Cheingsong-Popov, A. G. Dalgleish, C. A. Came, I. V. D. Weller, and R. S. Tedder. 1985. Neutralization of human T-lymphotropic virus type III by sera of AIDS and AIDS-risk patients. Nature 316:69-72. Weissman, I. L. 1988. AIDS research funding. Report presented to the President's Commission on the Human Immunodeficiency Virus Epidemic, New York, February 18. Wong-Staal, F., P. K. Chanda, and J. Ghrayeb. 1987. Human immunodeficiency virus: The eighth gene. AIDS Res. Hum. Retrovir. 3:33-39. Wright, C. M., B. K. Felber, H. Paskalis, and G. N. Pavlakis. 1986. Expression and characterization of the trans-activator of HTLV-III/LAV virus. Science 234:988-992. Yarchoan, K., and S. Broder. 1987. Development of antiretroviral therapy for the acquired immunodeficiency syndrome and related disorders: A progress report. N. Engl. J. Med. 316:557-564. Yarchoan, K., G. Berg, P. Brouwers, M. A. Fischl, A. R. Spitzer, A. Wichman, J. Grafman, R. V. Thomas, B. Safai, A. Brunetti, C. F. Perno, P. J. Schmidt, S. M. Larson, C. E. Myers, and S. Broder. 1987. Response of human-immunodeficiency-virus-associated neurological disease to 3'-azido-3'-deoxythyminine. Lancet 1:132-135. Young, F. E. 1987. Treatment use of experimental drugs. Address to the Council on Health Care Technology, Institute of Medicine, Washington, D.C., March 20. Zagury, D., J. Bernard, R. Leonard, R. Cheynier, M. Feldman, P. S. Sarin, and R. C. Gallo. 1986. Long-term cultures of HTLV-III-infected cells: A model of cytopathology of T-cell depletion in AIDS. Science 231:850-853.