TIBS 1 7 - M A Y 1 9 9 2

Outlook The structure of chromatin, particularly in the absence of linker histones, appears considerably more dynamic than previously thought. The intrinsically dynamic properties of nucleosome arrays provide a range of structural states, any of which can be stabilized either transiently or permanently by external influences. We have proposed that factors (e.g. histone modifications, non-histone proteins) associated with active chromatin, function at least in part by altering the distribution of structural states present. This in turn produces a specific state of biological activity. As such, the structural diversity intrinsic to nucleosome arrays provides the foundation for precisely coordinated control of genetic activity. It seems likely that much of what has been proposed may require modification in the future, particularly in terms of chromatin function in vivo. However, we hope that the concepts presented in this critique will focus more attention on the possible roles of chromatin structure in the modulation of genetic activity.

Acknowledgements We thank Drs A. Wolffe, J. Widom and K. van Holde for their thoughtful advice, and for providing copies of manuscripts prior to publication. We also acknowledge Dr D. Lohr for conversations leading to the concept presented in Fig. 3.

TRADITIONAL DEVELOPMENT of viral vaccines has generally been a process of trial and error: developing a method to propagate the virus, then either developing an inactivation strategy or selecting for an avirulent mutant, and using these preparations to induce protective immunity. Although effective for other vaccines such as that for polio virus, inactivation of HIV-1 can face the problem that procedures to inactivate nucleic acid may alter the structure of epitopes that could confer protective immunity. In addition, HIV-1, when propagated in culture, tends to lose its outer envelope protein, which is believed to be a major target of immunity. Attenuation, although effective for other viruses, is considered a poor

References TIBS editorial policy limits the number of references

that can be cited in the text. We apologize to those authors whose work has been cited through the reviews of others. 1 van Holde, K. E. (1988) Chromatin, SpringerVerlag 2 Richmond, T. J. et al. (1984) Nature 311, 532-537 3 Svaren, J. and Chalkley, R. (1990) Trends Genet. 6, 52-56 4 Elgin, S. C. R. (1988) J. Biol. Chem. 263, 19259-19262 5 Grunstein, M. (1990) Trends Genet. 6, 395-400 6 Wolffe, A. P. (1991) Trends Cell Biol. 1, 61-66 7 Felsenfeld, G. (1992) Nature 355, 219-223 8 Thoma, F. and Koller, T. (1977) Cell 12, 101-107 9 Thoma, F., Koller, T. and Klug, A. (1979) J. Cell Biol. 83, 403-427 10 Butler, P. J. G. and Thomas, J. O. (1980) J. Mol. Biol. 140, 505-529 11 Allan, J. et al. (1981) J. Cell Biol. 90, 279-288 12 Widom, J. (1989) Annu. Rev. Biophys. Biophys. Chem. 18, 365-395 13 Hansen, J. C., Ausio, J., Stanik, V. H. and van Holde, K. E. (1989) Biochemistry 28, 9129-9136 14 Yao, C., Lowary, J. and Widom, J. (1991) Biochemistry 30, 8408-8414 15 Clark, D. J. and Kimura, T. (1990) J. Mol. Biol. 211, 883-896 16 Ausio, J., Sasi, R. and Fasman, G. D. (1986) Biochemistry 25, 1981-1988 17 Hansen, J. C., van Holde, K. E. and Lohr, D. (1991) J. Biol. Chem. 266, 4276-4282 18 Stein, A. (1979) J. Mol. Biol. 130, 103-134 19 Burton, D. R. et al. (1978) Nucleic Acids Res. 5, 3643-3663 20 Yager, T. and van Holde, K. E. (1984) J. Biol. Chem. 259, 4212-4222 21 Ausio, J., Seger, D. and Eisenberg, H. (1984) J. Mol. Biol. 176, 77-104 22 Kamakaka, R. T. and Thomas, J. O. (1990)

EMBO J. 9, 3997-4006 23 Garrard, W. T. (1991) BioEssays 13, 87-88 24 Ausio, J. and van Holde, K. E. (1986) Biochemistry 25, 1421-1428 25 Imai, B. S. et al. (1986) J. Biol. Chem. 261,

8784~8792 26 Ausio, J., Dong, F. and van Holde, K. E. (1989) J. Mol. Biol. 206, 451-463 27 Libertini, L. J., Ausio, J., van Holde, K. E. and Small, E. (1988) Biophys. J. 53, 477-487 28 Norton, V. G., Imai, B. S., Yau, P. and Bradbury, E. M. (1989) Cell 57 449-457 29 Ridsdale, A. J., Hendzel, J. M., Decluve, P. G. and Davie, J. R. (1990) J. Biol. Chem. 265, 5150-5156 30 Churchill, M. E. A. and Travers, A. A. (1991) Trends Biochem. Sci. 16, 92-97 31 Grunstein, M. (1990) Annu. Rev. Cell Biol. 6, 643-678 32 Thoma, F. (1991) Trends Genet. 7,175-177 33 Morse, R. H. (1992) Trends Biochem. Sci. 17, 23-26 34 van Holde, K. E., Lohr, D. E. and Robert, C. R. (1992) J. Biol. Chem. 267, 2837-2840 35 Kornberg, R. D. and Lorch, Y. (1991) Cell 67, 833-836 36 Laskey, R. A. and Earnshaw, W. E. (1980) Nature 286, 763-767 37 Smith, S. and Stillman, B. (1991) EMBO J. 10, 971-980 38 Dilworth, S. M., Black, S. J. and Laskey, R. A. (1987) Cell 51, 1009-1018 39 Eickbush, T. H. and Moudrianakis, E. N. (1978) Biochemistry 17, 4955-4964 40 Hendzel, J. and Davie, J. R. (1990) Biochem. J. 271, 67-73 41 Almouzni, G., Mechali, M. and Wolffe, A. P. (1991) Mol. Cell. Biol. 11, 655-665 42 Corden, J. L. (1990) Trends Biochem. Sci. 15, 383-386 43 Hayes, J. J., Clark, D. J. and Wolffe, A. P. (1991) Proc. Natl Acad. Sci. USA 88, 6829-6833 44 Dong, F. and van Holde, K. E. (1991) Proc. Natl Acad. Sci. USA 88, 10596-10600 45 Hansen, J. C. and Wolffe, A. P. Biochemistry (in press)

How antibodies block HIV infection: paths to an AIDSvaccine Scott Putney We a r e b e g i n n i n g t o u n d e r s t a n d t h e m e c h a n i s m t h a t e n v e l o p e p r o t e i n s o f t h e h u m a n a n d s i m i a n i m m u n o d e f i c i e n c y v i r u s e s u s e t o g a i n e n t r y into h o s t c e l l s . A v a c c i n e t h a t c a n e l i c i t a n t i b o d i e s t h a t b i n d t o t h e viral epit o p e s i n v o l v e d in t h i s p r o c e s s w o u l d t h e r e b y p r e v e n t HIV i n f e c t i o n . This a r t i c l e o u t l i n e s o u r p r o g r e s s in t h e d e v e l o p m e n t o f p o s s i b l e c a n d i d a t e s f o r AIDS v a c c i n e s .

alternative for retroviruses such as HIV, which may integrate into host cell DNA. Such infection, which may persist for the life of the host, can cause malignant S. Putney is at Alkermes Inc., 26 Lansdowne transformation. St, Cambridge, MA 02139, USA. © 1992,ElsevierSciencePublishers, (UK) 0376-5067/92/$05.00

An additional problem is that HIV specifically infects T-helper cells, which are necessary for an effective immune response. Once infected, these cells contain an integrated proviral genome

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20 1

tween HIV-1, HIV-2 and the simian immunodeficiency virus (SIV), which is being used as a model for AIDS vaccine development. The elucidation of these functional determinants has allowed development of both candidate vaccines and therapeutic agents against these viruses.

The envelope protein: structure and CD4 binding Rgure 1

HIV-1 is surrounded by an envelope that is composed of a lipid bilayer, derived from the infected cell, and a glycosylated protein, encoded by the viral genome (Fig. 1). The HIV-1 envelope protein is translated as an 88 kDa precursor that is subsequently modified by glucosidase I to give a 160 kDa glycoprotein, gp160 is cleaved by a cellular protease during virus maturation to the external envelope protein, gp120 and the transmembrane glycoprotein, gp41. gp120 is anchored to the virus by non-covalent interactions with gp41. The first step in the infection of a cell by HIV-1 is the binding of gp120 to the cell-surface protein, CD4. CD4 is expressed on a subset of T lymphocytes (helper T cells) and is composed of an amino-terminal extracellular region that contains four tandem immunoglobulinlike domains, a carboxy-terminal transmembrane domain and a cytoplasmic domain. The region of CD4 that interacts with gp120 has been shown to be the amino-terminal half of the extracellular domain comprising the first two immunoglobulin-like domains. This specific interaction of gp120 and CD4 is the basis for the tropism of the virus for cells of the macrophage/monocyte and lymphoid lineages. Both gp120 purified from virus-infected cells and gp120 produced using recombinant expression vectors have been used to measure the gp120-CD4 dissociation constant, which was found to be approximately 10-9 (Ref. 1). The native conformation of gpl20 is necessary for binding of CD4, since reduced and alkylated or detergent-denatured gp120 does not bind with detectable affinity. gp120 contains 18 cysteine residues, all of which are specifically joined by disulfide crossbridges 2 (Fig. 2). At least

Model of HIV-1 showing the location of the external envelope protein (gp120), the transmembrane envelope protein, gp41, the core proteins (p17 and p24) and the viral RNA and reverse transcriptase. The recession and the knob on gp120 represent the CD4binding site and the V3 loop respectively (see Fig. 2). Each gp120/gp41 dimer is believed to be a trimer of three gp120 and three gp41 subunits.

and it thus seems likely that an effective HIV vaccine must prevent the virus from establishing infection. This criterion is not required for other vaccines such as those for polio virus, the hepatitis viruses or influenza virus, where there is some degree of virus infection and replication after exposure. This rather stringent requirement means that the vaccine will almost certainly have to elicit antibodies that prevent the virus from establishing infection and require that the titre of these antibodies be kept at or above the threshold to prevent infection from being established. Antibodies that prevent the establishment of the infection of a target cell are called neutralizing antibodies. These generally bind to the viral surface protein and all studies to date have shown that the envelope protein of HIV-1 is the principal target for such neutralizing antibodies. AIDS vaccine development efforts have focused principally on HIV-1 because this is the most prevalent virus in developed countries and because it appears to be more damaging than the related virus, HIV-2. Much work has been done over the past several years to determine the portions of the HIV-1 envelope protein that are necessary for virus entry and hence targets for neutralizing antibody. In this article, I summarize the work that has been done to define-the structure of the HIV-1 envelope and its role in allowing the virus to enter the cell and subsequently establish infection. In addition, 1 discuss the differences be-

192

some, if not all, of these crossbridges are apparently needed to maintain the necessary conformation for CD4 binding. Clearly, antibodies that prevent attachment of gp120 to CD4 should be able to prevent viral infec~vity. The CD4-binding site on gpl20 was localized in a study in which monoclonal antibodies to gp120 that block binding to CD4 were generatedL A peptide of gp120 that binds to one of these antibodies was isolated and sequenced. Although this peptide did not bind CD4, several mutations introduced in this domain in a clone expressing the envelope protein resulted in purified gp120 that could not bind CD4. The gp120-binding region of all three blocking antibodies were mapped using gp120 fragments. Separate experiments used in vitro mutagenesis to insert linkers and delete sequences randomly throughout the envelope protein 3. The mutants were introduced into a vector that expressed envelope protein after transfection of CD4 cells and the ability of gp120 to bind CD4 was measured. Mutations in three locations, about 50 amino acid residues apart, in the carboxy-terminal half of gp120 were found to interfere with binding (Fig. 2). The CD4-binding determinant, identified by mapping blocking monoclonal antibodies, is midway between the second and third of these positions. Recent experiments, in which a more extensive series of mutations were employed, show that mutations in several additional regions of gp120 disrupt CD4 binding4. The regions of gp120 implicated in CD4 binding are relatively conserved in amino acid sequence between different HIV-1 isolates, which seems logical given that gp120 from each virus isolate binds CD4. The regions of gpl20 defined by these mutations have an overall hydrophobic composition, suggesting that they are buried within the envelope. Although these regions are separated on the linear sequence of gp120, they may be brought together in the correctly folded molecule.

A model for the role of the envelope in virus entry Direct entry Or endocytosis?Once HIV binds CD4, viral entry might occur either by direct fusion of the viral envelope with the cell plasma membrane or by internalization of the virus-CD4 complex by CD4-mediated endocytosis (see Fig. 3). Although CD4 is internalized in response to phorbol esters or other sig-

TIBS 17 - MAY .1.992

rials, two findings show that endocytosis of CD4 is not necessary for HIV infection. (1) Cells expressing CD4 with mutations in the cytoplasmic domain that impair the ability of CD4 to undergo endocytosis are infected as efficiently as those expressing wild-type CD4S; and (2) neutralization of the low pH environment of the endosomal compartment does not interfere with viral entry6. Thus the low pH-dependent conformational changes that facilitate direct virus fusion with endosomal membranes are not necessary for entry of HIV and direct fusion of the virus envelope or the membrane of a virus-infected cell with the membrane of an uninfected CD4-bearing cell appears to be the principal mechanism of entry. Cleavage of gp160 is required for virus entry. The amino terminus of the F~ glycoprotein of paramyxoviruses is responsible for both envelope-cell fusion and cellcell fusion induced by these viruses. Cleavage of an F~ precursor by a trypsin-like enzyme exposes the F~ amino terminus which is hydrophobic and contains the peptide Phe-X-Gly. The amino terminus of HIV-1 gp41 has homology with the amino terminus of F~ glycoprotein; it is also hydrophobic and contains Phe-Leu-Gly 7. Mutations that affect the ability of the envelope to induce cell fusion map to this region 3. Endoproteolytic cleavage of gpl20 is required to generate infectious virus 8. A mutant virus in which the trypsin-like cleavage site is replaced with a chymotrypsin-like site is phenotypically wild type with respect to proviral replication, RNA processing, protein expression and viral assembly, but is noninfectious unless it is exposed to chymotrypsin. The ability of the envelope to induce syncitia is also inhibited when gpl60 cleavage is inhibited by the monovalent carboxylic ionophore monensin 9. A second cellular-bindingprotein. A cellular protein besides CD4 is probably required for virus entry into CD4-bearing cells. Human cell lines (e.g. HeLa) transfected with CD4 expression vectors are susceptible to HIV infection ~°. By contrast, although murine cell lines transfected with the human CD4 gene express CD4 and bind virus, the virus does not replicate. Thus, it is possible that a host factor in addition to CD4 is necessary for a step after binding and before replication. HIV infection of CD4-bearing cells is blocked by soluble CD4 but neither soluble CD4 nor anti-CD4 antibodies inhibit infection of non-CD4-expressing glial

NH 2 COOH

Rgure 2 Cartoon of HIV-1 gp120 showing the disulfide crossbridges (adapted from Leonard et al.2). The V3 region (upper right) and the regions 1,3,4 known to be important in binding to CD4 are shaded. The V3 loop is believed to be cleaved by a protease on the CD4 target cell during viral entry (shown by an arrow) and is the principal binding site for virus-neutralizing antibodies.

or muscle cells H, suggesting that virus entry is not mediated solely by CD4. It may be that entry is facilitated by the binding of gpl20 to galactosyi ceramide because antibodies to this liposaccharide prevent infection of neural cell lines ~2. One possible host cell protein required for infectivity is a cellular protease that specifically cleaves gpl20. The third variable domain of gpl20, known as the V3 loop, is a 36-aminoacid disulfide crossbridged loop that appears to be on the surface of gpl20, since V3-specific monoclonal antibodies bind HIV-1 infected cells 13. V3 does not appear to play a role in CD4 binding

because gpl20 efficiently binds CD4 when V3-specific antibodies are bound TM and because an insertion in V3 does not affect the binding of gpl20 to CD43. The overall length of V3 is relatively conserved, suggesting that large deletions or insertions cannot be tolerated. Several amino acid positions are also conserved, which indicates that a selective advantage is conferred by these sequences is. Also, envelope mutants with amino acid substitutions in the tip of the V3 loop bind CD4 and allow processing of gpl60 to gpl20 and gp41 but do not mediate cell fusion or virus infectivity3.~E These observations suggest that V3 has a critical function.

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Figure 3 Model of the role of the envelope protein in HIV-1 infection. It proposes that after gp120 binds to CD4, a cellular protease cleaves gp120 within the V3 loop and a conformational change of gp120 ensues. This change allows the amino terminus of gp41 to insert into the membrane of the CD4 cell and this causes the membrane of the cell and virus (or virus infected cell) to fuse. Membrane fusion thus allows the contents of the virus to spill into the CD4 cell. A similar model was originally proposed by Kowalski et which was supported and refined by numerous additional studies.

al.3,

Although the precise role of V3 in the steps leading to virus infection is unknown, there is evidence that proteolytic cleavage of V3 is necessary for infection. During preparation of recombinant gp120, some gp120 is found to be proteolytically cleaved between the Arg and the Ala in the sequence GPGRAF, which is located centrally within the loop 17. Furthermore, a seripe protease inhibitor, trypstatin, can block syncytia formation between infected ~nd uninfected cells, suggesting that such a protease is involved in virus infectivity~8. The model predicts that the cleavage of V3 is followed by a conformational

194

change in gpl20, which allows the amino terminus of gp41 to insert in the membrane of the CD4 cell. Recognition of V3 by a cellular protease can also explain the inability of particular HIV-1 isolates to infect certain CD4-bearing cell lines that can be infected by other HIV strains (cell tropism). Tropism may be explained by variations in the spectrum of cell-surface proteases expressed by different cell lines and the potential of V3-cleavage sites on the infecting virus. Consistent with this hypothesis, a single amino acid change at the tip of the V3 loop has been reported to alter

HIV tropism ~9. Envelope recombination studies between HIV clones from viruses with distinct cell tropisms suggest that the V3 loop determines host-cell range2°.

Neutralization determinants and vaccine development The model for virus entry presented above suggests there are at least three possible neutralizing antibody-binding regions (or determinants) on the gpl20gp41 complex: the amino terminus of gp41, the CD4 binding site on gpl20 and the V3 loop. Antibodies elicited by peptides containing the carboxyl terminus of gpl20

T I B S 1 7 - MAY 1992

and the amino terminus of gp41 have been reported to have a weak neutralizing effect2~. However, the amino terminus of gp41 is hydrophobic and thus is probably buried in the envelope structure and inaccessible to antibodies. The amino terminus of gp41 may therefore directly associate with the aminoterminal half of .gp120 because mutations in either of these two regions disrupt this association 3. In theory, the CD4-binding site on gp120 is attractive as a candidate for vaccine development; however, it has been difficult to exploit for this purpose for several reasons. First, the high affinity between gp120 and CD4 (Kd ~10-9) means that only a small subset of the antibodies elicited by this determinant will have sufficient affinity to neutralize virus infectivity. Second, the CD4-binding site is probably buried within the envelope, so neutralizing antibodies that block binding to CD4 are not readily elicited by purified envelope protein 22. No murine monoclonai antibodies elicited by purified gp120 that block CD4 binding have a neutralizing effect. It has only recently been possible to isolate, from HIV-l-infected individuals, human monoclonal antibodies that neutralize due to their ability to block binding to CD4 23'24. Although purified gp120 does not readily elicit these antibodies, HIV1-infected people generally have considerable titres of gp120-specific CD4blocking antibodies. It appears, therefore, that the CD4 binding determinant is more efficiently presented on infectious virus or virus-infected cells than on purified gp120, or that chronic exposure to gp120 (as is the case of HIV-1 infection) is required to elicit CD4-blocking antibodies. A particular conformation of gp120, perhaps formed when complexed with gp41 as exposed on the virus, may also be necessary to efficiently elicit antibodies to the CD4-binding site. Certainly the native conformation of gp120 is necessary for CD4 binding because reduced/denatured gp120 does not bind CD4 and CD4-blocking monoclonal antibodies do not bind denatured gp120. It is therefore difficult to envision mimicking the CD4 site on gp120 with synthetic peptides. In contrast, synthetic peptides of V3 elicit high titres of neutralizing antibodies 2s,26. V3specific monoclonal antibodies can prevent HIV-1 infection of chimpanzees (see below), also making V3 peptides an attractive avenue for vaccine development.

One problem that has plagued the development of any HIV-1 vaccine is the amino sequence variability of the envelope protein. Within gp120 there is a distinct pattern of such variation. Five hypervariable regions of gpl20 (V1-V5) have been defined as regions with 25% or less conservation of amino acids between several sequences, with V3 being the most variable. This variation does not occur during in vitro propagation of the virus, indicating the role of immune selection in the generation of variant virus strains. Mutants that have 'escaped' the effects of neutralizing antibody have been selected in vitro 27,28, demonstrating that immune selection can play a role in the generation of such variation. Experimental infection of chimpanzees has also shown that neutralization-resistant variants arise during the course of infection 29, lending further support to the importance of immune selection in the generation of antigenic diversity. If V3 is to be useful as a vaccine candidate, it is necessary to identify conserved sequences within V3 to which antibodies that can neutralize otherwise divergent virus isolates can bind. A survey of the sequence of the V3 loop of 245 HIV-1 isolates has revealed that some conservation of certain amino acid sequences and polypeptide secondary structure exits ~s. This indicates that antibodies that bind these conserved sequences or structures could neutralize a majority of HIV-1 isolates. In fact, polyclonal antisera elicited by one of these conserved hexapeptides neutralized all isolates tested (four of seven) that contain this hexapeptide in V3 (Ref. 30). in addition, a human monoclonal antibody to V3 neutralizes all isolates containing its binding site in V3 (Ref. 31). These experiments demonstrate that eliciting antibodies to such conserved V3 sequences can neutralize the majority of HIV-1 isolates. V3 has significant advantage as a vaccine candidate because V3-specific antibodies can neutralize virus infectivity after gp120 binds to CD432. Addition of V3-specific monoclonal antibodies neutralized infectivity up to 30 min after binding of virus to CD4 suggesting that, compared to CD4 binding, the proteolytic cleavage of V3 (Fig. 3) is slow. This indicates that, after exposure to HIV-1 and virus attachment to a CD4 cell, there is a 30 min window in which a V3 antibody can bind gp120 to prevent infection. As discussed below, a V3specific monoclonal antibody can pre-

vent infection of a chimpanzee even when administered after exposure to the virus.

V3 is not a neutralization determinant of SlY SIV causes an AIDS-like disease in several monkey species and is currently being used as a model for HIV-1 pathogenesis, therapy and vaccine development. Like HIV-1, SIV infects both human and monkey CD4 cells by binding to CD4. Experiments in which monkeys have been immunized with chemically inactivated SIV and subsequently challenged with infectious virus have shown that this immune response prevents infection33,34. Furthermore, passive immunization of monkeys with immune sera taken from protected monkeys confers protection 35. This suggests that antibody appears to be sufficient to confer protective immunity to SIV, although the nature of the immunity is unknown and the neutralization determinants have not been defined. By studying the HIV-1 gpl20 and gpl60 equivalents of SIV (gpll0 and gpl40) as well as fragments of gpll0, we have found that the V3 loop of SIV gpll0 is not a neutralization determinant3E In contrast to HIV-1, peptides of the V3 loop of SIV do not elicit neutralizing antibody and this region of the SIV envelope protein is far less variable among different isolates than its HIV-1 counterpart. This suggests that immunological selective pressure, such as neutralizing antibody, is not placed on this envelope sequence during infection. However, SIV gpll0 in its native conformation elicits high titers of neutralizing antibodies, indicating the presence of at least one potent neutralization determinant. Because the integrity of this determinant depends on the native tertiary structure, it probably consists of amino acids adjacent in the tertiary structure but distant in the primary sequence. Polio and rhinoviruses have such non-linear neutralization determinants. Whether neutralizing antibodies to native gpll0 act by blocking binding to CD4 has not been determined. Because the V3 loop of SIV (and presumably of HIV-2, which is much more similar to SIV than to HIV-1) is not a binding site for neutralizing antibody, it could be that the V3 loop of SIV is not exposed as it is in HIV-1 and hence not accessible to neutralizing antibodies. Alternatively, this difference could reflect significant differences in the mechanism of action of the HIV-1 and 195

TIBS

SlY envelope proteins. It has not been determined whether the SIV V3 loop is necessary for infectivity or, if so, whether it is acted upon by a cellular protease. It remains to be seen how useful SIWHIV-2 will be for HIV-1 vaccine development.

V3 antibodies prevent experimental infection of chimpanzees Although demonstration of the ability of envelope preparations or peptides to elicit antibodies that neutralize HIV-1 in culture is a prerequisite to their consideration as vaccine candidates, it is necessary to show that such antibodies prevent experimental infection of an animal by HIV-1. Chimpanzees are the only experimental animal that can be reliably infected with HIV-1 and four different chimpanzee challenge experiments have demonstrated protection from infection. In each case, protective immunity correlated with the presence and titre of V3-specific neutralizing antibodies 37-4°. The most convincing and recent of these showed that passive administration of a monoclonal antibody specific for the V3 loop prevented infection when administered either before or 10 min after virus challenge39. In the challenge experiments that demonstrated lack of protection, the challenged animals had either low or no measurable titres of V3-neutralizing antibody. There has been one published experiment in which neutralizing antibodies, contained in a polyclonal antisera obtained from HIV-l-infected people, were passively transferred to chimpanzees that were subsequently challenged 4°. Although these animals had significant titres of circulating neutralizing antibody, they were not protected. These neutralizing antibodies, however, did not bind V3, and the fact that the polyclonal antisera used in this experiment had antibodies that blocked binding of gpl20 to CD4 suggests that such non-V3-directed neutralizing antibody is not protective. These experiments are critical to vaccine development efforts because they not only show that protective immunity to HIV-1 can be induced, but also reveal that neutralizing antibody to the V3 determinant on the virus is protective.

Future prospects In spite of these advances: there is still much to be learned before a practical vaccine that can induce protective immunity to a majority of HIV-1 virus isolates is developed. The first of these

196

is that a protective level of neutralizing antibody will have to be maintained for a sufficient period of time to make a vaccine practical. Second, it is unknown whether the information (such as the protective titre) gained in chimpanzee challenge experiments will allow accurate prediction of the protective titre necessary to prevent infection of humans. The protective titre needed to protect humans when exposed to natural HIV-1 isolates by more natural infectious routes (for example, exposure at mucosal surfaces or in injection as would occur with a needle stick) is completely unknown. As was the case for hepatitis B, it may not be until completion of randomized double-blind clinical trials that this is known. And finally, both a neutralizing antibody response and a cytotoxic cellular immune response may be required to completely prevent establishment of permanent infection. For example, it may be eventually determined that it may not be possible to completely prevent initial infection, but rather we should aim to greatly reduce the level of free virus in the blood stream (thus possibly making the infected carrier less contagious) and delay onset of CD4 lymphocyte destruction and clinical symptoms. Other viral vaccines elicit or prime for immune responses that prevent or greatly attenuate disease following exposure or infection by the virus. It remains to be seen whether such an achievement will be acceptable, assuming it is against the majority of virus isolates, for an HIV-1 vaccine. In spite of these caveats, the demonstration of protective immunity to HIV-1 has been a major milestone and has given hope that a practical vaccine for AIDS can be developed.

Acknowledgements 1 thank my colleagues at Repligen Corporation (K. Javaherian, G. LaRosa, A. Profy, J. Rusche, S. Silver, G. Gray, W. Herlihy and R. Grimaila), at Duke University (T. Matthews, K. Weinhold, A. Longlois, B. Haynes, T. Palker and D. Bolognesi) and at Merck (E. Emini) who have contributed to much of the work summarized here, S. Mousterakis for preparing the figures, and J. Oskirko and D. Burke for typing the manuscript.

References Due to TIBS policy of short reference lists, the number of references cited in this article has been limited. Some work has therefere unfortunately been left unacknowledged.

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