Development of a vaccine for the prevention of AIDS, a critical appraisal.

Development of a vaccine for the prevention of AIDS, a critical appraisal David T. Karzon *~, Dani P. Bolognesi +, and Wayne C. Kofl~

The pathogenesis and clinical expression of HIV-1 infection in humans is considered in term's of classical pathogenetic studies of vival infections for which successful vaccines have been produced. The unique features of HIV pathogenesis are defined, and gaps in knowledge identified as a framework for considering designs for immune intervention. Envelope-derived candidate vaccines have been used in immunization and challenge experiments in SI V/macaque or HI V/chimpanzee models, presented either as vaccinia recombinant vectors or as subunits, singly or in sequence. These studies have been paralleled by clinical trials for safety and immunogenicity in seronegative individuals. Data generated will permit comparison of immune responses to specific antigens and delivery systems in animal models and in humans. In limited studies conducted under optimized conditions, non-human primates have been protected against virus challenge when immunized with some candidate vaccines or following passive transfer of tu'gh-titred antibody. Consideration of current information suggests that in order to prevent HIV infection it may be necessary to 41evise new strategies capable of inducing and maintaining high threshold titres of biologically relevant antibody, as well as persistence of active cytotoxic T cells recogmzmg multiple epitopes.

Keywords:AIDS; HIV; vaccine development; clinical trials; pathogenesis; animal models;ethical/legalissues T H E B I O L O G Y O F HIV-1 A N D P A T H O G E N E S I S O F AIDS HIV-1 was identified in 1983 and, despite the most expensive and determined international research effort ever mounted and the availability of new and critical tools of microbial genetics and molecular immunology, a safe and effective AIDS vaccine is not close to fruition. The development of an AIDS vaccine has received high priority in view of the massive toll of sickness and death, largely refractory to treatment or epidemiological control. HIV-I infection involves a chronic and intimate relationship with human cells and is adroitly successful in eluding the clearing processes of the immune system. Viral vaccines, in general, have been highly successful in preventing those infectious diseases characterized by * Department of Pediatrics and Microbiology and Immunology, Vanderbilt Medical School, Nashville, TN 37232, USA. + Department of Surgery, Duke University Medical Center, Durham, NC27710, USA.'tNIH/NIAID, Division of AIDS, Rockville, MD 20892, USA. ~To whom correspondence should be addressed 0264~410X/92/141039-14 (~ 1992 Butterworth-HeinemannLtd

a cluster of biological characteristics outlined in Table 1. Many important diseases fall into this category, including rabies, smallpox, yellow fever, poliomyelitis, measles, mumps, rubella and hepatitis B virus. Unfortunately, HIV-1 infection demonstrates few if any of the characteristics which mark infections amenable to vaccine prophylaxis. A central question guiding any vaccine development is 'what are the critical immune determinants required to prevent infection and disease when the host is later challenged through natural routes?' The quest for the definition of these immune elements in HIV infection and the rationale for appropriate vaccine design and clinical evaluation is the theme of this report. Since the virus was isolated, the structural and functional features involved in cell entry, replication, latency, activation and pathology have been addressed and a large body of information is now available. The surface envelope glycoprotein gpl60 has been sequenced, its variable and conserved regions, B and T epitopes, cell-binding and membrane-fusion sites are defined. The surface gp 160 and its cleavage products gp120 and gp41 prepared in various forms have been major vaccine candidate constituents based upon their recognition by the immune system and roles in receptor binding and entry functions. Other non-envelope HIV-components, gag and pol products and regulatory proteins have been explored to a lesser extent as immunogens. The pathogenesis and clinical expression of HIV infection in humans will be considered in terms of classical pathogenetic studies of other viral infections in animal models and humans. A considerable body of information has been acquired in the process of development of vaccines for several human infections on the basis of the classical model. Our intent is to define unique features of HIV pathogenesis as well as to identify gaps in knowledge, as a framework for considering immune intervention strategies. In diseases amenable to effective vaccination, a defined sequence of steps can usually be documented during the course of infection 1. Typically, exposure of the host to a viral pathogen at the portal of entry results in silent mucosal replication, although mucosa may be bypassed by direct parenteral entry. A low-grade primary viraemia ensues with dissemination to parenteral sites where viral expansion occurs. This produces a high titred secondary viraemia which seeds the target organ. The incubation period ends and disease is expressed when virus replicates sufficiently in the target organ to induce physiological damage. The incubation period is silent, although

Vaccine, Vol. 10, Issue 14, 1992 1039

Development of an AIDS vaccine, a critical appraisal: D. T. Karzon et ai. Table 1

1 2 3 4 5 6

7 8 9

Characteristics of viral infections with successful vaccines: features not shared by HIV-1

Successful viral vaccines

HIV-1 infection

Pathogenesis includes an obligatory viraemia before infecting target organ Pre-existing NT antibody prevents infection in an individual with intact immune system; infectious agent is free virus Monotypic or limited number of antigenic serotypes

Target organs, lymphocytes, monocytes macrophages, and mucosa probably infected as early event A high threshold titre of NT antibody may protect against free virus; CMI role uncertain; infectious agent may be cell associated Extensive genetic variation during individual persistent infection and regionally Challenge risks early integration and provirus state

Challenge is frequently accompanied by limited local replication; rapid anamnesis results in silent infection Latency not present Useful animal model generally available, may not reproduce human disease with fidelity Immune function not significantly compromised by infection Typically relatively benign and self-limiting illness; death or residua may occur Following recovery (i.e. elimination of virus) protection is complete, often tifelong

non-specific prodromata may accompany the secondary viraemia in poliomyelitis. Prophylactic vaccination is effective by interrupting the process at any point prior to significant viral replication in the target organ. In protected individuals, challenge virus can replicate at the portal of entry causing no disease, but providing time for recruitment and expansion of memory cells. It has been proposed that antibody is the first line of protection against reinfection, while cell-mediated mechanisms such as cytotoxic lymphocytes (CTL) serve as a secondary clean-up system for cells infected by virus escaping neutralization 2. Persistent circulating antibody protects against disease, whether that antibody is acquired by natural disease, passive administration, transplacentally or by vaccination. In this setting, circulating neutralizing antibody can be accepted as a surrogate marker of protection. Most viral vaccines currently in use, whether inactivated, live attenuated, or immunogenic viral proteins, involve organisms which fall into this pathogenetic model. By contrast, infectious diseases demonstrating other pathogenetic patterns are variably difficult to prevent. Infections limited to mucosal sites and those with latency states may require other immune elements, such as secretory antibody and/or cytotoxic lymphocytes to prevent or clear infection and for many such agents vaccine efforts remain unsuccessful. The pathogenetic events of HIV infection are only partially known. The natural portal of entry is the mucosal surface and evidence points to infection with a single or minimal number of clones 3. A stage of primary replication at the mucosal site has not been recognized. Access to blood may occur by direct transfusion or transplacentally, but the possible role of mucosal trauma or genital infection is not defined. Early on, either at the portal of entry or blood-borne sites, the virus invades the target cells, which in this case include rectal epithelial cells, CD4 and monocyte/macrophages, setting up latent infections with resident provirus and a low level of productive infection under complex regulatory controls. An acute prodromal syndrome resembling infectious mononucleosis appears in some individuals at variable times, 3-6 weeks or more, after known timed infection 4. This has been termcd primary HIV infection. Symptoms consist of fatigue, fever, adenopathy and occasionally rash and CNS involvement, all of which resolve spon-

1040

Vaccine, Vol. 10, Issue 14, 1992

Latent state inherent in replication HIV-f infection in chimpanzee induces infection with latency but no disease. SIV infection and disease in macaque but significant difference in antigen primary structure Immune system is the major target organ Regularly fatal Recovery not observed

taneously without residua. A period of rapid expansion of virus in the blood accompanied by a P24 antigenaemia peaks during the symptomatic period 5-v. The viraemia and antigenaemia are rapidly dampened by the appearance of antibody and presumably other immunological mechanisms not yet established. Rather than complete clearing with termination of viracmia as occurs in acute infections with other agents, rapid emergence of antigenic variants permits selection of escape mutants 8. Low-grade viraemia persists indefinitely, presumably by iterations of antigenic diversification and acquired immune containment. A steady state ensues, lasting years, during which time the patient is infectious. During this period, antibodies which recognize multiple determinants of all viral gone products and mediate neutralization, fusion inhibition, inhibition of gpl20 binding to CD4 and ADCC are present, as well as class I and II MHCrestricted cytotoxic lymphocytes. The failure of combined viral antibody and CTL clearing mechanisms may be explained by the continuous evolution of neutralizing and CTL recognition sites as well as the inability of cytolytic mechanisms to recognize non-productively infected cells. Functional alteration in immune elements and eventual destruction of the key regulatory CD 4+ cells result in marked immune deficits and appearance of opportunistic infections characterizing the AIDS syndrome. UNDERSTANDING THE IMMUNE DETERMINANTS OF PROTECTION

Implications for vaccine development Despite failure of clearance mechanisms in established HIV infection, it is possible to visualize primary prevention at the time of exposure by having in place antigenically appropriate immune barriers. While information regarding specific immunc determinants of protection is incomplete, prevention against HIV infection is most likely to be accomplished by maintaining threshold titres of biologically relevant antibodies and cell-mediated immunity. On challenge with HIV, the potential window of time available for effective anamnesis will depend upon the inoculum size, tissue site, rapidity of attaining proviral state and, possibly, the timing of rapid expansion of viraemia. If a single or minimal number of successful

Development of an AIDS vaccine, a critical appraisal: D. T. Karzon et al.

provirus hits determines irreversible persistent infection, reliance will be heavily dependent upon a preformed threshold of immune performance. While the HIV antigens used to date are only modestly immunogenic and appear to induce a short antibody half-life, it may be possible to maintain high neutralizing antibody titres by presenting immunogens in new forms or with new adjuvants. The prospects for maintaining active CTL is less clear, but it is notable that a vigorous peripheral CTL response is maintained in chronically infected patients. A vaccine inducing antigenically diverse neutralizing activities and CTL populations with multiple relevant recognition sites should enhance the probability of primary viral clearance. The strategy for defining the critical immune responses affording protection should be a part of all steps in vaccine development. (1) Protection should be correlated in the SIV/macaque and HIV/chimpanzee systems with multiple measures of immune functions. Primate challenge experiments should be completed with all candidate vaccines; failure to protect may or may not predict the human model, but the data for protection, correlates of immunity, and magnitude of immune responses should be available for comparison with humans. Additionally, immune determinants may be identified by passive transfer of monoclonal antibodies 9 or concentrated human immunoglobulin 1°. Passive transfer of immune cells or ablation of specific T-cell subsets is probably not practical in primate species. The SCID-hu mouse model could be explored further. (2) Human immune responses in phase 1/2 clinical trials should establish correlates with similarly studied primate data. (3) Clinical efficacy trials, if possible using several different vaccine constructs in different epidemiological settings (e.g. sexual, intravenous or perinatal transmission) and eventually in comparative trials, should ultimately provide definitive linkage of immune parameters with protection. (4) The

design of efficacy trials should be prospectively planned to include sufficient information to allow identification of surrogate markers. (5) Finally, the efficacy of a new vaccine could be predicted by documenting surrogate markers in primate studies and phase 1/2 clinical trials. Antigenic targets of HIV Despite their proven track record in other viral vaccines, it has been difficult to embrace strategies of vaccine development which are based on the use of whole inactivated or attenuated preparations of H1V. This is due principally to formidable safety considerations, although the concept of a safe virion product remains a goal of some investigators. Much effort has therefore been devoted to identifying the elements of HIV which would constitute the primary targets for humoral and cellular immunity. Fortunately, there are many tochoose from, including several which exhibit dominant characteristics (Figure 1). The aspects of viral infection that are most pertinent to vaccine strategies are represented by the initial interaction of the virus with its target cells. The HIV-1 exterior envelope glycoprotein, gpl20, and the transmembrane envelope glycoprotein, gp41, are derived by cleavage of the gpl60 envelope glycoprotein precursor 11-13. The primary mode of infection of CD4 + cells begins with a high-affinity interaction between gpl20 and the CD4 glycoprotein, which acts as the virus receptor 14-16. Following gpl20-CD4 binding, thc fusion of viral and host cell membranes, which involves both gpl20 and gp41, allows virus entry 17 19. Not unexpectedly, biologically important antibodies are probably restricted to the envelope glycoproteins. For example, binding antibody to p24 and other internal proteins which are present in infected individuals does not neutralize virus. Two principal neutralization sites are situated within gpl20. Each of these turns out to be an important

13I

,°v-l--I

v` ,

pol

pll

p66/ p51

gp120

p32

T1 V3

O

O

O

O

gpql

T2

DO0

0

0

cv V3

muu Figure 1 Selected domains of HIV gene products as possible targets for vaccine development. The diagram illustrates the approximate location of the epitopes for neutralizing antibodies (NA), cytotoxic lymphocytes (CTL) and antibody-dependent cell cytotoxicity (ADCC). For NA, the designations cv and cc reflect the emerging concepts of the existence of conformational epitopes affecting both variable and conserved domains. [], Structural (internal) epitopes; I , structural (external) epitopes; Iq, regulatory epitopes


1041

Development of an AIDS vaccine, a critical appraisal: D. T. Karzon et al. V3 loop (MN)

RIH

IGPG~A~

K

du~"I---CTL specificity to I f ":~T HIV stcain Class I MHC T recognition K

R

N

~

K N Y N

I I G IT

p

R R Zc.s.s.C HAQ I

IN

Domains of the V3 loop C C- ~

B -

I

A C

B'

Neutralizing antibodies ~ CrL ---4 epitope

C'

I

Figuce 2 The PND amino acid sequence of the HIVMN isolate. A high percentage of sera from HIV-1 infected individuals from North America as well as other parts of the world recognize this sequence. Variation within this domain occurs primarily at the sides of the loop while the crown and the regions near the cysteines are much less divergent from isolate to isolate. Neutralizing antibodies are principally targeted to the sides (isolate-restricted) or crown (cross-neutralizing). In addition, a CTL epitope has been mapped within this region. A, groupspecific (conserved); B, class-specific (semi-conserved); C, type-specific (variable)

functional domain in virus attachment and entry. One, designated the V3 loop, represents a continuous region of about 35 amino acids linked by a disulphide bond (Fiqure 2) 2°'2~. Antibodies to V3 exert their neutralization potential after virus has bound to its receptor by interfering with the process of fusion in which the V3 loop is thought to play a role. Indeed, antibodies to V3 effectively block the fusion process between infected and uninfected cells and thus represent a possible effective barrier for cell-to-cell transmission of H IV. This domain features both variable and semi-conserved neutralization cpitopes. The variable sites arc highly immunogenic and endow the virus with a mechanism to escape immune attack. The more conserved regions are believed to represent the functional domains of V3 which include elements that contribute to viral cell tropism 22 and possibly cytopathogenicity. Thc latter regions are poorly recognized by the human immune system during natural infection and this may be critical to the survival strategy of the virus. However, approaches have been devised to elicit immunity to such domains in a vaccine 23. Indeed, monoclonal antibody for the V3 loop has protected chimpanzees from homologous HIV challenge in studies of both pre -9 and postexposure immune prophylaxis 29. The second important region is related to the binding site of the virion to CD4 which is also situated on gp120. By definition, this site, termed fl for this discussion, is

1042


conserved among isolates: but, because it is not a continuous epitope 24"25, it is dependent on a number of conformational features of this complex glycoprotein and therefore not entirely similar from isolate to isolate. Thc best information available suggests that this site is recessed within the molecule and difficult to access by antibodies. However, during natural infection, antibodies which effectively block binding of gpl20 to CD4 do arise to a variety of epitopes which probably represent the rim of the binding pocket, as has been postulated for other viruses 26. Although such antibodies can be broader in neutralization potential than those directed to V327, they are still quite heterogeneous with respect to both specificity and potency, most probably reflecting the flexible configuration of the binding site. As might be expected, it remains quite difficult to induce such antibodies with subunit vaccine candidates in terms of both efficiency and potency 28 and this remains a major challenge in vaccine design. An interesting recent finding is that anti-V3 and anti-fi antibodies may act synergistically with one another in virus neutralization 293°. This is apparently the consequence of a unidirectional cooperative binding where anti-V3 antibodies facilitate the binding of anti-fl antibodies 3°, presumably by inducing conformational changes that make the binding site more accessible. While the simultaneous presence of both antibody classes would be attractive in a number of settings it is also known that viruses can be isolated at certain times during infection that are resistant to neutralization despite the fact that both antibody classes are present 3°. These presumably are escape mutants 31. One should also take note of the fact that HIV can also infect cells through mechanisms which are independent of CD4, as can best be evidenced on CD4-negative cell lines 32'33. It remains to be determined if anti-V3 or anti-fl antibodies will be effective in blocking such modes of viral infection. T-cell epitopes abound in most of the viral components of HIV including both structural and regulatory gene products 34. As might be expected, the number of CTL epitopes are fewer but multiple sites have been defined 35. Although a prominent CTL epitope exists in a variable domain of V3, most CTL epitopes are located within relatively conserved domains of several HIV gene products. However, recent cvidence indicates that variants arise during infection which can escape CTL attack. Escape variants display mutations within conserved CTL epitopes of the pl 7 and p24 internal virus (gag) proteins suggesting that these sites may be important in regulating viral replication and are under immunological pressure 3~'. This raises the possibility that dominant CTL epitopes may exist much in the same way as the neutralization sites discussed above. An important issue which pertains to these epitopes is that of allotypic restriction 36'37. The extent to which a given T-cell or CTL epitope is recognized by the multiple human M H C haplotypes which exist in diverse populations must be established. In all likelihood, a considerable number of such epitopes may well be needed in a vaccine in order to obtain good T-cell responses in all individuals. Another mechanism that might contribute to protective immunity is antibody-dependent cell cytotoxicity (ADCC) 38. This phenomenon is independent of M H C restriction and may thus be critical for rapid destruction of virus-infected donor cells transmitted during infection. As might be expected, the ADCC targets are present


principally within the envelope glycoproteins which are prominently exhibited on the surface of the infected cell. However, although the existence of this mechanism is recognized, definitive evidence of a biological function in vivo is lacking. Although very little is known about mucosal immunity to HIV, this mechanism is potentially of major importance in vaccine strategies. The hallmark of the mucosal immune system is the production of secretory immunoglobulin which is found in mucosal secretions and is capable of virus neutralization39. However, also associated with mucous membranes primarily of the bronchus and gut, one finds lymphoreticular tissue which upon stimulation by antigen can generate a variety of effector responses such as cytotoxic T cells, NK cells and ADCC, typical of the systemic immune response. In order to activate this entire system from the standpoint of vaccines, antigen delivery mechanisms must ensure that the immunogens find their way to the inductive sites within the mucosa 39. To this end, intranasal or oral immunization have been effective approaches in other viral systems. Only scant information regarding HIV-specific secretory immunity is available either from infected individuals or from vaccine trials 4°. While secretory IgA has been detected in various secretions in both adults and infants infected with HIV, its effects on virus or virus-infected cells remains unknown. However, the potential need for local immunity is emphasized by studies with SIV in macaques which demonstrated that vaccines given systemically and capable- of protecting against intravenous or intramuscular challenge, failed to protect against intravaginal administration of virus 41'4z. Vaccination protocols which can effectively induce secretory immunity are still under development. An important principle underlying these approaches is that primary stimulation of a particular mucosal site will enable the immunity to extend to other anatomical sites lined by mucous membranes 39. Thus, in the case of HIV, if oral immunization is to be effective, mucosal immunity must be extended to the various ports of viral entry such as the vaginal, urethral and rectal mucosa. A P P R O A C H E S TO P R O D U C T I O N O F AN IMMUNOGEN

Immunogenic qualities of HIV components Given that the rules for recognition of a determinant for humoral versus cellular immunity are different, the former relying more on native structure while the latter on primary sequence, it follows that in order to achieve recognition of both, a structure which approximates the epitopes on the virion or infected cell is desirable as at least one component of a vaccine regimen. It may also be important as to how such a native structure is seen by the immune system; i.e. whether it is secreted or anchored to a membrane. On the other hand, subcomponents in the form of recombinant fragments or synthetic peptides may be quite effective at stimulating or boosting responses to a particular epitope thought to be important in protection. Thus priming with a native structure for recognition followed by boosting with subunits or fragments to heighten the response may be an effective approach to achieving high levels of immunity to HIV.

One of the more striking advances in HIV vaccine research involved just such combinations of immunogens. It stems largely from work by Shiu-Lok Hu and colleagues and employed a recombinant vaccinia vector carrying the virus envelope gene to prime the immune system followed by a recombinant envelope subunit to boost the responses induced by the replicating vector43. Neutralizing antibody, as well as cytotoxic lymphocytes, was effectively generated with appropriate doses and spacing of recombinant vectors and subunits 44'45. The insights derived from such studies permit a number of new opportunities to enhance the potential efficacy of HIV vaccines. First, modifications of the live vector to include additional viral gene products (9a9, pol, regulatory products) to provide a broader arsenal of vaccine targets is easily achievable. The vector itself can be modified or substituted in a number of ways to be safer and more effective. Moreover, other viral or bacterial vectors with different properties such as ability to infect mucosal epithelial cells (e.g. adenovirus46, salmonella4v) or those endowed with strong adjuvant characteristics (e.g. BCG 4s) are under development. Viral subunits can also be improved in a number of ways. Their native structure can be preserved by recombinant systems employing mammalian cells49. This should enhance the recognition of conformational epitopes required for neutralization. They and the respective vectors might be modified to include multiple target epitopes (neutralization and CTL) in place of sites which are either neutral or representative of potentially undesirable reactivities such as regions which exhibit molecular mimicry with normal host cell components, notably MHC molecules5°. This of course presumes that such alterations do not influence the structure in a negative manner. Peptides can also be considered for immunization, especially as boosters following priming by live vectors or native subunits. Studies in animal models have attested boosting of neutralizing responses using V3 peptides following subunit priming 51. Particularly attractive in this regard are combinations of B- and T-cell epitopes (e.g. a V3 neutralizing epitope and a strong T-cell epitope from either the envelope or 9ag geneS2',). Peptides would also be useful to address the problem of HIV variability where cocktails might be employed to induce broadly neutralizing antibodies such as demonstrated by octameric forms of V353.

Strategies for immunopotentiation The emphasis on recombinant subunit and peptide strategies for HIV vaccines has stimulated a need to develop novel antigen presentation systems and adjuvants capable of potentiating levels of humoral, cellular and memory immune responses. Efforts to prevent establishment of HIV infection and subsequent HIV disease will probably require induction of HIV-specific neutralizing antibodies, cytotoxic T cells, T- and B-cell memory, and other immunological effector mechanisms, given the versatility by which HIV initiates and maintains infection. Paradigms of the past, suggesting that induction of cellular immune effector mechanisms require live vaccine approaches, have recently been challenged through a series of elegant antigen presentation and adjuvant studies 54. Similarly, biochemical purification of active components from bacterial cell walls and lipid A


1043

Development of an AIDS vaccine, a critical appraisal. D. T. Karzon et al.

derivatives ss have now p r o v i d e d a plethora of agents which augment effector B-cell functions. It is now clear that modification of recombinant protein and peptide immunogens, via chemical attachment of lipid moieties s6, presentation as multi-antigenic particles sv, presentation on liposomes ss and immunestimulating complexes 5'~ can render immunogens capable of eliciting quantitatively and qualitatively augmented cellular immune responses. Moreover, humoral immune responses can be similarly modified via covalent coupling of universal T-cell helper epitopes 6°, and through conjugation of antigen to protein carriers 6~. Similarly, adjuvants are being utilized to potentiate various immune responses. There arc three principal mechanisms through which adjuvants might enllance the immune responses of experimental HIV vaccines. First, the depot effect of numerous emulsions localizes the antigen to the injection site for long periods, thereby enabling recruitment and contact with antigen-presenting cells. In this regard, advances with methodologies for time-released drug delivery 6e might have similar potential. Secondly, adjuvants may activate antigen-presenting cells to process HIV antigens more effectively. Strategies incorporating cytokines as co-adjuvants expressed in recombinant vectors 63 are thus a particularly interesting approach. Lastly, adjuvants may be utilized for broadening immune responses to include induction of mucosal immunity. In this regard, strategies using cytokines °4, cholera toxin 6~, or orally administered vectors 47 are being evaluated for mucosal antibody targeting. Thus, approaches to modify antigen presentation and to potentiate anti-HIV immune responses have moved from empirical to designed formulations, with possibilities on the horizon to specifically modify immunoglobulin class subclass induction, enhance effector T-cell responses, induce local immunity, and broaden the duration and spectrum of neutralizing antibody. The remaining challenge is to determine the optimum strategy resulting in efficacy against natural HIV infection. In this regard, candidate vaccines must be developed which incorporate immunogenic epitopes which conform to the prevalent HIV isolates in sites where the testing is to occur. Indeed, it does appear as if distinct HIV families predominate in certain geographical areas with five to seven major virus subgroups now defined {Figure 3a and b). For example, in North America, Europe and certain parts of Africa, the isolates resemble a prototypic isolate designated as MN. Attributes of an ideal vaccine

Data concerning the qualitative, quantitative and site-specific immune responses to variously constituted antigens, adjuvants and presentations are only gradually becoming available and correlates with protection in primates are not definitive. There is, nevertheless, sufficient information from primate studies and phasc 1 c!inical trials to suggest some benchmarks by which to mt asure vaccine strategies and performance. It may be a useful exercise to set down goals to be met in at hypoth,:tical 'ideal vaccine'. Elements to be considered for entry in phase I/2 clinical trials are listed separately from .hose for entry into efficacy trials, the latter anticipating s:udies including developing countries (Tattles 2.4 apd 2B). Progression to efficacy trials awaits phase 1,'2 studies of optimization of immunogenicity (dose and regimen)and comparison of relative effectiveness of candidate vaccines

1044


its judged by standardized safety and imnaunological criteria. Initially, experimental ~accincs considered for efficacy trials cannot be expected to meet all desirable qualilications. Therefore, early trials will bc conducted with candidate vaccines judged to demonstrate the soundest safety and immunogenicity proliles, and which may be expected to have the greatest potential for demonstrating some level of protection, as well as to yield significant new scientific information 66. CONTRIBUTION OF ANIMAL MODELS Historically, animal models have played a key role in the development of vaccines currently important in public health practice. The pathogcnetic patterns of viral replication and immune response at the whole animal level have guided empirical and. more recently, designed immune intervention strategies. The models have been used for estimation of vaccine efficacy and. for some agents, still play a role in regulatory standards, e.g. live poliovirus vaccine. The animal models used for developing and testing vaccines often fell far short of duplicating human infection and clinical disease. Examples of unphysiological models used included exotic challenge routes which were convenient to induce and test clinical endpoints, rnodels with minimal or incomplete disease expression, or models involving manipulation of virulence or tissue tropism of highly adapated laboratory strains in order to manifest clinical markers 67 7o. Nonetheless, they proved useful. The discrepancy between these models and the current H IV systems is not the greater suitability of prior models, but that the biological questions asked were markedly simpler. All currently marketed viral vaccines have in common the fact that pre-existing circulating antibody (memory cells may be sufficient, as in measles) protects against clinical disease. The animal model wits not called upon to duplicate with lidelity in an exotic host the many nuances of viral behaviour and antibody and cellular immune responses of humans. On the contrary, prior models required only the demonstration of vaccineinduced circulating antibody, usually monoserotypic, which would regularly prevent or modify a clinical manifestation, however contrived. Antibody interference with the completion of the pathogenetic cycle could usually be shown equally well by passive antibody or observation of epidemiological behaviour of infection in the population. The absence of the clinical AIDS syndrome in the chimpanzee could be represented as a difference in virulence which theoretically could be manipulated by strain selection or adaptation, or more directly through knowledge of the molecular basis of virulence. Because an ultimate goal of an AIDS vaccine is to prevent the establishment of persistent HIV infection, the chimpanzee and the macaque represent reasonable preliminary indicators of this phenomenon. Once cells are infected, the task of clearing HIV from the host would appear to be a more formidable challenge. Whether a silent, transient and self-limiting HIV infection without the development of recognized antibody can occur in humans is not known. There are several animal models which have proven useful in guiding vaccine development against HIV and others being exploited that will have impact in the future. Notable among these is the simian immunodeficiency virus (SIV) .3 but also other lentiretroviruses such as the


a Subtype A

Subtype B

type E

Subtype C

b CT RPN N NTRKSI H I G P G R AFYTTGEI IGDI RQAHC /

~

...... ?..R.GPGQ...A..D

]

..........

.... YK...QRTP. GLGQ .....

........ ........

NIK ........

GPGK...A..D

~

........

R????I.G. . . .

ITIGPGQvF ......... IHIGPGRAF .........

?RGPGQ T..ATG? ........

. . . . . .

........

GWGR ...A .......

Figure 3 (a) The approximate locations of putative virus families based on initial information of prevalent envelope sequences. (b) Selected examples of consensus sequences of the crown region of the V3 loop from initial data. Note that virus groupings are not dictated by geography. (Courtesy of Dr Gerald Meyers, Los Alamos Data Base.)

feline immunodeficiency virus (FIV) 71. When one considers animal species which are directly infectable with HIV these include chimpanzees with HIV-1 and macaques with HIV-272. Recently it has been shown that one macaque species, Macaca nemestrina can also be infected with HIV-1 (M. Katze and L. Corey, personal communication). However, neither with HIV-1 nor HIV-2 is there any evidence that infection of non-human primates leads to disease. Finally immunodeficient mice into which human immune elements have been introduced (SCIDHu) can also support HIV-1 replication 73'74. The most extensive vaccine studies conducted to date have been in the SIV/HIV-2/macaque and HIV-1/

chimpanzee models. Such studies have demonstrated the all-important step of feasibility of developing a protective vaccine. Thus, vaccine candidates have demonstrated that infection can be prevented by prior vaccination, at least under optimal experimental conditions. In the chimpanzee model four distinct approaches have led to protection: (a) immunization with recombinant subunits 75; (b) combinations of recombinant vectors, boosted with subunits or peptidesS~; (c) passively administered monoclonal antibodies 9, and (d) passively administered high titred polyclonal antibodies from HIV-infected individuals 1°. With one exception the best correlate of protection is neutralizing antibody to the V3 loop. In the


1045

Development of an AIDS vaccine, a critical appraisal. D. T. Karzon et al. Table 2A Characteristics of an 'ideal vaccine: considerations for entry into phase 1 2 safety and immunogenicity clinical trials Product characterization and quality 1 Meet standards of national regulatory body

Immunogenicity 1 Documentation of HIV chimpanzee and SlV.'macaque immune responses, including biological assays: neutralization, binding of V3 loop, fusion inhibition, binding of CD4; ADCC; CTL 2 Neutralization and other biological activity versus homologous and heterologous variants 3 Primate challenge and protection

Safety 1 Vaccine non-replicating or non-transmissible 2 Satisfactory safety performance in laboratory animals including primates 3 Antibody-mediated enhancement assays performed

Schedule/delivery 1 Need for minimal number of primary and booster doses 2 Persistence of antibody and CMI

Table 2B Characteristics of an 'ideal vaccine'; considerations for entry into efficacy trials Product characterization and quality 1

Meet standards of international regulatory bodies

Immunogenicity 1 Documentation of HIV,,'chimpanzee and SlV,.'macaque immune responses, including biological assays: neutralization, binding of V3 loop, fusion inhibition, binding of CD4; ADCC; CTL versus homologous and regional variant strains 2 Primate challenge and protection, using intravenous and mucosal routes and free and cell-associated virus with homologous and variant strains 3 Documented immune responses in phase 1.'2 clinical trials paralleling responses in challenge-protected primates 4 Immune response induced by vaccine distinguishable from naturally acquired infection for scientific, ethical and legal purposes

Safety 1 2 3 4

Satisfactory Satisfactory Satisfactory Safety data

safety performances in phase 1/2 clinical trials performed in seronegatives in sponsor country safety performance in phase 1/2 seropositive clinical trials performed in sponsor country safety performance in clinical trials of seropositive pregnant women in sponsor country in immunodeficient primates (with replicating virus only)

Schedule/delivery 1 A minimal number of doses inducing durable antibody and CMI, demonstrated in phase 12 clinical trials and by protection in primate systems; no requirement for late booster doses 2 Feasibility and immune characterization of multi-antigen or sequential antigens 3 Feasibility and immune characterization of oral/nasal route of administration 4 Low cost 5 Heat-stable under field conditions

case of the polyclonal human antibodies which protected the chimpanzee, the most likely candidate was neutralizing antibody to the fl site, although the latter was not definitely proven 1°. The remaining tasks are to extend such studies to different routes of challenge infection, to determine the duration of protective immunity beyond the last booster and to evaluate if protection can be extended to diverse HIV isolates. Studies in the SIV/HIV-2 model which demonstrate protection are more difficult to interpret. Most of the experiments have employed whole inactivated SIV preparations which gave the unexpected result of little or no correlation of protection with the presence (or absence) of neutralizing antibodies 42. Subsequently, it was determined that the best correlates of protection were reactivities to cellular antigens associated with SIV vaccine preparations, including the demonstration that protection could be achieved by immunization with the uninfected cells used to grow the vaccine and challenge viruses 76. In contrast, an approach employing recombinant vectors as a priming immunogen followed by recombinant subunits for boosting also resulted in protection, but in this case neutralizing antibodies represented a positive correlate 43. This and other recombinant approaches, therefore, appear at this time

1046


to be the most direct avenue toward defining correlates of immunity in animal models. Some of the remaining challenges for improvement of animal models might be: the development of a model where HIV both infects and induces disease: the development of animal models amenable to natural transmission of infection: ensuring that the models can be infected with natural variants where relevant crossprotective immune responses can be evaluated: and the determination of duration of protection, where demonstrable, and its correlate with persistence of immune responses. Some unexplored features of both animal models also deserve attention. What can one learn from the chimpanzee model that is naturally resistant to development of disease? Similar considerations apply to macaque species which control SIV infection without development of disease while the same viruses can be highly pathogenic in other species. CLINICAL TRIALS FOR SAFETY AND IMMUNOGENICITY The process of developing safe and effective vaccines for the prevention of AIDS consists of preclinical studies aimed at defining, in animal models, the optimal

D e v e l o p m e n t of an AIDS vaccine, a critical appraisal: D. T. K a r z o n et al.

Table 3 HIV candidate vaccines in clinical trials; subjects: HIVseronegative volunteers Immunogen

Sponsor

Recombinant eubunit rgp160: Baculovirus expressiona'78 rgp160: Mammalian expression79 rgp120: Yeast/env 2-3, non-glycosylatede° rgp120: Mammalian8~ rgp120: Mammalian~

MicroGeneSys Immuno AG Biocine Biocine Genentech

Recombinant virus Vaccinia expressing HIV envb'Sa

Bristol-Myers Squibb

Synthetic peptide HGP-30: Peptide of pl ~,e~

Viral Technologies

Virus-like particle Ty-gag: Yeast retrotransposon~'87

British Biotechnology

Combinations Vaccinia expressing HIV env plus gp160~,~,ea Vaccinia expressing HIV env plus autologous Vac-env infected cells plus gp160~,9°

Bristol-Myers Squibb/ MicroGeneSys rgp160 D. Zagury, Paris, France

"C. Lane, personal communication bVaccinia-immune and vaccinia-naive subjects

approaches for antigen presentation, followed by clinical trials to evaluate the safety, immunogenicity, and finally efficacy of the experimental immunogens. The primary focus of HIV vaccine development is the prevention of HIV disease in uninfected, HIV-seronegative individuals. From March 1992, several experimental immunogens have been tested in phase 1/2 safety and immunogenicity trials (Table 3). In addition, two combinations have been evaluated in studies aimed at maximizing induction of both humoral and cellular immunity. A total of more than 500 HIV-seronegative volunteers have been immunized With experimental HIV immunogens. Participating volunteers have generally been at low risk for HIV infection, in an effort to minimize potential for intercurrent HIV infections during the trials. The theoretical potential for HIV envelope antigens to cause autoimmune phenomena 2 via molecular mimicry 5°, or to induce immune enhancement via complement or Fc receptors 9°'91, coupled with the current lack of identification of immune correlates of protection, has required HIV clinical trials to be more comprehensive than trials of other vaccines. An added hazard is the potential for transmission of replicating recombinant vectors particularly in regions of HIV endemicity. Thus, volunteers in HIV vaccine trials must be evaluated for a broad spectrum of physiological and immunological safety concerns, and immunogenicity studies encompass a wide range of humoral and cellular parameters. Several significant advances have already been achieved from the few completed phase 1 trials 9~. The logistics of conducting HIV vaccine trials has been confirmed, i.e. when clinical trials are well designed adequate numbers of HIV-seronegative volunteers can be recruited. HIV vaccines have demonstrated a strong record of safety in preliminary studies, comparable with that of other viral antigens, and no evidence of vaccine-related immunosuppression such as altered numbers or function of CD4 ceils has been observed. Dose-range studies of subunit HIV antigens in alum have been less than encouraging,

with short half-lives of humoral immune responses. Large quantities of HIV antigens are required to induce neutralizing antibody responses compared with other viral antigens 77, perhaps due to the extensive glycosylation of the HIV envelope antigens 93. Preliminary studies with gpl20 in adjuvants other than alum have demonstrated that anti-HIV immune responses may be potentiated by modifying antigen/adjuvant formulations 94. Comparative adjuvant studies planned for 1992 are aimed at optimizing the immune responses of subunit HIV antigens. Studies with non-envelope HIV antigens have not induced neutralizing antibody against HIV, but have elicited cellular immune responses which may be important as priming for immunological memory as well as in clearance of HIV-infected cells. This observation has led to the development of combination antigen vaccines i.e. env, oa 9, pol constructs which are planned for evaluation in safety and immunogenicity trials in the coming year. Trials of vaccinia-HIV env recombinant provided the first studies of a recombinant live vector in humans 44'82'87-89, and pave the way for expanded vector strategies for HIV and non-HIV antigens. Finally, combinations using recombinant vaccinia-HIV env for primary immunization followed by appropriate doses and spacing of recombinant subunit booster regimens induced both neutralizing antibody and anti-HIV cellular immune responses at levels not previously achieved by either vector or subunit alone 8s. These trials are of particular interest in light of the demonstration of protection in an SIV/macaque analogue system 44. None of the candidate HIV vaccines currently being evaluated has progressed thus far to large-scale efficacy trials (phase 3). E F F I C A C Y TRIALS O F HIV V A C C I N E S The challenges for conducting efficacy trials of HIV vaccines may be divided into three principal elements: 1 Scientific issues associated with development of the

vaccine and its progression through preclinical evaluation and pilot safety/immunogenicity clinical trials. 2 Operational issues associated with preparing populations, sites, and the ethical/legal/social/political infrastructure for vaccine efficacy trials. 3 Rationale for considering HIV vaccine efficacy trials which combines both scientific and political dimensions. Scientific issues

Several advisory panels and committees from national and international HIV vaccine development programmes have met during the past two years in attempts to delineate guidelines for progression of HIV vaccine candidates from pilot (phase 1/2) safety and immunogenicity studies to large-scale efficacy trials. This process is complicated by the following factors: (1) The limitations of results of animal model studies as discussed earlier in this review. (2) Natural history studies of humans infected with HIV have failed to elucidate correlates of disease progression and/or correlates of protective immunity. (3) Field isolates of HIV behave differently in immunological assays and in other characteristics from laboratory strains, thereby complicating evaluation of candidate vaccines. Thus, a vaccine which induces antibodies capable of inhibiting the replication


1047

Development of an AIDS vaccine, a critical appraisal. D. T. Karzon et al.

of laboratory strains of HIV but incapable of neutralizing field isolates from various regions of the world may not be as promising as the vaccine which inhibits a spectrum of HIV field isolates. (4) Finally, not all phase I HIV vaccine trials have been conducted using standard, reproducible assays for functional immune responses, due to a lack of consensus of assays, reagents and protocols, making comparison of candidate vaccines difficult. Resolution of the problem of antigenic diversity of H1V will require a clearer understanding of current and projected evolution of HIV in various parts of the globe 9s. In particular, one must determine with more certainty how many groups actually prevail and what degree of inter and intra-group variation exists 96. Implicit in this concept is the need to determine the relationship of scquencc diversity to the biology of the virus, in particular, its antigenic properties and its susceptibility to immune control. Thus, it will be necessary to evaluate immune responses of 'accines for their impact on infection with multiple endemic isolates representative of the population to be vaccinated. Moreover, these tests may need to be performed using relevant target cells such as T and macrophage lineages. Similarly, vaccine-induced cellular effector mechanisms such as cytotoxic lymphocytcs should also be tested on autologous tttrgets infected with representative natural isolates 97, as opposed to artificial systems using vaccinia constructs of prototypic isolates or pulsing with peptides representing prominent epitopes. For induction of broadly cross-reactive neutralizing antibodies, identification of conserved domains from diverse virus prototypes may be important; however, rendering such conserved sites immunogenic remains a challenge. Finally, the duration of threshold levels of humoral and/or cellular immunity requisite for protection must be established. This should be determined in trials where both infection and disease can be monitored. Operational issues

Perhaps the most important element in planning for efficacy trials is the identification of suitable populations where the annual transmission rate of HIV (incidence rate) is sufficiently high to distinguish efficacy of the vaccine when compared with a placebo control, coupled with a volunteer compliance rate which is high enough to conduct a multi-year follow-up 98. Additional relevant cofactors include the prevalence of other sexually transmitted diseases, common routes of HIV transmission, e.g. intravenous, rectal, vaginal and perinatal, and predominant circulating HIV isolates in a given population 99. The World Health Organization recently named Brazil, Thailand, Rwanda and Uganda as countries where it will fund the first round of site preparation for HIV vaccine efficacy trials. It is likely that other national and international health agencies will identify additional sites within the next few years, and that site preparedness for HIV vaccine efficacy trials will occur at multiple locations. Site preparation includes staff training, the establishment of laboratories for handling specimens and HIV serodiagnosis, as well as preparation of clinical settings for administering vaccines, conducting subject visits, and provi, fing medical care associated with administration of vaccines. Systems data collection require implementation. Pilot (phase 1/2) trials of promising HIV vaccines should be conducted in popu-

1048


lations where efficacy' trials will be undertaken, to test the feasibility of conducting vaccine trials and to compare safety and immunogenicity data with results from phasc 1 trials in the sponsoring country. Rationale

The rationale for initiating HIV vaccine efficacy trials combines both scientific and political dimensions. Scientific guidelines in the decision-making process would include preclinical data demonstrating protective efficacy in an animal model, and safety and immunogcnicity data from phase 1/2 human clinical trials. A more detailed consideration of these guidelines reveals many qucstions (Table 2). Optimally, animal protection should be assessed under real-world conditions, e.g. challenge with virus-infected cclls, heterologous strains and vaginal or rectal administration after peak immunity has waned. It is important to delineate efficacy endpoints, i.e. whether they prevent infection versus expression of clinical disease. In the event that a vaccine fails to prevent infection, but does delay or prcvent disease, a long period of observation is required (many years). The pattern of virus shedding and transmission should be documented to detect possible impact on the epidemiology of transmission. The selection of candidate vaccines for the costly and lengthy efficacy trials in a relatively limited number of sitcs thus poses issues and choiccs which will bc studied and debated carefully by scientists and public policy makers, as preparations for efficacy trials continue. The urgency of the HIV pandemic mandates that the opportunities to succeed be maximized, rather than merely minimizing the risks of failure 1°°. On balance, initiation of well designed and controlled trials will provide the stimulus for accelerating the timetable for successful HIV vaccine development. E T H I C A L , S O C I A L A N D L E G A L ISSUES Ethical and social considerations

When candidate vaccines initially became available in thc US (1986), the prospect of conducting clinical trials in man faced a series of complex scientific as well as policy issues. At the time, thc biomedical community held diverse opinions concerning the need, wisdom and timing of entry into human trials 1°1. Early clinical trials were prompted by the need for preliminary information about the human immune response to HIV components to serve as the basis for later trials, and to assist in validating the relevance and predictive value of non-human primate studies. Experience to date has borne out the value of this course. The NIAID, NIH is actively engaged in a national programme of discovery, development and phase 1/2 clinical trials in anticipation of identifying the most promising candidates for efficacy trials. Sound ethics begins with good scicncc. Vaccines to bc used in large population efficacy studies should represent the hig lest standards of international scientific thinking. A vaccine should have a substantial expectation of successful control of HIV infection and/or gaining important new knowledge and should carry minimal anticipated risk of causing harm. The WHO//Global Program for AIDS (GPA) is developing criteria for review and endorsement of proposed candidate vaccines 66. Clinical trials in the US have been conducted with relatively limited numbers of highly motivated, usually


well educated individuals and with close, one-to-one personalized monitoring. Large population efficacy trials, especially in developing countries with high HIV-1 seropositivity rates, will confront new challenges, for the participants may have very different educational levels, life-styles, customs, laws, language, perceptions and expectations. Individual informed consent is particularly important, and, when appropriate, community agreement must ensure that the volunteer understands the full nature and expectations of the study. Moreover, sponsoring investigators and manufacturers may encounter unaccustomed requests to share protocol and study design with local colleagues in host countries and to conform to new and multiple levels of review, oversight, safeguards and accountability as a result of guidelines under development by international bodies i o2-106 These agencies are currently formulating recommendations for ethical, social and legal aspects of the conduct of research in human subjects. The adoption of a memorandum of understanding between sponsors and collaborators in host countries has been suggested. The creation of local or regional multi-disciplinary ethical review committees with responsibility to review and oversee ethical aspects of proposals has also been recommended. The ethics committee is to consider scientific aspects as well, and may seek advice from technical bodies, but will reach its own decision on scientific soundness. It is hoped that the multi-level review process envisioned will be on an accelerated schedule. AIDS efficacy trials in developing countries will receive world scrutiny and any real or perceived physical or social injury may adversely impact global AIDS vaccine testing efforts. A vaccine-induced positive blood test for AIDS may impose social and personal discrimination. Induction of seropositivity by an envelope-based vaccine can be distinguished readily from acquired infection in the Western blot assay. However, vaccines based on combined 9ag-pol-env proteins may develop patterns indistinguishable from infection. It is essential to develop inexpensive and reliable assays or markers which can readily permit distinction between responses to immunization and naturally acquired infection. Detection of wild-type HIV 'breakthrough infection' will also be facilitated by such markers. Eventual development of acceptably safe and effective vaccines will be accompanied by a public health imperative to make vaccine available to sites participating in trials and, in a larger sense, to endemic areas generally. The latter is an international problem which will draw broadly on international expertise and financing. Medical liability considerations Medical liability remains an important issue and may be a disincentive to potential sponsors of trials in developing countries l°s'l°v. To date, no significant injury has been associated with US phase 1 trials in seronegative individuals. This is reassuring; however, this record is based upon immunization of several hundred low-risk, non-pregnant, healthy, cooperative volunteers with a relatively short follow-up period. They were immunized almost entirely with envelope-derived products and remain unchallenged with wild-type HIV. Vaccines tested in seropositive subjects have also appeared safe x°8. The longer term potential safety hazards, if any, of new vaccine constructs, replicating vectors and novel adjuvants, will continue to be tested. The risk of reactions

to vaccines, when used in large numbers of subjects against a background of endemic health problems in developing countries, including immunodeficient individuals with AIDS, remains to be carefully evaluated. International bodies 1°5 have proposed guidelines addressing unavoidable (non-negligent) injury associated with experimental trials, especially those conducted in developing countries. The guidelines suggest that injured subjects be compensated for temporary or permanent injury or death under an obligation to be agreed to by the sponsor prior to initiation of the study. This mandate for safeguarding subjects brings to the surface several issues that could pose formidable problems for vaccine trial sponsors, e.g. difficulty in obtaining insurance, questions of causal relationship between adverse events and vaccine, application of differing health care systems and standards, as well as potential problems to be encountered in differing legal systems in host and sponsoring countries. These obstacles must be weighed against the positive benefits which will accrue to such countries from the improved health infrastructure that will be a by-product of vaccine trials and the potential for ultimate control of AIDS in these highly endemic populations. Liability issues merit continued international discussion, with particular focus on alternative sources of compensation and innovative legal or administrative remedies.

A C C E L E R A T I N G HIV V A C C I N E DEVELOPMENT Mobilization of international efforts The urgency of the expanding global epidemic mandates that mobilization of international efforts be coordinated to optimize prospects for success. The effort would benefit from enhanced collaboration between private sector concerns (biotechnology and pharmaceutical companies); public sector concerns (World Health Organization, national AIDS programmes, UNICEF, UNDP, World Bank); and non-profit concerns (private foundations; community organizations; academic investigators). The following emerge as major avenues for accelerating HIV vaccine development: 1 International standardization of principal immunological assays utilized in preclinical and clinical testing of HIV vaccines. 2 Phenotypic (antigenic) and genotypic analyses of geographical regional isolates and preparation of viral and serological reagents, challenge stocks and vaccine components. 3 Establishment of mechanisms for regular review of emerging data concerning the status of AIDS vaccine development and availability of research resources to address new priorities and opportunities. 4 Linkage of HIV vaccine programmes with established non-HIV vaccine research and development programmes, to enable sharing of experience and expertise, e.g. STD and tropical diseases. 5 Development of economic incentives to promote greater pharmaceutical industry participation in HIV vaccine development. 6 Resolution of liability concerns in the conduct of HIV vaccine trials. Most of the above items are already on the agenda of


1049

Development

o f a n A I D S v a c c i n e , a c r i t i c a l a p p r a i s a l : D. T. K a r z o n et al.

international and national programmes. Their general recognition and implementation deserve universal support.

25

ACKNOWLEDGEMENTS The authors are grateful to Ms Pal Seitz for assistance in the preparation of the manuscript.

26

27

REFERENCES 1 White, D.O. and Fenner, F. Pathogenesis and pathology of viral infections. In: Medical Virology 3rd edn, Academic Press, Orlando, FL, 1986, pp 134 143 2 Ada, G.L. The immunological principles of vaccination. Lancet 1990, 335, 523-526 3 Wolinsky, S.M. Selective transmission of human immunodeficiency virus type-1 variants from mothers to infants. Science 1992, 255, 1134~1137 4 Cooper, D.A., Gold, J., Maclean, P. et al. Acute AIDS retrovirus infection; definition of a clinical illness associated with seroconversion. Lancet 1985, i, 537 540 5 Albert, J. Isolation of human immunodeficiency virus (HIV) from plasma during primary HIV infection. J. Med. Virol. 1987, 23, 67 73 6 Clark, S.J. High titres of cytopathic virus in plasma of patients with symptomatic primary HIV-1 infection. N. Engl. J. Med. 1991, 324, 9,54-960 7 Daar, E.S. Transient high levels of viremia in patients with primary human immunodeficiency virus type 1 infection. N. Engl. J. Med. 1991, 324, 961464 8 Von Gegerfelt, A., Albert, J., Morfeldt-Manson, L., et al. Isolatespecific neutralizing antibodies in patients with progressive HIV-l-related disease. Virology 1991, 185, 162 168 9 Emini, EA., Schleif, W.A., Nunberg, J.H. et al. Prevention of HIV-1 infection in chimpanzees by gp120 V3 domain-specific monoclonal antibody. Nature 1992, 355, 726-730 10 Prince, A.M., Reesink, H., Pascual, D. et al. Prevention of HIV infection by passive immunization with HIV immunoglobulin. AIDS Res. Hum. Retroviruses 1991, 7, 971-973 11 Robey, W.G., Safai, B., Oroszlan, S. et al. Characterization of envelope and core structural gene products of HTLV-III with sera from AIDS patients. Science 1985, 238, 593~595 12 Veronese, F.D., DeVico, A.L., Copeland, T.D. et al. Characterization of gp41 as the transmembrane protein coded by the HTLV-III/LAV envelope gene. Science 1985, 22g, 1402 1405 13 Willey, R.L., Bonifacino, J.S., Potts. B.J. et al. Biosynthesis, cleavage, and degradation of the human immunodeficiency virus 1 envelope glycoprotein gp160. Proc. Natl Acad. Sci. USA 1988, 85, 9580-9584 14 Dalgleish, A.G, Beverley, P.C., Clapham, P.R. et al. The CD4 (T4) antigen is an essential component of the receptor for the AIDS retrovirus. Nature 1984, 312, 763767 15 Klatzmann, D., Champagne, E., Chamaret, S. et al. T-lymphocyte T4 molecule behaves as the receptor for human retrovirus LAV. Nature 1984, 312, 767 771 16 McDougal, J.S., Kennedy, MS., Sligh, JM. et al. Binding of HTLV-III/LAV to T4÷T cells by a complex of the 110K viral protein and the T4 molecule. Science 1986, 231, 382-385 17 Lifson, J.D., Feinberg, M.B., Reyes, G.R. et al. Induction of CD4-dependent cell fusion by the HTLV-III/LAV envelope glycoprotein. Nature 1986, 323, 725-728 18 Kowalski, M., Potz, J., Basiripour, L. et al. Functional regions of the envelope glycoprotein of human immunodeficiency virus type 1. Science 1987, 237, 1351 1355 19 Stein, B.S., Gowda, S.D., Lifson, J.D. et al. pH-independent HIV entry into CD4-positive T cells via virus envelope fusion to the plasma membrane. Cell 1987, 49, 659-668 20 Putney, S.D. and McKeating, J.A. Antigenic variation in HIV. AIDS 1990, 4, $129 21 Moore, J.P. and Nara, P.L. The role of the V3 loop of gp120 in HIV infection. AIDS 1991, 5, $21~23 22 Hwang, S.S., Boyle, T.J., Lyerly, H.K. et al. Identification of the envelope V3 loop as the primary determinant of cell tropism in HIV-I. Science 1991, 253, 71 74 23 Javaherian, K., Langlois, A.J., LaRosa, GJ. et al. Broadly neutralizing antibodies elicited by the hypervariable neutralizing determinant of HIV-I. Science 1990, 250, 1590-1593 24 Ho, D.D, McKeating, J.A., Li, X.L. et al. Conformational epitope on gp120 important in CD4 binding and human immunodeficiency

1050


28

29

30

31

32

33

34 35 36

37

38 39

40

41

42 43

44

45

46

47

48

49

virus type 1 neutralization identified by a human monoclonal antibody. J. Virol. 1991, 65, 489~493 Thali, M., Olshevsky, U., Furman, C. et al. Characterization of a discontinuous human immunodeficiency virus type 1 gp120 epitope recognized by a broadly reactive neutralizing human monoclonal antibody. J. Virol. 1991, 65, 6188 6193 Colonno, R.J., Condra, J.H., Mizutani, S. et al. Evidence for the direct involvement of the rhinovirus canyon in receptor binding. Proc. Natl Acad. Sci. USA 1988, 85, 544~5453 Steimer, KS., Scandella, C.J., Skiles, P.V. et al. Neutralization of divergent HIV-1 isolates by conformation-dependent human antibodies to gp120. Science 1991, 254, 105-108 Kang, C.Y., Nara, P., Chamat, S. et al. Evidence for non-V3-specific neutralizing antibodies that interfere with gp120/CD4 binding in human immunodeficiency virus 1-infected humans. Prec. NaU Acad. Sci. USA 1991, 88, 6171-6175 Buchbinder, A., Karwowska, S., Gorny, M.K. etal. Synergy between human monoclonal antibodies to HIV extends their effective biologic activity against homologous and divergent strains. AIDS Res. Hum. Retroviruses 1992, 8, 425~.27 Tilley, S.A., Honnen, W.J., Racho, M.E. et al. Synergistic neutralization of HIV-1 by human monoclonal antibodies against the V3 loop and the CD4-binding site of gp120. AIDS Res. Hum. Retroviruses 1992, 8, 461 467 Nara, P.L., Smit, L., Dunlop, N. et al. Emergence of viruses resistant to neutralization by V3-specific antibodies in experimental human immunodeficiency virus type 1 IIIB infection of chimpanzees. J. Virol. 1990, 64, 3779-3791 Homsy, J., Meyer, M, Tateno, M e t al. The Fc and not CD4 receptor mediates antibody enhancement of HIV infection in human cells. Science 1989, 244, 1357 1360 McKeating, J.A., Griffiths, P.D. and Weiss, R.A. HIV susceptibility conferred to human fibroblasts by cytomegalovirus-induced Fc receptor. Nature 1990, 343, 659~661 Cease, K.B. Peptide component vaccine engineering: Targeting the AIDS virus. Intern. Rev. Immunol. 1990, 7, 85-107 Autran, B. and Letvin, NL. HIV epitopes recognized by cytotoxic T-lymphocytes. AIDS 1991, 5, $145 $150 Phillips, R.E., Rowland-Jones, S., Nixon, D.F. et al. Human immunodeficiency virus genetic variation that can escape cytotoxic T cell recognition. Nature 1991, 354, 453-459 Takahashi, H., Merli, S., Putney, SD. et al. A single amino acid interchange yields reciprocal CTL specificities for HIV-1 gp160. Science 1989, 246, 11& 124 Tyler, D.S., Lyerly, H.K. and Weinhold, K.J. Minireview: Anti-HIV-1 ADCC. AIDS Res. Hum. Retroviruses 1989, 5, 557563 McGhee, J.R., Mestecky, J., Dertzbaugh, M.T. et al. The mucosal immune system: from fundamental concepts to vaccine development. Vaccine 1992, 10, 75-88 McGhee, J.R. and Mestecky, J. The mucosal immune system in HIV infection and prospects for mucosal immunity to AIDS. Annu. Rev. AIDS Res. In press Sutjipto, S., Pedersen, NC., Miller, C.J. et al. Inactivated simian immunodeficiency virus vaccine failed to protect rhesus macaques from intravenous or genital mucosal infection but delayed disease in intravenously exposed animals. J. Virol. 1990, 64, 229(~2297 Gardner, MB. and Hu, S.L. SIV vaccines, 1991 a year in review. AIDS 1991, 5, $115-$127 Hu, S.L., Abrams, K., Barber, G.N. et al. Protection of macaques against SIV infection by subunit vaccine of SIV envelope glycoprotein gp160. Science 1992, 255, 4,56-459 Cooney, E.L., Corey, L., Hu, S.L. et al. Enhanced immunity to HIV envelope elicited by a combined vaccine regimen consisting of priming with a vaccinia recombinant expressing HIV envelope and boosting with gp160 protein. Science Submitted for publication Siliciano, R.F., Callahan, K., Liu, A.Y. et al. The role of HIV-specific T cells in the pathogenesis and prevention of AIDS. J. Cell. Biochem. 1992, 16E, 7 Chanda, P.K., Natuk, R.J., Dheer, S.K. et al. Helper independent recombinant adenovirus vectors: expression of HIV env or HBV surface antigen. Intern. Rev. Immunol. 1990, 7, 66-77 Chattfield, S., Strugnell, R. and Dougan, G. Live salmonella as vaccines and carriers of foreign antigenic determinants. Vaccine 1989, 7, 495~498 Aldovini, A. and Young, R.A. Humoral and cell-mediated immune responses to live recombinant BCG HIV vaccines. Nature 1991, 351, 479-482 Steimer, K.S., Klasse, P.J. and McKeating, J.A. HIV-1 neutralization directed to epitopes other than linear V3 determinants. AIDS 1991, 5, $135-$143

Development

56

51

52 53 54 55 56 57 56 59 60 61

62 63 64 65 56

67 68 69 70 71 72 73 74 75

76 77

78

o f a n A I D S v a c c i n e , a c r i t i c a l a p p r a i s a l : D. T. K a r z o n et al.

Golding, H., Robey, F.A., Gates, F.T. et al. Identification of homologous regions in human immunodeficiency virus gp41 and human MHC class II beta I domain. J. Exp. Med. 1988,167, 914-923 Girard, M., Kieny, M.P., Pinter, A. et al. Immunization of chimpanzees confers protection against challenge with human immunodeficiency virus. Proc. Natl Acad. Sci. USA 1991, 88,

79

542-546

80

Aldovini, A., Young, R.A. and Palker, T.J. New AIDS vaccine candidates: antigen delivery and design. AIDS 1991,5, $151-$158 Wang, C.Y., Looney, D.J., Li, M.L. et al. Long-term high-titre neutralizing activity induced by octameric synthetic HIV-1 antigen. Science 1991, 254, 285-288 Spriggs, D.R. and Koff, W.C. (eds) Topics in Vaccine Adjuvant Research. CRC Press, Boca Raton, FL, 1991 Warren, H.S., Vogel, F.R. and Chedid, L.A. Current status of immunological adjuvants. Annu. Rev. Immunol. 1986, 4, 369-388 Deres, K., Schild, H., Wiesmuller, K.-H. et al. In vivo priming of virus-specific cytotoxic T lymphocytes with synthetic lipopeptide vaccine. Nature 1989, 342, 561-564 Adams, S.E., Dawson, K.M., Gull, K. et al. The expression of hybrid HIV:Ty virus-like particles in yeast. Nature 1987, 329, 68-70 Gregoriadis, G., Davis, D. and Davies, A. Liposomes as immunological adjuvants: antigen incorporation studies. Vaccine 1987, 5, 145-151 Takahashi, H., Takeshita, T., Morein, B. et al. Induction of cytotoxic T cells by immunization with complexes of purified HIV-1 envelope protein. Nature 1990, 344, 873-875 Garcon, N. and Six, H.R. Universal vaccine carrier: liposomes that provide T dependent help to weak antigens. J. Immunol. 1991, 146, 3697-3702 Milich, D.R., Hughes, J.L., McLachlan, A. etal. Hepatitis B synthetic immunogen comprised of nucleocapsid T cell sites and an envelope B cell epitope. Proc. Natl Acad. Sci. USA 1988, 85, 1610-1614 Eldridge, J.H., Stass, J.K., Meulbroek, J.A. et al. Biodegradable microspheres as a vaccine delivery system. Mol. Immunol. 1991, 28, 287-294 Ruby, J. and Ramshaw, I.A. Expression of IL-1 by vaccinia virus: enhanced immune responses to vaccinia and co-expressed antigens. Cytokine 1991, 3, 1 Mosmann, T.R. and Coffman, R.L. Heterogeneity of cytokine secretion patterns by helper T cells. Adv. Immunology 1989, 46, 111-147 Dertzbaugh, M.T. and Elson, C.O. Cholera toxin as a mucosal adjuvant. In: Topics in Vaccine Adjuvant Research (Eds Spriggs, D.R. and Koff, W.C.). CRC Press, Boca Raton, FL, 1991, pp 119-131 Esparza, J., Osmanov, S., Kallings, L.O. et al. Planning for HIV vaccine trials: the World Health Organization perspective. AIDS 1991, 5, $159-$165 Horstmann, D,M. The use of primates in experimental viral infection~rubella and the rubella syndrome. Ann. NY Acad. Sci. 1969, 162, 594-597 Melnick, J.L. Live attenuated poliovaccines. In: Vaccines (Eds Plotkin, S.A. and Mortimer, E.A.) W.B. Saunders, Philadelphia, 1988, pp 11~157 Weibel, R.E., Mumps vaccine. In: Vaccines (Eds Plotkin, S.A. and Mortimer, E.A.) W.B. Saunders, Philadelphia, 1988, pp223-234 Gordon, J.E. The preparation and use of yellow fever vaccine. In: Virus and Rickettsial Diseases, Harvard University Press, Cambridge, MA, 1948, pp767-788 Jarrett, O., Yamamoto, J.K. and Neil, J.C. Feline immunodeficiency virus as a model for AIDS vaccination. AIDS 1990, 4, $163~$165 Biberfeld, G. and Emini, E.A. Progress with HIV vaccines. AIDS 1991, 5, $129~$133 NcCune, J.M., Namikawa, R., Kaneshima, H. et al. The SCID-hu mouse: Murine model for the analysis of human hematolymphoid differentiation and function. Science 1988, 241, 1632-1639 Mosier, D.E., Gulizia, R.J., Baird, S.M. et al. Transfer of a functional human immune system to mice with severe combined immunodeficiency. Nature 1988, 335, 256-259 Berman, P.W., Gregory, T.J., Riddle, L. et al. Protection of chimpanzees from infection by HIV-1 after vaccination with recombinant glycoprotein gp120 but not gp160. Nature 1990, 345, 622425 Stott, E.J. Anti-cell antibody in macaques. Nature 1991, 353, 393 Dolin, R., Graham, B.S., Greenberg, S.B. et al. The safety and immunogenicity of a human immunodeficiency virus type 1 (HIV-1) recombinant gp160 candidate vaccine in humans. Ann. Intern. Med. 1991, 114, 119-127 Mannhalter, J.W., Pure, M., Wolf, H.M. et al. Immunization of

81

82

83 84

85 86 87

88 89 90 91

92 93

94

95 96 97 98 99 100 101

102

chimpanzees with the HIV-1 glycoprotein gp160 induces longlasting T-cell memory. AIDS Res. Hum. Retroviruses, 1991, 7, 485-493 Wintsch, J., Chaignat, C., Braun, D.G. et al. Safety and immunogenicity of a genetically engineered human immunodeficiency virus vaccine. J. Infect. Dis. 1991, 163, 219-225 Haigwood, N.L., Nara, P.L., Brooks, E. et al. Native but not denatured recombinant human immunodeficiency virus type 1 gp120 generates broad-spectrum neutralizing antibodies in baboons. J. Virol. 1992, 661, 172-182 Berman, P.W., Gregoryo, T.J., Riddle, L. et al. Protection of chimpanzees from infection by HIV-1 after vaccination with recombinant glycoprotein gp120 but not gp160. Nature 1990, 345, 622425 Cooney, E.L., Collier, A.C., Greenberg, P.D. et al. Safety of and immunological response to a recombinant vaccinia virus vaccine expressing HIV envelope glycoprotein. Lancet 1991, 337, 567-572 Sarin, P.S., Sun, D.K., Thornton, A.H. et al. Neutralization of HTLV-III/LAV replication by antiserum to Thymosin ~. Science 1986, 232, 1135-1137 Papsidero, L.D., Sheu, M. and Ruscetti, F.W. Human immunodeficiency virus type-1 neutralizing monoclonal antibodies which react with p17 core protein: characterization and epitope mapping. J. Virol. 1989, 63, 267-272 Mills, K.H., Kitchin, P.A., Mahon, B.P. et al. HIV p24-specific helper T cell clones from immunized primates recognize highly conserved regions of HIV-I. J. Immunol. 1990, 1, 1677-1683 Gilmour, J.E., Senior, J.M., Burns, N.R. et al. A novel method for the purification of HIV-1 p24 protein from hybrid Ty virus-like particles (Ty-VLPs). AIDS 1989, 3, 717-723 Graham, B.S., Belshe, R.B., Clements, M.L. et al. Vaccination of vaccinia-naive adults with HIV-1 gp160 recombinant vaccinia (HIVAC-le) in a blinded controlled, randomized clinical trial. J. Infect. Dis. 1992, 166, 244-252 Zagury, D., Leonard, R., Fouchard, M. et al. Immunization against AIDS in humans, Nature 1987, 326, 249-256 Zagury, D. Bernard, J., Cheynier, R. et al. A group specific anamnestic immune reaction against HIV-1 induced by a candidate vaccine against AIDS. Nature 1988, 332, 728-731 Robinson, W.E. Jr, Montefiori, D.C. and Mitchell, W.M. Antibodydependent enhancement of human immunodeficiency virus type 1 infection. Lancet 1988, i, 790-794 Takeda, A., Tuazon, C.U. and Ennis, F.A. Antibody enhanced infection by HIV-1 via Fc receptor mediated entry. Science 1988, 2A.2, 580-583 Koff, W.C. and Fauci, A.S. Human trials of AIDS vaccines: current status and future directions. AIDS 1989, 3 (suppl), $125-$129 Matthews, T.J., Weinhold, K.J., Lyerly, H.K. et al. Interaction between the human T cell lymphotropic virus type IIIb envelope glycoprotein gp120 and the surface antigen CD4: role of carbohydrate in binding and cell fusion. Proc. NaU Acad. Sci. USA 1987, 84, 5424-5428 Steimer, K.S. and Haigwood, N.L. HIV-1 gp120 as a subunit vaccine. In: AIDS Research Reviews volume 2: (Eds Koff, W.C., Wong-Staal, F. and Kennedy, R.C.) Marcel Dekker, NY, 1992 Leigh Brown, A.J. Sequence variability in human immunodeficiency viruses: pattern and process in viral evolution. AIDS 1991, 5, $3.5~$42 Myers, G., Maclnnes, K. and Korber, B. The emergence of simian/human immunodeficiency viruses. AIDS Res. Hum. Retroviruses 1992, 8, 373-386 Ahearne, P.M., Sebastian, M.W., Morgan, R.A. et al. Identification of CTL reactivity against HIV-1 infected autologous B-cell lymphocyte cell lines. In preparation Koff, W.C. and Hoth, D.F. Development and testing of AIDS vaccines. Science 1988, 241,426-432 Vermund, S.H., Sheon, A.R., Ebner, S. et al. Transmission of the human immunodeficiency virus. In: AIDS Research Reviews Vol. 1, Marcel Dekker, NY, 1991, pp81-136 Koff, W.C. Advances in AIDS vaccine development: a global applied research strategy could expedite the development and testing of vaccine candidates. ASM News 1991, 57, 73 Prospects for Vaccines against HIV Infection. Report of the conference on promoting development of vaccines against human immunodeficiency virus infection and acquired immune deficiency syndrome, 14-15 December 1987. Institute of Medicine, National Academy Press, Washington, DC, 1988, pp iii-23 WHO global programme on AIDS: statement from the consultation on criteria for international testing of candidate HIV vaccines. Geneva, 27 February-2 March 1989, pp I 13


1051

D e v e l o p m e n t of an AIDS vaccine, a critical appraisal: D. T. K a r z o n et al. 103 104 105 106

1052

WHO global programme on AIDS: research priorities for the early 1990s. Geneva, 22 March 1991, pp 1-13 International Guidefines for Ethical Review of Epidemiological Studies. CIOMS, Geneva, 1991 International Ethical Guidelines for Biomedical Research Involving Human Subjects. CIOMS, January 1992, pp 1-37 World Medical Association Declaration of Helsinki. World Medical Association Inc. (amended Sept. 1989)


107

Keystone AIDS Vaccine Liability Project Final Report The Keystone Center, Keystone, CO, May 1990 108 Redfield, R., Birx, D.L., Ketter, N. et al. A phase I evaluation of the safety and immunogenicity of vaccination with recombinant gp160 in patients with early human immunodeficiency virus infection. Military Medical Consortium for Applied Retroviral Research. N. Engl. J. Med. 1991, 324, 1677 1684

The development of a critical appraisal tool for use in systematic reviews addressing questions of prevalence.

OnabotulinumtoxinA for chronic migraine: a critical appraisal.

Development of a novel AIDS vaccine: the HIV-1 transactivator of transcription protein vaccine.

Some notes on critical appraisal of prevalence studies: Comment on: "The development of a critical appraisal tool for use in systematic reviews addressing questions of prevalence".

The niche construction perspective: a critical appraisal.

The trainee year--a critical appraisal.

A checklist for critical appraisal of studies of biological variation.

A critical appraisal of microemulsions for drug delivery: part I.

A critical appraisal of hysteroscopic tubal fulguration for sterilization.

Classification of neoplasms: a critical appraisal.

A critical appraisal of microemulsions for drug delivery: part II.

AIDS.

A critical appraisal of the effectiveness of scientific presentations.

A critical appraisal of atomoxetine in the management of ADHD.

A critical appraisal of "Lokalisationslehre" in the brain.

Fluoroquinolones in the treatment of cystic fibrosis: a critical appraisal.

Clozapine and the dopamine hypothesis of schizophrenia, a critical appraisal.

Toward an effective AIDS vaccine development.

The imaging spectrum of pulmonary tuberculosis: a critical appraisal.

Rights to health care: a critical appraisal.

Chronic traumatic encephalopathy: a critical appraisal.

Smartphone applications for chronic pain management: a critical appraisal.

Guidelines for Reporting Medical Research: A Critical Appraisal.

AIDS vaccine development.