prospects for an aids vaccineaids viruses of animals and man inates the viral invader. the success...

AIDS Viruses of Animals and Man

chimpanzee remains functionally intactduring the persistent HIV I infection.

Studies following in vitro infectionwith HIV I show that all normal T4lymphocytes from the blood of unin-fected chimpanzees are capable of con-trolling the replication of the virus sothat only small numbers of completeinfectious virus particles are producedat any one time in these cells (Fig. 13).Also, the viral infection does not ap-pear to kill the lymphocytes. Moreimportant, the monocytes and mac-rophages derived from the blood- andbone-marrow stem cells of experimen-tally inoculated animals do not appearto be readily infected by the virus. Insupport of this in vivo observation is thefact that current attempts to infect cul-tured chimpanzee monocytes and mac-rophages with human monocyte HIV Ivariants have failed. One other interest-ing finding is the naturally larger con-centration of circulating T8 cells in theblood of chimpanzees. That populationof cells in uninfected chimpanzees maybe responsible for the antiviral control-ling effect against HIV I. In contrast,in vitro studies of lymphocytes fromthe blood of HIV-infected humans andSIV-infected monkeys show that thepresence of T8 lymphocytes seems toslow down but does not eliminate viralreplication in the other lymphocytes.

The fundamental differences in thechimpanzee’s response to HIV infec-tion are being actively explored for ap-plication to the prevention and treat-ment of the human condition. Studiesof the other SIV-adapted African pri-mates mentioned previously should alsocontain clues to the various factors re-sponsible for the carrier state present inthese animals. As described in the sec-tion on SIV, the African green monkeyand various other African primates ap-pear to be successfully adapted to theirvirus, whereas the same virus when putinto Asian primates leads to AIDS. TheHIV I-infected chimpanzees also appear

CONTROLLED VIRAL REPLICATIONIN CHIMP LYMPHOCYTES

Fig. 14. Transmission electron micrographs

of an HIV l-infected lymphocyte from a chim-

panzee. The cell was taken from a culture that

had been infected for 5 days and was at maxi-

mum virus production. (a) Only the boxed area

of the cell membrane of this peripheral blood

lymphocyte could be found budding HIV I par-

ticles (magnified 3500 times). (b) The boxed

area in (a) at a magnification of 10,000. (c) To

contrast with the chimp lymphocyte, we show

a completely degenerated portion of an HIV-infected human lymphocyte magnified 10,000

times. Note the remaining portions of nuclear

chromatin and cytoplasmic vacuoles as well

as the presence of numerous viral particles.

to be successfully adapted to HIV I.That fact suggests that an ancestralHIV I-like virus should be present inwild chimpanzee populations in cen-tral Africa. Furthermore, it looks likeHIV I-infected humans are the counter-part to the SIV- infected Asian mon-keys.

Prospects for an AIDS Vaccine

Given our current understanding oflentiviral infections, it appears that con-ventional vaccine strategies are unsuit-able for direct application to the pre-vention of lentiviral diseases. Since thetime of Jenner and Pasteur, all success-ful human and animal antiviral vaccineshave been made either from virus atten-uated in tissue cultures (called modified-“live” virus particles), or from a partor parts (termed subunit) of the virusthat activates the immune system. Thevaccines confer immunity by elicitingwhat is termed an anamnestic response,which basically means to “not forget.”On subsequent introduction of wild-typevirus (other than lentiviruses) into thebody, infection occurs, but the immunesystem, previously sensitized throughvaccination, rapidly responds and elim-

LoS Alamos Science Fall 1989 83


inates the viral invader. The success ofthese vaccines was due primarily to thenature of the viruses involved. Virusessuccessfully blocked by vaccine-inducedprotection generally do not integratethemselves into the host’s genome anddo not exclusively parasitize the im-mune system of the body. A prototypefor a subunit retroviral-based vaccinehas been developed for cats against fe-line leukemia virus infection. However,further work is still required to optimizeits usefulness and application to the pre-vention of lentiviral diseases. Recently,studies with a formalin-fixed Type Dretrovirus were found to completelyprotect primates from a simian TypeD retrovirus. As yet no vaccines againstany of the retroviruses have been madefrom approaches using modified-livevirus.

The immune activation induced by asuccessful vaccine is controlled by theimmune system’s activator and suppres-sor networks. With time, the inducedprotective state falls to levels of non-protection, but the state is permanentlyprogrammed into the immune system’smemory network. Reinfection of thehost cells with the real pathogenic viralagent causes release of chemical sig-nals that rapidly recruit and deploy theappropriate memory cells. These cellsproduce antibodies, T4 cells, and T8cells that eliminate the virus before itcan spread to a critical number of sus-ceptible target cells and cause significantlife-threatening disease.

A key point with regard to AIDS isthe fact that the immune response gen-erated by our current viral-based vac-cines does not always prevent the ini-tial infection. Since HIV is capable ofintegrating itself into the genetic mate-rial of infected cells, a vaccine wouldhave to produce a constant state of im-mune protection, which could totallyblock the initial infection of the hostcells at all times. Such complete andconstant protection has never before

84

Table 5

Problems with Vaccine Development against HIV

1. Integration of HIV genetic material into cellular DNA.

2. Regulatory genes responsible for controlled, low-level viral expression.

3. Similar cell populations serve as both targets of viral infection and vehicles ofimmune protection.

4. Viral activation and spread from antigen-presenting macrophages to T cells dur-ing normal immunogenic episodes.

5. Molecular mimicry between viral envelope proteins and MHC II molecules.

6. Rapid rate of envelope mutations.

7. Hypervariability of a single immunodominant neutralization epitope may act as adecoy to antibody producing cells.

8. Viral envelope proteins are poor immunogens due to high carbohydrate content.

9. Rapid shedding of viral envelope glycoproteins.

10. Cell-to-cell fusion, resulting in transmission of viral RNA without complete as-sembly of virus particles.

11. Presence of antibody induces viral latency.

12. Absence of complement-mediated cytolysis or direct complement activation.

13. No efficacious vaccine developed for any lentiviral infections of other species.

14. The need to maintain a constant level of protective immune activation in theface of an immune system suppressor network.

been accomplished and works in directopposition to the normal immune sup-pressor network, which dampens, orturns down, specific immune responses.A state of perpetual immune activationmay have as yet undefined detrimentalconsequences to the host. Only throughextensive and creative research will webe able to design the type of new gen-eration vaccines required to defeat theAIDS virus and its distant relatives.

Analysis of the comparative spec-trum of lentiviral diseases of animalsand man are providing important cluesto pathogenesis and host response. Thenonsusceptible versus susceptible statesthat occur, respectively, in adapted ver-sus nonadapted species need to be stud-ied further. For lentiviral diseases itappears that protective immunity will

not be conferred through the classicalimmune mechanisms but rather will re-quire a state similar to that of host adap-tation. The mechanisms of host adap-tation has never been investigated norunderstood in detail. Now the investiga-tion of such mechanisms seems urgent.We will have to understand novel viral-controlling mechanisms, the nature ofthe immune state that prevents the initialinfection, and methods for establishingpersistent but nondeleterious states ofprotective immune activation. As yet,no antiviral vaccine can claim such pro-tection. Nevertheless, continued stud-ies of all lentiviruses, as well as otherprimate retroviruses, will surely revealimportant clues to the understanding andcontrol of the disease process in the ani-

Los Alamos Science Fall 1989


The Search H ow does a host usually developa state of protection against aninvading virus? Three major

host responses to invading viruses in-clude activation of complement, produc-tion of neutralizing and complement-fixing antibodies, and cell-mediated im-mune responses. Traditionally, whena new viral disease is recognized in aspecies, efforts to understand the pro-tective immune states are derived fromits surviving members. These individ-uals serve as immune benchmarks, andsubsequent studies often reveal impor-tant clues to the eventual production ofa vaccine. Here we will review studiesof the major antiviral immune responsesto HIV and see that none of them arecompletely effective, although some av-enues of developing traditional vaccinesfor AIDS are still open.

ProtectiveHostResponses

Complement. One possible responseto HIV would be the activation of thecomplement system, known to be apowerful, continuous, ever-present,microbe-eliminating system (see Fig. 11in the main article). Complement is agroup of serum proteins circulating inthe bloodstream that bind to, becomeactivated, and destroy invading mi-crobes by creating holes in their surfacemembranes. Complement proteins aresynthesized by activated macrophages,liver cells, and epithelial cells. Com-plement inactivates some Type C on-coviruses directly due to the presenceof as yet undefined receptors on the vi-ral envelopes. Complement can alsowork in conjunction with antibodies.The antibodies produced in responseto a viral infection may bind both tocomplement and to the virus or virallyinfected cells, resulting in destructionof the intruder. The destruction of vi-rally infected cells through this mech-anism is called antibody-dependent,complement-mediated cytolysis (ACC).The lentiviruses as a group, unfortu-

nately, appear to be resistant to destruc-tion by complement.

Studies performed in my laboratoryin 1987 showed no evidence of ACCactivity in humans at various stagesof AIDS despite the presence of largeamounts of antibody directed againstboth HIV and HIV-infected cells. Theabsence of ACC is also documented invisna-maedi. No ACC activity has beenreported for the other animal-lentivirussystems mentioned here. Both my laband others have shown that human com-plement is incapable of inactivating HIVeither directly or in the presence of neu-tralizing antibody. Recently, we havediscovered a heat-sensitive serum factorin various laboratory and wild animalspecies that does inactivate the humanAIDS virus in vitro. Further studies areunderway to characterize this factor, orfactors, and to understand how to recruitits activity in humans and why humancomplement does not work against HIV.

Neutralizing Antibody. Neutralizingantibodies have been shown to be oneof the major lines of defense in viraldiseases of human and other animals.Following the infection of the host bythe AIDS virus, plasma cells produceantibodies directed against various partsof the virus. The antibodies are of twomajor types, functional (when they bindto the virus they inactivate or destroyit) and nonfunctional. A nonfunctionalantibody recognizes various parts of thevirus; however, they do not mediate anyantiviral effects in vitro or in vivo. Alsothe nonfunctional antibodies can coatthe virus and thereby block or inter-fere with otherwise effective antibodies,such as neutralizing or complement-fixing ones. An antibody that is coat-ing a virus can also, as previously de-scribed, facilitate entry of the virus intomonocytes and macrophages, thus in-fecting these cells. Some evidence forthis type of antibody-facilitated infectiv-

Los Alamos Science Fall 1989 85


ity has been reported in the visna-maedisystem.

Functional neutralizing antibodiesare produced by plasma cells derivedfrom B lymphocytes that have beenspecifically activated against a partic-ular antigen. We have already indicatedthat the ability of the neutralizing anti-body to inactivate lentiviruses is highlyvariable. Horses infected with the EIAvirus make antibody that is capable ofneutralizing the initial infecting viralstrain. However, antigenic drift even-tually produces viruses that can avoidthose neutralizing antibodies.

Our information on visna-maedi ismore detailed. In vitro, antibody againstvisna-maedi is capable of neutralizingthe virus so that it cannot infect a cell.However, neutralization of the virus oc-curs only if the virus and antibody allowed to interact for 15 minutes clonger before being introduced to thetarget cells. It appears that after thispreincubation the antibody prevents thevirus from attaching to the sheep’s cells.However, when the virus and antibodyare added to the target cells simulta-neously, no neutralization of the virusoccurs. These studies suggest that theantibody produced during the infectionis not biologically functional in vivo. Inthe host the virus probably encountersand infects target cells before neutral-izing antibody has sufficient time toneutralize it. The virus’s escape fromantibodies appears to be related to thehigh sugar content of the viral envelopeproteins, which conceal neutralizationepitopes (protein shapes that serve asantibody binding sites).

Fortunately, the neutralizing anti-body present in HIV-infected humans,HIV-infected chimpanzees, and animalsthat have been vaccinated with the vi-ral envelope protein gp 120 are moreeffective. Recent detailed kinetic stud-ies in my laboratory revealed that theserum from these hosts rapidly neutral-

izes the virus. Subsequent infectivitystudies with HIV I demonstrated thatthe virus can be neutralized at varioustimes, even after it has attached via theCD4 receptor to the host cell (Fig. 1).It appears that the virus binds to sus-ceptible lymphocytes at the diffusion-limited rate of 4.0 x 10-9M (see “TheKinetics of Viral Infectivity”). Afterbinding, however, the virus only slowlyenters the cell by the fusion process.Thus, neutralizing antibody is capable ofneutralizing the virus during the 30 to60 minutes between binding and entryinto the lymphocyte. This is a singu-larly encouraging finding for vaccinedevelopment. However, only the serafrom HIV-infected humans or HIV-infected chimpanzees were capable ofneutralizing more than one HIV I strain.Moreover, these strains may only be asubpopulation of the virus present inany one infected individual. Studiesof the role of neutralizing antibody inpreventing infection of monocytes andmacrophages will have to await the de-velopment of new assay methods.

Our studies also show that neutraliz-ing antibody derived from sera of goatsinfected with the purified envelope ofone HIV strain is also capable of neu-tralizing the virus either before or afterit has bound to a target cell. The ma-jor limiting feature however was thenarrow specificity of the neutralizing an-tibody produced. We, in collaborationwith Jaap Goudsmit, Scott Putney, andothers, have discovered that neutralizingantibody reacts only with the immun-odominant neutralizing epitope of gp 120shown in Fig. 2. Further, this portion ofgp120, which is about 30 amino acids inlength, appears to be changing its aminoacid content rapidly in infected humansand more slowly in chimpanzees. Inparticular, even the first viruses isolatedfrom chimps infected with a specific andwell-characterized human AIDS viruswere resistant to a typing sera made

(b) Binding of Antibody to HIV

MechanismUnknown

OF HIV

Fig. 1. In vitro studies suggest that neutral-

izing antibody against HIV can neutralize the

virus even after it has bound to a target-cell

membrane. The figure shows neutralizing an-

tibodies attaching to the viral envelope after

the virus has bound to and begun to fuse withthe cell membrane. The antibodies somehow

prevent infection, but the details of the neu-tralization mechanism are unknown.

86 Los Alamos Science Fall 1989


HIV ENVELOPE GLYCOPROTEINS

CD4 Binding Site

lmmunodominantLoop

Amino Acid Variability

Constant

Occasionally Altered

q Highly Variable

from goats immunized with gp 120 ofthe original innoculated virus as well assera from other chimps infected with theoriginal virus. We are currently studyingthe amino acid sequence of the relevantpieces of the viral envelope protein inan effort to identify the location andthe types of changes that occur duringviral replication. Additional collabo-rative studies in our lab now indicate

Fig. 2. The structure of the HIV envelope glycoproteins gp120 and gp41, showing constant

regions Ci, including the binding site for the CD4 receptor, and variable regions V i including

the immunodominant loop, a region that binds most of the known neutralizing antibodies.Both the CD4 binding site (c3) and the immunodominant loop V3, have very few sugar

molecules compared to most other regions of gp120 and are therefore exposed to the immune

system. The immunodominant loop consists of about 30 or so amino acids, many of which

are highly variable from one viral strain to another. The loop mediates all known neutralization

phenomena in infected humans, chimpanzees, and viral envelope-based vaccine constructs.

However, there are reports of other binding sites for neutralizing antibody on gp120, which

are not yet as well characterized. Studies show that the ability of the immunodominant loop

to bind neutralizing antibody is altered by the change of a single amino acid in either the

fusion domain of gp41 (see figure) or in the immunodominant loop. Such changes may alter

the conformation of the loop (indicated by the arrows). Also gp41 may contain an as yet

uncharacterized neutralization binding site.

that other sites in the viral envelopealso must contribute to the interactionbetween the neutralizing antibody andthe immunodominant loop (see Fig. 2).When completed, the molecular study ofthe viral-envelopes from chimp isolateswill provide a map of the mutation sitesand allow for a better understanding ofits complexity. Perhaps we will be ableto identify a limited number of locations

and variations that cover the spectrumof gp120 variations made during viralreplication. We might then be able tomanufacture an anti-gpl20 vaccine thatwould be effective against all these vari-ations.

Cell-mediated Immunity. We havejust discussed the ineffectiveness ofboth complement and neutralizing an-



tibody in preventing infection by cell-free HIV particles. Finally, we turn tocell-mediated responses. As mentionedearlier, T8 killer cells are designed todestroy infected cells and are activatedby T4 helper cells. The activation oc-curs when the T4 cells recognize anMHC-lentiviral antigen pair on the sur-face of infected macrophages and lym-phocytes. The T8 cells then circulatearound the body and kill any cells of thebody displaying both the MHC and viralproteins. K, or killer cells (a subset oflymphocytes), and certain T cells canalso destroy virally infected cells thatdo not present MHC antigens on theirsurface. One such mechanism, calledantibody-dependent cell-mediated cyto-toxicity, is the capacity of various an-tiviral antibodies to bind to the infectedcells and thus direct the viral-killing Kcells to them (see Fig. 10 in the mainarticle).

Most HIV-infected humans displayall these antiviral immune mechanismsand still progress to disease and death.One clue to their ineffectiveness may bethe discovery that parts of the envelopeof the feline leukemia virus, a mem-ber of the oncoviral subfamily, seem tosuppress these antiviral immune strate-gies, thus adding to the persistence ofthe virus in the cat’s body. Some re-ports suggest that the envelope of HIVmay have a similar effect on the humanimmune system. Thus we have one pos-sible explanation for the ineffectivenessof cell-mediated immune mechanismsagainst HIV. However, there have beenno reports of other similar immunosup-pressive effects for the other lentiviralinfections of animals.

This short review of protective im-mune responses suggests that protectionagainst HIV, if it can be developed, willprobably have to involve various un-defined elements of host-virus adaptiveresponses in addition to the known an-tiviral immune responses. n

Further ReadingScientific American October 1988. This entire issueis devoted to articles on AIDS, and each article hasa valuable reading list of its own.

The Control of Human Retrovirus Gene Expression,edited by B. Robert Franza, Jr., Bryan R. Cullen,and Flossie Wong-Stall. 1988. Cold Spring Harbor,New York: Cold Spring Harbor Laboratory.

Timothy B. Crawford, William P. Cheevers, PaulaKlevjer-Anderson, and Travis C. McCuire. 1978.Equine infectious anemia: virion characteristics,virus-cell interaction, and host responses. In Per-sistent Viruses: ICN-UCLA Symposia on Molecularand Cellular Biology, Volume XI, 1978, edited byJack G. Stevens, George J. Todaro, and C. FredFox, pp. 727-749. New York: Academic Press.

Alfred S. Evans. 1989. Does HIV cause AIDS? Anhistorical perspective. Journal of Acquired ImmuneDeficiency Syndromes 2: 107-113.

Anthony S. Fauci. 1988. The human immunodefi-ciency virus: infectivity and mechanisms of patho-genesis. Science 239:617-622.

Matthew A. Gonda. 1986. The natural history ofAIDS. Natural History 95:4.

Ashley T. Haase. 1986. Pathogenesis of lentivirusinfections. Nature 322:130-136.

William A. Haseltine, Ernest F. Terwilliger, CraigA. Rosen, and Joseph G. Sodroski. 1988. Structureand function of human pathogenic retroviruses. InRetrovirus Biology: An Emerging Role in HumanDiseases, edited by Robert C. Gallo and FlossieWong-Stall. New York: Marcel Dekker, Inc.

Peter L. Nara, W. Gerard Robey, Larry 0. Arthur,Matthew A. Gonda, David M. Asher, R. Yanagi-hara, Clarence J. Gibbs, Jr., D. Carleton Gajdusek,and Peter J. Fischinger. 1987. Simultaneous iso-lation of simian foamy virus and HTLV-III/LAVfrom chimpanzee lymphocytes following HTLV-IIor LAV inoculation. Archives of Virology 92:183-186.

Peter L. Nara, W. Gerard Robey, Matthew A.Gonda, Stephen G. Carter, and Peter J. Fischinger.1987. Absence of cytotoxic antibody to human im-munodeficiency virus-infected cells in humans andits induction in animals after infection or immu-nization with purified envelope glycoprotein gpl20.Proceedings of the National Academy of Sciences84:3797-3801.

Peter L. Nara, William G. Robey, Larry 0. Arthur,David M. Asher, Axe1 V. Wolff, Clarence J. Gibbs,Jr., D. Carleton Gajdusek, and Peter J. Fischinger.1987. Persistent infection of chimpanzees with hu-man immunodeficiency virus: serological responsesand properties of reisolated viruses. Journal of Vi-rology 61:3173-3180.

P. L. Nara, W. C. Hatch, N. M. Dunlop, W. G.Robey, and P. J. Fischinger. 1987. Simple, rapid,quantitative micro-syncytium forming assay for thedetection of neutralizing antibody against infectiousHTLV-III/LAV. AIDS Research and Human Retro-virus 3:283-302.

P. L. Nara and P. J. Fischinger. 1988. Quantitativeinfectivity microassay for HIV-l and -2. Nature332:469-470.

Peter L. Nara. 1989. HIV-1 neutralization: evi-dence for rapid, binding/postbinding neutralizationfrom infected humans, chimpanzees, and gpl20-vaccinated animals. In Vaccines 89: Modern Ap-proaches to New Vaccines Including Prevention ofAIDS, edited by Richard A. Lemer, Harold Gins-berg, Robert M. Channock, and Fred Brown, pp.137-144. Cold Spring Harbor, New York: ColdSpring Harbor Laboratory.

P. L. Nara, W. Hatch, J. Kessler, J. Kelliher, S.Carter, J. Ward, D. Looney, G. Ehrlich, H. Gendle-man, and R. C. Gallo. 1989. The biology of HIV-IIIB infection in the chimpanzee: in vivo and invitro correlations. Journal of Medical Primatology,in press.

0. Narayan, M. C. Zink, D. Huso, D. Sheffer, S.Crane, S. Kennedy-Stoskopf, P. E. Jolly, and J.E. Clements. 1988. Lentiviruses of animals arebiological models of the human immunodeficiencyviruses. Microbial Pathogenesis 5: 149-157.

G. Petursson, P. A. Palsson, and G. Georgsson.1989. Maedi-visna in sheep: host-virus interactionsand utilization as a model, Intervirology 30 (sup-plement 1):36-44.

Howard M. Temin. 1989. Is HIV unique or merelydifferent? Journal of Acquired Immune DeficiencySyndromes 2: l-9.

88 Los Alamos Science Fall 1989


Acknowledgments

The author wishes to acknowledge the veterinaryand medical researchers who have, over the past40 years, studied these persistent viral infectionsof animals and have contributed to the knowledgethat has rapidly advanced the research of humanAIDS: Bjorn Sigurdsson, Gudmundur Georgsson,Halldor Thormar, Pall Palsson, Opendra Narayan,Ashley Haase, Neal Nathanson, Gudmundur Peturs-son, Travis McGuire, Lane Perryman, Y. Kono, H.Sentsui, Leroy Coggins, and Timothy Crawford. Iwould also like to extend my deepest personal re-gard to Necia Cooper, Dixie McDonald, and Glo-ria Sharp, editor, office manager, and graphics de-

Peter Nara received a B.S. in zoology from Col-orado State University in 1977 and, by 1986, hadreceived an M.S., a D.V.M. (Doctor of VeterinaryMedicine), and a Ph.D. from Ohio State Univer-sity. For his doctoral work he studied mechanismsof viral inactivation, including identification of theresponsible proteins, species distribution, and devel-opmental biology, associated with a particular on-coviral inactivating factor. He is currently chiefof the Virus Biology Section, Laboratory of Tu-mor Cell Biology, at the National Cancer Institute-Frederick Cancer Research Facility in Frederick,Maryland and is a member of numerous scientificcommittees that deal with AIDS, HIV, and relatedviruses. Recently, he completed a four-year com-parative pathology residency at the Armed ForcesInstitute of Pathology. His major areas of researchinterest include comparative pathobiology, cornparative tumor biology, and comparative virology-especially virology that will shed light on the trou-blesome retroviruses.

signer, as well as the rest of the Los Alamos Sciencestaff, for their excellent technical and creative sup-port so critical in the production of a good reviewarticle. I would also like to thank my courageouswife, Brenda, and my children, DeAnna and KellyAnn, for their encouragement and support in theseendeavors. Finally, I would like to thank my staffat the Virus Biology Section for their dynamic andenthusiastic support.

The editor and author would like to thank the au-thors, designer, and publisher of Immunology (byIvan Roitt, Jonathan Brostoff, and David Male,Grove Medical Publishing, Ltd., London, 1985) forthe inspiration their beautifully illustrated volumeprovided in the creation of this article.


prospects for an aids vaccineaids viruses of animals and man inates the viral invader. the success...

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