AD-A237 303I I~n Ilii1111111111 11111 lll l 1iiiIHUMAN IMMUNE RESPONSE TO HTLV-III VIRUS INFECTION

IN ACQUIRED IMMUNODEFICIENCY SYNDROME

REPORT DTICA LECTTJUN 2 6 1991

FRANCIS A. ENNIS G

OCTOBER 28, 1990

Supported by

U.S. ARMY MEDICAL RESFARCH AND DEVELOPMENT COMMANDFort Detrick, Frederick, Maryland 21702-5012 ..r

Contract No. DAMDI7-86-C-62 ,o.

Cb7

University of Massachusetts Medicai'Center Avlabilty oaoeWorcester, MA 01605 fiii

i ;Dl % ISloifti~

Approved for public release; distribution unlimited

The findings in this report are not to be construed as an officialDepartment of the Army position unless so designated by other

authorized documents

91-0332791 6 24 132 itn u

f nitro AppoedREPORT DOCUMENTATION PAGE OMB NO 0704.0188

IA REPORT SECuRITY CLASSIFICATION b. RESTRICTIVE MARKINGS Corp Oate Jun 30, 196

Unclassified2,a SECURIY CLASSIFICAIION AUIIIORIIY 3 DISIRIB'UIION/AVAILABILIIY Or REPORT

2b. OECL ASSIFI(AIION/UOWNGIADING SCHIEDULE Approved for public releasedistribution unlimited

4 PERFORMING ORGANIZAIION REPORT NUMBER(S) 5. MONITORING ORGANIZATION REPORT NUMBER(S)

Ga. NAME OF PERFORMING ORGANIZATION 16b. OFFICE SYMBOL 7a. NAME OF MONITORING ORGANIZATION

University of Massachusetts (If applicable)Medical Center I

6c. ADDRESS (City, State, and ZIP Code) 7b. ADDRESS (City, State, and ZIP Code)

Worcester, MA 01655

Ba. NAME OF FUNDING I SPONSORING ob. OFFICE SYMBOL 9. PROCUREMENT INSTRUMENT IDENTIFICATION NUMBERORGANIZATION U.S. Army Medical [( (If applicable) DAMDI7-86-C-6279

Research & DevelopmentCommand

Bc. ADDRESS (City, Sta to., and ZIP Code) io. SOURCE OF FUNDING NUMBERS

Fort Detrick PROGRAM PROJECT I TASK WORK UNIT

Frederick, MD. 20702-5012 ELEMENT NO. NO. NO. ACCESSION NO

63105A 63105DH29 AD 019

11. TITLE (Include Security Classification,)

Human Immune Response to HTLV III Virus Infection in Acquired Immunodeficiency Syndrome

12. PERSONAL AUTHOR(S)

Ennis, Francis, A.13a. TYPE OF REPOR r ib TIME COVERED 14. DATE OF REPORT (Year Month,. 5ay) 11. PAGE COUNTAnnual/Final FROM 29/86 TO7/31/90 1990 October 28 11

16. SUPPLEMENTARY NOTATION

Annual report covers 29 September 1989 - 31 July 1990

17 (COSATI (CODES 18. SUBJECT ItERMS (Continue on reverse if necessary and identify by block number)

FIELD GROUP SUB-GROUP AIDS; IIIV-1, Retrovirus, Lentivirus, Immunology06 03 RI06 13

19. ABSTRACT (Continue on reverse if necessary and identify by block number)

This contract was awarded for a three year period to study aspects of the human cellular an(humoral antibody responses to HIV-1 infections.The major research findings during this study were:1. The memory lymphocytes of HIV-1 infected individuals fail to respond to recall viral -

antigens including HIV-1 antigen early in the course of HIV-1 infections. This findingmade aspects of these research investigations extremely. difficult especially the study of

T cell responses to HIV-1.2. We generated human CD4+ and CD8+ CTL clones to novel epitopes on the HIV-1 gag protein.3. We generated a human CD8+ CTL clone to a novel epitope on the envelope glycoprotein gp

160.4. We establsihed that the sera of HIV-1 infected patients contain antibodies which enhance

infection of FcR receptor bearing cells.5. We demonstrated the antibody dependent enhancement of HIV-I infection requires two

ree~tors,.Fc receptors aid CD4.20 DISTRIBUTION/AVAILABILITY or ABSTRACT 121 ABSTRACT SECURITY CLASSIFICATION

[ UtNCLASSIrIED/UNLIMIIED EN SAME AS RPT" U II USERS Unclassified72a NAME OF RIESPONSIBLE ITNDIVIDUAL 22 1ELEPIlQNE..fbtL Ide Area Code) 22c FILE SYMBOL

Mary Frances Bostian 0) 663-,, I SGRD-RMI-S

DU FORM 1473, 84 MAR 93 APR edition may he tried until exhausted ,,EcV.5ITY .$.AIrI MION . _ , 1_ PAGfAll other editions are obsolete,

Summary

This contract was awarded for a three year period to studyaspects of the human cellular and humoral antibody responses toHIV-l infections.

The major research findings during this study were:

1. The memory lymphocytes of HIV-1 infected individualsfail to respond to recall viral antigens including HIV-1antigen early in the course of HIV-I infections. Thisfinding made aspects of these research investigationsextremely difficult especially the study of T cellresponses to HIV-I.

2. We generated human CD4+ and CD8+ CTL clones to novelepitopes on the HIV-I gag protein.

3. We generated a human CD8+ CTL clone to a novel epitopeon the envelope glycoprotein gpl60.

4. We established that the sera of HIV-l infected patientscontain antibodies which enhance infection of FcRreceptor bearing cells.

5. We demonstrated the antibody dependent enhancement ofHIV-l infection requires two receptors, Fc receptors andCD4.

B. Foreword

1. Copyright material: NA

2. Limited distribution material: NA

3. Commercial organizations, etc.: NA

4. Animal experimentation: NA

5. For the protection of human subjects the investigatorshave adhered to policies of applicable Law 45CFR46.

6. The investigators have abided by the NIH Guidelines forResearch involving recombinant DNA Molecules (April1982) and the Administrative Practices Supplement.

Table of Contents

Page

A. Summary . . . . . . . . . . . . . . . . . . . .. . 1

B. Foreword ....... ................. . . . . . . . . 1

C. Body of Report........... . . . . . . . . . . 3

1. Memory T lymphocyte responses of HIV-linfected individuals to HIV-1 and otherviral antigens is reduced. . . . . . . . . . . . . 3

2. Generation of CD8+ human T cell clonesto HIV-l gag .................. . . . . . . . 6

3. HIV-specific CD4+ CTL clones to gag protein. . .. 6

4. Human CD8+ CTL clone to HIV-l gpl60 .... ........ 7

5. Sera of HIV-1 infected individuals containantibodies which bind to HIV-l virus andenhance infection of Fc receptor T cells . . . . . 7

6. Two receptors are required for antibodydependent enhancement of HIV-l infectiousCD4 and FcR ............ ................... 8

7. Conclusions ........ ..................... 9

8. Publications ........ ................. 11

2

C. Body of Report1. The memory T lymphoc]te reoponse of IIV-1 infected

individuals to HIV-I and other viral antigens is reduced.

Table 1. HIV induced proliferation of human peripheral bloodmononuclear cells is determined by 31, thymidine incorporation

Donor Meana Stim Donor Mean StimAb pos CPM Index Ab neg CPM Index

42 #HJMedia 254 1 Media 187 1HIV:5X 6,473 25 HIV 5X 192 1

IX 3,911 15 iX 210 1.1X 1,136 4 .IX 192 1.01X 537 2

PHA 33,731 PHA 48,206#12Media 263 1HIV 5X 1,453 5

X 682 2.6.1X 552 2.1

PHA 82,106aMean CPM of 4 microtiter wells each containing 2 x l05 cells/wellin 100 ul. On day 0 an equal volume of medium alone or ofinfectious HIV virus grown in MOLT-3 cells and concentrated on ametrizamide gradient (provided by Dr. C. Bruck) was added. A 3Hthymidine pulse was performed on day 5, and cells were harvestedon day 8 by a MASH harvester after overnight incubation.

This T cell response required concentrated preparations ofHIV-I virus. We determined that the PBMC of some HIV-I infecteddonors were stimulated by concentrated live HIV-l, and that thePBMC of antibody negative donors did not respond, however, mostHIV-I infected individuals' PBMC did not respond to HIV-I orinfluenza virus antigen.

Table 2: Stimulation of PBMC of HIV antibody positive donors

Stimulating Antigen # Tested Responders* % Responders

Concentrated live HIV-l 30 13 43gag p55 16 7 44A/PR/8 12 5 42PHA 26 25 96

*Responders to viral antigen were defined by a S.I. of >2.5:1.Responders to PHA were defined by S.I. >80:1.

PBMC were distributed into 96-well microplates.

Concentrated, live HIV-I or an HIV-I gag p55 construct was added

3

over a range of concentr-ations. PHA was added after dilution to1:200. The plates were then incubated at 370C for 5 days, pulsedwith CH3 ] thymidine for 18 hours and then harvested. In the caseof A/PR/8 the PBMC were pulsed with the live virus at a MOI of10:1 for 1.5 hours.

We found that the HIV-I antigen specific T cell proliferationresponses could be enhanced if low concentrations of IL-2 wereadded on day 5 after addition of antigen, with addition of 3H-thymidine on day 7 and harvesting on day 8, as shown in Fig. 1(antigen =___, media t72_A).

Day 0 Day 3 Day 5( 1.) (19)

(2.1)

(2.43) (1.7)

9,000 (1.3) 1.55)

4,000

2,000

CPM1.500/

(15)1,000 1.19)

1(1.49)

500- (.3)

0 0.1 1.0 10 50 0 0.1 1.0 10 50 0 0.1 1.0 10 50

Fig. 1 rlL-2 (units/ml)

The Figure 2 below demonstrates that the addition of 0.1-1.0u/ml of IL-2 enhances the specific T cell responses to HIV-lantigens, but does not affect the stimulation index of a non-responders PBMC.

7000'Donor #JR Donor #26

(HIV-) (HIV+)

6000- 14391 12261CPMi4000-

t 10711

2000 11.12)

1039)

0 0.1 1.0 0 0.1 1.0

Fig. 2 rI-2 (units/ml)

4

There was also an increase in the specific response tobaculovirus expressed p55 particles as shown below M mediumcontrol, l HIV-1 live virus antigen, WMI p55 antigen, when 0.1u/ml of IL-2 was added on day 5 after antigen to the PBMC of HIVantibody + donors (Fig. 3 below).

Donor #27 Donor #24 Donor #AJ(HIV +) (HIV +) (HIV -)

16,000 (20.88)

14.000

12,000ORM

10.000-

8.000 (9.47)

6.000

4,000 (5.22)

(2.2)

(3.54) 1 (1.51) (.33). (1.37) (1.48)

Fig. 3 No IL-2 + IL-2 No IL-2 + IL-2 No IL-2 + IL-2

These responses were dependent on IL-2 itself and not on somecontaminating material in the recombinant IL-2 because thespecific enhancement of T cell responses to virus antigen by theaddition of IL-2 was blocked if specific antisera to IL-2 wasadded to the culture (data not shown).

We conclude that these results demonstrate an early loss inantigen specific T cell responses of HIV-1 infected individuals.These results were not due to a decreased number of CD4+ cellsbecause these individuals were studied early in their HIV-linfections, and there were no significant differences in the CD4+cell numbers between the non-responders and the responders toviral antigen. The defect in response is not due to a lack ofmemory T cells, or to a failure by the antigen-specific T cell tobe activated by viral antigen, because the defect was corrected byIL-2 which could only specifically enhance proliferation of viralantigen specific T cells after activation and upregulation of IL-2receptors.

These results raise questions concerning the potential forimmunotherapy with IL-2 of HIV-l infected patients. To ourknowledge this was only performed in a few patients with fullblown AIDS before the cause of AIDS was defined. Our data suggestthe possibility that IL-2 therapy might improve the antigen-

5

specific T cell memory and subsequent effector cell responses toHIIV-i infected individuals to recall antigens. The failure of theimmune response to control infections is a hallmark of theacquired immunodeficient state which cause an inability of thehost to eliminate reactivated infections unlike theimmunocompetent host.

C2. Generation of CD4+ and CD8+ human T cell clones to 1IV-!a ag

It was extremely difficult to generate HIV-1 specific T cellclones despite our success in doing this with other viruses, e.g.,dengue and influenza. Undoubtedly this is due to the difficultyin generating antigen-specific T cell responses described above.This contract was requested for five years, but was funded forthree years. After much effort we finally succeeded in generatinghuman CD8+ and CD4+ T cell clones that lyse autologous targetcells infected with Vac gag or pulsed with E. coli derived p24antigen. Figure 4 below shows some results with clone #165.

100

80

60U

40 .020

Fig. 4 C VAC VAC/gag p24 1(562

This CTL clone is CD8+, and its HLA restriction pattern isbeing defined. We know that this is a novel HIV-l specific clonebecause our donor does not have either B7 or A2, the HLArestricting antigens for the only other two HIV-I gag specificepitopes that have been reported. Peptide mapping studies havealso ruled out the two sites reported early, and detailed mappingis almost complete. The site that appears to be recognized byclone 9165 is highly conserved.

C3. HIV-2 specific CD4+ CTL clones to Ql protein.

We have also generated a long-lived clone of CD4+ CTL whichrecognizes autologous target cells infected with Vac/gag, orpulsed with p24 antigen.

This is the first report that we are aware of describing ahuman CD4+ CTL clone to HIV-l gag. The only other CD4+ CTLreported is to an epitope in gp41 by Silaciano et al in a gpl60

6

vaccinated, not infected, individul.

Table 3 below is a summary of the results in the literatureand our results in the HIV-I gag specific human CD4+ and CD8+ Tcell responses.

Table 3

HIV-l ac CD4+ CD8+ HLA Laboratory

gag -- + B2 Nixongag -- + B8 Gotchgag -- + CW6 Ennisgag + Class II Ennis

C4. Human CD8+ CTL clone to gpl60

Recently we established a CTL clone which specifically killsautologous target cells infected with Vac/gp 160. This also is anovel CTL clone because it comes from a donor whose HLA antigensdo not contain the restrictive element for the only other CTLepitope on gpl6O that has been published to date. Berzofsky'sgroup has defined murine H-2d CD4+ and CD6+ T cell responses. Theonly human envelope specific CTL responses that have beenpublished is by Plata, et al. which is restricted by HLA, unlikethe CD+ T cell clone we have generated. Mapping of the epitope isnot completed.

Table 4 below is a summary of the literature on human CTL

responses to gpl60.

Table 4

HIV ag CD4+ CD8+ HLA Laboratory

env + A2 Plataenv + - DR4 Silicianienv + Class I Ennis

(non-A2)

In conclusion, we have generated three new human CTL epitopeson HIV-I

C5. We established that the sera of HIV-1 infected individual

infection of Fc recerptor T cells.

We were the first laboratory to report that the sera of HIV-1infected individuals contained antibodies which bound to HIV-1 andenhanced infection by Fc receptor mediated entry. The infectionwas detected at subneutralizing concentrations of antibodies inhuman sera (Table below).

7

The effect was Fc receptor mediated because it was blocked by1) addition of heat-aggregated human gamma globulin, and by 2)cleaving of the Fc from purified IgG, because the F(ab') couldneuLralize virus but not enhance infection (Table 5 & 6 elow).

TrABLE 53. EffeeL of blocking of FoR oin enhancement of IIIV-1 infection.

Treatment p24 (ng/ml)

HIV 65.2 z 0.1HIV + antiserum (10-5) 133.4 t 14.7HIV + AHG (1 mg/nd) 80.7 t 1.8HIV + antiserum + AHG 81.1 t 4.4

TABLE 6. Infection enhancement activity in IgG and F(ab') 2 fromHIV-I seropositive serum.

Serum p24 (ng/mJ)diludon

equivalent* IgG F(ab')2

go 96.0 t 3.6 (1.0) 90.6 ± 1.6 (1.0)10"3 49.3 t 1.2 (0.51)

72.7 ± 0. .76) 35.8 t 1.3 (0.40)10-' 115.0 ± 1.5 (1.20) 46.8 ± 0.4 (0.52)10 -41. 174.2 t 3.3 (1.81) 64.7 ± 2.7 (0.71)10-' 244.8 ± 6.Ot (2.55) 69.4 ± 3.1 (0.77)10" 5. 159.4 ± 0.8 (1.66) 93.1 ± 2.5 (1.03)10-1 121.1 t 3.9 (1.26) 81.0 ± 2.4 (0.89)

•HIV one. tPek cnhuncrnerL.

C6. Two receptors are required for antibody dependent enhancementof HIV-1 infection: CD4 and Fc R.

To define the entry mechanism of HIV-I complexed with anti-HIV-I antibody, we attempted to determine the receptor moleculesresponsible for mediating enhancement of HIV-1 infection ofmonocytic cells. Monoclonal antibodies to FcRI for immunoglobulininfection. Furthermore, we demonstrate a requirement for the CD4molecule in antibody-enhanced HIV-1 infection via FcR. SolubleCD4 prevented infection by HIV-1 antibody-treated virus, andenhancement of infection of virus-antibody complexes was abrogatedby monoclonal antibody to CD4 (anti-Lcu3a antibody). Treatmentof human macrophages with an anti-CD4 antibody also inhibitedantibody-enhanced HIV-I infection of macrophages, supporting ourcontention that antibody-dependent enhancement of HIV-1 infectionvia FoR requires CD4 interaction with the virus glycoprotein.

8

The figure below shows that a mfooclonal antibody to FcRI(mAbl97) blocks antibody dependent enhancement. A monoclonalantibody to FcRII (mAb 1 V-3) does not.

60 50

s o so so

C * 6040. 40-

C40

CL20 4020

-

0 20 0Fig. 5 -01t101110,o10to-0', 10o , t 0*1to" o7 to.. to0e to0

Serum Dilutionl Equlvalehlftc of SlAbt, Is c,l asid iFcjjit 06 lnresgtis enlmsncentemsl In list presence at arsilsOsile to Ill V.1. U937 cells (111.1 were

sintretslesi (A) or tsrcireated wis MAbI 197 to PC 1 I) or IAb Iv.3 to I~cRI I (C) at 5 ligmis for 30 into at 4C ands then isicubisted fosr 2 Itat 37T wills 10' ICID. or IIIV-1 (MoI. bi1l) or IlyV-1 compglesesl willh vaioas concentrautions of psuilflem IsO (ons an IIIV-lsslsiby-poillive Serunsk in tile presence of cacti NAb at 5 Ipglsss. Tile MAbs at thsis concentration saturate list bimslisg site% of Plc,Ils (7). AfterInfection. tile celis were wusahscs~i &Id cullted. Ssspcrnatats were hasrvestesd on dlay 7 four sietrnilpalios of p24. l)as are p'resented as thesticanss t standasrdi seiation$ of p24 vaueis floast dutplictle csslltsres. A repeatesd experiment liowed sinsihsr results. "it p24 yiss rolssutleclture of tile cells whsichm were Infeclesd In tile pbresenice Of lgI O intkIy to III V.1 Alijsesl @ tIII* serilm eqssllvslcmtt of 10" were signiicilssmsyillher Ilia itisc in tile absence or Igo2 (1, < 0.l)01) In Iretusscmst suss A ands C hill gmat In treatmuent trossp fl.

CD4 interaction with the gpl6O in the virus-antibody complexto infect the cell because sCD4 reduced infection of antibodycomplexed virus to very low levels, similar to its effect onblocking infection by free virus.

B10.*sC04 H-

so . sCDt 300ssgsCD4 15O0ssg

0

20

0 to", t0 10"' t0 10"I IV atone tujO sortm diuilion arpilvelont)

Fig. 6. Inhibition of infectivity of flIV-1 for U937 cells by sCD4.

07. In conclusion, the research performed under this contract hasprovided new, important, basic information concerning specifichuman T cell and humoral responses to HIV-1 infection which willimprove our understanding of:

1) the pathogenesis of HIV-1 infections and acquiredimmunodeficiency,

2) possible immunopathology mediated by CD4+ and CD8+ HIV-1specific CTL,

9

3) the definition of new CD4+ and CD8+ T cell epitopeswhich may be important for vaccine development efforts,

4) the definition of infection-enhancing antibodies to HIV-1 in infected patients, thus stimulating the need todefine such epitopes and to exclude them from vaccines,and

5) the demonstration that interaction by the gpl60 in theantibody-complexed virus must interact with the CD4 inthe cell, or to the endosome for antibody dependentenhancement of infection to occur. This may be relevantin other virus infections, e.g., dengue.

10

Publications

1. Takeda, A., Tuazon, C., Ennis, F.A. Antibody enhancedinfection by HIV-l via Fc receptor-mediated entry. Science242:580-583, 1988.

2. Takeda, A., Tuazon, C., and Ennis, F.A. Immune enhancementof HIV-I infection: Possible mechanism of Fc receptormediated infection enhancement in Vaccines 89, eds R.A.Lerner, H. Ginsberg, R.M. Chanock and F. Brown, Cold SpringHarbor Laboratory Press, 149-153, 1989.

3. Takeda, A. and Ennis, F.A. FcR-mediated enhancement of HIV-linfection by antibody. AIDS Res and Human Retroviruses6:999-1004, 1990.

4. Takeda, A., Sweet, R.W. and Ennis, F.A. Model forenhancement of HIV-l infection by antibody in Vaccines 90,Cold Spring Harbor Laboratory Press 333-337, 1990.

5. Takeda, A., Sweet, R.W., Ennis, F.A. Two receptors arerequired for antibody-dependent enhancement of HIV-Ineutralization and infection--enhancement by human monoclonalantibodies to gpl20. J. Virology, in press, 1990.

6. Takeda, A., Robinson, J.E., Ho, D.D., Debouck, C., Ennis,F.A. Distinction of HIV-I neutralization and infection--enhancement by human monoclonal antibodies to gpl20.Submitted 1990.

7. Littaua, P., Oldstone, M.B.A., Takeda, A., Debouck, C., Moss,B., Wong, J., and Ennis, F.A. An HLA-C-restricted CD8+ CTLclone recognizes a highly conserved epitope on HIV-1 gag. J.Virology, in press, Aug. 1991.

8. Takahaski, K., Dai, L-C., Fuerest, T.R., Biddison, W.E.,Earl, D.C., Moss, B., and F.A. Ennis. Characterization of aCTL epitope on HIV-l gag gp4l restricted by HLA-31 and lysisof HIV-I infected cells by a CD8+ CTL clone. Submitted forpublication.

9. Littaua, R.A., Oldstone, M.B.A., Takeda, A., and Ennis, F.A.CD4+ CTL clone to a conserved epitope on HIV-I p24:cytotoxic activity and secretion of IL-2 and IL-6. Submittedfor publication.

ii