J Med Primatol 2000: 29: 231–239Printed in Ireland - all rights reser6ed.

Enhanced safety and efficacy of liveattenuated SIV vaccines by prevaccinationwith recombinant vaccines

Jones L, Ahmad S, Chan K, Verardi P, Morton WR, Grant R, YilmaT. Enhanced safety and efficacy of live attenuated SIV vaccines byprevaccination with recombinant vaccines. J Med Primatol 2000;29:231–239. © Munksgaard, Copenhagen

Abstract: The only vaccines shown to be protective against intravenouschallenge with virulent virus in the simian immunodeficiency virus(SIV)/macaque model are attenuated live SIVs. However, these vaccineshave several disadvantages: 1) they persist indefinitely in vaccinatedmacaques; 2) they are pathogenic to neonatal macaques; and 3) theyare lethal in some adult macaques. To enhance the safety and efficacyof these vaccines, we immunized macaques first with recombinant vac-cines and then inoculated the animals with SIVDnef. In the first experi-ment, preimmunized macaques advanced to disease slower thancontrols after challenge with virulent SIV; five animals survived for 3years without disease and only the vaccine virus (SIVDnef) could beisolated at this time. In the second experiment, preimmunized animalshad lower virus loads and no disease compared to controls.

Leslie Jones1, Shabbir Ahmad1,Kenneth Chan1, Paulo Verardi1,William R. Morton2,Richard Grant2, Tilahun Yilma1

1International Laboratory of MolecularBiology for Tropical Disease Agents,Department of Veterinary Pathology,Microbiology, and Immunology, Universityof California, Davis, CA 95616, 2Universityof Washington Regional Primate ResearchCenter, Seattle, WA 98195

Key words: attenuation – live vaccines –macaques – subunit – virus-load

Accepted April 7, 2000.

T. Yilma, International Laboratory of Molec-ular Biology for Tropical Disease Agents,Department of Veterinary Pathology, Micro-biology, and Immunology, University of Cali-fornia, Davis, CA 95616.E-mail: tdyilma@ucdavis.edu.

Funding: This work was supported by NIHgrants UO1-AI29207, AI36197, andAI37182, and USA-DAMD 17-95-C-5054Nto T.Y. Other support included the Centerfor AIDS Research Grant AI27732 and theSVEU contract at the Washington RegionalPrimate Research Center.

Introduction

Many simian immunodeficiency virus (SIV) vac-cine strategies have targeted the envelope glyco-protein (gp160), the receptor-binding protein ofthe virus, as a key component in immunity. Inprevious work, we evaluated the potential ofSIVgp120 or gp160 as a vaccine against virulentvirus challenge in rhesus macaques immunizedwith various preparations of the antigen. Wewere the first to report that recombinant vaccinesdo not prevent infection, but do significantly re-duce virus load compared to unimmunized con-trols [2, 11]. This work has been corroborated bya number of laboratories [6, 15, 17], and it hasled to the current conclusion that the goal in

acquired immunodeficiency syndrome (AIDS)vaccine research may have to be the preventionof disease, but not infection [16].

The only vaccine that has been shown to beefficacious in this model is SIVDnef (SIV with adeletion in the nef gene) or SIVD3 (SIVDnef withan additional deletion of two more genetic ele-ments) [7, 23]. Although this vaccine providedlong-term protection even against challenge withhigh doses of virulent SIVmac251, it has severalsafety limitations: 1) it persists indefinitely in vac-cinated macaques; 2) SIVDnef and SIVD3 arepathogenic to neonatal macaques [4, 5, 24]; and3) the vaccine was pathogenic or lethal in somejuvenile macaques after a long period post-vacci-nation [5].

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To address the problems associated with limitedefficacy of subunit vaccines and the shortcoming ofthe safety of live attenuated vaccines (SIVDnef andSIVD3), we first proposed immunizing with subunitand then boosting with live attenuated vaccines.We hypothesized that primary immunization withsubunit vaccines would ameliorate or eliminateseveral of the safety problems associated with theuse of live attenuated SIV vaccines. To test thishypothesis, macaques were first vaccinated withvarious preparations of vaccinia virus recombi-nants (rVVs) expressing SIV antigens with or with-out interferon-gamma (IFN-g). They were thenboosted with SIV antigens expressed in baculovirusexpression vectors or CHO cells. Animals werethen inoculated with a live attenuated vaccine(SIVDnef) and finally challenged with unclonedSIVmac251 [12].

The SIV gp160 protein is the viral protein re-quired for binding to the cell receptor(s), and im-mune responses to these proteins can preventinfection; hence, this protein was included in all ofthe vaccines. A cytokine, IFN-g, was included toinvestigate its potential to increase the safety andefficacy of the vaccines. We have previouslydemonstrated the adjuvant activity of IFN-g [3]and its attenuating activity on the virulence of VVand SIV [9, 10]. Expression of the Gag protein inbaculovirus or VV expression systems results in theformation of virus-like particles (vlps) that shouldincrease the authentic presentation of antigen tothe immune system. The Nef protein was includedsince this protein has been implicated in SIV andHIV virulence. Also, there have been reports thatCD8+ cell responses to the Nef protein correlatedwith protection against virulent challenge [8, 18].

Materials and methodsCells and viruses

CEM-x-174 cells, rhesus peripheral blood mononu-clear cells (PBMCs), and lymph node cells (LNCs)were used for the propagation and isolation ofSIV. These cells were maintained in Roswell ParkMemorial Institute culture medium 1640 supple-mented with 10% fetal bovine serum (FBS). Hu-man A549, HeLa, TK−143B, and African greenmonkey kidney (BS-C-1) cells were propagated inDulbecco’s modified Eagle’s medium (DME;Gibco BRL, Rockville, MD) supplemented with5% FBS and antibiotics and used to propagate VVor encephalomyocarditis virus (EMCV). SIVDnef

was constructed by deleting a 186-base-pair frag-ment of the Nef coding sequences [9] of the patho-genic molecular clone SIVmac239 [18]. SIVmac251, a

pathogenic biological isolate, provided by R.Desrosiers (New England Regional Primate Re-search Center, Southborough, MA), was propa-gated in rhesus PBMCs and titered in rhesusmonkeys.

Construction of recombinant vaccinia virus vaccines

The Wyeth strain of VV was used to develop therVVs. Standard molecular biology techniques wereused to construct the plasmids used to generate therVVs. The rVVs generated included one expressingthe SIV gp120 gene (vSIVgp120), one expressingthe SIV gp160 gene (vSIVgp160), one expressingthe SIV gag, gp160, nef and human IFN-g genes(vSIVggen), and one expressing the SIV gag, gp160and nef genes (vSIVgen). Established transfectionprocedures using liposomes (Lipofectace, GibcoBRL) were used to deliver the plasmids to VV-in-fected BS-C-1 cells for the development of therVVs. Recombinants with a deletion in thethymidine kinase (TK) gene were plaque-purifiedin TK−143B cells in the presence of 5-bromo-deoxyuridine at 25 mg/ml to select for TK− mu-tants. Recombinants with a deletion in thehemagglutinin gene were selected and plaque-purified on the basis of expression of the b-galac-tosidase (lacZ) gene in the presence of thesubstrate 5-bromo-4-chloro-3-indoylyl b-D-galac-topyranoside.

Baculovirus constructs

Recombinant baculoviruses were utilized to ex-press the SIV antigens used for booster immuniza-tion and for coating enzyme-linked immuno-sorbent assay (ELISA) plates. These recombinantswere generated by standard procedures. In par-ticular, the gag antigen was found to form vlps.These recombinant antigens were purified by aseries of procedures, including precipitation bypolyethylene glycol followed by centrifugation(Gag) and membrane isolation on sucrose gradi-ents (GP160) [1].

Antiviral assay

The antiviral activity of IFN-g was determined bymeasuring inhibition of the cytopathic effects(CPEs) of EMCV on A549 cells [10]. Briefly, super-natants from BS-C-1 cells infected with rVVs andincubated for 24 hours were filtered through a0.45-micron filter to remove most of the virus. Thesamples (25 ml) were serially diluted in 50 ml ofDME in duplicate in 96-well plates. A549 cells(1.0×104) in 100 ml of DME/10% FBS were added

232

Prevaccination for attenuated live vaccines

to each well and the plates incubated for 24hours. EMCV (50 ml) was added to sample andvirus control wells at a concentration previouslydetermined to be the minimum amount of virusnecessary to produce 100% CPE in control wellsby 24 hours. Units of IFN-g are expressed as thereciprocal of the dilution giving 50% protectionagainst the challenge virus.

Vaccination and challenge of rhesus macaques

Colony-bred, juvenile rhesus macaques (Macacamulatta), seronegative for simian type D retro-viruses, simian T-cell leukemia virus, and SIVwere used in these experiments. They werehoused in accordance with the American Associa-tion for Accreditation of Laboratory AnimalCare guidelines. Monkeys were vaccinated intra-dermally (i.d.) with vSIVgp120 or vSIVgp160(Table 1) or intramuscularly (i.m.) with vSIVggenor vSIVgen (Table 2). Control animals were vac-cinated i.d. or i.m. with the parental Wyethstrain of VV. Monkeys were inoculated with bac-ulovirus or CHO-expressed antigens i.m. The ani-mals were inoculated intravenously (iv) with 1 mlof SIVDnef as a vaccine or SIVmac251 as a chal-lenge. Animals were euthanized if they met threeof four criteria and if their clinical presentationmerited euthanasia in the judgement of the at-tending veterinarian. The four criteria include: 1)Weight loss greater than 15% adjusted for age; 2)sustained PVC of less than 15; 3) a CD4+ cellcount less than 200; and 4) unresponsive clinicalillness.

Cell-associated viral load

Cell-associated virus was measured by a limiting-dilution assay (in replicates of four) of PBMCsor LNCs co-cultured with CEM-x-174 cells (105/well) in 24-well plates [19]. Aliquots of culturemedium were sampled twice weekly for the pres-ence of the SIV major core antigen (p27) byELISA [19]. When the p27 antigen was detectedat two consecutive time points, this was scored aspositive. Endpoint cultures were maintained andtested for 3–4 weeks before being scored as neg-ative. Virus levels were calculated according tothe method of Reed and Muench [21] and ex-pressed as TCID50 per 106 cells.

Detection of proviral SIV DNA

DNA was isolated from 2×105 cells (PBMCs,LNCs, or CEM-x-174 cells) using a DNA isola-tion kit (Qiagen, Chatsworth, CA) to recover the

DNA or RNA. The presence and identity of SIVproviral DNA was confirmed by polymerasechain reaction (PCR) amplification of the env-3%long terminal repeat region using primers A(5%GTACCATGGC-CCAAATGCAAG3%, senseprimer, nucleotide 8720) and E (5%AAATCCC-TTCCAGTCCCCC-C3%, antisense primer, nucle-otide 9710). The proviral DNA was hot-startedwith Mg2+ beads at 94°C for 5 minutes, an-nealed at 62°C for 1 minute, and extended at72°C for 2 minutes. The denaturation was thenreduced to 1 minute and the cycle repeated 35times.

Lymphocyte phenotyping

PBMCs were stained with anti-human mono-clonal antibodies to CD4 (phytocoerythrin-conju-gated anti-CD4 mAb OKT4); Ortho DiagnosticSystems, Inc, Raritan, NJ) or to CD8 (Leu 2a-flourescein isothiocyanate; Becton Dickinson Im-munocytometry Systems, San Jose, CA) asinstructed by the manufacturers, and immu-nofluorescence was measured by a dual-laser flowcytometer (FACScan).

Measurement of antibody responses by ELISA

Anti-Gag, anti-Nef, or anti-GP160 antibodieswere measured in the plasma of monkeys byELISA using 96-well plates coated with bac-ulovirus-expressed SIV Gag, Nef, or GP160 anti-gens, respectively, before the boost withbaculovirus produced vlps [1]. Antibody to wholeSIV was measured using plates coated with heat-inactivated SIVmac251. Titers were calculated asthe reciprocal of the dilution that produced anabsorbance of at least twice the value of the neg-ative control.

Measurement of antibody responses to VV

Antibody to VV was measured by a plaque-re-duction assay. Two-fold serial dilutions ofplasma in DME were incubated with an equalvolume of medium (100 ml) containing 50 plaque-forming units of VV at 37°C for 1 hour in dupli-cate. Each dilution was transferred to BS-C-1 cellmonolayers in well plates and incubated for 1hour at 37°C. Cells were then overlaid with 0.7ml of medium (DME with 2.5% FBS and 1%methylcellulose) and incubated for 48 hours at37°C before staining with crystal violet. Titers toVV were expressed as the reciprocal of thehighest dilution of plasma giving ]50% inhibi-tion of plaque formation.

233

Jones et al.

234

Tabl

e1.

Expe

rimen

t1:

Viru

slo

ads

Viru

stit

er(R

NA

copi

es/m

l)

Wee

k13

0W

eek

208

Wee

k22

5*St

atus

(199

8)An

imal

cVa

ccin

atio

nw

ithre

com

bina

nts

Wk

0–W

eek

11–W

eek

54W

eek

57W

eek

120

Wee

k80

(SIV

Dne

f)(S

IVD

nef)

(SIV

mac

251)

−Eu

than

ized

ND

ND

ND

−N

Dv1

20–v

120–

b120

A900

378.

25×

102

8.25

×10

31.

24×

103

Hea

lthy

v120

–v12

0–b1

20B

200

B20

0A9

0041

B20

06.

60×

103

Hea

lthy

A900

502.

64×

105

v120

–v12

0–b1

208.

25×

102

B20

06.

6×10

38.

25×

102

1.10

×10

4A9

1405

−Eu

than

ized

v120

–v12

0–b1

202.

06×

103

2.06

×10

23.

3×10

32.

06×

102

4.13

×10

2−

Euth

anize

dA9

0048

v120

–v12

0–c1

20B

200

B20

06.

19×

102

B20

0H

ealth

yA9

0052

v120

–v12

0–c1

20B

200

B20

0B

200

B20

0B

200

Euth

anize

d−

A900

57v1

20–v

120–

c120

6.86

×10

52.

11×

105

8.25

×10

23.

3×10

3

−A9

1397

Euth

anize

dv1

20–v

120–

c120

ND

ND

ND

ND

A914

04Eu

than

ized

v120

–v12

0–v1

20B

200

B20

05.

28×

104

5.28

×10

4

Euth

anize

d1.

03×

103

6.60

×10

54.

13×

102

B20

0B

200

v120

–v12

0–v1

20A8

8127

ND

A891

68−

Euth

anize

dv1

20–v

120–

v120

ND

ND

ND

−Eu

than

ized

ND

A913

91N

Dv1

20–v

120–

v120

ND

ND

A890

513.

30×

104

B20

0H

ealth

yv1

60–v

160–

b120

B20

0B

200

1.65

×10

3B

200

4.95

×10

3B

200

6.19

×10

2H

ealth

yA9

1386

v160

–v16

0–b1

20B

200

B20

03.

09×

102

ND

−Eu

than

ized

v160

–v16

0–b1

20N

DA9

1400

ND

ND

Euth

anize

d3.

3×10

31.

32×

104

−1.

06×

105

A914

02v1

60–v

160–

b120

1.32

×10

4

A900

44−

Euth

anize

dv1

60–v

160–

c120

ND

ND

ND

ND

A900

45B

200

B20

0H

ealth

yv1

60–v

160–

c120

B20

0B

200

B20

0B

200

Euth

anize

dN

D−

ND

ND

ND

v160

–v16

0–c1

20A9

1399

−A9

1401

Euth

anize

dv1

60–v

160–

c120

B20

0B

200

1.24

×10

31.

32×

104

Euth

anize

d1.

98×

104

A913

96vv

–vv–

vv−

1.65

×10

31.

32×

104

4.13

×10

2

−A9

1403

Euth

anize

dvv

–vv–

vvB

200

3.3×

103

4.13

×10

23.

30×

104

7.92

×10

4‘−

Euth

anize

dA8

9088

Nai

ve1.

24×

103

8.25

×10

21.

06×

105

4.22

×10

5−

Euth

anize

dN

aive

B20

0B

200

A911

832.

64×

105

Euth

anize

dN

D−

ND

ND

ND

A912

97N

aive

−A9

2163

Euth

anize

dN

aive

ND

ND

ND

ND

8.45

×10

5−

Euth

anize

dA9

1294

Nai

ve8.

45×

105

Euth

anize

dA9

2095

Nai

ve8.

45×

105

8.45

×10

5−

Euth

anize

dN

D−

5.28

×10

5A9

4101

Nai

ve−

A941

02Eu

than

ized

Nai

veN

DN

D

Mac

aque

sw

ere

vacc

inat

edw

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pres

sing

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gp12

0(v

120)

orgp

160

(v16

0)on

wee

ks0

and

11.O

nw

eek

54,t

hean

imal

sw

ere

boos

ted

with

bacu

lovir

us-e

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ssed

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0(b

120)

orC

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seha

mst

erov

ary

cells

(CH

O)-e

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ssed

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0(c

120)

.One

grou

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thre

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atio

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20.T

wo

anim

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wer

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yeth

VVas

aco

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l.O

nw

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56,t

hem

onke

ysw

ere

give

n10

AID

50of

SIV D

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ion

of10

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was

give

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plus

four

mor

ena

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ntro

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chal

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ofSI

V mac

251.

−,

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irus

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dete

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f.

Prevaccination for attenuated live vaccines

Table 2. Experiment 2: Virus loads

Virus titer (TCID50/106 cells)Vaccination withrecombinants

149 wpi65 wpv 117 wpi115wpi101 wpvAnimal c 0 wpv–8 wpv–26(SIVmac251) (2 wpc) (20 wpc)(38 wpi) (4 wpc) Statuswpv–61 wpv (SIVDnef)

vggen–vggen–vggen–bge 10 B1 21 B1 21 HealthyMMU27436vggen–vggen–vggen–bge 21 B1 100MMU27672 5 B1 Healthyvggen–vggen–vggen–bge 316 B1 B1 32MMU27991 316 Healthyvggen–vggen–vggen–bge 32 B1 1,000MMU28067 214 32 Healthyvggen–vggen–vggen–bge 100 2 21 5MMU28243 46 Healthy

vgen–vgen–vgen–bge 214 B1 21MMU28281 214 100 Healthyvgen–vgen–vgen–bge 46 B1 5MMU28358 B1 10 Healthyvgen–vgen–vgen–bge 32 B1 3,162MMU28401 B1 B1 Healthyvgen–vgen–vgen–bgeMMU28472 214 5 214 10 468 Healthyvgen–vgen–vgen–bge 32 B1 32MMU28703 B1 B1 Euthanized**

vv–vv–vv 46 B1 B1 B1MMU29116 B1 Healthyvv–vv–vv 46 B1 10MMU29117 5 10 Healthy

MMU28992 Naı̈ve 468 B1 4,677 316 468 DiarrheaNaı̈ve 46 1 21MMU28994 21 214 Healthy

MMU28785 Naı̈ve 10,000 214 468 HealthyNaı̈ve 468 468 1,000MMU29063 Rash

Macaques were vaccinated with rVVs expressing SIVGP160, Gag, Nef and human IFN-g (vggen) or SIVGP160, Gag, and Nef (vgen) onweeks 0, 8, and 26. On week 61, the animals were boosted with baculovirus-expressed GP160 and gag (bge). Two animals wereinoculated three times with the parental Wyeth VV as a control for the vaccine vector. On week 63, all monkeys were given 100 TCID50

of SIVDnef iv and two unvaccinated controls were included at this time as SIVDnef controls. All animals were positive for SIVDnef 1 week afterinoculation. On week 113, all animals plus two more naive controls were challenged iv with 10 TCID50 of SIVmac251. **MMU28703 waseuthanized due to an injury. This animal had no detectable virus in any tissue and was otherwise healthy at the time of euthanasia. wpi,weeks post-inoculation; wpv, weeks post-vaccination; wpc, weeks post-challenge.

Proliferation to viral antigens

PBMCs or LNCs from macaques were plated at105 cells/well in quadruplicate in 96-well plates.Each sample was stimulated with baculovirus-ex-pressed gag, gp160, vesicular stomatitis virus nu-cleocapsid protein (baculovirus control), ConA, orDME, and the incorporation of 3H-thymidine wasmeasured. Samples were collected using an Inotechcell harvester, washed and then counted using abeta scintillation counter (Wallac Trilux 1450).Samples were considered positive if the amount ofradioisotope incorporated in the cells was two ormore times that of the negative control (minus thebackground).

ResultsExperiment 1

Twenty rhesus macaques were vaccinated withvSIVgp120 or vSIVgp160 (Table 1). Eleven weeksafter primary vaccination, the animals wereboosted with the same. A second boost was given10 months later and consisted of baculovirus-ex-pressed gp120 (b120), CHO-expressed GP120(c120), or vSIVgp120. Two weeks after the secondboost, the animals were inoculated with 10 AID50

of attenuated live SIVDnef (iv). At this time, 11 of

20 vaccinated animals and one of two VV controlsbecame infected with the attenuated virus. After 6months, all the animals were re-inoculated with100 AID50 of attenuated live SIVDnef iv and allanimals became infected with the virus as shownby PBMC co-culture and/or western blot (data notshown). Finally, 8 months (34 weeks) after thesecond SIVDnef inoculation, all test animals andappropriate controls were challenged iv with 100AID50 of pathogenic SIVmac251.

Unvaccinated macaques and macaques inocu-lated with a single dose of SIVDnef became infectedand died of AIDS 24–73 weeks after SIVmac251

challenge (wpc). The mean survival time was 48.5weeks. Fourteen of the 20 animals that were ini-tially vaccinated with recombinant vaccines (VV,CHO, and baculovirus) followed by immunizationwith SIVDnef and challenged with SIVmac251 died ofAIDS 24–130 wpc (with a mean survival time of77 weeks). Six survived more than 3 years. Incontrast to the long-term survivors, all animalsthat died of AIDS early (24–37 wpc) had highplasma viral RNA levels (104–105 copies/ml), re-gardless of vaccination status. After challenge, allbut one (A90052) of the six long-term survivorswere infected with SIVmac251. The most significantfinding, however, was that SIVmac251 could not be

235

Jones et al.

isolated by PBMC co-culture and PCR from anyof these animals. The only virus detectable byco-culture of the PBMCs in these six animals wasSIVDnef, 3 years after challenge. The virus load inplasma of three of the animals was 6.19×102–6.6×103 RNA copies/ml; virus was undetectablein the plasma of the other three monkeys. Four ofthese animals had normal CD4+ cell counts at thattime (Table 3). These results suggest that immu-nization with recombinant subunit vaccines priorto vaccination with live attenuated SIV vaccinesmay enable the animals to eliminate or suppressvirulent challenge virus for long periods of time,even in the absence of sterilizing immunity.

Experiment 2

Twelve rhesus macaques (Table 2) were vaccinatedwith rVVs expressing SIVmac239 GP160, Gag, andNef, (vSIVgen), rVVs expressing GP160, Gag, Nef,and human IFN-g (vSIVggen), or the parental VV(Wyeth strain). The expressed Gag protein in all ofthe expression vectors is in the form of vlps in aneffort to increase the natural presentation of theantigens to the immune system. Vaccinia virusDNA was detectable in the LNCs 1 week post-vac-cination, but was undetectable at other time pointsor in PBMCs. The monkeys were revaccinatedwith rVVs at weeks 8 and 26 to increase theimmune response and then given a final inocula-

Table 3. Number of CD4+ T cells/ml after SIVmac251 challenge

Experiment 2Experiment 1

VaccinesAnimal c 20 wpc4 wpcVaccinesAnimal c105 wpc78 wpc

v120–v120–b120 − − MMU27436 vggen–vggen–vggen–bge 757A90037 884v120–v120–b120A90041 782 840 MMU27672 vggen–vggen–vggen–bge 816 489v120–v120–b120A90050 782 741 MMU27991 vggen–vggen–vggen–bge 924 694

393vggen–vggen–vggen–bgeMMU28067−562v120–v120–b120A91405MMU28243 vggen–vggen–vggen–bge 823 234

A90048 v120–v120–c120 117 −334574vgen–vgen–vgen–bgeMMU282819451,083v120–v120–c120A90052

A90057 v120–v120–c120 − − MMU22358 vgen–vgen–vgen–bge 1,705 1,092v120–v120–c120 − − MMU28401 vgen–vgen–vgen–bge 1,390 1,054A91397

808 453MMU28472 vgen–vgen–vgen–bgeA89051 v160–v160–b120 238 92 MMU28703 vgen–vgen–vgen–bge 815 1,336

v160–v160–b120A91386 874 279A91400 − MMU29116 vv–vv–vv 2,004 1,006−v160–v160–b120

− − MMU29117 vv–vv–vv 1,119A91402 1,231v160–v160–b120

A91404 v120–v120–v120 324 − MMU28992 SIVDnef control 381 484A88127 v120–v120–v120 132 − MMU28994 SIVDnef control 1,017 592

−−v120–v120–v120A89168v120–v120–v120 −A91391 − MMU28785 Naive 918 295

MMU29063 435Naive 437−A90044 −v160–v160–c120

7191,371v160–v160–c120A90045A91399 v160–v160–c120 −−

−1,242v160–v160–c120A91401

A91396 vv–vv–vv − −vv–vv–vv − −A91403

A89088 Naive 506 −−241NaiveA91183−−NaiveA91297

A92163 Naive − −

A91294 Naive − −−−NaiveA92095

A94101 Naive 102 −−−NaiveA94102

v120, rVV expressing SIVGP120; v160, rVV expressing SIV GP160; b120, baculovirus expressed SIV GP120; c120, CHO cell-expressedGP120; vggen, rVV expressing SIV GP160, Gag, Nef and IFN-g; vgen, rVV expressing SIV GP160, Gag and Nef; bge, baculovirusexpressed SIV GP160 and Gag.

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tion with baculovirus-expressed gag and gp160 onweek 61. The antibody titers to SIV GP160, Gag,and Nef were measured by ELISA, and VV anti-body was detected by a plaque-reduction assay.The first vaccination resulted in low antibody titersto all antigens, but each subsequent inoculation ofrVV increased the response, despite the presence ofantibody to VV (data not shown).

All animals were inoculated with a high dose ofattenuated live SIV; 100 TCID50 of SIVDnef peranimal, iv 63 weeks post-vaccination. Two controlanimals were added at this point to serve as SIVDnef

controls. There was no detectable difference inresistance to inoculation with SIVDnef in any of theexperimental groups or controls, probably due tothe high dose of virus given to the animals. All ofthe vaccinated and naive controls became infectedwith SIVDnef as determined by PBMC and CEM-x-174 co-culture assay and PCR by 1 week post-inoc-ulation. The viral titers in PBMCs and LNCs in allthe animals dropped to low or undetectable levelsby 14 weeks post-inoculation (Table 2). A slightincrease in antibody titers to SIV followed by adrop by 9 weeks post-inoculation paralleled thevirus titers.

All vaccinated macaques and two naive controlanimals were challenged with 10 TCID50 (60AID50) of SIVmac251 50 weeks post-inoculation (113weeks post-vaccination) with SIVDnef. By PCR, 6 of10 prevaccinated animals were found positive forSIVmac251 1 wpc. Nine of the 10 monkeys werepositive by 4 wpc and SIVmac251 was detectable inall animals by 6 wpc. The virus loads in most ofthe animals peaked by 2 wpc and then became lowto undetectable in 50% of the vaccinated animalsby 8 wpc and 79% by 12 wpc. The CD4 T countshave dropped on average (Table 3), but only halfof the vaccinated animals have had a drop of morethan 50%. The group of monkeys vaccinated withvSIVggen had the strongest proliferative responseto SIV gag, suggesting that the incorporation ofthe lymphokine may have increased this response.However, this response does not correlate with thelowest virus loads or highest CD4+ T cell counts.At 20 wpc, all of the vaccinated macaques werewithout signs of AIDS. The naive controls havehigher average virus loads and lower CD4 Tcounts than the vaccinated animals, and two mon-keys (one SIVDnef and one naive control) wereshowing early signs of AIDS. One of the vSIVgen-vaccinated animals (28703) was euthanized 35 wpcafter it suffered an unrelated injury that did notrespond to treatment. The animal had no de-tectable virus in any tissues and no signs of AIDSat the time of euthanasia.

Discussion

Live-attenuated vaccines are more immunogenicand offer more durable and broader protectionthan other types of vaccines, probably becauseattenuated viruses best mimic infection with thevirulent virus and, ideally, do not retain the capac-ity to cause disease. Accordingly, we believe thatlive-attenuated SIV vaccines will be more effectivethan vaccines based on inactivated whole virus,viral subunits, or live heterologous viral or bacte-rial vectors. However, the safety of attenuated livevaccine is a major concern. Although SIVDnef andSIVD3 appeared to be completely attenuated in thefirst trials [7, 8, 22], it has since been shown to bepathogenic to neonatal macaques and lethal insome adult macaques [4, 5, 24]. We have beenexploring the possibility of combining currentmodels of vaccines to improve the safety and effi-cacy of live attenuated vaccines. We hypothesizedthat by vaccinating with a recombinant vaccinefirst, the chances of the attenuated live vaccinecausing disease by itself might be decreased. Thisapproach may also decrease the time required forinduction of sterilizing immunity and protectionagainst infection with virulent virus.

In the first experiment, animals were vaccinatedwith rVVs expressing the SIV gp120 or gp160 geneand boosted with baculovirus or CHO-expressedSIVgp120 [12]. Out of 20 vaccinated monkeys, nineresisted infection from iv inoculation with 10AID50 of SIVDnef; however, one of the VV controlsalso did not become infected at this dose. Theanimals were then reinoculated with 100 AID50 ofSIVDnef and all of them became infected at thisdose. Thus, the vaccination regimen induced someresistance to infection with a low dose, but not ahigh dose of the attenuated live SIV vaccine.Thirty-four weeks later, macaques were challengedwith 100 AID50 of SIVmac251 to evaluate the level ofefficacy after vaccination with rVVs, boosting withsubunit antigens, and two inoculations withSIVDnef. This regimen did not prevent infectionwith SIVmac251, although the vaccinated group hadlower virus loads and longer survival times whencompared to the naive controls. In addition, the sixanimals that survived more than 3 years had nodetectable SIVmac251 or clinical disease. These re-sults clearly show that the immunization protocolsignificantly reduced virus load in vaccinated ani-mals and also eliminated the challenge virus toundetectable levels in 25% of macaques. Moreover,the length of survival of vaccinated animals wassignificantly increased compared to unvaccinatedcontrols.

In experiment 2, macaques were first immunizedwith rVVs expressing GP160, Gag, Nef, with or

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without IFN-g, and then boosted with bac-ulovirus-expressed GP160 and Gag. They werethen inoculated with 100 TCID50 of SIVDnef 50weeks before challenge with 10 TCID50 SIVmac251.Although none of the animals resisted infectionwith SIVmac251, they had, however, a significantreduction in virus loads. At 20 wpc, none of thevaccinated animals is exhibiting clinical signs ofdisease. In contrast, some of the animals in theunvaccinated and the SIVDnef control groups arebeginning to show early signs of AIDS.

Incorporation of lymphokines as a method toincrease safety and efficacy of recombinant vac-cines has been investigated thoroughly in our labo-ratory and others [3, 9, 10, 13, 14, 20]. We havepreviously shown that VVs and SIVs expressingIFN-g are highly attenuated and thus much saferfor use as live attenuated vaccines [9, 10]. Animalsvaccinated with the rVV-expressing IFN-g(vSIVggen) had an increased proliferative responseto SIV gag after challenge with SIVmac251. Thesignificance of increased proliferative response isnot clear since there was no correlation with virusload. These animals also have, on average, a lowervirus load in PBMCs and LNCs than macaquesvaccinated with vSIVgen; however, they also havelower mean CD4+ T cell counts. At this time, theeffect of expression of IFN-g in the prevaccinationprotocol on long-term survival is unknown sincethe experiment is still in progress.

In conclusion, vaccination with a recombinantvaccinia virus vaccine for SIV followed by inocula-tion with SIVDnef improved the ability of the atten-uated live vaccine to induce an immune responsecapable of decreasing virus load and increasing thesurvival time of macaques challenged with virulentSIV. However, it did not improve resistance toinfection with a high iv dose of SIVmac251 or de-crease the amount of time required for the attenu-ated virus to establish such resistance.

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