detection of anti-human cell antibodies in sera from macaques immunized with whole inactivated virus

AIDS RESEARCH AND HUMAN RETROVIRUSESVolume 8, Number 9, 1992Mary Ann Liebert, Inc., Publishers

Detection of Anti-Human Cell Antibodies in Sera from MacaquesImmunized with Whole Inactivated Virus

ALPHONSE J. LANGLOIS, KENT J. WEINHOLD, THOMAS J. MATTHEWS,MICHAEL L. GREENBERG, and DANI P. BOLOGNESI

ABSTRACT

More than 200 sera from macaques immunized with several different vaccine preparations were tested invarious assays with cells of human and macaque origin. Only in instances where whole inactivated SIVpreparations were used for immunization, were reactivities found with normal human cells, and this was thecase in every instance. Such sera produced a marked clumping of several normal human cell lines and exhibitedstrong staining of the cell surface in FACS analysis. In the presence of SIVDeltaB670, these sera also enhancedinfectivity and fusion formation. When similar tests were performed with macaque cells as targets, suchphenomena were not easily discernible. Likewise, there was no trace of such activities in sera from normalanimals, animals chronically infected with SIV, or in those from animals which received recombinant viralsubunits as vaccines. Finally, we show that in several instances where whole inactivated virus was used as a

vaccine, there is a strong correlation between the titer of anticellular activity with protection.

INTRODUCTION

THE CLOSE GENETIC RELATIONSHIP with HIV-1 coupled withthe ability of simian immunodeficiency virus (SIV) to

induce an AIDS-like disease in primates makes the SIV systeman ideal model for developing and testing candidate vaccines forhumans. Indeed, it was studies in this model which first showedthat vaccines against primate lenti viruses were feasible, an

accomplishment which provided a major impetus to pursuevaccine development against HIV-1.1'2 Vaccine research withSIV has since reached several other important milestones such as

cross-protective immunity against divergent viral strains anddevelopment of experimental approaches which can evaluate theeffects of vaccines against virus infection across mucosal sur¬

faces (reviewed in ref. 3).To date, the overwhelming majority of successful vaccine

experiments have been achieved using preparations of inacti¬vated virions or infected human T-cell lines. On the other hand,most attempts using viral subunits have failed.3 These resultshave led many investigators to propose that unique conforma-tional determinants present in the native virion structure or thepresence of all of the virion components may be essential for

efficient protection. Indeed, when SIV was disrupted andseparated into envelope and non-envelope fractions, much of theprotective activity was lost.4

These results with SIV are in stark contrast to those obtainedwith candidate vaccines in the HIV-1 chimpanzee model whereviral subunits have been successful and killed virus preparationshave failed to induce protection.5 Moreover, whereas the bestcorrelates for protection in the HIV model are antibodies to theprincipal neutralizing determinant (PND), protection in the SIVexperiments employing inactivated preparations have shownlittle or no correlation with neutralizing antibodies.2

Such dichotomies might be due not so much to differencesbetween the two models but rather to the protective mechanismsassociated with inactivated virus preparations versus subunitimmunogens. One issue to consider in this regard, relates to thepresence of nonviral material associated with virions either as

integral components or as contaminants difficult to remove bypurification. Indeed, there are ample precedents for cellularcomponents incorporated into retroviruses as they form at thecell membrane, including nucleic acids, cellular enzymes,major histocompatibility complex (MHC) products, etc. (re¬viewed in ref. 6). In essence, the virions often assume some of

Duke University Medical Center, Center for AIDS Research, Durham, NC.

1641

1642 LANGLOIS ET AL.

the signature of the cell surface through which they bud. Therecan also be contaminating material (cell debris) in viral prepara¬tions, especially when virus production is low. Of course, theuse of infected cells as immunogens would entail a pronouncedcontribution by cellular antigens.

This possibility was made a reality recently by Stott when theyannounced that sera from macaques receiving vaccines based on

inactivated simian immunodeficiency virus (SIV) or SIV-in-fected cells contain reactivities to normal human cell compo¬nents and that the anti-cell antibody titers correlate positivelywith protection.7 This was accompanied by the remarkablefinding that protection could be induced by vaccination withuninfected cells of human origin.7

Our previous studies6 and those of others8 which examinedsera from other SIV vaccine trials for the presence of anti-cellreactivity confirmed and extended the results of Stott7 using a

variety of approaches to both document and characterize theanticellular activity. In this report we present further details ofour findings on the reactivity of sera from immunized macaquesagainst normal cells and their effects on SIV infection.

MATERIALS AND METHODS

Cell cultures

The AA-5, a subclone of the AA cell line derived from an

Epstein-Barr virus- (EBV) infected B-cell line,9 was obtainedfrom Dr. S. Chaffee, Duke University Medical Center. Cellswere grown in RMPI-1640 containing 100 U/ml penicillin, 100pg/ml streptomycin, and 20% heat-inactivated (56°C/30 min)fetal calf serum (Grand Island Biological Co., Grand Island,NY). Cell cultures (20 ml) contained in 75 cm2 plastic flasks(Costar, Cambridge, MA) were grown at 37°C in a 5% C02humidified incubator. The population doubling time of this cellline is approximately 36 h under these conditions. Other celllines were as described earlier.10

Virus poolsThe SIVDeltaB670 strain of virus was provided by Dr.

Michael Murphy-Corb, Delta Regional Primate Research Cen¬ter, Covington, LA 18703. A pellet of 1.5 x 106 CEM cells was

resuspended in 1.0 ml volume of cell-free virus. The mixtureswere incubated at 37°C for 1 h, after which the suspension was

diluted to 10 ml of growth medium and transferred to 75 cm2 flasks. Cultures were continued for 10-20 days with cellsmaintained at 3-5 x 105/ml by addition of medium every 2-3days. When the reverse transcriptase level reached 40-50 x 103cpm per assay, the cells were pelleted and resuspended at8-9 x 105/ml in 100 ml of medium to obtain an overnightharvest of freshly released virions. Approximately 18-20 hlater, the cells were removed by centrifugation at 150 x gforlOmin, and the supernatant clarified by filtration through a 0.45µ millipore filter (Millipore Corp., Bedford, MA), aliquotedin 1.5 ml volumes and stored at —70°C.

Titration of virus poolsThe SIVDeltaB670 pools prepared in CEM were titrated on

AA-5 cells. Twofold serial dilutions of virus stock were made in

growth medium in 96-well plates (Costar 96 half area tissueculture A/2 clusters, Cambridge, MA). An additional 30 µ ofmedium, normal monkey serum, or serum from macaquesvaccinated with whole inactivated virus were then added to thewells and the plates incubated for 1 h at 37°C (to be consistentwith the format of the neutralization assay described below). Wethen added 30 µ of AA-5 target cells at 2 x 105/ml to the wells.Cultures were incubated in a 5% C02 atmosphere at 37°C andcell-free supernatant was assayed at 3-4 day intervals forreverse transcriptase (RT) activity using a modification of themicroassay already described. ' ' The highest virus dilution thatscored positive 14 days postinfection was defined as one infec¬tious unit.

Neutralization and fusion enhancement assayThe SIVDeltaB670 virus pool was diluted to contain approxi¬

mately 100 infectious units per 30 µ volume. Twofold serum

dilutions (heat-inactivated 56cC/30 min) were made in 96-wellhalf-area wells. A 30 µ volume of virus was added to the wellsand the cultures were incubated at 37°C for 1 h. AA-5 target cells(6 x 103/in 30µ1) were then added to the wells. This representsa multiplicity of infection (MOI) of 0.017. Assays for RT weremade at 7-10 days postinfection after two medium changesduring the culture period. The neutralization titer is expressed as

the reciprocal serum dilution which blocks infection as evi¬denced by a lack of reverse transcriptase activity in the superna¬tant 10 days postinfection. Wells were also scored for cellclumping 1 day postseeding and at 4-5 days postinfection forsyncytia formation. The number of syncytia per well was againserum concentration dependent. The control wells (no serum) or

wells containing dilutions of infected or normal macaque serum

contained very few syncytia (i.e., 0-12). The fusion enhance¬ment titer is expressed as the reciprocal of the serum dilution thatenhanced fusion by threefold compared with control wells.

Assays with S1VMAC25IPreparation of virus pools, neutralization and fusion assays

Clumping of target cells

Twofold serum dilutions (heat-inactivated 56°C/30 min) were

made in growth medium in 96 half-area wells. Targets cells(6 x 103 in 30 µ ) were then added to the wells. Cultures were

incubated in a 5% C02 atmosphere at 37°C. The wells were

examined one day postseeding with an inverted phase micro¬scope (X40) for cell clumping. Cell clumps consisting of 15 or

more cells were counted. The number of clumps per well was

serum concentration dependent and between 100 and 200 couldbe observed at high serum levels.

Flow cytometric analysesThe cell surface binding of various macaque sera were

analyzed by flow cytometry following conventional indirectfluorescent antibody staining.Briefly, 1 x 106 AA-5 cells were

pelleted and resuspended in 0.5 ml of a 1:250 dilution of serum

in phosphate-buffed saline (PBS, divalent cation free) contain¬ing 1% fetal calf serum, 0.1% sodium azide, and hereafter

ANTI-CELL ANTIBODIES IN IMMUNIZED MACAQUES 1643

referred to as PBS/FCS/NaN3. The cell suspensions were incu¬bated for 45 min at 5CC with occasional resuspension. Cells were

subsequently washed twice with 4 ml of PBS/FCS/NaN3 and thepellet resuspended in 0.5 ml of FITC-conjugated goat F(ab')?fragment anti-human IgG (Fc specific) (Organon-Teknika) di¬luted in PBS/FCS/NaN3. The cell suspensions were incubatedfor 45 min at 5°C with occasional resuspension. The pelletedcells were washed three times with 4 ml of PBS/FCS/NaN, andthe final pellet was resuspended in 1 ml of PBS/FCS/NaN3containing 1% formalin fixative. Cell suspensions were ana¬

lyzed on a Coulter Profile II flow cytometer and results were

plotted as the log-fluorescence intensity versus relative cellnumber.

Absorption of sera with normal cells

AA-5 cells were grown in large volume for absorption ofinfected and protected macaque sera. For each absorption 108cells were washed twice with RPMI-1640 without fetal calfserum. On the final wash, the cells were pelleted in a 15 mlconical centrifuge tube. The cell pellets were resuspended in 1ml of a 1:50 serum dilution, resuspended at 15 min intervals andmaintained at 4°C for 1 h. The cells were removed by centrifu-gation 150 x g/10 min and the supernatant filtered through a

0.45 µ spin-x filter (Costar, Cambridge, MA) to remove anyremaining cells. The absorbed sera were aliquoted and stored at70°C.

RESULTS

Effects of sera from vaccinated macaques on normaland SIV-infected human cells

We have recently described the establishment of neutraliza¬tion and fusion inhibition assays with SIVMAC251 where anumber of sera from experimentally infected and vaccinatedmacaques were evaluated.'u During the course ofour attempts todevelop a similar assay for SIVDeltaB670 on AA-5 cells, it was

observed that certain sera caused very noticeable clumping ofthe cells in the wells. We subsequently determined that this alsooccurred with uninfected AA-5 cells. A typical example isshown in Figure 1A, which depicts AA-5 cells, a humanB-lymphoblastoid cell line susceptible to SIV infection,10 in thepresence of serum from a macaque vaccinated with wholeinactivated SIV. Such clumping became evident after one day inculture and its degree was serum concentration dependent (seeTable 1). The clumps initially consisted of 15 or more cellswhich continued to divide, forming ever larger aggregates. Thisphenomenon was not observed when sera from either a normalor infected macaque were substituted (Fig. IB).

These observations prompted the examination of a largernumber of sera which included those from animals vaccinatedwith a variety of immunogens. To eliminate the problem ofsubjectivity, the analyses were conducted in blinded fashion andthe results for a total of 189 serum samples are summarized inTable 2. In essence, the clumping effect was restricted to sera

derived from animals immunized with whole inactivated viralpreparations. Furthermore, whereas all sera tested from suchvaccinated animals produced the phenomenon, none of the

animals vaccinated with recombinant subunits or recombinantvectors nor any of the control (infected and uninfected) animalsrevealed such an activity in their serum.

The same sera were examined for their effects on SIVinfection of susceptible target cells. To demonstrate this, we

initially used the AA-5 cell line as target and the SIVDeltaB670virus as the infecting agent. As illustrated in Figure IF,SIVDeltaB670 forms only occasional syncytia (< 10) within 4-5days of infection on the AA-5 cell line. However, in the presenceof a representative serum from a macaque immunized withwhole inactivated virus, a larger number of syncytia can bevisualized within the clumps (Fig. 1E). The number of syncytiaobserved, which could be enumerated after dispersal of theclumps by pipetting (Fig. IG), can be greater than 100 and isdependent on the serum dilution (Table 1 ). We have termed thismarked increase in the number of syncytia in the presence ofSIVDeltaB670 as fusion enhancement activity (FEA). Note thatat one day post infection, cells adhere similarly to uninfectedcells (Fig. 1 A, C). Thus cell clumping ( I day postinfection) andsyncytium formation (5 days postinfection) can be determined inthe same assay at different times.

Accompanying the FEA was a higher degree of SIV replica¬tion as measured by RT activity in the culture supernatant. Asshown in Figure 2, the intensity of the reaction in the dot blotmicroreverse transcriptase assay compared with virus alone isevident between a 1/40 and 1/320 dilution of the serum used.Note that in the same assay, serum from infected animalsneutralizes the infectivity of SIVDeltaB670 within a similar rangeof serum concentrations. To demonstrate that such sera actuallyenhance the infectious titer of the inoculum, the virus was

titrated by twofold dilutions in the presence of either medium,normal monkey serum or serum from a macaque immunizedwith whole inactivated SIV. The results (Fig. 3) demonstrate a

marked increase (100-fold) in the presence of serum from thevaccinated animal.

Although we have focused on the effects of sera on AA-5cells, similar observations were made on other human cell lines,particularly with cell clumping. Thus, H-9, HUT-78, and CEMcell lines aggregated similarly in the presence of sera frommacaques vaccinated with whole inactivated virus preparations.We also detected such reactivity in sera from macaques whichhad been immunized with a human glioma cell line. Weconclude from this that the antigen(s) in the vaccines whichinduce such reactivity are shared among a variety of human celltypes.

Cell surface reactivity of sera from vaccinated animals

The observations that only sera from animals immunized withwhole inactivated SIV preparations caused cell clumping, FEA,and enhancement of SIV replication suggested the presence of a

reactivity with the cell surface. To test this, we employedfluorescence-activated cell sorting (FACS) analysis, reactingsera from normal, infected, and immunized macaques withnoninfected AA-5 cells. A representative composite of theresulting FACS profiles is shown in Figure 4. Some of the seraused derive from vaccine studies summarized in Table 5. Serafrom normal and SIV-infected macaques had no detectablereactivity above background, whereas sera from animals immu¬nized with whole inactivated virus (MG23594) exhibited readily


e i,


Table 1. Titration of Cell Clumping'1 and FEA;'

Serumdilution

Cell clumps/well(day 1)

Syncytialwell(day 5)

2040801603206401,2802,5605,12010,24020,48040,960

1,920

1171119299

119124134117986025

60

Noneb148 (T)c163 (T)98 (T)6138201264300

"Numbers represent the average of two determinations.bEntire cell population is destroyed at high concentrations of

protective sera despite heat inactivation.CT = some toxic effect of sera is evident.

demonstrable cell surface binding activity against noninfectedAA-5 cells. Not all sera from this latter group of animals hadsignificant reactivity (MG23557). Likewise, sera frommacaques immunized with recombinant immunogens such as

SIV gpl 10 were devoid of detectable anti-cell reactivities.Curiously, a macaque inoculated with a human glioma cell linedeveloped antibodies capable of binding to AA-5 cells in a

manner similar to that seen with certain macaques immunizedwith whole inactivated SIV. The precise nature of the anti-cellspecificities in these animals is currently under investigation.

Reactivity of sera with normal and infectedmacaque cells

When the same sera were examined on normal macaqueperipheral blood mononuclear cells (PBMC) following phytohe-magglutinin (PHA) activation, we observed no evidence of cellclumping. By contrast, similar studies with human PBMCproduced clumping comparable to AA-5 cells. When macaquePBMC were infected with SIVDeltaB670 no detectable syncytiawere formed although virus infection could be verified by RTactivity. This may reflect the relatively low number of suscepti¬ble cells for SIV infection compared with cell lines such as

AA-5. However, in the presence of serum from animals receiv¬ing whole inactivated virus vaccines, we also observed no

evidence of syncytium formation and only a slight degree of

enhancement of SIV replication was detectable. As expected,sera from SIVDeltaB670-infected animals neutralized this infec¬tivity, but only at high serum levels.

We also studied the reactivity of the sera with macaque PBMCby FACS analysis. The detectable surface staining (not shown),although distinctly above that of sera from normal or infectedanimals, was markedly less pronounced than on human cells(see Fig. 4). This was surprising in that any cross reactivity withnormal macaque antigens presumably would have been removedthrough absorption by cells and tissues of the immunizedmacaque. Thus, it is uncertain if this cross reactivity has anyrelevance to the protection induced by the whole inactivatedvirus vaccines studied to date.

Relationship of cell aggregation and FEA activity tovirus neutralizing capacity of sera

A number of SIV isolates have been examined in virusneutralization assays with the outcome that some, such as

SIVMAC25|1012 and BK28 (13; E. Hoover, personal communi¬cation; our unpublished observations) are highly susceptible toneutralization while others, like 32H,14 appear to be quiterefractory. Such conclusions must be tempered somewhat sincethe assays used are not comparable. In our experience, serumfrom macaques infected with SIVMAC25, or SIVDeltaB67() neu¬

tralize SIVMAC25, with high titers (as high as 1:400,000). Whentested against SIVDeltaB()70, only marginal neutralizing activitywas observed (1:320 with the homologous serum). Because ofstriking differences in infectivity or cytopathicity of theseviruses for human cell lines, different target cells were used forthese assays (HUT-78 for SIVMAC251 and AA-5 forSIVDeltaB670) complicating comparison of these results. Nev¬ertheless, we have not found a target cell to date where thecapacity to neutralize SIVDeltaB670 is significantly improvedabove even the low degree in the AA-5 cell system. Furtherstudies are required with the available SIV strains, cell lines, andassay configurations in order to standardize SIV neutralizationassays in order to draw meaningful comparisons.

With this background, we examined sera from vaccinatedanimals for their ability to neutralize these two isolates. Serafrom animals vaccinated with whole inactivated SIVMAC25)neutralized the homologous virus, but did not block infection ofSIVDeltaB670 (Table 3). Thus the presence of anticellularreactivity did not improve the neutralizability of SIVDeltaB670.Furthermore, neither neutralization nor lack thereof was depen¬dent on the presence or absence of complement. Preincubationof virus with such sera with and without complement followedby sedimentation of the virus, washing and resedimentationallowed most of the infectivity to be recovered (data not shown).

FIG. 1. Phase micrographs ofcells inculture wells (X360). (A) Clumping ofAA-5 cells in the presence of serum from an animalvaccinated with whole inactivated SIV 1 day postseeding. Clumps consist of 15 or more cells. (B) Occasional clumping of AA-5cells with normal macaque serum one day postseeding. A similar pattern was observed when serum from an infected macaque wasused. (C) Clumping of AA-5 cells with serum from an animal vaccinated with whole inactivated SIV and 100 infectious units ofSIVDeltaB670 one day postseeding. Clumps are identical to those in frame A. (D) Little to no clumping of AA-5 cells with serumfrom infected macaques and 100 infectious units of SIVDeltaBft70 one day postseeding. Indistinguishable when serum from normalmacaques was used. (E) Large number of syncytia in clumps 5-6 following addition of 100 infectious units of SIVDeltaB670 andserum from a macaque vaccinated with whole inactivated SIV. (F) Occasional syncytia 5-6 days postchallenge with 100 infectiousunits of SIVDeltaB670 and normal macaque serum. (G) Same as (E) following vigorous pipetting. Note large number of syncytiacompared with (H). (H) Same as (F) following vigorous pipetting. An occasional syncytium can be seen.


Table 2. Large-Scale Blind Screening of Sera from Different Vaccine Studies

Vaccine typeNumber of

sera examinedNumber with

neutralizing AbNumber withcell clumping

Synthetic peptideSubunit proteinsVaccinia ± subunit boostVaccinia + WIV boostWIV alonePositive controlsNegative controlsTotal

62455

9351050

189

62455

922100

126

0009

3500

44

Sera from these studies were kindly provided by Dr. Alan Schultz, AIDS Vaccine Research andDevelopment Branch, Division of AIDS, National Institute for Allergy and Infectious Diseases.

WIV = whole, inactivated virus

Such sera also contained reactivities which caused clumpingof AA-5 cells, FEA, and enhanced infection with SIVDeltaB670(Table 4A). In contrast, sera from animals vaccinated withSIVDeltaB670 exhibited no more than marginal neutralizingactivity against SIVMAC251, no neutralizing activity againstSIVDeltaB670 (Table 3), but exhibited high titers of cell clumping,FEA, and enhanced infection of AA-5 cells by SIVDeltaB670(Table 4B). As with virus neutralization, none of these in vitroobservations were dependent on the presence of complement.

When one examines, in parallel, the reactivities of sera frommacaques immunized with recombinant envelope subunits or

Inf.mix G860 Serum

Dilution

• * 1

·«<• •1

• •1• 91• •1

2040

80

160

32064012802560

5120

10240

FIG. 2. Enhancement of SIVDeltaB670 infectivity on AA-5cells. Pooled sera from macaques infected with SIVDeltaB670(Inf. mix) reveal a neutralization titer of 1:320. In contrast,serum from a macaque vaccinated with whole inactivatedSIVDeltaBf)70 (G860) resulted in an increase in reverse tran¬scriptase activity over the same range of dilutions in comparisonto virus alone (v). (C) AA-5 cells without virus.

A Undiluted

24

81632

64

128256512

1,0242,0484,0968,19216,38432,76865,536131,072

TCIDS0 5.1xl03 9.2x10s 2.5x103FIG. 3. Titration of SIVDeltaB670 in the presence of macaquesera. Twofold dilutions of SIVDeltaB670 were incubated withserum from a normal macaque (1/100 dilution) (column A),serum from a macaque (G 860) vaccinated with whole inacti¬vated SIV (1/100 dilution) (column B), or medium (column C).Infectivity was assessed at 10 days postinfection as described inMaterials and Methods.


u

100

80

60

40

20

0

100

Normal Macaque Infected Macaque

MG 23594 MG 23557

J424

„ "U».

Glioma

12 3 12 3

Log Fluorescence IntensityFIG. 4. Flow cytometric analysis of cell surface binding byvarious macaque sera. Noninfected AA-5 cells were incubatedwith macaque sera, washed, and stained as detailed in theMethods section. All analyses were performed using identicalphotomultiplier settings and gating on viable CD45+ cells. Logfluorescence intensity is plotted versus relative cell number. Thenumbers on the x-axis denote the log domains of fluorescenceintensity. Specific details for the sera included in this compositecan be found in Table 4.

combinations of recombinant vectors followed by boosting withrecombinant subunits, varying degrees of neutralizing antibod¬ies were elicited to SIVMAC251, but no evidence of cell clump¬ing, FEA, or enhancement of SIV replication was detected(Table 4, CandD).

Based on studies carried out thus far, the enhancementphenomena have been clearly demonstrable only withSIVDeltaB670 on AA-5 target cells in the presence of sera frommacaques vaccinated with whole inactivated SIV. When SIV-MAC25I is used with AA-5 cells, the result is rapid killing of thecells in the absence of syncytium formation. Other isolates suchas SIVSM or SIVAGM infect this cell line very poorly. On theother hand, SIVMAC25I infects HUT-78 cells readily and alsoinduces syncytia.10 Sera possessing both anti-cell and neu-

Table 3. Neutralization Differences Between

Serum fromNeutralization Neutralization0fSlVMAC25, ofSlV/DeltaB670

Infected SIVMAC25IInfected SIV/DeltaB670Vaccinated SIVMAC251Vaccinated SIV/DeltaR,

1:400,0001:380,0001:44-1:7,6001:20-1:56

<1:201:320

<1:20<1:20

tralizing activities (Table 4A) showed only neutralization ofSIVMAC25I and sera with no neutralizing activity but displayinghigh anti-cell reactivity (Table 4B) showed no enhancement ofsyncytia with SIVMAC25, on HUT-78 cells. Thus, the FEAphenomenon was not evident with this virus/target cell combi¬nation employing the sera of this study. We have no clearexplanation for these differences between the SIVDeltaB670 andSIVMAC25I systems, except that they may relate to factors suchas the inherent neutralizability of the two viruses, or perhapsquantitative or qualitative features of the fusion reaction. Thus,the SIVDeltaB670/AA-5 cell combination emerges as a conve¬

nient, albeit artificial assay to detect anti-cell antibodies byscoring for cell clumping, FEA and enhancement of virus repli¬cation irrespective of the virus strain used for immunization.

Absorption analyses with AA-5 cells

It was important to determine how absorption of the varioussera affected their ability to induce cell aggregation and FEA.The results of these studies are summarized in Table 5. Whensera from animals immunized with SIVDeltaB670 were absorbedwith AA-5 cells and retested on AA-5 for cell clumpingreactivity, not surprisingly none was found. Likewise, absorbedsera lost their capacity to mediate FEA and enhance viralreplication.

To determine the effects of absorption on virus neutralization,we examined sera from animals immunized with whole inacti¬vated SIVMAC25] which possessed neutralizing activity againstthe homologous virus. Such sera also displayed cell clumpingactivity and enhanced infection of AA-5 cells by the poorlyneutralizable SIVDeltaB670. Upon absorption with normal AA-5cells, the neutralizing activity for SIVMAC25I was unaffectedwhile the cell clumping activity, FEA, and enhancement ofSIVDeltaB670 replication were removed. Similarly sera frominfected animals which possessed high titers of neutralizingantibodies for SIVM,sorption.

retained full activity following ab-

Relationships of serum reactivities to protectionWe now present four vaccine trials where each of these

reactivities noted above was examined on the day of challenge.Two of these studies employed whole inactivated virus vac¬

cines,212 one tested a recombinant envelope subunit,15 andanother evaluated a combination of a recombinant vector bear¬ing the SIVMNE envelope as a primer followed by a recombinantsubunit as booster.16

In the study by Murphey-Corb et al. ,2 which employed wholeinactivated SIVDeltaB670 as immunogen, all animals were

protected. As shown in Table 4B on the day of challenge, none

of the sera displayed more than barely detectable neutralizingreactivities against SIVMAC251 (or the SIVDeltaB67()) in contrastto serum from an infected animal. However, all sera possessedhigh cell clumping and cell surface reactivities on AA-5 cells as

well as pronounced FEA with SIVDeltaB670. Somewhat differ¬ent results were obtained from the study by Carlson et al.,12which employed whole inactivated SIVMAC251 (Table 4A).Here, various levels of neutralizing activity were evident againstthe homologous virus, the clumping titers on AA-5 were less


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Table 5. Absorption of Sera with Normal Human Cells"

Serum from MacaquesNeutralization of

ài VMAmt

FEA (5 days pi) withSIVIDeltaRf,yn

Infected with SIVMAC25]Infected with SIVMAC25]-ABSVacciniated with SIVMAC25,Vacciniated with SIVMAC25I-ABSInfected with SIV/DeltaB670Infected with SIV/DeltaB670-ABSVaccinated with SIV/DeltaRft7„Vaccinated with SNormal macaqueNormal macaque-ABS

1:100,0001:64,000

1:4201:400

1:160,0001:120,000

<100<100<I00<100

<100<100

1:6,600<100<100<100

1:4,500<100<100<100

"Sera were tested before and after absorption (ABS) with normal AA-5 cells as described inMethods.

pronounced and mixed values were obtained with surfacestaining of AA-5 cells and FEA with SIVDeltaB670.

Contrasting these results were those obtained with recombi¬nant subunit vaccines. Javaherian et al.I5 employed baculovirusexpressed envelope subunits; no protection was achieved. Someof the animals displayed high titers of neutralizing antibodies butthere was no evidence of anti-cell reactivity as evidenced by cellclumping, FEA, or FACS analysis (Table 4C). Surprisingly,although a similar pattern was discernible in the study by Huet al.,16 four of four vaccinated animals were protected (Table4D). In this instance, it is of interest that sera from animalsimmunized with virus envelope components as well as infectedanimals cross-neutralize the SIVMAC25,.

In searching for a correlate of protection from these limitedanalyses we find that for animals vaccinated with whole killedvirus preparations, 15 animals were protected and 3 were not. Ofthe 15 protected animals, 14 had cell clumping titers greater than1000 while none of the nonprotected animals had titers greaterthan 1000. Similar relationships pertain to the surface stainingand FEA reactivities. We derive from this that animals with hightiters of anti-cell reactivity (e.g., greater than 1000 in FEA) havea 93% probability of being protected against low challenge dosesof SIV grown in human cells. However, this does not precludeadditional correlations with antiviral antibodies such as in thestudy by Carlson et al.14 where neutralizing antibody titersexhibit some association with protection (Table 4A).

A similar analysis with the vaccine studies employing recom¬

binant subunits would conclude that none of the parametersmeasured can be used as correlates for protection except thatno anti-cell reactivities were detectable in any of the sera

studied. That protection was achieved in the absence of anti-cell activities in the protocol involving priming with a recom¬

binant vector followed by a subunit booster implies thatimmune responses to viral subunits were responsible. In¬deed, protection in this instance was associated with the pres¬ence of neutralizing antibodies, but other immune responses,particularly those involving the cellular arm also need to beconsidered.

DISCUSSION

We have described a numberof in vitro phenomena associatedwith serum from macaques receiving various SIV vaccinepreparations. Our findings indicate that vaccination with wholeinactivated SIV grown in human cell lines leads invariably to theproduction of serum reactivities which target surface elements ofhuman cells. This is consistent with the fact that the viruses usedto prepare the vaccine were grown in human cells. The degree ofanti-cell reactivity in the trials varies considerably and likelyreflects the nature of the virus preparation used for immuniza¬tion. This may depend on the properties of the human cells usedfor virus growth, the quantities of virus used for immunization,the purity of the preparations or the methods used for inactiva-tion. In addition to anti-cell reactivities, animals receivingwhole virus vaccines can display varying degrees of neutralizingantibodies; however, this also depends on both the virus strainused as immunogen and the target virus used for neutralization.

The pronounced clumping reaction of AA-5 cells signifies thepresence of anti-cell reactivities which contributes toward en¬

hancement of syncytium formation and SIV replication. Aggre¬gated cells may facilitate the phenomenon of cell/cell fusionwhich follows infection as well as the cell-to-cell transmission ofSIV. It is important to note that such enhancement of SIVinfectivity clearly evident with human cells, was only marginalwith macaque PBMC, rendering its relevance in vivo somewhatin question. Nonetheless, these phenomenon (cell clumping andFEA) represent a simple and efficient means to detect anti-cellreactivities which can be done in a single assay system. Theenhancement of infectivity reported here can be distinguishedfrom one form seen with HIV (for review see ref. 17) in that it iscomplement independent and mediated by cellular rather thanviral antigens.

We have not addressed the nature of the cellular antigenswhich are responsible for the reactivities observed. Since sera

from animals protected by vaccination with whole inactivatedSIV did not uniformly neutralize or lyse virus in vitro, themechanism of protection is likely to involve processes at the


cellular level which are somehow related to the anticellularreactivity. The powerful clumping effects mediated by sera fromvaccinated animals may be directed to any number of the celladhesion molecules present on human cells.18 A possible mech¬anism by which such reactivities might contribute to protectionis through deposition of such antigens on the surface of suscep¬tible target cells during the early events of SIV entry. Specifi¬cally SIV grown in human cells and bearing human transplanta¬tion antigens on its surface could target susceptible cells forimmune rejection in macaques immunized with similar inacti¬vated virus preparations.

The present results also emphasize that protection can beachieved with viral immunogens that do not induce anti-cellreactivities. Two studies using viral envelope subunits alone or

combined with recombinant vectors gave opposite results; theformer failing while the latter was successful. Comparison ofthese two studies is complicated by the fact that the virus strainsused for both immunization and challenge are distinct, particu¬larly in their pathogenicity and their biological properties.Indeed, attempts with similar protocols as used with the SIVMNEstudy (recombinant vector priming followed by subunit boost¬ing) failed when applied to the SIVMAC251 system. However,the differences could reside in the nature of the vector construc¬tions, the immunization protocols used and the virus used tochallenge including both dose and the homogeneity of the isolate(e.g., the SIVMNE challenge virus was from single cell clone).Nevertheless, clinical trials in humans with HIV using primeboost protocols analogous to those used with SIVMNE haveproduced impressive humoral and cellular responses. 19,2°

At this stage in studies with inactivated whole virus vaccinesone must consider both antiviral and anti-cell reactivities as

contributing to protection. It is tempting to suggest that it may bethe combination of the two that is responsible for the powerfulprotection mediated by such preparations. In order to addressthese questions more directly, experiments have been designedwhere the virus preparations used for immunization and chal¬lenge are matched or cross-matched according to their host cellorigin (human or macaque). Thus, whole inactivated viruspreparations grown in human cells did not protect againstchallenge with SIV grown in macaque cells.21'22 Barring ge¬netic changes in the SIV during passage in macaque cells, whichmight preclude protection,22 these results are consistent withsuggestions that anti-human cell reactivities can be a majorfactor in protection induced by whole inactivated SIV vaccines.They are also in keeping with observations of the present studythat sera from macaques immunized with whole inactivated SIVgrown in human cells reacted weakly with normal or infectedmacaque cells. This weak cross reaction would likely not besufficient to protect animals challenged with SIV grown inmacaque cells. That protection was indeed not achieved in theabove studies further suggests that the antiviral reactivitiesraised by the vaccine were not sufficient to prevent infection.However, we note that the relative proportions of antiviralversus anticellular reactivities vary considerably from study tostudy (see comparison in Table 4, and in some instances mighteven represent the dominant reactivity responsible for protection.23

It would appear from these observations that additionalstudies employing whole inactivated virus preparations originat¬ing from cells of the species where the vaccine is to be tested

(e.g., macaques) would minimize the anti-cell reactivity andenhance the antiviral reactivity. Such a study would provide a

better setting to determine the efficacy by which virion antigensare able to confer protection in a vaccine setting of a wholeinactivated virus vaccine. It may also more clearly establish therelevance of anti-cell reactivities in protection if the challengevirus is of macaque origin.

ACKNOWLEDGMENTS

These research studies were supported by Grant 5-POl-CA43447-07 from the National Cancer Institute and Grant1-P30-AI28662-04 from the National Institute of Allergy andInfectious Diseases.

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Address reprint requests to:

Alphonse J. LangloisDuke University Medical Center

Center for AIDS ResearchDurham, NC 27710

detection of anti-human cell antibodies in sera from macaques immunized with whole inactivated virus

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