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Successful vaccinations against retroviral infections in animalmodels using live attenuated viruses1–5 have prompted sugges-tions to use live attenuated human immunodefficiency virus(HIV) vaccines in humans6. However, the use of live retrovirusesas vaccines raises many safety concerns that may preclude theiruse in humans7,8. An alternative approach is to apply detailedknowledge about the requirements for protection by live attenu-ated retroviral vaccines to design safer vaccination strategies. Inthe simian immunodeficiency virus (SIV) model for AIDS re-search, virus-specific helper T cells9,10, cytotoxic T cells9,11,12

(CTLs) and B-cell responses13,14 have been described in macaquesvaccinated with live attenuated virus, but only correlative dataare available, and the role of these effectors in protection againstpathogenic viruses is still unknown15–17. To address basic ques-tions regarding the protective mechanisms of live attenuatedretroviruses, we have used Friend virus (FV) infection of mice.The use of this model allows experimental procedures such asadoptive transfers of immune cells that are now not possible innonhuman primate models.

FV is an immunosuppressive retroviral complex that induceslethal disease in adult immunocompetent mice18–20. It comprisesa replication-competent nonpathogenic helper virus, F-MuLV,and a replication-defective but pathogenic spleen focus-formingvirus, SFFV (ref. 21). Susceptible animals develop a rapid poly-clonal erythroblast proliferation after FV inoculation that ismanifested by acute and severe splenomegaly22–24. Within severalweeks, unresolved infections lead to fatal erythroleukemia25.

Live attenuated vaccines have been described in the FVmodel1,26,27. Here, the attenuated virus was a biologically clonedN-tropic F-MuLV helper virus, which replicates very poorly inadult mice of the Fv-1b/b genotype used here27–29. This vaccineelicits highly protective immune responses, even in mousestrains that otherwise have poor immunological responsivenessto FV because of their major histocompatibility complex (MHC)background1,27. Protection is mediated through immunologicalmechanisms rather than through viral interference27, and live at-tenuated FV induces virus-neutralizing antibody responses, CD4+

T-cell proliferative responses and CTL responses1. However, it is

not known which types of responses or immune cells are re-quired for protection. These studies address this using adoptivetransfer studies.

Transfer of spleen cells from F-MuL V-vaccinated miceInitial adoptive transfer experiments using titrated numbers ofimmune spleen cells were done to establish the quantity of cellsnecessary to transfer protective immunity into syngeneic naivehosts. Immune spleen cells were obtained from mice vaccinatedwith a live attenuated retrovirus, and all mice were challengedwith a lethal dose of a primary FV isolate, 1 day after cell transfer.As few as 1 × 106 transferred cells induced recovery fromsplenomegaly in half of the recipients, and increasing the cellnumber to 5 × 107 completely prevented induction of disease (Fig.1) and viremia (Fig. 2) in all recipients. Thus, transplantationwith 5 × 107 immune cells represents a level of protective immu-nity attainable by vaccination with a live attenuated retrovirus.

Requirement for multiple lymphocyte subsets in protectionby a live attenuated vaccine against retroviral infection

ULF DITTMER, DIANE M. BROOKS & KIM J. HASENKRUG

Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, NIAID, NIH, 903 S. 4th St., Hamilton, Montana 59840

Correspondence should be addressed to K.J.H.

Infection by live attenuated retroviruses provides excellent protection from challenge with path-ogenic viruses in several animal models, but little is known about which immune effectors arenecessary for protection. We examined this using adoptive transfer experiments in the Friendvirus mouse model. Transfers of immune spleen cells into naive mice conferred complete protec-tion, and transfers of purified lymphocyte subsets demonstrated that this effect required com-plex immune responses involving CD4+ and CD8+ T cells and also B cells. In addition, passiveimmunization experiments demonstrated that antibodies alone reduced virus loads but did notprevent infection. These findings may have implications for retroviral vaccine design in general.

Fig. 1 Titration ofspleen cells from F-MuLV-vaccinated micein naive recipients.Immune spleen cellswere obtained fromage- and sex-matched(B10.A × A.BY)F1 micevaccinated with live at-tenuated F-MuLV 30days before. Titratedimmune spleen cellswere transferred intonaive syngeneic recipi-ents and the mice werechallenged the nextday with pathogenicFV. After the virus chal-lenge, mice were palpated for splenomegaly24. Immune cells transferred:g, 1 × 106, n = 4; m, 1 × 107, n = 4; p, 5 × 107, n = 7; G, 1 × 108, n = 4. P,control mice (n = 7) received 5 × 107 spleen cells from naive mice. One ofthe seven control mice died with splenomegaly, and all control mice hadsevere splenomegaly at 8 weeks after infection.

Weeks after infection

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Fig. 2 Plasma viremia in FV-challenged re-cipients that received different lymphocytesubsets. Plasma samples were taken at 8 daysafter challenge to assay for the presence ofviremia and virus neutralizing antibodies.These data are from the same mice as shownin Figs. 1, 3 and 4. The lower limit of detectionfor viremia was 220 focus-forming units/ml ofplasma. Plasma samples that produced lessthan 75% neutralization at a dilution of 1:20were considered antibody (Ab)-negative.

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Based on the percentage of each lympho-cyte subpopulation present in 5 × 107

whole immune spleen cells (Table), it waspossible to determine the contribution ofeach lymphocyte subpopulation by trans-ferring the purified subpopulations at theappropriate cell numbers. For these experi-ments, different lymphocyte subsets werepurified by immunomagnetic bead separa-tion, and the purified fractions were thentransferred into naive recipients, 1 day be-fore virus challenge. All mice that receivedpurified CD19+ cells (B-cell lineage) devel-oped sustained splenomegaly, indicative ofFV-induced erythroleukemia (Fig. 3). In ad-dition, all mice were viremic at 1 week afterchallenge (Fig. 2). Transfers of purifiedCD4+ cells (T-cell lineage) also did not pre-vent FV-induced splenomegaly or viremia,but two of six recipients later recovered(Fig. 3). Finally, all mice receiving CD8+

cells (T-cell lineage) became splenomegalicand viremic as well, but six of seven mice later recovered fromsplenomegaly (Fig. 3). Thus, no single lymphocyte subset wassufficient to transfer protection from infection, but the immuneT cells, especially CD8+ T cells, facilitated recovery.

To determine if all lymphocyte subsets in the whole cell frac-tion were required for transfer of protection, the immune spleencells were depleted of either CD4+, CD8+ or CD19+ cells beforetransfer. Thus, each depleted fraction still contained two lym-phocyte subsets. Although all mice transplanted with the duallymphocyte subsets developed acute virus-induced splenomegalyand viremia, more than 80% of the animals in each group recov-ered within 8 weeks after challenge regardless of which subsetwas missing (Fig. 4). Thus, the spleen cell fractions containingany two lymphocyte subsets were generally able to induce recov-ery from disease, but did not protect against infection.

To ensure that the purification and depletion procedures didnot alter the physiological functions of the transferred cells, an‘addback’ experiment was done. Naive mice were inoculatedwith an admixture of immune CD4+, CD8+ and CD19+ lympho-cytes that had been purified and then remixed. After challengewith FV, these animals were protected from viremia (Fig. 2) andsplenomegaly (Fig. 3), demonstrating that the experimental pro-cedures did not abrogate the ability of the transferred cells to

confer immune protection. Furthermore, this result indicatedthat cells expressing T-cell and B-cell markers were the only cellsnecessary for transfer of protection.

Protection by treatment with virus-neutralizing antibodiesUnlike immunized mice challenged at 30 days after vaccination,the mice adoptively transferred with whole immune spleen cellsthe day before challenge did not have pre-existing high titers ofvirus-neutralizing antibodies. Although the full protection af-forded by such transfers indicated that pre-existing antibodytiters were not necessary for protection, the requirement for Bcells in the transfers indicated that antibodies might contributeto protection. Furthermore, protection from viremia showed a100% correlation with development of virus neutralizing anti-bodies by 8 days after challenge (Fig. 2). To determine whetherantibodies alone, in the absence of primed lymphocytes, couldaffect virus infection and recovery in vivo, we used passive trans-fer of a FV-neutralizing monoclonal antibody known to com-pletely neutralize the challenge virus in vitro and to mimic someof the effects of antiviral antisera in vivo30,31. Dosages were em-pirically determined such that plasma levels matched the titersfound in vaccinated mice at the timepoint of challenge (Fig. 5).

Passive immunization resulted in a substantial decrease in the

Table Quantitation of cells used in adoptive transfer studies

CD4+ cells CD8+ cells CD19+ cellsMean percentage of each 21.4% ± 1.2% 19.0% ± 0.5% 44.5% ± 1.1%subpopulation in whole immune spleen cells ± sd1

Mean cell number present 1.1 × 107 9.5 × 106 2.2 × 107

in 5 × 107 whole spleen cells

Cell number transferred per 1.0 × 107 9.2 × 106 2.1 × 107

mouse in single subpopulation transfers (Fig. 2)

CD4+ & CD8+ cells CD4+ & CD19+ cells CD8+ & CD19+ cellsMean cell number present 2.1 × 107 3.3 × 107 3.2 × 107

in 5 × 107 whole spleen

Cell number transferred per 2.0 × 107 3.9 × 107 3.2 × 107

mouse in dual subpopulation transfers (Fig. 3)1Spleens were analyzed by flow cytometry at the time of adoptive transfer for percentages of CD4+, CD8+ and CD19+

cells. CD25 and CD69 activation markers were also analyzed. The percentages of each lymphocyte subpopulationpositive for these markers were: CD4+ cells, 2.4% ± 0.15%; CD25+ and 3.9% ± 0.69% CD69+; CD8+ cells, 1.1% ±0.26% CD25+ and 2.6% ± 1.2% CD69+; CD19+ cells, 1.2% ± 0.15% CD25+ and 4.1% ± 0.15% CD69+.

Plasma viremia titer/ml


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Fig. 3 Transfer of enriched lymphocyte subsets from F-MuLV-vaccinated mice. Immune spleen cellswere enriched for CD4+, CD8+ or CD19+ cells and transferred into naive syngeneic recipients. Oneday later, the recipients were challenged with pathogenic Friend virus complex. The mice were pal-pated for splenomegaly at the indicated time points (horizontal axis). Mice with spleens greater thantwice the normal size were considered splenomegalic24. Each recipient received either CD4+ cells (p,n = 6), CD8+ cells (m, n = 6) or CD19+ cells (g, n = 5) at the cell numbers indicated in the Table. Fourmice (G) were transplanted with an admixture of enriched CD4+ (1.25 × 107), CD8+ (1.25 × 107) andCD19+ (2.5 × 107) cells. Eight mice (r) were passively immunized with monoclonal antibody 48. Forpassive immunization studies, virus neutralizing antibody titers in plasma samples from passively im-munized mice were determined on the day of challenge; the log2 mean geometric titer in these micewas 7.22, s.d. = 0.67 (n = 12) compared with 7.46, s.d. = 1.3 (n = 14) in F-MuLV-N-immunized miceat 30 days after vaccination. The difference between these titers was not significant as determined byMann-Whitney test (P = 1.000 based on a normal approximation).


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mean level of infectious centers (ICs) at 10 days after challenge(Fig. 5). In contrast, no ICs were detectable at this timepoint inmice immunized with attenuated virus. Although passive im-munization did not completely neutralize the input virus, alleight mice later recovered from FV-induced splenomegaly (Fig.3). When the same antibody transfer experiments were done infour recipients that had been previously depleted of both CD4+

and CD8+ T cells, none of the mice recovered. Thus, the anti-body-facilitated recovery was dependent on endogenous T-cellresponses.

Protection from persistent FVFV infection, even in resistant mouse strains that recover fromacute infection, is associated with persistent virus that can reacti-vate to cause disease upon immunosuppression32. To determinewhether recovery in transplanted mice was associated with com-plete elimination of virus, IC assays were done on spleen cellsharvested from mice at 2 months after infection. Nine of ninemice that were adoptively transferred with CD8+ cells and recov-ered from splenomegaly had persistent spleen virus rangingfrom 30 to 140 ICs per 1 × 107 spleen cells at this timepoint. Incontrast, six of seven mice that received 5 × 107 whole immunespleen cells had no detectable persistent virus (<1 IC per 1 × 107

cells), and one mouse had 8 ICs per 1 × 107 cells. Thus, although

CD8+ cells alone transferred recovery from acute disease, onlywhole immune cells protected against both initial splenomegalyand persistent infection.

DiscussionFor some viral infections, protective immunity can be achievedby priming either the humoral or the cellular arms of the im-mune system. For example, in rabies infections, only virus-spe-cific antibodies are essential for control of virus replication andspread, as has been shown in T cell-deficient mice33,34. In con-trast, protection against lymphocytic choriomeningitis virus canbe achieved without any detectable antibodies by priming CTLresponses35,36. In one retroviral model (Rauscher virus), it seemedthat cell-mediated immunity was sufficient for protection37.However, that study did not analyze protection from persistentvirus, so protection may have been incomplete. In our experi-ments here with FV, each lymphocyte subset seemed to haveunique and necessary functions that acted in concert to inducerapid and complete protection. For example, we observed betterprotection and quicker recovery after transfers of 1 × 107 wholeimmune spleen cells (Fig. 1) than was seen with transfers of200–400% as many cells depleted of any single lymphocyte sub-set (Fig. 4). Here we analyzed the complete protection providedby 5 × 107 whole immune spleen cells. Thus, it remains possiblethat single or dual lymphocyte subsets might be protective iftransferred in overwhelming numbers, but the efficacy of vacci-nation with live attenuated FV seems to be related to its ability tostimulate multiple arms of the immune system.

The recovery from splenomegaly of mice receiving only im-mune CD8+ cells or virus-neutralizing antibodies demonstratesthe potent protective capacity of these immunological effectors(Fig. 3). Transfers of CD8+ T cells alone reduced viremia titers(Fig. 2). The antiviral function of the CD8+ T cells might bethrough direct killing of infected target cells, as has been shownin vitro for vaccine-elicited FV-specific CTLs (ref. 1), or throughthe release of unknown antiviral factors, as has been found inHIV infection38,39. Antibody-mediated protection could also actthrough various mechanisms such as virus neutralization or an-tibody-dependent, cell-mediated cytolysis. Transfers of B cellsdid not result in detectable virus-neutralizing antibody titers un-less both CD4+ and CD8+ cells were also transferred (Fig. 2). Thisprobably reflects the inability of B cells alone to control viremiasuch that antibody titers could exceed the titer of available epi-topes on free virus and become detectable.

In addition to the CD8+ T-cell and the B-cell responses, primedCD4+ T cells were an important component for protection


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Fig. 4 Transferof dual subsets ofimmune spleencells. Immunespleen cells fromvaccinated micewere depleted foreither CD4+ cells(g, n = 8), CD8+

cells (m, n = 7) orCD19+ cells (p, n =7) before transferinto naive syn-genic recipients.The depleted pop-ulations contain-ing the twonon-depleted lym-phocyte subsets were transferred at the numbers indicated in the Table.One day later, the recipients were challenged with pathogenic Friend viruscomplex and were examined weekly for FV-induced splenomegaly. Sevencontrol mice in each group received 5 × 107 spleen cells from naive (P) orF-MuLV-vaccinated (L) mice.


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Fig. 5 Levels of antibody and of spleen infection in F-MuLV-vaccinated mice compared withthose in passively immunized mice. Single-cell suspensions from spleens taken at 10 days afterinfection were plated as infectious centers to determine levels of spleen virus (P). The mean in-fectious center levels per spleen were: unvaccinated mice, 5.1 × 108; mice passively immunizedwith monoclonal antibody (mAb) 48, 2.9 × 106; vaccinated mice vaccinated with F-MuLV, nonedetectable (< 1 focus per 107 spleen cells). Geometric mean titers of of virus-neutralizing anti-bodies (p) ± s.e.m. were measured on the day of challenge (30 days after vaccination for the F-MuLV group). There was no significant difference between vaccinated mice and mice passivelyimmunized with mAb 48 as determined by Mann-Whitney test (P = 1.000).

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against FV. CD4+ T cells probably provided help for both B cellsand CD8+ T cells, although B-cell responses were more depen-dent on primed CD4+ T cells than were CD8+ T-cell responses, asCD8+ T cells alone induced recovery (Fig. 3), whereas B cells re-quired CD4+ T cells (Figs. 3 and 4). CD4+ T cells may also havehad a direct antiviral effect as has been suggested in both FV (ref.32) and HIV infections40. None of the immune lymphocytestransferred in our experiments showed high levels of activationmarkers at the time of transfer (Table). This is not surprising, asthe donor mice had cleared the vaccine virus 1–2 weeks beforethe transfers (U.D. and K..J.H., unpublished results).

The question arises as to whether the findings here can be gen-eralized to other retroviral models such as SIV or HIV, especiallyas FV is an oncogenic retrovirus, whereas SIV and HIV are not.However, the host responses required for protective immunityare less related to late pathological events than they are to earlyfactors such as the types of cells infected, the mechanisms ofvirus spread, the route of infection, the virus dose and so on. Inthese areas, FV is both similar to and different from HIV. For ex-ample, both FV and HIV infect macrophages, although FV in-fects erythroid cells and B cells as well, with little infection of Tcells32. Both FV and HIV spread through free virus and cell-to-cellmechanisms, possibly requiring both humoral and cell-mediatedresponses for protection41. Both FV and HIV establish persistentinfections despite resolution of acute infection. In FV, relapse ofdisease is uncommon unless the mice have suppressed CD4+ T-cell responses32. In HIV, almost all untreated patients eventuallyrelapse with high viremia and develop AIDS. Our results here in-dicate that very early control over virus replication and spreadare required to prevent the establishment of persistent infection.

It may also be helpful to compare our results with studies ofprotection done with HIV and SIV. For example, passive immu-nization experiments in chimpanzees42 and hu-PBL-SCID mice43

have shown protection against HIV-1. Although these results in-dicate an important role for antibodies, they do not necessarilyindicate that humoral immunity alone is sufficient to protectagainst HIV, as virus replication was poor in these models andthe hosts do not usually develop AIDS-like symptoms. SIV vac-cines that have either elicited predominatly cell-mediated or hu-moral responses have generally not been protective against themore virulent, pathogenic strains of SIV (ref. 44). In contrast, re-cent vaccine strategies that induced complex effector mecha-nisms provided solid protection against SIV/HIV chimericviruses in macaques45,46. Thus, correlative data from other retro-viral models as well as our direct data from FV adoptive transferstudies support the idea that retroviral vaccine strategies de-signed to elicit both humoral and cell-mediated responses wouldbe more likely to succeed than narrowly focused strategies.

MethodsMice. Female (B10.A X A.BY)F1 mice 3–6 months old at experimentalonset were used for all experiments. Parental mouse strains for breeding F1mice were obtained from the Jackson Laboratories (Bar Harbor, Maine).Breeding of F1 strains was done at Rocky Mountain Laboratories. All micewere treated in accordance with NIH regulations and the guidelines of theAnimal Care and Use Committee of Rocky Mountain Laboratories.

Vaccination and virus challenge. The B-tropic, polycythemia-inducingFV complex used as challenge virus in all experiments was from unclonedvirus stocks obtained from 10% spleen cell homogenates as described24.The N-tropic F-MuLV vaccine virus (stock #29-51N)47 was a 24-hour su-pernatant from infected Mus dunni cells. Mice were vaccinated by inject-ing intravenously 0.5 ml of phosphate-buffered, balanced salt solutioncontaining 2% fetal bovine serum (FBS) and 1 × 104 focus-forming unitsof F-MuLV vaccine virus. In all virus challenge experiments, mice were in-jected intravenously with 0.5 ml phosphate-buffered, balanced salt solu-tion containing 2% FBS and 3,000 spleen focus-forming units of Friendvirus complex. After virus challenge, mice were palpated forsplenomegaly by observers ‘blinded’ to the experimental status of themice, as described24.

Viremia, infectious center and virus neutralizing antibody assays. Forviremia assays, freshly frozen plasma samples were titrated by focal infec-tivity assays as described27,48. Plasma samples were also tested for virus neu-tralizing antibodies as described27,49. The cut-off point for antibodytitrations was 75% virus neutralization. Infectious centers from spleenswere detected by titrations of single cell suspensions onto susceptible Musdunni cells as described27.

Passive transfers of monoclonal antibody 48. Monoclonal antibody 48 isa mouse monoclonal IgG2b specific for F-MuLV gp70 envelope50.Supernatants were prepared from a Cellmax® artificial capillary system(Cellco, Georgetown, Maryland). For the recovery experiments in Fig. 3,the supernatant was diluted 1:27 and 0.5 ml was injected intraperitoneally2 times, on days –5 and –3 relative to virus challenge.

Adoptive spleen cell transfer. Thirty days after vaccination, spleen cellsuspensions were pooled, depleted of erythroid cells by lysis in ammoniumchloride–Tris, and adoptively transferred to naive syngenic animals by in-travenous injection of 0.5 ml of phosphate-buffered, balanced salt solutioncontaining 15 U/ml heparin. The recipients were challenged 1 day aftertransfer.

Enrichment and depletion of lymphocyte subsets. The MidiMACS®Separation System (Miltenyi Biotech, Bergisch Gladbach, Germany) wasused for the enrichment and depletion of lymphocyte subsets using themanufacturer’s instructions. The purity of the cell fractions was determinedby flow cytometry by staining with FITC-labeled antibodies (Pharmingen,San Diego, California). Purities were 98.6% for enriched B cells, 96.4% forCD4+ cells and 97.2% for CD8+ cells. The depleted T-cell fractions bothcontained less than 0.5% of the depleted cell type, whereas the B-cell-de-pleted fraction contained 9.4% B220+ cells. All cell fractions contained lessthan 5% dead cells after the separation procedure.

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AcknowledgmentU.D. is supported by a fellowship from the ‘DeutscheForschungsgemeinschaft’.

RECEIVED 9 OCTOBER; ACCEPTED 23 DECEMBER 1998

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