Specific and non-specific immunity and protection of macaques against SlY infection Roger Le Grand *~, Guillaume Vogt*, Bruno Vaslin*, Pierre Roques*, Frrdrric Throdoro*, Anne Marie Aubertin t and Dominique Dormont *~ The simian immunodeficiency virus is a retrovirus closely related to the human immunodeficiency viruses; it induces an AIDS-like disease in macaques, and provides therefore an obvious animal model for anti-lentiviral drug and vaccine strategy assessments. In our experiment, we immunized rhesus macaques with a purified and formalin-inactivated whole SIV,,ac251 antigen preparation. Most of these monkeys were still protected for more than 4 months following a heterologous SIVs, . intravenous challenge. Both virus stocks, for vaccine preparation and challenge, were provided by culture supernatants of infected T cells of human origin. Four of the protected macaques were then reimmunized with the same antigen preparation and rechallenged intravenously with a homologous rhesus cell grown SIV,.~251. Unexpectedly, all animals developed clinical and biological evidence of infection by day 15 after the second challenge. Keywords: SIV vaccine, AIDS ammal model

INTRODUCTION The acquired immune deficiency syndrome (AIDS) is a lentiviral induced disease whose causative agents are the Human Immunodeficlency viruses, HIV-1 ~ and HIV-22. The worldwide dramatic epidemic spread since the first description of AIDS in the early eighties 3 emphasizes the urgent need for the development of efficient therapies and vaccines. Despite the difficulty of carrying out vaccine trials in human volunteers, different HIV antigen preparations have been demonstrated to elicit in vitro specific neutralizing antibodies and cellular immune responses 4-7. The search for anti-lentiviral drugs and vaccines, as well as the understanding of AIDS pathogenesls, is greatly facilitated by the availability of relevant animal models of human HIV infection. The simian immunodeficiency virus 8 (SIV) is a lentivirus closely related to HIV-1 and HIV-2 which induces in macaques an *Laboratolre de Neuropathologie Exp@rimentale et Neurovlrologie, Centre de Recherches du Service de Sant6 des Armees, Commissariat ~ I'Energie Atomlque/Dwectlon des Sciences du Vwant/D@partement de Toxlcologle Exp@nmentale, BP 6, 92 265 Fontenay aux Roses Cedex, France. tLaboratoire de V=rolog~e, Umvers~t6 Louis Pasteur, 3 rue Koeberle, 67 000 Strasbourg, France. tlnstitut Pasteur, 25-28 rue du Docteur Roux, 75 015 Paris, France °°To whom correspondence should be addressed. (Received 4 November 1991; revised 5 May 1992, accepted 6 May 1992) 0264-410)(/92/120873-07 ~, 1992 Butterworth-HememannLtd

AIDS-like disease in a time course suitable for experimental investigations. Extensive vaccine experiments have been achieved using the SIV-macaque model. Whole reactivated virus 9'1° and recombinant subunit vaccines 11 were demonstrated to successfully protect macaques against intravenous experimental SIV infection. However, most of these experiments may be reconsidered having regard to the recent report concerning the implication of anti-human cell responses in protective immunity 12. Indeed, Stott et al. reported that macaques immunized with uninfected human C8166 cells were protected against subsequent challenge with SIVmac251 (11/88 stock of the 32H isolate grown on C8166 human T-cell line). Moreover, their formalininactivated virus-vaccinated animals raised antibodies against cellular components whose ELISA titres seem to correlate with protection, while specific antl-SIV humoral immune response did not. In their experiment, viruses for vaccine preparation and challenge were provided by cell-free supernatants of SIV-infected C8166 human T cells. In our experiments, we have immunized rhesus macaques with a formalin-inactivated whole SiVmac251 grown on a human C8166 cell line. These animals were protected when challenged with a heterologous SIVsm grown on human PBMC, but protection could not be achieved against a homologous SlVma¢251 grown on simian cells x3. We therefore investigated the specific and non-specific humoral immune parameters of the vaccinated animals.

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Humoral tmmumty and SIV vaccme: R Le Grand et al

MATERIALS AND METHODS Animals Rhesus monkeys ( M a c a c a m u l a t t a ) , within a 3 to 4 kg weight range, one male and nine females, were imported from China, and housed in biosafety level 3 facilities. Animals were clinically examined every week under ketamine anaesthesia (M6rleux, France), and blood samples were drawn. All experimental procedures were done according to institutional guidelines for animal care.

Vaccine preparation The vaccine preparation (provided by M. Cranage and P. Greenaway, CAMR, U K ) was supphed by the European Vaccine against AIDS (EVA) programme. Briefly, viral antigens (11/88 stock of the 32H isolate of SIVmac251) were recovered from cell-free supernatants of infected C8166 human T cells 14 by ultracentrifugation at 18 000 rev mln- 1 for 30 min. Then the resuspended crude virus was loaded onto a Sepharose 4B column. The PBS eluted fractions were pelleted by ultracentrifugation ( 18000 rev min- ~ for 30 min ), and adjusted to 2 mg ml- 1 of final protein concentration in PBS prior to an incubation of 24 h at 0°C with 0.8% formaldehyde. The validation of inactivation of the product was done by a tissue culture infectivity assay.

Immunization and challenge infection of macaques Two groups of four animals received 0.5 mg or 0.1 mg of the vaccine preparation at days 0, 30, 60 and 135 Viral antigens were injected with 2 m l of aluminium hydroxide adjuvant (Alhydrogel, Superfos Blosector, Denmark), using the intramuscular route. As controls, two animals received 2 ml of the adjuvant alone at each step of the immunization schedule. At challenge, monkeys received by the intravenous route 1 ml of a tenfold dilution in phosphate-buffered saline (PBS) of an m v w o titrated virus stock containing 10 animal 50% infectious doses (AID5o). The SIV~m virus stock a5'~6 (SMM-3 Isolate, P. Fultz and H. McClure, Atlanta, USA, m VlVO titrated and kindly provided by P. Putkonen, N.B L., Sweden) was obtained from a cell-free supernatant of an infected human peripheral blood mononuclear cell ( PBMC ) culture. The SIVm, c251 virus stock was provided by a cell-free supernatant of a rhesus PBMC culture infected with a spleen homogenate obtained from an infected monkey.

Virus rescue from PBMC of infected monkeys Fresh monkey PBMC (2.106) isolated from FicollHypaque gradient ( MSL, France ) were cocultlvated with 2 x 10 6 C8166 human cells in the presence of 1/~g ml-1 of phytohaemagglutlnln-A (Dlfco, Detroit, USA) in a total volume of 3 ml of culture medium: RPMI 1640 (Boehringer Mannheim, Germany) supplemented with 10% fetal calf serum ( Boehrlnger Mannheim, Germany ), 2mM L-glutamme (Gibco, NY, USA), 2 # g m l - t polybrene (Sigma, St Louis, USA), 10-; IU anti-.~interferon antibody (Sigma, St Louis, USA), and 1% trlantlNOtlCS solution (Glbco, Grand Island, NY, USA). At day 3 of culture, the cell suspension was adjusted to 12 ml with fresh culture medium. The cell suspension was diluted 1:'4 in culture medium twice a week until detection of syncytla formation. Presence of viral particles m cell-free supernatants was confirmed by the detection of specific reverse transcrlptase activity 1.

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Polymerase chain reaction (PCR) amplification of integrated viral genome in monkey lymphocytes The presence of SIVsm DNA was determined using a primer pair specific for p o l gene (SdP4723N, SDP5199). The amplification of SIVmac2Sl DNA was performed using primer pairs for 9ag (1386-2129) and env genes (8125-9228), which do not allow SIV~m genome amplification. As described by Kitchln et al. 17, PCR was performed on both DNA extracted from fresh monkey PBMC and m w t r o SIV-infected PBMC. Molecular clone BK28 provided by EVA was used as positive control. The reaction mixture was as follows: 10 mM T r i s - H C l , pH 8.3, (at 25°C ); 50 mM KCI; 1.0, 1.5 or 2.5 mM MgCIz; 0.01% w/v gelatin; 200 mr~ each of dATP, dCTP, d G T P , dTTP; 0.25 mM of primer pair; 1 #g DNA template; 2.5 units o f T a q polymerase (Cetus Amplitaq). For ollgomer hybridization of the amplified DNA, 24 #1 of the PCR product were analysed on a 1.5% agarose gel. The DNA was transferred onto Hybond-N membrane by Southern blot. Oligonucleotlde probes specific for gao and env were 32p-radiolabelled using 5'-end labelling method. Membranes were washed at room temperature in SSPE 6X, 0.1% SDS (10 min) and SSPE, pH 7.4, (NaC1 3 M, Na2H2PO4 200 mu, EDTA 20 mM) 2X, 0.1% SDS at 42°C (10min). Autoradiography was carried out at - 7 0 ° C for 16 h.

Detection of viral and cellular specific antibodies Because of the high sequence homology between HIV-2 and SIV, the specific antl-SIV serum reactivity was determined as previously described 15, using an HIV-2 antigen detection assay. In our experiments we used the ELAVIA II kit (Diagnostics Pasteur, France). AntI-SIV specific responses were confirmed by Western blot using the New-Lav blot kit (Diagnostics Pasteur, France). For anti-cellular ELISA, 3 days pHa-p stimulated human or rhesus PBMC were maintained in culture for 7 days, and then extensively washed in PBS before they were lysed in Nonidet-P40 0.5% detergent solution, at a concentration of l0 T cells ml-1. After 30 min at 4°C, cell lysates were centrifuged at 120009 for 10 mm and the supernatants were diluted 1/50 in PBS for coating ELISA plates. In all the ELISA and Western blot described above, as second antibody a peroxidaselabelled anti-monkey lmmunoglobulin G was used (Organon Teknika, The Netherlands ). Tenfold dilutions starting at 10-1 were performed for each serum Tltres were determined as the last serum dilution giving a significant positive absorbance (AI at 490 nm when compared with ten measures of pre-immune sera at the same serum dilution (cut-off = A average of pre-immune sera + 2.26 s.d.).

Detection of neutralizing antibodies In a 96-well flat-bottomed culture microplate, twofold serial dilutions (in triplicates) of the tested serum were incubated with 100 tissue culture 50% infectious doses (TCIDso) of the virus stock m PBS supplemented with 10% complement depleted sera from healthy and uninfected rhesus monkeys. Controls were performed as above in the absence of tested sera. The two virus stocks were constituted by cell-free supernatants of SIVm,c2Sl (32H Isolate) infected HUTvs human T cells, and SIVmac251 infected rhesus PBMC (one more passage on rhesus cells of the virus used for the in vit, o challenge).

Humoral tmmumty and SIV vaccine R. Le Grand et al

the first challenge, all sera from the immunized monkeys recognized viral proteins. The stronger humoral immune responses were directed against 9a9 and pol proteins. In both groups of vaccinated animals, only faint bands were detected by envelope glycoproteins (Figure 2).

After 90 mln at 37°C, 3.104 HUT78 (Ref. 18) cells were added to each well. Following 1 h of infection at 37°C, the cell suspension was washed three times in PBS, and then maintained with culture medium in a 5% CO 2 humidified incubator at 37°C. At day 3 of culture, 100/A of the cell suspension of each well were treated for 30 mln at 4°C with 20/d of a 2.5% Nonidet-P 40 detergent solution, and were used to coat ELISA plates. An ELISA was performed for the detection of viral antigens, using a heat-inactivated serum of an SIV-lnfected and unvaccinated monkey as first antibody. The second antibody was a peroxidase-labelled anti-monkey IgG. The neutralization tltre of the tested serum was determined as the last dilution giving a mean A lower or equal to 50% of the mean A of positive controls.

In vitro neutralization assay. As for ELISA htres, the strongest in vitro neutralizing activities agamst the homologous SIVmac251 (32H isolate grown on human HUTTs T-cell line) were detected m the 2 mg vaccinated group ( Table 1 ). T~tres were respectively 64, 256 and 128 for monkeys 8715, 8744, 8770 of the high dose immunized group, and 128 for monkey 8758 of the low dose group. Titres remained undetectable for monkeys 8738 (high dose group) and 8778 and 8762 (low dose group ).

First challenge using a human PBMC-grown SIV

RESULTS

Ftfleen days after the last vaccine boost, animals were challenged by the intravenous route with 10 AID5o of an heterologous cell-free SIV~m stock obtained from a supernatant of infected human PBMC. Four out of four monkeys of the 2 mg vaccinated group and two out of three monkeys of the 0.4mg group (one died from unrelated dlsease prior to challenge) were stdl protected for more than 4 and 10 months, respectively, after the experimental infection (Table 2. Fujure 1 ). Two control animals, which received the adjuvant alone, and the vaccinated and unprotected monkey (8758) developed clinical and biological evidence of SIV infecuon by day 15 postchallenge: generalized lymphadenopathy, anamnestic humoral ~mmune response, wrus isolation by coculture of rhesus PBMC with C8166 human cells and PCR detection of integrated SIV DNA m fresh isolated rhesus PBMC ( Table 2. Figure I ).

Vaccine-induced anti-SIV humoral immunity before challenge The ant]-SIV humoral immune response induced by the immunization protocol was analysed by three distinct assays: determination of specific antl-SIV anhbody tltres by ELISA, ldentificatton by Western blot of the lentlvlral proteins recognized by these antibodies, and evaluation of the m t'ltro SIV refection neutralization capacity of the sera of vacclnees E L I S A titration. Sera drawn from the 2 mg vaccinated antmals showed higher tltres than that from the 0.4 mg vaccinated ones Humoral responses of the high dose vaccinated animals (8715, 8738, 8744, 8770 ) reached a peak at the day of challenge infection at 105 Within the low dose vaccinated monkeys (8758, 8778, 8762), only animal 8758 exhibited comparable tltres to the 2 mg group at 105 . The two remaining monkeys exhibited lower tltres at 104. Interestingly, in both groups, rares dropped significantly (about 1 loglo) after the second vaccine boost (Figure 1 ).

Outcome of the second challenge using a simian PBMC-grown SIV All four protected animals of the 2 mg immunized group were reboosted with 0.5 mg of the same vaccme preparation at day 141 after the first challenge. Two weeks later, the anti-SIV ELISA titres reached levels

Western blot. The anU-SIV antibody humoral response was confirmed by Western blot. On the day of

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(symbols represent monkeys A, 8744, II, 8715, O, 8770 and V, 8738, respectively)

Vaccme, Vol 10, Issue 12, 1992

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H u m o r a l t m m u m t y a n d SIV vaccme. R Le G r a n d et al IP)

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Figure 2

Table 1 Neutrahzatlon tltres at the day of challenge by m vttro neutrahzatlon assay High dose vaccinated monkeys

At day of first challenge SIVmac25, 32H Isolate HUT78 cells grown SIVmac25, Rhesus PBMC grown At day of second challenge SIVmac~s~ Rhesus PBMC grown

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8715

8738

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