Attenuated SIV imparts immunity to challenge with pathogenic spleen-derived SIV but cannot prevent repair of the nef deletion

Immunology Letters 51 (1996) 129-135

Attenuated SIV imparts immunity to challenge with pathogenic spleen-derived SIV but cannot prevent repair of the nef deletion Christiane Stahl-Hennig”%*, Ulf Dittmer”, T. NiBlein”, Katja Pekrun”, Harald Petry”, Elke Jurkiewicz”, Dietmar Fuchsb, Helmut Wachterb, Erling W. Rud”, Gerhard Hunsmann” “Department of Virology and Immunology, German Primate Centre, Kellnerweg 4, D-37077 Gijitingen? Germany bInstitute of Medical Chemistry and Biochemistry and Ludwig-Boltzmann-Institute for AIDS Research, University qf Innsbruck, Innsbruck, Austria ‘Health Canada, Laboratory Centre for Disease Control, Ottawa. Canada

Accepted 15 March 1996

Abstract

To date, some success has been achieved with several experimental vaccines against AIDS in the available animal models. In the simian immunodeficiency virus (SIV) macaque model protection against superinfection was obtained by preinfection with a virus attenuated by a deletion in neJ To investigate the efficacy of SIVmac32H(pC8), a nefdeletion mutant of SIVmac251, as a live-attenuated vaccine, rhesus monkeys were infected intravenously (i.v.) with this virus. All monkeys became productively infected by the pC8 virus. The animals had low cell-associated viral loads but developed a strong cellular and humoral antiviral immune response. Two out of eight preinfected monkeys developed signs of immunodeficiency and were excluded from the challenge. Sequence analysis of reisolates from one of them revealed a complete repair of the nef deletion. The remaining six monkeys, two preinfected for 42 weeks and four for 22 weeks, were challenged i.v. with a pathogenic SIV derived ex vivo from the spleen of a SIV infected macaque. Four of the monkeys challenged resisted the second infection whereas in two monkeys preinfected for 22 weeks full length nef was detectable. All monkeys maintained a virus-specific CD4-cell proliferative response after challenge. Thus, even after short preinfection periods with an attenuated SIV sterilising immunity against a challenge with a pathogenic SIV can be obtained. However, such a vaccine is unsafe since the attenuated virus frequently reverts to a more virulent form. Keywords:

Attenuated

SIV; Vaccine; Protection;

Molecular repair; Macaques

1. Introduction Deletions in some auxiliary genes of SIV can ameliorate pathogenicity [l-3]. Candidate vaccines consisting of inactivated whole virus or viral subunits have been tested in the SIV or HIV-2 macaque model with limited success with respect to sterilising immunity. Under optimised laboratory conditions, e.g. challenge at the peak of the vaccine-induced immunity, monkeys immunised with killed whole virus [4-61, split [4,7] or recombinantexpressed [S] or virion-derived subunit vaccines [9,10]

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author. Tel.: + 49 551 385 1154, fax: + 49 55 1

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were protected against challenge with SIV or HIV-2. However, the protective immunity only lasted for a few months and could be overcome with heterologous challenge viruses as well as high virus doses. Furthermore, so far resistance against challenge viruses not grown in human T-cell lines have been described only once [l 11. Besides, in neither of the described experiments clearcut immune correlates of protection could be demonstrated. The most promising results in the field of experimental AIDS vaccines yielded the ‘immunisation’ of macaques with attenuated SIV lacking part of the nef gene. With this virus protection was achieved after a 2.25 year waiting period and with high doses of challenge virus [12]. Nevertheless, nothing is known about

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the immunological mechanism(s) involved in this protective immunity. Even if recent data about pathogenicity of a triple deleted SIV in new-born macaques questions the potential use of similar live attenuated viruses in man [13], studying both the host’s cellular and humoral immune response against such viruses should give us more insight into the protective mechanism(s). Therefore, we have infected rhesus monkeys (Macaca mulatta) with a mutant of SIVmac251, SIVmac32H(pC8) containing an in-frame deletion of four amino acids in nef [14]. This virus has shown an attenuated phenotype in both rhesus and fascicularis macaques (Mucaca fuscicularis) [3]. Monkeys were challenged, 22 or 42 weeks after the pC8 infection, with a pathogenic spleen-derived SIVmac. Preinfection led to a complete protection in four out of six animals. However, reversion to virulence by molecular evolution was observed in two of the pC8 infected monkeys.

2. Materials and methods 2.1. Immunisation of macaques and challenge Four rhesus monkeys of Indian origin were immunised once with 200 ,ug of keyhole limpet hemocyanin (KLH) adsorbed onto aluminium hydroxide followed by intravenous infection with 104.’ TCID,, of cell-free virus of the molecular clone SIVmac32H(pC8) [14]. This virus has an in-frame nef deletion of four amino acids and two conservative amino acid changes in the same gene. It has an attenuated phenotype in rhesus and fascicularis macaques [3]. The inoculum propagated in the human T-cell line C81-66 was kindly provided by Dr. M. Cranage, Centre for Applied Microbiology and Research, Porton Down, U.K. Twenty weeks later another four monkeys of the same specification received the same dose of the pC8 virus. Twenty-two weeks thereafter all four monkeys from the second group (22-week-group) and two monkeys from the group infected earlier (42-week-group) were challenged intravenously with SIVmac32H/spl along with four naive control monkeys. This challenge virus was prepared ex vivo by homogenating the spleen of an immunised, but after challenge with SIVmac32H [15], infected rhesus monkey. This virus was never kept in cell culture and has been titrated in vitro and in vivo. The inoculum corresponded to 50 median monkey infectious doses (MID& Two monkeys from the 42week-group were not challenged, but observed further since they exhibited signs of immunodeficiency. Clinical follow-up during the whole investigation period was performed as reported [ 161.

Letters 51 (1996) 129-135

2.2. Monitoring of the immune status after the first infection and challenge The immune status of the monkeys after infection with the pC8 virus and after challenge with the spleenderived SIV was investigated by: (1) an enzyme-linked immunosorbent assay (ELISA) using whole inactivated SIVmac32H as the antigen [17]; (2) an assay for the detection of neutralising antibodies [ 18,191; (3) a CD4 + T-helper (Th) cell proliferation assay with monkey peripheral blood mononuclear cells (PBMC) stimulated with either purified heat-inactivated SIVmac32H or KLH or phytohaemagglutinin (PHA); [20] and (4) a cytotoxic T-lymphocyte (CTL) assay using autologous B-lymphoblastoid target cells infected with recombinant vaccinia viruses expressing SIV antigens [21]. Virus isolation was performed by cocultivation of PBMC with C81-66 cells. The cell-associated viral load was determined by a limiting dilution technique [21]. The threshold for positive virus isolation was defined at 3 x IO6 PBMC. If cultures initiated with this number of cells showed no signs of viral replication within a 4-week observation period they were regarded as virus negative. Urinary neopterin was measured in early morning urine [22]. Values obtained after infection were correlated to preinfection levels and expressed as x-fold of baseline. The T-cell subsets were analysed by flow cytometry with murine monoclonal antibodies against CD2, CD4 and CD8 [16]. 2.3. nef PCR umpliJication and sequence analysis A nested polymerase chain reaction (PCR) was performed in which each reaction was comprised of 40 cycles. The primers used in the first round were SN9044N (5’-GAC CTA CCT ACA ATA TGG G-3’) and SN9866C (5’-TCA GCG AGT TTC CTT CTT GT-3’). The primers used in the second round were SN9272N (S-GAA TAC TCC ATG GAG AAA CC-3’) and SN9763C (5’-GGG TAT CTA ACA TAT GCC TC-3’). The PCR products were analysed on a 2% agarose gel. A product of 480bp was amplified if the deletion in the nef gene was still present, while the product was 492bp long when wild type virus was detected. The DNA fragments were directly sequenced with the primer pair SN9272N and SN9763C [23].

3. Results 3.1. Infection with SIVmac32H@C8) Eight rhesus monkeys were infected with high doses of the attenuated SIVmac32H(pC8) containing a short

42 42 42 42 22 22 22 22 Control Control Control Control

1820* 1823* 1821 1822 1825 1828 1798 1199 7070 7071 7073 1839

Low Low Low Low Low Low Low Low

Low Low Low Low Low Low Low Low

before

Neopterinb

status

6.5 6.5 5.0 5.5 5.0 5.0 4.7 5.3 + + + + +

_ _ +

Th proliferationd

SIVmac32H(pC8):

Anti-SIV titres (Ig)’

challenge

with the attenuated

c-a viral load”

Immune

macaques

_ _

nd + + + + -

_ _

+ +

Thrombocytopenis’

+ +

__.._

CTL’

viral immunity

= = z = = z?

i

CD4/ CD@

before

+ + + _ _ _ _ _

_ +

nd

deletion”

nej

Indicators

of second

Low Low Low Low Mod Mod High High High High High High High High

= = =

zz

= =

Neopterinb

infection

with pathogenic

c-a viral load”

and after challenge

infectious

Anti-SIV titresC

after challenge

spleen-derived

-. Negative: +. positive; nd, not done; = , unchanged: mod. moderate: 1, declining: t. rising: h, healthy; d, dead; *, not challenged. .’ Cell-associated viral load determined in a limiting dilution assay: low corresponds to 0.3332, moderate to 4464 and high to 128-4096 ’ Neopterin increase IO- 15 days after infection compared to baseline levels. L‘Measured in ELISA using whole SIVmac32H as antigen. ’ Heat-inactivated whole SIV was used as antigen. e CTL against SIVGag/Pol, Env or Nef. r Thrombocytopenia. thrombocyte concentrations below I50 x lO’/~cl. g Measured by flow-cytometry. ” Determined by sequence analysis of PCR amplified rir/:

Waiting period (weeks)

of rhesus

Animal no.

Table 1 lmmunisation

_ _ _ _ _ _ + _

Thrombocytopenia’

cells/l x lo6 PBMC.

+ + _ + + + _ _ _ _

Th proliferatior+

SIVmac”

i

i

= = = = = =

CD4/ CDS@

h h h h h h

Clinical status

%

n 0, 6 z & s t. 3g :

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C. Stahl-Hennig et al. /Immunology

deletion of four amino acids in Nef. The immune status before challenge is summarised in Table 1. All monkeys became productively infected with this virus and exhibited an initially high cell-associated viral load (64 to 1028 infectious cells/l x lo6 PBMC) which rapidly declined thereafter. Virus isolation performed by cocultivation technique only became intermittently positive after week 12 of infection indicating declining viral loads. Four of the six monkeys selected for challenge were virus isolation positive at the time of challenge but had very low viral loads (0.33-l infectious cells/l x lo6 PBMC, data not shown). All infected monkeys rapidly generated SIV-specific antibodies by reaching titres between 50000 and 400000 at the time of challenge (Table 1). However, SIVmac32H neutralising antibodies were never detected. A SIV-specific Th activity appeared intermittently in all PCS-infected macaques by week 4 after inoculation. At the time of challenge the six monkeys with no signs of immunodeficiency exhibited a considerable Th-cell proliferative activity (stimulation indices ranged between 4 and 6, Fig. 1). Also, six of seven pC8-infected monkeys tested for CTL activity developed CTLs directed against all SIV structural proteins as well as against Nef (data not shown). 3.2. Follow-up after chaDtinge with the spleen-derived SI Vmac The six pC8-infected rhesus monkeys with no signs of immunodeficiency were challenged, two monkeys after a preinfection period of 42 weeks and four after 22 weeks of preinfection. The results after challenge are shown in Table 1. All control monkeys became invariably infected. The vaccinates, however, appeared to be protected. Their cell-associated viral load remained relatively low ( < 0.33-32 infectious cells/l x lo6 PBMC) compared to the unvaccinated controls. These had consistently higher viral loads exceeding those of the vaccinees by ten- to more than hundred-fold (data not shown). In addition, animals of the vaccine group did not develop an anamnestic antibody response, but the controls clearly seroconverted (except one monkey which had died 17 weeks after challenge). Furthermore, no typical initial neopterin increase was recorded in the preinfected monkeys conversely to the controls. Most interestingly, the SIV-specific Th proliferative activity was not abrogated in the vaccinees but remained detectable over the observation period of 28 weeks after challenge. In contrast, the infected controls never showed any substantial Th reactivity against SIV (Fig. 1). Finally, the CD4/CDX ratios remained on normal levels in the vaccinees, but dropped in the controls (data not shown). However, sequence analysis of PCR amplified nef from DNA extracted from the PBMC of all vaccinees 2

Letters 51 (1996) 129-135

weeks after the challenge revealed in two of the six animals a full-length nef gene. These two monkeys had a median level of viral load after challenge in contrast to the other four vaccinees which remained intermittently virus positive at a low level. Three of the control monkeys had to be sacrificed due to advanced AIDS-like symptoms between week 18 and 29 after challenge. The six vaccinees are still clinically healthy by week 36 post challenge. 3.3. Immunode~ciency in two monkeys infected with the pC8 virus Unexpectedly, two pC8-infected rhesus monkeys from the 42-week-group developed signs of immunodeficiency regularly observed in macaques infected with pathogenic SIV (data not shown). At 28 weeks after the pC8 infection one of these animals developed a thrombocytopenia simultaneously with a reduced CD4/CD8 ratio. A similar condition was noticed in the second monkey by week 40. In both monkeys these alterations coincided with a loss of their Th-cell responsiveness against SIV and KLH between 36 and 44 weeks post infection. At 44 weeks even their mitogen-induced proliferative activity also disappeared. However, these two animals are still clinically healthy 78 weeks after infection and their cell-associated viral load remained invariably low over 66 weeks until today. Investigation of nef at 50 weeks after infection showed in one animal a complete but partially incorrect repair of the deletion compared to the full length molecular clone pJ5 [14] of SIVmac32H (data not shown).

4. Discussion Impressive data on protection with an experimental AIDS vaccine have been obtained by ‘immunisation’ of macaques with cloned SIVmac239 containing a 182bp deletion in the nef gene. This virus was apathogenic for macaques [I]. Complete protection against challenge with pathogenic SIVmac was achieved after waiting periods of 2.25-3 years administering low to high challenge doses [12]. However, nothing was known about the mechanism(s) responsible for protection, especially since the cellular immune response had not been looked at in this particular experiment. Our aim was to investigate whether shorter intervals after the initial immunisation with a nef-deleted attenuated virus could confer protection against a pathogenic challenge virus. The mechanisms involved in mediating immunity should also be elucidated. We inoculated_ eight rhesus monkeys with a naturally occurring nef deletion mutant of SIVmac251 designated SIVmac32H(pC8). This virus has a Nef deletion of four

C. Stahl-Hennig et al. /

lmnamology

Letters 51 (1996) 1?9%135

133

10

8-

a.

0 0

2

4

8

12

16

20

24

28

32

2

4

8

12

16

20

24

28

32

28

32

b

8

6 t t 4 _ .I

2

0 ~ 0 10

8

TT

11.7

C

1.9

6

2

4

8

12

16

20

!4

Time after challenge (weeks) Fig. 1. SW-specific Th-proliferation of rhesus macaques after challenge. PBMC from (a) nonvaccinated control monkeys, (b) vaccinees preinfected with the pC8 virus for 42 weeks and (c) vaccinees preinfected for 22 weeks were stimulated with 0.25 pg purified heat-inactivated whole SIV antigen. Stimulation indices (SI) obtained after challenge are shown. The cut-off value is marked by a dotted line. Individual animal numbers are indicated. At some time points not all monkeys were examined (nd. not done). The last sample before the death of an animal is indicated (d, dead). The monkeys presenting with full length nef after challenge are marked by an asterisk.

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amino acids [14] much smaller than that of the mutated SIV described earlier [l]. pC8 had also lost its pathogenicity for rhesus and fascicularis macaques [3]. Four rhesus monkeys were observed for 42 weeks after pC8 infection, another four monkeys for 22 weeks. All monkeys became clearly infected by the pC8 virus. Virus could repeatedly be reisolated. The animals developed a strong virus-specific antibody response and a typical but moderate urinary neopterin increase. Interestingly, the initial cell-associated viral loads were as high as those measured in monkeys infected with pathogenic SIV. Such an early analysis has not yet been described with a nef deletion mutant. In the earlier report [l] viral load was only examined 14 weeks after infection when those monkeys were already virus negative. Similarly, viral loads declined in our animals and virus isolation became only intermittently positive 12 weeks after pC8 infection. Nevertheless, three of the eight monkeys maintained a persistent low level viraemia. Furthermore, we could measure a long-lasting cellular immunity in pC8-infected macaques not yet reported for attenuated SIV. Six out of seven monkeys examined had a CTL activity indicating virus replication [ 171. Most interestingly, all infected macaques generated a SIV-specific Th-cell response lasting over the respective waiting periods in four of the six monkeys. We have observed such a proliferative reactivity earlier in animals infected with HIV-2 [20] or SIVAvpr [24] but never after infection with highly pathogenic SIV strains. This underlines the attenuation of the pC8 virus. Unexpectedly, two pC8-infected monkeys lost their Th-cell responsiveness and developed thrombocytopenia. Their CD4/CD8 ratio declined indicating the development of an immunodeficiency. Therefore these monkeys were exempted from challenge. Sequence analysis performed 50 weeks after pCX-infection revealed a reversion to full length nef in one of these monkeys. Reversion to virulence by molecular evolution has been reported recently for rhesus monkeys of Indian origin [25], but has never been observed in pC8-infected fascicularis macaques (N. Almond, personal communication). In contrast, the other six pC8-infected monkeys remained normal by all laboratory and clinical parameters tested and were challenged with the pathogenic spleen-derived SIV. This virus was passaged once in vivo and thereafter had not been passaged in culture. Following challenge all preinfected monkeys were protected against the consequences of the challenge virus since no immunological, haematological or clinical changes were monitored. In contrast, all four control animals developed signs of immunodeficiency and three of them had to be euthanised within the first 6 months after challenge due to advanced stages of AIDS. By week 4 to 8 after challenge two animals of

the 22-week group had ‘median levels of cell-associated viral load lasting over an observation period of 32 weeks. In these two monkeys the nef gene had regained its full length by 2 weeks after challenge. However, the animals were apparently immune against disease development. Further sequence analysis will clarify whether the primary virus reverted or the challenge virus is replicating. Collectively, this is the first report on the induction of a sterile immunity by an attenuated SIV after a very short waiting period. Total protection was achieved 22 weeks as well as 42 weeks after the primary infection with the SIV nef deletion mutant thus confirming and extending earlier results on the protective potential of attenuated SIV [12]. In search for immune correlates of protection we have investigated several functional and nonfunctional parameters. In this experiment neutralising antibodies did not seem to be essential for protection. Instead, the live attenuated virus induced a virus specific Thcell immune response. We hypothesise that this response plays an important role in the protection against either primary challenge virus replication 1171 or at least the fatal consequences of the challenge with pathogenic SIV. This functional Th-cell response may have suppressed disease development in those animals in which primary challenge virus replication was not suppressed by vaccination [26,27] or preinfection with an apathogenic virus [28]. Consequently, interventive suppression of virus replication very early after infection would allow the build-up of an effective Th-cell response suppressing viral replication and thereby protecting against AIDS. This concept will be tested in the monkey model. If correct, this novel treatment strategy for HIV-infection and AIDS should be examined in man.

Acknowledgements

We are indebted to Dr. M.P. Cranage, Centre for Applied Microbiology and Research, Porton Down, U.K. for providing the pC8 virus. We thank K. Borchardt, K. Heinrichs, K. W&e and U. Zedler for excellent technical assistance. This work was funded by the European Community, Concerted Action on Monkey Models for AIDS Research (grant No. MR4*-042-D). The Programme EVA of the European Community is acknowledged for providing reagents.

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