AIDS RESEARCH AND HUMAN RETROVIRUSES Volume 8, Number 8, 1992 Mary Ann Liebert, Inc., Publishers

Prevention of HIV-2 and SIVSM Infection in Cynomolgus Monkeys by Active or Passive Immunization GUNNEL

BIBERFELD,1

PER

PUTKONEN,1

RIGMOR

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INTRODUCTION INFECTIONS of macaques with certain strains of human and simian immunodeficiency virus (HIV-2 and represent useful models for HIV vaccine studies (for review, see refs. 1.2). We summarize our results of active and passive immunizations against HIV-2 and of passive immunization against SIVsm in cynomolgus monkeys (Macaca fascicu-

Experimental SIV) laris).

EXPERIMENTAL INFECTION OF CYNOMOLGUS MONKEYS WITH HIV-2 AND SIVsm

THORSTENSSON,1

and ERLING

NORRBY2

We have prepared a stock of SIV^m (strain SMM-3 obtained from Yerkes Primate Center, USA) cultured in vitro on human PBMC and have titrated it in vivo in cynomolgus monkeys. Virus was recovered repeatedly from PBMC and all monkeys remained persistently infected until death. In contrast to the HIV-2SBl _6669-infected monkeys almost all SIVsm-infected monkeys (29 of 33) showed a significant decrease of CD4 lymphocytes. Thirteen of 33 SIV-infected monkeys have died or have been sacrificed within a year after inoculation because of severe clinical symptoms and 29 of 33 monkeys have died within 26 months. Thirteen of the 33 monkeys (39%) had malignant B-cell lymphomas6 and 18 (55%) had generalized giant cell

disease.7

We have established experimental infection in cynomolgus monkeys using the SBL-6669 and the SBL-K135 isolates of HIV-2 obtained from West African patients.3'4 In vivo passage of HIV-2SBL_666y in a cynomolgus monkey was found to increase the replicative capacity of this virus in monkeys.3'4 A cell-free stock was prepared of in vivo passaged SBL-6669 virus grown in vitro on monkey peripheral blood mononuclear cells (PBMC). All monkeys inoculated with 10-fold dilutions, up to 10 3, of this stock virus preparation developed infection demonstrable by repeated virus recovery from PBMC for at least 2 to 3 months after inoculation and thereafter by polymerase chain reaction (PCR) for viral DNA.4 Virus could also be persistently recovered from 50% of the animals during a follow-up period of more than one year. This stock virus preparation has successfully been used for challenge of immunized monkeys.4'5 Cynomolgus monkeys infected with HIV-2SBL.6669 have remained healthy and have not developed a decrease of CD4 T lymphocytes during a follow-up period of two years. Preliminary results using the SBL-K135 strain of HIV-2 indicate that in vivo passage can increase the pathogenicity of HIV-2 for macaques. Two of two monkeys infected with this strain previously passaged twice in vivo in monkeys developed an early and persistent decrease in CD4 lymphocytes (Per Putkonen and Gunnel Bibcrfeld, unpublished data).

CROSS-PROTECTION IN HIV-2 PREINFECTED MONKEYS AGAINST

SIVsm-INDUCED

DISEASE

We have taken advantage of the differences with regard to immunological and clinical outcome between the SIV and HIV-2 experimental models for studies of cross-protective immunity against SIVsm in HIV-2-infected monkeys.8 Three monkeys infected with HIV-2SBL_K|35 (not passaged in vivo), which were seropositive but from which virus could no longer be recovered, were superinfected with SIVsm (5 months after inoculation with HIV-2) and found to be protected against SIV-induced disease for at least two years. Virus could be isolated from these monkeys after SIV challenge but only for a limited time. Four naive control monkeys which received the same dose of SIV as the HIV-2-preinfected monkeys developed a decrease of the number of CD4 lymphocytes and clinical symptoms and died after 2 to 26 months. Viral antigen was abundant in lymph node biopsies from the SIV-infected control monkeys but was absent in the biopsies from the HIV-2preinfected and SIV-superinfected monkeys. All three HIV-2preinfected and SIV-superinfected monkeys are still healthy 29 months afterSIV inoculation. However, one monkey relapsed to virus isolation positive after 16 months and at the same time showed a decrease of CD4 lymphocytes. This study shows that

'Department of Immunology, National Bacteriological Laboratory. 105 21 Stockholm, Sweden. ^Department of Virology, Karolinska Institute, 104 01 Stockholm, Sweden. 1511

BIBERFELD ET AL.

1512 immunization with (live) HIV-2 gave protection against disease induced by a nonhomologous but related virus strain. The nucleotide homology between HIV-2 and SIVsm is approximately 70%.

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VACCINE INDUCED PROTECTION AGAINST HIV-2 INFECTION Our first vaccination experiment with killed HIV-2SB1 _hhm included four monkeys.4 Two of them received five intramuscular (im) injections of a viral preparation containing 100 or 300 p.g protein in incomplete Freund's adjuvant (IFA) and the two remaining received 4 im injections of a low dose (25 or 50 u.g)of viral protein in immunostimulating complexes (ISCOMS) (Table 1 ). All four immunized monkeys developed antibodies to HIV-2 envelope and core proteins but only the two animals vaccinated with virus in IFA developed detectable neutralizing antibodies. The four vaccinated monkeys along with four un vaccinated controls were challenged intravenously two weeks after the last booster with approximately 100 animal infectious doses of live HIV-2SBL6f)6y. The two monkeys immunized with killed virus were resistant to infection as shown by negative virus isolation attempts, negative PCR for viral DNA, and failure to transmit virus with blood and lymph node cells from the protected monkeys to naive monkeys. In contrast, virus was recovered repeatedly in all nonimmunized animals and in the two animals immunized with ISCOM-associated viral antigens. This study is the first demonstration of a successful vaccination against HIV-2 in a nonhuman primate. The failure to induce protective immunity with a low dose of HIV-2 proteins in ISCOMS does not imply that HIV-ISCOM preparations are unsuitable as vaccines. Preliminary results of ongoing studies indicate that repeated immunizations with a higher dose of HIV-2 proteins in ISCOMS followed by booster

Table 1

Outcome

of

immunization with synthetic peptides representing a dominating neutralizing region of HIV-2 gpl25 can induce protective

immunity. We have also immunized groups of monkeys with whole formalin-inactivated antigen of HIV-2SBL6669 combined with alum. RIBI (Immunochem Research Inc., Montana) or muramyldipeptide (MDP) (Sigma Chemical Company. St. Louis, MO) adjuvants. The immunization schedule and the outcome after challenge with homologous virus are shown in Table 1. Protection against infection was demonstrated in 2 of 4 monkeys immunized five or six times with vaccine in RIBI and in 1 of 2 monkeys immunized five times with vaccine in MDP but not in any of four monkeys which had received four immunizations with vaccine in alum. Comparison of vaccine preparations by Western blot analysis showed that the formalin-treated vaccines contained less HIV-2 gpl25 envelope glycoproteins than the originally used vaccine preparation in IFA that induced complete protection. Immunization of additional monkeys with a new formalin-killed HIV-2 vaccine preparation richer in gpl25 is ongoing. Determination of neutralizing antibodies to HIV-2 in our immunized monkeys has not shown a correlation between the titers of these antibodies and protective immunity. One reason for this might be that the SBL-6669 virus used in the neutralization assays had been grown in a human cell line whereas the SBL-6669 virus used for challenge of monkeys had been passaged in vivo in a monkey and then grown in monkey PBMC. There are several reports of induction of protective immunity in macaques by immunization with whole inactivated SIV vaccines (for review, see ref. 2). Recent observations indicate that immune responses against cellular proteins might contribute to vaccine-induced protection in SIV vaccine trials where the vaccine as well as the challenge virus were grown in human cells.9 It should be noted that in all our trials with inactivated HIV-2 vaccines the virus used for challenge had been grown in

Vaccine Trials in Cynomologus Monkeys Using Whole Inactivated HIV-2. ^SBL-6669 Total dose of viral

No.

Of

monkeys

Immunization

(months) 0,1,2,3,24 0,1,12,18 0,1,4,6 0,1,2,4,8 0,1,2,4,11,12 0,1,2,4 0,1,2,4,6,7

protein (mg)

Inaetivation

0.5 1.5 0.15 1.2 1.5 1.8 1.2

Trison X-100 X-100 MEGA Formalin Formalin Formalin Formalin Formalin

Adjuvant

Protectionc

FIA

2/2a

ISCOM Alum RIBI RIBI MDP MDP

0/2u

0/4h l/2b l/2b 0/2h l/2b

aThe animals were challenged intravenously (iv) with 100 MID5() of homologous virus. hThe animals were challenged iv with 10 MID50 of homologous virus. cAmong 50 nonimmunized control monkeys inoculated with 10 MID50 of the virus used for challenge experiments one moneky was resistant to infection. This monkey was resistant also upon reinoculation with the same virus.

PREVENTION OF INFECTION BY IMMUNIZATION

cynomolgus monkey PBMC whereas the homologous virus used for vaccine preparation had been grown in a human monocytoid cell line (U937). Therefore, it is unlikely that anticellular immune responses played a role in the vaccine-induced protective immunity demonstrated in our studies. PREVENTION OF HIV-2 AND SIVsm INFECTION BY PASSIVE IMMUNIZATION In order to elucidate the mechanisms of protective immunity HIV-2 and SIVsm we have done passive immunization experiments.5 Four monkeys were injected intravenously with a low dose (3 ml/kg) and three monkeys with a high dose (9 ml/kg) of anti-HIV-2 serum from a cynomolgus monkey which had been immunized with an inactivated whole virus vaccine and shown to be protected against a homologous HIV-2 challenge. At the high dose 2 of 3 monkeys and at the low serum dose 1 of 4 monkeys showed protection against infection after the homologous HIV-2 challenge. All 7 control animals became infected after inoculation with the same HIV-2 challenge dose. In another experiment 4 cynomolgus monkeys were given 9 ml/kg of anti-SIVMn serum from a SIV-infected cynomolgus monkey which had remained clinically healthy for more than one year. Three of the four monkeys pretreated with SIVsm antiserum showed no signs of infection following challenge with homologous SIVsm, whereas all 9 control monkeys inoculated with the same virus dose became infected. Thus, passively transferred antibodies can protect against experimental HIV-2 and SIVsm infections.

1513

ACKNOWLEDGMENTS This work was supported by the Swedish National Board for Technical Development, the Swedish Agency for Research Cooperation with Developing Countries (SAREC) and the Swedish Medical Research Council. SIVsm strain SMM-3 was kindly provided by Drs. P. Fultz and H. McClure, Yerkes Primate Research Center, Atlanta, GA (NIH Grant No.

kR00165).

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against

SUMMARY We have established experimental infection with HIV-2 and in cynomolgus monkeys and successfully used these models for vaccine experiments. Protection against homologous HIV-2 infection was demonstrated in two of two monkeys immunized with a Triton-X 100-treated whole HIV-2SBL.6669 vaccine in incomplete Freund's adjuvant and in 2 of 4 monkeys immunized with a formalin-inactivated whole HIV-2 vaccine in RIBI adjuvant. Monkeys preinfected with a live poorly replicating HIV-2 strain were shown to develop cross-protection against SIV-induced disease. We have shown also that HIV-2 and SIVsm infection in cynomolgus monkeys can be prevented by passive immunization. These results raise hope for effective immunization against HIV infections in humans.

SIVsm

REFERENCES 1. Biberfeld G. and Emini E: Progress with HIV vaccines. In: M.W. Adler, et al. (ed). AIDS 1991. A Year in Review. Current Science, Philadelphia, in press. 2. Gardner MB, and Hu SL: SIV vaccines. In: M.W. Adler, et al. (ed.). Current Science, Philadelphia, in press. 3. Putkonen P. Böttiger B. Warstedt K, Thorstensson R, Albert J. and Biberfeld G: Experimental infection of cynomolgus monkeys (Macaca fascicularis) with HIV-2. J AIDS 1989;2:366-373. 4. Putkonen P. Thorstensson R, Walther L. Albert J, Ákerblom L, Granquist O, Wadell G. Norrby E. and Biberfeld G: Vaccine protection against HIV-2 infection in cynomolgus monkeys. AIDS Res Human Retroviruses 1991:7:271-277. 5. Putkonen P. Thorstensson R, Ghavamzadeh L. Albert J, Hild K. Biberfeld G. and Norrby E: Prevention of HIV-2 and SIVsm infection by passive immunization in cynomolgus monkeys. Nature 1991; 352:436-438. 6. Feichtinger H, Putkonen P. Parravicini C, Li SL, Kaaya E, Böttiger D, Biberfeld G, and Biberfeld P: Malignant lymphomas in cynomolgus monkeys infected with SIV. Am J Pathol 1990:137:1311-1315. 7. Li SL. Kaaya E, Feichtinger H. Putkonen P. Parravicini C. Böttiger D, Biberfeld G, and Biberfeld P: Monocyte/macrophage giant cell disease in SIV-infected cynomolgus monkeys. Res Virol 1991:142:173-182. 8. Putkonen P, Thorstensson R, Albert J, Hild K. Norrby E. Biberfeld P, and Biberfeld G: Infection of cynomolgus monkeys with HIV-2 protects against pathogenic consequences of a subsequent simian immunodeficiency virus infection. AIDS 1990:4:783-789. 9. Stott J: Anti-cell antibody in macaques. Nature 1991:353:393.

reprint requests to: Gunnel Biberfeld Department of Immunology National Bacteriological Laboratory Address

105 21 Stockholm, Sweden

Prevention of HIV-2 and SIVSM infection in cynomolgus monkeys by active or passive immunization.

We have established experimental infection with HIV-2 and SIVsm in cynomolgus monkeys and successfully used these models for vaccine experiments. Prot...
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