Abstracts Vol. 1 [International Conference on AIDS (11th: 1996: Vancouver, Canada)]

Monday, July 8, 1996

Mo.A. 144 - Mo.A. 152

Only a small fraction (15-20%) of surface TNF-R was internalized, while sTNF-R75 was
already detectable in supernatants suggesting that early shedding ofTNF-R75 accounted for
the down-modulation of the cell-surface receptors. Endogenous TNF-a had no role in the
disappearance of its own receptor. Complete and stable restoration ofTNF-R expression at
the cell membrane, dependent on de novo protein synthesis, occurred after 5h, followed by
massive sTNF-R75 release.
Conclusions: These results demonstrate that infection of human monocytes with HIV- I LAI
triggers at an early stage a cascade of cellular events that lead to profound remodeling of
the cell TNF-R pool. Understanding the mechanisms of these receptor movements could be
of importance to document the central role of the TNF system in HIV infection.
F Zavala, INSERM U25, H6pital Necker, 161 rue de Sevres, 75743 PARIS Cedex 15, France
Telephone: 33- I -44-49-53-69 Fax: [phone]
Mo.A. 144
2-5A SYNTHETASE:ACTIVATION BY HIV-I TAR RNA AND RRE RNAAND
MODULATION BY PROPENTOFYLLINE IN HIV-I INFECTED CEM CELLS
Leuck, durgen, Scheffer U, MullerWEG, Schroder HC. Institute of Physiological Chemistry
University of Mainz, Mainz, Germany
Objective: To determine the structural elements of HIV- I RNAs (TAR RNA and RRE RNA)
that are able to activate the antiviral 2',5'-oligoadenylate (2-5A) system and to search for
compounds that prevent the decrease in interferon (IFN)-inducible 2-5A synthetase activity
during later stages of infection.
Methods: Human T lymphoblastoid CEM cells were infected with HIV- I (strain HTLVIIIB)
and cultivated in the absence or presence of compound and IFN.The number of viable cells
was monitored using the MTT colorimetric assay 2-5A synthetase activity was determined
in cell extracts after binding to immobilized poly(l)-poly(C). Formation of RNA-protein
complexes was detected in RNase protection and Northwestern assays using in vitro synthesized RRE RNA,TAR RNA and TAR RNA mutants.
Results: The TAR RNA sequence is among those RNAs which bind to and activate 2-5A
synthetase. Experiments with TAR mutants revealed that binding of 2-5A synthetase to TAR
RNA depends on the length but not the sequence of the double-stranded TAR RNA stem.
Binding of 2-5A synthetase isoenzymes (40-46 kDa, 67 kDa, and 100 kDa) to TAR RNA
could also be demonstrated in RNase protection and Northwestern assays with cytosolic
proteins from IFN-treated cells (CEM, HeLa, and other cell lines).The effect of TAR on 2-5A
synthetase was abolished in the presence of Tat protein.The 2-5A synthetase was also
found to be activated by the RRE sequence of HIV- I env RNA. Addition of recombinant
Rev protein suppressed complex formation between RRE RNA and 2-5A synthetase.
Previously we found that the activity of the 2-5A forming 2-5A synthetase and the levels of
2-5A transiently increase after infection of cells with HIV-I, followed by a strong decrease
simultaneous with the onset of virus production. In CEM cells the decrease in IFN-inducible
2-5A synthetase activity and protein could be retarded in the presence of the cAMP phosphodiesterase inhibitor propentofylline.
Conclusions: Both RRE RNA and TAR RNA were able to activate IFN-inducible 2-5A synthetase. Our results indicate that cAMP is involved in control of 2-5A metabolism.The
decrease in 2-5A synthetase activity during later stages of infection could be retarded by
xanthine derivatives.
J. Leuck, Inst. of Physiological Chemistry University Duesbergweg 6, 55099 Mainz, Germany
Telephone: 49 6 I 3 I-395789 Fax [phone] email: [email protected]
Mo.A. 145
SECRETION OF MULTIPLE CHEMOKINES IN UI CELLS:AUTO-CRINE
UPREGULATION OFVIRAL EXPRESSION BY MCP-I.
Biswas, Priscilla*, Delfanti F*,Vicenzi E*, Sozzani S^, More M*, Mantovani A^, Poll G*. * San
Raffaele Scientific Institute, Milan, Italy; ^ Mario Negri Institute for Pharmacological Research,
Milan, Italy.
Objective:To investigate the role of chemokines on HIV expression in the latently infected
promonocytic U I cell line that is characterized by virus inducibility by several cytokines.
Methods: HIV expression was measured in culture supernatants by the reverse transcriptase
(RT) activity assay Chemokine secretion: RANTES, MIP- Ia and MIP- Ib were measured by a
commercially available ELISA kit, whereas MCP- I and IL-8 were determined by home made
ELISAs. Standard Northern Blot analyses were performed to evaluate chemokine messages.
Results: Unstimulated U I cells produced detectable levels of RANTES, MCP- I and IL-8, but
not of MIP-I a and MIP-I b. PMA stimulation resulted in the secretion of all five chemokines,
whereas IL-6 and IFN-g selectively induced high levels of MCP- I, but not IL-8 or MIP-I a/b,
and downregulated RANTES accumulation in culture supernatants.These results were confirmed by Northern blot analyses of the messages for MCP- I and RANTES.We further
investigated whether MCP-I secretion induced by IL-6 and IFN-g was a mediator of HIV
expression. An anti-MCP- I neutralizing mAb inhibited at least in part HIV expression
induced by IL-6. However, exogenous recombinant MCP- I did not induce HIV expression
by U I cells, suggesting that IL-6 may also regulate MCP- I receptors, therefore allowing the
cells to respond to the endogenously produced chemokine.
Conclusions: Fndogenous secretion of MCP- I induced by IL6 and IFN-g msy be responsible for HIV expiession induced by these cytokines in U I cellsThese findings, together- with
the observation that MCP- I activates, whereas RANTES and MIP-Ia suppiess HIV replication in primary CUB-depleted PBMC from HIV(+) individuals (see abstiact by C. Poli et al.),
shed new light on the role of chemokines in the pathogenesis of HIV infection. A balance
between opposite regulatory effects exerted by chemokines of the same C-C family niay
contribute to determine the extent of virus replication in infected individuals.
Priscilla Biswas Via. Stamira A' Ancona N. 20, 20127, Milan, Italy Tel [phone],
Fax [phone]

Mo.A. 150
EFFICACY OF A DNA VACCINATION TO INDUCE NEUTRALIZING ANTIBODY
AND CYTOTOXIC CELLS AGAINST HIV- I
Fukushima Jun*, Hamajima K',Asakura Y5, Bukawa H*,TsujiT*, Xin K-Q*, Nishioka K",
Cullen B R**, Okuda K., *Yokohama City University School of Medicine,Yokohama, Japan,
**Hepatitis Virus Research Foundation of Japan, **Duke University Medical Center
Durham, USA
Objective: The efficacy of DNA vaccines for protecting HIV- I infection was studied by using
expression plasmids for Gag, Env, and Rev proteins.
Methods: We constructed expression vectors for HIV I genes driving cytomegalovirus promoter:These expression plasmids were injected with cationic liposomes into mice, rabbits
guinea pigs, and Japanese macaques intramuscularly or intraperitonealy Antibody titr e and
cell mediated immunity were monitored by enzyme-linked immunosorbent assay delayedtype hypersensitivity or cytotoxicT lymphocyte assays. We also evaluate the virus neutraliz
ing activity of serum antibody by p24 assay using several laboratory strains and clinical isolates.
Results: After three months of intramuscularly imrmunization, the antibody titre of 1:213 of
serum IgG in Japanese macaques was obtained when IIIB- env region expression vector was
injected.These antibodies neutralized not only laboratory strains but primary isolates
stronglyThis immunization was also induced certain level of V3 peptide specific cytotoxicT
cells and DTH activity in the mice when small amount of DNA (2 pg) was injected. In addi
tion, when Gag region expression vector was used for DNA immunizatlion, we could
observe the virus-like particles in the muscle. Humoral and cell -mediated immunity was
detected against Gag peptide.
Conclusions: Direct plasmid injection is a candidate for new type of vaccination methods
against HIV- I.The expression of Gag protein in the muscle may mimic the elicitation of anti
virus immunity of HIV- I infection. SCID/hu mice or chimpanzee should be used for the protection of HIV- I infection to evaluate the efficacy of these vaccines.
Jun Fukushima, 3-9 Fukuura, Kanazawa-kuYokohama 236, Japan,Telephone: [phone],
FAX: [phone], email: [email protected] jp
Mo.A.15 I
THE COMBINATION OF DNA AND PEPTIDE VACCINES INDUCES STRONG
IMMUNITIES AGAINST HIV-I IN BOTH HUMORAL AND CMI
Kenji Hamajima*, Fukushima J*, KanekoT*, Bukawa H*,Tsuji t*, Xin K -Q*, Asakura Y#
Okuda K**, Nishioka K**, Okuda K*. iYokohama City University 'Yokohama, Japan, " Tokyo
Dental Collage,Tiba, Japan, **Viral hepatitis Research Foundation of Japan,Tokyo, Japanr
Objective: We have reported that the synthetic peptide vaccine (VCI) against HIV induced
a high level of neutralizing antibodies for various HIV isolates, and the DNA vaccine against
HIV induced prolonged high CTI level. In the present study we examined whether the
combination of DNA vaccine and the peptide vaccine could induce a high level immune
responses against HIV- I in both humoral and CTI responses.
Materials and Methods: HIV- I strains, IIB and SF2, and three clinical isolates from Japan
were used for nuetrilizing assay, and mice, rabbits and Mclcoca f(uiscata were used for irnnmunization.VC I was the cycled form of the V3 loop peptides, which consisted of PND con
sensus sequences of Japan, IIII and Thai B, CD4 binding sites and a Gag region peptide
(HGP--30).The HIV- I DNA vaccine, a mixture of pCREV DNA and pCMV 160 IIIB DNA
was directly inoculated into the above animal muscles.
Results: The antisera derived from VCI or DNA plus VCI vaccination inhibited the growth
of the five HIV isolates. In addition, DNA vaccination or DNA plus VC I vaccination induced
a high level of CTL.
Discussion and Conclusions: Our results demonstrate that combination of our DNA vac
cine and peptide vaccine can induce high levels of both CMI and humoral immunities.This
vaccination design may be one of the ideal HIV I vaccines. However: HIV challenge i/n vo is
necessary for the final conclusion.
K Hamajima,Yokohama City University 3-9 Fukuura, Kanazawa-ku Yokohama 236 Japan.
Telephone: [phone] Fax: [phone]
Mo.A. 152
A MACROMOLECULAR MULTICOMPONENT PEPTIDE VACCINE CANDIDATE
INDUCES MUCOSAL IMMUNITY AGAINST HIV- I
Bukawa, Hiroki,Asakura Y Tsuji T Xin K-Q, Sasaki Shin, Hamajima K, Fukushima J, Kawamoto
S, Fujita K, Okuda K. Yokohama City University School of Medicine,Yokohama, Japan
Objective: To induice htIV- I-specific mucosal IgA antibody with neutralizing activity is of
importance for development of HIV vaccine. Induction of secretor IgA antibody against
HIV- I was studied by oral, rectal and vaginal immunization with a new synthetic peptide
vaccine candidate (VCI).
Method: VCI was composed of peptides fiom several third hypervriable regions (V3), a
CD4 binding site (helperT cell epitope) and a Gag region of HIV I.VCI + cholera tox//
(CT) were administered into BALB/c mice with oral, rectal and vaginal route. Fecal extract
solutions or vaginal washings were collected at a week after each immunization. HIV- I-spefic IgA antibody titers were determined using ELISA ssay. Delayed type hypi sensitity
IDrH an d cytotoxic F lymphocyte (GTE) weie also exanirned using footpad swelliis test,
VC l e n e assay, respectively.
Results Oil ectas and vaginal imiunization with VCI c elicited high tite/ of Hi ith
cific secretory IgA sntibody (I:27-). Secretory IgA antibody induced fry oral in/izast/on
neutr alized riot oisly iabo/ratoi y striss biat also prirsary isolastes. Rectal1 inmnunizacion ehicutedi
feral tgA astibody (I:2 ) and vaginal IgA antibody(1:2(.Vainl irnmsunizatioi ehicited fecaii
IgA asstbody (I:29) and vaginal1 IgA antibody (1:29)(. Rectal arsd vsyina ai dmin/st/rstion of
VP I+5CT slso iinduced DIV I-specific DED arsd CTL.
Conclusions: Mucosal irnsrnunizato ith VC I+CT irsdsced secretoryIlA antifbody wi/h
neutralization of HIV I and cell mediated immunity.
H. Bukawa, 3 9 Fukuura, Kanazawa ku,Yokohama 236, Japan. telephone: 8 I 45 787 2659,
Fax: [phone]

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