Raoul E. Benveniste

T-CELL PROLIFERATION TO SUBINFECTIOUS SIV CORRELATES WITH LACK OF INFECTION AFTER CHALLENGE OF MACAQUES

ObjectivesTo analyze correlates of protection in macaques exposed to SIV. MethodsPeripheral blood mononuclear cells (PBMC) from macaques inoculated intrarectally with various dilutions of SIV were examined for their in vitro proliferative response to SIV envelope peptides and generation of SIV-specific antibodies. Some macaques previously exposed intravenously to subinfectious doses of SIV were subsequently challenged 16 months later with an infectious intrarectal dose of SIV. ResultsThe viral-specific immune responses of macaques exposed to infectious doses of SIV were characterized by generation of antibodies and weak or undetectable T-cell-mediated responses. In contrast, macaques inoculated with doses of SIV below the threshold required for seroconversion and recovery of virus exhibited T-cell proliferation in response to SIV envelope synthetic peptides. The macaques that had previously been exposed to SIV resisted the subsequent virus challenge, whereas the naive macaques (never exposed to SIV) all became infected. ConclusionsThe inability to productively infect macaques previously exposed to subinfectious doses of SIV suggests that a T-cell-mediated response may confer long-term protection against infection, and that AIDS vaccines should be designed to optimize the cellular arm of the immune response.

Evolution of type C viral genes: origin of feline leukemia virus

Reiterated gene sequences related to the RNA of feline leukemia virus (FeLV) are detected in all tissues of domestic cats and their close Felis relatives but not in more distantly related Felis species. Partially homologous viral gene sequences are found in rodent, and particularly rat, DNA. Together with the immunologic relationships observed between FeLV and endogenous rodent type C viruses, the results lead to the conclusion that FeLV-related genes were transmitted from a rodent to cat ancestor and have been perpetuated in the germ line of cats.

Mammalian Cells in Culture Frequently Release Type C Viruses

Cell cultures commonly used in animal cell research, both cell strains and continuous cell lines from various mammalian species, spontaneously produce type C RNA viruses.

RNA in mammalian sarcoma virus transformed nonproducer cells homologous to murine leukemia virus RNA

Abstract Mammalian sarcoma virus transformed nonproducer clones of mouse and rat cells contain RNA which hybridizes to the DNA product made from virus preparations containing leukemia virus, or sarcoma and leukemia virus. The hybridization of this virus-specific [3H]thymidine-labeled DNA to either total cellular or polysomal RNA extracted from these cells was detected with an enzymatic assay using a nuclease preparation either from mung beans or from Aspergillus oryzae, or by centrifugation in Cs2SO4 density gradients. The enzymatic assays were found to be more sensitive than Cs2SO4 for detection of such hybrids.

Type C viruses of baboons: Isolation from normal cell cultures

Abstract Four new type C viruses were isolated from putatively virus-negative baboon lung, kidney, and testicular cells by cocultivation with several permissive host cell lines. The baboon type C viruses are infectious for cells from various mammalian species, but do not replicate in any baboon cell lines so far tested. These viruses can be distinguished from other major classes of mammalian type C viruses, including previous isolates from primates, but are most closely related to endogenous feline viruses of the RD-114/CCC group. By immunologic criteria, viral host range, and nucleic acid hybridization studies, the baboon type C viruses are highly related to one another and represent a distinct new class of endogenous primate type C viruses.

Passively transferred antibodies directed against conserved regions of SIV envelope protect macaques from SIV infection

Inactivated plasma collected from either SIV-infected or peptide-vaccinated macaques was transferred into 17 naive rhesus monkeys. Two additional macaques received normal plasma and served as controls. Following transfer all 19 monkeys were inoculated with SIV. While the controls became infected and were virus-isolation-positive, 3 of 6 recipients of SIV peptide vaccine plasma and 9 of 11 recipients of SIV-infected monkey plasma were protected. None of the 12 protected animals became virus-isolation-positive or seroconverted within 100 days of follow-up. One, however was SIV-PCR-positive. All 12 protected animals were rechallenged 100 days after the initial inoculation; 8 became infected and yielded virus as expected, but 4 remained uninfected. One of the latter was the SIV-PCR-positive monkey mentioned above, suggesting that cryptic SIV infection may be of significance in immunological protection. The results demonstrate that envelope anti-peptide antibodies have similar protective potential in vivo as antibodies directed to the whole virus. In vitro neutralization competition assays performed with sera from vaccinated macaques in the presence of the free peptides suggest that of the four conserved envelope peptides of the vaccine, the two originating from gp41 rather than the two from gp120 are responsible for inducing the neutralizing anti-syncytial activity.

Biologic and immunologic properties of porcine type C viruses

Six different porcine cell lines spontaneously begin to release type C viruses after long-term propagation in vitro. Reverse transcriptases from all of the viral isolates are inhibited by immune IgG directed against the polymerase from the endogenous porcine type C virus PK(15). Two of the isolates are capable of replicating in the pig cell line, ST-Iowa, and the third isolate, while unable to initiate productive infection, can rescue murine sarcoma virus genomes from transformed nonproducer cell lines.

Isolation and characterization of a new type D retrovirus from the Asian primate, Presbytis obscurus (spectacled langur)

Abstract A reverse transcriptase-containing virus (retrovirus) was isolated from a cocultivation of langur lung cells with bat and with human cells. This virus, PO-1-Lu, was first detected 6 months after the cocultivation began. Morphologically it resembles Mason-Pfizer monkey virus (MPMV), a type D retrovirus of primates. Study of its antigenic properties and its RNA genome show it to be related to but distinct from MPMV. An interspecies radioimmunoassay using labeled gp70 of baboon type C virus and antiserum to MPMV type D virus detects the PO-1-Lu envelope protein although both the baboon type C- and the MPMV-specific radioimmunoassays for gp70 do not.

Infectious primate type C viruses: Three isolates belonging to a new subgroup from the brains of normal gibbons

Abstract Three type C viruses (GBr-1, GBr-2, and GBr-3) were isolated by coculativation of normal gibbon brain tissues with cultured mammalian cell lines. The tissues, all frozen since 1968, were obtained from two animals inoculated with brain extracts from human patients with kuru and from one uninoculated cagemate. By viral interference tests and by immunologic studies of the viral polymerases and major internal structural proteins (p30), the new isolates are typical members of a group of mammalian type C viruses infectious for primates. By nucleic acid hybridization, the viruses isolated from the gibbon brains, while highly related to one another, can be readily distinguished from the previously isolated type C viruses of this group. The infectious primate type C viruses isolated to date can be classified into four distinct subgroups.

Protection from pathogenic SIV challenge using multigenic DNA vaccines

To assess DNA immunization as a strategy for protecting against HIV infection in humans, we utilized SIVmne infection of Macaca fascicularis as a vaccine challenge model with moderate pathogenic potential. We compared the efficacy of DNA immunization alone and in combination with subunit protein boosts. All of the structural and regulatory genes of SIVmne clone 8 were cloned into mammalian expression vectors under the control of the CMV IE-1 promoter. Eight M. fascicularis were immunized twice with 3 mg of plasmid DNA divided between two sites; intramuscular and intradermal. Four primed macaques received a further two DNA immunizations at weeks 16-36, while the second group of four were boosted with 250 microg recombinant gp160 plus 250 microg recombinant Gag-Pol particles formulated in MF-59 adjuvant. Half of the controls received four immunizations of vector DNA; half received two vector DNA and two adjuvant immunizations. As expected, humoral immune responses were stronger in the macaques receiving subunit boosts, but responses were sustained in both groups. Significant neutralizing antibody titers to SIVmne were detected in one of the subunit-boosted animals and in none of the DNA-only animals prior to challenge. T-cell proliferative responses to gp160 and to Gag were detected in all immunized animals after three immunizations, and these responses increased after four immunizations. Cytokine profiles in PHA-stimulated PBMC taken on the day of challenge showed trends toward Thl responses in 2/4 macaques in the DNA vaccinated group and in 1/4 of the DNA plus subunit vaccinated macaques; Th2 responses in 3/4 DNA plus subunit-immunized macaques; and Th0 responses in 4/4 controls. In bulk CTL culture, SIV specific lysis was low or undetectable, even after four immunizations. However, stable SIV Gag-Pol- and env-specific T-cell clones (CD3+ CD8+) were isolated after only two DNA immunizations, and Gag-Pol- and Nef-specific CTL lines were isolated on the day of challenge. All animals were challenged at week 38 with SIVmne uncloned stock by the intrarectal route. Based on antibody anamnestic responses (western, ELISA, and neutralizing antibodies) and virus detection methods (co-culture of PBMC and LNMC, nested set PCR- of DNA from PBMC and LNMC, and plasma QC-PCR), there were major differences between the groups in the challenge outcome. Surprisingly, sustained low virus loads were observed only in the DNA group, suggesting that four immunizations with DNA only elicited more effective immune responses than two DNA primes combined with two protein boosts. Multigenic DNA vaccines such as these, bearing all structural and regulatory genes, show significant promise and may be a safe alternative to live-attenuated vaccines.