Control of SHIV-89.6P-infection of cynomolgus monkeys by HIV-1 Tat protein vaccine.

Vaccine strategies aimed at blocking virus entry have so far failed to induce protection against heterologous viruses. Thus, the control of viral infection and the block of disease onset may represent a more achievable goal of human immunodeficiency virus (HIV) vaccine strategies. Here we show that vaccination of cynomolgus monkeys with a biologically active HIV-1 Tat protein is safe, elicits a broad (humoral and cellular) specific immune response and reduces infection with the highly pathogenic simian-human immunodeficiency virus (SHIV)-89.6P to undetectable levels, preventing the CD4+ T-cell decrease. These results may provide new opportunities for the development of a vaccine against AIDS.

Neutralizing cross-reactive and non-neutralizing monoclonal antibodies to HIV-1 gp120.

Amino acid sequences inducing neutralizing antibodies to HIV-1 were sought. Murine monoclonal antibodies (MAbs) were characterized by their reactivity with the envelope precursor gp160 or the Escherichia coli recombinant DNA products pB1 and pE3 representing the carboxy- and amino-terminal halves of mature envelope gp120. Fine mapping of the MAb determinants was performed using defined 15-mer synthetic peptides spanning the entire envelope gp120 region of HIV-1. One group of MAbs recognizes epitopes (amino acids 304-323) occurring in a small region with variable and conserved amino acid sequences of gp120. These MAbs mediate neutralization of the HIV-1 strain HTLV-IIIB (HIV-1IIIB) which was used for immunization. Nine out of 11 primary HIV-1 isolates were neutralized well or moderately well. In addition, prominent serological reactivity was noted with peptide sequences of strains of various European or American origins, but not with two HIV-1 strains of African origin. The cross-reactivity contrasts with previously described type-specific reactions to other sequences of this region. The reactivity to the short conserved site GPGR with its flanking amino acids may explain the broad sequence cross-reactivity seen with our neutralizing MAbs. Two other MAbs recognize conserved epitopes (amino acids 79-103) situated in the amino-terminal region of gp120. These MAbs did not neutralize HIV-1IIIB.

Azidocytidine is incorporated into RNA of 3T6 mouse fibroblasts.

Earlier work has shown that azidocytidine inhibits the growth and DNA synthesis of 3T6 mouse fibroblasts by inactivation of the enzyme ribonucleotide reductase. RNA synthesis, as measured by incorporation of [3H]cytidine was not affected. Here I show that azidocytidine is incorporated into RNA, but not into DNA. Incorporation of the analogue into RNA may under special circumstances contribute to the biological effect of the nucleoside.

Long-standing protection of macaques against cell-free HIV-2 with a HIV-2 iscom vaccine.

We investigated the capacity of two immunostimulating-complex (iscom) formulations including inactivated native HIV-2 viral proteins and selected peptides to induce protective immunity against HIV-2 in a nonhuman primate. Four cynomolgus monkeys were first immunized with five i.m. injections of purified detergent-disrupted HIV-2 virions (total dose, 0.7 mg) in iscoms over a period of 16 months. At months 18 and 20, all four macaques were given booster immunizations with iscom-coupled V3-derived synthetic peptides representing a dominating neutralizing region of HIV-2 gp125. Two weeks after the final dose of vaccine, the four vaccinated animals, together with four controls, were challenged i.v. with 10 monkey infectious doses (MID50) of monkey-cell-grown homologous cell-free virus, HIV-2SBL-6669/H5. After the challenge, the four control animals became readily infected; however, three of four vaccinated animals were protected as shown by repeated negative virus isolations and negative polymerase chain reaction for viral DNA and by failure to transmit HIV-2 infection with whole blood and lymph node cells into naive cynomolgus macaques. One of three protected animals showed an anamnestic antibody response to a dominating antigenic site, indicating possible limited virus replication. The vaccine-protected monkeys were subsequently resistant to rechallenge infection at 12, 15, and 18 months after the first challenge, suggesting that a reasonable duration of protective immunity had been induced by the vaccine.

Effects of adjuvants and multiple antigen peptides on humoral and cellular immune responses to gp160 of HIV-1.

The capacity of five different adjuvants, AlPO4, a muramyldipeptide formulation (MDP.TSL), Freund's adjuvant, immunostimulating complex and its matrix components to elicit humoral and cellular responses in rabbits immunized with the human immunodeficiency virus type 1 (HIV-1) envelope protein rgp160IIIB was compared. The highest antibody titers against gp160 and gp41/gp120 epitopes were seen with rgp160 in MDP.TSL or Freund's adjuvant, whereas the broadest responses were seen in rabbits immunized with rgp160 in matrix or MDP.TSL. The broadest spectrum of high-avidity antibodies was also induced by rgp160 in MDP.TSL. Neutralizing titers against HIV-1IIIB, low titers to HIV-1MN, and the most efficient inhibition of viral cell-to-cell spread was seen with rgp160 in MDP.TSL. The strongest and most persisting cellular responses were induced by rgp160 in AlPO4 or MDP.TSL. Using MDP.TSL as the adjuvant, we also improved the immune response against gp120 epitopes by boosting rgp160-primed rabbits with rgp160, multiple antigenic peptides (MAPs), or unconjugated peptides. The MAPs induced high neutralizing titers and were superior to rgp160 alone in inducing both humoral and cellular reactivity. MAPs are therefore strong candidates for inclusion into future HIV-1 vaccines.

HIV-1 vaccine-induced immune responses which correlate with protection from SHIV infection: compiled preclinical efficacy data from trials with ten different HIV-1 vaccine candidates.

The specific immune mechanisms necessary and/or sufficient to elicit HIV-vaccine protection remain undefined. Utilising the SHIV rhesus macaque model the immunogenicity as well as the efficacy of ten different HIV-1 vaccine candidates was evaluated. Comparison of the immune responses induced, with the ability of the vaccine to protect from SHIV infection provided a means to determine which type of immune responses were necessary for protection. Vaccine candidates included VLPs, DNA, subunit protein with novel adjuvant formulations, ISCOMs and pox-virus vectors. Protection from SHIV infection was achieved in approximately half of the animals which received a primary intravenous cell-free challenge. The presence of CTL in the absence of other effector responses did not correlate with protection from this route and type of challenge. Virus neutralising antibodies (Nab) appeared to be necessary but alone were insufficient for protection. If Ag-specific IFN-gamma and/or IL-4 as well as lymphoproliferative (LP) responses were found with the lack of a detectable IL-2 response, then protection was not observed. Immunity correlated with the magnitude of Nab responses, beta-chemokines and as well as balanced, qualitative T-helper responses.

Characterization of immunostimulating complexes (ISCOMS) of HIV-1.

HIV-1, strain HTLV-III, propagated in H9 cells and purified by sucrose gradient centrifugation, was used as native antigen source for the preparation of immunostimulating complexes, HIV-iscoms. The major antigen detected in the iscom was the cell-derived HLA-DR, which readily could be removed from the virus lysate by immunosorbent. In the iscoms the HIV structural proteins MA p17, p55 and TM gp41 were identified; SU gp120 was present in only minute amounts in the virus lysate. The iscom particles appeared well preserved after freeze drying with a round shape, approximately 35 nm in diameter, comprising morphological subunits, assembled with icosahedral symmetry. Immunization experiments in mice reflected the antigen content of the iscoms. High antibody response was induced to HLA-DR in non-depleted iscoms. Major humoral responses were observed to the viral structural proteins MA p17, CA p24, p55, and also to TM gp41. A low or negligible antibody response to SU gp120 was induced by the HIV-iscoms. The negligible response was, however, overcome by the addition of recombinant gp160 to the virus lysate prior to formation of iscoms, resulting in a preparation evoking a clear serum antibody to gp160.

Cross-neutralizing antibodies to HIV-1 in mice after immunization with gp160 iscoms. Dissection of the immune response.

Gp160 expressed in vaccinia and produced in vero cells was integrated into iscoms. Gp160 iscoms elicited a high serum antibody response in mice, and after two immunizations a ceiling was reached. The serum antibody response was dissected by the use of defined recombinant DNA products, representing different regions of the gp160 molecule. High antibody titers to the peptid RP135 (a.a. 296-332) correlated with induction of neutralizing serum antibodies. In some animals, gp160 (IIIB) iscoms elicited cross-neutralizing antibodies that also neutralized the distantly related RF isolate.

Comparison of protection afforded by whole virus ISCOM versus MDP adjuvanted formalin-inactivated SIV vaccines from IV cell-free or cell-associated homologous challenge.

A SIV-ISCOM and a SIV-MDP adjuvanted vaccine were tested for their potential to induce protection from intravenous cell-free or cell-associated homologous SIV challenge in rhesus monkeys (Macaca mulatta). Seven monkeys vaccinated four times over a four-month period with either the SIV-ISCOM or the SIV-MDP vaccine were challenged intravenously with approximately 10 MID50 cell-free SIVmac251 (32H). They all were protected from developing viremia during a three-month observation period. Two other groups of four monkeys were vaccinated essentially in the same way with either of these vaccines. They were challenged intravenously with approximately 10 MID50 of infected PBMC of a rhesus monkey that had been infected with SIVmac251 (32H) 11 months earlier (stock prepared by J. Heeney). Two monkeys of each of these two groups proved to be protected from developing viremia during a two-month observation period. For both the cell-free and the cell-associated SIV challenge, monkeys vaccinated with measles virus ISCOMS or MDP adjuvanted measles virus antigen, served as controls. They all became viremic within two weeks after SIV challenge. This is the first demonstration that vaccinated previously unchallenged nonhuman primates can be protected from infection with lentivirus-infected PBMC from another animal. Serological analysis indicated that SIV-specific serum antibody titers were considerably higher in SIV-ISCOM vaccinated animals than in the SIV-MDP vaccinated animals. The serology also confirmed the protection data, by showing the absence of increase in SIV-specific serum antibodies in apparently protected animals after challenge.

Vaccine protection against HIV-2 infection in cynomolgus monkeys.

The aim of this study was to determine if protection against an infectious human immunodeficiency virus type 2 (HIV-2) challenge could be obtained in cynomolgus macaques by active immunization using whole killed virus vaccine. Four monkeys were immunized with killed HIV-2SBL-6669, two of them with five intramuscular (im) injections of viral preparation containing 100 or 300 micrograms protein emulsified in incomplete Freund's adjuvant (IFA) and the two remaining received four im injections of 25-50 micrograms viral protein in iscoms. Each of the four vaccinated cynomolgus monkeys, along with four unvaccinated controls, were challenged intravenously two weeks after the last booster with approximately 100 animal infectious doses (ID50) of live HIV-2SBL-6669. All four immunized monkeys developed antibodies to HIV-2 envelope and core proteins before challenge exposure to HIV-2, but only the two animals vaccinated with virus in IFA developed detectable neutralizing antibodies. The two monkeys immunized with killed virus in IFA have shown no evidence of infection nine months after challenge with live virus. When blood and lymph node cells from these animals were transfused into naive cynomolgus monkeys, the recipients remained free of infection. In contrast, virus was recovered repeatedly in all nonimmunized animals and in the two animals immunized with iscom-associated viral antigens, which had a low content of envelope gp125 antigen. The demonstration of vaccine-induced protection against HIV-2 in a nonhuman primate raises hope for effective immunization against HIV infections in humans as well.

Monoclonal antibodies to native HIV type 1 reverse transcriptase and their interaction with enzymes from different subtypes.

Recombinant reverse transcriptase (RT) from HIV-1 subtype B was used to produce mouse anti-RT monoclonal antibodies (MAbs). Immunization was done by mixing RT with the ISCOM matrix-forming adjuvant saponin (Quil A). Two different assays, both based on the interaction of native RT and antibodies, were used to monitor the immune response in mice and for screening, selection, and characterization of the MAbs. The first assay measures the capacity of antibodies to inhibit the polymerase activity of the RT and the second assay measures the ability of antibodies to capture enzymatically active RT. Twelve clones with the capacity to inhibit at least 50% of the RT activity and 34 clones with high RT-capturing capacity were found. The MAb panel was utilized to evaluate the immunological properties of 18 different RTs representing 9 different HIV1 subtypes. The RT-inhibitory MAbs could be divided into two groups based on their pattern of cross-reactivity toward the different HIV-1 RTs. The degree of diversity recorded among MAbs with RT-capturing capacity was larger. At least seven groups of MAbs with distinct cross-reactivity patterns were identified. Thus, the degree of isoenzyme specificity varied greatly, from MAbs that were quite specific for subtype B RT to one MAb that was able to capture the RTs from all HIV-1 isolates tested except one of the two group O isolates. In conclusion, our study revealed that there exist surprisingly large immunological differences between RTs from different HIV-1 subtypes as well as from the same subtype.

Development of a rapid chromatographic strip test for the pen-side detection of foot-and-mouth disease virus antigen.

Foot-and-mouth disease (FMD) is the most contagious animal virus disease of cloven-hoofed livestock and requires reliable and accurate diagnosis for the implementation of measures to control effectively its spread. Routine diagnosis of FMD is carried out at the OIE/FAO World Reference Laboratory for Foot-and-Mouth Disease (WRL for FMD), Pirbright by the combined use of ELISA and virus isolation in cell culture supplemented by reverse transcription polymerase chain reaction (RT-PCR) methods. These techniques require skilled personnel and dedicated laboratory facilities which are expensive. The development of a rapid and simple test for the detection of FMD virus antigen using Clearview chromatographic strip test technology for field application is described. This device detected FMD viral antigen in nasal swabs, epithelial suspensions and probangs from clinical samples submitted from the field, from animals infected experimentally and in supernatant fluids resulting from their passage in cell culture. The test system was more sensitive than ELISA for the diagnosis of all seven serotypes of FMD virus in the epithelial suspensions and nasal swabs and had equivalent sensitivity to the ELISA for the detection of contemporary virus strains in cell culture supernatant fluids. The study demonstrated the potential for this device to confirm a clinical diagnosis at the site of a suspected FMD outbreak, thereby offering the possibility of implementing control procedures more rapidly. Such pen-side diagnosis would have particular benefits in FMD emergencies, relevance to FMD control programmes which operate in endemic regions of the world such as South East Asia and for increasing disease awareness in other areas where efforts to control disease may be difficult. In each circumstance the availability of a pen-side device for diagnosis would reduce the necessity for sending routine diagnostic samples to an FMD laboratory and thereby reduce the delay in diagnosis, which can in some areas be considerable.

A colorimetric reverse transcriptase assay optimized for Moloney murine leukemia virus, and its use for characterization of reverse transcriptases of unknown identity.

A non-radioactive reverse transcriptase (RT) assay, reported as useful for lentivirus RTs, was optimized for the measurement of Moloney murine leukemia virus (MMuLV) RT. The optimized assay could detect 0.3 microU of MMuLV RT. The specificities of the MMuLV and lenti RT assays were demonstrated using the RTs of human immunodeficiency virus type 1, simian immunodeficiency virus, feline immunodeficiency virus (FIV), visna virus, human T-cell lymphotropic virus type 1, MMuLV and feline leukemia virus (FeLV). An RT activity blocking antibody (RTb-ab) assay was standardized for Mn2+ dependent MuLV-related RTs. The assay was used to demonstrate the distinct antigenic properties of RTs from mammalian MuLV-related retroviruses and lentiviruses. Cross-reactivity between MMuLV RTb-ab and FeLV RT but not between MMuLV RTb-ab and e.g. FIV RT was demonstrated. An RT activity found in the murine myeloma cell line SP2/0 was found to have similar assay preferences as MMuLV RT, and the MMuLV-RT hyperimmune sera reacted strongly against this RT, indicating the RT to be of MuLV-related etiology. The use of the RT and RTb-ab assays for detection and characterization of RTs of known or unknown identity is discussed.

Deoxyribonucleoside triphosphate pool levels in three cell strains of human chromosome instability syndromes: ataxia telangiectasia (GM2052), Bloom's syndrome (GM1492), and Fanconi's anemia (GM368).

Deoxyribonucleoside triphosphate (dNTP) pool sizes were determined in cell strains derived from patients with the genetic diseases ataxia telangiectasia (GM2052), Bloom's syndrome (GM1492), and Fanconi's anemia (GM368), and were compared to the dNTP pools in a normal human fibroblast cell strain (253/79). In addition, the effect of deoxythymidine on both dNTP pool levels and cell growth was examined. The three mutant cell strains differed only slightly from the normal cell strain. The cellular characteristics of the cell strains, such as chromosome instability, are apparently not an effect of dNTP pool imbalance.

Azidocytidine is a specific inhibitor of deoxyribonucleotide synthesis in 3T6 cells.

2'-Azidocytidine is a specific inhibitor of DNA synthesis in cultured 3T6 mouse fibroblasts. Earlier work (Akerblom, L., Pontis, E., and Reichard, P. (1982) 257, 6776-6782) indicated that the nucleoside, after phosphorylation, acted by inhibiting both ribonucleotide reduction and DNA strand elongation. We now demonstrate that the effect on strand elongation was due to a contamination of azidocytidine with less than 0.3% of arabinosyl cytosine. Pure azidocytidine inhibits specifically ribonucleotide reductase and its effects on DNA synthesis are secondary to this inhibition. The results with azidocytidine concerning the size and turnover of deoxyribonucleoside triphosphate pools parallel those of hydroxyurea and are distinct from those of arabinosyl cytosine. Together with hydroxyurea, azidocytidine is a useful compound in studies aiming at a specific block of the production of deoxynucleoside triphosphates in intact cells. Comparisons of the effects of azidocytidine and arabinosyl cytosine complement earlier studies with hydroxyurea and aphidicolin concerning inter-relations between dNTP synthesis and DNA replication.

Effects of azidocytidine on DNA synthesis and deoxynucleotide pools of mouse fibroblast cell lines.

2'-Deoxy-2'-azidocytidine inhibits DNA synthesis of cells in tissue culture. In vitro, the diphosphate of azidocytidine was earlier shown to inhibit mammalian ribonucleotide reductase, while the triphosphate did not inhibit DNA replication in isolated cell nuclei. In intact cells, however, deoxyribonucleoside triphosphate pools were not decreased by azidocytidine in a manner expected from an inhibitor of ribonucleotide reductase. In order to differentiate between inhibition of precursor synthesis and DNA replication we studied in the present investigation the effects of azidocytidine on pool sizes of deoxynucleotides as well as on the incorporation of labeled cytidine or deoxycytidine into deoxycytidine triphosphate pools and DNA of cultured growing mouse fibroblasts. Azidocytidine was transformed to an active metabolite by phosphorylation with deoxycytidine kinase. Cell lines deficient in this enzyme showed an increased resistance against the analogue. Azidocytidine had a dual effect on the incorporation of labeled nucleosides. At low concentrations, the incorporation of cytidine into DNA was severely inhibited without a decrease of the specific activity of the dCTP pool or the size of the other three dNTP pools, suggesting a direct effect on DNA replication. At high concentrations of azidocytidine, incorporation into dCTP was also affected, demonstrating inhibition of CDP reduction. Our data also show that reduction of CDP is not necessarily tightly coupled to DNA replication since a rapid turnover of the dCTP pool occurred at the low concentrations of azidocytidine which severely inhibited DNA synthesis.

Detection of porcine MHC class II antigens in different immunoassays.

A monoclonal antibody (Mab), Mab 4-24-11, to human class II major histocompatibility antigens has been tested in commonly used immunoassays for detection of porcine class II major histocompatibility antigens (SLA-D). In a radioimmunoprecipitation assay, Mab 4-24-11 reacted with proteins from mitogen-stimulated porcine mononuclear cells (MNC). The molecular weights of the precipitated proteins were approximately 34 and 28 Kd. In indirect immunofluorescence, approximately 25% of porcine MNC in suspension reacted with Mab 4-24-11. This percentage diminished to 14% when Ig bearing MNC were removed, while it increased to 36% when adherent MNC were enriched. Thus, it was concluded that Mab 4-24-11 cross-reacts with SLA-D. In a immunohistochemical study, Mab 4-24-11 reacted with cells in acetone fixed cryostat sections from the gastric mucosa and endometrium. These properties of Mab 4-24-11 make it useful as a tool for further studies on the porcine immune system.

Free gp70 from FeLV: enrichment from cell culture fluid by ferric oxide-agarose chromatography.

A new chromatographic material based on beads of macroporous crosslinked agarose containing ferric oxide particles was used for enrichment of gp70--the envelope glycoprotein of feline leukemia virus (FeLV). Free gp70 was purified from cell culture fluid in one step with a recovery of 50 to 60% and a purification of about 60 times. The described procedure is a suitable first step for the purification of gp70 from large volumes of cell culture fluid.