RESUMEN

Development of curative approaches for HIV-1 infected patients requires novel approaches aimed at eliminating viral reservoirs and replacing potential target cells with infection-resistant immune cell populations. We have previously shown that autologous transplantation of genetically modified hematopoietic stem cells (HSCs) with lentiviral vectors encoding the mC46-fusion inhibitor results in a significant reduction in viral pathogenesis following challenge with the highly pathogenic dual tropic, SHIV89.6P strain. In this study, we used a combinatorial approach in which following engraftment of genetically modified HSCs, pigtailed macaques were vaccinated with a previously developed vaccinia-based vaccine expressing SIV-Gag, Pol. Using this dual therapy approach, lower viremia was detected in both the acute and chronic phase of disease with levels reaching near the lower limits of detection. In comparison with macaques receiving HSCT only, the combination approach resulted in a further log decrease in plasma viremia. Similar to our previous studies, positive selection of all CD4(+) T-cell subsets was observed; however, higher gene-modified CD4(+) T-cell levels were observed during the chronic phase when vaccination was included suggesting that combining vaccination with HSCT may lower the necessary threshold for achieving viremic control.

Asunto(s)

Trasplante de Células Madre Hematopoyéticas/métodos , Vacunas contra el SIDAS/farmacología , Síndrome de Inmunodeficiencia Adquirida del Simio/prevención & control , Animales , Linfocitos T CD4-Positivos/inmunología , Macaca nemestrina , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/inmunología , Subgrupos de Linfocitos T/inmunología , Vacunas de ADN/inmunología , Vacunas de ADN/farmacología , Carga Viral

RESUMEN

We evaluated four priming-boosting vaccine regimens for the highly pathogenic simian human immunodeficiency virus SHIV89.6P in Macaca nemestrina. Each regimen included gene gun delivery of a DNA vaccine expressing all SHIV89.6 genes plus Env gp160 of SHIV89.6P. Additional components were two recombinant vaccinia viruses, expressing SHIV89.6 Gag-Pol or Env gp160, and inactivated SHIV89.6 virus. We compared (i) DNA priming/DNA boosting, (ii) DNA priming/inactivated virus boosting, (iii) DNA priming/vaccinia virus boosting, and (iv) vaccinia virus priming/DNA boosting versus sham vaccines in groups of 6 macaques. Prechallenge antibody responses to Env and Gag were strongest in the groups that received vaccinia virus priming or boosting. Cellular immunity to SHIV89.6 peptides was measured by enzyme-linked immunospot assay; strong responses to Gag and Env were found in 9 of 12 vaccinia virus vaccinees and 1 of 6 DNA-primed/inactivated-virus-boosted animals. Vaccinated macaques were challenged intrarectally with 50 50% animal infectious doses of SHIV89.6P 3 weeks after the last immunization. All animals became infected. Five of six DNA-vaccinated and 5 of 6 DNA-primed/particle-boosted animals, as well as all 6 controls, experienced severe CD4(+)-T-cell loss in the first 3 weeks after infection. In contrast, DNA priming/vaccinia virus boosting and vaccinia virus priming/DNA boosting vaccines both protected animals from disease: 11 of 12 macaques had no loss of CD4(+) T cells or moderate declines. Virus loads in plasma at the set point were significantly lower in vaccinia virus-primed/DNA-boosted animals versus controls (P = 0.03). We conclude that multigene vaccines delivered by a combination of vaccinia virus and gene gun-delivered DNA were effective against SHIV89.6P viral challenge in M. nemestrina.

Asunto(s)

Vacunas contra el SIDA/inmunología , Linfocitos T CD4-Positivos/patología , Infecciones por VIH/prevención & control , Inmunización Secundaria , Vacunas contra el SIDAS/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/prevención & control , Vacunas de ADN/inmunología , Vacunas contra el SIDA/administración & dosificación , Vacunas contra el SIDA/genética , Animales , Anticuerpos Antivirales/sangre , Biolística , Linfocitos T CD4-Positivos/inmunología , Proteínas gp160 de Envoltorio del VIH/genética , Proteínas gp160 de Envoltorio del VIH/inmunología , Infecciones por VIH/inmunología , VIH-1/genética , VIH-1/inmunología , Humanos , Inmunidad Celular , Inmunización , Macaca nemestrina , Vacunas contra el SIDAS/administración & dosificación , Vacunas contra el SIDAS/genética , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Virus de la Inmunodeficiencia de los Simios/genética , Virus de la Inmunodeficiencia de los Simios/inmunología , Vacunas de ADN/administración & dosificación , Virus Vaccinia/genética , Virus Vaccinia/inmunología , Proteínas Virales/genética , Proteínas Virales/inmunología

RESUMEN

We previously showed that envelope (gp160)-based vaccines, used in a live recombinant virus priming and subunit protein boosting regimen, protected macaques against intravenous and intrarectal challenges with the homologous simian immunodeficiency virus SIVmne clone E11S. However, the breadth of protection appears to be limited, since the vaccines were only partially effective against intravenous challenge by the uncloned SIVmne. To examine factors that could affect the breadth and the efficacy of this immunization approach, we studied (i) the effect of priming by recombinant vaccinia virus; (ii) the role of surface antigen gp130; and (iii) the role of core antigens (Gag and Pol) in eliciting protective immunity. Results indicate that (i) priming with recombinant vaccinia virus was more effective than subunit antigen in eliciting protective responses; (ii) while both gp130 and gp160 elicited similar levels of SIV-specific antibodies, gp130 was not as effective as gp160 in protection, indicating a possible role for the transmembrane protein in presenting functionally important epitopes; and (iii) although animals immunized with core antigens failed to generate any neutralizing antibody and were infected upon challenge, their virus load was 50- to 100-fold lower than that of the controls, suggesting the importance of cellular immunity or other core-specific immune responses in controlling acute infection. Complete protection against intravenous infection by the pathogenic uncloned SIVmne was achieved by immunization with both the envelope and the core antigens. These results indicate that immune responses to both antigens may contribute to protection and thus argue for the inclusion of multiple antigens in recombinant vaccine designs.

Asunto(s)

Antígenos Virales/inmunología , Productos del Gen env/inmunología , Vacunas contra el SIDAS/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/prevención & control , Virus de la Inmunodeficiencia de los Simios/inmunología , Animales , Inmunidad , Inmunización , Macaca

RESUMEN

We previously reported that immunization with recombinant simian immunodeficiency virus SIVmne envelope (gp160) vaccines protected macaques against intravenous challenge by the cloned homologous virus E11S but that this protection was only partially effective against the uncloned virus, SIVmne. In the present study, we examine the protective efficacy of this immunization regimen against infection by a mucosal route. We found that the same gp160-based vaccines were highly effective against intrarectal infection not only with the E11S clone but also with the uncloned SIVmne. Protection against mucosal infection is therefore achievable by parenteral immunization with recombinant envelope vaccines. Protection appears to correlate with high levels of SIV-specific antibodies and, in animals protected against the uncloned virus, the presence of serum-neutralizing activities. To understand the basis for the differential efficacies against the uncloned virus by the intravenous versus the intrarectal routes, we examined viral sequences recovered from the peripheral blood mononuclear cells of animals early after infection by both routes. We previously showed that the majority (85%) of the uncloned SIVmne challenge stock contained V1 sequences homologous to the molecular clone from which the vaccines were made (E11S type), with the remainder (15%) containing multiple conserved changes (the variant types). In contrast to intravenously infected animals, from which either E11S-type or the variant type V1 sequences could be recovered in significant proportions, animals infected intrarectally had predominantly E11S-type sequences. Preferential transmission or amplification of the E11S-type viruses may therefore account in part for the enhanced efficacy of the recombinant gp160 vaccines against the uncloned virus challenge by the intrarectal route compared with the intravenous route.

Asunto(s)

Productos del Gen env/administración & dosificación , Productos del Gen env/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/prevención & control , Virus de la Inmunodeficiencia de los Simios , Vacunas Sintéticas/administración & dosificación , Vacunas Virales/administración & dosificación , Animales , Productos del Gen env/genética , Inmunidad Mucosa , Inmunización/métodos , Macaca , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/transmisión , Linfocitos T/inmunología , Linfocitos T/virología

RESUMEN

We previously reported that immunization with recombinant simian immunodeficiency virus SIVmne envelope (gp160) vaccines protected macaques against an intravenous challenge by the cloned homologous virus, E11S. In this study, we confirmed this observation and found that the vaccines were effective not only against virus grown on human T-cell lines but also against virus grown on macaque peripheral blood mononuclear cells (PBMC). The breadth of protection, however, was limited. In three experiments, 3 of 10 animals challenged with the parental uncloned SIVmne were completely protected. Of the remaining animals, three were transiently virus positive and four were persistently positive after challenge, as were 10 nonimmunized control animals. Protection was not correlated with levels of serum-neutralizing antibodies against the homologous SIVmne or a related virus, SIVmac251. To gain further insight into the protective mechanism, we analyzed nucleotide sequences in the envelope region of the uncloned challenge virus and compared them with those present in the PBMC of infected animals. The majority (85%) of the uncloned challenge virus was homologous to the molecular clone from which the vaccines were made (E11S type). The remaining 15% contained conserved changes in the V1 region (variant types). Control animals infected with this uncloned virus had different proportions of the two genotypes, whereas three of four immunized but persistently infected animals had >99% of the variant types early after infection. These results indicate that the protective immunity elicited by recombinant gp160 vaccines is restricted primarily to the homologous virus and suggest the possibility that immune responses directed to the V1 region of the envelope protein play a role in protection.

Asunto(s)

Vacunas contra el SIDAS/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/prevención & control , Vacunas Sintéticas/inmunología , Proteínas del Envoltorio Viral/inmunología , Secuencia de Aminoácidos , Animales , Humanos , Inmunización , Macaca fascicularis , Macaca nemestrina , Datos de Secuencia Molecular

RESUMEN

We have compared nef gene sequences isolated by PCR from peripheral blood lymphocyte DNA of macaques which had been inoculated with either biologically or molecularly cloned SIV(Mne). Two samples from each animal obtained either early after infection (week 2-8) or after significant CD4+ depletion (week 21-137) were analyzed. Three substitutions in the predicted Nef amino acid sequence were seen in all animals at the late time point, and two more in all but one. Two of the common exchanges are located about 40 residues apart in the Nef core sequence, but are in proximity on the tertiary structure as judged by computer modelling using the structure of the HIV Nef core protein as a guide. Most recurring in vivo changes replaced a residue found in the cloned Nef sequence with one present in a consensus derived by aligning the Nef sequences of the SIVsm/HIV-2 groups. Animals inoculated with virus already containing the "late version" nef gene developed a more aggressive disease. The macaque adapted (MA)nef conferred a threefold higher infectivity to the cloned virus, but had no effects on CD4 downregulation. Propagation of virus with MAnef in tissue culture resulted in the rapid emergence of variants with newly attenuated nef. These findings suggest that the selective pressure on nef in vivo and in vitro are different.

Asunto(s)

Productos del Gen nef/genética , Genes nef , Variación Genética , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/genética , Secuencia de Aminoácidos , Animales , Células Cultivadas , Cartilla de ADN , Productos del Gen nef/química , Linfocitos/virología , Macaca nemestrina , Modelos Moleculares , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Conformación Proteica , Alineación de Secuencia

RESUMEN

We explored the relationship between T cell activation signals and dendritic cells (DC) in the replication cycle of immunodeficiency viruses. First we analyzed the effect of two cell cycle inhibitors (mimosine and aphidicolin) on SIV reverse transcription, circularization, and integration in macaque resting T cells stimulated with anti-CD3 mAb at the time of infection. The formation of SIV LTR circles was blocked by the G1 inhibitor mimosine. The G1/S inhibitor aphidicolin neither affected circularization nor integration of SIV DNA. Therefore, the induction of SIV LTR circle production is likely to be mediated by signaling events normally regulating the G1 to S transition. We further characterized DC-dependent HIV-expression in human T cells. We examined the effect of ligating two novel receptors, IPO-3 and Bgp95, on DC-dependent HIV-1 expression. Activation of DCs through IPO-3 receptors, and to a lesser extent Bgp95 ligation, upregulated HIV spread in these cells. The mechanisms by which IPO-3 vs. Bgp95 increase HIV-1 levels appear to be different. In particular, IPO-3 ligation alone on T cells also increased HIV-1 levels. Activation of T cells via defined surface receptors or with DCs is required for establishing HIV/SIV cDNA synthesis in T cells.

Asunto(s)

ADN Viral/biosíntesis , Células Dendríticas/inmunología , VIH/fisiología , Activación de Linfocitos , Virus de la Inmunodeficiencia de los Simios/fisiología , Linfocitos T/inmunología , Linfocitos T/virología , Replicación Viral , Animales , Afidicolina/farmacología , Ciclo Celular/efectos de los fármacos , Replicación del ADN , ADN Complementario/biosíntesis , Citometría de Flujo , VIH/aislamiento & purificación , VIH/patogenicidad , Humanos , Macaca mulatta , Mimosina/farmacología , Reacción en Cadena de la Polimerasa , ADN Polimerasa Dirigida por ARN/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos , Transducción de Señal , Virus de la Inmunodeficiencia de los Simios/genética , Linfocitos T/citología , Integración Viral

RESUMEN

Using pathogenic simian immunodeficiency virus (SIV) infection of macaques as a model, we explored the limits of the protective immunity elicited by recombinant subunit vaccines and examined factors that affect their efficacy. Envelope gp 160 vaccines, when used in a live recombinant virus-priming and subunit-protein-boosting regimen, protected macaques against a low-dose, intravenous infection by a cloned homologous virus SIVmne E11S. The same regimen was also effective against intrarectal challenge by the same virus and against intravenous challenge by E11S grown on primary macaque peripheral blood mononuclear cells (PBMC). However, only limited protection was observed against uncloned SIVmne. Priming with live recombinant virus was more effective than immunization with subunit gp 160 alone, indicating a potential advantage of native antigen presentation and the possible role of cell-mediated immunity in protection. Whole gp 160 was more effective than the surface antigen (gp 130), even though both antigens elicited similar levels of neutralizing antibodies. Animals immunized with the core (gag-pol) antigens failed to generate any neutralizing antibody and were all infected following challenge. However, their proviral load was 10-100-fold lower than that of the control animals, indicating that immune mechanisms such as cytotoxic T lymphocytes (CTL) may play a role. Finally, animals immunized with both the core and the envelope antigens generated significant protective immunity, even with relatively low neutralizing antibodies. Taken together, these results indicate that multiple mechanisms may contribute to protection. It may therefore be advantageous to incorporate multiple antigens in the design of recombinant subunit vaccines against acquired immunodeficiency syndrome (AIDS).

Asunto(s)

Proteínas gp160 de Envoltorio del VIH/inmunología , Infecciones por Lentivirus/inmunología , Infecciones por Lentivirus/prevención & control , Fragmentos de Péptidos/inmunología , Virus de la Inmunodeficiencia de los Simios/inmunología , Vacunas Sintéticas/inmunología , Vacunas Virales/inmunología , Animales , Macaca fascicularis

RESUMEN

Although immunodeficiency viruses can enter resting CD4+ T lymphocytes, activation of T cells is required for complete viral cDNA synthesis and transport of double-stranded viral DNA to the nucleus. Cross-linking T cell receptors (TCRs) on resting CD4+ T cells induces reverse transcription of full-length simian immunodeficiency virus (SIV) genomes, but TCR engagement alone is insufficient to stimulate SIV DNA to move to the nucleus and form long terminal repeat (LTR) circles. Neither ligation of TCR or CD28 receptors nor interleukin 2 (IL-2) alone induces formation of LTR circles; however, the combination of TCR ligation with either CD28 ligation or IL-2 doses. Anti-IL-2 serum inhibits the formation of LTR circles induced by cross-linking CD3 and CD28, but has no effect on the induction of increased viral reverse transcription. Thus, two signals appear to be required for immunodeficiency viruses to move to the T cell nucleus, one from the TCR to promote reverse transcription of the viral genome, the other through an IL-2-dependent process leading to formation of LTR circles.

Asunto(s)

ADN Viral/genética , Regulación Viral de la Expresión Génica/efectos de los fármacos , Interleucina-2/farmacología , Activación de Linfocitos , Receptores de Antígenos de Linfocitos T/fisiología , Virus de la Inmunodeficiencia de los Simios/genética , Linfocitos T/microbiología , Animales , Antígenos CD28/fisiología , Núcleo Celular/microbiología , Técnicas In Vitro , Macaca nemestrina , Secuencias Repetitivas de Ácidos Nucleicos , Replicación Viral/efectos de los fármacos

RESUMEN

We investigated the role of blood dendritic cells (DC) in transmission of HIV-1 from infected to uninfected CD4+ T cells, and the accessory molecules involved. DC promoted transmission from infected to uninfected CD4+ cells, but blood DC themselves were not infectable. DC-mediated transmission was blocked by mAb to CD4 and MHC class II, but strongly increased by mAb to CD40 on DC or CD28 on T cells. The DC-dependent infection was inhibitable by anti-CD80 and a soluble fusion protein of the CD80 ligand, CTLA4; soluble CTLA4Ig also blocked infection augmented by crosslinking CD40. We also demonstrated that mAb to CD40 up-regulate the expression of CTLA4 ligands CD80 and B70/B7-2 (CD86) on DC. These data suggest that the dialog between CD40-CD40 ligand (CD40L) and CD28-CD80 counter-receptors on DC and T cells may be linked to HIV infection in vivo.

Asunto(s)

Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/virología , Células Dendríticas/inmunología , Células Dendríticas/virología , VIH-1/patogenicidad , Inmunoconjugados , Abatacept , Anticuerpos Bloqueadores/farmacología , Anticuerpos Monoclonales/farmacología , Antígenos CD/metabolismo , Antígenos de Diferenciación/metabolismo , Antígeno B7-1/metabolismo , Antígeno B7-2 , Células Sanguíneas/inmunología , Células Sanguíneas/virología , Antígenos CD28/metabolismo , Antígenos CD40/metabolismo , Ligando de CD40 , Antígeno CTLA-4 , Comunicación Celular , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/fisiología , Humanos , Técnicas In Vitro , Ligandos , Glicoproteínas de Membrana/metabolismo , Regulación hacia Arriba , Replicación Viral

RESUMEN

OBJECTIVES: To analyze correlates of protection in macaques exposed to SIV. METHODS: Peripheral 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. RESULTS: The 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. CONCLUSIONS: The 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.

Asunto(s)

Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Virus de la Inmunodeficiencia de los Simios/inmunología , Linfocitos T/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antivirales/sangre , Antígenos Virales/inmunología , División Celular , Células Cultivadas , Activación de Linfocitos , Macaca fascicularis , Macaca nemestrina , Datos de Secuencia Molecular , Péptidos/síntesis química , Péptidos/química , Péptidos/inmunología , Virus de la Inmunodeficiencia de los Simios/aislamiento & purificación , Timidina/metabolismo

RESUMEN

Macaca nemestrina and Macaca fascicularis were inoculated with various doses of a single-cell clone of SIVmne-infected HuT 78 cells (E11S) by both the intravenous and intrarectal routes. Animals inoculated intravenously at each dose seroconverted and virus was isolated from peripheral blood mononuclear cells, but only the high-dose intrarectally exposed macaques became viremic and seroconverted. However, some seronegative, virus isolation negative intrarectally inoculated macaques showed evidence of infection and disease.

Asunto(s)

Linfocitos T CD4-Positivos/inmunología , Depleción Linfocítica , Recto/virología , Síndrome de Inmunodeficiencia Adquirida del Simio/fisiopatología , Virus de la Inmunodeficiencia de los Simios , Animales , Anticuerpos Antivirales/sangre , Secuencia de Bases , Western Blotting , Células Clonales , Cartilla de ADN , Subgrupos Linfocitarios/inmunología , Macaca fascicularis , Macaca nemestrina , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa/métodos , Secuencias Repetitivas de Ácidos Nucleicos , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/transmisión , Virus de la Inmunodeficiencia de los Simios/genética , Virus de la Inmunodeficiencia de los Simios/aislamiento & purificación , Factores de Tiempo

RESUMEN

We investigated the role of blood dendritic cells (DCs) in transmission of HIV-1 from infected to uninfected CD4+ T cells, and the accessory molecules involved. DCs promoted transmission from infected to uninfected CD4+ cells, but DCs themselves were not infectable. DC-mediated transmission was blocked by MAb to CD4 and MHC class II, but strongly increased by MAb to CD40 on DCs or CD28 on T cells. The DC-dependent infection was inhibitable by anti-CD80 and a soluble fusion protein of the CD80 ligand, CTLA4; soluble CTLA4 immunoglobulin also blocked infection augmented by cross-linking CD40. These data suggest a linkage between CD40-CD40L and CD28-CD80 counterreceptors on DCs and T cells, and spread of HIV infection in vivo.

Asunto(s)

Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos B/metabolismo , Antígeno B7-1/metabolismo , Células Dendríticas/inmunología , Infecciones por VIH/inmunología , VIH-1 , Anticuerpos Monoclonales , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/virología , Antígenos CD28/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/virología , Antígenos CD40 , ADN Viral/biosíntesis , Células Dendríticas/virología , Infecciones por VIH/virología , VIH-1/fisiología , Humanos , Técnicas In Vitro , Regulación hacia Arriba , Replicación Viral

RESUMEN

The relationship between T-cell activation and early events in the replication cycle of simian immunodeficiency virus (SIV) was analyzed in resting T lymphocytes from macaques. We used the polymerase chain reaction to detect an early product of reverse transcription (R/U5) and almost complete viral DNA (long terminal repeat/gag). We found that SIV can enter resting T lymphocytes and initiate replication but that the reverse transcription process is not efficient and proceeds slowly in resting cells. Cross-linking the CD3/T-cell receptor complex with monoclonal antibodies, unlike cross-linking either the CD28 or CD2 accessory receptor and like phorbol myristate acetate, induced a rapid increase in viral R/U5 DNA detected within 3 to 6 h postinfection. Anti-CD3 or phorbol myristate acetate induced replication of full-length viral DNA within 6 to 9 h postinfection, but full-length SIV DNA was not detectable at earlier time points. We then compared various inhibitors of T-cell activation for their effects on viral initiation and complete replication. Cyclosporin A, an inhibitor of a distal step in T-cell activation, blocked anti-CD3-induced T-cell proliferation and completion of SIV DNA replication but had no effect on induced increases in SIV R/U5 DNA. By contrast, initial SIV DNA synthesis was partially blocked by inhibitors of very early steps in T-cell activation, including herbimycin A, an inhibitor of protein tyrosine kinases, and by two different inhibitors of protein kinase C, H7 and staurosporine. Since resting T cells do not efficiently complete SIV DNA synthesis and cyclosporin A can block the formation of complete viral DNA induced in activated T cells, a cellular factor(s) present in activated T cells appears to be required for the formation of full-length SIV DNA.

Asunto(s)

ADN Viral/biosíntesis , Activación de Linfocitos , Virus de la Inmunodeficiencia de los Simios/crecimiento & desarrollo , Linfocitos T/microbiología , Animales , Benzoquinonas , Complejo CD3/metabolismo , División Celular , Células Cultivadas , Ciclosporina/farmacología , Humanos , Interleucina-2/biosíntesis , Lactamas Macrocíclicas , Activación de Linfocitos/efectos de los fármacos , Macaca nemestrina , Proteína Quinasa C/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Quinonas/farmacología , Receptores de Antígenos/metabolismo , Rifabutina/análogos & derivados , Transducción de Señal , Acetato de Tetradecanoilforbol/farmacología

RESUMEN

A sensitive and specific procedure for the detection of Argentine isolates of bovine herpesvirus-1 was developed. The procedure was based on a dot-blot, nucleic acid hybridization, using 32P, nick-translated, plasmidic probes. The probes contained cloned Bam H1 restriction fragments in the left half of the viral genome. The detection limit of the procedure was 10 pg of viral DNA.

Asunto(s)

ADN Viral/análisis , Herpesvirus Bovino 1/genética , Animales , Secuencia de Bases , Bovinos , Clonación Molecular , Enzimas de Restricción del ADN , Desoxirribonucleasa BamHI , Desoxirribonucleasa EcoRI , Hibridación de Ácido Nucleico , Plásmidos

RESUMEN

Wild-type, virulent (A-24 Cruzeiro subtype) foot-and-mouth disease virus (FMDV), a related attenuated strain and revertants of the attenuated strain were examined by titration on primary bovine kidney (PBK) and baby hamster kidney (BHK-21) cells, as well as, by infection of unweaned mice. Wild type virus grew equally well in all three systems, whereas the attenuated strain had a titer 2-3 log lower in PBK cells than in the other 2 assays. Within 9 successive passages in BHK-21 cells the attenuated strain gave rise to revertants that had regained the growth properties of wild-type virus in PBK cells. After cloning of the attenuated strain by plaque isolations, the same revertant phenotype was obtained within 9 successive passages. Oligonucleotide mapping indicated that the attenuated strain differed from the wild-type and revertants by at least one additional oligonucleotide. Differences in poly(C) length were not found among any of the three strains of FMDV. These results correlate attenuation and virulence with point mutation(s) and not with deletions. Possible reversions in nature with this attenuated strain may be anticipated.

Asunto(s)

Aphthovirus/genética , ARN Viral/análisis , Animales , Aphthovirus/inmunología , Aphthovirus/patogenicidad , Bovinos , Genes Virales , Ratones , Oligonucleótidos/análisis , Vacunación/veterinaria , Vacunas Atenuadas , Virulencia

RESUMEN

An attenuated strain of foot-and-mouth disease virus (FMDV) of the A24 Cruzeiro subtype grew less well than wild-type virus in primary bovine fetal kidney (PBK) cells resulting in a 4-log lowered efficiency of plaque formation. Both wild-type and attenuated virus grew equally well in baby hamster kidney (BHK) cells and in suckling mice. Using PBK cells, virus-specific RNA of the wild-type accumulated up to 6 hours after infection. In contrast, PBK cells infected with the attenuated strain made less than 20 per cent of the RNA synthesized by wild-type virus. Infection of the cell with wild-type virus followed by superinfection with attenuated virus led to almost complete inhibition of viral RNA synthesis, an effect which is dependent on the concentration of input attenuated virus. Three subsequent undiluted successive passages of the attenuated virus resulted in a preparation which no longer interfered with wild-type RNA synthesis and which induced more of its own viral RNA synthesis in PBK cells. The basis of this interference was considered. Interference occurred intracellularly and was directed only against viruses within the genus FMDV. A role for interferon was ruled out. Attempts to demonstrate the physical presence of defective interfering (DI) particles of FMDV in the attenuated strain failed; but, cyclic patterns of infectivity were produced during successive undiluted passages.

Asunto(s)

Aphthovirus/patogenicidad , Interferencia Viral , Animales , Bovinos/microbiología , Células Cultivadas , Cricetinae , Efecto Citopatogénico Viral , Virus Defectuosos/fisiología , Interferones/fisiología , Riñón , ARN Viral/biosíntesis , Replicación Viral

RESUMEN

In the present study, evidence is presented for the existence of a morphogenetic intermediary that may be a precursor of the procapsids in the assembling process. BHK21 clone 13S cells were infected with Aphthovirus A24 (Cruzeiro strain), and pulse-chase experiments were carried out using 3H-leucine. Cytoplasmic extracts were then prepared at appropriate times, and analyzed by sucrose-gradient ultracentrifugation. After preliminary assays (Fig. 1), working conditions were standardized so as to obtain maximal recovery of the morphogenetic intermediary, as well as consistency of results. Only in the presence of DOC-Brij58 and Mg++ could a 10S sedimentation coefficient peak be seen (Fig. 1 a). A heterogeneous zone, with 4,5-5S as sedimentation coefficient, was also observed. The degree of labeling in the region 4,5-5S compared with that in the 10S portion, depends on the time within the infectious cycle when cells were pulse-labeled. Maximal levels for the ratio 10S/4,5-5S are reached when pulse-labeling takes place at the time when the amount of RNA viral synthesis reaches 80% of its total value (Fig. 2). Similar experiments, performed with third passage bovine fetal kidney cells, were confirmatory of the presence of a structure sedimenting at 10S, as well as of a heterogeneous zone of 4,5-5S (Fig. 3). It would appear that assembling of Aphthoviruses is accomplished through an intermediary unit which differs from that found for other Picornaviruses, the latter being the result of the union of 12 pentamers. The capsid of Aphthoviruses, also composed of 60 identical sub-units, would instead derive from the joining of 20 trimers.(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

Aphthovirus/ultraestructura , Animales , Aphthovirus/fisiología , Cápside/ultraestructura , Bovinos , Células Cultivadas , Cricetinae , Fibroblastos , Mesocricetus , Morfogénesis , Replicación Viral

RESUMEN

In the present study, evidence is presented for the existence of a morphogenetic intermediary that may be a precursor of the procapsids in the assembling process. BHK21 clone 13S cells were infected with Aphthovirus A24 (Cruzeiro strain), and pulse-chase experiments were carried out using 3H-leucine. Cytoplasmic extracts were then prepared at appropriate times, and analyzed by sucrose-gradient ultracentrifugation. After preliminary assays (Fig. 1), working conditions were standardized so as to obtain maximal recovery of the morphogenetic intermediary, as well as consistency of results. Only in the presence of DOC-Brij58 and Mg++ could a 10S sedimentation coefficient peak be seen (Fig. 1 a). A heterogeneous zone, with 4,5-5S as sedimentation coefficient, was also observed. The degree of labeling in the region 4,5-5S compared with that in the 10S portion, depends on the time within the infectious cycle when cells were pulse-labeled. Maximal levels for the ratio 10S/4,5-5S are reached when pulse-labeling takes place at the time when the amount of RNA viral synthesis reaches 80

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of its total value (Fig. 2). Similar experiments, performed with third passage bovine fetal kidney cells, were confirmatory of the presence of a structure sedimenting at 10S, as well as of a heterogeneous zone of 4,5-5S (Fig. 3). It would appear that assembling of Aphthoviruses is accomplished through an intermediary unit which differs from that found for other Picornaviruses, the latter being the result of the union of 12 pentamers. The capsid of Aphthoviruses, also composed of 60 identical sub-units, would instead derive from the joining of 20 trimers.(ABSTRACT TRUNCATED AT 250 WORDS)