Early suppression of SIV replication by CD8+ nef-specific cytotoxic T cells in vaccinated macaques.

In order to develop a successful subunit vaccine against infection with the human immunodeficiency virus (HIV), protective immune effector functions must be identified. Until now, there has been only indirect evidence that HIV-specific cytotoxic T lymphocytes (CTLs) fulfill this role. Using the macaque simian immunodeficiency virus (SIV) model, the protective potential of nef-specific CTLs, stimulated by vaccination, was examined in animals challenged with a high intravenous dose of the pathogenic simian immunodeficiency virus, SIVmac251(32H)(pJ5). An inverse correlation was found between the vaccine-induced nef-specific CTL precursor frequency and virus load measured after challenge. In addition, the early decline in viraemia, observed in both vaccinated and unvaccinated control animals was associated with the development of virus-specific CTL activity and not with the presence of virus-specific neutralizing antibodies. The results imply that vaccines that stimulate strong CTL responses could protect against HIV infection.

Control of SIV rebound through structured treatment interruptions during early infection.

In a randomized controlled trial with acute simian immunodeficiency virus (SIV)-infected macaques, both highly active antiretroviral therapy (HAART) and HAART with fixed-schedule structured treatment interruption (STI-HAART; alternating 3 weeks on and 3 weeks off therapy) suppressed viral load. In the STI-HAART group, T cell virus-specific immune response (VIR) and control of viral rebound increased concurrently during subsequent interruptions. In contrast, VIR did not increase and SIV rebounded after permanent treatment withdrawal in all animals on continuous HAART. Fixed-schedule STI-HAART appears to be an effective alternative to continuous HAART for the early treatment of retroviral infection.

Cytotoxic T-cell response to simian immunodeficiency virus by cynomolgus macaque monkeys immunized with recombinant vaccinia virus.

Simian immunodeficiency virus (SIV) gag-specific major histocompatibility complex (MHC)-restricted cytotoxic T-lymphocyte (CTL) activity was elicited in four out of six cynomolgus macaques after two immunizations with SIV gag recombinant vaccinia virus (rVV). No activity could be seen in three out of three non-immunized control animals. Low levels of anti-gag antibody were also seen in the same four responding animals. Virus-specific, MHC-restricted CTL are thought to give some protection and to assist in recovery in viral infection, and the induction of such CTL following vaccination with a single viral protein should act as an encouragement to those proposing similar vaccination studies in man.

Immunization with class I human histocompatibility leukocyte antigen can protect macaques against challenge infection with SIVmac-32H.

OBJECTIVE: To evaluate the efficacy of immunopurified class I human histocompatibility leukocyte antigen (HLA) to protect against SIV infection. METHODS: HLA class I antigens were immunopurified from a human B-lymphoblastoid cell line. Groups of four macaques were vaccinated subcutaneously with four doses of the immunogen in adjuvant, or with adjuvant alone and subsequently challenged intravenously with 10 median monkey infectious doses of cell-free SIVmac-32H. Infection was determined by polymerase chain reaction for SIVmac proviral DNA and by virus isolation. Antigen-specific humoral and cellular immune responses were monitored. RESULTS: Macaques immunized with the HLA molecules produced anti-HLA class I antibodies that inhibited SIV replication in vitro and downregulated autologous T-cell proliferation against irradiated C8166 cells. They were partially protected (two out of four) from virus infection for at least 33 weeks when challenged with SIV grown in human cells. All four control animals were infected. CONCLUSIONS: This demonstration of partial protection, together with our previous work reporting that vaccination with allogenic cynomolgus lymphocytes can protect against challenge infection with SIV grown in simian cells, suggests that allogenic immune response induced before or during establishment of HIV infection may have important implications for AIDS disease progression.

Production and of monoclonal antibodies to simian immunodeficiency virus envelope glycoproteins.

Eighteen monoclonal antibodies (MAb) to simian immunodeficiency virus (SIV) envelope have been characterized. All MAb were shown to bind to viral antigens on the surface of unfixed SIV-infected cells and to precipitate surface glycoproteins of SIVmac251. In Western blot 11 MAb bound to gp160 and gp120, five bound to gp160 and the transmembrane protein gp41 and two MAb did not react with denatured antigen. Preliminary competition assays identified the existence of six competition groups; two groups were within gp41 and four were within gp120. Of the latter four groups, three contained MAb with neutralizing activity. Two of the neutralizing MAb (KK5 and KK9) did not react with denatured antigen in Western blot suggesting that they may recognize conformational epitopes. Enzyme-linked immunosorbent-assay titres of MAb against SIVmac251 ranged from 10(2.4) to 10(5.6) and although similar titres were obtained with some MAb against other SIV and HIV antigens, the presence of isolate specific and shared group epitopes was demonstrated.

Fine analysis of humoral antibody response to envelope glycoprotein of SIV in infected and vaccinated macaques.

To characterize the serological response to SIV envelope, induced by vaccination with different envelope immunogens or by SIV infection, plasma samples from 11 cynomolgus macaques infected with simian immunodeficiency virus (SIV) and from 16 macaques vaccinated with three different recombinant envelope proteins were analyzed by (1) ELISA, using a variety of antigens including overlapping peptides encompassing the entire sequence of the envelope protein of SIV, and (2) competition assays, using neutralizing monoclonal antibodies to SIV gp120. Seven regions of SIV envelope were predicted to be antigenic. Peptides representing four of these, in the second and third variable regions (V2 and V3) and the fourth constant (C4) region of gp120 and the Gnann region of gp41, were recognized by the majority of sera from infected and vaccinated animals. Additional antigenic regions were identified in the first and fourth variable domains (V1 and V4) and the carboxy terminus (C5) of gp120 and in three additional regions of gp41. Most infected and vaccinated animals made antibodies that competed with the binding of the three conformational MAbs. Among the vaccinated groups, antibodies induced by vaccination with precursor glycoproteins (gp140 or gp160) recognized several additional gp120 epitopes when compared with antibodies induced by external glycoprotein gp130. Sera from infected animals showed a more restricted gp120 response (17 of 46 peptides recognized) compared to animals vaccinated with precursor glycoproteins (31 peptides recognized). The converse was true for antibodies to gp41. Sera from animals vaccinated with recombinant gp140, produced in insect cells, were the only group that failed to compete with the binding of conformational MAbs. Finally, the development of antibodies to specific epitopes of gp120 and gp41 revealed differences between long-term survivors and nonsurvivors, implying that responses to specific epitopes may be important in conferring resistance to disease progression.

Effect of PMPA and PMEA on the kinetics of viral load in simian immunodeficiency virus-infected macaques.

In this study we compared the effect of postexposure treatment of the acyclic nucleoside analogs 9-(2-phosphonylmethoxyethyl)-adenine (PMEA) and 9-(2-phosphonylmethoxypropyl)-adenine (PMPA) on the kinetics of viral load in the blood and lymph nodes of rhesus macaques chronically infected with SIVmac251 for 18 weeks. Two of the four macaques treated with PMPA (20 mg/kg per day) for 28 consecutive days had demonstrable reductions in viral loads of 1.5 and 3 logs. Three of four macaques given the same dosing regimen of PMEA had viral load reductions ranging from 1.25 to 2.8 logs. Furthermore, treatment with either drug caused a reduction in virus burden in the lymph nodes by 2 weeks posttreatment. However, in both PMEA- and PMPA-treated animals, viral loads rebounded to day of treatment levels by 2 weeks after termination of treatment. The extent to which viral load was suppressed was similar for both drugs. In contrast, viral loads in three of four mock-treated animals remained persistently high throughout the study. This study has demonstrated that postexposure treatment with these acyclic nucleoside analogs could modulate the kinetics of viral load reduction in some animals.

Passive immunization of cynomolgus macaques with immune sera or a pool of neutralizing monoclonal antibodies failed to protect against challenge with SIVmac251.

In the first of two passive transfer experiments, three groups of four macaques were injected intraperitoneally with a normal serum pool, an immune serum pool (pool 1) collected 132-172 weeks postinfection with the 11/88 pool of SIVmac251, or with a pool of four neutralizing monoclonal antibodies (KK9, 17, 54, and 56) raised against gp120 of the 11/88 pool. Sera were given at a dose of 13 ml/kg whereas the MAb pool was given at 30 ml/kg. In a second experiment, a further four macaques were injected with an immune serum pool (pool 2) collected 12 weeks postinfection with simian-grown SIVmac251 at a dose of 19 ml/kg. Animals in both experiments were challenged with SIVmac251 grown in simian peripheral blood lymphocytes. Despite high levels of circulating antibodies in the serum of animals that received either the immune serum pools or the MAbs, all macaques became infected following challenge. The results described are in contrast to a previous report in which passive transfer of sera from animals infected with SIVsm successfully protected against challenge with the homologous virus grown in human PBMCs. Challenge with SIVmac251 grown in simian PBMCs may be the reason for these conflicting results. Nevertheless, the results suggest that in this model the presence of circulating neutralizing antibodies alone does not necessarily confer protection against challenge with SIVmac251 grown in simian cells.

Mechanisms of protection induced by attenuated simian immunodeficiency virus. II. Lymphocyte depletion does not abrogate protection.

To determine the role that cellular immune responses play in the protection conferred by vaccination with attenuated SIVmac32H (pC8), we have attempted to deplete macaques of their CD8+ cells prior to challenge with wild-type SIVmac32H (pJ5). In two of four pC8-infected macaques, N109 and N112, a transient partial depletion of CD8+ cells by antibody treatment was achieved. On the day of challenge peripheral CD2+CD4-CD8+ cell counts were reduced by 92 and 95%, respectively, in animals N109 and N112 and their lymph nodes revealed a 46 and 58% reduction, respectively, in CD2+CD4-CD8+ cells. Two other pC8-immunized macaques, N110 and N111, treated in the same way, did not show significant depletion of CD8+ cells. None of these four pC8-immunized animals became infected when challenged with 50 MID50 of pJ5. Treatment of a further four pC8-infected and protected macaques and two naive control animals with Campath-1H antibody successfully depleted peripheral CD3+ cell counts by >99% in all treated animals. Campath-1H depletion resulted in enhanced, longer lasting lymphoid depletion. Yet subsequent challenge with 20 MID50 of pJ5 still failed to infect the pC8-immunized animals. All eight of the naive controls, including two Campath-1H-treated animals, became infected following challenge. In summary, partial depletion of circulating CD8+ cells or total lymphocytes prior to challenge failed to abrogate the protection conferred by vaccination with pC8.

Specific proliferative T cell responses and antibodies elicited by vaccination with simian immunodeficiency virus Nef do not confer protection against virus challenge.

The efficacy of immunizing with a combination of simian immunodeficiency virus (SIV) Nef vaccines was evaluated. Four vaccinates received three intradermal immunizations with recombinant vaccinia virus that expressed SIV Nef, followed by three intramuscular immunizations with rDNA also expressing SIV Nef. Finally, the four vaccinates received two subcutaneous boosts with recombinant SIV Nef protein. This immunization protocol elicited anti-Nef antibodies in all of the vaccinates as well as specific proliferative responses. However, specific cytotoxic T cell responses were not detected before virus challenge. All vaccinates were challenged intravenously with 10 MID(50) of SIVmacJ5 along with four controls. All eight subjects became infected after SIV challenge and there were no group-specific differences in virus load as measured by virus titration and vRNA analysis. The results of this study support indirectly the report from Gallimore and colleagues (Nat Med 1995;1:1667) suggesting that CD8(+) T lymphocyte responses are required for Nef-based vaccines to restrict SIV infection. If Nef-based vaccines are to be beneficial in controlling infection with immunodeficiency viruses, then it will be necessary to develop more effective immunization protocols that elicit potent CD8(+) cell responses reproducibly.

The development of PCR based assays for the detection and differentiation of simian immunodeficiency virus in vivo.

Polymerase chain reaction based assays, which amplify a region of the gag gene, have been developed for the direct detection of simian immunodeficiency virus (SIV) DNA sequences in the blood of experimentally infected cynomolgus macaques. In macaques infected with a characterised virus pool (11/88 pool SIVmac 32H), an assay employing a single round of amplification was found to be highly sensitive and specific. However, in animals infected with the SIV molecular clones J5 and C8 (Rud et al., J. Gen. Virol. 75, 529-543), it was necessary to use two rounds of amplification and nested primer pairs in order to achieve sensitivity > 90%. In order to differentiate macaques infected with either of the two genetically distinct SIV clones, J5 or C8, a third PCR based assay has been developed, which amplifies a 492 bp region of the nef gene. Sequence differences between the nef genes of the two molecular clones enabled the PCR product amplified from each virus to be distinguished by restriction analysis. These sensitive and specific assays complement virological detection of SIV and enable superinfection studies to be evaluated; a prerequisite for the testing of live attenuated immunodeficiency virus vaccines.

The use of the polymerase chain reaction for the detection of Simian immunodeficiency virus in experimentally infected macaques.

A rapid, non-radioactive assay for the detection of proviral Simian immunodeficiency virus (SIV) in tissue-culture cells is described. The assay is based on the co-amplification of the SIV env and gag genes by the polymerase chain reaction (PCR). When the gag PCR product is blotted onto a nylon membrane and hybridised to a radioactive oligonucleotide probe, the assay can also be used to detect the SIV gag gene in DNA isolated directly from experimentally infected cynomolgus macaque lymphocytes. This provides a valuable assay for the presence of proviral SIV during animal trials of AIDS vaccines and chemotherapeutics.

Macaques co-immunized with SIVgag/pol-HIVenv and IL-12 plasmid have increased cellular responses.

BACKGROUND: The cell mediated immune profiles following immunization with a recombinant DNA vaccine was assessed in the simian-human immunodeficiency virus (SHIV) and Macaque model. Earlier work demonstrated increased numbers of antigen specific CD8 and CD4 effector cells able to secrete IFN-gamma. METHOD: The vaccine strategy included co-immunization of a DNA based vaccine alone or in combination with a macaque IL-12 expressing plasmid (pmacIL12). Antigen activated lymphocytes were studied for activation of a set of immunological molecules. RESULTS: The current study demonstrates lymphocytes isolated and activated from the group that was immunized with DNA and pmacIL12 had a higher level of IFN-gamma producing cells. We also observed a different immunological profile when comparing the cells isolated from macaques immunized with DNA as compared to those animals that also received pmacIL12. CONCLUSION: The observed immune profiles are reflective of the co-delivery of pmacIL12 and demonstrates that IL-12 can increase the magnitude and polyfunctionality of the cellular immune response.

Preparation and characterization of new challenge stocks of SIVmac32H J5 following rapid serial passage of virus in vivo.

BACKGROUND: A new challenge stock of the simian immunodeficiency virus SIVmacJ5 has been produced following passage in vivo. METHODS: SIVmacJ5 3/92 (J5M), was passaged serially through cynomolgus macaques (Macaca fascicularis) by intravenous inoculation of infected spleen cells isolated and prepared 14 days post-infection. Two challenge stocks, SIVmacJ5 S61MLN and SIVmacJ5 S62spl, were prepared by culture of lymphoid tissue ex vivo. RESULTS: These virus stocks appeared better adapted for replication in M. fascicularis as demonstrated by a greater persistence of recoverable live virus from the periphery and increased pathology in lymphoid tissues 20 weeks post-challenge as detected by immunohistochemistry. Sequence analysis of the envelope gene from these stocks did not identify marked diversification of sequence as a result of this procedure. CONCLUSIONS: These stocks display more robust peripheral persistence and tissue pathology in cynomolgus macaques and should prove valuable analysing recombinant vaccines based upon SIVmacJ5 transgenes.

Flow cytometric study of T cell development in NOD mice reveals a deficiency in alphabetaTCR+CDR-CD8- thymocytes.

As a result of failed induction of T cell tolerance to pancreatic B cells, non-obese diabetic (NOD) mice develop spontaneous autoimmune insulin-dependent diabetes mellitus (IDDM). The thymic stroma, which plays a crucial role in thymic T cell maturation, undergoes extensive premature disorganization in NOD mice, so it is of interest to examine NOD T cell development. In this study, both major and minor developmental populations of thymocytes of NOD/Lt mice were studied and compared to those of BALB/c, C57BL/6 and CBA mice by multiparameter flow cytometry (FACS). These results are described in detail and reveal that most thymocyte subsets were normally represented, including alphaTcR-CD4-CD8- (triple negative; TN), alphabetaTcR-CD4+CD8- and alphabetaTcR-CD4-CD8+ (immature single positive; ISP), alphabetaTcR-/lowCD4+CD8+ (double positive; DP) and alphabetaTcR+CD4+CD8- and alphabetaTcR+CD4-CD8+ (mature single positive; SP) as well as gammadelta T cells. However, NOD mice exhibited a marked deficiency of thymic alphabetaTcR+CD4-CD8- (alphabeta+DN) T cells. alphabeta+DN T cells, which are included among NK1+ T cells in C57BL/6 mice, produce large amounts of IL-4 on primary stimulation. Given the potential significance of NKT cells in immunoregulation, it is possible that the scarcity of these cells in NOD mice plays a role in the pathogenesis of IDDM.

Mechanisms of protection induced by attenuated simian immunodeficiency virus. I. Protection cannot be transferred with immune serum.

To evaluate its role in protection, immune serum was collected from four macaques which were chronically infected with live attenuated simian immunodeficiency virus (SIVmacC8) and had resisted challenge with wild-type SIVmacJ5. The immune serum was transferred to two naive cynomolgus macaques by intraperitoneal injection (11 ml/kg). Four control macaques received an intraperitoneal injection of normal saline. One day later, all macaques were challenged with 10 MID50 of the J5M challenge stock of SIV. After challenge, all macaques became infected as determined by virus co-culture and diagnostic PCR. Virus loads in PBMC at 2 weeks post-challenge were indistinguishable between the two groups of macaques. Thus, the failure of passive immunization to transfer protection indicates that serum components alone are not sufficient to mediate the potent protection obtained using live attenuated vaccines. This is the first time that serum has been transferred from animals known to be protected against superinfection.

Mechanisms of protection induced by live attenuated simian immunodeficiency virus: III. Viral interference and the role of CD8+ T-cells and beta-chemokines in the inhibition of virus infection of PBMCs in vitro.

In this study, we investigated whether a type of retroviral interference might be one mechanism that mediates the powerful protection induced by live attenuated SIVC8. Our results show that retroviral interference could be demonstrated between SIV and SHIV-HXBc2 in human T-cell lines chronically infected with either SIVC8 or SIVJ5. Lymphocytes from macaques infected with live attenuated SIVC8 were significantly less sensitive (P < 0.05) to in vitro infection by virulent SIVJ5 and SHIV-HXBc2 than were lymphocytes from naive controls. However, this significant difference in the sensitivity of lymphocytes to virus infection was not observed for more efficiently replicating viruses such as SHIVSF33 and SIVsm3. Virus growth was significantly enhanced (P < 0.01) by depletion of CD8+ T-cells, suggesting a role for these cells in the control of SIV replication, both in vitro and in vivo. We found that levels of the beta-chemokines regulated upon activation, normal T-cell expressed and secreted, macrophage inflammatory protein-1alpha and macrophage inflammatory protein-1beta did not correlate with inhibition of virus replication. Taken together, our findings do not support the hypothesis that retroviral interference is the mechanism by which live attenuated SIVC8 induces protection.

Vaccine-induced CD4+ T cells against the simian immunodeficiency virus gag protein. Epitope specificity and relevance to protective immunity.

We have examined the induction and epitope specificity of T cells for the simian immunodeficiency virus (SIV) gag p27 protein in macaques immunized with either a recombinant SIV gag protein or an inactivated SIV vaccine. CD4+ MHC class II-restricted T cell lines and clones derived from five immunized macaques recognized a total of seven peptides in three immunodominant regions of p27. Two T cell clones generated from one of the lines, recognized a single 20 amino acid peptide that overlapped with a region previously shown to include a CTL epitope from SIV-infected macaques. Although this epitope is in a conserved region of the gag protein of SIV, its recognition by a CD4+ T cell clone was abrogated by sequence variation in the equivalent HIV protein. The specificity of the T cell lines for synthetic peptides demonstrated considerable overlap between T cells generated by immunization with the recombinant gag protein and inactivated SIV. However, in contrast to the protective efficacy of the whole virus vaccine in the syntex adjuvant formulation, immunization with the p27 protein with alum failed to generate a protective immune response. Furthermore, despite the consistent gag-specific T cell responses induced by the recombinant protein, there was no evidence of an enhanced antibody response to envelope (env) after live SIV challenge.

Immunisation of macaques with SIV env recombinants: specificity of T cell and antibody responses and evaluation of protective efficacy.

Macaques were immunised with lentil lectin purified recombinant SIVmac (BK28) derived gp160 (rgp160) with or without live vaccinia (vac)-env (BK28) priming, followed by a final boost with solid matrix antibody antigen (SMAA)-gp160 (J5) complexes and challenged with the SIVmac molecularly cloned virus J5M. Rgp160 and vac-env plus gp160 induced strong Ab responses against the homologous virus. Live vac-env did not enhance or prolong the antibody response, however, T cell responses were stronger. Analysis of the specificity of the immune response demonstrated that sequence variation within SIVmac viruses can affect antibody and T cell recognition. A single booster immunisation with the heterologous SIVmac J5 env recombinant protein was not sufficient to protect against the molecularly cloned virus J5M. These findings further illustrate the difficulty of generating protective immunity with immunogens based on single sequence recombinants.

Protection against SIV infection in macaques by immunization with inactivated virus from the BK28 molecular clone, but not with BK28-derived recombinant env and gag proteins.

Vaccination of cynomolgus macaques with beta-propiolactone inactivated SIVmacBK28 in Freund's adjuvant induced low but detectable levels of anti-SIV envelope (env) antibodies and T-cell responses and protected against challenge with the 32H isolate of SIVmac251 grown in C8166 cells. In contrast, purified recombinant SIV env and gag proteins derived from BK28 formulated in Syntex adjuvant generated consistent and long-lived cellular and humoral immune responses to SIV env, but failed to protect against infection with the 32H virus. Thus, protection against a heterogeneous challenge stock is possible by immunization with a molecularly-cloned virus, but not with recombinant proteins from the same molecular origin. High levels of anti-cell antibodies induced by the whole virus vaccine, but not by recombinant proteins, may have contributed to the protection observed.