Characteristics of a pathogenic molecular clone of an end-stage serum-derived variant of simian immunodeficiency virus (SIV(F359)).

End-stage simian immunodeficiency virus (SIV) isolates are suggested to be the most fit of the evolved virulent variants that precipitate the progression to AIDS. To determine if there were common characteristics of end-stage variants which emerge from accelerated cases of AIDS, a molecular clone was derived directly from serum following in vivo selection of a highly virulent SIV isolate obtained by serial end-stage passage in rhesus monkeys (Macaca mulatta). This dominant variant caused a marked cytopathic effect and replicated to very high levels in activated but not resting peripheral blood lymphocytes. Furthermore, although this clone infected but did not replicate to detectable levels in rhesus monocyte-derived macrophages, these cells were able to transmit infection to autologous T cells upon contact. Interestingly, although at low doses this end-stage variant did not use any of the known coreceptors except CCR5, it was able to infect and replicate in human peripheral blood mononuclear cells homozygous for the Delta 32 deletion of CCR5, suggesting the use of a novel coreceptor. It represents the first pathogenic molecular clone of SIV derived from viral RNA in serum and provides evidence that not only the genetic but also the biological characteristics acquired by highly fit late-stage disease variants may be distinct in different hosts.

Independence of herpesvirus-induced T cell lymphoma from viral cyclin D homologue.

Cyclin D family members are cellular protooncogenes, and their viral homologues in the Kaposi's sarcoma-associated herpesvirus (KSHV, human herpesvirus type 8 [HHV-8]) and the closely related Herpesvirus saimiri have been implicated as putative cofactors of viral transformation and pathogenesis. KSHV is regularly found in Kaposi's sarcoma and in the primary effusion B cell lymphoma and Castleman's disease associated with immunosuppression and AIDS. H. saimiri strain C488 transforms human and marmoset T cells in vitro and causes polyclonal T cell lymphoma in New World monkeys. The viral cyclins stimulate cell cycle progression of quiescent fibroblasts, and they form active cyclin-dependent kinase (CDK)6 complexes of broad substrate specificity that can resist and downregulate cellular CDK inhibitors. This study shows that the viral cyclin of H. saimiri strain C488 is not required for viral replication, T cell transformation, and pathogenicity in New World primates.

Safety and immunogenicity of ALVAC wild-type human p53 (vCP207) by the intravenous route in rhesus macaques.

p53 is over-expressed in approximately 50% of human cancers, and transfer of cytotoxic T lymphocytes (CTL) against wild-type p53 protects mice against p53-over-expressing tumors, suggesting that p53 might be an attractive target for immunotherapy. Immunization of mice with a recombinant canarypox virus, ALVAC, expressing human wild-type p53 (vCP207) prevented growth of p53-over-expressing tumors. Since intravenous administration induced better immune responses in mice than other routes, we have proposed to use this route in cancer patients. However, because this vector has never been administered intravenously to humans, and because of the possibility of inducing auto-immunity to a self-antigen, we felt it was necessary to first evaluate safety in rhesus macaques. We found that three intravenous administrations of vCP207 at proportional doses up to 10x those proposed for humans produced no abnormalities in hematologic or clinical chemistry parameters. Serologic markers of autoimmunity and inflammation were unaffected, despite the >95% amino acid identity between human and rhesus p53. Pathological examination of numerous tissues yielded findings comparable to those in animals given placebo. Some animals showed anti-p53 antibody responses following vaccination, indicating that tolerance could be broken to some extent. However, with the exception of one animal with a possible delayed type hypersensitivity reaction to p53 protein, we did not see evidence for a cell-mediated response. The safety profile in monkeys with ALVAC-p53 provides encouragement for using such live, modified vectors via the intravenous route for human immunotherapy.

Herpesvirus saimiri vFLIP provides an antiapoptotic function but is not essential for viral replication, transformation, or pathogenicity.

Apoptosis of infected cells is an important host defense mechanism, and many viruses have exploited antiapoptotic proteins that interfere with crucial cellular pathways. Viral FLICE inhibitory proteins (vFLIPs) are encoded by rhadinoviruses like herpesvirus saimiri, the related Kaposi's sarcoma-associated herpesvirus-human herpesvirus 8 (KSHV/HHV8), and the poxvirus responsible for molluscum contagiosum. The vFLIPs can block the interaction of the death receptor-adapter complex with the cellular effector FLICE (caspase-8), and this prevents the initiation of the downstream caspase cascade. KSHV/HHV8 vFLIP overexpression can confer resistance to T-cell-mediated apoptosis and acts as a tumor progression factor in a murine B-cell lymphoma model. To analyze the function of herpesvirus vFLIPs in the genetic background of the virus and in a model for viral pathogenesis, we deleted the vFLIP gene (open reading frame 71) from the genome of herpesvirus saimiri strain C488. The viral deletion mutant was viable and replicated like the wild-type virus. An antiapoptotic effect could be attributed to the vFLIP gene, but we also show that the vFLIP gene of herpesvirus saimiri is dispensable for viral transformation of T cells in vitro and for pathogenicity in cottontop tamarins in vivo.

A vaccine strategy utilizing a combination of three different chimeric vectors which share specific vaccine antigens.

A large number of recombinant of viral and bacterial systems have been engineered as vectors to express foreign genes for vaccination and/or gene therapy. A common problem is the immune response to the vector itself. The presence of anti-vector immune responses may preclude sufficient 'priming' or immunogenicity if pre-existing immune responses are present, or they may impair optimal 'boosting' upon repeated immunization or delivery with the same vector. To circumvent this problem we developed a strategy using different chimeric vectors which share only the expression of common specific antigens desired for immunization. This approach not only has the advantage of avoiding increased anti-vector responses, but allows the use of combinations of vectors which could subsequently present the same or related antigen differently to the immune system as well as at alternative sites to induce the optimal type of immunity against the pathogen of interest.

Antiretroviral therapy during primary immunodeficiency virus infection can induce persistent suppression of virus load and protection from heterologous challenge in rhesus macaques.

A limited period of chemotherapy during primary immunodeficiency virus infection might provide a long-term clinical benefit even if treatment is initiated at a time point when virus is already detectable in plasma. To evaluate this strategy, we infected rhesus macaques with the pathogenic simian/human immunodeficiency virus RT-SHIV and treated them with the antiretroviral drug (R)-9-(2-phosphonylmethoxypropyl)adenine (PMPA) for 8 weeks starting 7 or 14 days postinfection. PMPA treatment suppressed viral replication efficiently in all of the monkeys. After chemotherapy ended, virus replication rebounded and viral RNA in plasma reached levels comparable to that of the controls in four of the six monkeys. However, in the other two animals, virus loads peaked only moderately after withdrawal of the drug and then declined to low or even undetectable levels. These low levels of viremia remained stable for at least 31 weeks after cessation of therapy. At this time point, these two monkeys were challenged with SIV(8980) to evaluate whether the host responses which were able to keep RT-SHIV replication under control were also sufficient to protect against infection with a highly pathogenic heterologous virus. Both monkeys proved to be protected against the heterologous virus. In one of the two animals, low levels of SIV(8980) replication were detected. Thus, by chemotherapy during the acute phase of pathogenic virus replication, we could achieve not only persistent virus load suppression in two out of six monkeys but also protection from subsequent heterologous challenge. By this chemotherapeutic attenuation, the replication kinetics of attenuated viruses could be mimicked and a vaccination effect similar to that induced by live attenuated simian immunodeficiency virus vaccines was achieved.

Efforts to broaden HIV-1-specific immunity by boosting with heterologous peptides or envelope protein and the influence of prior exposure to virus.

In two previous studies, we have demonstrated the successful protection of human immunodeficiency virus type 1 (HIV-1)-vaccinated rhesus macaques from challenge with SHIV(SF13) with envelop immunogens derived from the closely related HIV-1(SF2) strain. Here we report on two follow-up studies in which we aimed to broaden immunity in order to elicit protection from a more diverse heterologous challenge with SHIV(SF33). In the first study, animals were boosted once with HIV-1(SF33) V2 and V3 peptides that were cross-linked to influenza immune-stimulating complexes (ISCOMs). In the second study, monkeys were boosted twice at 12-week intervals, using a heterologous recombinant gp120 derived from HIV-1(SF33) that was either incorporated into ISCOMs or mixed with the MF59 adjuvant. In both studies, the animals were challenged with 50 monkey infectious doses of SHIV(SF33) 4 weeks after the final boost. All controls became readily infected with the heterologous challenge virus SHIV(SF33). Neither boosting with heterologous SF33 peptides or gp120 afforded protection from infection to SF2-vaccinated animals that had previously resisted SHIV(SF13) challenge. These results demonstrate the importance of developing vaccine strategies that are capable of generating broad immune responses early in the immunization protocol. Furthermore, these findings may illustrate the potential pitfalls of early antigenic sin.

An anti-HIV strategy combining chemotherapy and therapeutic vaccination.

Combination chemotherapy using potent anti-retroviral agents has led to significant advances in the clinical management of human immunodeficiency virus (HIV) disease. However, the emergence of multiple drug-resistant mutants, the high need for compliance to adhere to demanding drug-dosing schemes, and the remaining toxic side-effects of drugs make the perspective of life-long treatment unattractive and possibly unrealistic. Therefore, means must be sought to shorten the time span during which treatment is necessary. Such means could be to stimulate an efficient immune response during the period of low virus load and restored CD4 + cell levels, which might be capable of keeping the virus under long-lasting control after treatment is stopped. Here we tested this concept of combined chemotherapy/ therapeutic vaccination in a non-human primate model. Rhesus macaques chronically infected with the chimeric simian/human immunodeficiency virus (SHIV) containing the HIV type 1 (HIV-1) HXBc2 gene for reverse transcriptase (RT) in the genomic background of simian immunodeficiency virus (SIV)(mac239) (RT-SHIV) were treated with (R)-9-(2-phosphonylmethoxypropyl)adenine (PMPA), a potent anti-HIV drug. When virus load had decreased significantly, we immunized with SIV genes env, gag/pol, rev, tat, and nef inserted in two different expression vector systems. Four weeks after the second immunization, drug treatment was stopped. Animals were monitored to determine if virus load stayed low or if it increased again to the original levels and if CD4+ T-cell levels remained stable. Humoral and cellular immune responses were also measured. This combined chemotherapy/ therapeutic vaccination regimen induced a significant reduction in the steady-state level of viremia in one out of two chronically infected rhesus macaques. Chemotherapeutic treatment alone did not achieve reduction of viremia in two chronically infected animals. The nature of the immune responses assumed to have been induced by vaccination in one out of the two monkeys remains to be elucidated.

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.

A pathogenic threshold of virus load defined in simian immunodeficiency virus- or simian-human immunodeficiency virus-infected macaques.

To determine if a specific pathogenic threshold of plasma viral RNA could be defined irrespective of virus strain, RNA levels in the plasma of more than 50 infected rhesus macaques (Macaca mulatta) were measured. Animals were inoculated intravenously with either simian immunodeficiency virus (SIV) or simian-human immunodeficiency virus (SHIV) strains of known pathogenic potential (SIV8980, SIVsmm-3, SIVmac32H/J5, SIVmac32H/1XC, reverse transcriptase-SHIV, SHIV89.6p) or with attenuated strains (SHIVW6.1D, SHIVsf13, SHIVhan-2, SIVmacDeltanef, SHIVsf33). In animals inoculated with nonpathogenic strains, shortly after the primary peak of viremia viral RNA levels declined and remained below 10(4) RNA equivalents/ml of plasma between 6 and 12 weeks postinoculation. Animals infected with documented pathogenic strains maintained viral RNA levels higher than 10(5) RNA equivalents/ml of plasma. In animals infected with strains with low virulence, a decline in plasma RNA levels was observed, but with notable individual variation. Our results demonstrate that the disease-causing potential was predicted and determined by a threshold plasma virus load which remained greater than 10(5) RNA equivalents/ml of plasma 6 to 12 weeks after inoculation. A threshold virus load value which remained below 10(4) RNA equivalents/ml of plasma was indicative of a nonpathogenic course of infection.

The interleukin-17 gene of herpesvirus saimiri.

In comparison to wild-type herpesvirus saimiri, viral interleukin-17 gene knockout mutants have unaltered behavior regarding viral replication, T-cell transformation in vitro, and pathogenicity in cottontop tamarins. Thus, this gene is not required for T-cell lymphoma induction but may contribute to apathogenic viral persistence in the natural host, the squirrel monkey.

T-cell lymphoma caused by herpesvirus saimiri C488 independently of ie14/vsag, a viral gene with superantigen homology.

The immediate-early gene ie14/vsag of herpesvirus saimiri has homology with murine superantigens. We compared the pathogenesis of infection with either ie14/vsag deletion mutants or wild-type virus C488 in cottontop tamarin monkeys (Saguinus oedipus). Two weeks after infection, all animals developed acute T-cell lymphomas independently of the presence of the viral ie14/vsag gene.

Comparison of in vitro and in vivo infectivity of different clade B HIV-1 envelope chimeric simian/human immunodeficiency viruses in Macaca mulatta.

The use of HIV-1 env/SIVmac chimeric viruses expressing divergent HIV-1 envelopes of clinical isolates, facilitates homologous and heterologous evaluation of various recombinant HIV-1 envelope vaccine candidates in lower primates. In this study we compare the in vitro and in vivo infectivity, via intravenous (IV) and intravaginal (IVAG) routes of infection, of stocks of chimeric viruses expressing env from four different clade B HIV-1 isolates. The TCID50/ml was 7.1 x 10(4), 1.0 x 10(4), 6.3 x 10(4), and 1.2 x 10(3) for SHIVsf13, SHIVHan2, SHIVNM-3rn, and SHIVW6.1D, respectively, with a MID50/ml upon IV inoculation of 3.2 x 10(3), 3.2 x 10(4), 3.2 x 10(4), and 3.2 x 10(3), respectively. The same SHIVsf13 stock was infectious after IVAG administration, requiring a 300-fold higher virus dose. Plasma antigenemia and cell-associated viremia were generally highest at weeks 2 or 4 after infection and decreased to subdetectable levels after 8-12 weeks. All infected animals tested developed anti-HIV-1 gp120 antibodies. Inoculated virus dose showed no (linear) quantitative correlation with cellular virus load, duration of viremia, plasma antigenemia, and anti-gp120 antibody titers. No significant changes in peripheral blood CD4 cell levels were observed and none of the animals has shown evidence of disease progression to date (i.e., 13 months postinfection). Four in vivo passages of cell-associated SHIVW6.1D did not result in increased virulence. Vaccine development studies in macaques monkeys have become feasible with the use of various clade B HIV-1 env SHIV chimeras.

The non-immunosuppressive cyclosporin A analogue SDZ NIM 811 inhibits cyclophilin A incorporation into virions and virus replication in human immunodeficiency virus type 1-infected primary and growth-arrested T cells.

SDZ NIM 811 is a cyclosporin A (CsA) analogue that is completely devoid of immunosuppressive capacity but exhibits potent and selective anti-human immunodeficiency virus type 1 (HIV-1) activity. Binding to cyclophilin A, the intracellular receptor for cyclosporins, is a prerequisite for HIV-1 inhibition by cyclosporins. Cyclophilin A was demonstrated to bind to HIV-1 p24gag and this cyclophilin-Gag interaction leads to the incorporation of cyclophilin A into HIV-1 virions. SDZ NIM 811 inhibits this protein interaction, and this is likely to be the molecular basis for its antiviral activity. Here, we show that in activated primary T cells SDZ NIM 811 interferes with two stages of the virus replication cycle: (i) translocation of pre-integration complexes into the nucleus and (ii) production of infectious virus particles. SDZ NIM 811 not only inhibits translocation of HIV-1 pre-integration complexes in primary T cells, but also in a growth-arrested T cell line. In vivo, most T lymphocytes are quiescent, but serve nevertheless as a major and inducible HIV-1 reservoir in infected individuals. Significant amounts of cyclophilin A were found to be associated with virus particles propagated in primary T cells. SDZ NIM 811 caused a strong reduction in the amount of incorporated cyclophilin A, thereby reducing infectivity. Thus, cyclophilin A seems to be necessary for HIV-1 replication in primary T cells.

Plasmodium vivax: in vitro antiparasitic effect of cyclosporins.

Plasmodium vivax is, next to P. falciparum, the second important human malaria parasite. In view of reports on developing chloroquine resistance, research on new drugs that are active against P. vivax is necessary. Due to a requirement for continuous addition of reticulocytes, long-term in vitro culture of P. vivax is not practicable. Conventional drug assays, i.e., culturing for a time equivalent to one asexual cycle of development in the presence of drugs, and then measuring propagation, are therefore not readily performed. In this report the in vitro susceptibility of P. vivax to cyclosporin A and a new, nonimmunosuppressive derivative, SDZ NIM 811, was investigated using parasite material obtained from an infected Aotus monkey. The assay was initiated with blood containing ring-stage parasites and was ended when parasites were multinucleate, but before merozoite release. The results were compared with the in vitro susceptibility of P. falciparum to these drugs in a conventional and a short assay. As an indicator of parasite propagation [3H]hypoxanthine incorporation was measured. The susceptibility of P. vivax to cyclosporins was found to be intermediate to that of P. falciparum in the conventional and in the much less sensitive short assay, and SDZ NIM 811 proved to be as active as cyclosporin A.

The use of cyclosporine, FK506, and SDZ NIM811 to prevent CD25- quiescent peripheral blood mononuclear cells from producing human immunodeficiency virus.

It has been shown that the combined use of two pharmacologic agents can inhibit human immunodeficiency virus (HIV) production by peripheral blood mononuclear cells in vitro. One, an anti-CD25 immunotoxin (IT), kills activated T cells that produce virus; the other, the immunosuppressive drug cyclosporine, prevents the quiescent cells, which harbor HIV, from becoming activated. The present study compares the antiviral activities of two agents, SDZ NIM811 and FK506, to that of cyclosporine. In combination with the anti-CD25 IT, these drugs significantly suppressed virus production. In the absence of prior addition of the IT, the ability of the drugs to inhibit virus production was much lower, suggesting that they work effectively in latently infected cells. In the case of SDZ NIM811, the inhibition of virus production was accompanied by a modest inhibition of cell proliferation. In contrast, FK506 exerted strong antiproliferative activity. Cyclosporine was both moderately antiproliferative and a potent antiviral agent.

Kinetics and isotype profile of rheumatoid factor production during viral infection: organ distribution of antibody secreting cells.

The kinetics and isotype profile of influenza virus-specific IgG antibodies were studied in correlation with the serum titre of IgG-reactive autoantibodies. An increased level of IgG isotype-specific, rheumatoid factor-type autoantibody secretion was observed in the late phase of the virus-specific memory response. These rheumatoid factors were specific for the IgG2a and IgG1 subclasses which dominated the anti-viral antibody response. As revealed by a preparative immunosorbent technique combined with isotype quantitation the majority of IgG2a- or IgG1-bound immunoglobulins isolated from the serum of virus-infected mice belonged to the same subclass as the target antibody. Comparison of the kinetics of appearance and the number of IgM-, IgG- and IgA-type IgG2a-reactive autoantibody secreting cells during the primary and memory anti-viral antibody responses showed isotype switch of IgM rheumatoid factor secreting cells predominantly to IgA. Localization of IgM and IgA antibody secreting cells demonstrated the wide organ distribution of IgM-type rheumatoid factor secreting cells. On the contrary, IgA rheumatoid factor production was observed only in Peyer's patches and at the site of the local virus-specific immune response, i.e. in mediastinal lymph nodes and in the lung. These results demonstrate that B cells specific for self IgG are activated and differentiated in concert with the viruspecific antibody response in similar microenvironments. The predominant involvement of the mediastinal lymph nodes and the spleen in the production of IgG2a-specific IgM-type autoantibodies suggest a regulatory function of this type of autoantibodies in modulating IgG2a production in both systemic and local anti-viral immune responses. The results also suggest a strictly regulated rheumatoid factor production which, however, can be unbalanced by repeated viral infections resulting in the escape of high affinity, isotype-switched autoantibodies.

Paracyclophanes: a novel class of water-soluble inhibitors of HIV proteinase.

A versatile synthesis of functionalized para- and metacyclophanes (macrocycles with one or more aromatic rings incorporated; ansa-compounds) has been developed. Cyclophanes constitute a novel building block for potent human immunodeficiency virus (HIV) protease inhibitors. The synthesis of the macrocyclic ring system was achieved by regio- and stereospecific ring opening of N-protected 4-amino-2,3-epoxy-5-phenylpentanoates with appropriate alpha, omega-diamines and consecutive ring closure under high dilution conditions. The resulting macrocyclic building blocks enabled further broad and flexible derivation. Paracyclophanes, containing oxyethylene substructures, were found to dissolve in phosphate-buffered saline at concentrations as high as 3 mg/mL at physiological pH. Several derivatives with Ki values lower than 10 nM and antiviral activities in the range of 15-50 nM have been obtained. The influence of the ring size and of the substitution pattern of the cyclophane moiety on enzyme inhibition, antiviral activity, and water solubility are discussed. Preliminary data on oral bioavailability in mice are given for selected compounds.

Inhibitors of human immunodeficiency virus type 1 protease containing 2-aminobenzyl-substituted 4-amino-3-hydroxy-5-phenylpentanoic acid: synthesis, activity, and oral bioavailability.

Systematic modifications of HIV protease inhibitor (2R,3S,4S)-4-[[(benzyloxycarbonyl)-L-valyl]-amino]-3-hydroxy-2-[(4- methoxybenzyl)amino]-5-phenylpentanoyl)-L-valine 2-(aminomethyl)- benzimidazole amide led to a novel series of inhibitors with shortened, modified carboxy terminus. Their synthesis, in vitro enzyme inhibitory data, and antiviral activities are reported. Of particular interest are derivatives featuring the (1S,2R)-1-amino-2-hydroxyindan moiety at the P2'-position since some of them exhibit substantial oral bioavailability in mice. The influence of aqueous solubility and structural parameters on the oral resorption of the inhibitors is discussed. Optimum enhancement of oral bioavailability was observed with L-tert-leucine in P2-position, resulting in the discovery of (2R,3S,4S)-4-[[(benzyloxycarbonyl)-L-tert-leucyl]- amino]-3-hydroxy-2-[(4-methoxybenzyl)amino]-5-phenylpentanoic acid (1S,2R)-1-amino-2-hydroxyindan amide which combines high antiviral activity (IC50 = 250 nM) with a good pharmacokinetic profile (AUC = 82.5 microM.h at a dose of 125 mg/kg po in mice).