Subcutaneous administration of a recombinant vaccinia virus vaccine expressing multiple envelopes of HIV-1.

A critical goal of HIV vaccine development is the identification of safe and immunogenic vectors. Recombinant vaccinia virus is a highly effective vaccine vector, with demonstrated capacity to protect animals from various viral pathogens, including rabies. Unlike many other candidate vaccine vectors, vast human experience exists with the parenteral smallpox vaccine. However, consideration of recombinant vaccinia virus as a modern vaccine is complicated by the relatively high prevalence of immunocompromised persons compared to such prevalence 4 or more decades ago (when smallpox vaccination was still routine). Administering vaccine by the subcutaneous (SQ) route, rather than the traditional scarification route, could address these concerns. SQ administration could prevent transmission of vaccinia virus to potentially vulnerable persons; it could also avoid the most common adverse events, which are cutaneous in nature. However, previous studies suggest that elicitation of immune response against passenger gene products following SQ administration requires development of a superficial pox lesion, defeating the intention of SQ administration. This is the first report to demonstrate that SQ administration of recombinant vaccinia virus does elicit immune response to the passenger protein in the absence of a cutaneous pox lesion. Results further show that a multi-envelope HIV vaccine can elicit antibody responses toward heterologous HIV-1 not represented by primary sequence in the vaccine. These findings have global implications because they support the consideration of recombinant vaccinia virus as a valuable HIV vaccine vector system.

MRP4: A previously unidentified factor in resistance to nucleoside-based antiviral drugs.

Dideoxynucleosides, which are potent inhibitors of HIV reverse transcriptase and other viral DNA polymerases, are a common component of highly active anti-retroviral therapy (HAART) (ref. 1). Six reverse transcriptase inhibitors have been approved for human use: azidothymidine; 2'3'-dideoxycytidine; 2'3'-dideoxyinosine; 2', 3'-didehydro-3'deoxythymidine; 2',3'-dideoxy-3'-thiacytidine; and 4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-++ +metha nol. Although drug-resistant HIV strains resulting from genetic mutation have emerged in patients treated with HAART (ref. 1), some patients show signs of drug resistance in the absence of drug-resistant viruses. In our study of alternative or additional mechanisms of resistance operating during antiviral therapy, overexpression and amplification of the MRP4 gene correlated with ATP-dependent efflux of PMEA (9-(2-phosphonylmethoxyethyl)adenine) and azidothymidine monophosphate from cells and, thus, with resistance to these drugs. Overexpression of MRP4 mRNA and MRP4 protein severely impaired the antiviral efficacy of PMEA, azidothymidine and other nucleoside analogs. Increased resistance to PMEA and amplification of the MRP4 gene correlated with enhanced drug efflux; transfer of chromosome 13 containing the amplified MRP4 gene conferred resistance to PMEA. MRP4 is the first transporter, to our knowledge, directly linked to the efflux of nucleoside monophosphate analogs from mammalian cells.

A novel vaccine regimen utilizing DNA, vaccinia virus and protein immunizations for HIV-1 envelope presentation.

Recombinant DNA and vaccinia virus (VV) vectors that express envelope (Env) proteins of the human immunodeficiency virus (HIV) have each been prominently utilized in vaccine development. These two vectors (termed DNA-Env and VV-Env) are attractive vaccine candidates due to their abilities to elicit both cytotoxic T-lymphocyte and B-cell responses. Our previous work demonstrated that DNA-Env primed animals, that were relatively unresponsive to DNA-Env boosters, could be immunized with VV-Env to yield more than a 100-fold increase in antibody responses. Here we show: (1) results with an optimized vaccine regimen that primes with DNA-Env, boosts with VV-Env, and re-boosts with purified Env proteins, (2) enhanced responses with 8 rather than 16 week intervals between VV-Env and protein immunizations, and (3) the failure of single Env vaccines to reproducibly elicit responses toward heterologous Env, regardless of the vaccination regimen utilized. Results encourage the use of poly-Env vaccine cocktails administered via DNA/VV/protein regimens in future non-human primate and clinical studies.

Human immunodeficiency virus protease inhibitors serve as substrates for multidrug transporter proteins MDR1 and MRP1 but retain antiviral efficacy in cell lines expressing these transporters.

The human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs)-saquinavir, ritonavir, nelfinavir, and indinavir-interact with the ABC-type multidrug transporter proteins MDR1 and MRP1 in CEM T-lymphocytic cell lines. Calcein fluorescence was significantly enhanced in MDR1(+) CEM/VBL100 and MRP1(+) CEM/VM-1-5 cells incubated in the presence of various HIV PIs and calcein acetoxymethyl ester. HIV PIs also enhanced the cytotoxic activity of doxorubicin, a known substrate for MDR1 and MRP1, in both VBL100 and VM-1-5 CEM lines. Saquinavir, ritonavir, and nelfinavir enhanced doxorubicin toxicity in CEM/VBL100 cells by approximately three- to sevenfold. Saquinavir and ritonavir also enhanced doxorubicin toxicity in CEM/VM-1-5 cells. HIV-1 replication was effectively inhibited by the various PIs in all of the cell lines, and the 90% inhibitory concentration for a given compound was comparable between the different cell types. Therefore, overexpression of MDR1 or MRP1 by T lymphocytes is not likely to limit the antiviral efficacy of HIV PI therapy.

Immunoprotective determinants in friend murine leukemia virus envelope protein.

Several immunological epitopes are known to be located within the Friend murine leukemia virus (F-MuLV) envelope protein, but their relative contributions to protection from Friend virus-induced disease are not known. To determine how expression of various immunological determinants affected protection, mice were immunized with recombinant vaccinia viruses expressing different portions of the F-MuLV envelope protein, and they were then challenged with a lethal dose of Friend virus complex. The disease parameters that were followed in the mice were early viremia, early splenomegaly, and late splenomegaly. Both the N-terminal and C-terminal portions of the F-MuLV gp70 were found to protect against late splenomegaly, the primary clinical sign associated with virus-induced erythroleukemia. However, neither region alone protected against early splenomegaly and early viremia, indicating poor immunological control over early virus replication and spread through the spleen and blood. In contrast, mice immunized with a vaccine expressing the entire F-MuLV envelope protein were protected against all three disease parameters. The results indicated that expression of multiple immunological determinants including both T-helper and B cell epitopes was necessary for full protection.

Antiviral activities of 9-R-2-phosphonomethoxypropyl adenine (PMPA) and bis(isopropyloxymethylcarbonyl)PMPA against various drug-resistant human immunodeficiency virus strains.

9-R-2-Phosphonomethoxypropyl adenine (PMPA) is an acyclic nucleoside phosphonate analog that has demonstrated efficacy against human immunodeficiency virus (HIV). We recently described the synthesis, metabolism, and biological activities of bis(isopropyloxymethylcarbonyl)PMPA [bis(poc)PMPA] as an orally bioavailable prodrug for PMPA. Among a large panel of drug-resistant HIV type 1 variants, only the K65R virus was resistant to PMPA. K65R virus also showed reduced susceptibility to bis(poc)PMPA, although the prodrug could still inhibit these viruses at submicromolar, nontoxic concentrations. Among a panel of seven primary clinical isolates from patients with diverse treatment histories, only one isolate showed reduced susceptibility to PMPA and was found to carry three mutations (M41L, T69N, R73K) in its reverse transcriptase catalytic domain.

Anti-human immunodeficiency virus activity and cellular metabolism of a potential prodrug of the acyclic nucleoside phosphonate 9-R-(2-phosphonomethoxypropyl)adenine (PMPA), Bis(isopropyloxymethylcarbonyl)PMPA.

Bis(isopropyloxymethylcarbonyl) 9-R-(2-phosphonomethoxypropyl)adenine [bis(POC)PMPA] has been identified as a novel prodrug of PMPA. The anti-human immunodeficiency virus activity of bis(POC)PMPA was >100-fold greater than that of PMPA in both an established T-cell line and primary peripheral blood lymphocytes. This improved efficacy was shown to be due to a rapid intracellular uptake of the prodrug resulting in an increased intracellular accumulation of PMPA diphosphate (PMPApp), the pharmacologically active metabolite. PMPApp levels in bis(POC)PMPA-treated cells exceeded by >1,000-fold the levels seen in cells treated with unmodified PMPA in both resting and activated peripheral blood lymphocytes. Significant differences in the intracellular catabolism of PMPA metabolites were noted between the resting and activated lymphocytes. The half-life for the disappearance of PMPApp, derived from either bis(POC)PMPA or PMPA, was 12 to 15 h in the activated lymphocytes and 33 to 50 h in the resting lymphocytes. This long persistence of PMPApp, particularly in resting lymphocytes, may be unique to the nucleoside phosphonate analogs and indicates that effective levels of the active metabolite can be achieved and maintained with relatively infrequent administration of the parent drug.

In vitro induction of human immunodeficiency virus type 1 variants resistant to 2'-beta-Fluoro-2',3'-dideoxyadenosine.

2'-beta-Fluoro-2',3'-dideoxyadenosine (F-ddA) is an acid-stable purine dideoxynucleoside analog active against a wide spectrum of human immunodeficiency virus type 1 (HIV-1) and HIV-2 strains in vitro. F-ddA is presently undergoing a phase I clinical trial at the National Cancer Institute. We induced HIV-1 variants resistant to F-ddA by exposing wild-type HIV-1 (HIV-1LAI) to increasing concentrations of F-ddA in vitro. After 18 passages, the virus was fourfold less sensitive to F-ddA than HIV-1LAI. Sequence analyses of the passage 18 virus revealed changes in three amino acids in the reverse transcriptase (RT)-encoding region of the pol gene: P to S at codon 119 (P119S; present in 3 of 13 and 28 of 28 molecular clones before and after F-ddA exposure, respectively), V179D (0 of 13 and 9 of 28, respectively), and L214F (9 of 13 and 28 of 28, respectively). Drug sensitivity assays using recombinant infectious clones confirmed that P119S was directly responsible for the reduced sensitivity of HIV-1 to F-ddA. Various infectious clones with single or multiple amino acid substitutions conferring viral resistance against nucleoside RT inhibitors, including HIV-1 variants with multi-dideoxynucleoside resistance, were generally sensitive to F-ddA. The moderate level of resistance of HIV-1 to F-ddA, together with the lack of conferment of significant cross-resistance by the F-ddA-associated amino acid substitutions, warrants further investigation of F-ddA as a potential antiviral agent for use in treatment of HIV-1 infection.

(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (HPMPC) inhibits HIV-1 replication in epithelial cells, but not T-lymphocytes.

PMEA [9-(2-phosphonylmethoxyethyl)adenine] inhibited both HSV-1 and HIV-1 replication in MT-2 and HeLa-CD4 cells. (S)-1-[3-hydroxy-2-(phosphonylmethoxy)propyl]cytosine (HPMPC) inhibited both these viruses in the epithelioid HeLa-CD4 cells, but did not inhibit either virus in the T-lymphocytic MT-2 cells. PMEA and HPMPC are metabolized to their diphosphorylated forms within cells, which then inhibit viral polymerases. We therefore compared the metabolism of PMEA and HPMPC in MT-2 and HeLa CD4 cells. PMEApp formation was efficient in both the cell types, whereas HPMPCpp levels were approximately 3-10 fold lower in MT-2 cells, compared to HeLa-CD4 cells. These results indicate that HPMPC can inhibt HIV replication in the appropriate cell types, and show that differences in their metabolism cannot account entirely for the lack of antiviral efficacy of HPMPC in MT-2 cells.

Inhibition of human immunodeficiency virus type 1 replication by a cellular transcriptional factor MBP-1.

A cellular transcriptional factor initially identified as the c-myc promoter binding protein (MBP-1) was subsequently characterized as a cell regulatory protein with multifunctional activities. In this study, the role of MBP-1 on human immunodeficiency virus type-1 (HIV-1) transcriptional activity was investigated. MBP-1 showed inhibition of HIV-1 long terminal repeat (LTR)-directed chloramphenicol acetyl transferase (CAT) activity in a transient cotransfection assay. Deletion of upstream elements of the HIV-1 LTR, including the nuclear factor kappa B (NF-kappa B) and Sp1 binding sites, did not affect the MBP-1 mediated suppression of HIV-1 LTR. The core promoter of the HIV-1 appeared to be the primary sequence involved in MBP-1 mediated inhibition. In the presence of HIV-1 TAR sequence and Tat protein, MBP-1 did not inhibit the viral promoter activity. In addition, cotransfection experiments with HIV-1 LTR and deletion mutants of MBP-1 suggested that the carboxyl terminal half of MBP-1 suppresses the HIV-1 promoter activity. Exogenous expression of MBP-1 showed suppression of HIV-1 replication in acutely infected cells and in cells cotransfected with a molecular clone of HIV-1. These results suggest that exogenous expression of MBP-1 plays an important role in the regulation of HIV-1 replication in infected cells.

Eliciting HIV-1 envelope-specific antibodies with mixed vaccinia virus recombinants.

Recombinant vaccinia virus (VV) vectors that express the envelope (Env) protein of the human immunodeficiency virus-type 1 (HIV-1) have been previously shown to elicit HIV-specific cytotoxic T-lymphocyte (CTL) and weak antibody responses in non-human primate studies and clinical trials. In first clinical trials, single Env proteins were presented to the immune system by VV recombinants and other vectors, but individuals were not protected against later exposures to heterologous HIV. It is likely that the generation of protective immune responses against diverse HIV will require that vaccines encompass proteins from not just one, but multiple distinct HIV isolates. Here is described the simple construction of numerous new VV, each expressing a unique, truncated, Env protein (VVenv). Mouse experiments were performed to evaluate the ability of these VVenv to elicit immune responses. HIV-1-specific antibodies appeared within one month following one intraperitoneal inoculation of mice with single or mixed VVenv, reaching plateau levels by 4 months. The magnitude of antibody production was poor at the dose of 10(5) p.f.u. VVenv per animal, but improved with increasing doses of VVenv up to 10(7) p.f.u. per animal. The subcutaneous route of VV immunization, previously proven safe in human trials, was also effective for administering VVenv. These results highlight the strengths of recombinant VV constructs as vehicles for the presentation of multiple HIV-1-Env proteins to the naive immune system.

Hepatitis C virus core protein inhibits human immunodeficiency virus type 1 replication.

We previously demonstrated that hepatitis C virus (HCV) core protein is a strong repressor of human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) basal transcription. In this study, we have localized the HCV core protein-response domain to a region between nucleotides -65 and +3 within the HIV-LTR. Thus, neither the upstream negative regulatory elements, or binding sites for various transcription factors (e.g. NF-kappa B, USF-1, IL2/IL-2R) nor the downstream TAR regions were involved in HCV core-mediated repression. HCV core protein mediated repression of the basal transcriptional activity of HIV-1 LTR was abrogated by the Tat protein. Furthermore, HeLa-T4 cells expressing HCV core protein showed inhibition of HIV-1 replication after acute infection with cell-free HIV. A similar observation was also noted in CD4+ and CD4-lymphocytic cell lines cotransfected with an infectious molecular clone of HIV-1 and the HCV core protein expression vector. Thus, a repression of basal transcription prior to the accumulation of threshold levels of Tat protein appears to restrict HIV-1 transcription and modulate viral replication.

Enhanced susceptibility to human immunodeficiency virus infection in CD4+ T lymphocytes genetically deficient in CD43.

CD43 is a cell surface sialoglycoprotein expressed by most cells of hematopoietic origin, including all T lymphocytes. Elimination of CD43 expression by gene targeting in the CEM T cell line results in its increased homotypic adhesion and binding to HIV-1 gp120. Here we report that the CD43-negative CEM cells show increased susceptibility to HIV-1 infection and increased viral replication compared with the parental CD43+ CEM cell line. Increased HIV-1 replication also was observed in CEM cells with diminished CD43 expression secondary to functional inactivation of a single CD43 allele. The CD43- CEM cells were more susceptible to HIV-1-induced cytopathicity than their CD43+ counterparts. HIV-1 replication also was increased in the CD43- CEM cells after transfection with the infectious HIV molecular clone pNL4-3. These data suggest that factors that diminish CD43 expression on T lymphocytes may enhance their susceptibility to HIV-1 infection.

Metabolic diversity and antiviral activities of acyclic nucleoside phosphonates.

The acyclic nucleoside phosphonates (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (HPMPC), (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine (HPMPA), and 9-(2-phosphonylmethoxyethyl)adenine (PMEA) inhibited herpes simplex virus-1 replication in Vero cells, and the IC50 values ranged from 4 microM (for HPMPC and HPMPA) to 40 microM (for PMEA). Pretreatment of cells with HPMPC for 12-24 hr induced an effective antiviral state, and the cells maintained this antiviral state for > 7 days. In contrast, much larger amounts (approximately 2.5-5 x IC50 doses) of PMEA or HPMPA were required to establish an antiviral state, which lasted for only approximately 24 or 72 hr, respectively. A 12-hr treatment of the cells with the phosphonates was required for the establishment of optimal antiviral activity; surprisingly, longer durations of exposure to PMEA (but not HPMPA or HPMPC) resulted in diminished antiviral effect. We investigated the metabolism of PMEA and HPMPC to determine the cellular basis for these differences. The cellular uptake of HPMPC was approximately 8-fold greater than that of PMEA. The levels of the PMEA metabolites PMEA monophosphate and PMEA diphosphate increased for approximately 12 hr and plateaued thereafter. PMEA and its metabolites were cleared from the cells with a half-life of 4.9 hr. In contrast, the HPMPC metabolites HPMPC monophosphate (HPMPCp) and HPMPC diphosphate (HPMPCpp) accumulated throughout the 24-hr study period and, at equimolar drug concentrations (25 microM), reached intracellular levels approximately 2-3-fold greater than those of the PMEA metabolites. HPMPC also differed from PMEA in its capacity to generate a phosphodiester metabolite (HMPCp-choline), which was a predominant metabolite in HPMPC-treated cells. In addition, the rates of disappearance of intracellular metabolites of the two drugs were significantly different. Thus, the decay of HPMPCpp was quite slow and biphasic (t1/2 = 24 and 65 hr) and that of HMPCp-choline was monophasic (t1/2 = 87 hr). Together, these factors can explain the differing antiviral potencies seen with PMEA and HPMPC. The possible role of the choline adduct in the expression of antiviral activity of the drug remains to be elucidated, but the adduct may serve as an intracellular store for the long term maintenance of active HPMPCpp in cells. The results also highlight the extent of diversity in the cellular pharmacology and antiviral activities of the acyclic nucleoside phosphonates.

A human T lymphoid cell variant resistant to the acyclic nucleoside phosphonate 9-(2-phosphonylmethoxyethyl)adenine shows a unique combination of a phosphorylation defect and increased efflux of the agent.

9-(2-Phosphonylmethoxyethyl)adenine (PMEA) is a new antiviral agent with activity against herpes viruses and retroviruses, including human immunodeficiency virus, but its metabolism and mechanism of action remain unclear. We have isolated a human T lymphoid cell line (CEMr-1) that is resistant to the antiproliferative effects of PMEA. The antiviral effects of PMEA against human immunodeficiency virus-1 infection were also greatly reduced in CEMr-1 cells, compared with the parental cells. This mutant showed cross-resistance to the related acyclic nucleoside phosphonates 9-(2-phosphonylmethoxyethyl)diaminopurine and 9-(2-phosphonylmethoxyethyl)guanine and the lipophilic prodrug bis(pivaloyloxymethyl)-9-(2-phosphonylmethoxyethyl)adenine-( bispome-PMEA), as well as partial resistance to the purine nucleosides 2-chlorodeoxyadenosine, 2-fluro-9-beta-D-arabinosylfuranosyladenine, and adenosine, but did not show resistance to 2'-deoxyadenosine or 9-beta-D-arabinosylfuranosyladenine. We compared the uptake and metabolism of [3H]PMEA and [3H]-bispom-PMEA in the mutant and parental cells. The analysis of radioactive products by high pressure liquid chromatography revealed marked alterations in the ability of the mutant cell line to accumulate PMEA and its anabolites, compared with the parental cells. Accumulation of PMEA, PMEA monophosphate, and PMEA bisphosphate (major metabolites formed with either PMEA or bispom-PMEA) decreased by 50, 95, and 97%, respectively. Compared with the parental cells, the variant cells showed a approximately 7-fold increase in the rate of efflux of PMEA and a 2-fold decrease in the activity of adenylate kinase. In contrast, other enzymes of nucleotide metabolism, such as adenosine kinase, deoxycytidine kinase, and 5-phosphoribosyl-1-pyrophosphate synthetase, showed no significant change in the two cell lines. Overall, these results suggest that the mutation in this resistant cell line is of a novel type, involving an alteration in the cellular efflux of PMEA as the major basis for the resistant phenotype.

Moloney leukemia virus-induced cell surface antigen mimicry by monoclonal antibodies.

We have investigated antigen-independent modulation of immune responses by monoclonal antibodies directed against both viral and nonviral antigens. BALB/c mice were immunized with monoclonal IgM (i.e. Ab1) specific for either Moloney murine leukemia virus-induced cell surface antigen (MCSA) or the hapten 2,4-dinitrophenyl (DNP). Injection with either Ab1 activated a functional idiotypic (Id) network as evidenced by production of both anti-Id (Ab2) antibodies and anti-anti-Id (Ab3) antibodies. A subset of induced Ab3 (designated Ab1'), exhibited specificity for antigen (virus or DNP). In mice immunized with anti-Id antibodies (Ab2), production of Ab3 and Ab1' was also observed. In the MCSA system, antibody-induced Ab1' responses were effective in protecting mice from tumor development upon subsequent challenge with live virus. Furthermore, antigen-independent modulation of immunity to both viral and nonviral antigens was found to be thymus-dependent. Similar findings in other viral systems suggest that antibody-induced activation of Id networks may prove a viable alternative vaccine strategy that can elicit antigen-specific responses, and in some cases protection, in the apparent absence of exposure to antigen.